Colorectal Cancer Microenvironment Research Articles

An inflammation-related subtype classification for analyzing tumor microenvironment and clinical prognosis in colorectal cancer

BackgroundThe inflammatory response plays an essential role in the tumor microenvironment (TME) of colorectal cancer (CRC) by modulating tumor growth, progression, and response to therapy through the recruitment of immune cells, production of cytokines, and activation of signaling pathways. However, the molecular subtypes and risk score prognostic model based on inflammatory response remain to be further explored.MethodsInflammation-related genes were collected from the molecular signature database and molecular subtypes were identified using nonnegative matrix factorization based on the TCGA cohort. We compared the clinicopathological features, immune infiltration, somatic mutation profile, survival prognosis, and drug sensitivity between the subtypes. The risk score model was developed using LASSO and multivariate Cox regression in the TCGA cohort. The above results were independently validated in the GEO cohort. Moreover, we explored the biological functions of the hub gene, receptor interacting protein kinase 2 (RIPK2), leveraging proteomics data, in vivo, and in vitro experiments.ResultsWe identified two inflammation-related subtypes (inflammation-low and inflammation-high) and have excellent internal consistency and stability. Inflammation-high subtype showed higher immune cell infiltration and increased sensitivity to common chemotherapeutic drugs, while inflammation-low subtype may be more suitable for immunotherapy. Besides, the two subtypes differ significantly in pathway enrichment and biological functions. In addition, the 11-gene signature prognostic model constructed from inflammation-related genes showed strong prognostic assessment power and could serve as a novel prognostic marker to predict the survival of CRC patients. Finally, RIPK2 plays a crucial role in promoting malignant proliferation of CRC cell validated by experiment.ConclusionsThis study provides new insights into the heterogeneity of CRC and provides novel opportunities for treatment development and clinical decision making.

Predicting prognosis and immunotherapy response in colorectal cancer by pericytes insights from single-cell RNA sequencing.

Immunotherapy has revolutionized the treatment of tumors, but there are still a large number of patients who do not benefit from immunotherapy. Pericytes play an important role in remodeling the immune microenvironment. However, how pericytes affect the prognosis and treatment resistance of tumors is still unknown. This study jointly analyzed single-cell RNA sequencing (scRNA-seq) data and bulk RNA sequencing data of multiple cancers to reveal pericyte function in the colorectal cancer microenvironment. Analyzing over 800 000 cells, it was found that colorectal cancer had more pericyte enrichment in tumor tissues than other cancers. We then combined the TCGA database with multiple public datasets and enrolled more than 1000 samples, finding that pericyte may be closely related to poor prognosis due to the higher epithelial-mesenchymal transition (EMT) and hypoxic characteristics. At the same time, patients with more pericytes have higher immune checkpoint molecule expressions and lower immune cell infiltration. Finally, the contributions of pericyte in poor treatment response have been demonstrated in multiple immunotherapy datasets (n = 453). All of these observations suggest that pericyte can be used as a potential biomarker to predict patient disease progression and immunotherapy response.

Relationship of FDG PET/CT imaging features with tumor immune microenvironment and prognosis in colorectal cancer: a retrospective study

