Total Neoadjuvant Therapy With PD-1 Blockade for High-Risk Proficient Mismatch Repair Rectal Cancer.

Importance: Total neoadjuvant therapy (TNT) is the standard treatment for locally advanced rectal cancer, especially for patients with high-risk factors. However, the efficacy of TNT combined with immunotherapy for patients with proficient mismatch repair (pMMR) rectal cancer is unknown. Objectives: To evaluate the safety and efficacy of TNT with induction chemoimmunotherapy followed by long-course chemoradiation in patients with high-risk, pMMR rectal cancer and to identify potential molecular biomarkers associated with treatment efficacy. Design, Setting, and Participants: This cohort study was a single-arm phase 2 trial conducted at Gastrointestinal Cancer Center, Peking University Cancer Hospital & Institute, from June 2020 to October 2021. Biopsies and plasma were collected before treatment for whole-exome sequencing and cell-free DNA sequencing, respectively. Data were analyzed from May 2022 to September 2022. Interventions: Participants received 3 cycles of induction oxaliplatin and capecitabine combined with camrelizumab and radiotherapy (50.6 Gy in 22 fractions) with concurrent capecitabine. Patients without disease progression received 2 cycles of consolidation oxaliplatin/capecitabine. Main Outcomes and Measures: The primary end point was pathologic complete response rate. Results: Of 25 patients enrolled (19 men [76%]; 6 women [24%]; median [IQR] age, 58 [48-64] years), 22 patients (88%) completed the TNT schedule. The pathologic complete response rate was 33.3% (7/21). Twelve patients (48%) achieved clinical complete response, and 4 patients (16%) chose to watch and wait. R0 resection was achieved in 21 of 21 patients, and the major pathologic response rate was 38.1% (8/21). The most common adverse event was nausea (80%, 20/25); grade 3 toxic effects occurred in 9 of 25 patients (36%). Patients with tumor shrinkage of 50% or greater after induction oxaliplatin/capecitabine and camrelizumab or clinical complete response had higher percentages of LRP1B mutation. Mutation of LRP1B was associated with high tumor mutation burden and tumor neoantigen burden. Patients with high tumor mutation burden all benefited from therapy. Conclusions and Relevance: This study found that TNT with induction chemoimmunotherapy followed by long-course chemoradiation was safe and effective for patients with high-risk rectal cancer with pMMR status. Longer follow-up and larger clinical studies are needed to validate this innovative regimen. There is also an urgent need to further validate the predictive value of LRP1B and discover other novel biomarkers with potential predictive value for rectal cancer.

Proteomic Profiling and Tumor Microenvironment Characterization Reveal Molecular and Immunological Hallmarks of Left-Sided and Right-Sided Colon Cancer Tumorigenesis.

Left-sided and right-sided colon cancer (LSCC and RSCC) display different biological and clinical characteristics. However, the differences in their tumorigenesis and tumor microenvironment remain unclear. In this study, we profiled the proteomic landscapes of LSCC and RSCC with data-independent acquisition mass spectrometry (DIA-MS) using fresh tumor and adjacent normal tissues from 24 patients. A total of 7403 proteingroups were primarily identified with DIA-MS. After quality control, 7212 proteingroups were used for further analysis. Through comparing the difference in proteomic profiles between LSCC and RSCC samples, 2556 commonly and 1982 region-type-specific regulated proteingroups were characterized. During the development of LSCC and RSCC, metabolic, growth, cell division, cell adhesion, and migration pathways were found to be significantly dysregulated (P < 0.05), which was further confirmed by transcriptome data from TCGA. Compared to RSCC, most parts of the immune-related signatures, immune cell infiltration scores, and overall immune scores of LSCC were higher. The systematic elucidation of proteomic and transcriptomic profiles in this work improves our understanding of tumorigenesis and immune microenvironment characteristics of LSCC and RSCC.

Multi-omics analysis uncovers tumor ecosystem dynamics during neoadjuvant toripalimab plus nab-paclitaxel and S-1 for esophageal squamous cell carcinoma: a single-center, open-label, single-arm phase 2 trial.

