Antibacterial Fusobacterium Nucleatum-Mimicking Nanomedicine to Selectively Eliminate Tumor-colonized Bacteria And Enhance Immunotherapy Against Colorectal Cancer.

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared to their subcutaneous tumor counterparts. Furthermore, treatment with FMS-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC. Citation Format: William W. Ho, Igor L. Gomes-Santos, Shuichi Aoki, Meenal Datta, Kosuke Kawaguchi, Nilesh P Talele, Sylvie Roberge, Jun Ren, Hao Liu, Ivy X Chen, Patrik Andersson, Sampurna Chatterjee, Ashwin S. Kumar, Zohreh Amoozgar, Qixian Zhang, Peigen Huang, Mei Rosa Ng, Vikash P Chauhan, Lei Xu, Dan G. Duda, Jeffrey W. Clark, Mikael J. Pittet, Dai Fukumura, Rakesh K Jain. Dendritic cell paucity in mismatch repair-proficient colorectal cancer liver metastases limits the efficacy of immune checkpoint blockade [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P061.

BackgroundDespite the curative potential of immune checkpoint blockade (ICB) therapy, only small subsets of patients achieve tumor regression while many responders relapse and acquire resistance. Monitoring treatment response and detecting...

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

Despite immune checkpoint blockade (ICB) therapy contributed to significant advances in cancer therapy, only a small percentage of patients with colorectal cancer (CRC) respond to it. Identification of these patients will facilitate ICB application in CRC. In this study, we integrated multiple CRC cohorts (2,078 samples) to construct tumor microenvironment (TME) subtypes using TME indices calculated by CIBERSORT and ESTIMATE algorithms. Furthermore, a surrogate quantitative indicator, a tumor microenvironment immune gene (TMEIG) score system, was established using the key immune genes between TME clusters 1 and 2. The subsequent analysis demonstrated that TME subtypes and the TMEIG score system correlated with clinical outcomes of patients in multiple CRC cohorts and exhibited distinct immune statuses. Furthermore, Tumor Immune Dysfunction and Exclusion (TIDE) analysis indicated that patients with low TMEIG scores were more likely to benefit from ICB therapy. A study on two ICB cohorts (GSE78220 and IMvigor210) also validated that patients with low TMEIG scores exhibited higher ICB response rates and better prognoses after ICB treatment. The biomarker evaluation module on the TIDE website revealed that the TMEIG score was a robust predictive biomarker. Moreover, differential expression analysis, immunohistochemistry, qPCR experiments, and gene set prioritization module on the TIDE website demonstrated that the five genes that constitute the TMEIG score system (SERPINE1, FABP4, SCG2, CALB2, and HOXC6) were closely associated with tumorigenesis, immune cells, and ICB response indices. Finally, TMEIG scores could accurately predict the prognosis and ICB response of patients with CRC. SERPINE1, FABP4, SCG2, CALB2, and HOXC6 might be potential targets related to ICB treatment. Furthermore, our study provided new insights into precision ICB therapy in CRC.

Background: Tumor progression and limited benefits of immune checkpoint blockade (ICB) therapy have been two major challenges in the clinical management of colorectal cancer (CRC). The objective of our research was to explore the role of PLCG2 in CRC progression, tumor microenvironment, and potentiating ICB therapy. Methods: Based on bioinformatics analysis and a prospective clinical observational study, the expression, prognostic significance, and clinical relevance of PLCG2 in CRC were unveiled. The single-cell and spatial transcriptome revealed the role of PLCG2 in shaping the heterogeneity of the CRC tumor microenvironment. The biological function of PLCG2 was validated by in vivo and in vitro experiments. The underlying mechanisms were elucidated by RNA-seq, western blotting, qRT-PCR, and multicolor immunofluorescence. The multiplex immunohistochemistry and flow cytometry were adopted to clarify the immunomodulatory role of PLCG2 in facilitating CRC immune escape. The translational value of targeting PLCG2 to potentiate the efficacy of ICB therapy and synergistic therapy to improve prognosis was explored in the preclinical animal models. Results: In CRC, PLCG2 exhibited high expression levels and was strongly associated with poor prognosis and advanced clinicopathological characteristics of patients. The single-cell transcriptome shed light on its important role in cell communication and the development and differentiation of immune cells. The spatial transcriptome described the spatial distribution of PLCG2 in CRC tissues. Further mechanistic analysis demonstrated that PLCG2 could promote proliferation, invasion, metastasis, epithelial-mesenchymal transition, and cell cycle regulation and inhibit apoptosis of CRC cells via the Akt-mTOR pathway activation. Furthermore, PLCG2 was found to contribute greatly to the immunosuppressive microenvironment and enhanced immune escape as it significantly suppressed the infiltration and functional activation of CD8+ T cells and promoted the infiltration of Treg cells as well as PD-1 and PD-L1 expression. Meanwhile, knockdown of PLCG2 could potentiate the efficacy of ICB therapy. Conclusion: In summary, we have identified for the first time that PLCG2 could be considered a precise biomarker and promising therapeutic target for predicting CRC prognosis, optimizing individualized treatment, reversing CRC immune escape, and overcoming resistance to ICB therapy.

