Murine Colorectal Cancer Research Articles

USP10 drives cancer stemness and enables super-competitor signalling in colorectal cancer.

The contribution of deubiquitylating enzymes (DUBs) to β-Catenin stabilization in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, and by using an unbiassed screen, we discovered that the DUB USP10 stabilizes β-Catenin specifically in APC-truncated CRC in vitro and in vivo. Mechanistic studies, including in vitro binding together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and is outcompeted by intact APC, favouring β-catenin degradation. However, in APC-truncated cancer cells USP10 binds to β-catenin, increasing its stability which is critical for maintaining an undifferentiated tumour identity. Elimination of USP10 reduces the expression of WNT and stem cell signatures and induces the expression of differentiation genes. Remarkably, silencing of USP10 in murine and patient-derived CRC organoids established that it is essential for NOTUM signalling and the APC super competitor-phenotype, reducing tumorigenic properties of APC-truncated CRC. These findings are clinically relevant as patient-derived organoids are highly dependent on USP10, and abundance of USP10 correlates with poorer prognosis of CRC patients. Our findings reveal, therefore, a role for USP10 in CRC cell identity, stemness, and tumorigenic growth by stabilising β-Catenin, leading to aberrant WNT signalling and degradation resistant tumours. Thus, USP10 emerges as a unique therapeutic target in APC truncated CRC.

Cabozantinib-encapsulated and maytansine-conjugated high-density lipoprotein for immunotherapy in colorectal cancer

Advanced colorectal cancer (CRC) responds poorly to current adjuvant therapies, partially due to its immunosuppressive intestinal microenvironment. We found that myeloid-derived suppressor cells (MDSCs) were enriched in orthotopic tumors due to treatment-induced succinate release, which activated tuft cells and upregulated interleukin 25 (IL-25) and interleukin 13 (IL-13). We engineered a cabozantinib (Cabo)-encapsulated and maytansine (DM1)-conjugated synthetic high-density lipoprotein (ECCD-sHDL) to modulate the tumor microenvironment. DM1 induced immunogenic cell death and promoted the maturation of dendritic cells. Meanwhile, Cabo alleviated DM1-induced succinate release, preventing tuft cell activation, downregulating IL-25 and IL-13 secretion, and reducing intratumoral MDSC infiltration. ECCD-sHDL increased the densities of active cytotoxic T lymphocytes (CTLs) and M1 macrophages in the tumors, effectively inhibiting tumor growth and metastasis, thereby prolonging survival in murine CRC models. Our study sheds light on the mechanism of treatment-induced immunosuppression in orthotopic CRC and demonstrates that this combinatorial therapy could be an effective treatment for CRC.

Peptides as modulators of FPPS enzyme: A multifaceted evaluation from the design to the mechanism of action

Bone diseases are medical conditions caused by the loss of bone homeostasis consecutive to increased osteoclast activity and diminished osteoblast activity. The mevalonate pathway (MVA) is crucial for maintaining this balance since it drives the post-translational prenylation of small guanosine triphosphatases (GTPases) proteins. Farnesyl pyrophosphate synthase (FPPS) plays a crucial role in the MVA pathway. Consequently, in the treatment of bone-related diseases, FPPS is the target of FDA-approved nitrogen-containing bisphosphonates (N-BPs), which have tropism mainly for bone tissue due to their poor penetration in soft tissues. The development of inhibitors targeting the FPPS enzyme has garnered significant interest in recent decades due to FPPS's role in the biosynthesis of cholesterol and other isoprenoids, which are implicated in cancer, bone diseases, and other conditions. In this study, we describe a multidisciplinary approach to designing novel FPPS inhibitors, combining computational modeling, biochemical assays, and biophysical techniques. A series of peptides and phosphopeptides were designed, synthesized, and evaluated for their ability to inhibit FPPS activity. Molecular docking was employed to predict the binding modes of these compounds to FPPS, while Surface Plasmon Resonance (SPR) and Nuclear Magnetic Resonance (NMR) spectroscopy experiments – based on Saturation Transfer Difference (STD) and an enzymatic NMR assay – were used to measure their binding affinities and kinetics. The biological activity of the most promising compounds was further assessed in cellular assays using murine colorectal cancer (CRC) cells. Additionally, genomics and metabolomics profiling allowed to unravel the possible mechanisms underlying the activity of the peptides, confirming their involvement in the modulation of the MVA pathway. Our findings demonstrate that the designed peptides and phosphopeptides exhibit significant inhibitory activity against FPPS and possess antiproliferative effects on CRC cells, suggesting their potential as therapeutic agents for cancer.