BackgroundImaging features of colorectal cancers on 2-deoxy-2-[18F]fluoro-d-glucose (FDG) positron emission tomography/computed tomography (PET/CT) have been considered to be affected by tumor characteristics and tumor immune microenvironment. However, the relationship between PET/CT imaging features and immune reactions in tumor tissue has not yet been fully evaluated. This study investigated the association of FDG PET/CT imaging features in the tumor, bone marrow, and spleen with immunohistochemical results of cancer tissue and recurrence-free survival (RFS) in patients with colorectal cancer.MethodsA total of 119 patients with colorectal cancer who underwent FDG PET/CT for staging work-up and received curative surgical resection were retrospectively enrolled. From PET/CT images, 10 first-order imaging features of primary tumors, including intensity of FDG uptake, volumetric metabolic parameters, and metabolic heterogeneity parameters, as well as FDG uptake in the bone marrow and spleen were measured. The degrees of CD4+, CD8+, and CD163 + cell infiltration and interleukin-6 (IL-6) and matrix metalloproteinase-11 (MMP-11) expression were graded through immunohistochemical analysis of surgical specimens. The relationship between FDG PET/CT imaging features and immunohistochemical results was assessed, and prognostic significance of PET/CT imaging features in predicting RFS was evaluated.ResultsCorrelation analysis with immunohistochemistry findings showed that the degrees of CD4 + and CD163 + cell infiltration and IL-6 and MMP-11 expression were correlated with cancer imaging features on PET/CT. Patients with enhanced inflammatory response in cancer tissue demonstrated increased FDG uptake, volumetric metabolic parameters, and metabolic heterogeneity. FDG uptake in the bone marrow and spleen was positively correlated with the degree of CD163 + cell infiltration and IL-6 expression, respectively. In multivariate survival analysis, the coefficient of variation of FDG uptake in the tumor (p = 0.019; hazard ratio, 0.484 for 0.10 increase) and spleen-to-liver uptake ratio (p = 0.020; hazard ratio, 24.901 for 1.0 increase) were significant independent predictors of RFS.ConclusionsThe metabolic heterogeneity of tumors and FDG uptake in the spleen were correlated with tumor immune microenvironment and showed prognostic significance in predicting RFS in patients with colorectal cancer.

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.

SDCBP modulates tumor microenvironment, tumor progression and anti-PD1 efficacy in colorectal cancer.

Anti-programmed cell death 1 (aPD1) therapy has yielded limited success in patients with colorectal cancer (CRC). Syndecan binding protein (SDCBP), encodes a PDZ domain-containing protein that is essential for cellular processes, including cell adhesion, migration, and signal transduction. Here, we investigated the effect of SDCBP on tumor progression, immunotherapy, and the tumor microenvironment (TME) in CRC. High expression of SDCBP is associated with non-response to immunotherapy and correlated with poorer disease-free survival (DFS) in CRC patients. Inhibiting SDCBP by transfecting shRNA or using its inhibitor zinc pyrithione (ZnPT) hindered proliferation and metastasis while enhancing the efficacy of aPD1 treatment in a mouse xenograft model and liver metastasis model. The TME of CRC was significantly altered following ZnPT treatment characterized by a reduced amount of M2 macrophages and a heightened percentage of M1 macrophages. The co-culture system of CRC cells and macrophages provided evidence that SDCBP silencing promoted the repolarisation of M2 macrophages into M1. SDCBP promotes the proliferation, metastasis, and immunotherapy resistance of CRC. Thus, ZnPT represents an effective SDCBP inhibitor and exhibits considerable potential for combination with aPD1 to enhance immunotherapy efficacy.

Characteristics of the Colorectal Cancer Microenvironment—Role in Cancer Progression and Therapeutic Possibilities

Colorectal cancer (CRC) is one of the most common and deadliest cancers worldwide. According to the GLOBOCAN (WHO) report in 2020, nearly 2 million patients were diagnosed globally. Despite the advances in cancer diagnosis and therapy, CRC remains a global challenge. Recently, attention has been paid to the tumor microenvironment (TME), which constitutes a significant part of the tumor and mainly includes various immune cells, fibroblasts, vascular cells, and extracellular elements, such as the extracellular matrix (ECM). Many components of the stroma initially exert an anti-tumor effect, but over time, they undergo functional transformation into elements that promote tumor growth. As a result, conditions conducive to further cancer development, invasion into local tissues, and distant metastasis arise. The microenvironment of colorectal cancer (CRC) may be an important direction in the search for therapeutic targets, but it requires further understanding. The main purpose of our review is to explain the role of the complex CRC microenvironment in the progression of this cancer and highlight the potential of targeted therapy directed at the TME. Therefore, continued research into its components and typical biomarkers is necessary to improve therapy and enhance the quality of life for patients.

The Tumor Microenvironment Mediates the HIF-1α/PD-L1 Pathway to Promote Immune Escape in Colorectal Cancer.