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.

Colorectal cancer with chromosomal instability (CIN+) phenotype is immunosuppressive and refractory to immune checkpoint blockade (ICB) therapy. Recently, KIF18A is found to be a mitotic vulnerability in chromosomally unstable cancers, but whether targeting KIF18A affects antitumor immunity in CIN+ colorectal cancer is unknown. In our study, western blot, cell viability assay, transwell migration and invasion assays, flow cytometry, animal model, immunohistochemistry (IHC) staining, reverse transcription–quantitative PCR (RT-qPCR) and ELISA assay were conducted to evaluate the potential function of KIF18A in CIN+ colorectal cancer. We found that KIF18A inhibition by short hairpin RNAs (ShRNAs) or small inhibitor AM-1882 suppressed proliferation, migration, invasion and tumor growth and metastasis of CIN+ colorectal cancer cells in vitro and in vivo. Moreover, targeting KIF18A disrupted cell-cycle progression and induced G2/M arrest in CIN+ colorectal cancer cells. In addition, KIF18A inhibition promoted immune infiltration and activation in CIN+ colorectal tumors. KIF18A inhibition suppressed proliferation of Tregs and increased infiltration and activation of cytotoxic CD8+ T cells in CIN+ colorectal tumors. Mechanically, KIF18A inhibition stimulated type I IFN signaling and cGAS-STING activation in CIN+ colorectal tumors. Finally, targeting KIF18A enhanced PD-1 blockade efficiency in CIN+ colorectal tumors through T cells. Our data elucidated a novel role of KIF18A in antitumor immunity of CIN+ colorectal cancer.

Tumor-Secreted Extracellular Vesicles Regulate T-Cell Costimulation and Can Be Manipulated To Induce Tumor-Specific T-Cell Responses

Loss of the Ras Regulator RASAL1: Another Route to Ras Activation in Colorectal Cancer

e14582 Background: Tumor mutational burden (TMB) is considered a promising predictive biomarker for immune checkpoint blockade (ICB) therapy response . However, the measurement of TMB relies on the whole-exome sequencing (WES), which is technically limited by sample volume and freshness, thus restricts its use in clinical practice. Since TMB is controlled by the balance between DNA damage and the repair, it is a rational approach to identify a subset of DNA repair/replication gene mutations associated with increased TMB as a surrogate, which could be monitored in clinic to predict ICB efficacy even busing limited archived tissues. Methods: The mutation status of 471 genes associated with DNA repair and replication were analyzed for the correlation with TMB and/or ICB therapeutic responses in three cohorts of NSCLC patients, which are: Cohort A, WES data of 100 Chinese NSCLC patients; Cohort B, WES data of NSCLC patients from TCGA database; Cohort C, WES data and therapeutic response to PD-1 mAb from a Science paper (Rizvi et al. 2015, Science). Results: We identified somatic mutations in 32 DNA repair/replication genes associated with high TMB in both Cohort A and Cohort B. In Cohort C, mutations were found in 20 out of these 32 genes, and the mutation status of 17/20 genes were associated with the clinical benefit of ICB therapy. Noteworthy, mutations of TP53, POLQ, MMS22L, HERC2, CEP164, BRCA1/2, FANCM and MSH4 were mostly common mutations associated with TMB in all 3 cohorts, and ERCC2 and ERCC6 were uniquely high mutated and associated with high TMB in Chinese Han people from Cohort A. Interestingly, EGFR activating mutations were inversely correlated with both high TMB and good ICB response. Conclusions: This retrospective study identified 17 DNA repair/replication-related genes, whose mutation status was associated with high TMB and/or ICB response. Mutational patterns of DNA repair genes are likely different between western and Chinese NSCLC patient population. The data also suggested that NSCLC patients carrying EGFR activating mutations are not preferential population for ICB therapy. Further data is needed to verify the validity of these observations.