Chinese herbal Jianpi Jiedu formula suppressed colorectal cancer growth in vitro and in vivo via modulating hypoxia-inducible factor 1 alpha-mediated fibroblasts activation

Ethnopharmacological relevanceJianpi Jiedu Formula (JPJDF) is a traditional Chinese medicinal decoction clinically used for its anti-cancer properties, particularly in colorectal cancer (CRC). Aim of the studyThis study aims to investigate the therapeutic effects of JPJDF on CRC and elucidate its potential molecular mechanisms, with a focus on its impact on hypoxia-inducible factor 1 alpha (HIF1α) and cancer-associated fibroblasts (CAFs) both in vitro and in vivo. Materials and methodsUPLC-Q-TOF-MS was used to identify the constituents of JPJDF. A chemical-induced colorectal cancer model was established and treated with JPJDF to evaluate its effects. Tumor size was measured, and histopathological analyses were performed to examine JPJDF's regulatory potential on CRC. The functional mechanism of JPJDF was predicted through network pharmacology, molecular docking, and transcriptomics. Co-culture techniques involving CRC cells and CCD-18Co fibroblasts were used to assess JPJDF's impact on fibroblast activation. The effects of HIF1α on CAFs were evaluated using CCK-8 proliferation, clonal formation, and apoptotic assays, with differential marker expression quantified via qPCR and Western blotting. ResultsPharmacodynamic assessment demonstrated that JPJDF reduced tumor size without affecting body weight, indicating its safety in the chemical-induced murine CRC model. Network pharmacology analysis, combined with molecular docking and transcriptomics, revealed that JPJDF regulates HIF-1 signaling pathways and identified HIF1α as a potential target for JPJDF's anti-CRC effect. JPJDF effectively suppressed CRC growth in vivo by attenuating fibroblast activation, reducing α-SMA expression and POSTN secretion through HIF1α inhibition. HIF1α knockdown in CRC cells inhibited fibroblast proliferation and clonal formation, while overexpression promoted these processes. Additionally, downregulating HIF1α suppressed α-SMA and POSTN expression in fibroblasts, whereas overexpression enhanced fibroblast activation. ConclusionJPJDF emerges as a promising therapeutic candidate for inhibiting CAFs activation by targeting HIF1α, offering potential avenues for modulating fibroblast activation towards CAFs in CRC therapy.

E-cigarettes increase the risk of adenoma formation in murine colorectal cancer model.

E-cigarettes (E.cigs) cause inflammation and damage to human organs, including the lungs and heart. In the gut, E.cig vaping promotes inflammation and gut leakiness. Further, E.cig vaping increases tumorigenesis in oral and lung epithelial cells by inducing mutations and suppressing host DNA repair enzymes. It is well known that cigarette (cig) smoking increases the risk of colorectal cancer (CRC). To date, it is unknown whether E.cig vaping impacts CRC development. A mouse model of human familial adenomatous polyposis (CPC-APC) was utilized wherein a mutation in the adenomatous polyposis coli (APC) gene, CDX2-Cre-APCMin/+, leads to the development of colon adenomas within 16 weeks. Mice were exposed to air (controls), E.cig vaping, cig, or both (dual exposure). After 4 weeks of 2-hour exposures per day (1 hour of each for dual exposures), the colon was collected and assessed for polyp number and pathology scores by microscopy. Expression of inflammatory cytokines and cancer stem cell markers were quantified. DNA damage such as double-strand DNA breaks was evaluated by immunofluorescence, western blot and gene-specific long amplicon qPCR. DNA repair enzyme levels (NEIL-2, NEIL-1, NTH1, and OGG1) were quantified by western blot. Proliferation markers were assessed by RT-qPCR and ELISA. CPC-APC mice exposed to E.cig, cig, and dual exposure developed a higher number of polyps compared to controls. Inflammatory proteins, DNA damage, and cancer stemness markers were higher in E-cig, cig, and dual-exposed mice as well. DNA damage was found to be associated with the suppression of DNA glycosylases, particularly with NEIL-2 and NTH1. E.cig and dual exposure both stimulated cancer cell stem markers (CD44, Lgr-5, DCLK1, and Ki67). The effect of E.cigs on polyp formation and CRC development was less than that of cigs, while dual exposure was more tumorigenic than either of the inhalants alone. E.cig vaping promotes CRC by stimulating inflammatory pathways, mediating DNA damage, and upregulating transcription of cancer stem cell markers. Critically, combining E.cig vaping with cig smoking leads to higher levels of tumorigenesis. Thus, while the chemical composition of these two inhalants, E.cigs and cigs, is highly disparate, they both drive the development of cancer and when combined, a highly common pattern of use, they can have additive or synergistic effects.

