Lymph node metastasis, the leading cause of mortality in esophageal squamous carcinoma (ESCC) with a highly complex tumor microenvironment, remains underexplored. Here, the transcriptomes of 85 263 single cells are analyzed from four ESCC patients with lymph node metastases. Strikingly, it is observed that the metastatic microenvironment undergoes the emergence or expansion of interferon induced IFIT3 T, B cells, and immunosuppressive cells such as APOC1 APOE macrophages and myofibroblasts with highly expression of immunoglobulin genes (IGKC) and extracellular matrix component and matrix metallopeptidase genes. A poor-prognostic epithelial-immune dual expression program regulating immune effector processes, whose activity is significantly enhanced in metastatic malignant epithelial cells and enriched in CD74 CXCR4 and major histocompatibility complex (MHC) class II genes upregulated malignant epithelia cells is discovered. Comparing with primary tumor, differential intercellular communications of metastatic ESCC microenvironment are revealed and furtherly validated via multiplexed immunofluorescence and immunohistochemistry staining, which mainly rely on the crosstalk of APOC1 APOE macrophages with tumor and stromal cell. The data highlight potential molecular mechanisms that shape the lymph-node metastatic microenvironment and may inform drug discovery and the development of new strategies to target these prometastatic nontumor components for inhibiting tumor growth and overcoming metastasis to improve clinical outcomes.

Emerging data have shown that previously defined noncoding genomes might encode peptides that bind human leukocyte antigen (HLA) as cryptic antigens to stimulate adaptive immunity. However, the significance and mechanisms of action of cryptic antigens in anti-tumour immunity remain unclear. Here mass spectrometry of the HLA class I (HLA-I) peptidome coupled with ribosome sequencing of human breast cancer samples identified HLA-I-binding cryptic antigenic peptides that were noncanonically translated by a tumour-specific circular RNA (circRNA): circFAM53B. The cryptic peptides efficiently primed naive CD4 and CD8 T cells in an antigen-specific manner and induced anti-tumour immunity. Clinically, the expression of circFAM53B and its encoded peptides was associated with substantial infiltration of antigen-specific CD8 T cells and better survival in patients with breast cancer and patients with melanoma. Mechanistically, circFAM53B-encoded peptides had strong binding affinity to both HLA-I and HLA-II molecules. In vivo, administration of vaccines consisting of tumour-specific circRNA or its encoded peptides in mice bearing breast cancer tumours or melanoma induced enhanced infiltration of tumour-antigen-specific cytotoxic T cells, which led to effective tumour control. Overall, our findings reveal that noncanonical translation of circRNAs can drive efficient anti-tumour immunity, which suggests that vaccination exploiting tumour-specific circRNAs may serve as an immunotherapeutic strategy against malignant tumours.

Recent identification of oncogenic cells within healthy tissues and the prevalence of indolent cancers found incidentally at autopsies reveal a greater complexity in tumor initiation than previously appreciated. The human body contains roughly 40 trillion cells of 200 different types that are organized within a complex three-dimensional matrix, necessitating exquisite mechanisms to restrain aberrant outgrowth of malignant cells that have the capacity to kill the host. Understanding how this defense is overcome to trigger tumorigenesis and why cancer is so extraordinarily rare at the cellular level is vital to future prevention therapies. In this review, we discuss how early initiated cells are protected from further tumorigenesis and the non-mutagenic pathways by which cancer risk factors promote tumor growth. By nature, the absence of permanent genomic alterations potentially renders these tumor-promoting mechanisms clinically targetable. Finally, we consider existing strategies for early cancer interception with perspectives on the next steps for molecular cancer prevention.

