Immune Microenvironment In Colorectal Cancer Research Articles

Type I Diabetes Mellitus impairs cytotoxic immunity through CEACAM5 upregulation in colorectal cancer : Exploring the intersection of autoimmune dysfunction and cancer progression: the role of NF-κB p65 in colorectal cancer.

Type 1 diabetes (T1D) is characterized by an autoimmune-mediated destruction of pancreatic beta cells and a chronic inflammatory state, which may influence the progression of colorectal cancer (CRC) through immune system dysregulation and enhanced tumor immune evasion. This study aims to elucidate the role of p65 in modulating the tumor microenvironment in CRC within the context of T1D and to determine how this modulation affects tumor growth, immune cell infiltration, and the expression of immune evasion molecules such as CEACAM5. NOD mice, which model T1D, were inoculated with MC38 colon carcinoma cells engineered to knock down p65. Tumor growth was monitored, and the tumor microenvironment was analyzed using flow cytometry to assess the infiltration of immune cells. The expression of Ki-67 and CEACAM5 in tumor cells was also evaluated. Additionally, in vitro assays were conducted to study the proliferation and activation of T cells co-cultured with tumor cells. Knockdown of p65 in tumor cells significantly inhibited tumor growth in NOD mice. This was accompanied by an increased infiltration of cytotoxic CD8+ T cells and no significant change in CD4+ or Foxp3 + T regulatory cells within the tumor microenvironment. There was a notable reduction in the expression of Ki-67 and CEACAM5, indicating decreased proliferation and potential immune evasion capabilities of the tumor cells. Our findings demonstrate that the NF-κB p65 subunit plays a crucial role in promoting tumor growth and modulating the immune microenvironment in CRC, particularly in the context of T1D. Knocking down p65 not only reduces tumor progression but also enhances the anti-tumor immune response by decreasing immune evasion mechanisms. These results suggest that targeting the NF-κB pathway may be a viable strategy to improve the efficacy of cancer immunotherapy, especially in patients with autoimmune diseases like T1D. Physical activity enhances the effect of immune checkpoint blockade by inhibiting the intratumoral HIF1-α/CEACAM5 axis.

Functional gene signature offers a powerful tool for characterizing clinicopathological features and depicting tumor immune microenvironment of colorectal cancer

BackgroundColorectal cancer, a prevalent malignancy worldwide, poses a significant challenge due to the lack of effective prognostic tools. In this study, we aimed to develop a functional gene signature to stratify colorectal cancer patients into different groups with distinct characteristics, which will greatly facilitate disease prediction.ResultsPatients were stratified into high- and low-risk groups using a prediction model built based on the functional gene signature. This innovative approach not only predicts clinicopathological features but also reveals tumor immune microenvironment types and responses to immunotherapy. The study reveals that patients in the high-risk group exhibit poorer pathological features, including invasion depth, lymph node metastasis, and distant metastasis, as well as unfavorable survival outcomes in terms of overall survival and disease-free survival. The underlying mechanisms for these observations are attributed to upregulated tumor-related signaling pathways, increased infiltration of pro-tumor immune cells, decreased infiltration of anti-tumor immune cells, and a lower tumor mutation burden. Consequently, patients in the high-risk group exhibit a diminished response to immunotherapy. Furthermore, the high-risk group demonstrates enrichment in extracellular matrix-related functions and significant infiltration of cancer-associated fibroblasts (CAFs). Single-cell transcriptional data analysis identifies CAFs as the primary cellular type expressing hub genes, namely ACTA2, TPM2, MYL9, and TAGLN. This finding is further validated through multiple approaches, including multiplex immunohistochemistry (mIHC), polymerase chain reaction (PCR), and western blot analysis. Notably, TPM2 emerges as a potential biomarker for identifying CAFs in colorectal cancer, distinguishing them from both colorectal cancer cell lines and normal colon epithelial cell lines. Co-culture of CAFs and colorectal cancer cells revealed that CAFs could enhance the tumorigenic biofunctions of cancer cells indirectly, which could be partially inhibited by knocking down CAF original TPM2 expression.ConclusionsThis study introduces a functional gene signature that effectively and reliably predicts clinicopathological features and the tumor immune microenvironment in colorectal cancer. Moreover, the identification of TPM2 as a potential biomarker for CAFs holds promising implications for future research and clinical applications in the field of colorectal cancer.

Colorectal Cancer-Derived Exosomes Impair CD4+ T Cell Function and Accelerate Cancer Progression via Macrophage Activation.

