THBS3 Functions as a Novel Biomarker for Prognosis and Immunotherapeutic Response in Colorectal Cancer: An Integrative Analysis and Validation of the Thrombospondin Gene Family

The correlation between gut microbiota dysbiosis and inflammation-associated colorectal cancer (CRC) has generated considerable interest in dietary interventions targeting immune modulation. Our prior study demonstrated the colon cancer protective effects of rice bran (RB) and ex vivo Bifidobacterium longum fermented rice bran (FRB) diet intervention against azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC in gut microbiota-intact specific pathogen-free mice (SPF). RB showed higher levels of protection compared to FRB against CRC, as evidenced by diminished neoplastic lesion size and restored colonic epithelial integrity. The major objective of the present study was to leverage cutting-edge multispectral imaging technique for simultaneous detection of multiple immune cells markers and for a comprehensive analysis of distinct immune landscapes. We also performed spatial correlations within the tumor microenvironment of AOM/DSS-SPF mice undergoing RB and FRB dietary interventions. The primary focus was on T cell subtypes including cytotoxic T cells, helper T cells, regulatory T (Treg) cells, natural killer (NK) cells, NKT cells, B cells and macrophages (MΦ) subtypes (M1 MΦ, and M2 MΦ) to uncover the intricacies of the immune landscape and the interplay for CRC prevention through dietary intervention. The results indicated that RB intervention for 15 weeks led to reductions in overall T cell subtypes in the colonic tissue, particularly Treg cells, which are associated with protecting tumors against immune surveillance in CRC. On the other hand, FRB intervention also resulted in reductions in overall T cell subtypes but exhibited higher infiltration of NK cells and proinflammatory M1 macrophages in the colonic tissue after 15 weeks. While an increased presence of NK cells might be beneficial in countering CRC, the infiltration of M1 macrophages could potentially lessen the impact of FRB compared to RB intervention in CRC protection. The outcomes suggest that the better protective efficacy of non-fermented RB diets is linked to the modulation of Treg cell infiltration, whereas the efficacy of the FRB diet is to some extent limited by the presence of M1 macrophages. Overall, this study provides valuable insights into the complex immune landscape influenced by RB and FRB interventions in a murine model of inflammation-induced CRC, and a novel mechanism for the immunomodulatory effects of dietary RB interactions with gut microbiota. In the future, observing immune modulation throughout diet intervention would enhance our understanding of the role played by these immune cells. Citation Format: Robin Kumar, Lakshmi Sai Pratyusha Bugata, Md. Imtiazul Kabir, Rajesh Agarwal, Elizabeth P. Ryan, Komal Raina. Modulation of immune infiltration patterns in inflammation-associated colorectal cancer through rice bran-based dietary strategies [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 2180.

Background: Fusobacterium nucleatum (Fn) appears to play a role in colorectal cancer carcinogenesis through suppression of antitumor immune response. This study aims to characterize immune-related signatures, including the expression of multiple immune checkpoint molecules in tumor-infiltrating T cells. Methods: We measured Fn DNA by quantitative PCR in a total of 155 colorectal cancer tissues. Gene expression profile was studied using microarray technology to determine the consensus molecular subtypes (CMS) and immune-related signatures using CIBERSORT and Gene Set Enrichment Analysis (GSEA), respectively. Expression of immune checkpoint molecules (PD-1, TIM-3, and TIGIT) on CD8+ cytotoxic T cells and the ratio of FOXP3-CD4-/FOXP3+CD4 were examined using multicolor flow cytometric analysis. Results: All patients had stage III disease underwent curative surgery. Fn infection was observed in 43% of patients and was associated with recurrence (Fisher’ exact test, P=0.002) and decreased overall survival (log-rank test, P<0.001). The distribution of CMS subtypes was CMS1/2/3/4/mixed (14%/24%/15%/27%/20%), which is comparable to previous data. Fn infection was associated with the platinum-resistant signature, hypoxia pathway, and stem cell pathways in the GSEA. Interestingly, Patients with Fn infection showed a trend toward enrichment in CMS4, mesenchymal subtype with the inflamed immune phenotype (9% vs. 17% with Fn-infection, P=0.24). The number of cytotoxic CD8+ T cells showed a trend toward decreased expression in patients with Fn infection. Furthermore, higher expression of inhibitory immune checkpoints (PD-1, P<0.01; TIM-3, P<0.05; TIGIT, P<0.05) was observed on CD8+ cytotoxic T cells of patients with Fn infection, compared to that of Fn negative cases. Besides, the regulatory FOXP3+CD4+ cells were increased in patients with Fn infection, supporting the suppressive role of Fn infection in the tumor immune microenvironment (P<0.05). Conclusions: The adverse prognostic impact of Fn infection depends on the suppression of T cell-mediated immune response by increased expression of inhibitory immune checkpoints on cytotoxic T cell and by increased regulatory T cells. Citation Format: Han Sang Kim, Wonkyu Kim, Chang Gon Kim, Jinseon Yoo, Soonmyung Paik, Eui-Cheol Shin, Tae-Min Kim, Hoguen Kim, Joong Bae Ahn. Fusobacterium nucleatum in colorectal carcinoma and expression of multiple immune checkpoint molecules on T cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C132. doi:10.1158/1535-7163.TARG-19-C132