Neoadjuvant immune checkpoint blockade (ICB) demonstrates promise in operable esophageal squamous cell carcinoma (ESCC), but lacks available efficacy biomarkers. Here, we perform single-cell RNA-sequencing of tumors from patients with ESCC undergoing neoadjuvant ICB, revealing a subset of exhausted CD8 T cells expressing SPRY1 (CD8 Tex-SPRY1) that displays a progenitor exhausted T cell (Tpex) phenotype and correlates with complete response to ICB. We validate CD8 Tex-SPRY1 cells as an ICB-specific predictor of improved response and survival using independent ICB-/non-ICB cohorts and demonstrate that expression of SPRY1 in CD8 T cells enforces Tpex phenotype and enhances ICB efficacy. Additionally, CD8 Tex-SPRY1 cells contribute to proinflammatory phenotype of macrophages and functional state of B cells, which thereby promotes antitumor immunity by enhancing CD8 T cell effector functions. Overall, our findings unravel progenitor-like CD8 Tex-SPRY1 cells' role in effective responses to ICB for ESCC and inform mechanistic biomarkers for future individualized immunotherapy.

BACKGROUND & AIMS:The immune landscape of hepatocellular carcinoma (HCC) following transarterial chemoembolisation (TACE) remains to be clarified. This study aimed to characterise the immune landscape following TACE and the underlying mechanism of HCC progression. METHODS:Tumour samples from five patients with treatment-naive HCC and five patients who received TACE therapy were collected and subjected to single-cell RNA sequencing. Another 22 paired samples were validated using immunofluorescence staining and flow cytometry. To clarify the underlying mechanisms, in vitro co-culture experiments and two types of TREM2-KO/WT mouse models, namely, an HCC cell orthotopic injection model and a spontaneous HCC model, were used. RESULTS:A reduced number of CD8 T cells and an increased number of tumour-associated macrophages (TAMs) were observed in the post-TACE microenvironment. TACE therapy reduced the cluster CD8_C4, which was highly enriched with tumour-specific CD8 T cells of pre-exhausted phenotype. TREM2 was found to be highly expressed in TAMs following TACE, which was associated with a poor prognosis. TREM2 TAMs secreted less CXCL9 but more galectin-1 than did TREM2 TAMs. Galectin-1 promoted PD-L1 overexpression in vessel endothelial cells, impeding CD8 T cell recruitment. TREM2 deficiency also increased CD8 T cell infiltration, which inhibited tumour growth in both in vivo HCC models. More importantly, TREM2 deficiency enhanced the therapeutic effect of anti-PD-L1 blockade. CONCLUSIONS:This study shows that TREM2 TAMs play an important role in suppressing CD8 T cells. TREM2 deficiency increased the therapeutic effect of anti-PD-L1 blockade by enhancing antitumour activity of CD8 T cells. These findings explain the reasons for recurrence and progression after TACE and provide a new target for HCC immunotherapy after TACE. IMPACT AND IMPLICATIONS:Studying the immune landscape in post-TACE HCC is important to uncover the mechanisms of HCC progression. By using scRNA sequencing and functional assays, we discovered that both the number and function of CD8 T cells are compromised, whereas the number of TREM2 TAMs is increased in post-TACE HCC, correlating with worse prognosis. Moreover, TREM2 deficiency dramatically increases CD8 T cell infiltration and augments the therapeutic efficacy of anti-PD-L1 blockade. Mechanistically, TREM2 TAMs display lower CXCL9 and increased Gal-1 secretion than do TREM2 TAMs, with Gal-1 mediating the overexpression of PD-L1 in vessel endothelial cells. These results suggest that TREM2 could be a novel immunotherapeutic target for patients treated with TACE in HCC. This provides an opportunity to break the plateau of limited therapeutic effect. This study has the value of understanding the tumour microenvironment of post-TACE HCC and thinking a new strategy of immunotherapy in the field of HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

