Immune Response In Colorectal Cancer Research Articles

Co-Colorectal cancer stem cells employ the FADS1/DDA axis to evade NK cell-mediated immunosuppression after co-cultured with NK cells under hypoxia

Colorectal cancer (CRC) ranks as China’s second most common cancer and fifth top cancer death cause. The study highlights the role of Natural Killer (NK) cells in targeting cancer stem cells (CSCs) that evade immune responses in CRC. Colorectal cancer stem cells (CCSCs) were stem from HT-29 cells and co-cultured with NK cells under normoxic or hypoxic conditions. The impact of this co-culture was evaluated using CCK8 assays for NK cell viability, ELISA for cytokine level changes, and flow cytometry for assessing NK cell apoptosis and activation. Comprehensive metabolomic and transcriptomic analyses were also performed to identify key genes and metabolites involved in the interaction between CCSCs and NK cells Co-culture of CCSCs with NK cells under hypoxia reduced NK cytotoxicity, increased NK apoptosis, and altered cytokine secretion by decreasing IFN-γ and TNF-α levels while increasing IL-6. Transcriptomic and metabolomic analysis identified 4 genes (FADS1, ALDH3A2, GCSH, MTCL1) and 3 metabolites (glyoxylic acid, spermine, DDA) as significant. Interfering with FADS1 counteracted the suppression of IFN-γ and TNF-α induced by CSC cells. Curiously, this inhibition caused by si-FADS1 could be neutralized by the addition of exogenous DDA. Co-culturing with NK cells notably increased spermine levels. Exogenous spermine resulted in a significant reduction in HT-29 cell death rates at 32 µM, 64 µM, and 128 µM, compared to NK cells without spermine. Our research explored CCSCs employed the FADS1/DDA axis to evade NK cell-mediated immunosuppression after co-cultured with NK cells under hypoxia.

Machine learning-based model for CD4+ conventional T cell genes to predict survival and immune responses in colorectal cancer

Globally, CRC ranks as a principal cause of mortality, with projections indicating a substantial rise in both incidence and mortality by the year 2040. The immunological responses to cancer heavily rely on the function of CD4Tconv. Despite this critical role, prognostic studies on CRC-related CD4Tconv remain insufficient. In this investigation, transcriptomic and clinical data were sourced from TCGA and GEO. Initially, we pinpointed CD4TGs using single-cell datasets. Prognostic genes were then isolated through univariate Cox regression analysis. Building upon this, 101 machine learning algorithms were employed to devise a novel risk assessment framework, which underwent rigorous validation using Kaplan-Meier survival analysis, univariate and multivariate Cox regression, time-dependent ROC curves, nomograms, and calibration plots. Furthermore, GSEA facilitated the examination of these genes’ potential roles. The RS derived from this model was also analyzed for its implications in the TME, and its potential utility in immunotherapy and chemotherapy contexts. A novel prognostic model was developed, utilizing eight CD4TGs that are significantly linked to the outcomes of patients with CRC. This model’s RS showcased remarkable predictive reliability for the overall survival rates of CRC patients and strongly correlated with malignancy levels. RS serves as an autonomous prognostic indicator, capable of accurately forecasting patient prognoses. Based on the median value of RS, patients were categorized into subgroups of high and low risk. The subgroup with higher risk demonstrated increased immune infiltration and heightened activity of genes associated with immunity. This investigation’s establishment of a CD4TGs risk model introduces novel biomarkers for the clinical evaluation of CRC risks. These biomarkers may enhance therapeutic approaches and, in turn, elevate the clinical outcomes for patients with CRC by facilitating an integrated treatment strategy.

UCN-Centric Prognostic Model for Predicting Overall Survival and Immune Response in Colorectal Cancer.

