Malignant-ascites-derived small extracellular vesicles in advanced ovarian cancer patients: insights into the dynamics of the extracellular matrix.
Bortot Barbara,Apollonio Maura,Rampazzo Enrico,Valle Francesco,Brucale Marco,Ridolfi Andrea,Ura Blendi,Addobbati Riccardo,Di Lorenzo Giovanni,Romano Federico,Buonomo Francesca,Ripepi Chiara,Ricci Giuseppe,Biffi Stefania
The exact role of malignant ascites in the development of intraperitoneal metastases remains unclear, and the mechanisms by which extracellular vesicles (EVs) promote tumor progression in the pre-metastatic niche have not been fully discovered. In this study, we characterized ascites from high-grade epithelial ovarian cancer patients. Small-EVs (30-150 nm) were isolated from two sources - the bulk ascites and the ascitic-fluid-derived tumor cell cultures - and assessed with a combination of imaging, proteomic profiling and protein expression analyses. In addition, gene ontology and pathways analysis were performed using different databases and bioinformatic tools. The results proved that the small-EVs derived from the two sources exhibited significantly different stiffness and size distribution. The bulk-ascitic-fluid-derived small-EVs were predominantly involved in the complement and coagulation cascade. Small-EVs derived from ascites cell cultures contained a robust proteomic profile of extracellular matrix remodeling regulators, and we observed an increase in transforming growth factor-β-I (TGFβI), plasminogen activator inhibitor 1 (PAI-1) and fibronectin expression after neoadjuvant chemotherapy. When measured in the two sources, we demonstrated that fibronectin exhibited opposite expression patterns in small-EVs in response to chemotherapy. These findings highlight the importance of an ascites cells isolation workflow in investigating the treatment-induced cancer adaption processes.
Functional expression of TRPV channels in T cells and their implications in immune regulation.
Majhi Rakesh K,Sahoo Subhransu S,Yadav Manoj,Pratheek Belluru M,Chattopadhyay Subhasis,Goswami Chandan
The FEBS journal
The importance of Ca(2+) signalling and temperature in the context of T cell activation is well known. However, the molecular identities of key players involved in such critical regulations are still unknown. In this work we explored the endogenous expression of transient receptor potential vanilloid (TRPV) channels, a group of thermosensitive and non-selective cation channels, in T cells. Using flow cytometry and confocal microscopy, we demonstrate that members belonging to the TRPV subfamily are expressed endogenously in the human T cell line Jurkat, in primary human T cells and in primary murine splenic T cells. We also demonstrate that TRPV1- and TRPV4-specific agonists, namely resiniferatoxin and 4α-phorbol-12,13-didecanoate, can cause Ca(2+) influx in T cells. Moreover, our results show that expression of these channels can be upregulated in T cells during concanavalin A-driven mitogenic and anti-CD3/CD28 stimulated TCR activation of T cells. By specific blocking of TRPV1 and TRPV4 channels, we found that these TRPV inhibitors may regulate mitogenic and T cell receptor mediated T cell activation and effector cytokine(s) production by suppressing tumour necrosis factor, interleukin-2 and interferon-γ release. These results may have broad implications in the context of cell-mediated immunity, especially T cell responses and their regulations, neuro-immune interactions and molecular understanding of channelopathies.
IL-23/IL-17A/TRPV1 axis produces mechanical pain via macrophage-sensory neuron crosstalk in female mice.
Luo Xin,Chen Ouyang,Wang Zilong,Bang Sangsu,Ji Jasmine,Lee Sang Hoon,Huh Yul,Furutani Kenta,He Qianru,Tao Xueshu,Ko Mei-Chuan,Bortsov Andrey,Donnelly Christopher R,Chen Yong,Nackley Andrea,Berta Temugin,Ji Ru-Rong
Although sex dimorphism is increasingly recognized as an important factor in pain, female-specific pain signaling is not well studied. Here we report that administration of IL-23 produces mechanical pain (mechanical allodynia) in female but not male mice, and chemotherapy-induced mechanical pain is selectively impaired in female mice lacking Il23 or Il23r. IL-23-induced pain is promoted by estrogen but suppressed by androgen, suggesting an involvement of sex hormones. IL-23 requires C-fiber nociceptors and TRPV1 to produce pain but does not directly activate nociceptor neurons. Notably, IL-23 requires IL-17A release from macrophages to evoke mechanical pain in females. Low-dose IL-17A directly activates nociceptors and induces mechanical pain only in females. Finally, deletion of estrogen receptor subunit α (ERα) in TRPV1 nociceptors abolishes IL-23- and IL-17-induced pain in females. These findings demonstrate that the IL-23/IL-17A/TRPV1 axis regulates female-specific mechanical pain via neuro-immune interactions. Our study also reveals sex dimorphism at both immune and neuronal levels.
