In Silico Modeling of Immunotherapy and Stroma-Targeting Therapies in Human Colorectal Cancer.

<div>Abstract<p>Despite the fact that the local immunological microenvironment shapes the prognosis of colorectal cancer, immunotherapy has shown no benefit for the vast majority of colorectal cancer patients. A better understanding of the complex immunological interplay within the microenvironment is required. In this study, we utilized wet lab migration experiments and quantitative histological data of human colorectal cancer tissue samples (<i>n</i> = 20) including tumor cells, lymphocytes, stroma, and necrosis to generate a multiagent spatial model. The resulting data accurately reflected a wide range of situations of successful and failed immune surveillance. Validation of simulated tissue outcomes on an independent set of human colorectal cancer specimens (<i>n</i> = 37) revealed the model recapitulated the spatial layout typically found in human tumors. Stroma slowed down tumor growth in a lymphocyte-deprived environment but promoted immune escape in a lymphocyte-enriched environment. A subgroup of tumors with less stroma and high numbers of immune cells showed high rates of tumor control. These findings were validated using data from colorectal cancer patients (<i>n</i> = 261). Low-density stroma and high lymphocyte levels showed increased overall survival (hazard ratio 0.322, <i>P</i> = 0.0219) as compared with high stroma and high lymphocyte levels. To guide immunotherapy in colorectal cancer, simulation of immunotherapy in preestablished tumors showed that a complex landscape with optimal stroma permeabilization and immune cell activation is able to markedly increase therapy response <i>in silico</i>. These results can help guide the rational design of complex therapeutic interventions, which target the colorectal cancer microenvironment. <i>Cancer Res; 77(22); 6442–52. ©2017 AACR</i>.</p></div>

<div>Abstract<p>Despite the fact that the local immunological microenvironment shapes the prognosis of colorectal cancer, immunotherapy has shown no benefit for the vast majority of colorectal cancer patients. A better understanding of the complex immunological interplay within the microenvironment is required. In this study, we utilized wet lab migration experiments and quantitative histological data of human colorectal cancer tissue samples (<i>n</i> = 20) including tumor cells, lymphocytes, stroma, and necrosis to generate a multiagent spatial model. The resulting data accurately reflected a wide range of situations of successful and failed immune surveillance. Validation of simulated tissue outcomes on an independent set of human colorectal cancer specimens (<i>n</i> = 37) revealed the model recapitulated the spatial layout typically found in human tumors. Stroma slowed down tumor growth in a lymphocyte-deprived environment but promoted immune escape in a lymphocyte-enriched environment. A subgroup of tumors with less stroma and high numbers of immune cells showed high rates of tumor control. These findings were validated using data from colorectal cancer patients (<i>n</i> = 261). Low-density stroma and high lymphocyte levels showed increased overall survival (hazard ratio 0.322, <i>P</i> = 0.0219) as compared with high stroma and high lymphocyte levels. To guide immunotherapy in colorectal cancer, simulation of immunotherapy in preestablished tumors showed that a complex landscape with optimal stroma permeabilization and immune cell activation is able to markedly increase therapy response <i>in silico</i>. These results can help guide the rational design of complex therapeutic interventions, which target the colorectal cancer microenvironment. <i>Cancer Res; 77(22); 6442–52. ©2017 AACR</i>.</p></div>

Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer

BACKGROUNDImproving and predicting tumor response to immunotherapy remains challenging. Combination therapy with a transforming growth factor-β receptor (TGF-βR) inhibitor that targets cancer-associated fibroblasts (CAFs) is promising for the enhancement of efficacy of immunotherapies. However, the effect of this approach in clinical trials is limited, requiring in vivo methods to better assess tumor responses to combination therapy.METHODSWe measured CAFs in vivo using the 68Ga-labeled fibroblast activation protein inhibitor-04 (68Ga-FAPI-04) for PET/CT imaging to guide the combination of TGF-β inhibition and immunotherapy. One hundred thirty-one patients with metastatic colorectal cancer (CRC) underwent 68Ga-FAPI and 18F-fluorodeoxyglucose (18F-FDG) PET/CT imaging. The relationship between uptake of 68Ga-FAPI and tumor immunity was analyzed in patients. Mouse cohorts of metastatic CRC were treated with the TGF-βR inhibitor combined with KN046, which blocks programmed death ligand 1 (PD-L1) and CTLA-4, followed by 68Ga-FAPI and 18F-FDG micro-PET/CT imaging to assess tumor responses.RESULTSPatients with metastatic CRC demonstrated high uptake rates of 68Ga-FAPI, along with suppressive tumor immunity and poor prognosis. The TGF-βR inhibitor enhanced tumor-infiltrating T cells and significantly sensitized metastatic CRC to KN046. 68Ga-FAPI PET/CT imaging accurately monitored the dynamic changes of CAFs and tumor response to combined the TGF-βR inhibitor with immunotherapy.CONCLUSION68Ga-FAPI PET/CT imaging is powerful in assessing tumor immunity and the response to immunotherapy in metastatic CRC. This study supports future clinical application of 68Ga-FAPI PET/CT to guide precise TGF-β inhibition plus immunotherapy in CRC patients, recommending 68Ga-FAPI and 18F-FDG dual PET/CT for CRC management.TRIAL REGISTRATIONCFFSTS Trial, ChiCTR2100053984, Chinese Clinical Trial Registry.FUNDINGNational Natural Science Foundation of China (82072695, 32270767, 82272035, 81972260).

Immunotherapy in colorectal cancer: an unmet need deserving of change.

Background: NOTCH, as an oncogene, has been shown to be closely associated with radiation and chemotherapy, hormone therapy, and other molecular targeted therapies (such as anti-EGFR and anti-PI3K molecular targeted therapies) in a variety of tumors, and even in drug resistance mechanisms. However, the correlation between NOTCH mutations and immunotherapy in colorectal cancer (CRC) has not been reported. Methods: We retrospectively identified NOTCH mutation and treatment outcomes. The relationship between clinical pathologic features and NOTCH were analyzed with using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics associated with overall survival using Cox regression and the Kaplan-Meier method. Results: We retrospectively analyzed 108 CRC patients who received immunotherapy, including 31 patients with NOTCH mutation (MUT) and 78 patients with NOTCH wide-type (WT). NOTCH mutation was discovered to be enriched in MSI instable and associated with significant better overall survival (HR, 0.4; 95% CI, 018-0.9; P=0.0272). Although compared with NOTCH WT, no association was observed between NOTCH1/2/4 MUT and overall survival, Kaplan-Meier survival analysis showed that patients with NOTCH3 MUT obtained better OS (not reach vs. 14 months, HR, 0.17; 95% CI 0.04-0.74; P=0.0176). In addition, in an in-house Chinese CRC cohort (n=1968), we found 322 (16.4%) patients with NOTCH mutations, including NOTCH1/2/3/4 mutation frequency were 43.5%, 32.9%, 46.9% and 12.1%, respectively. 206 (60.4%) patients had one NOTCH mutation, and only 20 (6.2%) patients had more than four mutations. NOTCH mutations were significantly associated with higher TMB levels (P<0.0001) and PD-L1 positive (P=0.0011) in the in-house Chinese CRC cohorts. Conclusions: Our results suggest that NOTCH1/2/3/4 mutation may be a potential predictor to favorable ICI response in CRC patients. The exact mechanisms underlying NOTCH mutations are needed to be further evaluated. Citation Format: Xiaoyang Xia, DongShan You, Jing Cao, JingHui Huang, Junling Zhang, Mengli Huang, Yanan Chen, Xihua Xia, Yuezong Bai. Identification of NOTCH mutation as novel predictor to efficacious immunotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5088.

There is increasing evidence in a range of cancer types that the microbiome plays a direct role in modulating the anti-cancer immune response both at the gut level and systemically. Differences in the gut microbiota have been shown to correlate with differences in immunotherapy responses in a range of non-gastrointestinal tract cancers. DNA mismatch repair-deficient (dMMR) colorectal cancer (CRC) is radically different to DNA mismatch repair-proficient (pMMR) CRC in clinical phenotype and in its very good responses to immunotherapy. While this has usually been thought to be due to the high mutational burden in dMMR CRC, the gut microbiome is radically different in dMMR and pMMR CRC in terms of both composition and diversity. It is probable that differences in the gut microbiota contribute to the varied responses to immunotherapy in dMMR versus pMMR CRC. Targeting the microbiome offers a way to boost the response and increase the selection of patients who might benefit from this therapy. This paper reviews the available literature on the role of the microbiome in the response to immunotherapy in dMMR and pMMR CRC, explores the potential causal relationship and discusses future directions for study in this exciting and rapidly changing field.

