IGSF6 is a novel biomarker to evaluate immune infiltration in mismatch repair-proficient colorectal cancer

IGSF6 is a novel biomarker to evaluate immune infiltration in mismatch repair-proficient colorectal cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IGSF6 is a novel biomarker to evaluate immune infiltration in mismatch repair-proficient colorectal cancer Yu-cheng Xu, Zhao-liang Yu, Xiao-chuan Chen, Min-er Zhong, Yu-fan Liang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2194686/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background Immunotherapy has dramatically changed the landscape of treatment for colorectal cancer (CRC), but there is lack of effective predictive biomarker, especially for tumors with mismatch repair (MMR) proficiency. Immune response relies to cell surface receptors and their interactions, such as cell-cell recognition, binding and adhesion. However, the function of immunoglobulin superfamily (IGSF) genes in tumor immune microenvironment remains uncharacterized. Methods This study quantified the immune using the gene expression matrix obtained from the public database. Also the associations between IGSF6 gene expression and immune cell infiltration were assessed. The expression levels of IGSF6, CD8 + and CD4 + T cells in cancer tissues from CRC patients were evaluated. Results IGSF6 was more highly expressed in CRC tumor tissues than corresponding adjacent normal tissues. And IGSF6 was significantly correlated with immune cell infiltration in MMR-proficient patients. Remarkably, MMR-proficient patients with high IGSF6 expression showed more sensitive to immunotherapy and chemotherapy than those with low IGSF6 expression. Conclusions In summary, IGSF6 could be a novel biomarker to evaluate immune infiltration and predict therapeutic effect for MMR-proficient CRC. IGSF6 Immunotherapy Colorectal cancer Biomarker Immune checkpoint Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Immunotherapy is demonstrated as a promising strategy for many types of cancers with long-term durable responses, such as for breast cancer [ 1 ] , lung cancer and melanoma [ 2 , 3 ] . But there are still lots of challenges in immunotherapy for colorectal cancer (CRC) patients. CRC are classified into MMR-deficient (microsatellite instability) and MMR-proficient (microsatellite stability) subtypes. Programmed death 1 (PD-1) blockade is verified as breakthrough therapy for MMR-deficient CRC, but less effective in MMR-proficient CRC [ 4 – 6 ] . MMR-deficient tumors are characterized by a high tumor mutational burden (TMB) and high infiltration of activated CD8 + cytotoxic T lymphocytes (CTL) and activated Th1 cells with IFN-γ production [ 7 ] . These features enable MMR-deficient tumor to be a good target for immunotherapy [ 8 ] . On the contrast, MMR-proficient CRC has been long thought to have an inactive response to immune checkpoint inhibitors. The lack of immune infiltration and low TMB in MMR-proficient CRC decrease the potential of obtaining benefits from immunotherapy, which is defined as an “immune resistant” phenotype [ 9 ] . Interestingly, about 45% of MMR-proficient tumors had a high immunoscore, which means that some of MMR-proficient tumors may be sensitive to immunotherapy [ 10 ] . Consistently, a previous clinical study reported that 27% (4/15) of MMR-proficient patients had pathological responses when treating with ipilimumab plus nivolumab, indicating that immunotherapy may be effectivity in some of MMR-proficient patients [ 6 ] . Furthermore, immune hot tumors with extensive immune infiltration also had better responses to chemotherapy [ 11 , 12 ] . However, there is currently insufficient information to routinely utilise predictive biomarkers for MMR-proficient patients. As more than 90% CRC are MMR-proficient, it is an urgent clinical need to identify effective immune checkpoints for MMR-proficient patients to predict sensibility of immunotherapy. Increasing evidence show that the tumor immune contexture, including spatial organization and density, directly influence the clinical outcome of cancer [ 13 – 16 ] . The immunoglobulin superfamily (IGSF) consists of the immunoglobulins (IG), T cell receptors (TR) and proteins that have the common feature of having at least one Ig-like domain [ 17 ] . It has been reported that IGSF genes are frequently overexpressed in several cancer types and plays a role in promoting cancer cell growth in the progression of cancer, such as thyroid cancer [ 18 ] and prostate cancer [ 19 ] . Recently, a study reported that IGSF protein signatures are associated with distinct tumor immunophenotypes and clinical outcome [ 20 ] . However, it is unclear whether IGSF genes could be used as biomarkers in CRC. In this study, we evaluated a group of IGSF genes in CRC and found IGSF6 may be a novel prognostic biomarker for MMR-proficient CRC. As far as we know, the function of IGSF6 in cancer has not yet to be clarified. We identified IGSF6 was upregulated in CRC tissues, which was correlated with the immune checkpoint genes and immune cell infiltration. Moreover, we demonstrated overexpression of IGSF6 was associated with a high density of CD8 + T cell and CD4 + T cell tumor-infiltrating lymphocytes. Importantly, MMR-proficient tumors with high IGSF6 expression showed a better response to immunotherapy and chemotherapy. Materials And Methods Study design and patient selection Two public cohorts, The Cancer Genome Atlas (TCGA) CRC cohort and GSE39582 dataset from the Gene Expression Omnibus (GEO) database, with gene expression data derived from fresh-frozen CRC samples were evaluated retrospectively. A total of 303 patients diagnosed as colon or rectal cancer and treated in the Sixth Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China), 6 MMR-proficient CRC patients treated by immune checkpoint inhibitors (ICI) plus chemotherapy and 21 MMR-proficient CRC patients underwent neoadjuvant chemotherapy at the Sixth Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China), were retrospectively studied. Patients with infectious diseases, autoimmune diseases, or multiple primary cancers were excluded. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (IRB) of the Sixth Affiliated Hospital, Sun Yat-sen University (approval number: 2021ZSLYEC-099). Immunofluorescence Sections of 5 µm thickness were obtained from CRC tumors. Deparaffinization of slides was done with xylene followed by rehydration in histological-grade ethanol and fixation with 3% hydrogen peroxide in methanol, before antigen retrieval using pH 6.0 citrate buffer or pH 9.0 EDTA buffer. Then the slides were incubated with the primary antibody overnight at 4℃. Slides were scanned and digitalized with and visualized with CASEVIEWER software. Immunohistochemistry Staining Immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining were performed using standard immunoperoxidase staining on CRC tissue sections of 5 µm thickness from resected tumors. Sections were stained against anti-IGSF6 (SANTACRUZ, sc-377053, 1:200), anti-CD4(ZSGB-Bio, ZA-0519), anti-CD8(ZSGB-Bio, ZA-0508). Paraffin sections were deparaffinized with xylene in the stainer and then underwent heat-mediated antigen retrieval with pH 9.0 EDTA buffer. Then the slides were incubated with the primary antibody overnight at 4℃, and the sections were stained with diaminobenzidine. Sections were counterstained with hematoxylin, dehydrated and mounted with coverslips. Slides were scanned and digitalized with the TEKSQRAY image analysis system and visualized with ImageViewerG software. A board-certified pathologist evaluated the staining digitally to ensure the appropriate quality of tumor tissue. Evaluation Of Immunohistochemical And Immunofluorescence Analysis Immunoreactivity for IHC and immunofluorescence staining was evaluated by a semiquantitative method, as described previously. Each TMA spot was assigned an intensity score from 1 to 4 (1, 2, 3, or 4) by two trained researchers. The percentage of positive tumor cells divided by the total number of tumor cells was assigned using 25% increments (25%, 50%, 75%, and 100%). IHC and immunofluorescence scores were determined by the intensity score and the proportion of area positively stained tumor cells. A final score was determined as the average