Colorectal cancer (CRC), a prevalent malignancy, ranks third in global incidence and second in mortality rates. Despite advances in screening methods such as colonoscopy, the accurate diagnosis of CRC remains challenging due to the absence of reliable biomarkers. This study aimed to develop a robust prognostic model for precise CRC outcome prediction. Employing weighted co-expression network analysis (WGCNA) and Cox regression analysis on data from The Cancer Genome Atlas (TCGA), we identified a panel of 12 genes strongly associated with patient survival. This gene panel facilitated accurate CRC outcome predictions, which is also validated via the external validation cohort GSE17536. We conducted further investigations into the key gene, urocortin (UCN), using single-cell transcriptomic data and immune infiltration analysis in CRC patients. Our results revealed a significant correlation between high UCN expression and the reduced prevalence of key immune cells, including B cells, CD4+ cytotoxic T cells, CD8+ T cells, and NKT cells. Functional experiments showed that UCN gene interference in the CRC cell lines significantly decreased cancer cell proliferation, underscoring UCN's role in intestinal immunity modulation. The UCN-centric prognostic model developed enhances prognosis prediction accuracy and offers critical insights for CRC diagnosis and therapeutic interventions.

Stromal CD4 (+) T Cell Subsets Mediate Antitumor Cytotoxic Immune Responses in Human Colorectal Carcinoma.

The local immune response in colorectal cancer is closely related to prognosis and therapeutic efficacy. In this study, histological analyses were performed to determine the phenotype of tumor-infiltrating lymphocytes (TILs) and their infiltration in the stromal and intratumoral regions, aiming to elucidate their interactions and prognostic effects. Multiplex fluorescent labeling was performed using surgically resected colorectal cancer specimens to investigate the infiltration of CD45RO (+) TILs, which exhibit cytotoxicity, and subsets of CD4 (+) TILs, identified by their characteristic transcription factor expression. The degree of CD45RO (+) TIL infiltration in the entire observation field or stromal area was not associated with prognosis. However, a high degree of infiltration in the tumor nest (intratumoral area) was significantly associated with a favorable prognosis. CD4 (+) TILs and their subsets were not associated with prognosis. However, stratified analyses revealed that a high degree of infiltration of stromal CD4 (+) TILs and the subsets T helper (Th)1, Th2, Th17, and regulatory T cells is necessary for the association between high intratumoral CD45RO (+) TIL infiltration and favorable prognosis. A sufficient degree of infiltration of stromal CD4 (+) TIL subsets is required for intratumoral CD45RO (+) TILs to exert toxicity against cancer cells. This highlights the significance of stromal immune reactions in achieving effective cytotoxic immune responses in the intratumoral area and demonstrates the critical role of the spatial distribution pattern of TILs in exerting their functions.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

Correlation of gasdermin B staining patterns with prognosis, progression, and immune response in colorectal cancer

BackgroundPyroptosis is a type of programmed cell death mediated by the gasdermin family. Gasdermin B (GSDMB), as a member of gasdermin family, can promote the occurrence of cell pyroptosis. However, the correlations of the GSDMB expression in colorectal cancer with clinicopathological predictors, immune microenvironment, and prognosis are unclear.MethodsSpecimens from 267 colorectal cancer cases were analyzed by immunohistochemistry to determine GSDMB expression, CD3+, CD4+, and CD8+ T lymphocytes, CD20+ B lymphocytes, CD68+ macrophages, and S100A8+ immune cells. GSDMB expression in cancer cells was scored in the membrane, cytoplasm, and nucleus respectively. GSDMB+ immune cell density was calculated. Univariate and multivariate survival analyses were performed. The association of GSDMB expression with other clinicopathological variables and immune cells were also analyzed. Double immunofluorescence was used to identify the nature of GSDMB+ immune cells. Cytotoxicity assays and sensitivity assays were performed to detect the sensitivity of cells to 5-fluorouracil.ResultsMultivariate survival analysis showed that cytoplasmic GSDMB expression was an independent favorable prognostic indicator. Patients with positive cytoplasmic or nuclear GSDMB expression would benefit from 5-fluorouracil based chemotherapy. The assays in vitro showed that high GSDMB expression enhanced the sensitivity of colorectal cancer cells to 5-fluorouracil. Patients with positive membranous or nuclear GSDMB expression had more abundant S100A8+ immune cells in the tumor invasive front. Positive nuclear GSDMB expression indicated more CD68+ macrophages in the tumor microenvironment. Moreover, GSDMB+ immune cell density in the stroma was associated with a higher neutrophil percentage but a lower lymphocyte counts and monocyte percentage in peripheral blood. Furthermore, the results of double immunofluorescence showed that GSDMB co-expressed with CD68 or S100A8 in stroma cells.ConclusionThe GSDMB staining patterns are linked to its role in cancer progression, the immune microenvironment, systemic inflammatory response, chemotherapeutic efficacy, and prognosis. Colorectal cancer cells with high GSDMB expression are more sensitive to 5-fluorouracil. However, GSDMB expression in immune cells has different effects on cancer progression from that in cancer cells.

