Immune Microenvironment In Colorectal Cancer Research Articles

Early administration of Wumei Wan inhibit myeloid-derived suppressor cells via PI3K/Akt pathway and amino acids metabolism to prevent Colitis-associated Colorectal Cancer

Ethnopharmacological relevanceWumei Wan (WMW), a traditional Chinese medicine prescription, has been proved to be effective in treating Colitis-associated colorectal cancer (CAC), but it has not been proven to be effective in different stages of CAC. Aim of the studyThe purpose of our study is to investigate the therapeutic effect and mechanism of WMW on the progression of CAC. Materials and methodsAzioximethane (AOM) and dextran sulfate sodium (DSS) were used to treat mice for the purpose of establishing CAC models. WMW was administered in different stages of CAC. The presentative chemical components in WMW were confirmed by UHPLCQTOF/MS under the optimized conditions. The detection of inflammatory cytokines in the serum and colon of mice were estimated by qRT-PCR and ELISA. The changes of T cells and myeloid-derived suppressor cells (MDSCs) in each group were detected by flow cytometry. The metabolic components in serum of mice were detected by UPLC-MS/MS. Expression of genes and proteins were detected by eukaryotic transcriptomics and western blot to explore the key pathway of WMW in preventing CAC. ResultsWMW had significant effect on inhibiting inflammatory responses and tumors during the early development stage of CAC when compared to other times. WMW increased the length of mice's colons, reduced the level of IL-1β, IL-6, TNF-α in colon tissues, and effectively alleviated colonic inflammation, and improved the pathological damage of colon tissues. WMW could significantly reduce the infiltration of MDSCs in the spleen, increase CD4+ T cells and CD8+ T cells in the spleen of CAC mice, and effectively reform the immune microenvironment in CAC mice.Transcriptomics analysis revealed that 2204 genes had different patterns of overlap in the colon tissues of mice between control group, AOM+DSS group, and early administration of WMW group. And KEGG enrichment analysis showed that PI3K/Akt signaling pathway, ECM-receptor interaction, IL-17 signaling pathway, MAPK signaling pathway, pancreatic secretion, thermogenesis, and Rap1 signaling pathway were all involved.The serum metabolomics results of WMW showed that the metabolic compositions of the control group, AOM+DSS group and the early stage of WMW were different, and 42 differential metabolites with the opposite trends of changes were screened. The metabolic pathways mainly included pyrimidine metabolism, glycine, serine and threonine metabolism, tryptophan metabolism, and purine metabolism. And amino acids and related metabolites may play an important role in WMW prevention of CAC. ConclusionWMW can effectively prevent the occurrence and development of CAC, especially in the initial stage. WMW can reduce the immune infiltration of MDSCs in the early stage. Early intervention of WMW can improve the metabolic disorder caused by AOM+DSS, especially correct the amino acid metabolism. PI3K/Akt signaling pathway was inhabited in early administration of WMW, which can regulate the amplification and function of MDSCs.

Abstract 1519: Effect of obesity on CD4+ T cells in the tumor immune microenvironment of colorectal cancer

Abstract Background: The number of new cases and deaths from colorectal cancer (CRC) is increasing rapidly, and obesity is considered one of the risk factors. Obesity-associated cancers are related to the overproduction of hormones, induction of reactive oxygen species (ROS) production by free fatty acids, and changes in the gut microbiome. Obesity also promotes immune cell dysfunction in the tumor immune microenvironment (TIME). The increase of fatty acid levels due to obesity causes metabolic reprogramming and decreases the cytotoxic activity of natural killer (NK) cells and CD8+ T cells, which accelerate tumor progression. However, the impact of obesity on CD4+ T cells in the TIME remains unclear. Therefore, this study aimed to clarify how obesity affected the number and the anti-tumor activity of CD4+ T cells in CRC using mouse models. Methods: Seven-week-old male C57BL/6J mice were randomly divided into two groups and fed the control diet (CD) or high-fat diet (HFD) for nine weeks. Blood samples were collected from the buccal vein of the mice every four weeks after feeding for analysis. MC38 CRC cells of 4 × 106 were subcutaneously inoculated in the middle of the backs of mice to analyze tumor progression. Three weeks after tumor cell inoculation, the mice were sacrificed, and the immune cells of the tumor, blood, and various organs were analyzed. Results: After nine weeks of feeding, the HFD-fed mice had a significantly increased body weight compared to CD-fed mice. Flow cytometry results revealed that the number and frequency of CD4+ T cells in the blood were significantly reduced in HFD-fed mice compared to those in CD-fed mice, and the expression of programmed death-1 (PD-1) in CD4+ T cells in HFD-fed mice was significantly higher than that in CD-fed mice at eight weeks. Three weeks after tumor inoculation, HFD feeding accelerated tumor growth and decreased the survival rate. The number of CD4+ T cells in the tumors was significantly reduced in HFD-fed mice compared to that in CD-fed mice (55.9 vs 18.2 cells/mg; 95% CI 14.9-60.3 cells/mg; P = 0.006). The number of CD8+ T cells in the tumors was also reduced, which was more pronounced for CD4+ T cells than for CD8+ T cells. In addition, the production of cytokines such as IFN-γ and TNF-α was significantly reduced in CD4+ T cells and CD8+ T cells of HFD-fed mice. In a depletion analysis of CD4+ T cells, the feeding-dependent acceleration of tumor growth was not observed in each group. This result indicated that CD4+ T cells were involved in accelerating tumor growth due to HFD-induced obesity. Conclusion: We showed that the reduced number and dysfunction of CD4+ T cells due to obesity led to a decreased anti-tumor response of both CD4+ and CD8+ T cells, ultimately accelerating the progression of colorectal cancer. Our findings may elucidate the pathogenesis for poor outcomes of colorectal cancer associated with obesity. Citation Format: Masayuki Ando, Kota Yamada, Masafumi Saito, Kimihiro Yamashita, Takao Tsuneki, Yuri Adachi, Tomoki Abe, Tomosuke Mukoyama, Ryuichiro Sawada, Yasufumi Koterazawa, Hitoshi Harada, Naoki Urakawa, Hironobu Goto, Hiroshi Hasegawa, Shingo Kanaji, Takeru Matsuda, Yoshihiro Kakeji. Effect of obesity on CD4+ T cells in the tumor immune microenvironment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1519.