Effects of fecal microbiota transplantation combined with selenium on intestinal microbiota in mice with colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in the world. With the development of high-throughput gene sequencing technology, homeostasis imbalance of the intestinal microbiota has been proven to play a key role in the pathogenesis of CRC. Furthermore, fecal bacteria transplantation (FMT) has been shown to alter the intestinal microecology, and is potentially an effective treatment for CRC. Sodium selenite plays an important role in anticancer adjuvant therapy due to its high pro-oxidation characteristics. In this study, a murine CRC tumor model was induced by AOM/DSS, and CRC mice were treated by FMT, sodium selenite, and FMT combined with sodium selenite. The results showed that FMT, sodium selenite, and FMT combined with sodium selenite inhibited the occurrence of CRC in mice, increased the abundance of beneficial intestinal bacteria, produced different microorganisms, and changed the metabolic pathways of the intestinal microbiota. In summary, FMT, sodium selenite, and FMT combined with sodium selenite can inhibit the occurrence of CRC by increasing the abundance of beneficial bacteria and regulating phenotypes and metabolic pathways. Notably, the effect of FMT combined with sodium selenite in reducing the number of tumors, protecting intestinal tissues, and restoring the diversity and richness of the intestinal microbiota is superior to that of FMT alone or sodium selenite alone. The results of this study provide new ideas for the application of FMT and selenium in the treatment of CRC.

Irreversible Electroporation has More Synergistic Effect with Anti‐PD‐1 Immunotherapy than Thermal Ablation or Cryoablation, in a Colorectal Cancer Model

AbstractBoosting the response rate of immune checkpoint blockade (ICB) therapy to improve treatment efficacy is a primary goal in cancer immunotherapy. One of the promising approaches involves focal tumor ablation to reduce tumor burden and trigger the in situ vaccination. Even though this combination strategy has demonstrated enhanced therapeutic outcomes in both preclinical research and clinical trials, limited research has comparatively investigated diverse ablation techniques. The optimal choice among focal therapy techniques remains largely unknown. In a murine colorectal cancer model (MC‐38), the therapeutic efficacy of anti‐PD‐1 in combination with thermal ablation, cryoablation, and irreversible electroporation (IRE) is evaluated, utilizing well‐characterized miniature probes. In this model, ICB monotherapy has limited effect in controlling tumor growth. IRE exhibits the most favorable synergistic effect with anti‐PD‐1 immunotherapy than thermal ablation or cryoablation, leading to the greatest primary tumor growth delay, longest tumor‐free survival, and highest protection against secondary tumor challenge. Furthermore, the co‐administration of IRE and anti‐PD‐1 significantly fosters the infiltration of CD8+ T cells into the tumor coupled with a remarkable stem‐like progenitor phenotype. The findings demonstrate that IRE stands as a promising modality that can potentiate the antitumor efficacy when the tumor is poorly responding to ICB monotherapy.

Targeting Nuclear Receptor Coactivator SRC-1 Prevents Colorectal Cancer Immune Escape by Reducing Transcription and Protein Stability of PD-L1.

Programmed death-ligand 1 (PD-L1) is overexpressed in multiple cancers and critical for their immune escape. It has previously shown that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability, yet its role in CRC immune escape is unclear. Here, we demonstrate that SRC-1 is positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibits PD-L1 expression in CRC cells and retards murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration of CD8+ T cells. Genetic ablation of SRC-1 in mice also decreases PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, these findings highlight a crucial role of SRC-1 in regulating PD-L1 expression and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment.