Abstract 3084: Aldob regulates fructose metabolism to confer ferroptosis and immune response in colorectal cancer harboring microsatellite instability

Abstract Microsatellite instability (MSI) is a genetic alteration that occurs during cancer progression, specifically in colorectal cancer. Objective evidence demonstrates that colorectal cancer patients with MSI have better survival rates than those without MSI. Moreover, patients with both dMMR and MSI respond equally well to immunotherapy. We evaluated the MSI status of colorectal cancer specimens and identified an activation of aldolase B (ALDOB), the primary regulator of fructose metabolism, in the MSI group. Our analysis of the cancer cell panel revealed that the expression level of ALDOB in MSI+ cells (HCT116 and LoVo) was significantly higher than that in the microsatellite stable (MSS) group (SW480 and HT29). We also discovered a significant negative correlation between ALDOB and numerous dMMR-related genes, such as MLH1 and MSH2. We investigated how fructose concentration and pathway activation affected MSI occurrence. Our results demonstrated that abnormal fructose metabolism leaded to SLC7A11-mediated ferroptosis. SLC7A11 functioned as the active subunit and generated glutathione, GPX4, and lipid epoxides in response to transported cystine. This may explain the better clinical outcomes observed in colorectal patients with MSI. On the other hand, we also observed that ALDOB could increase certain immune ratios, such as CD4+ T cells and B cells. This aligns with the implementation of immunotherapy in the MSI community, which has been proven to yield positive effects. Consistent results were still being shown. Our study revealed significant increases in various chemokines and cytokines in media conditioned with fructose. It was interesting to note that fructose resulted in more significant changes than glucose. Based on the gathered evidence, it could be posited that ALDOB plays a crucial role as a promoter of fructose metabolism, leading to the induction of ferroptosis and immune responses in MSI colorectal cancer. The findings shed new light on known consequences and provide new avenues of information. Citation Format: Yu-Chan Chang, Chi-Long Chen. Aldob regulates fructose metabolism to confer ferroptosis and immune response in colorectal cancer harboring microsatellite instability [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 3084.

Abstract 5492: Spatial organization and cellular composition of immunity hubs in human colorectal cancer