Exosomes in tumor-stroma crosstalk: Shaping the immune microenvironment in colorectal cancer.

Colorectal cancer (CRC) is a multifaceted disease characterized by a complex interaction between tumor cells and the surrounding microenvironment. Within this intricate landscape, exosomes have emerged as pivotal players in the tumor-stroma crosstalk, influencing the immune microenvironment of CRC. These nano-sized vesicles, secreted by both tumoral and stromal cells, serve as molecular transporters, delivering a heterogeneous mix of biomolecules such as RNAs, proteins, and lipids. In the CRC context, exosomes exert dual roles: they promote tumor growth, metastasis, and immune escape by altering immune cell functions and activating oncogenic signaling pathways and offer potential as biomarkers for early CRC detection and treatment targets. This review delves into the multifunctional roles of exosomes in the CRC immune microenvironment, highlighting their potential implications for future therapeutic strategies and clinical outcomes.

Prognostic and predictive role of immune microenvironment in colorectal cancer.

Colorectal cancer (CRC) represents a molecularly heterogeneous disease and one of the most frequent causes of cancer-related death worldwide. The traditional classification of CRC is based on pathomorphological and molecular characteristics of tumor cells (mucinous, ring-cell carcinomas, etc.), analysis of mechanisms of carcinogenesis involved (chromosomal instability, microsatellite instability, CpG island methylator phenotype) and mutational statuses of commonly altered genes (KRAS, NRAS, BRAF, APC, etc.), as well as expression signatures (CMS 1-4). It is also suggested that the tumor microenvironment is a key player in tumor progression and metastasis in CRC. According to the latest data, the immune microenvironment can also be predictive of the response to immune checkpoint inhibitors. In this review, we highlight how the immune environment influences CRC prognosis and sensitivity to systemic therapy.

Comparative analysis of single-cell transcriptome reveals heterogeneity and commonality in the immune microenvironment of colorectal cancer and inflammatory bowel disease.

During aging, chronic inflammation can promote tumor development and metastasis. Patients with chronic inflammatory bowel diseases (IBD) are at an increased risk of developing colorectal cancer (CRC). However, the molecular mechanism underlying is still unclear. We conducted a large-scale single-cell sequencing analysis comprising 432,314 single cells from 92 CRC and 24 IBD patients. The analysis focused on the heterogeneity and commonality of CRC and IBD with respect to immune cell landscape, cellular communication, aging and inflammatory response, and Meta programs. The CRC and IBD had significantly different propensities in terms of cell proportions, differential genes and their functions, and cellular communication. The progression of CRC was mainly associated with epithelial cells, fibroblasts, and monocyte-macrophages, which displayed pronounced metabolic functions. In particular, monocyte-macrophages were enriched for the aging and inflammation-associated NF-κB pathway. And IBD was enriched in immune-related functions with B cells and T cells. Cellular communication analysis in CRC samples displayed an increase in MIF signaling from epithelial cells to T cells, and an increase in the efferent signal of senescence-associated SPP1 signaling from monocyte-macrophages. Notably, we also found some commonalities between CRC and IBD. The efferent and afferent signals showed that the pro-inflammatory cytokine played an important role. And the activity of aging and inflammatory response with AUCell analysis also showed a high degree of commonality. Furthermore, using the Meta programs (MPs) with the NMF algorithm, we found that the CRC non-malignant cells shared a substantial proportion of the MP genes with CRC malignant cells (68% overlap) and IBD epithelial cells (52% overlap), respectively. And it was extensively involved in functions of cell cycle and immune response, revealing its dual properties of inflammation and cancer. In addition, CRC malignant and non-malignant cells were enriched for the senescence-related cell cycle G2M phase transition and the p53 signaling pathway. Our study highlights the characteristics of aging, inflammation and tumor in CRC and IBD at the single-cell level, and the dual property of inflammation-cancer in CRC non-malignant cells may provide a more up-to-date understanding of disease transformation.