The unsatisfactory efficacy of immunotherapy for colorectal cancer (CRC) remains a major challenge for clinicians and patients. The tumor microenvironment may promote CRC progression by upregulating the expression of hypoxia-inducing factor (HIF) and PD-L1. Therefore, this study explored the expression and correlation of HIF-1α and PD-L1 in the CRC microenvironment. The expression and correlation of HIF-1α and PD-L1 in CRC were analyzed using bioinformatics and Western blotting (WB). The hypoxia and inflammation of the CRC microenvironment were established in the CT26 cell line. CT26 cells were stimulated with two hypoxia mimics, CoCl2 and DFO, which were used to induce the hypoxic environment. Western blotting was used to assess the expression and correlation of HIF-1α and PD-L1 in the hypoxic environment.LPS stimulated CT26 cells to induce the inflammatory environment. WB and bioinformatics were used to assess the expression and correlation of TLR4, HIF-1α, and PD-L1 in the inflammatory environment. Furthermore, the impact of curcumin on the inflammatory environment established by LPS-stimulated CT26 cells was demonstrated through MTT, Transwell, molecular docking, network pharmacology and Western blotting assays. In this study, we found that the HIF-1α/PD-L1 pathway was activated in the hypoxic and inflammatory environment and promoted immune escape in CRC. Meanwhile, curcumin suppressed tumor immune escape by inhibiting the TLR4/HIF-1α/PD-L1 pathway in the inflammatory environment of CRC. These results suggest that combination therapy based on the HIF-1α/PD-L1 pathway can be a promising therapeutic option and that curcumin can be used as a potent immunomodulatory agent in clinical practice.

DNA hypomethylation patterns and their impact on the tumor microenvironment in colorectal cancer.

Recent research underscores the pivotal role of immune checkpoints as biomarkers in colorectal cancer (CRC) therapy, highlighting the dynamics of resistance and response to immune checkpoint inhibitors. The impact of epigenetic alterations in CRC, particularly in relation to immune therapy resistance, is not fully understood. We integrated a comprehensive dataset encompassing TCGA-COAD, TCGA-READ, and multiple GEO series (GSE14333, GSE37892, GSE41258), along with key epigenetic datasets (TCGA-COAD, TCGA-READ, GSE77718). Hierarchical clustering, based on Euclidean distance and Ward's method, was applied to 330 primary tumor samples to identify distinct clusters. The immune microenvironment was assessed using MCPcounter. Machine learning algorithms were employed to predict DNA methylation patterns and their functional enrichment, in addition to transcriptome expression analysis. Genomic mutation profiles and treatment response assessments were also conducted. Our analysis delineated a specific tumor cluster with CpG Island (CGI) methylation, termed the Demethylated Phenotype (DMP). DMP was associated with metabolic pathways such as oxidative phosphorylation, implicating increased ATP production efficiency in mitochondria, which contributes to tumor aggressiveness. Furthermore, DMP showed activation of the Myc target pathway, known for tumor immune suppression, and exhibited downregulation in key immune-related pathways, suggesting a tumor microenvironment characterized by diminished immunity and increased fibroblast infiltration. Six potential therapeutic agents-lapatinib, RDEA119, WH.4.023, MG.132, PD.0325901, and AZ628-were identified as effective for the DMP subtype. This study unveils a novel epigenetic phenotype in CRC linked to resistance against immune checkpoint inhibitors, presenting a significant step toward personalized medicine by suggesting epigenetic classifications as a means to identify ideal candidates for immunotherapy in CRC. Our findings also highlight potential therapeutic agents for the DMP subtype, offering new avenues for tailored CRC treatment strategies.