Understanding tumor microenvironment (TME) reprogramming in gastric adenocarcinoma (GAC) progression may uncover novel therapeutic targets. Here, we performed single-cell profiling of precancerous lesions, localized and metastatic GACs, identifying alterations in TME cell states and compositions as GAC progresses. Abundant IgA plasma cells exist in the premalignant microenvironment, whereas immunosuppressive myeloid and stromal subsets dominate late-stage GACs. We identified six TME ecotypes (EC1-6). EC1 is exclusive to blood, while EC4, EC5, and EC2 are highly enriched in uninvolved tissues, premalignant lesions, and metastases, respectively. EC3 and EC6, two distinct ecotypes in primary GACs, associate with histopathological and genomic characteristics, and survival outcomes. Extensive stromal remodeling occurs in GAC progression. High SDC2 expression in cancer-associated fibroblasts (CAFs) is linked to aggressive phenotypes and poor survival, and SDC2 overexpression in CAFs contributes to tumor growth. Our study provides a high-resolution GAC TME atlas and underscores potential targets for further investigation.

BACKGROUND:The proteome is a major source of therapeutic targets. We conducted a proteome-wide Mendelian randomization (MR) study to identify candidate protein markers and therapeutic targets for colorectal cancer (CRC). METHODS:Protein quantitative trait loci (pQTLs) were derived from seven published genome-wide association studies (GWASs) on plasma proteome, and summary-level data were extracted for 4853 circulating protein markers. Genetic associations with CRC were obtained from a large-scale GWAS meta-analysis (16,871 cases and 26,328 controls), the FinnGen cohort (4957 cases and 304,197 controls), and the UK Biobank (9276 cases and 477,069 controls). Colocalization and summary-data-based MR (SMR) analyses were performed sequentially to verify the causal role of candidate proteins. Single cell-type expression analysis, protein-protein interaction (PPI), and druggability evaluation were further conducted to detect the specific cell type with enrichment expression and prioritize potential therapeutic targets. RESULTS:Collectively, genetically predicted levels of 13 proteins were associated with CRC risk. Elevated levels of two proteins (GREM1, CHRDL2) and decreased levels of 11 proteins were associated with an increased risk of CRC, among which four (GREM1, CLSTN3, CSF2RA, CD86) were prioritized with the most convincing evidence. These protein-coding genes are mainly expressed in tissue stem cells, epithelial cells, and monocytes in colon tumor tissue. Two interactive pairs of proteins (GREM1 and CHRDL2; MMP2 and TIMP2) were identified to be involved in osteoclast differentiation and tumorigenesis pathways; four proteins (POLR2F, CSF2RA, CD86, MMP2) have been targeted for drug development on autoimmune diseases and other cancers, with the potentials of being repurposed as therapeutic targets for CRC. CONCLUSIONS:This study identified several protein biomarkers to be associated with CRC risk and provided new insights into the etiology and promising targets for the development of screening biomarkers and therapeutic drugs for CRC.

Although polymorphic microbiomes have emerged as hallmarks of cancer, far less is known about the role of the intratumor mycobiome as living microorganisms in cancer progression. Here, using fungi-enriched DNA extraction and deep shotgun metagenomic sequencing, we have identified enriched tumor-resident Aspergillus sydowii in patients with lung adenocarcinoma (LUAD). By three different syngeneic lung cancer mice models, we find that A. sydowii promotes lung tumor progression via IL-1β-mediated expansion and activation of MDSCs, resulting in suppressed activity of cytotoxic T lymphocyte cells and accumulation of PD-1 CD8 T cells. This is mediated by IL-1β secretion via β-glucan/Dectin-1/CARD9 pathway. Analysis of human samples confirms that enriched A. sydowii is associated with immunosuppression and poor patient outcome. Our findings suggest that intratumor mycobiome, albeit at low biomass, promotes lung cancer progression and could be targeted at the strain level to improve patients with LUAD outcome.