BACKGROUND: Immune checkpoint inhibitors combined with chemotherapy as a neoadjuvant therapy have been applied to the treatment of esophageal squamous cell carcinoma (ESCC). However, the optimal regimen needs to be further explored, particularly for older patients, and the mechanisms by which the immune checkpoint inhibitor combined with chemotherapy modulates the evolution of ESCC are unknown. METHODS: In this single-arm phase 2 trial, patients with resectable (stage II/III/IV without metastasis) ESCC were enrolled and received nanoparticle albumin-bound (nab) paclitaxel for two cycles and oral S-1 for 2 weeks, combined with intravenous toripalimab for two cycles before surgery. Combination postoperative adjuvant therapy was administered. The primary outcome was the major pathological response (MPR). Secondary outcomes included pathological complete response (pCR), overall response rate (ORR), disease control rate (DCR), disease-free survival (DFS), overall survival (OS), improvement in Stooler's dysphagia score and degree of daily living ability (dADL). Biopsies and plasma pre- and post-neoadjuvant therapy were performed using whole-exome sequencing, transcriptome sequencing, immunohistochemistry (IHC) for PD-L1, multiplex immunofluorescence (mIF) and proximity extension assay technology (PEA) for 92 proteins. FINDINGS: From November 2019 to July 2021, 60 patients were enrolled. After neoadjuvant therapy, R0 resection was achieved in 55 (98.21%) patients. MPR was identified in 27 patients (49.09%), and 16 patients (29.09%) achieved pCR. Patients with PR, SD and PD were 37 (61.67%), 21 (35.00%) and 2 (3.33%), respectively. The overall staging, Stooler dysphagia scores and dADL were significantly decreased after treatment. 11 patients (18.3%) experienced grade ≥3 AEs. Compared to PD-L1-Low patients, PD-L1-High patients had a significantly higher ratio of PR. During therapy, the tumor mutation burden (TMB) and tumor neoantigen burden (TNB) were significantly decreased in patients with PR. Differential clonal evolution within tumors was demonstrated by analysis of intratumoral heterogeneity. Transcriptome analyses revealed that the infiltration of CD4+ T lymphocytes at baseline was associated with clinical outcome. During therapy, CD8+ T cells and CD4+ T cells were increased in all patients; however, exhausted cells, nTregs and iTregs were significantly increased in patients with non-MPR. Protein analyses revealed that the levels of IFN-γ, Gal.1 and LAMP3 can predict the clinical benefit. In addition, the expression of CD83, TNFRSF4, TNFSF14, VEGFR2, ADA, ARG1, and HO-1 was associated with serious AEs. More importantly, the integration of CD4+ T cells with plasma protein of IFN-γ, Gal.1 or LAMP3 could further distinguish responders from non-responders. INTERPRETATION: In this study, neoadjuvant therapy with toripalimab, nab-paclitaxel and S-1 was less toxic and showed promising antitumor activity in patients with resectable ESCC. Changes in the genome, transcriptome, PD-L1 expression and serum proteins were comprehensively analyzed and correlated with clinical outcomes, which provides insight into the mechanism of action of toripalimab combined with nab-paclitaxel and S-1 in patients with ESCC. FUNDING: This study was funded by Major projects of the ministry of science and technology of the 13th five-year plan of China [grant number: 2018ZX09201013].

Spatial multi-omics revealed the impact of tumor ecosystem heterogeneity on immunotherapy efficacy in patients with advanced non-small cell lung cancer treated with bispecific antibody.

BACKGROUND: Immunotherapy for malignant tumors has made great progress, but many patients do not benefit from it. The complex intratumoral heterogeneity (ITH) hindered the in-depth exploration of immunotherapy. Conventional bulk sequencing has masked intratumor complexity, preventing a more detailed discovery of the impact of ITH on treatment efficacy. Hence, we initiated this study to explore ITH at the multi-omics spatial level and to seek prognostic biomarkers of immunotherapy efficacy considering the presence of ITH. METHODS: Using the segmentation strategy of digital spatial profiling (DSP), we obtained differential information on tumor and stromal regions at the proteomic and transcriptomic levels. Based on the consideration of ITH, signatures constructed by candidate proteins in different regions were used to predict the efficacy of immunotherapy. RESULTS: Eighteen patients treated with a bispecific antibody (bsAb)-KN046 were enrolled in this study. The tumor and stromal areas of the same samples exhibited distinct features. Signatures consisting of 11 and 18 differentially expressed DSP markers from the tumor and stromal areas, respectively, were associated with treatment response. Furthermore, the spatially resolved signature identified from the stromal areas showed greater predictive power for bsAb immunotherapy response (area under the curve=0.838). Subsequently, our stromal signature was validated in an independent cohort of patients with non-small cell lung cancer undergoing immunotherapy. CONCLUSION: We deciphered ITH at the spatial level and demonstrated for the first time that genetic information in the stromal region can better predict the efficacy of bsAb treatment. TRIAL REGISTRATION NUMBER: NCT03838848.