Centrosomal Protein 55 (CEP55) Drives Immune Exclusion and Resistance to Immune Checkpoint Inhibitors in Colorectal Cancer.

Colorectal cancer (CRC) currently ranks as the third most common cancer in the United States, and its incidence is on the rise, especially among younger individuals. Despite the remarkable success of immune checkpoint inhibitors (ICIs) in various cancers, most CRC patients fail to respond due to intrinsic resistance mechanisms. While microsatellite instability-high phenotypes serve as a reliable positive predictive biomarker for ICI treatment, the majority of CRC patients with microsatellite-stable (MSS) tumors remain ineligible for this therapeutic approach. In this study, we investigated the role of centrosomal protein 55 (CEP55) in shaping the tumor immune microenvironment in CRC. CEP55 is overexpressed in multiple cancer types and was shown to promote tumorigenesis by upregulating the PI3K/AKT pathway. Our data revealed that elevated CEP55 expression in CRC was associated with reduced T cell infiltration, contributing to immune exclusion. As CRC tumors progressed, CEP55 expression increased alongside sequential mutations in crucial driver genes (APC, KRAS, TP53, and SMAD4), indicating its involvement in tumor progression. CEP55 knockout significantly impaired tumor growth in vitro and in vivo, suggesting that CEP55 plays a crucial role in tumorigenesis. Furthermore, the CEP55 knockout increased CD8+ T cell infiltration and granzyme B production, indicating improved anti-tumor immunity. Additionally, we observed reduced regulatory T cell infiltration in CEP55 knockout tumors, suggesting diminished immune suppression. Most significantly, CEP55 knockout tumors demonstrated enhanced responsiveness to immune checkpoint inhibition in a clinically relevant orthotopic CRC model. Treatment with anti-PD1 significantly reduced tumor growth in CEP55 knockout tumors compared to control tumors, suggesting that inhibiting CEP55 could improve the efficacy of ICIs. Collectively, our study underscores the crucial role of CEP55 in driving immune exclusion and resistance to ICIs in CRC. Targeting CEP55 emerges as a promising therapeutic strategy to sensitize CRC to immune checkpoint inhibition, thereby improving survival outcomes for CRC patients.

Prolyl isomerase Pin1 sculpts the immune microenvironment of colorectal cancer

Pin1, a peptide prolyl cis-trans isomerase, is overexpressed and/or overactivated in many human malignancies. However, whether Pin1 regulates the immunosuppressive TME has not been well defined. In this study, we detected the effect of Pin1 on immune cells and immune checkpoint PD-L1 in the TME of CRC and explored the anti-tumor efficacy of Pin1 inhibitor ATRA combined with PD-1 antibody. We found that Pin1 facilitated the immunosuppressive TME by raising the proportion of myeloid-derived suppressor cells (MDSCs) and declining the percentage of CD8+ T cells and CD4+ T cells. Pin1 restrained PD-L1 protein expression in CRC cells and the effect was tempered by endoplasmic reticulum (ER) stress inducers. Mechanically, Pin1 overexpression decreased the stability of PD-L1 and promoted its degradation by mitigating ER stress. Silencing or inhibiting Pin1 promoted PD-L1 protein expression by inducing ER stress. Hence, Pin1 inhibitor ATRA enhanced the anti-tumor efficacy of PD-1 antibody in the CRC allograft by upregulating PD-L1. Our results reveal the critical and pleiotropic effects of Pin1 on managing the immune cells and immune checkpoint PD-L1 in the TME of CRC, providing a new promising candidate for combination with immunotherapy.

The impact of SLC10A3 on prognosis and immune microenvironment in colorectal adenocarcinoma

BackgroundSLC10A3, a gene upregulated in pan-cancer, lacks full understanding regarding its prognostic implications and association with immune infiltration in colorectal cancer (CRC). This study comprehensively analyzed SLC10A3 in CRC, evaluating its prognostic significance and influence on the tumor's immune microenvironment.MethodsTranscriptomic data from TCGA were obtained to compare SLC10A3 expression in both colorectal cancer (CRC) and normal tissues. Prognostic value was assessed for overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI). DNA methylation patterns of SLC10A3 and correlation with DNA mismatch repair (MMR) were explored. Genetic alterations in SLC10A3 were scrutinized. The study also delved into the influence of SLC10A3 on the immune microenvironment of CRC, including immune cell infiltration and chemokines. Involvement of cancer-associated fibroblasts (CAFs) was explored. Methylation status of specific CpG islands in the SLC10A3 gene correlated with CRC patient prognosis. CRC tissue microarray was performed to verify the expression of SLC10A3 and its relationship with prognosis.ResultsThe research revealed that SLC10A3 is significantly upregulated in CRC and holds promise as a potential diagnostic marker. Elevated SLC10A3 expression was linked to poorer OS, DSS, and PFI. Methylation patterns of SLC10A3 displayed prognostic relevance, and genetic alterations in the gene were identified. SLC10A3 was shown to impact the immune microenvironment, with significant correlations observed between its expression and various immune cell types, chemokines, and markers associated with CAFs. Furthermore, an inverse relationship between SLC10A3 and MMR molecules was established. Methylation status of specific CpG islands within the SLC10A3 gene was associated with CRC patient prognosis. Tissue microarray showed that SLC10A3 was highly expressed in CRC and significantly correlated with poor prognosis.ConclusionThe study underscores the importance of elevated SLC10A3 in CRC, associating it with decreased survival and immune infiltration, proposing it as a diagnostic biomarker and appealing immunotherapy target, given its significant overexpression and influence on the immune microenvironment and prognosis through methylation patterns.Graphical