Colorectal cancer (CRC) is regarded as one of the commonest cancer types around the world. Due to the poor understanding on the causes of CRC formation and progression, this study sets out to investigate the physiological mechanisms by which Astragalus mongholicus Bunge-Curcuma aromatica Salisb. (ARCR) regulates CRC growth and metastasis, and the role in which M2 macrophage polarization plays in this process. An orthotopic-transplant model of CRC was established to evaluate the influence of ARCR on the polarization of M2 macrophage and the growth and metastasis of tumors. Next, the binding affinity among Sp1, ZFAS1, miR-153-5p, and CCR5 was identified using multiple assays. Finally, after co-culture of bone marrow-derived macrophages (BMDM) with CRC cell line CT26.WT, the cell proliferative, invasive, and migrated abilities were assessed in gain- or loss-of-function experiments. ARCR inhibited the infiltration of M2 macrophages into tumor microenvironment to suppress the CRC growth and metastasis in vivo. Additionally, ARCR inhibited the transcription of ZFAS1 by reducing Sp1 expression to repress M2 macrophage polarization. Moreover, ZFAS1 competitively binds to miR-153-3p to upregulate the CCR5 expression. Finally, ARCR suppressed the polarization of M2 macrophages to inhibit the tumor growth and tumor metastasis in CRC by mediating the Sp1/ZFAS1/miR-153-3p/CCR5 regulatory axis. Collectively, ARCR appears to suppress the CRC cell growth and metastasis by suppressing M2 macrophage polarization via Sp1/ZFAS1/miR-153-3p/CCR5 regulatory axis. 1. ARCR suppress the CRC cell growth and metastasis 2. ZFAS1 promotes CCR5 expression by competitively binding to miR-153-3p. 3. Sp1 promotes M2 macrophage polarization by activating ZFAS1 via miR-153-3p/CCR5. 4. The study unveiled a protective target against CRC.

NG03: Marine omega-3 polyunsaturated fatty acid and colorectal cancer prevention and treatment

Due to the loss of DNA repair mechanisms in colorectal cancer (CRC) with microsatellite instability (MSI), somatic mutations accumulate within DNA; making them more prone to attack by tumor infiltrating lymphocytes (TIL) and macrophages. We hypothesize that MSI-High (MSI-H) patients have favorable survival due to increased tumor immunogenicity. The Cancer Genome Atlas (TCGA) was used to evaluate gene expression from 283 patients with CRC, comparing MSI-H and microsatellite stable (MSS) patients. CIBERSORT algorithm estimated the fraction of immune cell types. We found that low expression of DNA repair genes (MLH1, MLH3, PMS1, PMS2, ATR, PRKDC, ATM, BRCA2) associated with MSI-H. MSI-H was directly associated with Helper T-cells (p = 0.034) and M1 macrophages (p < 0.0001). MSI-H tumors associated with diminished intra-tumoral heterogeneity as well as higher expression of checkpoint molecules PD-1, PD-L1, CTLA4, LAG3 and TIM3 (p < 0.0001). Improved OS was seen in patients with low ATM, PMS2 and MLH3. In the TCGA CRC cohort, decreased expression of DNA repair genes associated with MSI-H. MSI-H patients had improved survival, likely due to higher TIL and M1 macrophage infiltration as well as lower intra-tumoral heterogeneity. MSI-H also associates with expression of immune checkpoint molecules with potential for development of therapeutic targets.