Pexidartinib and Immune Checkpoint Inhibitors Combine to Activate Tumor Immunity in a Murine Colorectal Cancer Model by Depleting M2 Macrophages Differentiated by Cancer-Associated Fibroblasts.

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are known to play supportive roles in tumor development and progression, but their interactions in colorectal cancer (CRC) remain unclear. Here, we investigated the effects of colon-cancer-derived CAFs on TAM differentiation, migration, and tumor immunity, both in vitro and in vivo. When co-cultured with monocytes, CAFs attracted monocytes and induced their differentiation into M2 macrophages. Immunohistology of surgically resected human CRC specimens and orthotopically transplanted mouse tumors revealed a correlation between numbers of CAFs and numbers of M2 macrophages. In a mouse model of CRC orthotopic transplantation, treatment with an inhibitor of the colony-stimulating factor-1 receptor (PLX3397) depleted M2 macrophages and increased CD8-positive T cells infiltrating the tumor nest. While this treatment had a minor effect on tumor growth, combining PLX3397 with anti-PD-1 antibody significantly reduced tumor growth. RNA-seq following combination therapy showed activation of tumor immunity. In summary, CAFs are involved in the induction and mobilization of M2 macrophage differentiation in the CRC tumor immune microenvironment, and the combination of cancer immunotherapy and PLX3397 may represent a novel therapeutic option for CRC.

Assessment of the Anti-cancer Effects of Camel Milk Exosomes (CMEXOs) on Murine Colorectal Cancer Cell Line (CT-26).

Today, camel milk consumption in the Middle East is trendy because it is believed that it reduces the risk of cancer. Recently, studies have discovered that most of milk's beneficial effects are because of its nanoparticles, especially exosomes. The objective of the present research was to investigate the anti-cancer effects of camel milk exosomes (CMEXOs) in the murine colorectal cancer cell line (CT-26). Our findings verified the existence of exosomes measuring approximately 114.1±3.4 nm in diameter. Through MTT and migration assays, we established that CMEXOs exhibit dose-dependent anti-proliferative and anti-migration effects on the CT-26 cell line. Furthermore, our study showed that treatment with CMEXOs led to a reduction in TNF-α and IL-6 gene expression in CT-26 cells. While additional in vivo studies are required, our data demonstrate that CMEXOs have anti-proliferative and anti-migration effects on CT-26, possibly by influencing crucial genes within the inflammation pathway.

Effect of TT-816, a novel immune response modifier targeting cannabinoid CB2 receptor, on antitumor immunity and cancer growth.

e14595 Background: The endocannabinoid system is widely expressed in the human body, including the innate and adaptive immune system, where endocannabinoids, Δ9-tetrahydrocannabinol and synthetic ligands regulate immune suppression. The effects of endocannabinoids on immune regulation are primarily mediated by G-protein coupled cannabinoid CB2 receptors (CB2R) via several mechanisms, including immune cell development, migration, proliferation and T and NK cell effector functions. The upregulated expression of CB2R and elevated levels of endocannabinoids have been observed in a variety of tumor microenvironments and are associated with the aggressiveness of cancer. Methods: NK cell function was determined by co-culturing TT-816 pretreated NK cells with K562 cancer cells for 24 hours. The mixed lymphocyte reaction assay was conducted by co-culturing human CD4+ T cells with monocyte-derived dendritic cells. Cell viability was measured by FACS and IFN-g by MSD. The in vivo efficacy of TT-816 was determined in the murine syngeneic B16F10 melanoma and MC38 colorectal cancer models. Tumor-bearing C57BL/6 mice were randomized 6 days after inoculation and treated with vehicle (0.5% CMC, QD) or with TT-816 at 1, 4 or 10 mg/kg (QD) orally, or anti-PD1 (RMP1 14) at 5 mg/kg (Q3D), intraperitoneally. Tumor size and body weight were determined every 2 to 3 days. Data represent mean±SEM. Results: TT-816 concentration-dependently enhanced the functions of NK cells, dendritic cells and T cells. It increased NK cell killing of the human cancer cells and IFN-g production, significantly stimulated the expression of CD86, HLA-DR, IL-12 and TNF-a in monocyte-derived dendritic cells, and enhanced CD4+ T cell proliferation and IFN-g production in a mixed lymphocyte reaction assay. In B16F10 melanoma and MC38 colorectal cancer models, TT-816 dose-dependently inhibited the tumor growth. Furthermore, combination of TT-816 with anti-PD1 synergistically inhibited both tumor growth. In B16F10 model, treatment with TT 816 (4 mg/kg, QD) alone significantly inhibited the tumor growth by 45%. By contrast, anti-PD1 had little effect (TGI 5%). However, the combination of TT-816 with anti-PD1 resulted in a TGI of 72%. In the MC38 model, treatment with TT-816 (4 mg/kg, QD) and anti-PD1 alone, achieved TGI of 50% and 36%, respectively. When combined, TGI was increased to 88%. Conclusions: TT-816 is a novel, oral small molecule immune response modifier that selectively blocks CB2R on immune cells and cancer cells. Preclinical data have demonstrated that it stimulates antitumor immune response and inhibits cancer growth in both hot and cold tumor models. TT-816 is currently undergoing phase 1 clinical trials for the treatment of a broad range of solid tumors.

Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 μg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25–3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.

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.

Abstract LB267: Disrupting cytoplasmic proton motive force induces ferroptotic cell death in colorectal cancer: A novel anti-cancer mechanism

Abstract Introduction: Chemotherapeutic resistance and tumor recurrence remain the leading causes of poor prognosis in colorectal cancer (CRC). Herein, we evaluated the anti-cancer effect of a novel repurposed drug HLN* in CRC. Methods: The changes in membrane potential were studied using DiSC(3) fluorescence by confocal microscopy. Cytotoxic activity of compound was studied using Sulphorhodamine-B (SRB) assay. Changes in mitochondrial membrane potential were studied using JC-1 dye. ROS accumulation was analyzed using DCFDA fluorescence using FACS analysis. Ferrorange assay was used to check the ferrous ion influx within the cell. Western blot was used to identify mode of cell death enacted. Results: Our results show that HLN treatment reduced the survival of human and murine CRC cells (CT26, HCT116, Colo 205 and MC38) with IC50 ranging 1-4 µM. HLN further inhibited proton motive force (PMF) by sequentially depolarizing and hyperpolarizing the cell membrane, as examined by cellular membrane potential using DiSC(3) dye by confocal microscopy. Disruption of the PMF leads to changes in various cellular signaling cascades including ROS production and mitochondrial membrane potential (MMP) changes. The MMP was significantly reduced by HLN treatment in CRC cells. Furthermore, we also observed an increase in ROS production by HLN. Pretreatment with N-acetyl cysteine (NAC), an antioxidant, blocked the effects of HLN. Interestingly, ferroptotic cell death markers GPX4, GPX1, SLC7A11/xCT, NRF2 and KEAP1 were significantly inhibited by HLN treatment in CRC cells as observed by western blotting. Ferroptotic induction was then confirmed using ferrorange assay wherein we observed a concentration dependent increase in ferrorange intensity indicating an increase in Fe+2. Overall, HLN dissipates ΔpH by modulating iron influx leading to ferroptotic cell death through ROS. Conclusion: Taken together, our results demonstrate the anti-cancer activity of a novel repurposed drug HLN and its unique mechanism of action. Our results provide a promise for developing this drug as a potential treatment option for CRC, through a novel anti-cancer mechanism. * Due to Intellectual property issued, the name of the drug is not provided Citation Format: Shreyas R. Gaikwad, Mohamed A. Eltokhy, Morgan Moore, Keerthi Kailath, Sanjay K. Srivastava. Disrupting cytoplasmic proton motive force induces ferroptotic cell death in colorectal cancer: A novel anti-cancer mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB267.

Tissue-specific reprogramming leads to angiogenic neutrophil specialization and tumor vascularization in colorectal cancer.