MYL9 expressed in cancer-associated fibroblasts regulate the immune microenvironment of colorectal cancer and promotes tumor progression in an autocrine manner

BackgroundThe tumor microenvironment (TME) is an important factor that regulates the progression of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) are the main mesenchymal cells in the TME and play a vital role in tumor progression; however, the specific underlying mechanisms require further study.MethodsMultiple single-cell and transcriptome data were analyzed and validated. Primary CAFs isolation, CCK8 assay, co-culture assay, western blotting, multiple immunofluorescence, qRT-PCR, ELISA, immunoprecipitation, ChIP, double luciferase, and animal experiments were used to explore the potential mechanism of MYL9 regulation in CRC.ResultsOur findings revealed that MYL9 was predominantly localized and expressed in CAFs rather than in CRC cells, and bioinformatics analysis revealed that high MYL9 expression was strongly associated with poor overall and disease-free survival in various tumors. In addition, high MYL9 expression is closely associated with M2 macrophage infiltration, which can lead to an immunosuppressive microenvironment in CRC, making it insensitive to immunotherapy. Mechanically, MYL9 can regulate the secretion of CAFs on CCL2 and TGF-β1, thus affecting the immune microenvironment and progression of CRC. In addition, MYL9 bounded with IQGAP1 to regulate CCL2 and TGF-β1 secretion through the ERK 1/2 pathway, and CCL2 and TGF-β1 synergistically promoted CRC cells progression through the PI3K-AKT pathway. Furthermore, MYL9 promotes epithelial-mesenchymal transition (EMT) in CRC. During the upstream regulation of MYL9 in CAFs, we found that the EMT transcription factor ZEB1 could bind to the MYL9 promoter in CAFs, enhancing the activity and function of MYL9. Therefore, MYL9 is predominantly expressed in CAFs and can indirectly influence tumor biology and EMT by affecting CAFs protein expression in CRC.ConclusionsMYL9 regulates the secretion of cytokines and chemokines in CAFs, which can affect the immune microenvironment of CRC and promote CRC progression. The relationship between MYL9 expression and CRC clinical staging and immunotherapy is closer in CAFs than in tumor cells; therefore, studies using CAFs as a model deserve more attention when exploring tumor molecular targets in clinical research.

Exploring the relationship between lactate metabolism and immunological function in colorectal cancer through genes identification and analysis.

Introduction: Metabolic dysregulation is a widely acknowledged contributor for the development and tumorigenesis of colorectal cancer (CRC), highlighting the need for reliable prognostic biomarkers in this malignancy. Methods: Herein, we identified key genes relevant to CRC metabolism through a comprehensive analysis of lactate metabolism-related genes from GSEA MsigDB, employing univariate Cox regression analysis and random forest algorithms. Clinical prognostic analysis was performed following identification of three key genes, and consistent clustering enabled the classification of public datasets into three patterns with significant prognostic differences. The molecular pathways and tumor microenvironment (TME) of these patterns were then investigated through correlation analyses. Quantitative PCR was employed to quantify the mRNA expression levels of the three pivotal genes in CRC tissue. Single-cell RNA sequencing data and fluorescent multiplex immunohistochemistry were utilized to analyze relevant T cells and validate the correlation between key genes and CD4+ T cells. Results: Our analysis revealed that MPC1, COQ2, and ADAMTS13 significantly stratify the cohort into three patterns with distinct prognoses. Additionally, the immune infiltration and molecular pathways were significantly different for each pattern. Among the key genes, MPC1 and COQ2 were positively associated with good prognosis, whereas ADAMTS13 was negatively associated with good prognosis. Single-cell RNA sequencing (scRNA-seq) data illustrated that the relationship between three key genes and T cells, which was further confirmed by the results of fluorescent multiplex immunohistochemistry demonstrating a positive correlation between MPC1 and COQ2 with CD4+ T cells and a negative correlation between ADAMTS13 and CD4+ T cells. Discussion: These findings suggest that the three key lactate metabolism genes, MPC1, COQ2, and ADAMTS13, may serve as effective prognostic biomarkers and support the link between lactate metabolism and the immune microenvironment in CRC.