of two cores from the same representative tumor area. Statistical analysis All statistical analyses were accomplished by R software V.4.2.1 ( http://www.r-project.org ) and Graphpad Prism 9 software. Data were presented as the mean ± SD, unless otherwise stated. Statistical significance between two groups was evaluated by two-tailed Student’s t-test. Statistical significance was considered at p < 0.05. Results IGSF6 is highly expressed in CRC tumor tissues and could be a novel biomarker to evaluate immune infiltration To characterize the potential function of IGSF genes in CRC, we first investigated the expression of IGSF genes in CRC tumor tissue and adjuvant tissue from Gene Expression Profiling Interactive Analysis (GEPIA, cancer-pku.cn). Interestingly, only IGSF6 was significative up-regulated in both colon and rectal tumor versus normal tissue ( Fig. 1 A ) . To confirm the findings in public database, we detected IGSF6 expression in 16 pairs of CRC tissues and adjacent normal colorectum tissues. Unexpectedly, IGSF6 levels are highly expressed in tumor tissues as compared with adjacent normal tissues ( p < 0.0001, Fig. 1 B-C ) . Since IGSF genes play a central role in cell-cell recognition as cell surface receptors, these results suggest that IGSF6 may be a neoantigen generated during carcinogenesis and involving in immune infiltration. Tumor immune microenvironments are important factors to effect immunotherapy in clinic, but currently lock of effective biomarker to evaluate immune infiltration and the effective of immunotherapy. To evaluate IGSF6 can be used as a biomarker for immune infiltration, we assessed the association between IGSF6 expression and tumor-infiltrating lymphocytes in TCGA datasets. The results showed that the expression of IGSF6 was positive correlated with tumor-infiltrating lymphocytes in various cancers, especially for CRC (Fig. 1 D). Furthermore, only IGSF6, among IGSF family genes, showed a strong positive correlation with tumor-infiltrating lymphocytes in CRC patients, including MMR-deficient and MMR-proficient tumors (Fig. 1 E). Interestingly, a strong positive correlation between IGSF6 expression and tumor-infiltrating lymphocytes can be found in MMR-proficient tumors, indicating that IGSF6 could be an immunotherapy biomarker for MMR-proficient CRC patients (Fig. 1 F). What’s attractive, IGSF6 was strongly positive correlated with immune checkpoints in MMR-proficient CRC, such as PD-1, PD-L1, CTLA-4, LAG3 and TIGIT (Fig. 1 G-H). Moreover, expression of IGSF6 correlated with tumor-infiltrating lymphocytes were confirmed in GSE39582 dataset ( Supplementary Fig. 1 ), which supported that IGSF6 could be a novel biomarker to improve clinical applications of current immunotherapies. MMR-deficient tumors which have highly tumor mutational burden (TMB), for accumulation of insertions–deletions (indels) which given rise to more neoantigens, are more beneficial from immunotherapy [ 21 – 25 ] . Therefore, we evaluated the association between IGSF6 expression and TMB in CRC, and found a positive correlation between IGSF6 genes and TMB (Fig. 1 I). Furthermore, we investigated the relationship between IGSF6 expression and the tumor immune microenvironment. We calculated single-sample gene set enrichment analysis (ssGSEA) scores for patients using known CRC immune signatures. Given that MMR-deficient tumors have a distinct immunologic profile, we separated them into their own group and performed unsupervised hierarchical clustering on the MMR-proficient tumors (Fig. 1 J). We found high IGSF6 expression tumors were associated with extensive immune infiltration, which also be confirmed in another CRC dataset (Fig. 1 K). Based on the public database studies of IGSF6, we hypothesized that IGSF6 may be associated with CD4 + and CD8 + T cell infiltration. We constructed a tissue microarray (TMA) on colon or rectal tumor tissues containing a large cohort of CRC patients (n = 303) and detected the expression of IGSF6, CD4 + and CD8 + T cell with multiple immunofluorescences. Consistent with previous studies [ 6 ] , the infiltrated CD4 + and CD8 + T lymphocytes were different in various cases of CRC tissues, which was highly correlated with tumor response to treatment (Fig. 2 A). Interestingly, multiple immunofluorescences analysis indicated a strong positive correlation between IGSF6 expression and tumor-infiltrating lymphocytes, including CD4 + and CD8 + T cell, in CRC tumors (Fig. 2 B). High Igsf6 Expression Is Associated Benefit From Immunotherapy And Chemotherapy As IGSF6 expression was associated with immune infiltration, we next investigated whether IGSF6 could be used as a biomarker to predict therapeutic effect for CRC. A total of 6 MMR-proficient CRC patients treated with immunotherapy were studied (Table 1 ). After systematic ICI treatment, the patients received staged or simultaneous complete surgical resection for primary tumor. A thorough pathological examination of the resected tumors before and after ICI was conducted. Immuno-sensitive MMR-proficient CRC showed a sharp tumor burden reduction, massive necrosis tissue and lymphocytes infiltration after ICI treatment, such as Case 1 (Fig. 3 A, Ⅰ-Ⅻ ). On the contrary, there was no response or even bad response in immuno-resistance tumors, such as Case 2 ( Fig. 3 A, a-l ) . We next detected IGSF6 expression, CD4 + T cell and CD8 + T cell from pathological tumor specimens with IHC. Interestingly, high IGSF6 expression were observed in resected specimens in immuno-sensitive tumors, while with high CD4 + and CD8 + T cell infiltration ( Fig. 3 A ) . As expected, IGSF6 levels are more highly expressed in immuno-sensitive tumors than immuno-resistance tumors (Fig. 3 B). To further clarify whether the IGSF6 could predict benefit in CRC patients, we focused on the clinical outcome in the IGSF6 high and IGSF6 low groups. The OS of CRC patients with IGSF6 high expression was better than those with IGSF6 low expression in TCGA cohorts, especially for MMR-proficient patients ( Fig. 3 C ) . The same tendency can also be found in GSE39582 cohorts ( Fig. 3 D ) . Table 1 Patient Characteristics of 6 MMR-proficient CRC patients treat with ICI. Characteristics Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 PRIMARY TUMOR SITE rectum Rectum Rectum Rectum Rectum Colon STAGE T2N0M0 T3N0M0 T3N0M0 T3N1M0 T4aN1aM0 T3N0M0 METASTAIC SITE NO NO NO NO NO NO Treatment before ICI Systematic (response ¶) none FOLFOX + radiotherapy (PR) FOLFOX (PR) none FOLFOX (PD) XELOX (PD) Surgery none none none none hartmann right hemicolectomy ICI combined treatment Regimen sintilimab + Avastin + FOLFOX FOLFOX + sintilimab FOLFOX + sintilimab sintilimab + FOLFOX camrelizumab nivolumab Systematic (response ¶) PR PR PR PD PD PD Radiological response PR PR PR PD ξ ξ Surgical treatment (after ICI) Dixon TaTME Dixon Dixon ξ ξ Pathological response Primary tumor PR PR PR PD ξ ξ Reginal lymph nodes PR PR PR PD ξ ξ Postoperative treatment NONE NONE FOLFOX FOLFOX camrelizumab nivolumab TRG score (NCCN Guidelines) 1 1 1 2 ξ ξ ¶ Assessed by the Response Evaluation Criteria in Solid Tumors 1.1 criteria. ξ Colectomy was conducted before ICI combined treatment. FOLFOX, fluorouracil + oxaliplatin; XELOX, capecitabine + oxaliplatin; ICI, immune checkpoint inhibitor; pMMR, mismatch repair (MMR) proficient; dMMR, mismatch-repair (MMR) deficient; PD, progressive disease; PR, partial response. Chemotherapy remains a relatively common and effective treatment for most MMR-proficient CRC patients. To further explore the relationship between IGSF6 and patients’ response to chemotherapy, we collected the fresh tissues of chemosensitive and chemoresistance MMR-proficient CRC patients before treatment (Table 2 ). As expected, IGSF6 levels are more highly expressed in tumor tissues from chemosensitive patients than from chemoresistance patients (Figs. 4 A-B). There were more tumor-infiltrating immune cells in tumor tissues of the IGSF6 High group than in those of the IGSF6 Low group, indicating that MMR-proficient CRC tissues with high levels of IGSF6 had a tumor microenvironment with an activated adaptive immune phenotype (Figs. 4 A-B). Table 2 Patient Characteristics of 21 MMR-proficient CRC patients treat with chemotherapy. TRG treatment STAGE PRIMARY TUMOR