Role of sensory neurons, neuroimmune pathways, and transient receptor potential vanilloid 1 (TRPV1) channels in a murine model of breast cancer metastasis.
Cancer immunology, immunotherapy : CII
Sensory nerves sensitive to capsaicin are afferent nerve fibers which contain TRPV1 channels. Activation of these channels induces release of neuropeptides which regulate local blood flow and immune response. Inactivation of sensory neurons either with high-dose capsaicin treatment or local ablation of vagal sensory nerve activity markedly increases metastasis of breast carcinoma formed by 4T1 derivative cells. These cancer cells also induce an extensive systemic inflammatory response. Further findings have documented that lack of local sensory neuromediators alters phenotype of cancer cells within primary tumor leading to overgrowth of metastatic subsets. This might be due to decreases in local and systemic immune response to growing tumor. Specifically, Substance P, one of the most abundant sensory neuropeptides, enhances anti-tumoral immune response evoked by radiotherapy under in vivo conditions. These findings further suggest that activation of TRPV1 channels on sensory neurons may induce an anti-tumoral immune response. We are testing this hypothesis. Our initial results as reported here demonstrate anti-inflammatory consequences of low-dose systemic capsaicin treatment. In conclusion, sensory nerve fibers sensitive to capsaicin have important roles in defense against metastatic breast carcinoma; hence, controlled activation of these neural pathways might be effective in cancer therapy. Specifically, activation of sensory fibers of left vagus nerve using a perineuronal stimulation may inhibit metastasis of breast carcinoma. Likewise, pharmacological modulators of TRPV1 channels may induce anti-tumoral immune response. Exact players of this newly explored defense system are, however, only partly validated, and further studies are required.
Malignant effusions and immunogenic tumour-derived exosomes.
Andre Fabrice,Schartz Noel E C,Movassagh Mojgan,Flament Caroline,Pautier Patricia,Morice Philippe,Pomel Christophe,Lhomme Catherine,Escudier Bernard,Le Chevalier Thierry,Tursz Thomas,Amigorena Sebastian,Raposo Graca,Angevin Eric,Zitvogel Laurence
Lancet (London, England)
BACKGROUND:Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supernatants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells. METHODS:We isolated exosomes by ultracentrifugation on sucrose and D(2)O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes. FINDINGS:Malignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules. INTERPRETATION:Exosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.
Loss of host tissue transglutaminase boosts antitumor T cell immunity by altering STAT1/STAT3 phosphorylation in ovarian cancer.
Sima Livia Elena,Chen Siqi,Cardenas Horacio,Zhao Guangyuan,Wang Yinu,Ivan Cristina,Huang Hao,Zhang Bin,Matei Daniela
Journal for immunotherapy of cancer
BACKGROUND:Tissue transglutaminase (TG2), an enzyme overexpressed in cancer cells, promotes metastasis and resistance to chemotherapy. Its distinct effects in cancer versus the host compartments have not been elucidated. METHODS:Here, by using a TG2 syngeneic ovarian cancer mouse model, we assessed the effects of TG2 deficiency in the host tissues on antitumor immunity and tumor progression. Multicolor flow cytometry was used to phenotype immune cell populations in the peritoneal environment. Cancer cells recovered from malignant ascites were characterized by RNA sequencing, proliferation, and apoptosis assays. RESULTS:We observed that host TG2 loss delayed tumor growth and ascites accumulation and caused increased infiltration of CD8 T cells and decreased numbers of myeloid cells in the peritoneal fluid. Tumor antigen-specific CD8 T cell cytotoxic responses were enhanced in ascites from TG2 versus TG2 mice and CD8 T cell depletion caused accelerated ascites accumulation in TG2 mice. CD8 T cells from tumor-bearing TG2 mice displayed an effector T cell phenotype, differentiated toward effector memory (T). Mechanistically, absence of TG2 augmented signals promoting T cell activation, such as increased cytokine-induced STAT1 and attenuated STAT3 phosphorylation in T cells. Additionally, immune-suppressive myeloid cell populations were reduced in the peritoneal milieu of TG2 tumor-bearing mice. In response to the more robust immune response caused by loss of TG2, cancer cells growing intraperitoneally exhibited an interferon-γ(IFN-γ) responsive gene signature and underwent apoptosis. In human specimens, stromal, not tumor, TG2 expression correlated indirectly with numbers of tumor-infiltrating lymphocytes. CONCLUSIONS:Collectively, our data demonstrate decreased tumor burden, increased activation and effector function of T cells, and loss of immunosuppressive signals in the tumor microenvironment of TG2 mice. We propose that TG2 acts as an attenuator of antitumor T cell immunity and is a new immunomodulatory target.