Integrated tumor genomic and immune microenvironment analysis identifies predictive biomarkers associated with the efficacy of neoadjuvant therapy for triple-negative breast cancer.

BACKGROUND: Although neoadjuvant chemotherapy (NAC) is currently the best therapy for triple-negative breast cancer (TNBC), resistance still occurs in a considerable proportion, thus it is crucial to understand resistance mechanisms and identify predictive biomarkers for patients selection. METHODS: Biopsy samples were collected from 21 patients with TNBC who underwent NAC. Whole-exome sequencing (WES), targeted sequencing, and multiplex immunohistochemistry (mIHC) were carried out on the clinical samples and used to identify and validate potential biomarkers associated with response to NAC. In addition, data on 190 TNBC patients who had undergone chemotherapy were obtained from The Cancer Genome Atlas (TCGA) and analyzed to further validate our findings. RESULTS: Both the tumor mutational burden (TMB) and tumor neoantigen burden (TNB) were significantly higher in responders than in non-responders. Higher response rates and longer survival rates were observed in patients with higher TMB. Patients with higher ratios of CD8 to M2 macrophages had higher response rates and improved survival rates. Finally, the integrated analysis demonstrated that the combination of TMB and the ratio of CD8 T cells to M2 macrophages could further distinguish patients who benefitted from the treatment in both enrolled patients and public data. CONCLUSIONS: The findings of this study indicated that the combination of TMB and the ratio of CD8 T cells to M2 macrophages may be a potential biomarker for improving the recognition of NAC responders, thereby providing a basis for developing precision NAC regimens.

Integrated investigation of the prognostic role of HLA LOH in advanced lung cancer patients with immunotherapy.

Although multiple studies have shown that loss of heterozygosity (LOH) at the human leukocyte antigen (HLA) locus is one of the mechanisms of immune escape, the effect of HLA LOH on the immunotherapy response of patients is still unclear. Based on the data of 425 Chinese lung cancer patients, the genomic characteristics with different HLA LOH statuses were analyzed. The driver genes mutation frequency, oncogenic signaling pathways mutation frequency, tumor mutational burden (TMB) and chromosomal instability (CIN) score in the HLA LOH high group was significantly higher than in the HLA LOH negative group. Transcriptome analyses revealed that pre-existing immunologically active tumor microenvironment (TME) was associated with HLA LOH negative patients. Non-small cell lung cancer (NSCLC) patients, especially for lung squamous cell carcinomas (LUSC), with HLA LOH negative have a longer survival period than those with HLA LOH. In addition, the combination of HLA LOH with TMB or programmed cell death-Ligand 1 (PD-L1) expression can further distinguish responders from non-responders. Furthermore, a comprehensive predictive model including HLA LOH status, TMB, PD-L1 expression and CD8+ T cells was constructed and exhibited a higher predictive value, which may improve clinical decision-making.

Systematic assessment and optimizing algorithm of tumor mutational burden calculation and their implications in clinical decision-making.

Tumor mutation burden (TMB) has been validated as a biomarker to predict the response of immune checkpoint inhibitors (ICIs) treatment in various cancers. However, the effects of different sequencing platforms, cancer types, and calculation algorithms on TMB as well as its cut-off value for predicting immunotherapy efficacy in the East Asian population still need to be further investigated. In this study, the data of 4126 samples generated by targeted panel sequencing or whole-exome sequencing (WES) in different platforms and public sequencing data from 3680 samples that contained targeted panel sequencing, WES and whole-genome sequencing (WGS) were obtained. The impact of different sequencing platforms and methods on TMB calculation was assessed. No significant bias was found in TMB calculated by different platforms. However, TMB calculated from WGS was significantly lower than those calculated from targeted panel sequencing and WES. The distribution of TMB at different sequencing depths and tumor purity were analyzed. There was no significant difference in the distribution of TMB when the sequencing depth was greater than 500, the tumor purity estimated by hematoxylin-eosin (HE) staining was between 0.1-1.0 or estimated by next-generation sequencing (NGS) was greater than 0.4. In addition, the somatic-germline-zygosity (SGZ) algorithm was optimized to calculate TMB from tumor-only sequencing samples in the East Asian population. The correlation coefficient of TMB calculated with the optimized SGZ algorithm and paired normal-tumor sequencing is 0.951. Furthermore, the optimal cut-off value of TMB in East Asian lung cancer patients treated with ICIs was determined to be 7 mut/Mb instead of 10 mut/Mb through the ROC curve and Log-rank analysis in the training cohort and validated in the test cohort. Patients with TMB ≥ 7 mut/Mb had better outcomes than patients with TMB<7 mut/Mb. In conclusion, this study systematically analyzed the factors that influenced the TMB calculation and optimized the SGZ algorithm to calculate TMB from tumor-only sequencing samples in the East Asian population. More importantly, the cut-off value of TMB for predicting immunotherapy efficacy was determined to be 7 mut/Mb instead of 10 mut/Mb in East Asian lung cancer patients, which can help in clinical decision-making.