SITE Patient 1 0 FOLFOX T3N1aM0 Rectum Patient 2 0 FOLFOX T4aN2aM0 Rectum Patient 3 0 FOLFOX T3N0M0 Rectum Patient 4 3 FOLFOX T3N1aM0 Rectum Patient 5 0 FOLFOX T4aN2aM1 Rectum Patient 6 0 FOLFOX T3N2aM0 Rectum Patient 7 0 UNKNOWED T4aN2bM0 Rectum Patient 8 0 Avastin + FOLFOXIRI T3N2aM1 Rectum Patient 9 3 FOLFOXIRI T3N1aM0 Rectum Patient 10 3 FOLFOX T3N1bM0 Rectum Patient 11 0 FOLFOX T3N2aM0 Rectum Patient 12 3 FOLFOX T3N2bM0 Rectum Patient 13 3 FOLFOXIRI T3N2bM0 Rectum Patient 14 0 FOLFOXIRI T3N2aM0 Rectum Patient 15 3 FOLFOX T3N1bM0 Rectum Patient 16 3 FOLFOX T3N1aM0 Rectum Patient 17 3 XELOX T4aN2bM1 Rectum Patient 18 0 FOLFOXIRI T3N1aM0 Rectum Patient 19 3 FOLFOX T3N0M0 Rectum Patient 20 0 FOLFOX T3N2bM0 Rectum Patient 21 0 FOLFOXIRI T3N2aM0 Rectum FOLFOX, fluorouracil + oxaliplatin; XELOX, capecitabine + oxaliplatin; FOLFOXIRI, fluorouracil + oxaliplatin + Irinotecan. Discussion CRC is one of the most common cancer types in the world due to its high prevalence [ 26 ] . Immunotherapy has shown as an effectively therapeutic strategy for many types of cancers, but its clinical application in CRC only for MMR-deficient patients. A large amount of MMR-proficient tumors had shown a high proportion of inter-tumoral tumor-infiltrating lymphocytes as “hot tumors”, which may be sensitive to immunotherapy [ 11 , 12 ] . Biomarkers, including CEA, CA199, and CA125, in clinical practice are significance for tumor cell proliferation and tumor recurrence. But lock of effectively biomarker which can predict immune infiltration. Therefore, it is critical for identifying novel predictive markers for MMR-proficient tumors who may achieve benefits from immunotherapy. Recent studies point out the importance of the tumor immune contexture for the prognosis of patients with CRC [ 13 – 16 ] . Immunotherapy targeting immune checkpoints (such as PD1/PD-L1) has become an approved treatment option for patients with CRC with mismatch repair deficiency or high microsatellite instability [ 27 ] . Interestingly, about 45% of MMR-proficient and 65% of MMR-deficient CRC had a high immunoscore, while 55% of MMR-proficient and 35% of MMR-deficient CRC had a low immunoscore, suggesting that some of the MMR-deficient tumors are unable to mount an antitumor response while the reverse may apply for some of the MMR-proficient population [ 10 ] . Indeed, the 27% pathological response in MMR-proficient early-stage CRC treated with neoadjuvant ipilimumab plus nivolumab provide further support that MMR-proficient CRC is not an immune desert and can be targeted with immunotherapy [ 6 ] . Apart from MMR-deficiency and MMR-proficiency, we speculate that IGSF6 gene is another potential biomarker for responsiveness to immunotherapy due to its significant association with TMB, which is an effective indicator for response prediction to ICI. In our study, IGSF6 may be a new biomarker which may promote the expression of immune cells, such as CD8 + T cells associated with improved survival of patients with MMR-proficient CRC [ 28 , 29 ] , contributing to a better immune function. The complex interactions between tumor and immune cells play out in the TME and dictate clinical outcomes, including among patients with CRC. No data are available on CRC, but in several tumor types an increased T cell infiltration (TCI) has been shown to increase the probability of response to ICI [ 30 , 31 ] . A number of inhibitory immunoreceptors have been identified and studied in cancer in past decades, including but not limited to PD-1, PD-L1, CTLA-4, LAG3, HAVCR2, TIGIT, CD69 and CD40. They are viewed as “immune checkpoints” referring to molecules that act as gatekeepers of immune responses. In the evolutionary process, immune checkpoints have co-evolved with stimulatory immunoreceptors and appear as early as in fish [ 32 ] . These receptors often use monotyrosine signaling motifs, such as immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM), to deliver inhibitory signals. As surface molecules, their activity can be easily inhibited by blocking antibodies that prevent ligand-receptor engagement. The most successful immune checkpoint blockade therapy, one of anticancer immunotherapies, is anti-PD-1/PD-L1 therapy that has been approved to treat a wide variety of cancer types, such as blood, skin, lung, liver, bladder and kidney cancers [ 33 ] . Immune checkpoint blockade therapy often leads to more durable response than chemo or targeted therapies, perhaps reflecting the memory feature of the immune system. But as clinical data accumulates all over the world, drawbacks and side effects have begun to be revealed. The major bottleneck of immune checkpoint blockade therapy is its low response rate in most cancers, with a range of 10–30% [ 33 ] . For some major cancer types such as MMR-proficient CRC, anti-PD-1/PD-L1 therapy shows nearly no effect [ 21 ] . In this study, we identified a crucial involvement of IGSF6 in CRC promotion immune cells. IGSF6 expression was upregulated in CRC tissues and high expression of IGSF6 correlated with an active immune microenvironment and a favorable prognosis and furthermore it may facilitate immune mediated tumor killing. IGSF6 could be a new biomarker to design next generation therapies and to improve clinical protocols of current immunotherapies. But this should be validated in further study before its clinical application. Here, we elucidated that IGSF6 high expression promoted more immune cells in CRC, so as to facilitate immune mediated tumor killing. IGSF6 high expression was an independent predictor of better prognosis, and these results may provide insight into effective strategies for therapy in MMR-proficient CRC, which enables IGSF6 to be a novel prognostic biomarker and target of immunotherapies. Declarations Acknowledgments: Not appliable. Author Contributions: Yu-cheng Xu, Zhao-liang Yu, Xiao-chuan Chen and Min-er Zhong contributed to study concept and design, acquisition, analysis, interpretation of data and drafting of the manuscript. Yu-fan Liang and Jing-rong Weng contributed to data collections and manuscript review. Dan-dong Luo and Yi-ran Bie contributed to study concept and design, analysis and interpretation of data and critical revision of the manuscript for important intellectual content. Xi Chen, Jia-wei Cai, Yu-ming Rong and Yi-feng Zou supervised the study. All authors read and approved the final manuscript. Conflict of Interest Statement: All authors declare no conflict of interests. Running Title: IGSF6 is a biomarker for immune infiltration Ethics approval: This study was approved by the ethics committees of Sixth Affiliated Hospital of Sun Yat-sen University. Consent for publication: All authors approve to publish this paper. Data availability: All data that supporting the findings of this study are available from the corresponding author upon request. Funding: This work was supported by the program of Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004), National Natural Science Foundation of China (No.82203072), National Key Clinical Discipline, Natural Science Foundation of Guangdong Province, China (2020A1515010428), Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515111201), Guangdong Basic and Applied Basic Research Foundation (SL2022A04J01718), the National Natural Science Foundation of China (82102475, MZ), Guangdong Basic and Applied Basic Research Foundation (2020A1515110489, MZ) and the Fundamental Research Funds for the Central Universities, Sun Yat-sen University. References Franzoi M A, Romano E, Piccart M. Immunotherapy for early breast cancer: too soon, too superficial, or just right?[J]. Ann Oncol, 2021, 32(3): 323–336. Samstein R M, Lee C H, Shoushtari A N, et al. Tumor mutational load predicts survival after immunotherapy across multiple cancer types[J]. 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(C, D) Correlation between immune check points and IGSF6 expression in MMR-proficient patients by Spearman’s correlation coefficient, n=444. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2194686","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":175681936,"identity":"dbfc31f8-98a0-47c9-8827-492b68bab18a","order_by":0,"name":"Yu-cheng Xu","email":"data:image/png;base64,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","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yu-cheng","middleName":"","lastName":"Xu","suffix":""},{"id":175681937,"identity":"dc5becdb-8019-4964-b1da-d571baf1a0bf","order_by":1,"name":"Zhao-liang Yu","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhao-liang","middleName":"","lastName":"Yu","suffix":""},{"id":175681938,"identity":"45140828-da3e-4090-910c-a29d9ad01d8d","order_by":2,"name":"Xiao-chuan Chen","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiao-chuan","middleName":"","lastName":"Chen","suffix":""},{"id":175681939,"identity":"4533c3bd-ac0f-48d9-b35e-ec467b79ce91","order_by":3,"name":"Min-er Zhong","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min-er","middleName":"","lastName":"Zhong","suffix":""},{"id":175681940,"identity":"dae7d79a-06f4-44b7-8e8c-10fbcf1ea127","order_by":4,"name":"Yu-fan Liang","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-fan","middleName":"","lastName":"Liang","suffix":""},{"id":175681941,"identity":"11b0bddd-916c-4706-b329-82b0a924db27","order_by":5,"name":"Jing-rong Weng","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing-rong","middleName":"","lastName":"Weng","suffix":""},{"id":175681942,"identity":"c4d83aa0-eec8-4b1b-a98a-d94ff94b8e4e","order_by":6,"name":"Dan-dong Luo","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan-dong","middleName":"","lastName":"Luo","suffix":""},{"id":175681943,"identity":"2fde93fe-6f3e-4503-9eaf-1555403e5183","order_by":7,"name":"Yi-ran Bie","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi-ran","middleName":"","lastName":"Bie","suffix":""},{"id":175681944,"identity":"03746bb2-bbb2-4a75-8813-97e09ad5e962","order_by":8,"name":"Xi Chen","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Chen","suffix":""},{"id":175681945,"identity":"118d9835-72a2-4950-99ff-cb1c0b78d98f","order_by":9,"name":"Jia-wei Cai","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jia-wei","middleName":"","lastName":"Cai","suffix":""},{"id":175681946,"identity":"f702c7e9-db18-4fe4-be93-26897719d896","order_by":10,"name":"Yu-ming Rong","email":"","orcid":"","institution":"Cancer Center of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-ming","middleName":"","lastName":"Rong","suffix":""},{"id":175681947,"identity":"c9d97ebd-2361-465d-856b-e9553c7ac7e7","order_by":11,"name":"Yi-feng Zou","email":"","orcid":"","institution":"The Sixth Affiliated Hospital of Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi-feng","middleName":"","lastName":"Zou","suffix":""}],"badges":[],"createdAt":"2022-10-23 03:44:16","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-2194686/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-2194686/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32919342,"identity":"e0c4c386-5226-4d83-be7e-6e4741b03026","added_by":"auto","created_at":"2023-02-14 15:24:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5180982,"visible":true,"origin":"","legend":"\u003cp\u003eIGSF6 is highly expressed in CRC tumor tissues and could be a novel biomarker to evaluate immune infiltration. (A) Expression of IGSF from the GEPIA database. (B) Representative immunofluorescence images of IGSF6 expression in CRC and adjacent normal tissues from the patient. (C) Quantification of the score for IGSF6 level in tumor tissue versus adjacent tissue assessed by immunofluorescence assay. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, *** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001. p values were determined by paired-t test. (D) Correlation between immune cell infiltration and IGSF6 in pan-cancer, * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (E) Correlation between immune cell infiltration and IGSF in CRC patients from TCGA database, n=383. (F) Correlation between immune cell infiltration and IGSF in MMR-proficient CRC patients, n=249. (G, H) Correlation between immune check points and IGSF6 expression in MMR-proficient patients by Spearman’s correlation coefficient. n=249. (I) Correlation between TMB and IGSF6 expression in CRC patients by Spearman’s correlation coefficient, n=359. (J, K) Unsupervised hierarchical clustering of colorectal tumors using ssGSEA scores for immune signatures identifies increasing levels of immune infiltrates. J from TCGA database, n=367, K from GSE39582 database, n=519.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2194686/v2/45b78e3b3d115628bb4b1986.png"},{"id":32919343,"identity":"6721c8b7-a78e-4568-8ed6-dc98b38ffa5a","added_by":"auto","created_at":"2023-02-14 15:24:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8875948,"visible":true,"origin":"","legend":"\u003cp\u003eIGSF6 is highly correlated with immune infiltration in CRC tissues. (A) Representative immunofluorescence images of IGSF6, CD4+ T cell and CD8+ T cell expression in CRC tissues from the patients, red arrowheads for IGSF6, green arrowheads for CD4+ T cell, purple arrowheads for CD8+ T cell. (B) Correlation between immune cell infiltration and IGSF6 in CRC patients by Spearman’s correlation coefficient, n=303.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2194686/v2/e6d98df7dc92c2b74823b4c7.png"},{"id":32917569,"identity":"94e247e1-d616-4881-86b7-d6b0d8c91e98","added_by":"auto","created_at":"2023-02-14 15:16:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11162132,"visible":true,"origin":"","legend":"\u003cp\u003eHigh IGSF6 expression is benefit from immunotherapy. (A) Radiological and pathological response to FOLFOX plus sintilimab in a patient with stage T3N0M0 (Case 1) and FOLFOX plus sintilimab in a patient with stage T3N1M0 (Case 2). Radiographic imaging shows the tumor in rectum (Ⅰ, a) at initial diagnoses. A notable tumor regression could be seen in primary tumor from Case 1 (Ⅱ). But there was no response in Case 2 after ICI (b). Primary tumor was observed using colonoscopy (Ⅲ, c) at initial diagnosis and after ICI treatment (Ⅳ, d). H\u0026amp;E staining shows primary tumor at initial diagnosis (Ⅴ, e) and pathological response after ICI treatment (VI, f). Fibrosis and an infiltration with viable density of many lymphocytes (VI, arrowheads) can be found, which cannot be found in case 2 (f). IHC staining showed CD4+ T cells, CD8+ T cells and IGSF6 expression with pretreatment tumor samples (Ⅶ, Ⅸ, Ⅺ for Case 1; g, i, k for Case 2) and posttreatment tumor tissues (Ⅷ, Ⅹ, Ⅻ for Case 1; h, j, l for Case 2). (B) Quantification of the score for CD4+ T cells, CD8+ T cells and IGSF6 staining before treatment in CRC tissue from immunotherapy sensitive versus immunotherapy resistance assessed by IHC assay, n=6. (C, D) OS curve of patients with high IGSF6 and low IGSF6 group in TCGA database (C) and GSE39582 database (D).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2194686/v2/475c8d796a133d18cdabd97a.png"},{"id":32917568,"identity":"bd7f1e05-eef1-444f-8a84-cda30c82cf5c","added_by":"auto","created_at":"2023-02-14 15:16:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6664376,"visible":true,"origin":"","legend":"\u003cp\u003eHigh IGSF6 expression is benefit from chemotherapy. (A) Representative micrographs of CD4+ T cells, CD8+ T cells and IGSF6 staining from endoscopic pathological tumor specimens before patients underwent chemotherapy, n=21. (B) Quantification of the score for CD4+ T cells, CD8+ T cells and IGSF6 staining from chemosensitive CRC versus chemoresistance CRC by IHC assay, n=21. pMMR, mismatch repair (MMR) proficiency; dMMR, mismatch repair (MMR) deficient. * \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003e p\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2194686/v2/453159b8ce354fea86de35cf.png"},{"id":37454332,"identity":"23c36571-a055-443d-b93a-12db5620f031","added_by":"auto","created_at":"2023-05-24 18:44:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3060491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2194686/v2/30fbaa22-c47f-4f61-8043-f44625a25756.pdf"},{"id":32917572,"identity":"c4df9e90-dc4f-46a5-92e9-428a45cc1ef8","added_by":"auto","created_at":"2023-02-14 15:16:30","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4008762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 1. \u003c/strong\u003e(A) Correlation between immune cell infiltration and IGSF in CRC patients from GSE39582 database, n=566. (B) Correlation between immune cell infiltration and IGSF in MMR-proficient CRC patients from GSE39582 database, n=444. (C, D) Correlation between immune check points and IGSF6 expression in MMR-proficient patients by Spearman’s correlation coefficient, n=444.