Esophageal squamous-cell carcinoma (ESCC) develops through multistage epithelial cancer formation, i.e., from normal epithelium, low- and high-grade intraepithelial neoplasia to invasive carcinoma. However, how the precancerous lesions progress to carcinoma remains elusive. Here, we report a comprehensive single-cell RNA sequencing and spatial transcriptomic study of 79 multistage esophageal lesions from 29 patients with ESCC. We reveal a gradual and significant loss of ANXA1 expression in epithelial cells due to its transcription factor KLF4 suppression along the lesion progression. We demonstrate that ANXA1 is a ligand to formyl peptide receptor type 2 (FPR2) on fibroblasts that maintain fibroblast homeostasis. Loss of ANXA1 leads to uncontrolled transformation of normal fibroblasts into cancer-associated fibroblasts (CAFs), which can be enhanced by secreted TGF-β from malignant epithelial cells. Given the role of CAFs in cancer, our study underscores ANXA1/FPR2 signaling as an important crosstalk mechanism between epithelial cells and fibroblasts in promoting ESCC.

OBJECTIVE:The role of N-methyladenosine (mA) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC) TIME. DESIGN:Clinical significance of YTHDF1 was assessed in tissue microarrays (N=408) and TCGA (N=526) cohorts. function was determined in syngeneic tumours, intestine-specific knockin mice, and humanised mice. Single-cell RNA-seq (scRNA-seq) was employed to profile TIME. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) were used to identify YTHDF1 direct targets. Vesicle-like nanoparticles (VNPs)-encapsulated -siRNA was used for silencing in vivo. RESULTS: expression negatively correlated with interferon-γ gene signature in TCGA-CRC. Concordantly, YTHDF1 protein negatively correlated with CD8 T-cell infiltration in independent tissue microarrays cohorts, implying its role in TIME. Genetic depletion of augmented antitumour immunity in CT26 (MSS-CRC) and MC38 (MSI-H-CRC) syngeneic tumours, while knockin promoted an immunosuppressive TIME facilitating CRC in azoxymethane-dextran sulphate-sodium or models. scRNA-seq identified reduction of myeloid-derived suppressor cells (MDSCs), concomitant with increased cytotoxic T cells in knockout tumours. Integrated MeRIP-seq, RNA-seq and Ribo-seq revealed p65/Rela as a YTHDF1 target. YTHDF1 promoted p65 translation to upregulate CXCL1, which increased MDSC migration via CXCL1-CXCR2 axis. Increased MSDCs in turn antagonised functional CD8 T cells in TIME. Importantly, targeting YTHDF1 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or VNPs-si boosted anti-PD1 efficacy in MSI-H CRC, and overcame anti-PD1 resistance in MSS CRC. CONCLUSION:YTHDF1 impairs antitumour immunity via an mA-p65-CXCL1/CXCR2 axis to promote CRC and serves as a therapeutic target in immune checkpoint blockade therapy.

Immune checkpoint inhibitor (ICI) therapy can induce complete responses in mismatch repair-deficient and microsatellite instability-high (d-MMR/MSI-H) colorectal cancers (CRCs). However, the underlying mechanism for pathological complete response (pCR) to immunotherapy has not been completely understood. We utilize single-cell RNA sequencing (scRNA-seq) to investigate the dynamics of immune and stromal cells in 19 patients with d-MMR/MSI-H CRC who received neoadjuvant PD-1 blockade. We found that in tumors with pCR, there is a concerted decrease in CD8 Trm-mitotic, CD4 Tregs, proinflammatory IL1B Mono and CCL2 Fibroblast following treatment, while the proportions of CD8 Tem, CD4 Th, CD20 B, and HLA-DRA Endothelial cells increase. Proinflammatory features in the tumor microenvironment mediate the persistence of residual tumors by modulating CD8 T cells and other response-associated immune cell populations. Our study provides valuable resources and biological insights into the mechanism of successful ICI therapy and potential targets for improving treatment efficacy.