Abstract Background: Among colorectal cancer (CRC) tumors, mismatch repair deficient (MMRd) tumors have more immunogenic neo-antigens, hence more cytotoxic T-cell mediated anti-tumor immunity than mismatch repair proficient (MMRp) tumors. Previously1, we transcriptionally profiled over 300,000 cells from 62 MMRd and MMRp CRC tumors and found an MMRd-unique network of immunologically active transcriptional states enriched in interferon-stimulated genes (ISGs). With 4-color imaging, we showed that this program arises from spatially organized hubs of at least 3 cell types: IFNã-secreting T and interferon-responsive CXCL9/10/11+ myeloid and tumor cells. To characterize the breadth of cells involved in tumor immunity hubs, we must simultaneously measure the expression of 100s of genes in situ. Methods: To describe the cellular composition, structural organization and intercellular spatial interactions of these immunity hubs, we profiled primary untreated resection specimens from 17 CRC donors (n=12 MMRd, 5 MMRp) using multiplexed error-robust FISH (MERFISH) mRNA microscopy. Efforts were made to sample both the tumor body and the invasive border of the tumor. We designed a panel of 479 genes to capture key transcriptional programs from our single-cell atlas1. We wrote analytical pipelines to segment transcripts into cells, select high-quality cells, and organize cells into distinct zones in tissue. To leverage the rich information in the scRNA-seq atlas1, we designed a robust label transfer analysis to assign cell state labels to MERFISH cells by comparison to scRNAseq cells. Results: We profiled 7.5 million high quality MERFISH cells across 21 CRC tissue sections and labeled the cells into 7 major lineages: 63.19% epithelial, 22.52% stromal, 7.75% myeloid, 3.72% T/NK/ILCs, 1.45% plasma, 1.2% B, and 0.17% mast cells. We visualized the spatial location of these cell types. T cells aggregated into 3 distinct regions of tissue: tertiary lymphoid structures (TLS) outside the tumor, and smaller clusters near the invasive border and in the tumor center. We identified immunity hubs defined by colocalized CXCL9/10/11 expressing cells and T cells near the invasive border and in the center of MMRd tumors. As expected, we found enrichment of CXCL13, IFNG, and ISGs such as CD74 and MHCII genes at these locations. In contrast, we found evidence of CCL17+CCL19+LAMP3+ activated dendritic cells and B cells enriched in the TLS. These results support a distinct composition and localization of immunity hubs in tumors, in contrast to TLS outside tumors. Conclusions: Our study defines the composition, spatial organization, and intercellular interactions of immunity hubs, shedding light on the anti-tumor immune response in CRC. Ethics Approval: Approved by the DF/HCC Institutional Review Board (protocol #02-240).(1) Pelka, K. et al. Spatially organized multicellular immune hubs in human colorectal cancer. Cell 184, 4734-4752.e20 (2021) Citation Format: Mukta G. Palshikar, Maxwell Spurrell, Jack Demaray, Han Chen, Scott Engels, Roopa Madhu, Milan Parikh, Izabella Gazmora, Arnav Mehta, Nicolas Fernandez, Colles Price, Jiang He, Sonia Cohen, Karin Pelka, Nir Hacohen, Jonathan Chen, Ilya Korsunsky. Spatial organization and cellular composition of immunity hubs in human 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 5492.

Three-in-One Peptide Prodrug with Targeting, Assembly and Release Properties for Overcoming Bacterium-Induced Drug Resistance and Potentiating Anti-Cancer Immune Response.

The presence of bacteria in tumor results in chemotherapeutic drug resistance and weakens the immune response in colorectal cancer. To overcome bacterium-induced chemotherapeutic drug resistance and potentiate antitumor immunity, herein a novel molecule Biotin-Lys(SA-Cip-OH)-Lys(SA-CPT)-Phe-Phe-Nap (Biotin-Cip-CPT-Nap) is rationally designed containing four functional motifs (i.e., a biotin motif for targeting, Phe-Phe(-Nap) motif for self-assembly, ciprofloxacin derivative (Cip-OH) motif for antibacterial effect, and camptothecin (CPT) motif for chemotherapy). Using the designed molecule, a novel strategy of intracellular enzymatic nanofiber formation and synergistic antibacterium-enhanced chemotherapy and immunotherapy is achieved. Under endocytosis mediated by highly expressed biotin receptor in colorectal cancer cell membrane and the catalysis of highly expressed carboxylesterase in the cytoplasm, this novel molecule can be transformed into Biotin-Nap, which self-assembled into nanofibers. Meanwhile, antibiotic Cip-OH and chemotherapeutic drug CPT are released, overcoming bacterium-induced drug resistance and enhancing the therapeutic efficacy of immunotherapy towards colorectal cancer. This work offers a feasible strategy for the design of novel multifunctional prodrugs to improve the efficiency of colorectal cancer treatment.