Abstract 99: Versican regulates T cell abundance within the colorectal cancer microenvironment

Abstract Background: Clinical benefit from immunotherapy is seen in a minority of patients with metastatic colorectal cancer (CRC). Identifying how the tumor microenvironment (TME) influences immune infiltration and treatment response is an unmet need. Versican (VCAN), a chondroitin sulfate proteoglycan, common in the CRC TME has immunoregulatory properties and can be cleaved releasing an immunostimulatory product, versikine (Vkine). Here we evaluate the impact of the abundance of VCAN and Vkine on CD8+ T cell infiltration in CRC. Methods: Samples include 306 primary colon tumors, an additional 176 primary CRCs with matched liver metastases (175 samples), and 328 normal samples (238 colon and 90 liver). Samples were stained via immunohistochemistry (IHC) for VCAN, Vkine, and CD8. CD8+ tumor infiltrating lymphocytes (TILs) were counted per high powered field (HPF, 400X magnification) and presented as averages. Intensity of VCAN and Vkine expression were scored from 0-3+. Samples designated a 0/1 were considered low accumulation and samples scored as a 2/3+ were designated high. Samples scored low for VCAN but high for Vkine were designated VCAN proteolytic predominant (VPP) and all other combinations considered VCAN proteolytic weak (VPW). To create an in vitro model, the mouse MC38 cell line was CRISPR engineered to over-express VCAN. Conditioned media was then concentrated and added to a collagen matrix in a transwell with activated T cells. The number of cells and their subtype that migrated through the matrix were evaluated with flow cytometry. Results: VCAN accumulation was highest in cancerous stroma with 58% of cancers being VCAN high and only 14% of adjacent normal tissues (p<0.001). Vkine accumulation was similar between groups, with 67% of tumors and 54% of adjacent normal samples scored as Vkine high. The VPP phenotype was least common in tumor tissues with only 27% of tumor samples designated VPP versus 43% of adjacent normal samples (p<0.001). VCAN high tumors had significantly fewer TILs with 4 TILs/HPF vs 13 in the VCAN low tumors (p<0.001). High levels of Vkine correlated with greater TIL abundance (Vkine high: 10 TILs/HPF, Vkine low: 5 TILs/HPF; p=0.02). Versican proteolysis had the strongest correlation with TIL abundance in cancers (VPP: 18 TILs/HPF, VPW: 5 TILS/HPF; p<0.001). In vitro, the addition of VCAN containing media into transwell matrices reduced both CD4+ and CD8+ T cell migration through the matrix (Control: 4277 CD8+ T cells, VCAN: 2603 CD8+ T cells; p<0.001 and Control: 7770 CD4+ T cells, VCAN: 5340 CD4+ T cells; p=0.007). Conclusions: VCAN accumulation in human CRCs is common and correlates with reduced TIL abundance, whereas VCAN proteolysis correlates with increased TILs. In vitro, VCAN demonstrated the ability to reduce T cell migration. Future studies will explore the mechanism by which VCAN abundance leads to decreased T cell infiltration and the importance of this as an immunotherapy biomarker. Citation Format: Sean Kraus, Philip Emmerich, Mitch Hayes, Kristina Matkowskyj, Wei Zhang, Cheri Pasch, Fotis Asimakopoulos, Dustin A. Deming. Versican regulates T cell abundance within the colorectal cancer microenvironment [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 99.