Dissecting and understanding the cancer ecosystem, especially that around the tumor margins, which have strong implications for tumor cell infiltration and invasion, are essential for exploring the mechanisms of tumor metastasis and developing effective new treatments. Using a novel tumor border scanning and digitization model enabled by nanoscale resolution-SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we identified a 500 µm-wide zone centered around the tumor border in patients with liver cancer, referred to as "the invasive zone". We detected strong immunosuppression, metabolic reprogramming, and severely damaged hepatocytes in this zone. We also identified a subpopulation of damaged hepatocytes with increased expression of serum amyloid A1 and A2 (referred to collectively as SAAs) located close to the border on the paratumor side. Overexpression of CXCL6 in adjacent malignant cells could induce activation of the JAK-STAT3 pathway in nearby hepatocytes, which subsequently caused SAAs' overexpression in these hepatocytes. Furthermore, overexpression and secretion of SAAs by hepatocytes in the invasive zone could lead to the recruitment of macrophages and M2 polarization, further promoting local immunosuppression, potentially resulting in tumor progression. Clinical association analysis in additional five independent cohorts of patients with primary and secondary liver cancer (n = 423) showed that patients with overexpression of SAAs in the invasive zone had a worse prognosis. Further in vivo experiments using mouse liver tumor models in situ confirmed that the knockdown of genes encoding SAAs in hepatocytes decreased macrophage accumulation around the tumor border and delayed tumor growth. The identification and characterization of a novel invasive zone in human cancer patients not only add an important layer of understanding regarding the mechanisms of tumor invasion and metastasis, but may also pave the way for developing novel therapeutic strategies for advanced liver cancer and other solid tumors.

Breast cancer is now the most frequently diagnosed malignancy, and metastasis remains the leading cause of death in breast cancer. However, little is known about the dynamic changes during the evolvement of dissemination. In this study, 65 968 cells from four patients with breast cancer and paired metastatic axillary lymph nodes are profiled using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics. A disseminated cancer cell cluster with high levels of oxidative phosphorylation (OXPHOS), including the upregulation of cytochrome C oxidase subunit 6C and dehydrogenase/reductase 2, is identified. The transition between glycolysis and OXPHOS when dissemination initiates is noticed. Furthermore, this distinct cell cluster is distributed along the tumor's leading edge. The findings here are verified in three different cohorts of breast cancer patients and an external scRNA-seq dataset, which includes eight patients with breast cancer and paired metastatic axillary lymph nodes. This work describes the dynamic metabolic evolvement of early disseminated breast cancer and reveals a switch between glycolysis and OXPHOS in breast cancer cells as the early event during lymph node metastasis.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40 Th1-like cells and CD8 memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

BACKGROUND:Immunotherapy has revolutionized cancer treatment, but most patients are refractory to immunotherapy or acquire resistance, with the underlying mechanisms remaining to be explored. METHODS:We characterized the transcriptomes of ~92,000 single cells from 3 pre-treatment and 12 post-treatment patients with non-small cell lung cancer (NSCLC) who received neoadjuvant PD-1 blockade combined with chemotherapy. The 12 post-treatment samples were categorized into two groups based on pathologic response: major pathologic response (MPR; n = 4) and non-MPR (NMPR; n = 8). RESULTS:Distinct therapy-induced cancer cell transcriptomes were associated with clinical response. Cancer cells from MPR patients exhibited a signature of activated antigen presentation via major histocompatibility complex class II (MHC-II). Further, the transcriptional signatures of FCRL4+FCRL5+ memory B cells and CD16+CX3CR1+ monocytes were enriched in MPR patients and are predictors of immunotherapy response. Cancer cells from NMPR patients exhibited overexpression of estrogen metabolism enzymes and elevated serum estradiol. In all patients, therapy promoted expansion and activation of cytotoxic T cells and CD16+ NK cells, reduction of immunosuppressive Tregs, and activation of memory CD8+T cells into an effector phenotype. Tissue-resident macrophages were expanded after therapy, and tumor-associated macrophages (TAMs) were remodeled into a neutral instead of an anti-tumor phenotype. We revealed the heterogeneity of neutrophils during immunotherapy and identified an aged CCL3+ neutrophil subset was decreased in MPR patients. The aged CCL3+ neutrophils were predicted to interact with SPP1+ TAMs through a positive feedback loop to contribute to a poor therapy response. CONCLUSIONS:Neoadjuvant PD-1 blockade combined with chemotherapy led to distinct NSCLC tumor microenvironment transcriptomes that correlated with therapy response. Although limited by a small patient sample size subjected to combination therapy, this study provides novel biomarkers to predict therapy response and suggests potential strategies to overcome immunotherapy resistance.