Abstract A025: Adaptive therapy: Leveraging evolutionary dynamics for colorectal cancer management in vivo

Abstract Therapeutic resistance to chemotherapy is frequently the proximate cause of cancer treatment failure and, ultimately, patient mortality. Evolutionary therapies have been developed to address this problem and potentially reduce toxicities. The most successful strategy to date, adaptive therapy (AT), is designed to use chemosensitive cells to out-compete and suppress the chemoresistant cells. However, detailed observations to elucidate the mechanisms of AT's success are lacking. Typically, AT involves lowering the dose of the chemotherapy, thereby reducing toxicity and preserving the immune system. We are evaluating AT for the first time in colorectal cancer, using syngeneic orthotopic xenograft models. This project marks several firsts: 1) sensitive and resistant clones will be tracked longitudinally, 2) AT will be tested with a targeted therapy (MRTX1133), 3) AT will be tested in an immune competent model, 4) multiple drugs will be used for AT, and 5) extinction therapy will be tested in mice. We will employ genetic barcodes and differential luciferase tagging to track the tumor cell subpopulations longitudinally. Currently, we are conducting an in-vivo pilot experiment to validate our protocol, emphasizing tumor engraftment efficacy and tracking cell subpopulations. Additionally, we are investigating the potential of Lactobacillus acidophilus to inhibit tumor growth, augment chemotherapy, and bolster immune response in colorectal cancer. Citation Format: Harley I. Richker, Gissel Marquez-Alcaraz, Carlo Maley. Adaptive therapy: Leveraging evolutionary dynamics for colorectal cancer management in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A025.

Oral probiotics microgel plus Galunisertib reduced TGF-β blockade resistance and enhanced anti-tumor immune responses in colorectal cancer

Transforming growth factor β (TGF-β), a versatile immunosuppressive cytokine, has gained increasing attention as a potential target for cancer immunotherapy. However, current strategies are constrained by tumor heterogeneity and drug resistance. Therapeutic probiotics, such as Escherichia coli Nissle1917 (EcN), not only regulate the gut microbiota to increase beneficial bacteria with anti-tumor effects, but also modulate immune factors within the body, thereby enhancing immunity. In this study, we developed an oral microgel delivery system of EcN@(CS-SA)2 by electrostatic interaction between chitosan (CS) and sodium alginate (SA), aiming to enhance its bioavailability in the gastrointestinal tract (GIT). Notably, EcN@(CS-SA)2 microgel showed a synergistic enhancement of the anti-tumor efficacy of Galunisertib (Gal, a TGF-β inhibitor) by inducing apoptosis and immunogenic cell death (ICD) in tumor cells, as well as promoting increased infiltration of CD8+ T cells into the tumor microenvironment (TME).

Dual role of CD73 as a signaling molecule and adenosine-generating enzyme in colorectal cancer progression and immune evasion.

Metastasis and limited benefits of immune checkpoint blockade are two obstacles to the battle against colorectal cancer (CRC). CD73, encoded by the gene 5'-Nucleotidase Ecto (NT5E), is a major enzyme that generates extracellular adenosine. However, whether CD73 affects cancer progression and immune response in CRC remains unclear. Here, the clinical significance of CD73 was assessed in human CRC specimens using immunohistochemistry and bioinformatic analyses. We demonstrated that CD73 is elevated in CRC tissues, particularly in those with metastasis, and correlates with poor prognosis. Gain- and loss-of-function experiments demonstrate that tumor CD73 supports tumor progression and impairs the viability and effector functions of CD8+ T cells. Targeting CD73 on CRC cells reduces their malignant phenotypes and improves the anti-cancer response of CD8+ T cells in the tumor microenvironment (TME). Moreover, the combination of CD73 blockade and PD-1 inhibitors exhibited enhanced anti-cancer effects when compared to a single-agent treatment. Thus, CD73 may be a promising target in the treatment of CRC.

