The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements.
Procaccini Claudio,Carbone Fortunata,Di Silvestre Dario,Brambilla Francesca,De Rosa Veronica,Galgani Mario,Faicchia Deriggio,Marone Gianni,Tramontano Donatella,Corona Marco,Alviggi Carlo,Porcellini Antonio,La Cava Antonio,Mauri Pierluigi,Matarese Giuseppe
Human CD4(+)CD25(hi)Foxp3(+)CD127(-) Treg and CD4(+)CD25(-)Foxp3(-) Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells.
Tumour-associated macrophages as treatment targets in oncology.
Mantovani Alberto,Marchesi Federica,Malesci Alberto,Laghi Luigi,Allavena Paola
Nature reviews. Clinical oncology
Macrophages are crucial drivers of tumour-promoting inflammation. Tumour-associated macrophages (TAMs) contribute to tumour progression at different levels: by promoting genetic instability, nurturing cancer stem cells, supporting metastasis, and taming protective adaptive immunity. TAMs can exert a dual, yin-yang influence on the effectiveness of cytoreductive therapies (chemotherapy and radiotherapy), either antagonizing the antitumour activity of these treatments by orchestrating a tumour-promoting, tissue-repair response or, instead, enhancing the overall antineoplastic effect. TAMs express molecular triggers of checkpoint proteins that regulate T-cell activation, and are targets of certain checkpoint-blockade immunotherapies. Other macrophage-centred approaches to anticancer therapy are under investigation, and include: inhibition of macrophage recruitment to, and/or survival in, tumours; functional re-education of TAMs to an antitumour, 'M1-like' mode; and tumour-targeting monoclonal antibodies that elicit macrophage-mediated extracellular killing, or phagocytosis and intracellular destruction of cancer cells. The evidence supporting these strategies is reviewed herein. We surmise that TAMs can provide tools to tailor the use of cytoreductive therapies and immunotherapy in a personalized medicine approach, and that TAM-focused therapeutic strategies have the potential to complement and synergize with both chemotherapy and immunotherapy.
Infarcted Myocardium-Primed Dendritic Cells Improve Remodeling and Cardiac Function After Myocardial Infarction by Modulating the Regulatory T Cell and Macrophage Polarization.
Choo Eun Ho,Lee Jun-Ho,Park Eun-Hye,Park Hyo Eun,Jung Nam-Chul,Kim Tae-Hoon,Koh Yoon-Seok,Kim Eunmin,Seung Ki-Bae,Park Cheongsoo,Hong Kwan-Soo,Kang Kwonyoon,Song Jie-Young,Seo Han Geuk,Lim Dae-Seog,Chang Kiyuk
BACKGROUND:Inflammatory responses play a critical role in left ventricular remodeling after myocardial infarction (MI). Tolerogenic dendritic cells (tDCs) can modulate immune responses, inducing regulatory T cells in a number of inflammatory diseases. METHODS:We generated tDCs by treating bone marrow-derived dendritic cells with tumor necrosis factor-α and cardiac lysate from MI mice. We injected MI mice, induced by a ligation of the left anterior descending coronary artery in C57BL/6 mice, twice with tDCs within 24 hours and at 7 days after the ligation. RESULTS:In vivo cardiac magnetic resonance imaging and ex vivo histology confirmed the beneficial effect on postinfarct left ventricular remodeling in MI mice treated with tDCs. Subcutaneously administered infarct lysate-primed tDCs near the inguinal lymph node migrated to the regional lymph node and induced infarct tissue-specific regulatory T-cell populations in the inguinal and mediastinal lymph nodes, spleen, and infarcted myocardium, indicating that a local injection of tDCs induces a systemic activation of MI-specific regulatory T cells. These events elicited an inflammatory-to-reparative macrophage shift. The altered immune environment in the infarcted heart resulted in a better wound remodeling, preserved left ventricular systolic function after myocardial tissue damage, and improved survival. CONCLUSIONS:This study showed that tDC therapy in a preclinical model of MI was potentially translatable into an antiremodeling therapy for ischemic tissue repair.
Liver metastasis restrains immunotherapy efficacy via macrophage-mediated T cell elimination.
Yu Jiali,Green Michael D,Li Shasha,Sun Yilun,Journey Sara N,Choi Jae Eun,Rizvi Syed Monem,Qin Angel,Waninger Jessica J,Lang Xueting,Chopra Zoey,El Naqa Issam,Zhou Jiajia,Bian Yingjie,Jiang Long,Tezel Alangoya,Skvarce Jeremy,Achar Rohan K,Sitto Merna,Rosen Benjamin S,Su Fengyun,Narayanan Sathiya P,Cao Xuhong,Wei Shuang,Szeliga Wojciech,Vatan Linda,Mayo Charles,Morgan Meredith A,Schonewolf Caitlin A,Cuneo Kyle,Kryczek Ilona,Ma Vincent T,Lao Christopher D,Lawrence Theodore S,Ramnath Nithya,Wen Fei,Chinnaiyan Arul M,Cieslik Marcin,Alva Ajjai,Zou Weiping
Metastasis is the primary cause of cancer mortality, and cancer frequently metastasizes to the liver. It is not clear whether liver immune tolerance mechanisms contribute to cancer outcomes. We report that liver metastases diminish immunotherapy efficacy systemically in patients and preclinical models. Patients with liver metastases derive limited benefit from immunotherapy independent of other established biomarkers of response. In multiple mouse models, we show that liver metastases siphon activated CD8 T cells from systemic circulation. Within the liver, activated antigen-specific FasCD8 T cells undergo apoptosis following their interaction with FasLCD11bF4/80 monocyte-derived macrophages. Consequently, liver metastases create a systemic immune desert in preclinical models. Similarly, patients with liver metastases have reduced peripheral T cell numbers and diminished tumoral T cell diversity and function. In preclinical models, liver-directed radiotherapy eliminates immunosuppressive hepatic macrophages, increases hepatic T cell survival and reduces hepatic siphoning of T cells. Thus, liver metastases co-opt host peripheral tolerance mechanisms to cause acquired immunotherapy resistance through CD8 T cell deletion, and the combination of liver-directed radiotherapy and immunotherapy could promote systemic antitumor immunity.
Human tumor-associated monocytes/macrophages and their regulation of T cell responses in early-stage lung cancer.
Singhal Sunil,Stadanlick Jason,Annunziata Michael J,Rao Abhishek S,Bhojnagarwala Pratik S,O'Brien Shaun,Moon Edmund K,Cantu Edward,Danet-Desnoyers Gwenn,Ra Hyun-Jeong,Litzky Leslie,Akimova Tatiana,Beier Ulf H,Hancock Wayne W,Albelda Steven M,Eruslanov Evgeniy B
Science translational medicine
Data from mouse tumor models suggest that tumor-associated monocyte/macrophage lineage cells (MMLCs) dampen antitumor immune responses. However, given the fundamental differences between mice and humans in tumor evolution, genetic heterogeneity, and immunity, the function of MMLCs might be different in human tumors, especially during early stages of disease. Here, we studied MMLCs in early-stage human lung tumors and found that they consist of a mixture of classical tissue monocytes and tumor-associated macrophages (TAMs). The TAMs coexpressed M1/M2 markers, as well as T cell coinhibitory and costimulatory receptors. Functionally, TAMs did not primarily suppress tumor-specific effector T cell responses, whereas tumor monocytes tended to be more T cell inhibitory. TAMs expressing relevant MHC class I/tumor peptide complexes were able to activate cognate effector T cells. Mechanistically, programmed death-ligand 1 (PD-L1) expressed on bystander TAMs, as opposed to PD-L1 expressed on tumor cells, did not inhibit interactions between tumor-specific T cells and tumor targets. TAM-derived PD-L1 exerted a regulatory role only during the interaction of TAMs presenting relevant peptides with cognate effector T cells and thus may limit excessive activation of T cells and protect TAMs from killing by these T cells. These results suggest that the function of TAMs as primarily immunosuppressive cells might not fully apply to early-stage human lung cancer and might explain why some patients with strong PD-L1 positivity fail to respond to PD-L1 therapy.
Use of PD-1 Targeting, Macrophage Infiltration, and IDO Pathway Activation in Sarcomas: A Phase 2 Clinical Trial.
IMPORTANCE:There is a strong rationale for treating sarcomas with immunotherapy. OBJECTIVE:To assess the efficacy and safety of programmed cell death protein 1 (PD-1) targeting in combination with metronomic chemotherapy in sarcomas. DESIGN, SETTING, AND PARTICIPANTS:This was an open-label, multicenter, phase 2 study of 4 cohorts of patients with advanced soft-tissue sarcoma (STS), including leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), other sarcomas (others), and gastrointestinal stromal tumor (GIST). All patients received 50 mg twice daily cyclophosphamide 1 week on and 1 week off and 200 mg of intravenous pembrolizumab every 3 weeks. INTERVENTION OR EXPOSURE:Pembrolizumab in combination with metronomic cyclophosphamide. MAIN OUTCOMES AND MEASURES:There was a dual primary end point, encompassing both the nonprogression and objective responses at 6 months per Response Evaluation Criteria in Solid Tumours (RECIST) v1.1 for LMS, UPS, and others and 6-month nonprogression for GIST. An objective response rate of 20% and/or a 6-month nonprogression rate of 60% were determined as reasonable objectives for treatment with meaningful effect. Correlative studies of immune biomarkers were planned from patient tumor and plasma samples. RESULTS:Between June 2015 and July 2016, 57 patients were included (median [range] age, 59.5 [18.5-84.0] years; 24 women [42%]); 50 patients were assessable for the efficacy end point. Three patients experienced tumor shrinkage, resulting in a partial response in a single solitary fibrous tumor. The 6-month nonprogression rates were 0%, 0%, 14.3% (95% CI, 1.8%-42.8%) for LMS, UPS, and others, respectively, and 11.1% (95% CI, 2.8%-48.3%) for GIST. The most frequent adverse events were grade 1 or 2 fatigue, diarrhea, and anemia. The only patient who experienced partial response was the only one with strong programmed cell death 1 ligand 1-positive staining in immune cell. Strong infiltration by macrophage expressing the inhibitory enzyme indoleamine 2,3-dioxygenase 1 (IDO1) was observed in the majority of cases. Moreover, a significant increase in the kynurenine to tryptophan ratio was observed in patient plasma samples during the study treatment. CONCLUSIONS AND RELEVANCE:We found that PD-1 inhibition has limited activity in selected STS and GIST. This may be explained by an immunosuppressive tumor microenvironment resulting from macrophage infiltration and IDO1 pathway activation. TRIAL REGISTRATION:clinicaltrials.gov Identifier: NCT02406781.
Control of Metastases via Myeloid CD39 and NK Cell Effector Function.
Yan Juming,Li Xian-Yang,Roman Aguilera Amelia,Xiao Christos,Jacoberger-Foissac Celia,Nowlan Bianca,Robson Simon C,Beers Courtney,Moesta Achim K,Geetha Nishamol,Teng Michele W L,Smyth Mark J
Cancer immunology research
Natural killer (NK) cell protection from tumor metastases is a critical feature of the host immune response to cancer, but various immunosuppression mechanisms limit NK cell effector function. The ectoenzyme, CD39, expressed on tumor-infiltrating myeloid cells, granulocytes, and lymphocytes, including NK cells, converts extracellular ATP (eATP) into AMP and, thus, potentially suppresses eATP-mediated proinflammatory responses. A CD39-targeting monoclonal antibody (mAb) that inhibits the mouse ectoenzyme CD39 suppressed experimental and spontaneous metastases in a number of different tumor models and displayed superior antimetastatic activity compared with the CD39 inhibitor POM1 and inhibitors and mAbs that block other members of the adenosinergic family (e.g., A2AR and CD73). The antimetastatic activity of anti-CD39 was NK cell and IFNγ dependent, and anti-CD39 enhanced the percentage and quantity of IFNγ produced and CD107a expression in lung-infiltrating NK cells following tumor challenge and anti-CD39 therapy. Using conditional gene-targeted mouse strains and adoptive NK cell transfers, we showed that CD39 expressed on bone marrow-derived myeloid cells was essential for anti-CD39's antimetastatic activity, but NK cell expression of CD39 was not critical. The eATP receptor P2X7 and the NALP3 inflammasome, including downstream IL18, were critical in the mechanism of action of anti-CD39, and the frequency of P2X7 and CD39 coexpressing lung alveolar macrophages was specifically reduced 1 day after anti-CD39 therapy. The data provide a mechanism of action involving NK cells and myeloid cells, and anti-CD39 combined with anti-PD-1, NK cell-activating cytokines IL15 or IL2, or an inhibitor of A2AR to effectively suppress tumor metastases.
Targeting an adenosine-mediated "don't eat me signal" augments anti-lymphoma immunity by anti-CD20 monoclonal antibody.
Nakamura Kyohei,Casey Mika,Oey Harald,Vari Frank,Stagg John,Gandhi Maher K,Smyth Mark J
A growing body of evidence suggests that macrophage immune checkpoint molecules are potential targets in the era of cancer immunotherapy. Here we showed that extracellular adenosine, an abundant metabolite in the tumor microenvironment, critically impedes the therapeutic efficacy of anti-CD20 monoclonal antibodies (mAbs) against B-cell lymphoma. Using a syngeneic B-cell lymphoma model, we showed that host deficiency of adenosine 2A receptor (A2AR), but not A2BR, remarkably improved lymphoma control by anti-CD20 mAb therapy. Conditional deletion of A2AR in myeloid cells, and to a lesser extent in NK cells, augmented therapeutic efficacy of anti-CD20 mAb. Indeed, adenosine signaling impaired antibody-mediated cellular phagocytosis (ADCP) by macrophages and limited the generation of anti-lymphoma CD8 T cells. Pharmacological inhibition of A2AR overcame the adenosine-mediated negative regulation of ADCP by rituximab in a xeno-transplanted lymphoma model. Moreover, aberrant overexpression of CD39, an apical ecto-enzyme for adenosine generation, showed a negative impact on prognosis in patients with diffuse large B-cell lymphoma, as well as on preclinical efficacy of rituximab. Together, adenosine acts as a "don't eat me signal", and may be a potential target to harness anti-lymphoma immunity.