The Tumor microenvironment (TME) plays a crucial role in colorectal cancer (CRC) prognosis and treatment response. However, comprehensive understandings of TME-related immune subtypes and their mechanisms for precision medicine remain insufficient. This study aims to identify immune subtypes in CRC, develop a prognostic model, and explore the role of microbial diversity in tumor progression. Multi-omics data and non-negative matrix factorization (NMF) were used to classify CRC into immune subtypes. Differentially expressed TME-related genes were identified, and a prognostic risk model was developed using Cox and LASSO regression. Single-cell RNA sequencing (scRNA-seq) assessed cellular interactions and gene set variations. Microbiome profiling was integrated to evaluate the impact of microbial diversity on CRC progression and immune modulation. Key findings were validated using immunohistochemistry, external datasets, and qPCR in patient-derived organoids. Four TME-related immune subtypes were identified: immune-exhausted C1 (poor prognosis, high immune infiltration), immune-activated C2/C3 (better prognosis), and immune-desert C4 (worst prognosis). A risk model based on genes (SOX9, CLEC10A, RAB15, RAB6B, PCOLCE2, FUT1) stratified patients into high- and low-risk groups. High-risk groups exhibited increased Enterobacteriaceae and Clostridium, while low-risk groups showed higher Porphyromonadaceae and Peptostreptococcaceae, correlating with better immunotherapy responses. scRNA-seq revealed distinct cell-cell communication patterns across subtypes. The study highlights the complexity of CRC's TME and its role in prognosis and treatment. Findings support personalized treatment strategies, considering immune and microbial factors. This research integrates TME subtyping, risk modeling, single-cell analysis, and microbiome profiling to advance CRC prognosis and precision therapy, emphasizing personalized strategies for better outcomes.

Background Patients with relapsed metastatic osteosarcoma have no effective treatments available to them,1 and immunotherapy thus far has not succeeded in improving outcomes.2–5 We aim to understand the immune architecture...

Immune dysregulation plays a vital role in colorectal cancer initiation and progression. Long noncoding RNAs (lncRNA) exhibit multiple functions including regulation of gene expression. Here, we identified an immune-related lncRNA, MIR17HG, whose expression was gradually upregulated in adjacent, adenoma, and colorectal cancer tissue. MIR17HG promoted tumorigenesis and metastasis in colorectal cancer cells both in vitro and in vivo. Mechanistically, MIR17HG increased the expression of NF-κB/RELA by competitively sponging the microRNA miR-375. In addition, RELA transcriptionally activated MIR17HG in a positive feedback loop by directly binding to its promoter region. Moreover, miR-17-5p, one of the transcribed miRNAs from MIR17HG, reduced the expression of the tumor suppressor B-cell linker (BLNK), resulting in increased migration and invasion of colorectal cancer cells. MIR17HG also upregulated PD-L1, indicating its potential role in immunotherapy. Overall, these findings demonstrate that MIR17HG plays an oncogenic role in colorectal cancer and may serve as a promising therapeutic target. SIGNIFICANCE: These findings provide mechanistic insight into the role of the lncRNA MIR17HG and its miRNA members in regulating colorectal cancer carcinogenesis and progression.