Targeting acid ceramidase enhances antitumor immune response in colorectal cancer

IntroductionAcid ceramidase (hereafter referred as ASAH1) is an enzyme in sphingolipid metabolism that converts pro-survival ceramide into sphingosine. ASAH1 has been shown to be overexpressed in certain cancers. However, the role of ASAH1 in colorectal cancer still remain elusive. ObjectiveThe present study is aimed to understand how ASAH1 regulates colorectal cancer (CRC) progression and resistance to checkpoint inhibitor therapy. MethodsBoth pharmacological and genetic silencing of ASAH1 was used in the study. In vitro experiments were done on human and mouse CRC cell lines. The in vivo studies were conducted in NOD-SCID and BALB/c mice models. The combination of ASAH1 inhibitor and checkpoint inhibitor was tested using a syngeneic tumor model of CRC. Transcriptomic and metabolomic analyses were done to understand the effect of ASAH1 silencing. ResultsASAH1 is overexpressed in human CRC cases, and silencing the expression resulted in the induction of immunological cell death (ICD) and mitochondrial stress. The ASAH1 inhibitor (LCL-521), either as monotherapy or in combination with an anti-PD-1 antibody, resulted in reduction of tumors and, through induction of type I and II interferon response, activation of M1 macrophages and T cells, leading to enhanced infiltration of cytotoxic T cells. Our findings supported that the combination of LCL-521 and ICIs, which enhances the antitumor responses, and ASAH1 can be a druggable target in CRC.

Curcumin/L-OHP co-loaded HAP for cGAS-STING pathway activation to enhance the natural immune response in colorectal cancer.

Insufficient immune cell infiltration into the tumor microenvironment (TME) greatly compromises the clinical application of immune-checkpoint inhibitors (ICIs)-based immunotherapy. Recent findings have shown that activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway can enhance natural immunity and increase lymphocyte infiltration into the TME, which presents a promising strategy for cancer immunotherapy. In this study, we constructed hydroxyapatite nanoparticles co-loaded with curcumin and L-oxaliplatin (Cur/L-OHP@HAP NPs). We analyzed the particle-size distribution, zeta potential, spectral characteristics (Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, ultraviolet-visible spectroscopy), and drug-release properties of the Cur/L-OHP@HAP NPs. The cellular uptake of the Cur/L-OHP@HAP NPs detected by flow cytometry and confocal laser-scanning microscopy. We comprehensively evaluated the anti-tumor properties and immune-activating effects of the NPs, both in vitro and in vivo. Physicochemical characterizations demonstrated that the Cur/L-OHP@HAP NPs were successfully synthesized and were capable of pH-dependent drug release. Notably, the Cur/L-OHP@HAP NPs efficiently entered cancer cells, after which the released L-OHP induced nuclear DNA (nDNA) damage to some extent. HAP promoted the release of intracellular Ca2+ stores in cancer cells, and curcumin inhibited Ca2+ efflux, resulting in intracellular Ca2+ overload and the release of mitochondrial DNA (mtDNA). Damage to both nDNA and mtDNA greatly stimulated the cGAS-STING pathway, thereby activating natural immunity, accompanied by immune cell recruitment to the TME. In summary, the Cur/L-OHP@HAP NPs show good prospects for improving cancer immunotherapy.

Senescence risk score: a multifaceted prognostic tool predicting outcomes, stemness, and immune responses in colorectal cancer.

Colorectal cancer (CRC) remains a primary cause of cancer mortality globally, necessitating precise prognostic indicators for effective clinical management. Our study introduces the Senescence Risk Score (SRRS), based on several senescence-related genes (SRGs), a potent prognostic tool designed to measure cellular senescence in CRC. The higher SRRS predicts a poorer prognosis, providing a novel and efficient approach to patient stratification. Notably, we found that SRRS correlates with methylation and mutation variations, and increased immune infiltration in the tumor microenvironment, thus revealing potential therapeutic targets. We also discovered an inverse relationship between SRRS and cell stemness, which could have significant implications for cancer treatment strategies. Utilizing bioinformatics resources and machine learning, we identified LIMK1 and WRN as key genes associated with SRRS, further enhancing its prognostic value. Importantly, the modulation of these genes significantly impacts cellular senescence, proliferation, and stemness in CRC cells. In summary, our development of SRRS offers a powerful tool for CRC prognosis and paves the way for novel therapeutic strategies, underscoring its potential in transforming CRC patient management.