Unexpected favorable outcome to sintilimab monotherapy in a relapse pancreatic ductal adenocarcinoma patient with high tumor mutational burden: a case report.

The reason that immune checkpoint inhibitors have not been widely applied to pancreatic cancer treatment is probably because of low immunogenicity or dense stromal fibrosis. Recently, only pembrolizumab was recommended for DNA mismatch repair deficiency or high microsatellite instability by National Comprehensive Cancer Network guideline. Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of pancreatic cancer, with a poor overall survival rate, the value of immunotherapy for PDAC needs more research. Here, we report a 56-year-old man suffered from PDAC with liver metastasis after radical surgery. The next-generation sequencing result demonstrated that it had remarkably high tumor mutational burden (TMB) of 49.92 Muts/Mb and microsatellite stability. Sintilimab (anti-PD-1) monotherapy was continuously administrated after failure of combined chemotherapy in second line, achieving stable disease within 22 months and few immunotherapy-related adverse events. To our knowledge, this is the first time to report a good outcome achieving 22 months with progression-free survival after PDAC metastasis with monotherapy of sintilimab. TMB may serve as a potential efficacy-related predictor in PDAC patients with sintilimab and help physicians make optimum clinical strategy.

Predicting Durable Responses to Immune Checkpoint Inhibitors in Non-Small-Cell Lung Cancer Using a Multi-Feature Model.

Due to the complex mechanisms affecting anti-tumor immune response, a single biomarker is insufficient to identify patients who will benefit from immune checkpoint inhibitors (ICIs) treatment. Therefore, a comprehensive predictive model is urgently required to predict the response to ICIs. A total of 162 non-small-cell lung cancer (NSCLC) patients undergoing ICIs treatment from three independent cohorts were enrolled and used as training and test cohorts (training cohort = 69, test cohort1 = 72, test cohort2 = 21). Eight genomic markers were extracted or calculated for each patient. Ten machine learning classifiers, such as the gaussian process classifier, random forest, and support vector machine (SVM), were evaluated. Three genomic biomarkers, namely tumor mutation burden, intratumoral heterogeneity, and loss of heterozygosity in human leukocyte antigen were screened out, and the SVM_poly method was adopted to construct a durable clinical benefit (DCB) prediction model. Compared with a single biomarker, the DCB multi-feature model exhibits better predictive value with the area under the curve values equal to 0.77 and 0.78 for test cohort1 and cohort2, respectively. The patients predicted to have DCB showed improved median progression-free survival (mPFS) and median overall survival (mOS) than those predicted to have non-durable clinical benefit.

Corrigendum: The Identification of Two RNA Modification Patterns and Tumor Microenvironment Infiltration Characterization of Lung Adenocarcinoma.

[This corrects the article DOI: 10.3389/fgene.2022.761681.].

Expression Patterns of Glycosylation Regulators Define Tumor Microenvironment and Immunotherapy in Gastric Cancer.