\u003c/p\u003e","description":"","filename":"SupplymentaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-2194686/v2/25c0ee06ba57402950d905f2.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"IGSF6 is a novel biomarker to evaluate immune infiltration in mismatch repair-proficient colorectal cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmunotherapy is demonstrated as a promising strategy for many types of cancers with long-term durable responses, such as for breast cancer \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, lung cancer and melanoma \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. But there are still lots of challenges in immunotherapy for colorectal cancer (CRC) patients. CRC are classified into MMR-deficient (microsatellite instability) and MMR-proficient (microsatellite stability) subtypes. Programmed death 1 (PD-1) blockade is verified as breakthrough therapy for MMR-deficient CRC, but less effective in MMR-proficient CRC \u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. MMR-deficient tumors are characterized by a high tumor mutational burden (TMB) and high infiltration of activated CD8\u0026thinsp;+\u0026thinsp;cytotoxic T lymphocytes (CTL) and activated Th1 cells with IFN-γ production \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. These features enable MMR-deficient tumor to be a good target for immunotherapy \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. On the contrast, MMR-proficient CRC has been long thought to have an inactive response to immune checkpoint inhibitors. The lack of immune infiltration and low TMB in MMR-proficient CRC decrease the potential of obtaining benefits from immunotherapy, which is defined as an \u0026ldquo;immune resistant\u0026rdquo; phenotype \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Interestingly, about 45% of MMR-proficient tumors had a high immunoscore, which means that some of MMR-proficient tumors may be sensitive to immunotherapy \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Consistently, a previous clinical study reported that 27% (4/15) of MMR-proficient patients had pathological responses when treating with ipilimumab plus nivolumab, indicating that immunotherapy may be effectivity in some of MMR-proficient patients \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Furthermore, immune hot tumors with extensive immune infiltration also had better responses to chemotherapy \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. However, there is currently insufficient information to routinely utilise predictive biomarkers for MMR-proficient patients. As more than 90% CRC are MMR-proficient, it is an urgent clinical need to identify effective immune checkpoints for MMR-proficient patients to predict sensibility of immunotherapy.\u003c/p\u003e \u003cp\u003eIncreasing evidence show that the tumor immune contexture, including spatial organization and density, directly influence the clinical outcome of cancer \u003csup\u003e[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. The immunoglobulin superfamily (IGSF) consists of the immunoglobulins (IG), T cell receptors (TR) and proteins that have the common feature of having at least one Ig-like domain \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. It has been reported that IGSF genes are frequently overexpressed in several cancer types and plays a role in promoting cancer cell growth in the progression of cancer, such as thyroid cancer \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e and prostate cancer \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Recently, a study reported that IGSF protein signatures are associated with distinct tumor immunophenotypes and clinical outcome \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. However, it is unclear whether IGSF genes could be used as biomarkers in CRC. In this study, we evaluated a group of IGSF genes in CRC and found IGSF6 may be a novel prognostic biomarker for MMR-proficient CRC. As far as we know, the function of IGSF6 in cancer has not yet to be clarified. We identified IGSF6 was upregulated in CRC tissues, which was correlated with the immune checkpoint genes and immune cell infiltration. Moreover, we demonstrated overexpression of IGSF6 was associated with a high density of CD8\u0026thinsp;+\u0026thinsp;T cell and CD4\u0026thinsp;+\u0026thinsp;T cell tumor-infiltrating lymphocytes. Importantly, MMR-proficient tumors with high IGSF6 expression showed a better response to immunotherapy and chemotherapy.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and patient selection\u003c/h2\u003e \u003cp\u003eTwo public cohorts, The Cancer Genome Atlas (TCGA) CRC cohort and GSE39582 dataset from the Gene Expression Omnibus (GEO) database, with gene expression data derived from fresh-frozen CRC samples were evaluated retrospectively. A total of 303 patients diagnosed as colon or rectal cancer and treated in the Sixth Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China), 6 MMR-proficient CRC patients treated by immune checkpoint inhibitors (ICI) plus chemotherapy and 21 MMR-proficient CRC patients underwent neoadjuvant chemotherapy at the Sixth Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China), were retrospectively studied. Patients with infectious diseases, autoimmune diseases, or multiple primary cancers were excluded. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (IRB) of the Sixth Affiliated Hospital, Sun Yat-sen University (approval number: 2021ZSLYEC-099).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eSections of 5 \u0026micro;m thickness were obtained from CRC tumors. Deparaffinization of slides was done with xylene followed by rehydration in histological-grade ethanol and fixation with 3% hydrogen peroxide in methanol, before antigen retrieval using pH 6.0 citrate buffer or pH 9.0 EDTA buffer. Then the slides were incubated with the primary antibody overnight at 4℃. Slides were scanned and digitalized with and visualized with CASEVIEWER software.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry Staining\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry (IHC) and hematoxylin and eosin (H\u0026amp;E) staining were performed using standard immunoperoxidase staining on CRC tissue sections of 5 \u0026micro;m thickness from resected tumors. Sections were stained against anti-IGSF6 (SANTACRUZ, sc-377053, 1:200), anti-CD4(ZSGB-Bio, ZA-0519), anti-CD8(ZSGB-Bio, ZA-0508). Paraffin sections were deparaffinized with xylene in the stainer and then underwent heat-mediated antigen retrieval with pH 9.0 EDTA buffer. Then the slides were incubated with the primary antibody overnight at 4℃, and the sections were stained with diaminobenzidine. Sections were counterstained with hematoxylin, dehydrated and mounted with coverslips. Slides were scanned and digitalized with the TEKSQRAY image analysis system and visualized with ImageViewerG software. A board-certified pathologist evaluated the staining digitally to ensure the appropriate quality of tumor tissue.\u003c/p\u003e\n\u003ch3\u003eEvaluation Of Immunohistochemical And Immunofluorescence Analysis\u003c/h3\u003e\n\u003cp\u003eImmunoreactivity for IHC and immunofluorescence staining was evaluated by a semiquantitative method, as described previously. Each TMA spot was assigned an intensity score from 1 to 4 (1, 2, 3, or 4) by two trained researchers. The percentage of positive tumor cells divided by the total number of tumor cells was assigned using 25% increments (25%, 50%, 75%, and 100%). IHC and immunofluorescence scores were determined by the intensity score and the proportion of area positively stained tumor cells. A final score was determined as the average of two cores from the same representative tumor area.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were accomplished by R software V.4.2.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.r-project.org\u003c/span\u003e\u003cspan address=\"http://www.r-project.