BackgroundLong noncoding RNAs (lncRNAs) are emerging as critical regulatory elements and play fundamental roles in the biology of various cancers. However, we are still lack of knowledge about their expression patterns and functions in human colorectal cancer (CRC).MethodsDifferentially expressed lncRNAs in CRC were identified by bioinformatics screen and the level of MIR22HG in CRC and control tissues were determined by qRT-PCR. Cell viability and migration capacities were examined by MTT and transwell assay. Mouse model was used to examine the function and rational immunotherapy of MIR22HG in vivo.ResultsWe systematically investigated the expression pattern of lncRNAs and revealed MIR22HG acts as a tumor suppressor in CRC. The expression of MIR22HG was significantly decreased in CRC, which was mainly driven by copy number deletion. Reduced expression of MIR22HG was significantly associated with poor overall survival. Silencing of MIR22HG promoted cell survival, proliferation and tumor metastasis in vitro and in vivo. Mechanistically, MIR22HG exerts its tumor suppressive activity by competitively interacting with SMAD2 and modulating the activity of TGFβ pathway. Decreased MIR22HG promoted the epithelial-mesenchymal transition in CRC. Importantly, we found that MIR22HG expression is significantly correlated with CD8A and overexpression of MIR22HG triggers T cell infiltration, enhancing the clinical benefits of immunotherapy.ConclusionMIR22HG acts as a tumor suppressor in CRC. Our data provide mechanistic insights into the regulation of MIR22HG in TGFβ pathway and facilitates immunotherapy in cancer.

Colon cancer is one of the most prevalent malignancies of the digestive system, characterized by high morbidity and mortality rates. Its development is driven by a complex interplay of factors within cancer cells and the tumor microenvironment (TME). Microsatellite-stable (MSS) colorectal cancer, often referred to as a “cold tumor, ” is characterized by chromosomal stability and a lack of neoantigen production. As a result, MSS tumors are generally unresponsive to immunotherapy. Post-translational modifications of proteins are critical in tumor remodeling and progression. Histone deacetylase 6 (HDAC6), a member of the HDAC family, plays a key role in regulating the deacetylation of both histone and non-histone substrates. Overexpression of HDAC6 has been reported in multiple tumor types and is associated with tumor growth and metastasis. While immune checkpoint blockade (ICB) therapies, such as those targeting programmed cell death protein 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1), have achieved clinical success in several malignancies, the majority of colorectal cancer patients particularly those with MSS tumors fail to respond to these treatments. Recent evidence indicates that the intratumoral immune context strongly influences the efficacy of ICB therapies. Patients with high levels of intratumoral CD8+ T cell infiltration, elevated tumor mutational burden (TMB), and increased PD-L1 expression within the TME are more likely to respond favorably to immunotherapy. In this study, we focused on enhancing the immunogenicity of the TME and upregulating PD-L1 expression through selective HDAC6 inhibition to improve the efficacy of anti-PD-L1 immunotherapy. Analysis of The Cancer Genome Atlas (TCGA) data revealed that HDAC6 expression is inversely correlated with several immune-related genes, including PD-L1. This inverse relationship suggests a potential role for HDAC6 in regulating immune responses in MSS colorectal cancer. These findings imply that HDAC6 may influence the TME and immune evasion mechanisms, particularly by modulating immune checkpoint molecules such as PD-L1. In the CT26 syngeneic mouse model, inhibition of HDAC6 led to an enhanced immune response characterized by increased PD-L1 expression, improved antigen presentation, and upregulation of MHC-II. This was accompanied by a polarization toward M1 macrophages and elevated levels of pro-inflammatory cytokines, including TNF-α and IFN-γ, which are crucial for T cell activation. Furthermore, HDAC6 inhibition resulted in a reduction of TGF-β, a key immunosuppressive cytokine, thereby moderating immune suppression within the tumor microenvironment. These results highlight the potential of HDAC6 inhibitors to reprogram the tumor immune landscape and sensitize MSS colorectal cancer to immune checkpoint therapies. Citation Format: Ahran Yu, Jeong Eun Kang, Ju Yeon Park, Seijong Kim, Hye Kyung Hong, Yong Beom Chov. Targeting HDAC6 to modulate the tumor microenvironment and enhance anti-PD-L1 immunotherapy in MSS colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2168.