Background: Exosomal programmed death ligand 1 (PD-L1), an exosomal membrane protein found in many tumor types, is reckoned to help regulate the immune microenvironment. However, the functions and the mechanisms underlying the exosome-mediated regulation of the immune microenvironment in colorectal cancer (CRC) remain unknown. Methods: Western blotting was used to investigate the levels of exosomal PD-L1 in the peripheral blood of patients with CRC and healthy controls. A CRC mouse model was constructed by administering 10 mg/kg azoxymethane (AOM) and dextrane sodium sulfate (DSS) intraperitoneally. The mice were then administered the control or CRC-derived exosomes to examine the regulatory effect of the exosomes on macrophage infiltration and CRC development. In vitro studies, using a coculture system, and flow cytometry analysis were conducted to examine the relationship between the regulatory effect of CRC-derived exosomes on CD4+ T cells and tumor-associated macrophages. RNA-seq and reverse transcription-quantitative polymerase chain reaction assays were used to investigate the mechanisms underlying the regulatory effect of the CRC-derived exosomes on macrophage proliferation and the regulation of the immune microenvironment during CRC development. Results: In patients with CRC, higher levels of exosomal PD-L1 were associated with a more severe form of disease. The treatment of mice with AOM/DSS-induced CRC with CRC-derived exosomes resulted in high levels of macrophage proliferation, increased PD-L1 levels in macrophages, and accelerated CRC progression. Importantly, analysis of an in vitro coculture system and flow cytometry analysis showed that the CRC-derived exosomes transported PD-L1 into macrophages and impaired CD4+ T cell function. Preliminary data suggest that the NF-κb signaling pathway regulates the function of CRC-derived exosomal PD-L1-dependent macrophages. Conclusion: CRC-derived exosomes induce the proliferation of macrophages and increase their PD-L1 levels. They also impair CD4+ T cell function and promote CRC progression. Our findings reveal a novel exosomal PD-L1-mediated crosstalk between the CRC cells and immune cells in the CRC microenvironment.

Dual inhibitors of DNMT and HDAC remodels the immune microenvironment of colorectal cancer and enhances the efficacy of anti-PD-L1 therapy

Colorectal cancer is the second most prevalent and deadly cancer worldwide. The emergence of immune checkpoint therapy has provided a revolutionary strategy for the treatment of solid tumors. However, less than 5 % of colorectal cancer patients respond to immune checkpoint therapy. Thus, it is of great scientific significance to develop “potentiators” for immune checkpoint therapy. In this study, we found that knocking down different DNMT and HDAC isoforms could increase the expression of IFNs in colorectal cancer cells, which can enhance the effectiveness of immune checkpoint therapy. Therefore, the combined inhibition of DNMT and HDAC cloud synergistically enhance the effect of immunotherapy. We found that dual DNMT and HDAC inhibitors C02S could inhibit tumor growth in immunocompetent mice but not in immunocompromised nude mice, which indicates that C02S exerts its antitumor effects through the immune system. Mechanistically, C02S could increase the expression of ERVs, which generated the intracellular levels of dsRNA in tumor cells, and then promotes the expression of IFNs through the RIG-I/MDA5-MAVS signaling pathway. Moreover, C02S increased the immune infiltration of DCs and T cells in microenvironment, and enhanced the efficacy of anti-PD-L1 therapy in MC38 and CT26 mice model. These results confirmed that C02S can activate IFNs through the RIG-I/MDA5-MAVS signaling pathway, remodel the tumor immune microenvironment and enhance the efficacy of immune checkpoint therapy, which provides new evidence and solutions for the development of “potentiator” for colorectal cancer immunotherapy.

Action of circulating and infiltrating B cells in the immune microenvironment of colorectal cancer by single-cell sequencing analysis.

The complexity of the immune microenvironment has an impact on the treatment of colorectal cancer (CRC), one of the most prevalent malignancies worldwide. In this study, multi-omics and single-cell sequencing techniques were used to investigate the mechanism of action of circulating and infiltrating B cells in CRC. By revealing the heterogeneity and functional differences of B cells in cancer immunity, we aim to deepen our understanding of immune regulation and provide a scientific basis for the development of more effective cancer treatment strategies. To explore the role of circulating and infiltrating B cell subsets in the immune microenvironment of CRC, explore the potential driving mechanism of B cell development, analyze the interaction between B cells and other immune cells in the immune microenvironment and the functions of communication molecules, and search for possible regulatory pathways to promote the anti-tumor effects of B cells. A total of 69 paracancer (normal), tumor and peripheral blood samples were collected from 23 patients with CRC from The Cancer Genome Atlas database (https://portal.gdc.cancer.gov/). After the immune cells were sorted by multicolor flow cytometry, the single cell transcriptome and B cell receptor group library were sequenced using the 10X Genomics platform, and the data were analyzed using bioinformatics tools such as Seurat. The differences in the number and function of B cell infiltration between tumor and normal tissue, the interaction between B cell subsets and T cells and myeloid cell subsets, and the transcription factor regulatory network of B cell subsets were explored and analyzed. Compared with normal tissue, the infiltrating number of CD20+B cell subsets in tumor tissue increased significantly. Among them, germinal center B cells (GCB) played the most prominent role, with positive clone expansion and heavy chain mutation level increasing, and the trend of differentiation into memory B cells increased. However, the number of plasma cells in the tumor microenvironment decreased significantly, and the plasma cells secreting IgA antibodies decreased most obviously. In addition, compared with the immune microenvironment of normal tissues, GCB cells in tumor tissues became more closely connected with other immune cells such as T cells, and communication molecules that positively regulate immune function were significantly enriched. The role of GCB in CRC tumor microenvironment is greatly enhanced, and its affinity to tumor antigen is enhanced by its significantly increased heavy chain mutation level. Meanwhile, GCB has enhanced its association with immune cells in the microenvironment, which plays a positive anti-tumor effect.