&lt;div&gt;Abstract&lt;p&gt;Immune dysregulation plays a vital role in colorectal cancer initiation and progression. Long noncoding RNAs (lncRNA) exhibit multiple functions including regulation of gene expression. Here, we identified an immune-related lncRNA, MIR17HG, whose expression was gradually upregulated in adjacent, adenoma, and colorectal cancer tissue. MIR17HG promoted tumorigenesis and metastasis in colorectal cancer cells both &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. Mechanistically, MIR17HG increased the expression of NF-κB/RELA by competitively sponging the microRNA miR-375. In addition, RELA transcriptionally activated MIR17HG in a positive feedback loop by directly binding to its promoter region. Moreover, miR-17-5p, one of the transcribed miRNAs from MIR17HG, reduced the expression of the tumor suppressor B-cell linker (BLNK), resulting in increased migration and invasion of colorectal cancer cells. MIR17HG also upregulated PD-L1, indicating its potential role in immunotherapy. Overall, these findings demonstrate that MIR17HG plays an oncogenic role in colorectal cancer and may serve as a promising therapeutic target.&lt;/p&gt;Significance:&lt;p&gt;These findings provide mechanistic insight into the role of the lncRNA MIR17HG and its miRNA members in regulating colorectal cancer carcinogenesis and progression.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Immune dysregulation plays a vital role in colorectal cancer initiation and progression. Long noncoding RNAs (lncRNA) exhibit multiple functions including regulation of gene expression. Here, we identified an immune-related lncRNA, MIR17HG, whose expression was gradually upregulated in adjacent, adenoma, and colorectal cancer tissue. MIR17HG promoted tumorigenesis and metastasis in colorectal cancer cells both &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. Mechanistically, MIR17HG increased the expression of NF-κB/RELA by competitively sponging the microRNA miR-375. In addition, RELA transcriptionally activated MIR17HG in a positive feedback loop by directly binding to its promoter region. Moreover, miR-17-5p, one of the transcribed miRNAs from MIR17HG, reduced the expression of the tumor suppressor B-cell linker (BLNK), resulting in increased migration and invasion of colorectal cancer cells. MIR17HG also upregulated PD-L1, indicating its potential role in immunotherapy. Overall, these findings demonstrate that MIR17HG plays an oncogenic role in colorectal cancer and may serve as a promising therapeutic target.&lt;/p&gt;Significance:&lt;p&gt;These findings provide mechanistic insight into the role of the lncRNA MIR17HG and its miRNA members in regulating colorectal cancer carcinogenesis and progression.&lt;/p&gt;&lt;/div&gt;

Cancer immunotherapy using monoclonal antibodies targeting inhibitory immune checkpoint molecules is being established as a new paradigm in cancer treatment. This crucial strategy involves inhibition of the interaction between immune checkpoint receptors, expressed on the immune cells, and their respective ligands, expressed on antigen-presenting cells. While this receptor-ligand concept is well accepted in infection immunology, there have been numerous reports on the expression of immune checkpoint molecules in tumor cells. In addition to well-characterized tumor-intrinsic expression of PD11 and PD-L12, many other checkpoint molecules are associated with tumor cells3, conferring important impact on tumor biology and clinical consequences. In this study, we evaluated in situ single-cell expression profiles of immune checkpoint molecules in the tumor microenvironment (TME) of archived FFPE tissues from multiple solid cancers, including breast, kidney, ovarian, stomach, colorectal, and non-small cell lung cancer. Applying the RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, tissue microarrays consisting of 20-35 FFPE tumors per each cancer type were stained for key therapeutic targets including PD1, PD-L1, TIM3, and LAG3. We observed that, in addition to PD-L1, all evaluated immune checkpoint molecules were expressed in the tumor cells of multiple tumor types though at a lower level than in immune cells. While there was notable heterogeneity of expression in different tumor regions in some samples, the single-cell expression pattern of each checkpoint molecule across all tumor cells in each region suggested a potential tumor-intrinsic mechanism for the expression of these molecules. In selected lung and ovarian cancers, fluorescence multiplex ISH-IHC staining confirmed that multiple different checkpoint molecules were co-expressed together in individual tumor cells. These findings add to the growing evidence that immune checkpoint genes can be expressed in tumor cells in addition to immune cells, which will be missed by gene expression analysis techniques using bulk tissue. The RNAscope® ISH platform is especially well suited for this type of analysis since it allows for assessing cell type-specific expression of any gene within the native tissue architecture even when it is expressed at very low levels. The approach illustrated in this study may help to better understand the anti-tumor activities of immune checkpoint inhibitors and provide new insights into better predictive biomarkers and therapeutic strategies.