Tumour microenvironment and metabolic plasticity in cancer and cancer stem cells: Perspectives on metabolic and immune regulatory signatures in chemoresistant ovarian cancer stem cells.
Ahmed Nuzhat,Escalona Ruth,Leung Dilys,Chan Emily,Kannourakis George
Seminars in cancer biology
Cancer stem cells (CSCs) are a sub-population of tumour cells, which are responsible to drive tumour growth, metastasis and therapy resistance. It has recently been proposed that enhanced glucose metabolism and immune evasion by tumour cells are linked, and are modulated by the changing tumour microenvironment (TME) that creates a competition for nutrient consumption between tumour and different sub-types of cells attracted to the TME. To facilitate efficient nutrient distribution, oncogene-induced inflammatory milieu in the tumours facilitate adaptive metabolic changes in the surrounding non-malignant cells to secrete metabolites that are used as alternative nutrient sources by the tumours to sustain its increasing energy needs for growth and anabolic functions. This scenario also affects CSCs residing at the primary or metastatic niches. This review summarises recent advances in our understanding of the metabolic phenotypes of cancer cells and CSCs and how these processes are affected by the TME. We also discuss how the evolving TME modulates tumour cells and CSCs in cancer progression. Using previously described proteomic and genomic platforms, ovarian cancer cell lines and a mouse xenograft model we highlight the existence of metabolic and immune regulatory signatures in chemoresistant ovarian CSCs, and discuss how these processes may affect recurrence in ovarian tumours. We propose that progress in cancer control and eradication may depend not only on the elimination of highly chemoresistant CSCs, but also in designing novel strategies which would intervene with the tumour-promoting TME factors.
Activated regulatory and memory T-cells accumulate in malignant ascites from ovarian carcinoma patients.
Landskron Johannes,Helland Øystein,Torgersen Knut Martin,Aandahl Einar Martin,Gjertsen Bjørn Tore,Bjørge Line,Taskén Kjetil
Cancer immunology, immunotherapy : CII
Invasive ovarian cancer is associated with poor outcome. The presence of infiltrating regulatory T-cells (Tregs) suppresses protective anti-tumor immune responses, and their accumulation into the tumor microenvironment correlates with reduced survival in ovarian cancer patients. Here, we conducted a detailed characterization of CD4(+) T-cells, CD8(+) T-cells and Treg subsets in the peripheral blood and malignant ascites fluid from seventeen patients with ovarian carcinoma of epithelial origin. Cell distribution, activation status and proliferation status were assessed by multi-color flow cytometry. In ascites fluid, a significant accumulation of CD8(+) cytotoxic T-cells and Tregs was observed compared to peripheral blood. Furthermore, a skewing toward the CD45RA(-) effector/memory compartment was observed in all T-cell subsets in the ascites fluid, but was most pronounced in the Treg population. Regulatory T-cells in the malignant ascites were more activated and had a higher proliferation rate compared to blood-derived cells from the same patient, and their number in ascites was positively correlated with the number of epithelial cells in effusion. In summary, we demonstrate an accumulation of activated CD4(+), CD8(+) and regulatory T-cells in the cancer microenvironment of ovarian carcinoma.
Computational modeling of malignant ascites reveals CCL5-SDC4 interaction in the immune microenvironment of ovarian cancer.
Kim Soochi,Han Youngjin,Kim Se Ik,Lee Juwon,Jo HyunA,Wang Wenyu,Cho Untack,Park Woong-Yang,Rando Thomas A,Dhanasekaran Danny N,Song Yong Sang
Fluid accumulation in the abdominal cavity is commonly found in advanced-stage ovarian cancer patients, which creates a specialized tumor microenvironment for cancer progression. Using single-cell RNA sequencing (scRNA-seq) of ascites cells from five patients with ovarian cancer, we identified seven cell types, including heterogeneous macrophages and ovarian cancer cells. We resolved a distinct polarization state of macrophages by MacSpectrum analysis and observed subtype-specific enrichment of pathways associated with their functions. The communication between immune and cancer cells was predicted through a putative ligand-receptor pair analysis using NicheNet. We found that CCL5, a chemotactic ligand, is enriched in immune cells (T cells and NK cells) and mediates ovarian cancer cell survival in the ascites, possibly through SDC4. Moreover, SDC4 expression correlated with poor overall survival in ovarian cancer patients. Our study highlights the potential role of T cells and NK cells in long-term survival patients with ovarian cancer, indicating SDC4 as a potential prognostic marker in ovarian cancer patients.