Bacterial lipopolysaccharide modulates immune response in the colorectal tumor microenvironment

Immune responses can have opposing effects in colorectal cancer (CRC), the balance of which may determine whether a cancer regresses, progresses, or potentially metastasizes. These effects are evident in CRC consensus molecular subtypes (CMS) where both CMS1 and CMS4 contain immune infiltrates yet have opposing prognoses. The microbiome has previously been associated with CRC and immune response in CRC but has largely been ignored in the CRC subtype discussion. We used CMS subtyping on surgical resections from patients and aimed to determine the contributions of the microbiome to the pleiotropic effects evident in immune-infiltrated subtypes. We integrated host gene-expression and meta-transcriptomic data to determine the link between immune characteristics and microbiome contributions in these subtypes and identified lipopolysaccharide (LPS) binding as a potential functional mechanism. We identified candidate bacteria with LPS properties that could affect immune response, and tested the effects of their LPS on cytokine production of peripheral blood mononuclear cells (PBMCs). We focused on Fusobacterium periodonticum and Bacteroides fragilis in CMS1, and Porphyromonas asaccharolytica in CMS4. Treatment of PBMCs with LPS isolated from these bacteria showed that F. periodonticum stimulates cytokine production in PBMCs while both B. fragilis and P. asaccharolytica had an inhibitory effect. Furthermore, LPS from the latter two species can inhibit the immunogenic properties of F. periodonticum LPS when co-incubated with PBMCs. We propose that different microbes in the CRC tumor microenvironment can alter the local immune activity, with important implications for prognosis and treatment response.

P-306 Bacterial lipopolysaccharide modulates immune responses in colorectal cancer

P-306 Bacterial lipopolysaccharide modulates immune responses in colorectal cancer

Tigecycline reduces tumorigenesis in colorectal cancer via inhibition of cell proliferation and modulation of immune response

Backgroundand Purpose: Colorectal cancer (CRC) is one of the cancers with the highest incidence in which APC gene mutations occur in almost 80% of patients. This mutation leads to β-catenin aberrant accumulation and an uncontrolled proliferation. Apoptosis evasion, changes in the immune response and microbiota composition are also events that arise in CRC. Tetracyclines are drugs with proven antibiotic and immunomodulatory properties that have shown cytotoxic activity against different tumor cell lines. Experimental approachThe effect of tigecycline was evaluated in vitro in HCT116 cells and in vivo in a colitis-associated colorectal cancer (CAC) murine model. 5-fluorouracil was assayed as positive control in both studies. Key resultsTigecycline showed an antiproliferative activity targeting the Wnt/β-catenin pathway and downregulating STAT3. Moreover, tigecycline induced apoptosis through extrinsic, intrinsic and endoplasmic reticulum pathways converging on an increase of CASP7 levels. Furthermore, tigecycline modulated the immune response in CAC, reducing the cancer-associated inflammation through downregulation of cytokines expression. Additionally, tigecycline favored the cytotoxic activity of cytotoxic T lymphocytes (CTLs), one of the main immune defenses against tumor cells. Lastly, the antibiotic reestablished the gut dysbiosis in CAC mice increasing the abundance of bacterial genera and species, such as Akkermansia and Parabacteroides distasonis, that act as protectors against tumor development. These findings resulted in a reduction of the number of tumors and an amelioration of the tumorigenesis process in CAC. Conclusion and implicationsTigecycline exerts a beneficial effect against CRC supporting the use of this antibiotic for the treatment of this disease.