Glycosylation (Glyc) is prevalently related to gastric cancer (GC) pathophysiology. However, studies on the relationship between glycosylation regulators and tumor microenvironment (TME) and immunotherapy of GC remain scarce. We extracted expression data of 1,956 patients with GC from eight cohorts and systematically characterized the glycosylation patterns of six marker genes into phenotype clusters using the unsupervised clustering method. Next, we constructed a Glyc. score to quantify the glycosylation index of each patient with GC. Finally, we analyzed the relationship between Glyc. score and clinical traits including molecular subtype, TME, and immunotherapy of GC. On the basis of prognostic glycosylation-related differentially expressed genes, we constructed the Glyc. score and divided the samples into the high- and low-Glyc. score groups. The high-Glyc. score group showed a poor prognosis and was validated in multiple cohorts. Functional enrichment analysis revealed that the high-Glyc. score group was enriched in metabolism-related pathways. Furthermore, the high-Glyc. score group was associated with the infiltration of immune cells. Importantly, the established Glyc. score would contribute to predicting the response to anti-PD-1/L1 immunotherapy. In conclusion, the Glyc. score is a potentially useful tool to predict the prognosis of GC. Comprehensive analysis of glycosylation may provide novel insights into the epigenetics of GC and improve treatment strategies.

The Identification of Two RNA Modification Patterns and Tumor Microenvironment Infiltration Characterization of Lung Adenocarcinoma.

Background: RNA modification plays an important role in many diseases. A comprehensive study of tumor microenvironment (TME) characteristics mediated by RNA modification regulators will improve the understanding of TME immune regulation. Methods: We selected 26 RNA modification "writers" of lung adenocarcinoma (LUAD) samples and performed unsupervised clustering analysis to explore RNA modification patterns in LUAD. Differentially expressed genes (DEGs) with RNA modification patterns were screened to develop a "writers" of RNA modification score (WM score) system. The infiltration ratio of TME cell subsets was analyzed by CIBERSORT. Results: We identified two RNA modification modes showing different characteristics of overall survival (OS) and TME cell infiltration. According to WM score, LUAD patients were divided into a high-WM score group and a low-WM score group. High-scored patients had a poor prognosis and higher tumor mutation burden (TMB), they were more sensitive to four LUAD therapies (erlotinib, XA V939, gefitinib, and KU-55933) and more clinically responsive to PD-L1 treatment. Those with a low WM score showed higher stromal scores, ESTIMATE scores, and survival chance. Conclusion: Our work revealed the potential role of RNA modification patterns in TME, genetic variation, targeted inhibitor therapy, and immunotherapy. Identifying RNA modification pattern of LUAD patients help understand the characteristics of TME and may promote the development of immunotherapy strategies.

CDK2 Inhibition Enhances Antitumor Immunity by Increasing IFN Response to Endogenous Retroviruses.

Inhibitors of cyclin-dependent kinase-2 (CDK2) are commonly used against several solid tumors, and their primary mechanisms of action were thought to include cell proliferation arrest, induction of cancer cell apoptosis and induction of differentiation. Here, we found that CDK2 inhibition by either small molecular inhibitors or genetic Cdk2 deficiency promoted antitumor immunity in murine models of fibrosarcoma and lung carcinoma. Mechanistically, CDK2 inhibition reduced phosphorylation of RB protein and transcription of E2F-mediated DNA methyltransferase 1 (DNMT1), which resulted in increased expression of endogenous retroviral RNA and type I IFN (IFN-I) response. The increased IFN-I response subsequently promoted antitumor immunity by enhancing tumor antigen presentation and CD8+ T-cell infiltration. Our studies provide evidence that inhibition of CDK2 in cancer cells suppresses tumor growth by enhancing antitumor immune responses in the tumor microenvironment, suggesting a new mechanism to enhance antitumor immunity by CDK2 inhibitors.

Histone deacetylase 3 contributes to the antiviral innate immunity of macrophages by interacting with FOXK1 to regulate STAT1/2 transcription.

It is well known that interferon (IFN)-α/-ß activates the JAK/STAT signaling pathway and suppresses viral replication through the induction of IFN stimulated genes (ISGs). Here, we report that knockout of HDAC3 from macrophages results in the decreased expression of STAT1 and STAT2, leading to defective antiviral immunity in cells and mice. Further studies show that HDAC3 interacts with a conserved transcription factor Forkhead Box K1 (FOXK1), co-localizes with FOXK1 at the promoter of STAT1 and STAT2, and is required for protecting FOXK1 from lysosomal system-mediated degradation. FOXK1-deficient macrophages also show low STAT1 and STAT2 expression with defective responses to viruses. Thus, our studies uncover the biological importance of HDAC3 in regulating the antiviral immunity of macrophages through interacting with FOXK1 to regulate the expression of STAT1 and STAT2.