BACKGROUND & AIMS:The tumour microenvironment (TME) is a crucial mediator of cancer progression and therapeutic outcome. The TME subtype correlates with patient response to immunotherapy in multiple cancers. Most previous studies have focused on the role of different cellular components in the TME associated with immunotherapy efficacy. However, the specific structure of the TME and its role in immunotherapy efficacy remain largely unknown. METHODS:We combined spatial transcriptomics with single-cell RNA-sequencing and multiplexed immunofluorescence to identify the specific spatial structures in the TME that determine the efficacy of immunotherapy in patients with hepatocellular carcinoma (HCC) receiving anti-PD-1 treatment. RESULTS:We identified a tumour immune barrier (TIB) structure, a spatial niche composed of SPP1 macrophages and cancer-associated fibroblasts (CAFs) located near the tumour boundary, which is associated with the efficacy of immune checkpoint blockade. Furthermore, we dissected ligand‒receptor networks among malignant cells, SPP1 macrophages, and CAFs; that is, the hypoxic microenvironment promotes SPP1 expression, and SPP1 macrophages interact with CAFs to stimulate extracellular matrix remodelling and promote TIB structure formation, thereby limiting immune infiltration in the tumour core. Preclinically, the blockade of SPP1 or macrophage-specific deletion of Spp1 in mice led to enhanced efficacy of anti-PD-1 treatment in mouse liver cancer, accompanied by reduced CAF infiltration and increased cytotoxic T-cell infiltration. CONCLUSIONS:We identified that the TIB structure formed by the interaction of SPP1 macrophages and CAFs is related to immunotherapy efficacy. Therefore, disruption of the TIB structure by blocking SPP1 may be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockade in HCC. IMPACT AND IMPLICATIONS:Only a limited number of patients with hepatocellular carcinoma (HCC) benefit from tumour immunotherapy, which significantly hinders its application. Herein, we used multiomics to identify the spatial structure of the tumour immune barrier (TIB), which is formed by the interaction of SPP1+ macrophages and cancer-associated fibroblasts in the HCC microenvironment. This structure constrains immunotherapy efficacy by limiting immune cell infiltration into malignant regions. Preclinically, we revealed that blocking SPP1 or macrophage-specific deletion of Spp1 in mice could destroy the TIB structure and sensitize HCC cells to immunotherapy. These results provide the first key steps towards finding more effective therapies for HCC and have implications for physicians, scientists, and drug developers in the field of HCC.

The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs. The human EC continues to develop during childhood, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5RELN inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimer's disease, and previous work has linked cognitive decline to the loss of LAMP5RELN cells. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

Immune checkpoint blockade (ICB) is a promising therapy for solid tumors, but its effectiveness depends on biomarkers that are not precise. Here, we utilized genome-wide association study to investigate the association between genetic variants and tumor mutation burden to interpret ICB response. We identified 16 variants (p < 5 × 10) probed to 17 genes on 9 chromosomes. Subsequent analysis of one of the most significant loci in 19q13.11 suggested that the rs111308825 locus at the enhancer is causal, as its A allele impairs KLF2 binding, leading to lower carbohydrate sulfotransferase 8 (CHST8) expression. Breast cancer cells expressing CHST8 suppress T cell activation, and Chst8 loss attenuates tumor growth in a syngeneic mouse model. Further investigation revealed that programmed death-ligand 1 (PD-L1) and its homologs could be sulfated by CHST8, resulting in M2-like macrophage enrichment in the tumor microenvironment. Finally, we confirmed that low-CHST8 tumors have better ICB response, supporting the genetic effect and clinical value of rs111308825 for ICB efficacy prediction.