New insight on the correlation of metabolic status on F-FDG PET/CT with immune marker expression in patients with non-small cell lung cancer.
Wang Yang,Zhao Ning,Wu Zhanbo,Pan Na,Shen Xuejie,Liu Ting,Wei Feng,You Jian,Xu Wengui,Ren Xiubao
European journal of nuclear medicine and molecular imaging
BACKGROUND:Metabolic information obtained through F-flurodeoxyglucose positron emission tomography/computed tomography (F-FDG PET/CT) is used to evaluate malignancy by calculating the glucose uptake rate, and these parameters play important roles in determining the prognosis of non-small cell lung cancer (NSCLC). The expression of immune-related markers in tumor tissue reflects the immune status in the tumor microenvironment. However, there is lack of reports on the association between metabolic variables and intra-tumor immune markers. Herein, we investigate the correlation between metabolic status on F-FDG PET/CT and intra-tumor immunomarkers' expression in NSCLC patients. METHODS:From April 2008 to August 2014, 763 patients were enrolled in the analysis to investigate the role of maximum standardized uptake value (SUVmax) in lung cancer. One hundred twenty-two tumor specimens were analyzed by immunohistochemistry (IHC) to intra-tumor immune cells and programmed death protein ligand 1(PD-L1) expression on tumor cells. The correlation between metabolic variables and the expression of tissue immune markers were analyzed. RESULTS:SUVmax values have significant variations in different epidermal growth factor receptor (EGFR) statuses (wild type vs mutant type), high/low neutrophil-to-lymphocyte ratio (NLR) groups, and high/low platelets-to-lymphocyte ratio (PLR) groups (p < 0.001, p < 0.001, p = 0.003, respectively). SUVmax was an independent prognostic factor in lung cancer patients (p = 0.013). IHC demonstrated a statistically significant correlation between SUVmax and the expression of CD8 tumor-infiltrating lymphocytes (p = 0.015), CD163 tumor-associated macrophages (TAMs) (p = 0.003), and Foxp3-regulatory T cells (Tregs) (p = 0.004), as well as PD-1 and PD-L1 (p = 0.003 and p = 0.012, respectively). With respect to patient outcomes, disease stage, BMI, SUVmax, metabolic tumor volume (MTV), TLG (tumor lesion glycolysis), CD163-TAMs, CD11c-dendritic cells (DCs), PD-L1, and Tregs showed a statistically significant correlation with progression-free survival (PFS) (p < 0.001, 0.023, < 0.001, 0.007, 0.005, 0.004, 0.008, 0.048, and 0.014, respectively), and disease stage, SUVmax, MTV, TLG, CD163-TAMs, CD11c-DCs, and PD-L1 showed a statistically significant correlation with overall survival (OS) (p < 0.001, < 0.001, 0.014, 0.012, < 0.001, 0.001, and < 0.001, respectively). CONCLUSION:This study revealed an association between metabolic variable and immune cell expression in the tumor microenvironment and suggests that SUVmax on F-FDG PET/CT could be a potential predictor for selecting candidates for immunotherapy.
A risk model developed based on tumor microenvironment predicts overall survival and associates with tumor immunity of patients with lung adenocarcinoma.
Wu Jie,Li Lan,Zhang Huibo,Zhao Yaqi,Zhang Haohan,Wu Siyi,Xu Bin
Tumor microenvironment (TME) has been reported to exhibit a crucial effect in lung cancer. Therefore, this study was aimed to investigate the genes associated with TME and develop a risk score to predict the overall survival (OS) of patients with lung adenocarcinoma (LUAD) based on these genes. The immune and stromal scores were generated by the ESTIMATE algorithm for LUAD patients in The Cancer Genome Atlas (TCGA) database. Differentially expressed gene and weighted gene co-expression network analyses were used to derive immune- and stromal-related genes. The Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression was applied for further selection and the selected genes were inputted into stepwise regression to develop TME-related risk score (TMErisk) which was further validated in Gene Expression Omnibus (GEO) datasets. TMErisk-related biological phenotypes were analyzed in function enrichment, tumor immune signature, and tumor mutation signature. The patient's response to immunotherapy was inferred by the tumor immune dysfunction and exclusion (TIDE) score and immunophenoscore (IPS). According to our results, TMErisk was developed based on SERPINE1, CX3CR1, CD200R1, GBP1, IRF1, STAP1, LOX, and OR7E47P. Furthermore, high TMErisk was identified as a poor factor for OS in TCGA and GEO datasets, as well as in subgroup analysis with different gender, smoking status, age, race, anatomic site, therapies, and tumor-node-metastasis (TNM) stages. Higher TMErisk is also associated negatively with the abundance of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and other stromal or immune cells. Several genes of the human leukocyte antigen (HLA) family and immune checkpoints were less expressed in the high-TMErisk group. Mutations of 19 genes occurred more frequently in the high-TMErisk group. These mutations may be associated with TME change and indicate patients' response to immunotherapy. According to our analyses, a lower TMErisk score may indicate better response and OS outcome of immunotherapy.
Tumor-associated macrophages: potential therapeutic strategies and future prospects in cancer.
Journal for immunotherapy of cancer
Macrophages are the most important phagocytes in vivo. However, the tumor microenvironment can affect the function and polarization of macrophages and form tumor-associated macrophages (TAMs). Usually, the abundance of TAMs in tumors is closely associated with poor prognosis. Preclinical studies have identified important pathways regulating the infiltration and polarization of TAMs during tumor progression. Furthermore, potential therapeutic strategies targeting TAMs in tumors have been studied, including inhibition of macrophage recruitment to tumors, functional repolarization of TAMs toward an antitumor phenotype, and other therapeutic strategies that elicit macrophage-mediated extracellular phagocytosis and intracellular destruction of cancer cells. Therefore, with the increasing impact of tumor immunotherapy, new antitumor strategies to target TAMs are now being discussed.
M1 tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer.
Garrido-Martin Eva M,Mellows Toby W P,Clarke James,Ganesan Anusha-Preethi,Wood Oliver,Cazaly Angelica,Seumois Gregory,Chee Serena J,Alzetani Aiman,King Emma V,Hedrick Catherine C,Thomas Gareth,Friedmann Peter S,Ottensmeier Christian Hermann,Vijayanand Pandurangan,Sanchez-Elsner Tilman
Journal for immunotherapy of cancer
BACKGROUND:The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor's anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer. METHODS:Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8 tissue-resident memory T cells (T) in tumors and survival data from an independent cohort of 393 patients with lung cancer. RESULTS:TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1). Importantly, there was a strong association between the density of M1 TAMs and T cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1 TAMs may recruit T cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which T depend. CONCLUSIONS:We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1 TAMs was associated with a strong T tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1 phenotype are likely to augment the adaptive antitumor responses.
Differential intratumoral distributions of CD8 and CD163 immune cells as prognostic biomarkers in breast cancer.
Fortis Sotirios P,Sofopoulos Michael,Sotiriadou Nectaria N,Haritos Christoforos,Vaxevanis Christoforos K,Anastasopoulou Eleftheria A,Janssen Nicole,Arnogiannaki Niki,Ardavanis Alexandros,Pawelec Graham,Perez Sonia A,Baxevanis Constantin N
Journal for immunotherapy of cancer
BACKGROUND:Tumor immune cell infiltrates are essential in hindering cancer progression and may complement the TNM classification. CD8+ and CD163+ cells have prognostic impact in breast cancer but their spatial heterogeneity has not been extensively explored in this type of cancer. Here, their potential as prognostic biomarkers was evaluated, depending on their combined densities in the tumor center (TC) and the tumor invasive margin (IM). METHODS:CD8+ and CD163+ cells were quantified by immunohistochemistry of formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples from a cohort totaling 162 patients with histologically-confirmed primary invasive non-metastatic ductal breast cancer diagnosed between 2000 and 2015. Clinical follow-up (median 6.9 years) was available for 97 of these patients. RESULTS:Differential densities of CD8+ and CD163+ cells in the combined TC and IM compartments (i.e., high(H)/low(L), respectively for CD8+ cells and the reverse L/H combination for CD163+ cells) were found to have significant prognostic value for survival, and allowed better patient stratification than TNM stage, tumor size, lymph node invasion and histological grade. The combined evaluation of CD8+ and CD163+ cell densities jointly in TC and IM further improves prediction of clinical outcomes based on disease-free and overall survival. Patients having the favorable immune signatures had favorable clinical outcomes despite poor clinicopathological parameters. CONCLUSIONS:Given the important roles of CD8+ and CD163+ cells in regulating opposing immune circuits, adding an assessment of their differential densities to the prognostic biomarker armamentarium in breast cancer would be valuable. Larger validation studies are necessary to confirm these findings. TRIAL REGISTRATIONS:Study code: IRB-ID 6079/448/10-6-13 Date of approval: 10/06/2013 Retrospective study (2000-2010) First patient prospectively enrolled 14/2/2014.
Evaluating the Polarization of Tumor-Associated Macrophages Into M1 and M2 Phenotypes in Human Cancer Tissue: Technicalities and Challenges in Routine Clinical Practice.
Jayasingam Sharmilla Devi,Citartan Marimuthu,Thang Thean Hock,Mat Zin Anani Aila,Ang Kai Cheen,Ch'ng Ewe Seng
Frontiers in oncology
Tumor-associated macrophages (TAMs) as immune cells within the tumor microenvironment have gained much interests as basic science regarding their roles in tumor progression unfolds. Better understanding of their polarization into pro-tumoral phenotype to promote tumor growth, tumor angiogenesis, immune evasion, and tumor metastasis has prompted various studies to investigate their clinical significance as a biomarker of predictive and prognostic value across different cancer types. Yet, the methodologies to investigate the polarization phenomena in solid tumor tissue vary. Nonetheless, quantifying the ratio of M1 to M2 TAMs has emerged to be a prevailing parameter to evaluate this polarization phenomena for clinical application. This mini-review focuses on recent studies exploring clinical significance of M1/M2 TAM ratio in human cancer tissue and critically evaluates the technicalities and challenges in quantifying this parameter for routine clinical practice. Immunohistochemistry appears to be the preferred methodology for M1/M2 TAM evaluation as it is readily available in clinical laboratories, albeit with certain limitations. Recommendations are made to standardize the quantification of TAMs for better transition into clinical practice and for better comparison among studies in various populations of patients and cancer types.
Murray Peter J
Annual review of physiology
Macrophage polarization refers to how macrophages have been activated at a given point in space and time. Polarization is not fixed, as macrophages are sufficiently plastic to integrate multiple signals, such as those from microbes, damaged tissues, and the normal tissue environment. Three broad pathways control polarization: epigenetic and cell survival pathways that prolong or shorten macrophage development and viability, the tissue microenvironment, and extrinsic factors, such as microbial products and cytokines released in inflammation. A plethora of advances have provided a framework for rationally purifying, describing, and manipulating macrophage polarization. Here, I assess the current state of knowledge about macrophage polarization and enumerate the major questions about how activated macrophages regulate the physiology of normal and damaged tissues.
Macrophage Polarization States in the Tumor Microenvironment.
International journal of molecular sciences
The M1/M2 macrophage paradigm plays a key role in tumor progression. M1 macrophages are historically regarded as anti-tumor, while M2-polarized macrophages, commonly deemed tumor-associated macrophages (TAMs), are contributors to many pro-tumorigenic outcomes in cancer through angiogenic and lymphangiogenic regulation, immune suppression, hypoxia induction, tumor cell proliferation, and metastasis. The tumor microenvironment (TME) can influence macrophage recruitment and polarization, giving way to these pro-tumorigenic outcomes. Investigating TME-induced macrophage polarization is critical for further understanding of TAM-related pro-tumor outcomes and potential development of new therapeutic approaches. This review explores the current understanding of TME-induced macrophage polarization and the role of M2-polarized macrophages in promoting tumor progression.
Immune Cell Infiltration May Be a Key Determinant of Long-Term Survival in Small Cell Lung Cancer.
Muppa Prasuna,Parrilha Terra Simone Barreto Siqueira,Sharma Anurag,Mansfield Aaron S,Aubry Marie-Christine,Bhinge Kaustubh,Asiedu Michael K,de Andrade Mariza,Janaki Nafiseh,Murphy Stephen J,Nasir Aqsa,Van Keulen Virginia,Vasmatzis George,Wigle Dennis A,Yang Ping,Yi Eunhee S,Peikert Tobias,Kosari Farhad
Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer
INTRODUCTION:Although most patients with SCLC die within a few months of diagnosis, a subgroup of patients survive for many years. Factors determining long-term survivorship remain largely unknown. We present the first comprehensive comparative genomic and tumor microenvironment analyses of SCLC between patients with long-term survivorship and patients with the expected survivorship. METHODS:We compared surgically resected tumors of 23 long-term SCLC survivors (survival >4 years) and 18 SCLC survivors with the expected survival time (survival ≤2 years). There were no significant differences in clinical variables, including TNM staging and curative- versus non-curative-intent surgery between the groups. Gene expression profiling was performed by using microarrays, and tumor microenvironment analyses were performed by immunohistochemistry of prominent immune-related markers. RESULTS:Immune-related genes and pathways represented the majority of the differentially overexpressed genes in long-term survivorship compared with in expected survivorship. The differences in the immunological tumor microenvironment were confirmed by quantitative immunostaining. Increased numbers of tumor-infiltrating and associated lymphocytes were present throughout tumors of long-term survivors of SCLC. Several differentiating patterns of enhanced antitumor immunity were identified. Although some areas of the tumors of long-term survivors of SCLC also harbored higher numbers of suppressive immune cells (monocytes, regulatory lymphocytes, and macrophages), the ratios of these suppressive cells to CD3-positive lymphocytes were generally lower in the tumors of long-term survivors of SCLC, indicating a less tumor-suppressive microenvironment. CONCLUSIONS:Our data demonstrate that long-term survivorship of patients with SCLC is strongly influenced by the presence of the immune cells in the tumor microenvironment. Characterization of the antitumor immune responses may identify opportunities for individualized immunotherapies for SCLC.