Abstract 2332: VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction

Abstract Background: Checkpoint immunotherapy using humanized antibody is a new therapeutic strategy against advanced colorectal cancer (CRC). Nonetheless, it shows low tumor penetration due to the large molecule size. Therefore, small immune regulatory molecules are actively sought. Our previous study showed that a small secretory protein VSTM2A suppressed Wnt signalling pathway through inducing LRP6 lysosome dependent degradation. However, its role in immune modulation is largely unknown. Methods: Clinicopathological features were analyzed using CRC patient cohort. Functional biology of VSTM2A was investigated using T cell-antigen presenting cell (APC) co-culture models and knockout mice model. Liquid chromatography with tandem mass spectrometry (LC-MS) was carried out for mechanistic investigation. Results: Gene ontology analysis of colon RNA-seq dataset from TCGA/GTEx revealed significant enrichment of adaptive immune response and lymphocyte activation with high VSTM2A expression. CRC patients with inferior VSTM2A expressions were associated with lower immunoscores in both early and late stages of CRC. In addition, our immunohistochemical examinations of 208 in-house CRC cases further pinpoints to a considerable fraction of VSTM2A-negative cases showing avoidance of spontaneous CD8a+ cells infiltration (Pearson's chi-squared test P < 0.001, φ = 0.24) and worse clinical outcome. LC-MS assay followed by reciprocal immunoprecipitation revealed that VSTM2A interacted with the IgV motif of immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1). We further found that VSTM2A recognized PD-L1 with a dissociation constant (Kd) of 2.6 nM. Recombinant VSTM2A dose dependently suppressed the interaction of PD-L1 and PD-1 with IC50 of 4.51 nM, indicating VSTM2A is a potential intrinsic PD-L1 antagonist. Moreover, our in-vitro mechanistic studies revealed that VSTM2A protein could block the interaction of PD-1/PD-L1, and induced NFAT-RE luciferase activity dose-dependently using a bioluminescent cell-based PD-1/PD-L1 blockade bioassay. VSTM2A could dramatically activate the interleukin-2 production from DO11.10 T cell in the presence non-T cell spherocyte isolated from BALB/c mice and OVA323-339 peptide. Furthermore, our in-vivo study revealed that VSTM2A triggered a distinct increase in CD45+CD3+ T cells infiltration in the syngeneic MC38 mouse model. Vstm2a+/- knockout mice challenged with azoxymethane and dextran sodium sulfate showed significantly high CRC incidence and tumor burden in comparison to wild-type littermates. Tumor from Vstm2a+/- mice showed significantly less CD8+ T cell infiltration. Conclusions: our study demonstrate the tumor extrinsic role of VSTM2A in modulation of microenvironment through abrogating PD-L1/PD-1 interaction. Silencing of VSTM2A in CRC could suppress T cell activation and infiltration in the tumor sites. Citation Format: Yujuan Dong, Yunfei Zhou, Nathalie Wong, Jun Yu, Siu Man Simon Ng. VSTM2A modulation of immune response in colorectal cancer through abrogating PD-L1 and PD-1 interaction [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 2332.

Targeting CD43 optimizes cancer immunotherapy through reinvigorating antitumor immune response in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies worldwide, with dramatically increasing incidence and mortality for decades. However, current therapeutic strategies for CRC, including chemotherapies and immunotherapies, have only demonstrated limited efficacy. Here, we report a novel immune molecule, CD43, that can regulate the tumor immune microenvironment (TIME) and serves as a promising target for CRC immunotherapy. The correlation of CD43 expression with CRC patient prognosis was revealed by public data analysis. CD43 knockout (KO) CRC cell lines were generated by CRISPR-Cas9 technology, and a syngenetic murine CRC model was established to investigate the in vivo function of CD43. The TIME was analyzed via immunohistochemical staining, flow cytometry and RNA-seq.Immune functions were investigated by depletion of immune subsets in vivo and T-cell functional assays in vitro, including T-cell priming, cytotoxicity, and chemotaxis experiments. In this study, we found that high expression of CD43 was correlated with poor survival of CRC patients and the limited infiltration of CD8+ T cells in human CRC tissues. Importantly, CD43 expressed on tumor cells, rather than host cells, promoted tumor progression in a syngeneic tumor model. Loss of CD43 facilitated the infiltration of immune cells and immunological memory in the TIME of CRC tumors. Mechanistically, the protumor effect of CD43 depends on T cells, thereby attenuating T-cell-mediated cytotoxicity and cDC1-mediated antigen-specific T-cell activation. Moreover, targeting CD43 synergistically improved PD-L1 blockade immunotherapy for CRC. Our findings revealed that targeting tumor-intrinsic CD43 could activate the antitumor immune response and provide particular value for optimized cancer immunotherapy by regulating the TIME in CRC patients.