Bifidobacterium adolescentis induces Decorin+ macrophages via TLR2 to suppress colorectal carcinogenesis

BackgroundThe interplay between gut microbiota and tumor microenvironment (TME) in the pathogenesis of colorectal cancer (CRC) is largely unknown. Here, we elucidated the functional role of B. adolescentis and its possible mechanism on the manipulation of Decorin+ macrophages in colorectal cancer.MethodsThe relative abundance of B. adolescentis in tumor or para-tumor tissue of CRC patients was analyzed. The role of B. adolescentis was explored in the CRC animal models. The single cell-RNA sequencing (scRNA-seq) was used to investigate the myeloid cells subsets in TME. The expression level of TLR2/YAP axis and its downstream Decorin in macrophages were tested by Western blot and qRT-PCR. Knockdown of Decorin in Raw264.7 was performed to investigate the effect of Decorin+ macrophages on subcutaneous tumor formation. Multi-immunofluorescence assay examined the number of Decorin+ macrophages on the CRC tissue.ResultsWe found that the abundance of B. adolescentis was significantly reduced in tumor tissue of CRC patients. Supplementation with B. adolescentis suppressed AOM/DSS-induced tumorigenesis in mice. ScRNA-seq and animal experiment revealed that B. adolescentis increased Decorin+ macrophages. Mechanically, Decorin was activated by TLR2/YAP axis in macrophages. The abundance of B. adolescentis was correlated with the number of Decorin+ macrophages and the expression level of TLR2 in tumor tissue of CRC patients.ConclusionsThese results highlight that B. adolescentis induced Decorin+ macrophages and provide a novel therapeutic target for probiotic-based modulation of immune microenvironment in CRC.

A novel anoikis-related gene prognostic signature and its correlation with the immune microenvironment in colorectal cancer.

Background: Anoikis is a type of apoptosis associated with cell detachment. Resistance to anoikis is a focal point of tumor metastasis. This study aimed to explore the relationship among anoikis-related genes (ARGs), immune infiltration, and prognosis in colorectal cancer (CRC). Methods: The transcriptome profile and clinical data on patients with CRC were retrieved from The Cancer Genome Atlas and Gene Expression Omnibus databases. Patients were divided into two clusters based on the expression of ARGs. Differences between the two ARG molecular subtypes were analyzed in terms of prognosis, functional enrichment, gene mutation frequency, and immune cell infiltration. An ARG-related prognostic signature for predicting overall survival in patients with CRC was developed and validated using absolute value convergence and selection operator (LASSO) regression analysis. The correlation between the signature risk score and clinicopathological features, immune cell infiltration, immune typing, and immunotherapy response was analyzed. The risk score combined with clinicopathological characteristics was used to construct a nomogram to assess CRC patients' prognosis. Results: Overall, 151 ARGs were differentially expressed in CRC. Two ARG subtypes, namely, ARG-high and ARG-low groups, were identified and correlated with CRC prognosis. The gene mutation frequency and immune, stromal, and ESTIMATE scores of the ARG-high group were higher than those of the ARG-low group. Moreover, CD8, natural killer cells, M1 macrophages, human leukocyte antigen (HLA), and immune checkpoint-related genes were significantly increased in the ARG-high group. An optimized 25-gene CRC prognostic signature was successfully constructed, and its prognostic predictive ability was validated. The high-risk score was correlated with T, N, M, and TNM stages. Risk scores were negatively correlated with dendritic cells, eosinophils, and CD4 cells, and significantly positively correlated with regulatory T cells. Patients in the high-risk group were more likely to exhibit immune unresponsiveness. Finally, the nomogram model was constructed and showed good prognostic predictive power. Conclusion: ARGs are associated with clinicopathological features and the prognosis of CRC, and play important roles in the immune microenvironment. Herein, we underpinned the usefulness of ARGs in CRC to develop more effective immunotherapy techniques.