Expression Analysis and Significance of PD-1, LAG-3, and TIM-3 in Human Non-Small Cell Lung Cancer Using Spatially Resolved and Multiparametric Single-Cell Analysis.
Datar Ila,Sanmamed Miguel F,Wang Jun,Henick Brian S,Choi Jungmin,Badri Ti,Dong Weilai,Mani Nikita,Toki Maria,Mejías Luis D,Lozano Maria D,Perez-Gracia Jose Luis,Velcheti Vamsidhar,Hellmann Matthew D,Gainor Justin F,McEachern Kristen,Jenkins David,Syrigos Konstantinos,Politi Katerina,Gettinger Scott,Rimm David L,Herbst Roy S,Melero Ignacio,Chen Lieping,Schalper Kurt A
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN:Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment. RESULTS:PD-1, LAG-3, and TIM-3 were detected in tumor-infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progression-free survival. CONCLUSIONS:PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.
The non-small cell lung cancer immune contexture. A major determinant of tumor characteristics and patient outcome.
Remark Romain,Becker Christian,Gomez Jorge E,Damotte Diane,Dieu-Nosjean Marie-Caroline,Sautès-Fridman Catherine,Fridman Wolf-Herman,Powell Charles A,Altorki Nasser K,Merad Miriam,Gnjatic Sacha
American journal of respiratory and critical care medicine
Solid tumors, beyond mere accumulation of cancer cells, form a complex ecosystem consisting of normal epithelial cells, fibroblasts, blood and lymphatic vessels, structural components, and infiltrating hematopoietic cells including myeloid and lymphoid elements that impact tumor growth, tumor spreading, and clinical outcome. The composition of the immune microenvironment is diverse, including various populations of T cells, B cells, dendritic cells, natural killer cells, myeloid-derived suppressor cells, neutrophils, or macrophages. The immune contexture describes the density, location, and organization of these immune cells within solid tumors. In lung cancer, which is the deadliest type of cancer, and particularly in non-small cell lung cancer, its most prevalent form, reports have described some of the interactions between the tumor and the host. These data, in addition to articles on various types of tumors, provide a greater understanding of the tumor-host microenvironment interaction and stimulate the development of prognostic and predictive biomarkers, the identification of novel target antigens for therapeutic intervention, and the implementation of tools for long-term management of patients with cancer.
Prognostic impact of tumor-associated macrophage infiltration in non-small cell lung cancer: A systemic review and meta-analysis.
Mei Jiandong,Xiao Zhilan,Guo Chenglin,Pu Qiang,Ma Lin,Liu Chengwu,Lin Feng,Liao Hu,You Zongbing,Liu Lunxu
Tumor-associated macrophages (TAMs) are important components of cancer microenvironment. In the present study, we searched PubMed, Embase, Cochrane library and Web of Science to perform a meta-analysis of 20 studies including a total of 2,572 non-small cell lung cancer (NSCLC) patients, in order to determine the association between TAMs and NSCLC prognosis. The combined hazard ratio (HR) of 9 studies showed that the density of total CD68+ TAMs in the tumor islet and stroma was not associated with overall survival (OS) of the patients. However, the pooled HR of 4 studies showed that high density of CD68+ TAMs in the tumor islet predicted better OS, while the pooled HR of 6 studies showed that high density of CD68+ TAMs in the tumor stroma was associated with poor OS. A high islet/stroma ratio of CD68+ TAMs was associated with better OS. A high density of M1 TAMs in the tumor islet was associated with better OS, while a high density of M2 TAMs in the tumor stroma predicted poor OS. These findings suggest that, although the density of total CD68+ TAMs is not associated with OS, the localization and M1/M2 polarization of TAMs are potential prognostic predictors of NSCLC.
PD-L1 expression on tumor-infiltrating immune cells is highly associated with M2 TAM and aggressive malignant potential in patients with resected non-small cell lung cancer.
Sumitomo Ryota,Hirai Tatsuya,Fujita Masaaki,Murakami Hiroaki,Otake Yosuke,Huang Cheng-Long
Lung cancer (Amsterdam, Netherlands)
OBJECTIVES:PD-L1 expression on tumor cells (TCs) and tumor-infiltrating immune cells (ICs) plays important roles in regulating the antitumor T cell response. However, the mechanistic and clinical significance of the effect of PD-L1 on TCs versus ICs remains unclear. On the other hand, tumor-associated macrophages (TAMs), M2 macrophages in particular, can promote tumor progression. METHODS:We evaluated PD-L1 expression on TCs and ICs using Ventana SP263 assay and the stromal M2 TAM distribution using CD163 staining in 160 consecutive patients with resected non-small cell lung cancer (NSCLC). RESULTS:PD-L1 expression on TCs and ICs was significantly higher in stromal M2 TAM-high group than in stromal M2 TAM-low group (p < 0.001 and p < 0.001, respectively). Regarding the clinical significance of PD-L1, PD-L1 expression on TCs was significantly associated with histology (p = 0.001), tumor differentiation (p < 0.001) and nodal status (p = 0.029). Furthermore, PD-L1 expression on ICs was significantly associated with histology (p < 0.001), tumor differentiation (p < 0.001), tumor status (p = 0.024), nodal status (p = 0.016), and pathologic stage (p = 0.004). The disease-free survival rate was significantly lower in patients with PD-L1-positive TC than in those with PD-L1-negative TC (p = 0.023), as well as in patients with PD-L1-positive IC than in those with PD-L1-negative IC (p < 0.001). Furthermore, the overall survival rate was significantly lower in patients with PD-L1-positive IC than in those with PD-L1-negative IC (p = 0.023). CONCLUSIONS:During tumor progression in NSCLC, the presence of M2 TAMs might affect PD-L1 expression both on TCs and ICs. In patients with NSCLC, PD-L1 expression both on TCs and ICs was associated with malignant behaviors, which was more in case of ICs.
M2 macrophage infiltration into tumor islets leads to poor prognosis in non-small-cell lung cancer.
Cao Lili,Che Xiaofang,Qiu Xueshan,Li Zhi,Yang Bowen,Wang Shuo,Hou Kezuo,Fan Yibo,Qu Xiujuan,Liu Yunpeng
Cancer management and research
Lung cancer is the leading cause of cancer-related death worldwide. Although the macrophages can affect the development of tumor, the contribution of macrophages to the prognosis of non-small-cell lung cancer (NSCLC) is still controversial. Moreover, anti-PD-1 therapy can redirect macrophages from an M2 to an M1 phenotype, suggesting that tumor PD-L1 may affect the prognostic role of macrophages. Therefore, in this study, we aimed to display a macrophage landscape to clarify the function of macrophages, considering the localization and polarization of the macrophages, and evaluate the effect of M2 macrophages and tumor PD-L1 in combination on the prognosis of NSCLC. We performed multiplex quantitative immunofluorescence staining of pan-cytokeratin (CK), CD68, CD163, PD-L1, and DAPI on one tissue specimen simultaneously from 137 NSCLC patients. M2 macrophages, involved marginM2 (M2 macrophages in tumor stroma), and centralM2 (M2 macrophages infiltrating into tumor islets) increased as the tumor stage increased. More macrophages were found in lung squamous cell carcinoma (LUSC) patients, patients with wild-type EGFR, and smokers than in patients with lung adenocarcinoma (LUAD), patients with EGFR mutations, and non-smokers. Infiltration of centralM2 was an independent prognostic factor of poor overall survival (OS) and disease-free survival (DFS) for NSCLC patients (<0.05), which was superior to total macrophages and total M2 macrophages. Moreover, patients with centralM2PD-L1 tumors showed the best OS and DFS, while the patients with centralM2PD-L1 tumors showed the worst OS and DFS, and the two groups with centralM2PD-L1 and centralM2PD-L1 were in the middle (=0.002, 0.034, respectively). Tumor islet-infiltrating M2 macrophages influence the prognosis of NSCLC patients. The analysis of M2 macrophages and tumor PD-L1 in combination may enhance the accuracy of prognostic prediction. This study provides a new understanding of macrophages in the development of NSCLC through the analysis of macrophage landscape.
Prognostic Value of Macrophage Phenotypes in Resectable Non-Small Cell Lung Cancer Assessed by Multiplex Immunohistochemistry.
Rakaee Mehrdad,Busund Lill-Tove Rasmussen,Jamaly Simin,Paulsen Erna-Elise,Richardsen Elin,Andersen Sigve,Al-Saad Samer,Bremnes Roy M,Donnem Tom,Kilvaer Thomas K
Neoplasia (New York, N.Y.)
Macrophages are important inflammatory cells that regulate innate and adaptive immunity in cancer. Tumor-associated macrophages (TAMs) are thought to differentiate into two main phenotypes: proinflammatory M1 and protumorigenic M2. Currently, the prognostic impact of TAMs and their M1 and M2 phenotypes is unclear in non-small cell cancer (NSCLC). The present study was set up to evaluate an approach for identifying common M1 and M2 macrophage markers and explore their clinical significance in NSCLC. Using multiplex chromogenic immunohistochemistry, tissue microarrays of 553 primary tumors and 143 paired metastatic lymph nodes of NSCLC specimens were stained to detect various putative macrophage phenotypes: M1 (HLA-DR/CD68), M2 (CD163/CD68), M2 (CD204/CD68), and pan-macrophage (CD68/CK). Correlation analyses were performed to examine the relationship between TAMs and adaptive/innate immune infiltrates. HLA-DR/CD68M1 TAM level significantly decreased from pathological stage I to III. In a compartment-specific correlation analysis, moderate to strong correlations were observed between both TAM subsets (M1 and M2) with CD3-, CD8-, CD4-, and CD45RO-positive immune cells. Survival analyses, in both stromal and intratumoral compartments, revealed that high levels of HLA-DR/CD68M1 (stroma, hazard ratio [HR] = 0.73, P = .03; intratumor, HR = 0.7, P = .04), CD204M2 (stroma, HR = 0.7, P = .02; intratumor, HR = 0.6, P = .004), and CD68 (stroma, HR = 0.69, P = .02; intratumor, HR = 0.73, P = .04) infiltration were independently associated with improved NSCLC-specific survival. In lymph nodes, the intratumoral level of HLA-DR/CD68M1 was an independent positive prognostic indicator (Cox model, HR = 0.38, P = .001). In conclusion, high levels of M1, CD204M2, and CD68 macrophages are independent prognosticators of prolonged survival in NSCLC.
Distribution of M1 and M2 macrophages in tumor islets and stroma in relation to prognosis of non-small cell lung cancer.
Jackute Jurgita,Zemaitis Marius,Pranys Darius,Sitkauskiene Brigita,Miliauskas Skaidrius,Vaitkiene Simona,Sakalauskas Raimundas
BACKGROUND:Non-small cell lung cancer (NSCLC) remains the most common cause of cancer related death worldwide. Tumor-infiltrating macrophages are believed to play an important role in growth, progression, and metastasis of tumors. In NSCLC, the role of macrophages remains controversial; therefore, we aimed to evaluate the distribution of macrophages (M1 and M2) in tumor islets and stroma and to analyze their relations to patients' survival. METHODS:Lung tissue specimens from 80 NSCLC patients who underwent surgical resection for NSCLC (pathological stage I-III) and 16 control group subjects who underwent surgery because of recurrent spontaneous pneumothorax were analyzed. Immunohistochemical double staining of CD68/iNOS (markers for M1 macrophages) and CD68/CD163 (markers for M2 macrophages) was performed and evaluated in a blinded manner. The numbers of M1 and M2 macrophages in tumor islets and stroma were counted manually. RESULTS:Predominant infiltration of M1 and M2 macrophages was observed in the tumor stroma compared with the tumor islets. M2 macrophages predominated over M1 macrophages in the tumor tissue. Tumor islets-infiltrating M1 macrophages and the number of total tumor-infiltrating M2 macrophages were independent predictors of patients survival: high infiltration of M1 macrophages in tumor islets was associated with increased overall survival in NSCLC (P < 0.05); high infiltration of total M2 macrophages in tumor (islets and stroma) was associated with reduced overall survival in NSCLC (P < 0.05). CONCLUSIONS:This study demonstrated that high infiltration of M1 macrophages in the tumor islets and low infiltration of total tumor-infiltrating M2 macrophages were associated with improved NSCLC patients' survival. TRIAL REGISTRATION:ClinicalTrials.gov NCT01955343 , registered on September 27, 2013.
Detailed resolution analysis reveals spatial T cell heterogeneity in the invasive margin of colorectal cancer liver metastases associated with improved survival.
Berthel Anna,Zoernig Inka,Valous Nektarios A,Kahlert Christoph,Klupp Fee,Ulrich Alexis,Weitz Juergen,Jaeger Dirk,Halama Niels
On a broader scale, T cell density and localization in colorectal cancer liver metastases have prognostic and predictive implications. As T cell distribution at higher resolutions has not been fully investigated, a detailed resolution analysis of T cell distribution was performed. Patient tissues were divided into 10 µm distance classes between the tumor border and adjacent normal liver. Thereby, distinct density patterns of T cell localization in relation to the malignant tissue could be detected. At a distance of 20 to 30 µm to the tumor, a decrease of CD3 T cells is common. Within this area, cytotoxic Granzyme B and CD8 T cells were found to be significantly reduced as well as CD163 macrophages were increased and identified to be in close contact with T cells. Our data suggests a physical or functional border within this region. Survival analysis revealed improved overall survival in patients with high T cells numbers at the direct tumor border. Interestingly, the decreased T cells in the 20 to 30 µm region were also found to be significantly associated with improved survival. Consequently, the detailed localization of T cells, despite blockade, could be associated with improved clinical outcome. The high-resolution analysis represents new insights into relevant heterogenous T cell distributions especially related to clinical responses. As the paradoxical observation of localization-dependent prognostic relevance of T cell densities is only detectable by detailed spatial analyses, this investigation of spatial profiles at higher resolutions is suggested as a new biomarker for survival and response to therapies.
Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells.
Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8 T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.
Prognostic Effect of Tumor Lymphocytic Infiltration in Resectable Non-Small-Cell Lung Cancer.
Brambilla Elisabeth,Le Teuff Gwénaël,Marguet Sophie,Lantuejoul Sylvie,Dunant Ariane,Graziano Stephen,Pirker Robert,Douillard Jean-Yves,Le Chevalier Thierry,Filipits Martin,Rosell Rafael,Kratzke Robert,Popper Helmut,Soria Jean-Charles,Shepherd Frances A,Seymour Lesley,Tsao Ming Sound
Journal of clinical oncology : official journal of the American Society of Clinical Oncology
PURPOSE:Tumor lymphocytic infiltration (TLI) has differing prognostic value among various cancers. The objective of this study was to assess the effect of TLI in lung cancer. PATIENTS AND METHODS:A discovery set (one trial, n = 824) and a validation set (three trials, n = 984) that evaluated the benefit of platinum-based adjuvant chemotherapy in non-small-cell lung cancer were used as part of the LACE-Bio (Lung Adjuvant Cisplatin Evaluation Biomarker) study. TLI was defined as intense versus nonintense. The main end point was overall survival (OS); secondary end points were disease-free survival (DFS) and specific DFS (SDFS). Hazard ratios (HRs) and 95% CIs associated with TLI were estimated through a multivariable Cox model in both sets. TLI-histology and TLI-treatment interactions were explored in the combined set. RESULTS:Discovery and validation sets with complete data included 783 (409 deaths) and 763 (344 deaths) patients, respectively. Median follow-up was 4.8 and 6 years, respectively. TLI was intense in 11% of patients in the discovery set compared with 6% in the validation set (P < .001). The prognostic value of TLI in the discovery set (OS: HR, 0.56; 95% CI, 0.38 to 0.81; P = .002; DFS: HR, 0.59; 95% CI, 0.42 to 0.83; P = .002; SDFS: HR, 0.56; 95% CI, 0.38 to 0.82; P = .003) was confirmed in the validation set (OS: HR, 0.45; 95% CI, 0.23 to 0.85; P = .01; DFS: HR, 0.44; 95% CI, 0.24 to 0.78; P = .005; SDFS: HR, 0.42; 95% CI, 0.22 to 0.80; P = .008) with no heterogeneity across trials (P ≥ .38 for all end points). No significant predictive effect was observed for TLI (P ≥ .78 for all end points). CONCLUSION:Intense lymphocytic infiltration, found in a minority of tumors, was validated as a favorable prognostic marker for survival in resected non-small-cell lung cancer.
ConvPath: A software tool for lung adenocarcinoma digital pathological image analysis aided by a convolutional neural network.
Wang Shidan,Wang Tao,Yang Lin,Yang Donghan M,Fujimoto Junya,Yi Faliu,Luo Xin,Yang Yikun,Yao Bo,Lin ShinYi,Moran Cesar,Kalhor Neda,Weissferdt Annikka,Minna John,Xie Yang,Wistuba Ignacio I,Mao Yousheng,Xiao Guanghua
BACKGROUND:The spatial distributions of different types of cells could reveal a cancer cell's growth pattern, its relationships with the tumor microenvironment and the immune response of the body, all of which represent key "hallmarks of cancer". However, the process by which pathologists manually recognize and localize all the cells in pathology slides is extremely labor intensive and error prone. METHODS:In this study, we developed an automated cell type classification pipeline, ConvPath, which includes nuclei segmentation, convolutional neural network-based tumor cell, stromal cell, and lymphocyte classification, and extraction of tumor microenvironment-related features for lung cancer pathology images. To facilitate users in leveraging this pipeline for their research, all source scripts for ConvPath software are available at https://qbrc.swmed.edu/projects/cnn/. FINDINGS:The overall classification accuracy was 92.9% and 90.1% in training and independent testing datasets, respectively. By identifying cells and classifying cell types, this pipeline can convert a pathology image into a "spatial map" of tumor, stromal and lymphocyte cells. From this spatial map, we can extract features that characterize the tumor micro-environment. Based on these features, we developed an image feature-based prognostic model and validated the model in two independent cohorts. The predicted risk group serves as an independent prognostic factor, after adjusting for clinical variables that include age, gender, smoking status, and stage. INTERPRETATION:The analysis pipeline developed in this study could convert the pathology image into a "spatial map" of tumor cells, stromal cells and lymphocytes. This could greatly facilitate and empower comprehensive analysis of the spatial organization of cells, as well as their roles in tumor progression and metastasis.
CD68, CD163, and matrix metalloproteinase 9 (MMP-9) co-localization in breast tumor microenvironment predicts survival differently in ER-positive and -negative cancers.
Pelekanou Vasiliki,Villarroel-Espindola Franz,Schalper Kurt A,Pusztai Lajos,Rimm David L
Breast cancer research : BCR
BACKGROUND:The role of tumor-associated macrophages (TAMs) in the cancer immune landscape and their potential as treatment targets or modulators of response to treatment are gaining increasing interest. TAMs display high molecular and functional complexity. Therefore their objective assessment as breast cancer biomarkers is critical. The aims of this study were to objectively determine the in situ expression and significance of TAM biomarkers (CD68, CD163, and MMP-9) in breast cancer and to identify subclasses of patients who could benefit from TAM-targeting therapies. METHODS:We measured CD68, CD163, and MMP-9 protein expression in formalin-fixed paraffin-embedded tissues of breast carcinomas represented in tissue microarray format using multiplexed quantitative immunofluorescence (QIF) in two independent Yale cohorts: cohort A-n = 398, estrogen receptor-positive (ER) and ER cases-and the triple-negative breast cancer (TNBC)-only cohort B (n = 160). Associations between macrophage markers, ER status, and survival were assessed. Protein expression measured by QIF was compared with mRNA expression data from the METABRIC study. RESULTS:All three macrophage markers were co-expressed, displaying higher expression in ER cancers. High pan-macrophage marker CD68 correlated with poorer overall survival (OS) only in ER cases of cohort A (P = 0.02). High expression of CD163 protein in TAMs was associated with improved OS in ER cases (cohort A, P = 0.03 and TNBC cohort B, P = 0.04, respectively) but not in ER cancers. MMP-9 protein was not individually associated with OS. High expression of MMP-9 in the CD68/CD163 TAMs was associated with worse OS in ER tumors (P <0.001) but not in ER cancers. In the METABRIC dataset, mRNA levels followed the co-expression pattern observed in QIF but did not always show the same trend regarding OS. CONCLUSIONS:Macrophage activity markers correlate with survival differently in ER and ER cancers. The association between high co-expression and co-localization of MMP-9/CD163/CD68 and poor survival in ER cancers suggests that these cancers may be candidates for macrophage-targeted therapies.
Immunity, inflammation and cancer: a leading role for adenosine.
Antonioli Luca,Blandizzi Corrado,Pacher Pál,Haskó György
Nature reviews. Cancer
Cancer is a complex disease that is dictated by both cancer cell-intrinsic and cell-extrinsic processes. Adenosine is an ancient extracellular signalling molecule that can regulate almost all aspects of tissue function. As such, several studies have recently highlighted a crucial role for adenosine signalling in regulating the various aspects of cell-intrinsic and cell-extrinsic processes of cancer development. This Review critically discusses the role of adenosine and its receptors in regulating the complex interplay among immune, inflammatory, endothelial and cancer cells during the course of neoplastic disease.
Immune landscape in Burkitt lymphoma reveals M2-macrophage polarization and correlation between PD-L1 expression and non-canonical EBV latency program.
Infectious agents and cancer
BACKGROUND:The Tumor Microenviroment (TME) is a complex milieu that is increasingly recognized as a key factor in multiple stages of disease progression and responses to therapy as well as escape from immune surveillance. However, the precise contribution of specific immune effector and immune suppressor components of the TME in Burkitt lymphoma (BL) remains poorly understood. METHODS:In this paper, we applied the computational algorithm CIBERSORT to Gene Expression Profiling (GEP) datasets of 40 BL samples to draw a map of immune and stromal components of TME. Furthermore, by multiple immunohistochemistry (IHC) and multispectral immunofluorescence (IF), we investigated the TME of additional series of 40 BL cases to evaluate the role of the Programmed Death-1 and Programmed Death Ligand-1 (PD-1/PD-L1) immune checkpoint axis. RESULTS:Our results indicate that M2 polarized macrophages are the most prominent TME component in BL. In addition, we investigated the correlation between PD-L1 and latent membrane protein-2A (LMP2A) expression on tumour cells, highlighting a subgroup of BL cases characterized by a non-canonical latency program of EBV with an activated PD-L1 pathway. CONCLUSION:In conclusion, our study analysed the TME in BL and identified a tolerogenic immune signature highlighting new potential therapeutic targets.
Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-Cell Analyses.
Lavin Yonit,Kobayashi Soma,Leader Andrew,Amir El-Ad David,Elefant Naama,Bigenwald Camille,Remark Romain,Sweeney Robert,Becker Christian D,Levine Jacob H,Meinhof Klaus,Chow Andrew,Kim-Shulze Seunghee,Wolf Andrea,Medaglia Chiara,Li Hanjie,Rytlewski Julie A,Emerson Ryan O,Solovyov Alexander,Greenbaum Benjamin D,Sanders Catherine,Vignali Marissa,Beasley Mary Beth,Flores Raja,Gnjatic Sacha,Pe'er Dana,Rahman Adeeb,Amit Ido,Merad Miriam
To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single-cell analysis of the tumor, non-involved lung, and blood cells, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor-infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single-cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies. VIDEO ABSTRACT.
Annexin-A1 enhances breast cancer growth and migration by promoting alternative macrophage polarization in the tumour microenvironment.
Moraes Leonardo A,Kar Shreya,Foo Sok Lin,Gu Tong,Toh Yi Qian,Ampomah Patrick B,Sachaphibulkij Karishma,Yap Gracemary,Zharkova Olga,Lukman Hakim M,Fairhurst Anna-Marie,Kumar Alan Prem,Lim Lina H K
Macrophages are potent immune cells with well-established roles in the response to stress, injury, infection and inflammation. The classically activated macrophages (M1) are induced by lipopolysaccharide (LPS) and express a wide range of pro-inflammatory genes. M2 macrophages are induced by T helper type 2 cytokines such as interleukin-4 (IL4) and express high levels of anti-inflammatory and tissue repair genes. The strong association between macrophages and tumour cells as well as the high incidences of leukocyte infiltration in solid tumours have contributed to the discovery that tumour-associated macrophages (TAMs) are key to tumour progression. Here, we investigated the role of Annexin A1 (ANXA1), a well characterized immunomodulatory protein on macrophage polarization and the interaction between macrophages and breast cancer cells. Our results demonstrate that ANXA1 regulates macrophage polarization and activation. ANXA1 can act dually as an endogenous signalling molecule or as a secreted mediator which acts via its receptor, FPR2, to promote macrophage polarization. Furthermore, ANXA1 deficient mice exhibit reduced tumour growth and enhanced survival in vivo, possibly due to increased M1 macrophages within the tumor microenvironment. These results provide new insights into the molecular mechanisms of macrophage polarization with therapeutic potential to suppress breast cancer growth and metastasis.
ERK1/2 signaling regulates the immune microenvironment and macrophage recruitment in glioblastoma.
Lailler Claire,Louandre Christophe,Morisse Mony Chenda,Lhossein Thomas,Godin Corinne,Lottin Marine,Constans Jean-Marc,Chauffert Bruno,Galmiche Antoine,Saidak Zuzana
The tumor microenvironment is an important determinant of glioblastoma (GBM) progression and response to treatment. How oncogenic signaling in GBM cells modulates the composition of the tumor microenvironment and its activation is unclear. We aimed to explore the potential local immunoregulatory function of ERK1/2 signaling in GBM. Using proteomic and transcriptomic data (RNA seq) available for GBM tumors from The Cancer Genome Atlas (TCGA), we show that GBM with high levels of phosphorylated ERK1/2 have increased infiltration of tumor-associated macrophages (TAM) with a non-inflammatory M2 polarization. Using three human GBM cell lines in culture, we confirmed the existence of ERK1/2-dependent regulation of the production of the macrophage chemoattractant CCL2/MCP1. In contrast with this positive regulation of TAM recruitment, we found no evidence of a direct effect of ERK1/2 signaling on two other important aspects of TAM regulation by GBM cells: (1) the expression of the immune checkpoint ligands PD-L1 and PD-L2, expressed at high mRNA levels in GBM compared with other solid tumors; (2) the production of the tumor metabolite lactate recently reported to dampen tumor immunity by interacting with the receptor GPR65 present on the surface of TAM. Taken together, our observations suggest that ERK1/2 signaling regulates the recruitment of TAM in the GBM microenvironment. These findings highlight some potentially important particularities of the immune microenvironment in GBM and could provide an explanation for the recent observation that GBM with activated ERK1/2 signaling may respond better to anti-PD1 therapeutics.
Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment.
Azizi Elham,Carr Ambrose J,Plitas George,Cornish Andrew E,Konopacki Catherine,Prabhakaran Sandhya,Nainys Juozas,Wu Kenmin,Kiseliovas Vaidotas,Setty Manu,Choi Kristy,Fromme Rachel M,Dao Phuong,McKenney Peter T,Wasti Ruby C,Kadaveru Krishna,Mazutis Linas,Rudensky Alexander Y,Pe'er Dana
Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.
Prognostic nomograms based on immune scores for head-neck squamous cell carcinoma patients.