Colorectal cancer (CRC) is a common and lethal disease with a high therapeutic need. For most patients with metastatic CRC, chemotherapy is the only viable option. Currently, immunotherapy is restricted to the particular genetic subgroup of mismatch-repair deficient (MMRd)/microsatellite instable (MSI) CRC. Anti-PD1 therapy was recently FDA-approved as a second-line treatment in this subgroup. However, in a metastatic setting, these MMRd/MSI tumors are vastly outnumbered by mismatch-repair proficient (MMRp)/microsatellite stable (MSS) tumors. These MMRp/MSS tumors do not meaningfully respond to any traditional immunotherapy approach including checkpoint blockade, adoptive cell transfer and vaccination. This resistance to immunotherapy is due to a complex tumor microenvironment that counteracts antitumor immunity through a combination of poorly antigenic tumor cells and an immunosuppressive tumor microenvironment. To find ways of overcoming immunotherapy resistance in the majority of CRC patients, it is necessary to analyze the immunological makeup in an in-depth and personalized way and in the context of their tumor genetic makeup. Flexible, biomarker-guided early-phase immunotherapy trials are needed to optimize this workflow. In this review, we detail key mechanisms for immune evasion and emerging immune biomarkers for personalized immunotherapy in CRC. Also, we present a template for biomarker-guided clinical trials that are needed to move new immunotherapy approaches closer to clinical application.

Repurposing the Pentameric B-Subunit of Shiga Toxin for Gb3-Targeted Immunotherapy of Colorectal Cancer by Rhamnose Conjugation

To date, anticancer immunotherapy has presented some clinical benefits to most of advanced mismatch repair deficient (dMMR)/microsatellite instability-high (MSI-H) colorectal cancer (CRC) patients. In addition to MSI status, we aimed to reveal the potential predictive value of adenomatous polyposis coli (APC) gene mutations in CRC patients. A total of 238 Chinese CRC patients was retrospectively identified and analyzed for clinical features and gene alternations in APC-mutant type (MT) and APC-wild-type (WT) groups. Clinical responses were then evaluated from the public TCGA database and MSKCC immunotherapy database. Although programmed cell death ligand 1 (PD-L1) level, MSI status, loss of heterogeneity at the human leukocyte antigen (HLA LOH), and tumor neoantigen burden (TNB) level were not statistically different between the APC-MT group and APC-WT group, tumor mutation burden (TMB) level was significantly higher in APC-MT patients (P < 0.05). Furthermore, comutation analysis for APC mutations revealed co-occurring genomic alterations of PCDHB7 and exclusive mutations of CTNNB1, BRAF, AFF3, and SNX25 (P < 0.05). Besides, overall survival from MSKCC-CRC cohort was longer in the APC-WT group than in the APC-MT group (HR 2.26 (95% CI 1.05–4.88), P < 0.05). Furthermore, most of patients in the APC-WT group were detected as high-grade immune subtypes (C2–C4) comparing with those in the APC-MT group. In addition, the percentages of NK T cells, Treg cells, and fibroblasts cells were higher in APC-WT patients than in APC-MT patients (P < 0.05). In summary, APC mutations might be associated with poor outcomes for immunotherapy in CRC patients regardless of MSI status. This study suggested APC gene mutations might be a potential predictor for immunotherapy in CRC.