Abstract 1268: The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus

Abstract Background: Although metformin (Met) is well known to suppress tumor growth, the detailed mechanisms of its anti-tumor effect remain unknown. In recent years, it has been pointed out that tumor-associated neutrophils (TAN) can produce neutrophil extracellular traps (NETs) in the tumor microenvironment (TME) which can tumor invasion and metastasis. Here, in this study, we investigated the effect of Met on tumor-associated NETs and its effect on the prognosis of cancer patients. Method: We retrospectively examined the outcome of colorectal cancer (CRC) patients with type 2 diabetes mellitus (T2DM) who received curative surgery in Jichi Medical University Hospital and asked the impact of Met intake on their outcome. In addition, we performed multicolor immunohistochemistry to examine the phenotypes of tumor-infiltrating lymphocytes (TIL), TAN and NETs in surgically removed tissues specimens in 40 (colorectal cancer) CRC patients who had taken Met before surgery and propensity score-matched 40 patients who had not. Results: A total of 1858 patients underwent curative colectomy from April 2010 to March 2021. Among them, 283 patients had T2DM at the time of surgery, and 62 patients had been treated with drugs including Met. The postoperative disease-free survival rate (DFS) was significantly improved in the Met intake group compared with non-intake group (HR=0.21, p<0.01). The number of CD66b(+) TANs significantly decreased in Met-intake group (Median(M)=169 (36-553)/mm2 vs 99 (34-322)/mm2, p<0.01). The number of tumor-associated NETs defined as CD66b(+) cit-H3(+) cells significantly decreased in Met-intake group (M=36 (8-272)/mm2 vs 20 (0-104)/mm2, p<0.01), and tumor-associated NETs/TANs ratio significantly decreased in Met-intake group (29.9% vs 19.8%, p<0.01). The number of CD3(+) TILs significantly increased in Met intake group (M=122 (28-257)/mm2 vs 142 (48-386)/mm2, p<0.05). The number of CD8(+) TILs significantly increased in Met-intake group (M=60 (14-180)/mm2 vs 99 (52-318)/mm2, p<0.01), and CD8/CD3 ratio significantly increased in Met-intake group (51.2% vs 72.1%, p<0.01). In addition, there was a negative correlation between TANs or tumor-associated NETs and CD8 positive TILs in all 80 patients (r=-0.26, p<0.05). Conclusion: Met can inhibit neutrophil infiltration and NETs formation while promote the infiltration of cytotoxic T cells in TME, which may lead to the improvement of the outcome of the patients with CRC. Citation Format: Akira Saito, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Yuki Kimura, Rie Kawashima, Hideyo Miyato, Joji Kitayama, Naohiro Sata. The effect of metformin for neutrophil extracellular traps (NETs) on tumor immune microenvironment of colorectal cancer with type 2 diabetes mellitus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1268.

Abstract 2242: KRAS mutations suppress the tumor immune microenvironment in colorectal cancer

Abstract Background: KRAS mutations (KRASmut) drive tumorigenesis through various signaling pathways that are associated with worse survival in colorectal cancer patients (CRC). Immune evasion is a hallmark of KRAS-driven cancer, but the role of KRASmut in the regulation of the immune response is unclear, particularly in the tumor microenvironment (TME), which often assists cancer cells to thrive and successfully escape immune surveillance. Here, we explore the immunobiological impacts of KRASmut in CRC patients. Methods: A total of 97 fresh frozen tumor samples were collected from each CRC patient with stage I-IV who underwent surgical treatment at the Department of Surgery, Faculty of Medicine Siriraj Hospital between October 2010 and March 2011. First, all patients were identified KRAS mutational status and compared overall survival (OS) with wild-type KRAS (KRASwt). Next, we analyzed the gene expression profiling of KRASmut which was performed using the Nanostring platform. Finally, we performed the biological network analysis focusing on immunological pathways associated with KRASmut using the Ingenuity Pathway Analysis (IPA). Results: We observed KRASmut in 41.24% (n=40) of the total cases. Mutations occurred primarily in exon 2 (total n=36; codon 12, n=28; codon 13, n=8) and rarely in exon 3 (n=4). Patients with KRASmut CRC in exon 2 and exon 3, showed significantly worse OS compared to KRASwt (HR 1.77, 95% CI 1.07-2.93; P<0.05). Comparing gene expression profiles between KRASmut and KRASwt, found that the survival outcome of patients with KRASmut was correlated with immunosuppressive TME by activation of genes encoded in TGFβ signaling pathway. In addition to activating TGFβ pathway, the gene expression profile of KRASmut also interrupted gene encoded pro-inflammatory cytokines, including TNFRSF12, IRF6, CCR2, CCL8, CCL11, CXCR3 and CXCL10. IPA analysis revealed that differentially expressed genes of KRASmut were significantly enriched in regulation of epithelial mesenchymal transition (EMT) and macrophage-stimulating protein receptor d'origine nantais (MSP-RON) signaling pathways (P value<0.001), which are strongly associated with maintaining the stability of the TME and contributing to immune escape in the immune TME. Conclusions: Our results suggested that KRASmut activated TGFβ and interrupted pro-inflammatory cytokine to suppress tumor immune microenvironment in CRC. Future studies and clinical trials in patients with CRC with KRASmut should take these transcriptional profiles into account. Citation Format: Pariyada Tanjak, Amphun Chaiboonchoe, Tharathorn Suwatthanarak, Onchira Acharayothin, Kullanist Thanormjit, Jantappapa Chanthercrob, Thanawat Suwatthanarak, Bundit Wannasuphaphol, Kemmapon Chumchuen, Bhoom Suktitipat, Somponnat Sampattavanich, Krittiya Korphaisarn, Ananya Pongpaibul, Naravat Poungvarin, Harald Grove, Woramin Riansuwan, Atthaphorn Trakarnsanga, Asada Methasate, Manop Pithukpakorn, Vitoon Chinswangwatanakul. KRAS mutations suppress the tumor immune microenvironment in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2242.

Identification of angiogenesis-related subtypes, the development of prognostic models, and the landscape of tumor microenvironment infiltration in colorectal cancer.