Li Wenfei,Zhao Kun,Wang Zhanqiu
European archives of oto-rhino-laryngology : official journal of the European Federation of Oto-Rhino-Laryngological Societies (EUFOS) : affiliated with the German Society for Oto-Rhino-Laryngology - Head and Neck Surgery
PURPOSE:We aim to develop an immune-score nomograms for predicting overall survival (OS) of patients with HNSCC and assess the association of immune scores with prognosis. METHODS:The data of 530 patients used in this study were retrieved from The Cancer Genome Atlas database. The optimization cut-point for immune scores was expressed by X-tile 3.6.1 tool. Possible prognostic factors from univariate Cox analysis were further included in a multivariate Cox proportional hazards analysis to obtain significant risk factors. Prognostic nomograms were constructed based on the factors of significant multivariate prognostic using R version 3.5.1. A calibration map was generated by comparing the nomogram prediction probability and the observation for the 3-year and 5-year OS rates. RESULTS:We retrospectively analyzed 462 patients downloaded from TCGA dataset. Prognostic nomograms was integrated following risk factors of significant multivariate prognostic, such as age, angiolymphaic invasion (AI), perineura invasion(Per_invasion),tumor site, immune score, tumor-node-metastasis(TNM) stage. The concordance Index (C-index) for OS predictions was 0.723 (95% CI 0.671-0.785). Moreover, we compared the powerful efficiency of the nomograms with that of the TNM staging system. OS prediction determined on immune score set compared with the TNM staging with C-index = 0.723 vs 0.612. The calibration curves for the probability of OS of 3-year or 5-year showed no deviations between the prediction by nomograms and actual reference line. CONCLUSION:The present study indicate that high and intermediate immune scores are as independent prognostic variables for OS of head-neck squamous cell carcinoma patients. We constructed novel nomograms may has the potential to provide individualized survival risk assessments and guide treatment decisions.
The density and spatial tissue distribution of CD8 and CD163 immune cells predict response and outcome in melanoma patients receiving MAPK inhibitors.
Massi Daniela,Rulli Eliana,Cossa Mara,Valeri Barbara,Rodolfo Monica,Merelli Barbara,De Logu Francesco,Nassini Romina,Del Vecchio Michele,Di Guardo Lorenza,De Penni Roberta,Guida Michele,Sileni Vanna Chiarion,Di Giacomo Anna Maria,Tucci Marco,Occelli Marcella,Portelli Francesca,Vallacchi Viviana,Consoli Francesca,Quaglino Pietro,Queirolo Paola,Baroni Gianna,Carnevale-Schianca Fabrizio,Cattaneo Laura,Minisini Alessandro,Palmieri Giuseppe,Rivoltini Licia,Mandalà Mario,
Journal for immunotherapy of cancer
BACKGROUND:Clinical response to MAPK inhibitors in metastatic melanoma patients is heterogeneous for reasons still needing to be elucidated. As the patient immune activity contributes to treatment clinical benefit, the pre-existing level of immunity at tumor site may provide biomarkers of disease outcome to therapy. Here we investigated whether assessing the density and spatial tissue distribution of key immune cells in the tumor microenvironment could identify patients predisposed to respond to MAPK inhibitors. METHODS:Pretreatment tumor biopsies from a total of 213 patients (158 for the training set and 55 for the validation set) treated with BRAF or BRAF/MEK inhibitors within the Italian Melanoma Intergroup were stained with selected immune markers (CD8, CD163, β-catenin, PD-L1, PD-L2). Results, obtained by blinded immunohistochemical scoring and digital image analysis, were correlated with clinical response and outcome by multivariate logistic models on response to treatment and clinical outcome, adjusted for American Joint Committee on Cancer stage, performance status, lactate dehydrogenase and treatment received. RESULTS:Patients with high intratumoral, but not peritumoral, CD8 T cells and concomitantly low CD163 myeloid cells displayed higher probability of response (OR 9.91, 95% CI 2.23-44.0, p = 0.003) and longer overall survival (HR 0.34, 95% CI 0.16-0.72, p = 0.005) compared to those with intratumoral low CD8 T cells and high CD163 myeloid cells. The latter phenotype was instead associated with a shorter progression free survival (p = 0.010). In contrast, PD-L1 and PD-L2 did not correlate with clinical outcome while tumor β-catenin overexpression showed association with lower probability of response (OR 0.48, 95% CI 0.21-1.06, p = 0.068). CONCLUSIONS:Analysis of the spatially constrained distribution of CD8 and CD163 cells, representative of the opposite circuits of antitumor vs protumor immunity, respectively, may assist in identifying melanoma patients with improved response and better outcome upon treatment with MAPK inhibitors. These data underline the role of endogenous immune microenvironment in predisposing metastatic melanoma patients to benefit from therapies targeting driver-oncogenic pathways.
The Prognostic Landscape of Tumor-Infiltrating Immune Cells and Immune Checkpoints in Glioblastoma.
Wu Shiman,Yang Wenli,Zhang Hua,Ren Yan,Fang Ziwei,Yuan Chengjie,Yao Zhenwei
Technology in cancer research & treatment
Tumor-infiltrating immune cells are part of a complex microenvironment and associated with improved clinical outcomes in a broad range of tumor types. However, a detailed map for the prognostic landscape of tumor-infiltrating immune cells and immune checkpoint modulators in glioblastoma is still lacking. Here, with the web-accessible resource, The Cancer Immunome Archive, 28 types of both adaptive and innate tumor-infiltrating immune cells were characterized in glioblastoma. Tumors lacking central memory CD4 T cells or natural killer cells were associated with better prognosis in glioblastoma, as verified by immunohistochemical analysis. Moreover, Kaplan-Meier analysis for a total of 71 key immune checkpoint molecules revealed that the expression level of inducible T cell costimulators, tumor necrosis factor superfamily member 14, and UL16 binding protein 1 were negatively correlated with the clinical outcome of patients with glioblastoma. In addition, there was a significant difference between nontumor and glioblastoma samples of several immune checkpoint modulators based on the expression level of their corresponding gene. Collectively, the annotation of tumor-infiltrating immune cells and immune checkpoint modulators in glioblastoma provides a valuable resource for identifying their involvement in tumor escape mechanisms and response to therapy.
Topographical Distribution and Spatial Interactions of Innate and Semi-Innate Immune Cells in Pancreatic and Other Periampullary Adenocarcinoma.
Lundgren Sebastian,Micke Patrick,Elebro Jacob,Heby Margareta,Hrynchyk Ina,Nodin Björn,Leandersson Karin,Mezheyeuski Artur,Jirström Karin
Frontiers in immunology
Background:The clinical management of pancreatic and other periampullary neoplasms remains challenging. In contrast to other cancer types, immunotherapies are largely ineffective, and the reason for the deprived immune response and the immune inhibiting cellular composition is only fragmentarily understood. The aim of this study was to comprehensively map the abundance, topographic distribution and spatial interaction of innate and innate-like immune cells in the tumor microenvironment of periampullary adenocarcinoma. Methods:Multiplexed immunofluorescent imaging was performed on tissue microarrays with tumors from a consecutive cohort of 175 patients with resected periampullary adenocarcinoma. To obtain a detailed spatial analysis of immune cell infiltration, two multiplex immune panels including antibodies against CD3, NKp46, CD56, CD68, CD163 and CD1a, CD208, CD123, CD15, CD68 and pan-cytokeratin were applied. Results:The infiltration of natural killer (NK) and NK-like T (NKT) cells was lower in malignant compared to benign tissue. NKT cells were more abundant in intestinal type compared to pancreatobiliary type tumors, and were associated with more favorable clinicopathological features and a prolonged survival. The interaction of NKp46 NKT cells with macrophages was also associated with a prolonged survival. Conclusions:This study provides a comprehensive map of the innate immune landscape in periampullary adenocarcinoma. NK cells, and even more so NKT cells, are revealed to be central players in the local immune response in a clinically relevant context.
A multiscale signalling network map of innate immune response in cancer reveals cell heterogeneity signatures.
Kondratova Maria,Czerwinska Urszula,Sompairac Nicolas,Amigorena Sebastian D,Soumelis Vassili,Barillot Emmanuel,Zinovyev Andrei,Kuperstein Inna
The lack of integrated resources depicting the complexity of the innate immune response in cancer represents a bottleneck for high-throughput data interpretation. To address this challenge, we perform a systematic manual literature mining of molecular mechanisms governing the innate immune response in cancer and represent it as a signalling network map. The cell-type specific signalling maps of macrophages, dendritic cells, myeloid-derived suppressor cells and natural killers are constructed and integrated into a comprehensive meta map of the innate immune response in cancer. The meta-map contains 1466 chemical species as nodes connected by 1084 biochemical reactions, and it is supported by information from 820 articles. The resource helps to interpret single cell RNA-Seq data from macrophages and natural killer cells in metastatic melanoma that reveal different anti- or pro-tumor sub-populations within each cell type. Here, we report a new open source analytic platform that supports data visualisation and interpretation of tumour microenvironment activity in cancer.
Cellular heterogeneity map of diverse immune and stromal phenotypes within breast tumor microenvironment.
BACKGROUND:Cellular heterogeneity within the tumor microenvironment is essential to tumorigenesis and tumor development. A high-resolution global view of the tumor-infiltrating immune and stromal cells in breast tumors is needed. METHODS:xCell was used to create a cellular heterogeneity map of 64 cell types in 1,092 breast tumor and adjacent normal tissues. xCell digitally dissects tissue cellular heterogeneity based on gene expression. Integrated statistical analyses were then performed. RESULTS:There were noticeable differences between the cell fractions in tumor tissues and normal tissues. Tumors displayed higher proportions of immune cells, including CD4+ Tem, CD8+ naïve T cells, and CD8+ Tcm compared with normal tissues. Immune inhibitory receptors (PD1, CTLA4, LAG3 and TIM3) were co-expressed on certain subtypes of T cells in breast tumors, and PD1 and CTLA4 were both positively correlated with CD8+ Tcm and CD8+ T cells. 28 cell types were significantly associated with overall survival in univariate analysis. CD4+ Tem, CD8+ Tcm, CD8+ T-cells, CD8+ naive T-cells, and B cells were positive prognostic factors but CD4+ naive T-cells were negative prognostic factors for breast cancer patients. TDRD6 and TTK are promising T cell and B cell targets for tumor vaccines. Endothelial cells and fibroblasts were significantly less prevalent in tumor tissues; astrocytes and mesangial cells were negatively correlated with the T stage. Mesangial cells and keratinocytes were found to be favorable prognostic factors and myocytes were negative prognostic factors. Five cell types were found to be independent prognostic factors and we used these to create a reliable prognostic model for breast cancer patients. Cellular heterogeneity was discovered among different breast cancer subtypes by Her2, ER, and PR status. Tri-negative patients had the highest fraction of immune cells while luminal type patients had the lowest. The various cells may have diverse or opposing roles in the prognosis of breast cancer patients. CONCLUSIONS:We created a uniquecellular map for the diverse heterogeneity of immune and stromal phenotypes within the breast tumor microenvironment. This map may lead to potential therapeutic targets and biomarkers with prognostic utility.
Challenges and Opportunities in the Statistical Analysis of Multiplex Immunofluorescence Data.
Immune modulation is considered a hallmark of cancer initiation and progression. The recent development of immunotherapies has ushered in a new era of cancer treatment. These therapeutics have led to revolutionary breakthroughs; however, the efficacy of immunotherapy has been modest and is often restricted to a subset of patients. Hence, identification of which cancer patients will benefit from immunotherapy is essential. Multiplex immunofluorescence (mIF) microscopy allows for the assessment and visualization of the tumor immune microenvironment (TIME). The data output following image and machine learning analyses for cell segmenting and phenotyping consists of the following information for each tumor sample: the number of positive cells for each marker and phenotype(s) of interest, number of total cells, percent of positive cells for each marker, and spatial locations for all measured cells. There are many challenges in the analysis of mIF data, including many tissue samples with zero positive cells or "zero-inflated" data, repeated measurements from multiple TMA cores or tissue slides per subject, and spatial analyses to determine the level of clustering and co-localization between the cell types in the TIME. In this review paper, we will discuss the challenges in the statistical analysis of mIF data and opportunities for further research.
COX-2 expression positively correlates with PD-L1 expression in human melanoma cells.
Botti Gerardo,Fratangelo Federica,Cerrone Margherita,Liguori Giuseppina,Cantile Monica,Anniciello Anna Maria,Scala Stefania,D'Alterio Crescenzo,Trimarco Chiara,Ianaro Angela,Cirino Giuseppe,Caracò Corrado,Colombino Maria,Palmieri Giuseppe,Pepe Stefano,Ascierto Paolo Antonio,Sabbatino Francesco,Scognamiglio Giosuè
Journal of translational medicine
BACKGROUND:The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma. METHODS:Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAF A375 and NRAS SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro. RESULTS:BRAF and NRAS were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAF A375 and NRAS SK-MEL-2 melanoma cell lines. CONCLUSIONS:COX-2 expression correlates with and modulates PD-L1 expression in melanoma cells. These findings have clinical relevance since they provide a rationale to implement novel clinical trials to test COX-2 inhibition as a potential treatment to prevent melanoma progression and immune evasion as well as to enhance the anti-tumor activity of PD-1/PD-L1 based immunotherapy for the treatment of melanoma patients with or without BRAF/NRAS mutations.
Concordance of immune checkpoints within tumor immune contexture and their prognostic significance in gastric cancer.
Dai Congqi,Geng Ruixuan,Wang Chenchen,Wong Angela,Qing Min,Hu Jianjun,Sun Yu,Lo A W I,Li Jin
Checkpoint blockade therapy has emerged as a novel approach for cancer immunotherapy in several malignancies. However, patient prognosis and disease progression relevant to immune checkpoints in gastric tumor microenvironment are not defined. This study aims to investigate the expression and prognostic significance of immune checkpoints within gastric cancer. In the study, a cohort of 398 cancer tissues from stage I to IV gastric cancer patients were assessed for programmed cell death 1 ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) infiltration using immunohistochemistry to ascertain their survival correlation. The data revealed that higher TIL density correlated with less risk of disease progression, and exhibited survival benefits in gastric cancer patients, and PD-L1 positivity showed a significant association with the presence of high TIL infiltration. Furthermore, real-time quantitative polymerase chain reaction was performed to detect expression of multiple immune checkpoints with the relation to clinical outcome in 139 samples randomly selected from the same cohort, and higher messenger RNA levels of most immune checkpoints were associated with favorable outcome, while consistently showing a positive correlation with interferon gamma levels. In situ hybridization was used to determine the localization of Epstein-Barr virus (EBV) in 97 specimens, and showed EBV-positive gastric cancer samples correlated with PD-L1 expression and increased TIL density. These results suggest that induction of immune checkpoint within gastric cancer patients reflects a high immune infiltration density, especially in those with EBV-associated gastric cancer, which may direct patient selection for checkpoint blockade therapy.
Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation.
Zhang Wei,Zhou Shumin,Liu Guoping,Kong Fanqiang,Chen Song,Yan Hua
Physiologically, retinal pigment epithelium (RPE) expresses high levels of CD73 in their membrane, converting AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. However, after being exposed to inflammatory factors, RPE rapidly becomes CD73-negative cells, which render RPE's immune suppressive function and accelerate local inflammation. Here, we investigated the mechanism leading to the loss of membrane CD73 in RPE. We found the controversy that when membrane CD73 was significantly diminished in inflammatory RPE, Cd73 mRNA levels were not changed at all. It was further verified that, matrix metalloproteinase-9 (MMP-9) mediated the shedding of CD73 from the cell membrane of inflammatory RPE by catalyzing its K547/F548 site. However, MMP-9 could not catalyze uncomplexed CD73, the interaction of CD73 with adenosine receptor A1 subtype (ARA1) is necessary for being catalyzed by MMP-9. After being treated by LPS and TNF-α, the formation of CD73/ARA1 complex in RPE was verified by co-immunoprecipitation and FRET-based assays. It was also revealed that CD73 need to be localized in lipid rafts to be capable of interacting with ARA1, since CD73/ARA1 interaction and CD73 shedding were completely blocked by the addition of lipid raft synthesis inhibitor. As a conclusion, multiple steps are involved in CD73 shedding in RPE, including up-regulation of MMP-9 activity, localization of CD73 in lipid rafts, and the formation of CD73/ARA1 complex. Lipid rafts committed CD73 with high mobility, shuttled CD73 to ARA1 to form a complex, which was capable of being recognized and catalyzed by MMP-9.
Beyond immune density: critical role of spatial heterogeneity in estrogen receptor-negative breast cancer.
Nawaz Sidra,Heindl Andreas,Koelble Konrad,Yuan Yinyin
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
The abundance of tumor-infiltrating lymphocytes has been associated with a favorable prognosis in estrogen receptor-negative breast cancer. However, a high degree of spatial heterogeneity in lymphocytic infiltration is often observed and its clinical implication remains unclear. Here we combine automated histological image processing with methods of spatial statistics used in ecological data analysis to quantify spatial heterogeneity in the distribution patterns of tumor-infiltrating lymphocytes. Hematoxylin and eosin-stained sections from two cohorts of estrogen receptor-negative breast cancer patients (discovery: n=120; validation: n=125) were processed with our automated cell classification algorithm to identify the location of lymphocytes and cancer cells. Subsequently, hotspot analysis (Getis-Ord Gi*) was applied to identify statistically significant hotspots of cancer and immune cells, defined as tumor regions with a significantly high number of cancer cells or immune cells, respectively. We found that the amount of co-localized cancer and immune hotspots weighted by tumor area, rather than number of cancer or immune hotspots, correlates with a better prognosis in estrogen receptor-negative breast cancer in univariate and multivariate analysis. Moreover, co-localization of cancer and immune hotspots further stratified patients with immune cell-rich tumors. Our study demonstrates the importance of quantifying not only the abundance of lymphocytes but also their spatial variation in the tumor specimen for which methods from other disciplines such as spatial statistics can be successfully applied.
Multiplexed immunohistochemistry for immune cell phenotyping, quantification and spatial distribution in situ.
Methods in enzymology
It is increasingly recognized that a deep characterization of the immune microenvironment is required for the identification of prognostic and predictive immune biomarkers. Recent advances in the field of tissue imaging resulted in the development of fluorescence multiplex IHC technologies enabling quantitative assessment of immune phenotypes and functional orientation of immune cells in a way similar to flow cytometry, while simultaneously providing tissue context and spatial distribution. Multiplex immunofluorescent technology to FFPE tumor tissue is applied to characterize immune infiltration and PD-L1 expression. A panel consists of five protein markers: CD8, CD68, PD-L1, CK, and SOX10. The assay workflow is fast, optimized and compatible with existing instrumentation. The resulting images can be analyzed with routinely used software for digital pathology enabling the quantification of dynamic range of expression, co-localization and co-expression of markers in the whole tissue. In this chapter, we provide the protocol for the use of the UltiMapper™ I/O PD-L1 multiplex assay, from the bench to the image analysis, as well as an overview of the current multiplex image analysis solutions. Such deep profiling could guide the development of strategies to better select immune checkpoint molecules and a better stratification of patients who will potentially benefit from immunotherapies.
Localization and density of immune cells in the invasive margin of human colorectal cancer liver metastases are prognostic for response to chemotherapy.
Halama Niels,Michel Sara,Kloor Matthias,Zoernig Inka,Benner Axel,Spille Anna,Pommerencke Thora,von Knebel Doeberitz Magnus,Folprecht Gunnar,Luber Birgit,Feyen Nadine,Martens Uwe M,Beckhove Philipp,Gnjatic Sacha,Schirmacher Peter,Herpel Esther,Weitz Juergen,Grabe Niels,Jaeger Dirk
Analysis of tumor-infiltrating lymphocytes (TIL) in primary human colorectal cancer (CRC) by in situ immunohistochemical staining supports the hypothesis that the adaptive immune response influences the course of human CRC. Specifically, high densities of TILs in the primary tumor are associated with good prognosis independent of other prognostic markers. However, the prognostic role of TILs in metastatic CRC lesions is unknown, as is their role in response or resistance to conventional chemotherapy. We analyzed the association of TIL densities at the invasive margin of CRC liver metastases with response to chemotherapy and progression-free survival in a set of 101 large section samples. High-resolution automated microscopy on complete tissue sections was used to objectively generate cell densities for CD3, CD8, granzyme B, or FOXP3 positive immune cells. A predictive scoring system using TIL densities was developed in a training set and tested successfully in an independent validation set. TIL densities at the invasive margin of liver metastases allowed the prediction of response to chemotherapy with a sensitivity of 79% and specificity of 100%. The association of high density values with longer progression-free survival under chemotherapy was statistically significant. Overall, these findings extend the impact of the local immune response on the clinical course from the primary tumor also to metastatic lesions. Because detailed quantification of TILs in metastatic lesions revealed a strong association with chemotherapy efficacy and prognosis, we suggest that the developed scoring system may be used as a predictive tool for response to chemotherapy in metastatic CRC.
The localization and density of immune cells in primary tumors of human metastatic colorectal cancer shows an association with response to chemotherapy.
Halama Niels,Michel Sara,Kloor Matthias,Zoernig Inka,Pommerencke Thora,von Knebel Doeberitz Magnus,Schirmacher Peter,Weitz Jürgen,Grabe Niels,Jäger Dirk
In situ immunohistochemical staining of tumor-infiltrating immune cells in large cohorts of human colorectal cancers has recently supported the hypothesis that the adaptive immune response influences the behavior of human tumors. Tumor-infiltrating immune cells therefore represent a valuable prognostic marker for patients with colorectal cancer, with a high density of immune cells being associated with a good outcome independently of other established prognostic markers. The aim of the present study was to investigate the correlation between infiltrates of immune cells, in either the primary tumor or (where available) the corresponding liver metastases, with the response to chemotherapy in patients with metastatic colorectal cancer. The analysis consisted of 32 samples from 22 patients with metastasized colorectal cancer, including ten pairs of primary tumors and corresponding liver metastases. In primary tumors the ratio of stained immune cells in the epithelial portion of the tumor as compared to the total number of immune cells staining for CD3, CD8 and Granzyme B showed a relationship to the response to chemotherapy and the time to progression under chemotherapy. The primary tumors showed marked intra-tumoral heterogeneity with respect to immune cell densities. Infiltrate densities differed significantly between corresponding primary tumors and liver metastases, a variability that was also observed at the invasive margin of liver metastases. This suggests that immune infiltrates at the invasive margin of liver metastases could be predictive with respect to response to treatment. This is currently being evaluated in a larger patient cohort.
Co-localization of CD169 macrophages and cancer cells in lymph node metastases of breast cancer patients is linked to improved prognosis and PDL1 expression.
Björk Gunnarsdottir Frida,Auoja Nathalie,Bendahl Pär-Ola,Rydén Lisa,Fernö Mårten,Leandersson Karin
Breast cancer is the most common form of cancer in women worldwide. Although the survival among breast cancer patients has improved, there is still a large group of patients with dismal prognosis. One of the most important prognostic factors for poor prognosis is lymph node metastasis. Increasing knowledge concerning the lymph nodes of breast cancer patients indicates that they are affected by the primary tumor. In this study we show that presence of CD169 subcapsular sinus macrophages in contact with lymph node metastases in breast cancer patients, is related to better prognosis after adjuvant tamoxifen treatment, but only in patients with PDL1 primary tumors. This is in contrast to the prognostic effect of CD169 primary tumor-associated macrophages (TAMs). We further show that CD169 macrophages were spatially associated with expression of PDL1 on nearby cells, both in primary tumors and metastatic lymph node, although PDL1 expression in metastatic lymph node as such did not have further prognostic impact. Our data suggest that CD169 resident lymph node macrophages have a unique function in targeting immune responses against breast cancer and should be further investigated in detail.
The spatial structure of cell signaling systems.
The spatial structure of the cell is highly organized at all levels: from small complexes and assemblies, to local nano- and microclusters, to global, micrometer scales across and between cells. We suggest that this multiscale spatial cell organization also organizes signaling and coordinates cellular behavior. We propose a new view of the spatial structure of cell signaling systems. This new view describes cell signaling in terms of dynamic allosteric interactions within and among distinct, spatially organized transient clusters. The clusters vary over time and space and are on length scales from nanometers to micrometers. When considered across these length scales, primary factors in the spatial organization are cell membrane domains and the actin cytoskeleton, both also highly dynamic. A key challenge is to understand the interplay across these multiple scales, link it to the physicochemical basis of the conformational behavior of single molecules and ultimately relate it to cellular function. Overall, our premise is that at these scales, cell signaling should be thought of not primarily as a sequence of diffusion-controlled molecular collisions, but instead transient, allostery-driven cluster re-forming interactions.
Spatial Metrics of Tumour Vascular Organisation Predict Radiation Efficacy in a Computational Model.
Scott Jacob G,Fletcher Alexander G,Anderson Alexander R A,Maini Philip K
PLoS computational biology
Intratumoural heterogeneity is known to contribute to poor therapeutic response. Variations in oxygen tension in particular have been correlated with changes in radiation response in vitro and at the clinical scale with overall survival. Heterogeneity at the microscopic scale in tumour blood vessel architecture has been described, and is one source of the underlying variations in oxygen tension. We seek to determine whether histologic scale measures of the erratic distribution of blood vessels within a tumour can be used to predict differing radiation response. Using a two-dimensional hybrid cellular automaton model of tumour growth, we evaluate the effect of vessel distribution on cell survival outcomes of simulated radiation therapy. Using the standard equations for the oxygen enhancement ratio for cell survival probability under differing oxygen tensions, we calculate average radiation effect over a range of different vessel densities and organisations. We go on to quantify the vessel distribution heterogeneity and measure spatial organization using Ripley's L function, a measure designed to detect deviations from complete spatial randomness. We find that under differing regimes of vessel density the correlation coefficient between the measure of spatial organization and radiation effect changes sign. This provides not only a useful way to understand the differences seen in radiation effect for tissues based on vessel architecture, but also an alternate explanation for the vessel normalization hypothesis.
Multi-Omics Profiling of the Tumor Microenvironment: Paving the Way to Precision Immuno-Oncology.
Frontiers in oncology
The tumor microenvironment (TME) is a multifaceted ecosystem characterized by profound cellular heterogeneity, dynamicity, and complex intercellular cross-talk. The striking responses obtained with immune checkpoint blockers, i.e., antibodies targeting immune-cell regulators to boost antitumor immunity, have demonstrated the enormous potential of anticancer treatments that target TME components other than tumor cells. However, as checkpoint blockade is currently beneficial only to a limited fraction of patients, there is an urgent need to understand the mechanisms orchestrating the immune response in the TME to guide the rational design of more effective anticancer therapies. In this Mini Review, we give an overview of the methodologies that allow studying the heterogeneity of the TME from multi-omics data generated from bulk samples, single cells, or images of tumor-tissue slides. These include approaches for the characterization of the different cell phenotypes and for the reconstruction of their spatial organization and inter-cellular cross-talk. We discuss how this broader vision of the cellular heterogeneity and plasticity of tumors, which is emerging thanks to these methodologies, offers the opportunity to rationally design precision immuno-oncology treatments. These developments are fundamental to overcome the current limitations of targeted agents and checkpoint blockers and to bring long-term clinical benefits to a larger fraction of cancer patients.
Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment.
Flores Molina Manuel,Fabre Thomas,Cleret-Buhot Aurélie,Soucy Geneviève,Meunier Liliane,Abdelnabi Mohamed N,Belforte Nicolas,Turcotte Simon,Shoukry Naglaa H
Journal of visualized experiments : JoVE
The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.
INFORM: INFrared-based ORganizational Measurements of tumor and its microenvironment to predict patient survival.
The structure and organization of a tumor and its microenvironment are often associated with cancer outcomes due to spatially varying molecular composition and signaling. A persistent challenge is to use this physical and chemical spatial organization to understand cancer progression. Here, we present a high-definition infrared imaging-based organizational measurement framework (INFORM) that leverages intrinsic chemical contrast of tissue to label unique components of the tumor and its microenvironment. Using objective and automated computational methods, further, we determine organization characteristics important for prediction. We show that the tumor spatial organization assessed with this framework is predictive of overall survival in colon cancer that adds to capability from clinical variables such as stage and grade, approximately doubling the risk of death in high-risk individuals. Our results open an all-digital avenue for measuring and studying the association between tumor spatial organization and disease progression.