Neutrophil (PMN) tissue accumulation is an established feature of ulcerative colitis (UC) lesions and colorectal cancer (CRC). To assess the PMN phenotypic and functional diversification during the transition from inflammatory ulceration to CRC we analyzed the transcriptomic landscape of blood and tissue PMNs. Transcriptional programs effectively separated PMNs based on their proximity to peripheral blood, inflamed colon, and tumors. In silico pathway overrepresentation analysis, protein-network mapping, gene signature identification, and gene-ontology scoring revealed unique enrichment of angiogenic and vasculature development pathways in tumor-associated neutrophils (TANs). Functional studies utilizing ex vivo cultures, colitis-induced murine CRC, and patient-derived xenograft models demonstrated a critical role for TANs in promoting tumor vascularization. Spp1 (OPN) and Mmp14 (MT1-MMP) were identified by unbiased -omics and mechanistic studies to be highly induced in TANs, acting to critically regulate endothelial cell chemotaxis and branching. TCGA data set and clinical specimens confirmed enrichment of SPP1 and MMP14 in high-grade CRC but not in patients with UC. Pharmacological inhibition of TAN trafficking or MMP14 activity effectively reduced tumor vascular density, leading to CRC regression. Our findings demonstrate a niche-directed PMN functional specialization and identify TAN contributions to tumor vascularization, delineating what we believe to be a new therapeutic framework for CRC treatment focused on TAN angiogenic properties.

Abstract 1688: Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability

Abstract Programmed death-ligand 1 (PD-L1), one of the immunosuppressive molecules, overexpresses in multiple cancers and is critical for their immune escape. We previously showed that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability; however, the role of SRC-1 in CRC immune escape is unclear. Here, we demonstrated that SRC-1 was positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibited PD-L1 expression in both human and murine CRC cells and retarded murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration and antitumor activity of effector CD8+ T cells. Genetic ablation of SRC-1 in mice also decreased PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, our findings highlight a crucial role of SRC-1 in facilitating CRC immune escape and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment. Citation Format: Yilin Hong, Qiang Chen, Zinan Wang, Yong Zhang, Bei Li, Xu Kong, Pingli Mo, Nengming Xiao, Jianming Xu, Yunbin Ye, Chundong Yu. Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability [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 1688.

Abstract 4044: Potentiated antitumor activity of poly-γ-glutamic acid combined with anti PD-L1 blockade on murine models of colorectal cancer

Abstract Objective: Immune checkpoint inhibitors(ICIs) have been shown to be key role in cancer therapy. Especially, antibodies targeting programmed cell death ligand1(PD-L1) promoted T cells to activate immune response against cancer cells. However, the current ICIs still have insignificant to motivate effective antitumor immunity by blocking PD-1/PD-L1 pathway and the regulatory network of PD-L1 by cytokines in the tumor microenvironment remains to be uncovered. Previously, we have reported the anti-cancer effects of combination treatment with a natural product, poly-γ-glutamic acid(γ-PGA), and PD-1 blockade. Now, we show that combination therapy with anti PD-L1 and γ-PGA can overcome the limits of ICIs and enhance anticancer effect in murine colorectal cancer. Design: In vivo efficacy studies were performed by intraperitoneal injection of antibody targeting PD-L1 and oral administration of γ-PGA in C57BL/6 model transplanted with mouse tumor cell lines. Systemic changes in immunophenotype was measured by tumor volume size and body weight. The splenocyte was isolated from mice and the populations of myeloid-driven suppressor cells(MDSCs) and CD4/CD8 T cells were analyzed by FACS. Also, the significant cytokines activated by T- cell, IFN-γ and IL-2, were measured by ELISA. Results and Conclusions: In murine model of colorectal cancer, γ-PGA and anti PD-L1 blockade combination therapy significantly improved anti-tumor activity compared with single agents. In addition, tumoral infiltration of CD8+ T cells and IFN-γ productions were increased by γ-PGA, and its antitumoral activity was attenuated by antibody-mediated depletion of these cells, indicating that g-PGA suppressed tumor growth in a CD8 T cell-dependent manner. Therefore, our results suggest that γ-PGA promote ICI-mediated antitumor responses by the transformation of "cold tumors" into “hot tumors” Citation Format: Jae-Pyung Jang, Chan-Hee Yoo, Kyung-Soo Hahm, Do Young Lee, Young-Chul Park, Jae Chul Choi. Potentiated antitumor activity of poly-γ-glutamic acid combined with anti PD-L1 blockade on murine models of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4044.