Background: Colorectal cancer (CRC) is the third most common malignancy in the United States and United Kingdom and deadly in one third of patients. Tumor lymphocytic infiltration is associated with improved survival and analysis of some lymphocyte subsets has revealed that their prognostic ability rivals the TNM tumor staging system. Regulatory T cells (Tregs) are known to be enriched in CRC and it is postulated that they promote immunological tumor escape by suppression of effector T cell responses. Little is known however about the signals controlling entry of Tregs into CRC. Methods: Matched CRC, distal colonic tissue, draining lymph node and blood were obtained from patients undergoing resection of CRC having given informed consent. The study was approved by the Local Research and Ethics Committee. Tumor-infiltrating lymphocytes were isolated from fresh tissue and phenotyped for chemokine receptors and various other markers using multicolor flow cytometry. The presence of tissue chemokines was analyzed using real-time PCR and Western blotting. CD3+ cells were isolated from tumor tissue and placed in a transwell system to measure chemotaxis in response to various chemokines. Migrated and non-migrated T cells were phenotyped to establish differential migration of T cell subsets. CD4+CD127lowCD25+ T cells were isolated by fluorescent-activated cell sorting from tumor tissue and incubated with fluorescent-labeled responder cells in standard suppression assays. Peripheral blood lymphocytes were co-cultured with tumor supernatant and effects on lymphocyte phenotype and proliferation were analyzed by flow cytometry. Results: The proportion of T cells with a suppressive phenotype (Treg, CD4+CD127lowCD25+) was significantly increased in CRC compared to matched distal colon. More than 95% of this cell population expressed the transcription factor, foxp3. The chemokine receptor CCR5 was found to be markedly upregulated on Treg compared to other effector T cells and in CRC compared to distal colon. CCR4 and to a lesser extent, CCR6, were also upregulated on Treg compared to other T cells. The ligands for CCR5 (CCL3, CCL4 and CCL5) were overexpressed in CRC tissue compared to matched colon. CCL4 was found to localize to the tumor endothelium. The ligands for other chemokine receptors were not overexpressed in the tumor tissue compared to the colon and were therefore not investigated further. Tumor-resident Treg migrated in response to CCR5 ligands and blockade of CCR5 inhibited this migration. Co-culture experiments demonstrated that CCR5 was upregulated on peripheral blood lymphocytes, and especially Treg, in mixed lymphocyte reactions. This effect that could be augmented by the addition of tumor supernatant. Conclusion: Conditions exist to actively recruit CCR5+ Treg into colorectal cancer tissue in humans. CCR5 upregulation is also promoted by the tumor microenvironment that may provide a tumor-retention signal. CCR5 inhibition may prove to be a novel immunotherapy for colorectal cancer by blocking the selective recruitment and/or egress of suppressive Treg, thereby promoting an anti-tumor immune response. Citation Format: Stephen T. Ward, Elizabeth A. Hepburn, Ka-kit Li, Stuart M. Curbishley, Rahul K. Hejmadi, Tariq Ismail, Roy Bicknell, Antal Rot, David H. Adams. The selective recruitment and retainment of regulatory T cells in human colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A73.

IDO in Colorectal Tumorigenesis: Involvement of Immune Tolerance and Significance in Prevention and Therapy

Colorectal cancer is a common malignant disease, which, despite some progress, still requires improved therapeutic options. Several clinical studies have used active specific immunotherapy (i.e., vaccination) in colorectal cancer. However, the literature still lacks a comprehensive meta-analysis of this approach in advanced colorectal cancer. We did a systematic review with a meta-analysis of clinical studies to evaluate the objective clinical and immunologic response to active specific immunotherapy in patients with colorectal cancer. We conducted a search of Medline and the Web of Science, manually reviewed the literature, and consulted with experts. Criteria for including studies were colorectal cancer patients, active specific immunotherapy to induce a response directed against cancer or cancer antigens, an evaluable tumor burden (i.e., advanced or metastatic colorectal cancer), and precise classification of the patient, disease, and response. Response rates were assessed according to WHO criteria. Primary end points were the objective clinical response rate and the rate of immunologic responses. The secondary end point was the distribution of immune and clinical responses in relation to the route of vaccination and the type of vaccine. Thirty-two phase I/II studies reporting on 527 patients with advanced or metastatic colorectal cancer met all inclusion criteria. Pooled analysis showed an overall response rate (complete response + partial response) of 0.9% for advanced/metastatic colorectal cancer patients who underwent active specific immunization with a broad variety of substances (e.g., autologous tumor cells, peptide vaccine, dendritic cells, idiotypic antibody, and virus-based vaccine). Humoral immune responses were reported in 59%, and cellular ones were reported in 44% of the cases. Mixed or minor responses and disease stabilization are described in 1.9% and 8.3% of colorectal cancer patients, respectively. Pooled results of clinical trials reveal a very weak clinical response rate of <1% for active specific immunization procedures currently available for advanced colorectal cancer. Immune response induction is described in approximately half the patients.