Abstract 636: Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts

Abstract The tumor microenvironment (TME) is well recognized for its role in tumor progression, chemoresistance, and recurrence. The TME consists of various components, such as extracellular matrix (ECM), chemical and mechanical cues, as well as stromal cells and their interactions. As the most abundant cells in tumor stroma, cancer-associated fibroblasts (CAFs) have drawn a lot of attention. It has been reported that CAFs not only directly interact with cancer cells via surface proteins and cytokines, but also influence their behaviors by remodeling the ECM. However, due to their heterogenous characteristics and diverse functions, the influence of tumor-CAF interactions on drug response is not fully understood. Here, we present a coculture model for investigating CAF effects on patient-derived colorectal tumor organoids (CTOs) growth, differentiation, and drug response. It consists of normal colon fibroblasts, CCD18-Co, or patient-derived CAFs seeded with CTOs in a mixed hydrogel containing 90% basement membrane extract (BME) and 10% collagen type I. Media conditions were optimized (1:1 mix of CTO culture media plus reduced media excluding growth factors and inhibitors) to achieve similar organoid sizes in the monoculture and coculture conditions at the onset of drug testing. To support higher throughput investigations, our workflow incorporates a liquid handler for automated CTO seeding, media and drug exchange, and an automated microscope with a robotic arm for multi-plate imaging. Briefly, single cell CTOs and fibroblasts were seeded at a 1:1 ratio (3000 total cells/well) in 96-well plates. After 4 days in culture, anti-cancer drugs were administrated in reduced media to the samples. Brightfield images were acquired on days 0, 3, and 5 after drug administration. The collected images were analyzed with a neural network (NN) to dynamically monitor the growth and viability of individual organoids. The CellTiter-Glo assay was performed on day 5 to quantify the total CTO cell viability. The normalized drug response (NDR) was calculated from the NN processed images to determine the drug response. An immunostaining panel, which includes protein markers such as CK20, Ki67, α-SMA, Vimentin, and FAP, was used to evaluate the fibroblast phenotypes and CTO differentiation. Additionally, the cultures were subjected to fluorescent lifetime imaging microscopy (FLIM), a label-free imaging technique, to measure the metabolic adaptations of each cell population. Preliminary studies revealed a CAF-dependent increase in CTO growth and glycolytic signatures in the co-culture setting; however, minimal effects on drug response to common chemotherapy agents such as 5-fluorouracil and irinotecan were observed. In the future, we will use this integrated pipeline as a screening method to identify new treatment strategies aimed at targeting the stromal microenvironment in colorectal cancer. Citation Format: Yuyuan Zhou, Yu-Kai Huang, Emma J. Fong, Elizabeth Elton, Erin McManus, Brandon Choi, Seungil Kim, Shannon M. Mumenthaler. Coculture model for investigating cellular interactions between colorectal tumor organoids and cancer-associated fibroblasts [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 636.

Abstract 6806: Single cell RNA sequencing of human and murine cancers demonstrates metastases-specific neutrophil phenotypes driven by the IL-1β/CXCR2 signaling axis - Disruption of this pathway abrogates neutrophil immunosuppressive effects and reduces tumor burden

Abstract Introduction: Neutrophils are a highly heterogenous, plastic cell population, known to play a vital role in the tumor microenvironment and progression of CMS4 colorectal cancer (CRC) metastases. We have performed a comprehensive examination of different neutrophil transcriptional states, through utilization of single-cell RNA sequencing of publicly available and independently generated data across health and malignancy in human and murine disease, and validated findings using in vivo and in vitro models of CMS4 subtype CRC. Methods: Publicly available and independently generated breast, lung and CRC datasets from human disease mouse models were integrated to establish and validate reproducibility of neutrophil-gene signatures, and pseudo-time analysis was used to identify genes driving neutrophil development along a lineage from health to tumor using Seurat on R. Findings were validated in healthy and KrasG12D/+, Trp53fl/fl, Rosa26N1CD/+ (KPN) tumor-bearing wild type or Mrp8CreCxcr2fl/fl genetically engineered mouse models with neutrophil-specific loss of Cxcr2. Results: We have identified distinct neutrophil subtypes in health and primary tumors, preserved in murine and human cancer and across multiple tumor types. In CRC metastases, discrete neutrophil transcriptomic subtypes are identified, with phenotypes from health to those identified in metastases driven by the IL-1β/CXCL8/CXCR2 signalling axis. Assessment of global signalling of single-cell transcripts has identified CD4+ T cells and macrophages as dominant regulators of the metastatic niche. Neutrophils sorted from the metastatic niche strongly suppress T cell proliferation, identifying them as immunosuppressive. Loss of neutrophil-specific Cxcr2 attenuates suppression of T cell proliferation in co-culture with neutrophils from the metastatic niche of tumor-bearing mice. T cell proliferation is unaffected in co-culture with healthy wild type or Cxcr2fl/fl neutrophils. We currently are running orthotopic intrasplenic models to assess the importance of loss of neutrophil-specific Cxcr2 in generation of metastases in vivo in CMS4 CRC. Conclusions: We speculate that ablation of metastasis specific neutrophil subsets in CMS4 CRC in combination with other therapies, may alter the immunosuppressive nature of the immune microenvironment and allow for reduction in tumor burden in metastatic disease. Citation Format: Alistair S. McLaren, Rana Fetit, Mark White, Megan L. Mills, Tamsin Lannagan, John Falconer, Xabier Cortes-Lavaud, Kathryn Gilroy, Rachel Ridgway, Colin Nixon, Varushka Naiker, Renee Njunge, Cassie J. Clarke, Declan Whyte, Kristina Kirschner, Rene Jackstadt, Jim C. Norman, Leo M. Carlin, Andrew D. Campbell, Owen J. Sansom, Colin W. Steele. Single cell RNA sequencing of human and murine cancers demonstrates metastases-specific neutrophil phenotypes driven by the IL-1β/CXCR2 signaling axis - Disruption of this pathway abrogates neutrophil immunosuppressive effects and reduces tumor burden [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 6806.