Recent studies indicated that intratumor microbes are an essential part of the tumor microenvironment. Here, we performed an integrated analysis of genetic, epigenetic, and intratumor microbial factors to unravel the potential remodeling mechanisms of immune-cell infiltration (ICI) and tumorigenesis of colorectal cancer (CRC). We identified the components and structure of the intratumor microbiome as primary contributors to the difference in survival between ICI subtypes. Multiple tumor-infiltrating immune cells (TIICs) and immune-related genes were associated with intratumor microbial abundance. Additionally, we found that Clostridium was enriched in CRC patients who were nonsensitive to immune checkpoint blockade (ICB) therapy. We further provided clues that the intratumor microbes might influence the response to ICB therapy by mediating TIICs, especially MAIT (mucosa-associated invariant T) cells. Finally, three ICB-related TIICs and 22 of their associated microbes showed the potential to predict the response to ICB therapy (area under the receiver operating characteristic curve [AUC] = 89%). Our findings highlight the crucial role of intratumor microbes in affecting immune-cell infiltration patterns, prognosis, and therapy response of CRC and provide insights for improving current immunotherapeutic treatment strategies and prognosis for CRC patients. IMPORTANCE Using the multi-omics data from The Cancer Genome Atlas (TCGA) colorectal cancer (CRC) cohort, we estimated the tumor microenvironment (TME) infiltration patterns of patients and unraveled the interplay of gene expression, epigenetic modification, and the intratumor microbiome. This study suggests the impact of intratumor microbes on maintaining the tumor immune microenvironment in the pathogenesis of CRC and modulating the response to immune checkpoint blockade (ICB) therapy. We identified a set of combined features, including 3 ICB-related tumor-infiltrating immune cells (TIICs) and 22 of their associated microbes, that are predictive of ICB responses.

e14566 Background: Immune checkpoint blockade (ICB) therapy transformed clinical oncology by inducing durable responses and increasing survival rates in many types of cancers. However, ICB is effective in only in a subset of patients. Recent studies delineated the role of gut microbiome as both a biomarker and a therapeutic in ICB responsiveness. We aimed to increase understanding of the microbiome-immune system axis in ICB therapy by using antibiotics to knock out certain components of gut microbiome. Methods: We treated MC38 colon adenocarcinoma-bearing mice with a widely-used antibiotic, metronidazole, that is effective against anaerobic and protozoal infections including Clostridioides difficile – a major mediator of colitis. Metronidazole was administered via oral gavage or mixed in drinking water before and after tumor injections. Mice received twice weekly treatment with a-CTLA4 ICB. Results: Metronidazole treatment alone slowed the growth rate of MC38 tumors, consistent with the current literature regarding colon cancer murine models. When metronidazole treatment was combined with a-CTLA4 therapy, we found ̃90% complete tumor regression. In the metronidazole and a-CTLA4 combination group, we also observed an increase in the number of CD103+ type 1 conventional dendritic cells (cDC1s) in colon lamina propria, which suggests enhanced antigen sampling from lumen. Also, in the mesenteric lymph nodes (mLN), we detected upregulation of CD80 and CD86 co-stimulatory molecule expression on CX3CR1+ antigen presenting cells. Multiplex analysis of colon cytokines and colon pathology evaluation was comparable among groups, which implies a non-inflammatory environment in colon. Analysis of tumor-draining lymph nodes eight days after tumor injections showed higher expression of CD86 on CD103+ cDC1s implying superior anti-tumor immunity. 16S rRNA gene sequencing analysis of fecal samples revealed loss of Lachnospiraceae and enrichment of Bifidobacteriaceae and Sutterellaceae families in metronidazole-treated mice. Conclusions: Shifting microbiome composition with metronidazole treatment elicits a favorable anti-tumor immune response to a-CTLA4 treatment in murine colon adenocarcinoma.