Computational Staining of Pathology Images to Study the Tumor Microenvironment in Lung Cancer.
Wang Shidan,Rong Ruichen,Yang Donghan M,Fujimoto Junya,Yan Shirley,Cai Ling,Yang Lin,Luo Danni,Behrens Carmen,Parra Edwin R,Yao Bo,Xu Lin,Wang Tao,Zhan Xiaowei,Wistuba Ignacio I,Minna John,Xie Yang,Xiao Guanghua
The spatial organization of different types of cells in tumor tissues reveals important information about the tumor microenvironment (TME). To facilitate the study of cellular spatial organization and interactions, we developed Histology-based Digital-Staining, a deep learning-based computation model, to segment the nuclei of tumor, stroma, lymphocyte, macrophage, karyorrhexis, and red blood cells from standard hematoxylin and eosin-stained pathology images in lung adenocarcinoma. Using this tool, we identified and classified cell nuclei and extracted 48 cell spatial organization-related features that characterize the TME. Using these features, we developed a prognostic model from the National Lung Screening Trial dataset, and independently validated the model in The Cancer Genome Atlas lung adenocarcinoma dataset, in which the predicted high-risk group showed significantly worse survival than the low-risk group ( = 0.001), with a HR of 2.23 (1.37-3.65) after adjusting for clinical variables. Furthermore, the image-derived TME features significantly correlated with the gene expression of biological pathways. For example, transcriptional activation of both the T-cell receptor and programmed cell death protein 1 pathways positively correlated with the density of detected lymphocytes in tumor tissues, while expression of the extracellular matrix organization pathway positively correlated with the density of stromal cells. In summary, we demonstrate that the spatial organization of different cell types is predictive of patient survival and associated with the gene expression of biological pathways. SIGNIFICANCE: These findings present a deep learning-based analysis tool to study the TME in pathology images and demonstrate that the cell spatial organization is predictive of patient survival and is associated with gene expression..
Characterizing cell-cell interactions induced spatial organization of cell phenotypes: application to density-dependent protein nucleocytoplasmic distribution.
Han Fujun,Zhang Biliang
Cell biochemistry and biophysics
Cell-cell interactions play an important role in spatial organization (pattern formation) during the development of multicellular organisms. An understanding of these biological roles requires identifying cell phenotypes that are regulated by cell-cell interactions and characterizing the spatial organizations of the phenotypes. However, conventional methods for assaying cell-cell interactions are mainly applicable at a cell population level. These measures are incapable of elucidating the spatial organizations of the phenotypes, resulting in an incomplete view of cell-cell interactions. To overcome this issue, we developed an automated image-based method to investigate cell-cell interactions based on spatial localizations of cells. We demonstrated this method in cultured cells using cell density-dependent nucleocytoplasmic distribution of β-catenin and aryl hydrocarbon receptor as the phenotype. This novel method was validated by comparing with a conventional population-based method, and proved to be more sensitive and reliable. The application of the method characterized how the phenotypes were spatially organized in a population of cultured cells. We further showed that the spatial organization was governed by cell density and was protein-specific. This automated method is very simple, and will be applicable to study cell-cell interactions in different systems from prokaryotic colonies to multicellular organisms. We envision that the ability to extract and interpret how cell-cell interactions determine the spatial organization of a cell phenotype will provide new insights into biology that may be missed by traditional population-averaged studies.
Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images.
Saltz Joel,Gupta Rajarsi,Hou Le,Kurc Tahsin,Singh Pankaj,Nguyen Vu,Samaras Dimitris,Shroyer Kenneth R,Zhao Tianhao,Batiste Rebecca,Van Arnam John, ,Shmulevich Ilya,Rao Arvind U K,Lazar Alexander J,Sharma Ashish,Thorsson Vésteinn
Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumor-infiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment.
Analysis of Spatial Organization of Suppressive Myeloid Cells and Effector T Cells in Colorectal Cancer-A Potential Tool for Discovering Prognostic Biomarkers in Clinical Research.
Zwing Natalie,Failmezger Henrik,Ooi Chia-Huey,Hibar Derrek P,Cañamero Marta,Gomes Bruno,Gaire Fabien,Korski Konstanty
Frontiers in immunology
The development and progression of solid tumors such as colorectal cancer (CRC) are known to be affected by the immune system and cell types such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are emerging as interesting targets for immunotherapy and clinical biomarker research. In addition, CD3 and CD8 T cell distribution in tumors has shown positive prognostic value in stage I-III CRC. Recent developments in digital computational pathology support not only classical cell density based tumor characterization, but also a more comprehensive analysis of the spatial cell organization in the tumor immune microenvironment (TiME). Leveraging that methodology in the current study, we tried to address the question of how the distribution of myeloid derived suppressor cells in TiME of primary CRC affects the function and location of cytotoxic T cells. We applied multicolored immunohistochemistry to identify monocytic (CD11bCD14) and granulocytic (CD11bCD15) myeloid cell populations together with proliferating and non-proliferating cytotoxic T cells (CD8Ki67). Through automated object detection and image registration using HALO software (IndicaLabs), we applied dedicated spatial statistics to measure the extent of overlap between the areas occupied by myeloid and T cells. With this approach, we observed distinct spatial organizational patterns of immune cells in tumors obtained from 74 treatment-naive CRC patients. Detailed analysis of inter-cell distances and myeloid-T cell spatial overlap combined with integrated gene expression data allowed to stratify patients irrespective of their mismatch repair (MMR) status or consensus molecular subgroups (CMS) classification. In addition, generation of cell distance-derived gene signatures and their mapping to the TCGA data set revealed associations between spatial immune cell distribution in TiME and certain subsets of CD8 and CD4 T cells. The presented study sheds a new light on myeloid and T cell interactions in TiME in CRC patients. Our results show that CRC tumors present distinct distribution patterns of not only T effector cells but also tumor resident myeloid cells, thus stressing the necessity of more comprehensive characterization of TiME in order to better predict cancer prognosis. This research emphasizes the importance of a multimodal approach by combining computational pathology with its detailed spatial statistics and gene expression profiling. Finally, our study presents a novel approach to cancer patients' characterization that can potentially be used to develop new immunotherapy strategies, not based on classical biomarkers related to CRC biology.
Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region.
Moffitt Jeffrey R,Bambah-Mukku Dhananjay,Eichhorn Stephen W,Vaughn Eric,Shekhar Karthik,Perez Julio D,Rubinstein Nimrod D,Hao Junjie,Regev Aviv,Dulac Catherine,Zhuang Xiaowei
Science (New York, N.Y.)
The hypothalamus controls essential social behaviors and homeostatic functions. However, the cellular architecture of hypothalamic nuclei-including the molecular identity, spatial organization, and function of distinct cell types-is poorly understood. Here, we developed an imaging-based in situ cell-type identification and mapping method and combined it with single-cell RNA-sequencing to create a molecularly annotated and spatially resolved cell atlas of the mouse hypothalamic preoptic region. We profiled ~1 million cells, identified ~70 neuronal populations characterized by distinct neuromodulatory signatures and spatial organizations, and defined specific neuronal populations activated during social behaviors in male and female mice, providing a high-resolution framework for mechanistic investigation of behavior circuits. The approach described opens a new avenue for the construction of cell atlases in diverse tissues and organisms.
B cell signatures and tertiary lymphoid structures contribute to outcome in head and neck squamous cell carcinoma.
Ruffin Ayana T,Cillo Anthony R,Tabib Tracy,Liu Angen,Onkar Sayali,Kunning Sheryl R,Lampenfeld Caleb,Atiya Huda I,Abecassis Irina,Kürten Cornelius H L,Qi Zengbiao,Soose Ryan,Duvvuri Umamaheswar,Kim Seungwon,Oesterrich Steffi,Lafyatis Robert,Coffman Lan G,Ferris Robert L,Vignali Dario A A,Bruno Tullia C
Current immunotherapy paradigms aim to reinvigorate CD8 T cells, but the contribution of humoral immunity to antitumor immunity remains understudied. Here, we demonstrate that in head and neck squamous cell carcinoma (HNSCC) caused by human papillomavirus infection (HPV), patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with tertiary lymphoid structures (TLS) with GCs, both of which correlate with favorable outcome. GC TIL-Bs in HPV HNSCC are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. Semaphorin 4a expression is enhanced on GC TIL-Bs present in TLS of HPV HNSCC and during the differentiation of TIL-Bs. Our study suggests that therapeutics to enhance TIL-B responses in HNSCC should be prioritized in future studies to determine if they can complement current T cell mediated immunotherapies.
An ecological measure of immune-cancer colocalization as a prognostic factor for breast cancer.
Maley Carlo C,Koelble Konrad,Natrajan Rachael,Aktipis Athena,Yuan Yinyin
Breast cancer research : BCR
INTRODUCTION:Abundance of immune cells has been shown to have prognostic and predictive significance in many tumor types. Beyond abundance, the spatial organization of immune cells in relation to cancer cells may also have significant functional and clinical implications. However there is a lack of systematic methods to quantify spatial associations between immune and cancer cells. METHODS:We applied ecological measures of species interactions to digital pathology images for investigating the spatial associations of immune and cancer cells in breast cancer. We used the Morisita-Horn similarity index, an ecological measure of community structure and predator-prey interactions, to quantify the extent to which cancer cells and immune cells colocalize in whole-tumor histology sections. We related this index to disease-specific survival of 486 women with breast cancer and validated our findings in a set of 516 patients from different hospitals. RESULTS:Colocalization of immune cells with cancer cells was significantly associated with a disease-specific survival benefit for all breast cancers combined. In HER2-positive subtypes, the prognostic value of immune-cancer cell colocalization was highly significant and exceeded those of known clinical variables. Furthermore, colocalization was a significant predictive factor for long-term outcome following chemotherapy and radiotherapy in HER2 and Luminal A subtypes, independent of and stronger than all known clinical variables. CONCLUSIONS:Our study demonstrates how ecological methods applied to the tumor microenvironment using routine histology can provide reproducible, quantitative biomarkers for identifying high-risk breast cancer patients. We found that the clinical value of immune-cancer interaction patterns is highly subtype-specific but substantial and independent to known clinicopathologic variables that mostly focused on cancer itself. Our approach can be developed into computer-assisted prediction based on histology samples that are already routinely collected.
Composition, Spatial Characteristics, and Prognostic Significance of Myeloid Cell Infiltration in Pancreatic Cancer.
Väyrynen Sara A,Zhang Jinming,Yuan Chen,Väyrynen Juha P,Dias Costa Andressa,Williams Hannah,Morales-Oyarvide Vicente,Lau Mai Chan,Rubinson Douglas A,Dunne Richard F,Kozak Margaret M,Wang Wenjia,Agostini-Vulaj Diana,Drage Michael G,Brais Lauren,Reilly Emma,Rahma Osama,Clancy Thomas,Wang Jiping,Linehan David C,Aguirre Andrew J,Fuchs Charles S,Coussens Lisa M,Chang Daniel T,Koong Albert C,Hezel Aram F,Ogino Shuji,Nowak Jonathan A,Wolpin Brian M
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:Although abundant myeloid cell populations in the pancreatic ductal adenocarcinoma (PDAC) microenvironment have been postulated to suppress antitumor immunity, the composition of these populations, their spatial locations, and how they relate to patient outcomes are poorly understood. EXPERIMENTAL DESIGN:To generate spatially resolved tumor and immune cell data at single-cell resolution, we developed two quantitative multiplex immunofluorescence assays to interrogate myeloid cells (CD15, CD14, ARG1, CD33, HLA-DR) and macrophages [CD68, CD163, CD86, IFN regulatory factor 5, MRC1 (CD206)] in the PDAC tumor microenvironment. Spatial point pattern analyses were conducted to assess the degree of colocalization between tumor cells and immune cells. Multivariable-adjusted Cox proportional hazards regression was used to assess associations with patient outcomes. RESULTS:In a multi-institutional cohort of 305 primary PDAC resection specimens, myeloid cells were abundant, enriched within stromal regions, highly heterogeneous across tumors, and differed by somatic genotype. High densities of CD15ARG1 immunosuppressive granulocytic cells and M2-polarized macrophages were associated with worse patient survival. Moreover, beyond cell density, closer proximity of M2-polarized macrophages to tumor cells was strongly associated with disease-free survival, revealing the clinical significance and biologic importance of immune cell localization within tumor areas. CONCLUSIONS:A diverse set of myeloid cells are present within the PDAC tumor microenvironment and are distributed heterogeneously across patient tumors. Not only the densities but also the spatial locations of myeloid immune cells are associated with patient outcomes, highlighting the potential role of spatially resolved myeloid cell subtypes as quantitative biomarkers for PDAC prognosis and therapy.
Towards the introduction of the 'Immunoscore' in the classification of malignant tumours.
Galon Jérôme,Mlecnik Bernhard,Bindea Gabriela,Angell Helen K,Berger Anne,Lagorce Christine,Lugli Alessandro,Zlobec Inti,Hartmann Arndt,Bifulco Carlo,Nagtegaal Iris D,Palmqvist Richard,Masucci Giuseppe V,Botti Gerardo,Tatangelo Fabiana,Delrio Paolo,Maio Michele,Laghi Luigi,Grizzi Fabio,Asslaber Martin,D'Arrigo Corrado,Vidal-Vanaclocha Fernando,Zavadova Eva,Chouchane Lotfi,Ohashi Pamela S,Hafezi-Bakhtiari Sara,Wouters Bradly G,Roehrl Michael,Nguyen Linh,Kawakami Yutaka,Hazama Shoichi,Okuno Kiyotaka,Ogino Shuji,Gibbs Peter,Waring Paul,Sato Noriyuki,Torigoe Toshihiko,Itoh Kyogo,Patel Prabhu S,Shukla Shilin N,Wang Yili,Kopetz Scott,Sinicrope Frank A,Scripcariu Viorel,Ascierto Paolo A,Marincola Francesco M,Fox Bernard A,Pagès Franck
The Journal of pathology
The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).