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Graphpad Prism 9 software. Data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, unless otherwise stated. Statistical significance between two groups was evaluated by two-tailed Student\u0026rsquo;s t-test. Statistical significance was considered at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIGSF6 is highly expressed in CRC tumor tissues and could be a novel biomarker to evaluate immune infiltration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo characterize the potential function of IGSF genes in CRC, we first investigated the expression of IGSF genes in CRC tumor tissue and adjuvant tissue from Gene Expression Profiling Interactive Analysis (GEPIA, cancer-pku.cn). Interestingly, only IGSF6 was significative up-regulated in both colon and rectal tumor versus normal tissue \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. To confirm the findings in public database, we detected IGSF6 expression in 16 pairs of CRC tissues and adjacent normal colorectum tissues. Unexpectedly, IGSF6 levels are highly expressed in tumor tissues as compared with adjacent normal tissues \u003cb\u003e(\u003c/b\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C\u003cb\u003e)\u003c/b\u003e. Since IGSF genes play a central role in cell-cell recognition as cell surface receptors, these results suggest that IGSF6 may be a neoantigen generated during carcinogenesis and involving in immune infiltration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTumor immune microenvironments are important factors to effect immunotherapy in clinic, but currently lock of effective biomarker to evaluate immune infiltration and the effective of immunotherapy. To evaluate IGSF6 can be used as a biomarker for immune infiltration, we assessed the association between IGSF6 expression and tumor-infiltrating lymphocytes in TCGA datasets. The results showed that the expression of IGSF6 was positive correlated with tumor-infiltrating lymphocytes in various cancers, especially for CRC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Furthermore, only IGSF6, among IGSF family genes, showed a strong positive correlation with tumor-infiltrating lymphocytes in CRC patients, including MMR-deficient and MMR-proficient tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Interestingly, a strong positive correlation between IGSF6 expression and tumor-infiltrating lymphocytes can be found in MMR-proficient tumors, indicating that IGSF6 could be an immunotherapy biomarker for MMR-proficient CRC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). What\u0026rsquo;s attractive, IGSF6 was strongly positive correlated with immune checkpoints in MMR-proficient CRC, such as PD-1, PD-L1, CTLA-4, LAG3 and TIGIT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). Moreover, expression of IGSF6 correlated with tumor-infiltrating lymphocytes were confirmed in GSE39582 dataset (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e), which supported that IGSF6 could be a novel biomarker to improve clinical applications of current immunotherapies.\u003c/p\u003e \u003cp\u003eMMR-deficient tumors which have highly tumor mutational burden (TMB), for accumulation of insertions\u0026ndash;deletions (indels) which given rise to more neoantigens, are more beneficial from immunotherapy \u003csup\u003e[\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Therefore, we evaluated the association between IGSF6 expression and TMB in CRC, and found a positive correlation between IGSF6 genes and TMB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eFurthermore, we investigated the relationship between IGSF6 expression and the tumor immune microenvironment. We calculated single-sample gene set enrichment analysis (ssGSEA) scores for patients using known CRC immune signatures. Given that MMR-deficient tumors have a distinct immunologic profile, we separated them into their own group and performed unsupervised hierarchical clustering on the MMR-proficient tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). We found high IGSF6 expression tumors were associated with extensive immune infiltration, which also be confirmed in another CRC dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eBased on the public database studies of IGSF6, we hypothesized that IGSF6 may be associated with CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell infiltration. We constructed a tissue microarray (TMA) on colon or rectal tumor tissues containing a large cohort of CRC patients (n\u0026thinsp;=\u0026thinsp;303) and detected the expression of IGSF6, CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell with multiple immunofluorescences.\u003c/p\u003e \u003cp\u003eConsistent with previous studies \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, the infiltrated CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T lymphocytes were different in various cases of CRC tissues, which was highly correlated with tumor response to treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, multiple immunofluorescences analysis indicated a strong positive correlation between IGSF6 expression and tumor-infiltrating lymphocytes, including CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell, in CRC tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n\u003ch3\u003eHigh Igsf6 Expression Is Associated Benefit From Immunotherapy And Chemotherapy\u003c/h3\u003e\n\u003cp\u003eAs IGSF6 expression was associated with immune infiltration, we next investigated whether IGSF6 could be used as a biomarker to predict therapeutic effect for CRC. A total of 6 MMR-proficient CRC patients treated with immunotherapy were studied (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After systematic ICI treatment, the patients received staged or simultaneous complete surgical resection for primary tumor. A thorough pathological examination of the resected tumors before and after ICI was conducted. Immuno-sensitive MMR-proficient CRC showed a sharp tumor burden reduction, massive necrosis tissue and lymphocytes infiltration after ICI treatment, such as Case 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eⅠ-Ⅻ\u003c/b\u003e). On the contrary, there was no response or even bad response in immuno-resistance tumors, such as Case 2 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, a-l\u003cb\u003e)\u003c/b\u003e. We next detected IGSF6 expression, CD4\u0026thinsp;+\u0026thinsp;T cell and CD8\u0026thinsp;+\u0026thinsp;T cell from pathological tumor specimens with IHC. Interestingly, high IGSF6 expression were observed in resected specimens in immuno-sensitive tumors, while with high CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cell infiltration \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. As expected, IGSF6 levels are more highly expressed in immuno-sensitive tumors than immuno-resistance tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To further clarify whether the IGSF6 could predict benefit in CRC patients, we focused on the clinical outcome in the IGSF6\u003csup\u003ehigh\u003c/sup\u003e and IGSF6\u003csup\u003elow\u003c/sup\u003e groups. The OS of CRC patients with IGSF6 high expression was better than those with IGSF6 low expression in TCGA cohorts, especially for MMR-proficient patients \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. The same tendency can also be found in GSE39582 cohorts \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient Characteristics of 6 MMR-proficient CRC patients treat with ICI.