Enhancing the HSV-1-mediated antitumor immune response by suppressing Bach1.

BACKGROUND: In 2015, herpes simplex virus 1 (HSV-1)-derived talimogene laherparepvec (T-VEC) was the first oncolytic virus approved by the US Food and Drug Administration as a therapeutic agent for cancer treatment. However, its antitumor application is limited to local treatment of melanoma, and there is a lack of understanding of the mechanisms underlying the regulation of HSV-1 replication in cancer cells and the associated antitumor immunity. We hypothesized that increasing the replication capacity of HSV-1 in tumor cells would enhance the antitumor effect of this virus. METHODS: We systematically identified IFN-stimulated genes induced by HSV-1 by performing functional screens and clarified the mechanism by which BACH1 acts against HSV-1. Then, we tested the effect of BACH1 deficiency on immunogenic cell death induced by HSV-1. Furthermore, we investigated the antitumor effect of BACH1 deficiency on HSV-1 in MCA205 and B16 murine tumor models. RESULTS: We identified eight IFN-stimulated genes (ISGs) controlling HSV-1 replication, among which BTB and CNC homology 1 (BACH1) suppressed HSV-1 replication by inhibiting the transcription of ICP4, ICP27, and UL39. Loss of Bach1 function not only increased HSV-1 proliferation but also promoted HSV-1-induced cell apoptosis, HMGB1 secretion, and calreticulin exposure in tumor cells. More importantly, hemin, an FDA-approved drug known to downregulate BACH1, significantly enhanced HSV-1-mediated antitumor activity with increased T lymphocyte infiltration at the tumor site. CONCLUSIONS: Our studies uncovered a novel antiviral activity of BACH1 and provided a new strategy for improving the clinical efficiency of the oncolytic virus HSV-1.

An enhanced genetic mutation-based model for predicting the efficacy of immune checkpoint inhibitors in patients with melanoma.

Background: Programmed death ligand 1 (PD-L1) and tumor mutation burden (TMB) have been developed as biomarkers for the treatment of immune checkpoint inhibitors (ICIs). However, some patients who are TMB-high or PD-L1-high remained resistant to ICIs therapy. Therefore, a more clinically applicable and effective model for predicting the efficacy of ICIs is urgently needed. Methods: In this study, genomic data for 466 patients with melanoma treated with ICIs from seven independent cohorts were collected and used as training and validation cohorts (training cohort n = 300, validation cohort1 n = 61, validation cohort2 n = 105). Ten machine learning classifiers, including Random Forest classifier, Stochastic Gradient Descent (SGD) classifier and Linear Support Vector Classifier (SVC), were subsequently evaluated. Results: The Linear SVC with a 186-gene mutation-based set was screened to construct the durable clinical benefit (DCB) model. Patients predicted to have DCB (pDCB) were associated with a better response to the treatment of ICIs in the validation cohort1 (AUC=0.838) and cohort2 (AUC=0.993). Compared with TMB and other reported genetic mutation-based signatures, the DCB model showed greater predictive power. Furthermore, we explored the genomic features in determining the benefits of ICIs treatment and found that patients with pDCB were associated with higher tumor immunogenicity. Conclusion: The DCB model constructed in this study can effectively predict the efficacy of ICIs treatment in patients with melanoma, which will be helpful for clinical decision-making.

Homeoprotein SIX1 compromises antitumor immunity through TGF-ß-mediated regulation of collagens.

The tumor microenvironment (TME), including infiltrated immune cells, is known to play an important role in tumor growth; however, the mechanisms underlying tumor immunogenicity have not been fully elucidated. Here, we discovered an unexpected role for the transcription factor SIX1 in regulating the tumor immune microenvironment. Based on analyses of patient datasets, we found that SIX1 was upregulated in human tumor tissues and that its expression levels were negatively correlated with immune cell infiltration in the TME and the overall survival rates of cancer patients. Deletion of Six1 in cancer cells significantly reduced tumor growth in an immune-dependent manner with enhanced antitumor immunity in the TME. Mechanistically, SIX1 was required for the expression of multiple collagen genes via the TGFBR2-dependent Smad2/3 activation pathway, and collagen deposition in the TME hampered immune cell infiltration and activation. Thus, our study uncovers a crucial role for SIX1 in modulating tumor immunogenicity and provides proof-of-concept evidence for targeting SIX1 in cancer immunotherapy.