Abstract 5153: Pyramidobacter-DMMR synergy in colorectal cancer: Unveiling prognostic impact, mechanistic insights, and tumor microenvironment dynamics

Abstract Background: Colorectal cancer (CRC) exhibits a heightened presence of intratumoral bacteria, and deficient DNA mismatch repair (dMMR) contributes to nucleotide mismatch accumulation, fostering mutagenesis. However, the combined impact of intratumoral bacteria and dMMR on the tumor immune microenvironment (TIME) in CRC, and its consequential influence on patient prognosis, remains elusive. Methods: We utilized samples from the Cancer Microbiome Atlas project (TCMA), collecting tumor and matched paracancer tissues from colorectal cancer patients. Phyloseq in R was employed to scrutinize microbial abundance differences. The effects of microbial enrichment on prognosis were assessed through survival analysis, and GSEA enrichment analysis provided mechanistic insights. Results: A total of 173 samples, including 28 dMMR tumors, 112 proficient MMR (pMMR) tumors, and 22 paired para-cancer tissues, were collected. Following stringent filtration, we analyzed 230 bacterial genera. Notably, 26 genera, including Fusobacterium and Streptococcus, showed significant enrichment in dMMR tumors compared to para-cancer tissues. Furthermore, 18 genera were enriched in dMMR tumors compared to pMMR tumors. Of these, 8 genera like Desulfovibrio and Pyramidobacter consistently exhibited enrichment in dMMR CRC compared to either para-cancer tissues or pMMR tumors. Survival analysis highlighted Pyramidobacter as the only genus significantly associated with prognosis in dMMR CRC. Increased Pyramidobacter abundance correlated with decreased overall survival (OS) (P value: 0.049). Stratifying dMMR CRC patients based on Pyramidobacter abundance further emphasized the significantly worse survival in the Pyramidobacter-High group (HR 0.93, 95%CI 1.03-9.60, log-rank p = 0.015). Exploring the impact of Pyramidobacter infection on tumor progression, we assessed TIME and oncogenic pathways between Pyramidobacter-Low and Pyramidobacter-High groups. While overall immune infiltration was higher in the Pyramidobacter-High group, it primarily stemmed from increased B cell and naive CD8+ T cell infiltration. Importantly, pathways enriched in the Pyramidobacter-High group included inflammatory response, epithelial-mesenchymal transition (EMT), and KRAS signaling, while those enriched in the Pyramidobacter-Low group encompassed IFNα response, IFNγ response, and DNA damage repair. These findings suggest Pyramidobacter's role in inducing inflammation, promoting tumor proliferation and metastasis, and suppressing interferon response. Conclusion: Pyramidobacter emerges as a key player in the development of dMMR CRC, exerting a pivotal influence on patient prognosis through the induction of inflammatory responses and EMT. Citation Format: Shisen LI, Yunlong LI, Hongjiang Ma, Shihao Qin, Feilong Zhao, Jipeng Li. Pyramidobacter-DMMR synergy in colorectal cancer: Unveiling prognostic impact, mechanistic insights, and tumor microenvironment dynamics [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 5153.

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor 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 287.

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.