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePatient 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePatient 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePatient 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePatient 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePatient 5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePatient 6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePRIMARY TUMOR SITE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003erectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eColon\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSTAGE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT2N0M0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N0M0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT3N0M0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eT3N1M0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eT4aN1aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT3N0M0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMETASTAIC SITE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTreatment before ICI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSystematic (response \u0026para;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFOLFOX\u0026thinsp;+\u0026thinsp;radiotherapy (PR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFOLFOX (PR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFOLFOX (PD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eXELOX (PD)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ehartmann\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eright hemicolectomy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eICI combined treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegimen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003esintilimab\u0026thinsp;+\u0026thinsp;Avastin\u0026thinsp;+\u0026thinsp;FOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFOLFOX\u0026thinsp;+\u0026thinsp;sintilimab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFOLFOX\u0026thinsp;+\u0026thinsp;sintilimab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003esintilimab\u0026thinsp;+\u0026thinsp;FOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ecamrelizumab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enivolumab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSystematic (response \u0026para;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRadiological response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurgical treatment (after ICI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDixon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTaTME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDixon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDixon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePathological response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimary tumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReginal lymph nodes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePostoperative treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNONE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNONE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ecamrelizumab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enivolumab\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTRG score (NCCN Guidelines)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eξ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u0026para; Assessed by the Response Evaluation Criteria in Solid Tumors 1.1 criteria.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eξ Colectomy was conducted before ICI combined treatment.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eFOLFOX, fluorouracil\u0026thinsp;+\u0026thinsp;oxaliplatin; XELOX, capecitabine\u0026thinsp;+\u0026thinsp;oxaliplatin; ICI, immune checkpoint inhibitor; pMMR, mismatch repair (MMR) proficient; dMMR, mismatch-repair (MMR) deficient; PD, progressive disease; PR, partial response.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChemotherapy remains a relatively common and effective treatment for most MMR-proficient CRC patients. To further explore the relationship between IGSF6 and patients\u0026rsquo; response to chemotherapy, we collected the fresh tissues of chemosensitive and chemoresistance MMR-proficient CRC patients before treatment (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As expected, IGSF6 levels are more highly expressed in tumor tissues from chemosensitive patients than from chemoresistance patients (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). There were more tumor-infiltrating immune cells in tumor tissues of the IGSF6\u003csup\u003eHigh\u003c/sup\u003e group than in those of the IGSF6\u003csup\u003eLow\u003c/sup\u003e group, indicating that MMR-proficient CRC tissues with high levels of IGSF6 had a tumor microenvironment with an activated adaptive immune phenotype (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePatient Characteristics of 21 MMR-proficient CRC patients treat with chemotherapy.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTRG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003etreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSTAGE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePRIMARY TUMOR SITE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N1aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT4aN2aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N0M0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N1aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT4aN2aM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUNKNOWED\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT4aN2bM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAvastin\u0026thinsp;+\u0026thinsp;FOLFOXIRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2aM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOXIRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N1aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N1bM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2bM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOXIRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2bM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOXIRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N1bM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N1aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXELOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT4aN2bM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOXIRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N1aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N0M0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2bM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePatient 21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFOLFOXIRI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT3N2aM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRectum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eFOLFOX, fluorouracil\u0026thinsp;+\u0026thinsp;oxaliplatin; XELOX, capecitabine\u0026thinsp;+\u0026thinsp;oxaliplatin; FOLFOXIRI, fluorouracil\u0026thinsp;+\u0026thinsp;oxaliplatin\u0026thinsp;+\u0026thinsp;Irinotecan.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCRC is one of the most common cancer types in the world due to its high prevalence \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Immunotherapy has shown as an effectively therapeutic strategy for many types of cancers, but its clinical application in CRC only for MMR-deficient patients. A large amount of MMR-proficient tumors had shown a high proportion of inter-tumoral tumor-infiltrating lymphocytes as \u0026ldquo;hot tumors\u0026rdquo;, which may be sensitive to immunotherapy \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Biomarkers, including CEA, CA199, and CA125, in clinical practice are significance for tumor cell proliferation and tumor recurrence. But lock of effectively biomarker which can predict immune infiltration. Therefore, it is critical for identifying novel predictive markers for MMR-proficient tumors who may achieve benefits from immunotherapy.