Background: Angiogenesis is one of the most prominent markers of cancer progression and contributes to tumor metastasis and prognosis. Anti-angiogenic drugs have proven effective in treating metastatic colorectal cancer. However, there is some uncertainty regarding the potential role of angiogenesis-related genes in the tumor microenvironment. Methods: We analyzed 1,214 colorectal cancer samples to identify alterations in angiogenesis-related genes (ARGs), and then correlated angiogenesis with clinical features, prognosis, and TME. The ARGs expression profiles in colorectal cancer were analyzed using three computational methods (CIBERSORT, ssGSEA, and MCPcounter) and provided a systematic immune landscape. Patients with CRC were classified into two subtypes based on consensus clustering analysis of angiogenesis-related genes. The revealed differentially expressed genes between the two subtypes were used to create and validate ARGscore prognostic models. In addition, we collected eight colorectal cancer patient specimens and performed RT-qPCR to validate the signature gene expression. Results: We assessed the expression patterns of ARGs in colorectal cancer. We identified two molecular subtypes and confirmed that the expression of ARGs was associated with prognosis and TME characteristics. Based on differentially expressed genes between subtypes, we constructed ARGscore and evaluated their predictive power for the survival of colorectal cancer patients. We also developed an accurate nomogram to make the ARGscore more clinically useful. In addition, ARGscore was significantly correlated with microsatellite instability, cancer stem cells, and chemotherapeutic drug sensitivity. Patients with ARGscore-low characterized by immune activation and microsatellite instability high had a better prognosis. Conclusion: ARGs expression influenced the prognosis, clinicopathological features, and tumor stromal immune microenvironment in colorectal cancer. We developed a new risk model, ARGscore, for the treatment and prognosis of CRC patients and validated its promising predictive power. These findings will enable us to understand colorectal cancer better, assess prognoses, and develop more effective treatment options.

Periostin deficiency reduces PD-1+ tumor-associated macrophage infiltration and enhances anti-PD-1 efficacy in colorectal cancer.

Periostin, a multifunctional extracellular protein, plays an important role in inflammatory disorders and tumorigenesis. Our previous work has demonstrated that periostin deficiency inhibits colorectal cancer (CRC) progression. Here, we aim to clarify the role of periostin in the immune microenvironment of CRC. We find that periostin deficiency significantly decreases the infiltration of programmed death receptor 1 (PD-1)+ tumor-associated macrophages (TAMs) in CRC tissues. Periostin promotes the expression of PD-1 on TAMs by integrin-ILK-nuclear factor κB (NF-κB) signaling, and PD-1+ TAMs produce interleukin-6 (IL-6) and interferon γ (IFN-γ) to induce the expression of PD-L1 on colorectal tumor cells. Moreover, combined inhibition of periostin and PD-1 significantly suppresses CRC progression compared with the inhibition of periostin or PD-1 alone. In summary, our results suggest that periostin deficiency reduces the infiltration of PD-1+ TAMs and enhances the efficacy of anti-PD-1 treatment in CRC.

High endothelial venules proportion in tertiary lymphoid structure is a prognostic marker and correlated with anti-tumor immune microenvironment in colorectal cancer

Background High endothelial venules (HEV) and tertiary lymphoid structures (TLS) are associated with clinical outcomes of patients with colorectal cancer (CRC). However, because HEV are components of TLS, there have been few studies of the role of the HEV proportion in TLS (HEV/TLS). This study investigated the role of the HEV/TLS and its relationship with the tumor immune microenvironment in CRC. Methods A retrospective analysis of 203 cases of tissue pathologically diagnosed as CRC after general surgery was performed at the First Affiliated Hospital of Jinan University from January 2014 to July 2017. Paraffin sections were obtained from the paracancerous intestinal mucosal tissues. The area of HEV and TLS and immune cells were detected by immunohistochemistry. We further divided the positive HEV expression group into the high HEV/TLS group and the low HEV/TLS group by the average area of HEV/TLS. After grouping, the data were also analyzed using the chi-square test, Kaplan-Meier method, and univariate and multivariate Cox proportional risk regression analyses. A correlation analysis of the HEV/TLS and immune cells as well as angiogenesis was performed. Results Patients with a high HEV/TLS in CRC tissue were associated with longer OS, DFS and lower TNM stage. Meanwhile, CRC tissue with a high HEV/TLS showed a greater ability to recruit the CD3+ T cells, CD8+ T cells and M1 macrophages and correlated with less angiogenesis. Conclusively, high HEV/TLS links to the favorable prognosis of CRC patients and correlated with anti-tumor immune microenvironment, which can be a potential biomarker for prognosis of CRC patients. Conclusion A high HEV/TLS is associated with a favorable prognosis for CRC and is correlated with the anti-tumor immune microenvironment. Therefore, it is a potential biomarker of the CRC prognosis. KEY MESSAGES High HEV/TLS is associated with a favorable prognosis for CRC. High HEV/TLS correlated with the anti-tumor immune microenvironment of CRC and can serve as a novel prognostic biomarker.

Efficacy of zinc carnosine in the treatment of colorectal cancer and its potential in combination with immunotherapy in vivo.