Abstract 5271: Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS colorectal cancer

Abstract Purpose: Microsatellite-stable (MSS) colorectal cancer is resistant for immune therapy, necessitating the exploration of novel modalities for its conquest. Recent revelations have revealed the genetic upregulation of HIF-1α as a potential activator of CD8+ T-cells, enhancing an anti-tumor effect. Pseudohypoxia, characterized by normoxic upregulation of Hypoxia-Inducible Factor (HIF-1α), is known to be induced by iron chelators, including HIF-PHD inhibitors. In this study, we aimed to examine whether pseudohypoxia by HIF-PHD inhibitor can enhance immune response and elucidate the underlying mechanism. Method: Colon 26, SW480 and HT29 were used as murine and human MSS colorectal cancer model. Human and murine CD8+ T-cells were represented by Jurkat cells and isolated splenic CD8+ T-cells, respectively. HIF-PHD inhibitors Roxadustat and Vadadustat were used in the study, and a subcutaneous Colon 26 tumor model was established in BALB/c WT and nude mice. Hypoxic culture conditions were maintained for 48 hours at 2% O2. Results: Roxadustat and Vadadustat did not suppress the proliferation of cancer cells under conditions marked by HIF-1α upregulation. Roxadustat and Vadadustat suppress the colon 26 subcutaneous tumor growth in WT mice, the effect was cancelled in the nude mice. The treatment regimen was associated with an augmentation in the total number and effector-like subtypes of CD8+ T-cells (Tex) infiltrating the tumor. Combination therapy involving Roxadustat and PD-1 antibody synergistically inhibited tumor growth and was accompanied by an increase in PD-1lo, Ly108-, TIM-3+ CD8+ T-cells within the tumor microenvironment. Immunohistochemistry revealed that Interleukin-2 (IL-2) expression of murine spleen was stronger in Roxadustat than control group. Interestingly, HIF-PHD inhibitors induced the secretion of IL-2 from isolated CD3+ T-cells of murine spleens in ELISA, a phenomenon uniquely observed under pseudo-hypoxic conditions created by HIF-PH inhibitors, rather than actual hypoxia. This result indicates that pseudo-hypoxia may induce more robust hypoxic response than real hypoxia. Conclusion: The induction of pseudohypoxia by HIF-PHD inhibitors activates the tumor immune response for MSS colorectal cancer via induction of splenic IL-2. Citation Format: Toshiaki Ohara, Yuehua Chen, Yusuke Hamada, Seitaro Nishimura, Hotaka Kawai, Kazuhiro Noma, Hiroshi Tazawa, Toshiyoshi Fujiwara, Akihiro Matsukawa. Pseudohypoxia by HIF-PHD inhibitors activates tumor immune response for MSS 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 5271.

Abstract 5264: KRASG12C inhibition synergizes with checkpoint blockade in KRASG12C-mutated tumor model