\u003c/p\u003e \u003cp\u003eRecent studies point out the importance of the tumor immune contexture for the prognosis of patients with CRC \u003csup\u003e[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Immunotherapy targeting immune checkpoints (such as PD1/PD-L1) has become an approved treatment option for patients with CRC with mismatch repair deficiency or high microsatellite instability \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Interestingly, about 45% of MMR-proficient and 65% of MMR-deficient CRC had a high immunoscore, while 55% of MMR-proficient and 35% of MMR-deficient CRC had a low immunoscore, suggesting that some of the MMR-deficient tumors are unable to mount an antitumor response while the reverse may apply for some of the MMR-proficient population \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Indeed, the 27% pathological response in MMR-proficient early-stage CRC treated with neoadjuvant ipilimumab plus nivolumab provide further support that MMR-proficient CRC is not an immune desert and can be targeted with immunotherapy \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Apart from MMR-deficiency and MMR-proficiency, we speculate that IGSF6 gene is another potential biomarker for responsiveness to immunotherapy due to its significant association with TMB, which is an effective indicator for response prediction to ICI. In our study, IGSF6 may be a new biomarker which may promote the expression of immune cells, such as CD8\u0026thinsp;+\u0026thinsp;T cells associated with improved survival of patients with MMR-proficient CRC \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e, contributing to a better immune function. The complex interactions between tumor and immune cells play out in the TME and dictate clinical outcomes, including among patients with CRC. No data are available on CRC, but in several tumor types an increased T cell infiltration (TCI) has been shown to increase the probability of response to ICI \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA number of inhibitory immunoreceptors have been identified and studied in cancer in past decades, including but not limited to PD-1, PD-L1, CTLA-4, LAG3, HAVCR2, TIGIT, CD69 and CD40. They are viewed as \u0026ldquo;immune checkpoints\u0026rdquo; referring to molecules that act as gatekeepers of immune responses. In the evolutionary process, immune checkpoints have co-evolved with stimulatory immunoreceptors and appear as early as in fish \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. These receptors often use monotyrosine signaling motifs, such as immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM), to deliver inhibitory signals. As surface molecules, their activity can be easily inhibited by blocking antibodies that prevent ligand-receptor engagement. The most successful immune checkpoint blockade therapy, one of anticancer immunotherapies, is anti-PD-1/PD-L1 therapy that has been approved to treat a wide variety of cancer types, such as blood, skin, lung, liver, bladder and kidney cancers \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Immune checkpoint blockade therapy often leads to more durable response than chemo or targeted therapies, perhaps reflecting the memory feature of the immune system. But as clinical data accumulates all over the world, drawbacks and side effects have begun to be revealed. The major bottleneck of immune checkpoint blockade therapy is its low response rate in most cancers, with a range of 10\u0026ndash;30% \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. For some major cancer types such as MMR-proficient CRC, anti-PD-1/PD-L1 therapy shows nearly no effect \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In this study, we identified a crucial involvement of IGSF6 in CRC promotion immune cells. IGSF6 expression was upregulated in CRC tissues and high expression of IGSF6 correlated with an active immune microenvironment and a favorable prognosis and furthermore it may facilitate immune mediated tumor killing. IGSF6 could be a new biomarker to design next generation therapies and to improve clinical protocols of current immunotherapies. But this should be validated in further study before its clinical application.\u003c/p\u003e \u003cp\u003eHere, we elucidated that IGSF6\u003csup\u003ehigh\u003c/sup\u003e expression promoted more immune cells in CRC, so as to facilitate immune mediated tumor killing. IGSF6\u003csup\u003ehigh\u003c/sup\u003e expression was an independent predictor of better prognosis, and these results may provide insight into effective strategies for therapy in MMR-proficient CRC, which enables IGSF6 to be a novel prognostic biomarker and target of immunotherapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e Not appliable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Yu-cheng Xu, Zhao-liang Yu, Xiao-chuan Chen and Min-er Zhong contributed to study concept and design, acquisition, analysis, interpretation of data and drafting of the manuscript. Yu-fan Liang and Jing-rong Weng contributed to data collections and manuscript review. Dan-dong Luo and Yi-ran Bie contributed to study concept and design, analysis and interpretation of data and critical revision of the manuscript for important intellectual content. Xi Chen, Jia-wei Cai, Yu-ming Rong and Yi-feng Zou supervised the study. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u003c/strong\u003e All authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRunning Title:\u003c/strong\u003e IGSF6 is a biomarker for immune infiltration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e This study was approved by the ethics committees of Sixth Affiliated Hospital of Sun Yat-sen University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll authors approve to publish this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e All data that supporting the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the program of Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004), National Natural Science Foundation of China (No.82203072), National Key Clinical Discipline, Natural Science Foundation of Guangdong Province, China (2020A1515010428), \u0026nbsp; Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515111201), Guangdong Basic and Applied Basic Research Foundation (SL2022A04J01718), the National Natural Science Foundation of China (82102475, MZ), Guangdong Basic and Applied Basic Research Foundation (2020A1515110489, MZ) and the Fundamental Research Funds for the Central Universities, Sun Yat-sen University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFranzoi M A, Romano E, Piccart M. 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Science, 2018, 359(6382): 1350\u0026ndash;1355.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"IGSF6, Immunotherapy, Colorectal cancer, Biomarker, Immune checkpoint","lastPublishedDoi":"10.21203/rs.3.rs-2194686/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2194686/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eImmunotherapy has dramatically changed the landscape of treatment for colorectal cancer (CRC), but there is lack of effective predictive biomarker, especially for tumors with mismatch repair (MMR) proficiency. Immune response relies to cell surface receptors and their interactions, such as cell-cell recognition, binding and adhesion. However, the function of immunoglobulin superfamily (IGSF) genes in tumor immune microenvironment remains uncharacterized.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study quantified the immune using the gene expression matrix obtained from the public database. Also the associations between IGSF6 gene expression and immune cell infiltration were assessed. The expression levels of IGSF6, CD8\u0026thinsp;+\u0026thinsp;and CD4\u0026thinsp;+\u0026thinsp;T cells in cancer tissues from CRC patients were evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIGSF6 was more highly expressed in CRC tumor tissues than corresponding adjacent normal tissues. And IGSF6 was significantly correlated with immune cell infiltration in MMR-proficient patients. Remarkably, MMR-proficient patients with high IGSF6 expression showed more sensitive to immunotherapy and chemotherapy than those with low IGSF6 expression.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, IGSF6 could be a novel biomarker to evaluate immune infiltration and predict therapeutic effect for MMR-proficient CRC.\u003c/p\u003e","manuscriptTitle":"IGSF6 is a novel biomarker to evaluate immune infiltration in mismatch repair-proficient colorectal cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2023-02-14 15:16:25","doi":"10.21203/rs.3.rs-2194686/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2022-10-27 15:00:40","doi":"10.21203/rs.3.rs-2194686/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6606ae5d-21d5-4b3a-9625-0316ed6044c0","owner":[],"postedDate":"February 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-24T18:44:23+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-14 15:16:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-2194686","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2194686","identity":"rs-2194686","version":["v2"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}