A complex of Zn and carnosine, called Zinc-L-carnosine (ZnC), enjoys a wide application as part of a Zn supplement therapeutic method as well as in treating peptic ulcers. However, researches fail to confirm the biological functions possessed by ZnC as well as tumor immune microenvironment in colorectal cancer (CRC). Cell counting kit 8(CCK8), 5-ethynyl-2'-deoxyuridine (EdU), transwell and wound healing assays were conducted to study the influence of ZnC in the proliferating, invading and migrating processes of CRC cell lines (HCT116, LOVO) in vitro. The antitumor activity ZnC as well as its effects on tumor immune microenvironment were then assessed using CRC subcutaneous tumors in the C57BL/6 mouse model. According to CCK8, EdU, transwell and wound healing assays, ZnC inhibited CRC cell lines in terms of proliferation, invasion and migration. ZnC could inhibit miR-570 for up-regulating PD-L1 expression. In vivo experiments showed that gavage (100 mg/kg, once every day) of ZnC inhibited the tumor growth of CRC, and the combination of ZnC and anti-PD1 therapy significantly improved the efficacy exhibited by anti-PD1 in treating CRC. In addition, mass cytometry results showed that immunosuppressive cells including regulatory T cells (tregs), bone marrow-derived suppressor cells (MDSC), and M2 macrophages decreased whereas CD8+ T cells elevated after adding ZnC. The present study reveals that ZnC slows the progression of CRC by inhibiting CRC cells in terms of proliferation, invasion and migration, meanwhile up-regulating PD-L1 expression via inhibiting miR-570. The ZnC-anti-PD1 co-treatment assists in synergically increasing anti-tumor efficacy in CRC therapy.

A mitophagy-related gene signature associated with prognosis and immune microenvironment in colorectal cancer

Colorectal cancer (CRC) is a heterogeneous disease and one of the most prevalent malignancies worldwide. Previous research has demonstrated that mitophagy is crucial to developing colorectal cancer. This study aims to examine the association between mitophagy-related genes and the prognosis of CRC patients. Gene expression profiles and clinical information of CRC patients were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Univariate Cox regression and the least absolute shrinkage and selection operator (LASSO) regression analysis were applied to establish a prognostic signature using mitophagy related genes. Kaplan–Meier and receiver operating characteristic (ROC) curves were used to analyze patient survival and predictive accuracy. Meanwhile, we also used the Genomics of Drug Sensitivity in Cancer (GDSC) database and Tumor Immune Dysfunction and Exclusion (TIDE) algorithm to estimate the sensitivity of chemotherapy, targeted therapy and immunotherapy. ATG14 overexpression plasmid was used to regulate the ATG14 expression level in HCT116 and SW480 cell lines, and cell counting kit-8, colony formation and transwell migration assay were performed to validate the function of ATG14 in CRC cells. A total of 22 mitophagy-driven genes connected with CRC survival were identified, and then a novel prognostic signature was established based on 10 of them (AMBRA1, ATG14, MAP1LC3A, MAP1LC3B, OPTN, VDAC1, ATG5, CSNK2A2, MFN1, TOMM22). Patients were divided into high-risk and low-risk groups based on the median risk score, and the survival of patients in the high-risk group was significantly shorter in both the training cohort and two independent cohorts. ROC curve showed that the area under the curves (AUC) of 1-, 3- and 5-year survival were 0.66, 0.66 and 0.64, respectively. Multivariate Cox regression analysis confirmed the independent prognostic value of the signature. Then we constructed a Nomogram combining the risk score, age and M stage, which had a concordance index of survival prediction of 0.77 (95% CI 0.71–0.83) and more robust predictive accuracy. Results showed that CD8+ T cells, regulatory T cells and activated NK cells were significantly more enriched in the high-risk group. Furthermore, patients in the high-risk group are more sensitive to targeted therapy or chemotherapy, including bosutinib, elesclomol, lenalidomide, midostaurin, pazopanib and sunitinib, while the low-risk group is more likely to benefit from immunotherapy. Finally, in vitro study confirmed the oncogenic significance of ATG14 in both HCT116 and SW480 cells, whose overexpression increased CRC cell proliferation, colony formation, and migration. In conclusion, we developed a novel mitophagy-related gene signature that can be utilized not only as an independent predictive biomarker but also as a tool for tailoring personalizing treatment for CRC patients, and we confirmed ATG14 as a novel oncogene in CRC.

Fusobacterium and Colorectal Cancer: Important Association or Random Coincidence?

The role of the microbiome in human cancer has become an area of intensive research and controversy. Many reports have highlighted the physical association of Fusobacterium with colorectal cancer. This association has provided diagnostic and therapeutic promise but has also given rise to several controversies regarding the influence of Fusobacterium species on human colorectal cancer. Here, we discuss two areas of controversy surrounding this emerging pathogen: the influence of Fusobacterium on colorectal cancer proliferation and the effect of Fusobacterium on the immune microenvironment of colorectal cancer.