Abstract 6682: Bile acid profiles and racial disparities in colorectal cancer: Exploring the protective role of secondary bile acid derivatives

Abstract Background: Colorectal cancer (CRC) is the third leading cause of cancer death in the United States, with diet and gut microbiota-derived bile acids implicated as long-term risk factors. The interplay between bile acids and immune cell differentiation, particularly of macrophages and T-cells, is a crucial aspect of the CRC microenvironment. Given the high-animal-fat diets' influence on the microbiome’s production of hydrophobic secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid (LCA), this study investigates the potential immunomodulatory effects of their lower concentrations and their isomers in possibly fostering M2-like macrophage expansion. Methods: Clinical samples were obtained during colonoscopies with no tumor findings at Rush University Medical Center, Chicago. RNA was extracted from colonoscopy biopsies for NanoString RNA expression analysis, while bile acids were isolated from both lyophilized stool and frozen serum samples. To explore the immunological effects of secondary bile acids, the macrophage RAW 264.7 cell line, alongside peritoneal macrophages and bone marrow-derived macrophages (BMDMs) from C57BL/6J mice, were treated with LCA and LCA isomers. Following the bile acid treatment, LPS (100 ng/mL) or IL-4 (40 ng/ml) was added to elicit pro-inflammatory or anti-inflammatory states, respectively. After 48 hours, conditioned media were collected for enzyme-linked immunosorbent assay (ELISA), while cells were analyzed by flow cytometry and rt-qPCR. Results: Contrary to initial hypotheses, the levels of DCA and LCA levels were not significantly different between African American and non-Hispanic White subjects. Notably, allo-isomers and oxo-derivatives of these bile acids, which may promote Treg and M2-like macrophage expansion, were substantially lower in African American subjects. Ex vivo treatments with isoalloLCA and IsoLCA led to notable changes in the M2 macrophage differentiation and shifts in PGE2 production upon exposure to inflammatory or anti-inflammatory stimuli, suggesting a nuanced alteration in the macrophage-mediated inflammatory response. Conclusion: This study highlights a significant ethnic variation in bile acid profiles, with a potential impact on immune modulation and CRC risk. The reduced presence of certain bile acid isomers in African American subjects may influence the risk of CRC through altered macrophage differentiation and function. These findings emphasize the need for personalized dietary and therapeutic strategies to modulate the bile acid metabolome, potentially reducing CRC disparities. Citation Format: Hanchu Dai, Patricia G. Wolf, Lisa Tussing-Humphreys, Ece Mutlu, Jason M. Ridlon, Rex Gaskins. Bile acid profiles and racial disparities in colorectal cancer: Exploring the protective role of secondary bile acid derivatives [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 6682.

Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.

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.

HIF-1α Mediates Immunosuppression and Chemoresistance in Colorectal Cancer by Inhibiting CXCL9, −10 and −11

Uncertainty exists regarding the mechanisms by which hypoxia-inducible factors (HIFs) control CD8+T-cell migration into tumor microenvironments. Here, we found that HIF-1α knockdown or overexpression resulted in increased or decreased CXCL9, −10, and −11 expression in vitro, respectively. Gene Set Variation Analysis revealed that elevated HIF-1α levels correlated with a poor prognosis, severe pathological stage, and an absence of CD8+ T cells in the tumor microenvironment in colorectal cancer (CRC) patients. HIF-1α was inversely associated with pathways beneficial to anti-tumor immunotherapy and cytokine/chemokine function. In vivo, inhibiting HIF-1α or its upstream regulator BIRC2 significantly suppressed tumor growth and promoted CD8+ T-cell infiltration. CXCR3 neutralizing antibodies reversed these effects, implicating the involvement of CXCL9, −10, and −11/CXCR3 axis. The presence of HIF-1α weakened the upregulation of CXCL9, −10, and −11 by bleomycin and doxorubicin. Combining HIF-1α inhibition with bleomycin promoted CD8+ T-cell infiltration and tumor suppression in vivo. Moreover, doxorubicin could upregulate CXCL9, −10 and −11 by suppressing HIF-1α. Our findings highlight the potential of HIF-1α inhibition to improve CRC microenvironments and increase chemotherapy sensitivity.

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.