Abstract Introduction: The KRASG12C mutation is found in 14% of non-small cell lung cancer (NSCLC) and 3% of colorectal cancer (CRC). Sotorasib (AMG510), which covalently binds to the mutated cysteine 12 of KRASG12C protein, became the first KRASG12C inhibitors approved by the FDA for KRASG12C-mutated NSCLC treatment. AMG510 not only increased apoptosis in cancer cells harboring KRASG12C mutation, but also re-modelled tumor microenvironment. Thus, to evaluate novel therapeutic strategies for treating KRASG12C-mutated tumors, we established a CT26-KRASG12C tumor model and explored the combination of AMG510 with anti-PD1 therapy. Methods: We knocked in KRASG12C mutation in murine CT26 colorectal cancer cells using CRISPR/Cas9 to generate CT26-KRASG12C cells. The mutation was confirmed by PCR sequencing. In vitro, CT26-KRASG12C cells were treated with AMG510 for 2h or 72h, and then p-ERK and cell viability were analyzed. In vivo, 3×105 CT26-KRASG12C cells were subcutaneously injected into BALB/c mice, and the tumor bearing mice were treated with AMG510 (30 mg/kg or 100 mg/kg, QD×21), anti-PD-1 (10 mg/kg, BIW×3) and combination of AMG510 and anti-PD-1. On day 5 after randomization, the composition of immune cells, including CD4+ T cells, CD8+ T cells, dendritic cells (DC), and natural killer (NK) cells in tumors were analyzed by flow cytometry. The mice with complete tumor regression were re-challenged with bilateral tumors of CT26-KRASG12C (3×105) and unrelated mouse breast tumor cells, 4T1 (3×105) to evaluate the effect on tumor growth. Results: Following confirmation of KRASG12C mutation, AMG510 treatment decreased p-ERK expression by ~50% and impaired the cell growth with IC50 value of 0.1802 μM in CT26-KRASG12C cells. In vivo, single treatment of AMG510 at 30 mg/kg or 100 mg/kg significantly inhibited tumor growth with TGI of 60% or 99% on day 15 after randomization, respectively (both p<0.001). Combined treatment of AMG510 (30 mg/kg or 100 mg/kg) and anti-PD-1 also led to significant tumor inhibition with TGI of 64% or 99% on the same day, respectively (both p<0.001), although PD-1 blockade didn’t show anti-tumor effect alone. Noticeably, combined treatment of AMG510 (100 mg/kg) and anti-PD-1 induced complete tumor regression in 3 of 10 mice, which remained cured 124 days later. Moreover, the combined treatment significantly improved survival compared to the AMG510 treatment alone. Notably, all 4T1 tumors grew, but none of the CT26-KRASG12C tumors were palpable in the cured mice after the re-challenge. In the control group, all tumors were well established. Additionally, combined treatment of AMG510 (100mg/kg) and anti-PD-1 markedly increased the infiltration of CD4+ T cells, DC, and NK cells in the tumors. Conclusion: The successfully established CT26-KRASG12C model is a useful tool for investigating therapeutic strategies to combine KRASG12C inhibitors with immunotherapy. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Chenfei Zhang, Ludovic Bourre, Jingjing Wang. KRASG12C inhibition synergizes with checkpoint blockade in KRASG12C-mutated tumor model [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 5264.

Abstract 6690: Gut microbial metabolite butyrate facilitates antitumor efficacy of telomerase-specific oncolytic adenovirus via MHC-I and cGAS-STING pathway activation

Abstract Introduction: Gut microbiota has been reported to have a potential to modulate tumor sensitivity to chemo- or immuno-therapies although the underlying mechanism is still unclear. Oncolytic virotherapy is a promising antitumor treatment that selectively induces oncolytic cell death in tumor cells. We have previously developed OBP-702, a telomerase-specific oncolytic adenovirus armed with the p53 tumor suppressor gene, highlighting potent antitumor effects across different cancer types. To explore the potential of gut microbial metabolites, particularly butyrate, in augmenting the antitumor efficacy of oncolytic virotherapy. Materials and Methods: We assessed the synergistic antitumor effects of butyrate and OBP-702 in cytotoxic assay using two human (HCT116, SW48) and two murine (CT26, MC38) colorectal cancer cell lines in vitro, and the in vivo antitumor effects in subcutaneous tumor models using HCT116 and CT26. We explored underlying mechanisms of the synergistic effects, focusing on the influences of butylate on viral infectivity via Coxsackie-Adenovirus Receptor (CAR) and integrins, and antitumor immunity via tumor MHC-I expression and CD8-positive T cells. Results: Butyrate and OBP-702 showed potent synergistic effects in all four cell lines in vitro, and the combination suppressed tumor growth significantly in HCT116 and CT26 subcutaneous tumors compared to each monotherapy. These synergistic effects were produced by butyrate increasing the potential of OBP-702 by enhancing viral infection efficiency via upregulating expressions of CAR and integrins on tumor cells. Butyrate also increased MHC-I expression on tumor cells by activating the cGAS-STING pathway, leading to increased CXCL10 expression, which led to activation of antitumor immunity through recruitment of CD8-positive T cells in tumor tissues. The synergistic effects of butyrate and OBP-702 were proved in an orthotopic colorectal tumor model with liver metastases using CT26 cells expressing luciferase, in which this combination not only reduced tumor growth, but also improved survival by activating antitumor immunity via CD8-positive T cells. Conclusion: Butyrate and OBP-702 showed synergistic antitumor effects via activation of systemic antitumor immunity in addition to direct effects of butyrate on viral cytotoxic potential, which provides valuable insights into the development of innovative cancer treatment strategies. Citation Format: Masaki Sakamoto. Gut microbial metabolite butyrate facilitates antitumor efficacy of telomerase-specific oncolytic adenovirus via MHC-I and cGAS-STING pathway activation [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 6690.