BackgroundZinc finger protein 132 (ZNF132) has emerged as a potential tumor suppressor, with its dysregulation closely associated with the initiation and progression of various malignancies. However, a comprehensive assessment of ZNF132’s expression patterns across diverse cancer types, its clinical prognostic implications, and its immunoregulatory role in colorectal cancer remains insufficiently characterized. This study aims to elucidate the biological functions of ZNF132 within the context of colorectal cancer.MethodsWe utilized transcriptomic data from 33 cancer types obtained from The Cancer Genome Atlas (TCGA) and the TCGA-COADREAD dataset to systematically analyze the expression characteristics of ZNF132 and its differential expression in colorectal cancer. Validation of protein expression was conducted using data from the Human Protein Atlas (HPA). The relationship between ZNF132 and immune infiltration was examined through single-sample gene set enrichment analysis (ssGSEA) and Spearman correlation analysis. We evaluated the diagnostic, prognostic, and independent risk prediction capabilities of ZNF132 using receiver operating characteristic (ROC) curves, Kaplan-Meier survival analysis, and multivariable Cox regression models. Additionally, we explored the biological pathways associated with ZNF132-related differentially expressed genes through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), supplemented by in vitro experiments to investigate the functional roles of ZNF132 in colorectal cancer.ResultsPan-cancer analysis demonstrated a significant downregulation of ZNF132 in colorectal and rectal cancers, accompanied by reduced protein levels in colorectal cancer tissues. Immune infiltration analysis revealed significant correlations between ZNF132 and various immune cell subtypes in colorectal cancer, notably a negative correlation with Th17 cells and NK CD56bright cells, while exhibiting positive correlations with T helper cells, central memory T cells, macrophages, and Th2 cells. The expression of ZNF132 was significantly associated with clinical pathological features in colorectal cancer patients, including age, M stage, and pathological grade. ROC curve analysis indicated that ZNF132 possesses high sensitivity and specificity for colorectal cancer diagnosis (AUC = 0.845). Kaplan-Meier analysis further confirmed that elevated ZNF132 expression correlates with extended survival in colorectal cancer patients. Multivariable Cox regression analysis identified ZNF132 as an independent risk factor for overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in these patients. Enrichment analysis suggested that ZNF132 modulates tumor progression by influencing calcium signaling, PPAR pathways, and apoptosis-related genes. Functional assays demonstrated that ZNF132 significantly inhibits the proliferation, migration, and invasion of colorectal cancer cells.ConclusionZNF132 functions as a novel tumor suppressor, influencing colorectal cancer progression through the regulation of the immune microenvironment and malignant cellular phenotypes. Its diagnostic and prognostic potential offers new therapeutic targets for clinical application.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12885-025-14810-9.

Colorectal cancer (CRC) remains one of the most common and lethal malignancies worldwide, with limited effective biomarkers for predicting prognosis and guiding immunotherapy response. The present study aimed to investigate the potential of DDB1- and CUL4-associated factor 13 (DCAF13) as a biomarker for CRC. Bioinformatics analyses were performed using public datasets from The Cancer Genome Atlas and Gene Expression Omnibus; ESTIMATE, CIBERSORT, immune checkpoint, TIDE score and Kaplan-Meier survival analyses were performed to assess the prognostic value and implications of DCAF13 expression in CRC. In silico findings were validated through immunohistochemistry (IHC), in vitro cell-based assays and transcriptomic analysis. Increased DCAF13 expression levels were associated with reduced overall survival in patients with CRC based on the bioinformatics analysis, and this was validated using samples from patients using IHC. Immune profiling demonstrated an increased infiltration of M0 and M1 macrophages, activation of mast cells, neutrophils and CD4+ memory T cells in the DCAF13-high expression group compared with the DCAF13-low expression group. DCAF13 expression was correlated with immune modulators and checkpoint genes. High DCAF13 expression was associated with lower TIDE scores. In vitro assays and transcriptomic analyses confirmed the pro-tumourigenic effects of DCAF13, which demonstrated roles in regulating cell proliferation, migration, clonogenicity, adhesion, metastasis, epithelial-to-mesenchymal transition and homologous recombination in CRC cells. The present study demonstrated that DCAF13 was upregulated in CRC and served a role in tumour progression, thus providing novel insights into the pro-tumourigenic functions of DCAF13 and its potential as a critical regulator in CRC.

Integrated molecular analysis demonstrated that colorectal cancer can be classified into four molecular subtypes (proliferative, immunomodulatory, immunosuppressed, and immune-excluded subtypes), providing valuable insight into the intricate relationship between tumor microenvironment heterogeneity and various clinical phenotypes.

Icariin modulates the tumor microenvironment in colorectal cancer by targeting M2 macrophage polarization via PI3K/AKT pathway.