The realm of cancer therapy has been profoundly altered with the emergence of immune checkpoint blockade (ICB) therapy, providing improved survival prospects for many patients with some cancers. However, the challenge of achieving efficient or sustained therapeutic benefits underscores the critical imperative to optimize ICB strategies. This review elucidates the pivotal role of predictive biomarkers in optimizing precision ICB therapy, deciphering the intricate dynamics associated with the response heterogeneity. Furthermore, it critically examines the application of nanotechnology-driven drug delivery as a promising avenue to amplify ICB efficacy, facilitating controlled and targeted drug release. Recognizing the comprehensive and dynamic interplay among tumor cells, immune cells, and stromal cells has catalyzed the transformative advances in reverse translational research. This approach enables researchers to gain insights into the underlying mechanisms of ICB therapy, therapeutic responses, and resistance mechanisms. The convergence of predictive biomarkers, revolutionary nanotechnology, and reverse translational research emerges as an indispensable focal point, propelling the frontiers of precision oncology within the complex landscape of ICB therapy.

Immunotherapies such as immune checkpoint blockade (ICB) therapies have been developed in the last years as a promising strategy for the treatment of cancer, however there remains a need to improve their efficacy. The most critical factor that affects the efficacy of ICB therapies, is the frequency of tumor mutations, the associated neoantigens generated and the T-cell response against them. Therefore, it is expected that neoantigen vaccinations would improve ICB therapy efficacy by boosting the neoantigen-specific T cell response. The aim of this study is to develop an effective vaccine using a combination of TLR9 and STING agonists together with synthetic long peptides to induce a potent neoantigen-specific cytotoxic CD8+ T cell response. We first analyzed the efficacy of the vaccine formulation containing 20-mer OVA peptide and found that mice immunized with OVA peptide together with the adjuvant combination induced potent antigen-specific T cell responses and are capable of controlling tumor growth and improving survival in B16-F10-OVA tumor bearing mice. Moreover, we found that the vaccine together with OVA peptide is able to synergize with anti-PD-1 treatment in controlling tumor growth. Finally, we examined the immunogenicity of the vaccine formulation with neopeptides identified from two different tumor models. The vaccine formulation induced potent neoantigen-specific T cell responses in vivo. Our findings demonstrate that the vaccine formulation using the combination of TLR9 and STING agonists induces potent T cell immunity against synthetic long peptides and can serve as a promising immunogenic neoantigen vaccine platform. Supported by the Department of Immunology of the Erasmus MC the Dutch Cancer Society (KWF grant 12837) and in part by a grant from International Joint Usage/Research Center, the Institute of Medical Science, the University of Tokyo, and Japan Agency for Medical Research and Development (AMED).

Colorectal cancer (CRC) is a malignant tumor that poses a significant threat to human health due to rising incidence and mortality rates. In recent years, immune checkpoint blockade (ICB) therapy, represented by Programmed death receptor 1 (PD-1), T-lymphocyte-associated protein 4 (CTLA-4), and others, has been widely applied in CRC and has achieved encouraging results in some patients and has become a hot topic in both clinical and basic research. This study undertakes a comprehensive bibliometric analysis of ICB research in CRC, aiming to evaluate the current status, identify future trends, and provide scientific insights for researchers and decision-makers. Utilizing the Web of Science Core Collection (WoSCC), articles focusing on ICB in CRC from 2000 to 2022 were retrieved. Knowledge mapping and bibliometric analysis were conducted using tools such as CiteSpace, VOSviewer, SCImago Graphicay, and the R package bibliometrix. 6,718 publications were analyzed from 24,846 institutions across 639 regions. Temporally, ICB research in CRC is rapidly advancing, led by the USA and China with extensive global collaborations. Sun Yat-sen University from China stands out as the institution with the highest number of publications. Professor Thierry Andre from Sorbonne University in France is identified as a prolific author in this field, engaging in extensive collaboration for clinical trials on a global scale. Publications related to this research topic were published in 1,142 academic journals, demonstrating a positive co-citation relationship. Key clustering and burst terms analysis indicate that current research on ICB in CRC has shifted from basic experiments to clinical trials and from universal healthcare to precision medicine. ICB therapies have shown substantial progress in CRC, highlighting their therapeutic potential. Research trends emphasize deeper drug mechanisms, treatment efficacy prediction, managing immune-related adverse events, and exploring novel drug delivery methods. Collaboration across borders remains crucial for further advancements.