Prediction of lymph node metastasis by tumor-infiltrating lymphocytes in T1 breast cancer.
Takada Koji,Kashiwagi Shinichiro,Asano Yuka,Goto Wataru,Kouhashi Rika,Yabumoto Akimichi,Morisaki Tamami,Shibutani Masatsune,Takashima Tsutomu,Fujita Hisakazu,Hirakawa Kosei,Ohira Masaichi
BACKGROUND:Lymph node metastasis is more likely in early-stage breast cancer with lower tumor-infiltrating lymphocyte (TIL) density. Therefore, we investigated the correlation between TILs and lymph node metastasis in cT1 breast cancer patients undergoing surgery and the usefulness of TILs in predicting sentinel lymph node metastasis (SLNM) in cT1N0M0 breast cancer. METHODS:We investigated 332 breast cancer patients who underwent surgery as the first-line treatment after preoperative diagnosis of cT1. A positive diagnosis of SLNM as an indication for axillary clearance was defined as macrometastasis in the sentinel lymph node (SLN) (macrometastasis: tumor diameter > 2 mm). Semi-quantitative evaluation of lymphocytes infiltrating the peritumoral stroma as TILs in primary tumor biopsy specimens prior to treatment was conducted. RESULTS:For SLN biopsy (SLNB), a median of 2 (range, 1-8) SLNs were pathologically evaluated. Sixty cases (19.4%) of SLNM (macrometastasis: 46, micrometastasis: 16) were observed. Metastasis was significantly greater in breast cancers with tumor diameter > 10 mm than in those with diameter ≤ 10 mm (p = 0.016). Metastasis was significantly associated with lymphatic invasion (p < 0.001). These two clinicopathological factors correlated with SLNM even in patients diagnosed with cN0 (tumor size; p = 0.017, lymphatic invasion; p = 0.002). Multivariate analysis for SLNM predictors revealed lymphatic invasion (p = 0.008, odds ratio [OR] = 2.522) and TILs (p < 0.001, OR = 0.137) as independent factors. CONCLUSIONS:Our results suggest a correlation between lymph node metastasis and tumor immune-microenvironment in cT1 breast cancer. TIL density may be a predictor of SLNM in breast cancer without lymph node metastasis on preoperative imaging.
Characterization of the immune microenvironment of NSCLC by multispectral analysis of multiplex immunofluorescence images.
Surace Michael,Rognoni Lorenz,Rodriguez-Canales Jaime,Steele Keith E
Methods in enzymology
Multiplex immunofluorescence (MIF) staining of tumor sections combined with computational pathology quantifies phenotypic variants of tumor and immune cells and assesses their spatial relationships. Here, we discuss a MIF panel composed of cytokeratin, PD-L1, PD1, CD8, CD68, and Ki67 applied to non-small cell lung cancer (NSCLC) to demonstrate key components of the immune response to this cancer. We also describe a method of whole-slide multiplex imaging and digital multispectral image analysis. Key aspects of marker labeling and digital tissue and cellular classification are highlighted. We then illustrate how digital analysis can measure the spatial relationships among important cell types. This approach is presented in the context of a multidisciplinary team of scientists who together can optimize the combined methods to increase the impact of the study findings. Recommendations are provided to assist others to apply similar methods to further understand the immune response to NSCLC.
Close proximity of immune and tumor cells underlies response to anti-PD-1 based therapies in metastatic melanoma patients.
Gide Tuba N,Silva Ines P,Quek Camelia,Ahmed Tasnia,Menzies Alexander M,Carlino Matteo S,Saw Robyn P M,Thompson John F,Batten Marcel,Long Georgina V,Scolyer Richard A,Wilmott James S
Immune checkpoint blockade has greatly improved the clinical outcomes of many patients with metastatic melanoma, however, almost half do not respond. Whether the interspatial distribution of immune and tumor cells predicts response to anti-PD-1-based therapies and patient outcomes in any cancer, including melanoma, is currently unknown. Here, we examined the spatial distribution of immune and tumor cells via multiplex immunofluorescence. Pre-treatment melanoma specimens from 27 patients ( = 18 responders; = 9 non-responders) treated with anti-PD-1 monotherapy and 34 patients ( = 22 responders; = 12 non-responders) treated with combined ipilimumab and anti-PD-1 immunotherapy were studied. Responders displayed significantly higher densities of CD8 tumor-infiltrating lymphocytes within a 20 µM distance from a melanoma cell compared to non-responders in both anti-PD-1 alone ( = .0024) and combination-treated patients ( = .0096), that were associated with improved progression-free survival for both therapies (anti-PD-1 = .0158; combination therapy = .0088). In multivariate analysis, the best model for 12-month progression-free survival for anti-PD-1 monotherapy included PD-L1 cells within proximity to tumor cells and intratumoral CD8 density (AUC = 0.80), and for combination therapy included CD8 cells in proximity to tumor cells, intratumoral PD-L1 density and LDH (AUC = 0.85). Assessment of the spatial distribution of immune cells in relation to tumor cells provides insight into their role in modulating immune response and highlights their potential role as predictors of response to anti-PD-1 based therapies.
Relevance of Spatial Heterogeneity of Immune Infiltration for Predicting Risk of Recurrence After Endocrine Therapy of ER+ Breast Cancer.
Heindl Andreas,Sestak Ivana,Naidoo Kalnisha,Cuzick Jack,Dowsett Mitchell,Yuan Yinyin
Journal of the National Cancer Institute
Background:Despite increasing evidence supporting the clinical utility of immune infiltration in the estrogen receptor-negative (ER-) subtype, the prognostic value of immune infiltration for ER+ disease is not well defined. Methods:Quantitative immune scores of cell abundance and spatial heterogeneity were computed using a fully automated hematoxylin and eosin-stained image analysis algorithm and spatial statistics for 1178 postmenopausal patients with ER+ breast cancer treated with five years' tamoxifen or anastrozole. The prognostic significance of immune scores was compared with Oncotype DX 21-gene recurrence score (RS), PAM50 risk of recurrence (ROR) score, IHC4, and clinical treatment score, available for 963 patients. Statistical tests were two-sided. Results:Scores of immune cell abundance were not associated with recurrence-free survival. In contrast, high immune spatial scores indicating increased cell spatial clustering were associated with poor 10-year, early (0-5 years), and late (5-10 years) recurrence-free survival (Immune Hotspot: LR-χ2 = 14.06, P < .001, for 0-10 years; LR-χ2 = 6.24, P = .01, for 0-5 years; LR-χ2 = 7.89, P = .005, for 5-10 years). The prognostic value of spatial scores for late recurrence was similar to that of IHC4 and RS in both populations, but was not as strong as other tests in comparison for recurrence across 10 years. Conclusions:These results provide a missing link between tumor immunity and disease outcome in ER+ disease by examining tumor spatial architecture. The association between spatial scores and late recurrence suggests a lasting memory of protumor immunity that may impact disease progression and evolution of endocrine treatment resistance, which may be exploited for therapeutic advances.
Tumor-Infiltrating Lymphocytes-Location for Prognostic Evaluation.
Peled Michael,Onn Amir,Herbst Roy S
Clinical cancer research : an official journal of the American Association for Cancer Research
Tumor-infiltrating lymphocytes (TIL) are crucial for the success of immunotherapy, and their density can predict prognosis. It has now been shown that computer-based analysis of the spatial organization of TILs adds prognostic value in early-stage non-small cell lung cancer, possibly improving the treatment algorithm to prevent recurrence..
The non-small cell lung cancer immune landscape: emerging complexity, prognostic relevance and prospective significance in the context of immunotherapy.
Anichini Andrea,Tassi Elena,Grazia Giulia,Mortarini Roberta
Cancer immunology, immunotherapy : CII
Immunotherapy of non-small cell lung cancer (NSCLC), by immune checkpoint inhibitors, has profoundly improved the clinical management of advanced disease. However, only a fraction of patients respond and no effective predictive factors have been defined. Here, we discuss the prospects for identification of such predictors of response to immunotherapy, by fostering an in-depth analysis of the immune landscape of NSCLC. The emerging picture, from several recent studies, is that the immune contexture of NSCLC lesions is a complex and heterogeneous feature, as documented by analysis for frequency, phenotype and spatial distribution of innate and adaptive immune cells, and by characterization of functional status of inhibitory receptor T cells. The complexity of the immune landscape of NSCLC stems from the interaction of several factors, including tumor histology, molecular subtype, main oncogenic drivers, nonsynonymous mutational load, tumor aneuploidy, clonal heterogeneity and tumor evolution, as well as the process of epithelial-mesenchymal transition. All these factors contribute to shape NSCLC immune profiles that have clear prognostic significance. An integrated analysis of the immune and molecular profile of the neoplastic lesions may allow to define the potential predictive role of the immune landscape for response to immunotherapy.
Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.
Joshi Kroopa,de Massy Marc Robert,Ismail Mazlina,Reading James L,Uddin Imran,Woolston Annemarie,Hatipoglu Emine,Oakes Theres,Rosenthal Rachel,Peacock Thomas,Ronel Tahel,Noursadeghi Mahdad,Turati Virginia,Furness Andrew J S,Georgiou Andrew,Wong Yien Ning Sophia,Ben Aissa Assma,Sunderland Mariana Werner,Jamal-Hanjani Mariam,Veeriah Selvaraju,Birkbak Nicolai J,Wilson Gareth A,Hiley Crispin T,Ghorani Ehsan,Guerra-Assunção José Afonso,Herrero Javier,Enver Tariq,Hadrup Sine R,Hackshaw Allan,Peggs Karl S,McGranahan Nicholas,Swanton Charles, ,Quezada Sergio A,Chain Benny
Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8 tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.
Spatial Architecture and Arrangement of Tumor-Infiltrating Lymphocytes for Predicting Likelihood of Recurrence in Early-Stage Non-Small Cell Lung Cancer.
Corredor Germán,Wang Xiangxue,Zhou Yu,Lu Cheng,Fu Pingfu,Syrigos Konstantinos,Rimm David L,Yang Michael,Romero Eduardo,Schalper Kurt A,Velcheti Vamsidhar,Madabhushi Anant
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:The presence of a high degree of tumor-infiltrating lymphocytes (TIL) has been proven to be associated with outcome in patients with non-small cell lung cancer (NSCLC). However, recent evidence indicates that tissue architecture is also prognostic of disease-specific survival and recurrence. We show a set of descriptors (spatial TIL, SpaTIL) that capture density, and spatial colocalization of TILs and tumor cells across digital images that can predict likelihood of recurrence in early-stage NSCLC. EXPERIMENTAL DESIGN:The association between recurrence in early-stage NSCLC and SpaTIL features was explored on 301 patients across four different cohorts. Cohort D ( = 70) was used to identify the most prognostic SpaTIL features and to train a classifier to predict the likelihood of recurrence. The classifier performance was evaluated in cohorts D ( = 119), D ( = 112), and D ( = 112). Two pathologists graded each sample of D and D; intraobserver agreement and association between manual grading and likelihood of recurrence were analyzed. RESULTS:SpaTIL was associated with likelihood of recurrence in all test sets (log-rank < 0.02). A multivariate Cox proportional hazards analysis revealed an HR of 3.08 (95% confidence interval, 2.1-4.5, = 7.3 × 10). In contrast, agreement among expert pathologists using tumor grade was moderate (Kappa = 0.5), and the manual TIL grading was only prognostic for one reader in D ( = 8.0 × 10). CONCLUSIONS:A set of features related to density and spatial architecture of TILs was found to be associated with a likelihood of recurrence of early-stage NSCLC. This information could potentially be used for helping in treatment planning and management of early-stage NSCLC..
Hyperspectral cell sociology reveals spatial tumor-immune cell interactions associated with lung cancer recurrence.
Enfield Katey S S,Martin Spencer D,Marshall Erin A,Kung Sonia H Y,Gallagher Paul,Milne Katy,Chen Zhaoyang,Nelson Brad H,Lam Stephen,English John C,MacAulay Calum E,Lam Wan L,Guillaud Martial
Journal for immunotherapy of cancer
BACKGROUND:The tumor microenvironment (TME) is a complex mixture of tumor epithelium, stroma and immune cells, and the immune component of the TME is highly prognostic for tumor progression and patient outcome. In lung cancer, anti-PD-1 therapy significantly improves patient survival through activation of T cell cytotoxicity against tumor cells. Direct contact between CD8+ T cells and target cells is necessary for CD8+ T cell activity, indicating that spatial organization of immune cells within the TME reflects a critical process in anti-tumor immunity. Current immunohistochemistry (IHC) imaging techniques identify immune cell numbers and densities, but lack assessment of cell-cell spatial relationships (or "cell sociology"). Immune functionality, however, is often dictated by cell-to-cell contact and cannot be resolved by simple metrics of cell density (for example, number of cells per mm). To address this issue, we developed a Hyperspectral Cell Sociology technology platform for the analysis of cell-cell interactions in multi-channel IHC-stained tissue. METHODS:Tissue sections of primary tumors from lung adenocarcinoma patients with known clinical outcome were stained using multiplex IHC for CD3, CD8, and CD79a, and hyperspectral image analysis determined the phenotype of all cells. A Voronoi diagram for each cell was used to approximate cell boundaries, and the cell type of all neighboring cells was identified and quantified. Monte Carlo analysis was used to assess whether cell sociology patterns were likely due to random distributions of the cells. RESULTS:High density of intra-tumoral CD8+ T cells was significantly associated with non-recurrence of tumors. A cell sociology pattern of CD8+ T cells surrounded by tumor cells was more significantly associated with non-recurrence compared to CD8+ T cell density alone. CD3+ CD8- T cells surrounded by tumor cells was also associated with non-recurrence, but at a similar significance as cell density alone. Cell sociology metrics improved recurrence classifications of 12 patients. Monte Carlo re-sampling analysis determined that these cell sociology patterns were non-random. CONCLUSION:Hyperspectral Cell Sociology expands our understanding of the complex interplay between tumor cells and immune infiltrate. This technology could improve predictions of responses to immunotherapy and lead to a deeper understanding of anti-tumor immunity.