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 LC-MS/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.

Relationship Between Farnesoid X Receptor and Macrophage Polarization Status in Colorectal Cancer

Objective: Most colorectal cancer patients are insensitive to immunotherapy, which is associated with the polarization state of macrophages in the immune microenvironment. Research indicates that the farnesoid X receptor can modulate the immune microenvironment of colorectal cancer, but its relationship with the polarization status of macrophages requires further investigation. This study aims to investigate the expression of farnesoid X receptor in colorectal cancer, and analyze its correlation with the clinical pathological features of colorectal cancer and the polarization status of macrophages. Methods: Pathological specimens from 31 cases diagnosed with colorectal adenocarcinoma and undergoing surgery at Wuhan Central Hospital between January 2022 and December 2022 were collected. Immunohistochemistry was employed to assess the expression of farnesoid X receptor in tumor cells and tumor stroma. The quantity of CD86-positive and CD206-positive cells in the samples was also measured to reflect the infiltration of M1 and M2 macrophages in the tumor immune microenvironment. Describe the expression of farnesoid X receptor in colorectal cancer tumor cells and tumor stroma, and analyze the relationship between farnesoid X receptor expression and clinical pathological features and macrophage polarization status. Results: The positive rates of farnesoid X receptor expression in tumor cells and tumor stroma were 29.03% and 45.16%, respectively. The expression of farnesoid X receptor in tumor cells was associated with tumor location (P=0.015), with a higher incidence of farnesoid X receptor expression loss in left-sided colon cancer compared to right-sided colon cancer. The expression of farnesoid X receptor in tumor stroma was correlated with macrophage polarization status. The positive expression of farnesoid X receptor in stroma was associated with a higher infiltration of M1-type macrophages (P=0.008) and a higher M1/M2 ratio compared to the farnesoid X receptor expression loss group (P=0.003), suggesting a better response to immunotherapy and a favorable prognosis. Conclusion: Farnesoid X receptor is expressed in both colorectal cancer tumor cells and tumor stroma, showing a close association with the primary site of colorectal cancer and the polarization status of macrophages. The positive expression of farnesoid X receptor in tumor cells is predominantly observed in right-sided colorectal cancer, while the positive expression of farnesoid X receptor in tumor stroma is associated with macrophage polarization towards the M1 phenotype.

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

A Pyroptosis-Related Gene Signature Predicts Prognosis and Tumor Immune Microenvironment in Colorectal Cancer.

Pyroptosis is a programmed cell death, which garners increasing attention by relating to immune and therapy response. However, the role of pyroptosis in colorectal cancer (CRC) remains unclear. Our study mainly to explore the role of pyroptosis in CRC. The mRNA expression data and corresponding clinical information of CRC patients were achieved from The Cancer Genome Atlas (TCGA). Pyroptosis-related genes (PRGs) were identified using DESeq2 R package and biological function was analyzed using cluster Profiler R package. A PRGs-based prognosis model was constructed by a univariate Cox and LASSO regression analyses. Then, the affecting of risk signature to clinicopathological characteristics, immune status and infiltrated immune cells, immune checkpoint and chemotherapy sensitivity was analyzed. qRT-PCR and IHC were performed for the expression level of PRGs. Moreover, a nomogram predict model was constructed. Total 57 PRGs were identified between 500 CRC samples and 44 normal samples. Those PRGs mainly enriched in immune-related and pyroptosis-related pathways. GABRD, NADK, TMEM240, RER1, AGRN, UBE2J2, CALML6, PLCH2, TMEM88B have been identified as gene signature and a prognostic model was constructed and validated. CRC patients with high-risk score showed poor survival, high TMB score, high proportion of CD4 + memory T cells, common lymphoid progenitors, cancer associated fibroblasts, mast cells, and neutrophils. The immune checkpoint related genes, CD160, CD200R1, CD244, CD28, CD40LG, CD44, CD48, CD80, CD86, HHLA2, ICOS, IDO1, TIGIT, TNFRSF25, TNFRSF4, TNFRSF9, TNFSF15, TNFSF18 also increased in high-risk score group. CRC patients with high-risk score more sensitive to docetaxel and rapamycin but resistance to gemcitabine and mitomycin. Moreover, a predictive nomogram for 1-, 3-, 5-year for CRC patients was established and validated. In the study, a PRGs-based prognostic model and a predictive model were constructed. These models are effective and robust in prediction the 1-, 3-, and 5-year survival of CRC patients.

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.