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

doi:10.21203/rs.3.rs-2194686/v2

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

https://doi.org/10.21203/rs.3.rs-2194686/v2

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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

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.

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.

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. 1A). 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. 1B-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. 1D). 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. 1E). 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. 1F). 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. 1G-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. 1I).

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. 1J). We found high IGSF6 expression tumors were associated with extensive immune infiltration, which also be confirmed in another CRC dataset (Fig. 1K).

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. 2A).

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. 2B).

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. 3A, Ⅰ-Ⅻ). On the contrary, there was no response or even bad response in immuno-resistance tumors, such as Case 2 (Fig. 3A, 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. 3A). As expected, IGSF6 levels are more highly expressed in immuno-sensitive tumors than immuno-resistance tumors (Fig. 3B). 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. 3C). The same tendency can also be found in GSE39582 cohorts (Fig. 3D).

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. 4A-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. 4A-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.

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.

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.

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No competing interests reported.

SupplymentaryFigure1.tif
Supplementary figure 1. (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.

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

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials And Methods

Study design and patient selection

Immunofluorescence

Immunohistochemistry Staining

Evaluation Of Immunohistochemical And Immunofluorescence Analysis

Statistical analysis

Results

High Igsf6 Expression Is Associated Benefit From Immunotherapy And Chemotherapy

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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