Murine Colon Adenocarcinoma Research Articles

A first-in-human, phase I, open-label study of a novel cancer vaccine labvax 3(22)-23 and adjuvant GM-CSF in patients with advanced stage adenocarcinomas.

TPS2700 Background: Adenocarcinoma is the most common histologic type of solid tumors and can arise from almost anywhere in the body. Labyrinthin is a novel tumor-specific protein expressed on the cell surface of the majority of adenocarcinomas of various cancer types. Several therapeutic strategies targeting labyrinthin are under development. We hypothesize that vaccination against labyrinthin can elicit strong immune responses against the labyrinthin-positive adenocarcinomas in cancer patients. LabVax 3(22)-23 is a novel anti-tumor vaccine that contains 4 synthetic labyrinthin-based peptides designed to elicit both B-cell and T-cell responses. Sargramostim, a recombinant granulocyte macrophage colony-stimulating factor (GM-CSF), is given as an immunostimulator with LabVax 3(22)-23 to boost the antitumor immune response. Preclinical studies showed that LabVax 3(22)-23 significantly inhibited tumor growth that was augmented by GM-CSF without any significant toxicity in C57/BL6 transgenic mice expressing human PD-1/PD-L1 implanted with the murine colon adenocarcinoma cell line MC-38-huPD-L1. This single institution, first-in-human, phase I trial (UCDCC#296) evaluates LabVax 3(22)-23 and adjuvant sargramostim in patients with labyrinthin-positive metastatic or recurrent adenocarcinoma of any primary tumor site after all standard-of-care therapies. Methods: The primary objective is to assess the toxicity of LabVax 3(22)-23 and adjuvant sargramostim according to NCI CTCAE V5.0. The primary endpoint is dose limiting toxicity (DLT), which is defined as grade ≥ 2 allergic and autoimmune reaction, grade ≥ 3 injection site reaction, any grade 3 toxicities lasting >1 week, or any grade ≥ 4 toxicities. With a sample size of 10 patients, there is 89-97% of chance to observe ≥1 DLT if the true event rate is 20-30%. A secondary endpoint is the preliminary assessment of tumor response rate by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Exploratory objectives measure the effect of LabVax 3(22)-23 on various immune responses (cytokines, anti-labyrinthin antibody production) and the correlation between the level of labyrinthin expression and the efficacy of LabVax 3(22)-23. Eligible patients are required to have labyrinthin expression on their tumor cells by immunohistochemistry and adequate organ function. Patients receive sargramostim subcutaneously and LabVax 3(22)-23 intradermally on weeks 1, 2, 4, 8, and 12 in the absence of disease progression or unacceptable toxicity. After completion of study treatment, patients are followed every 3 months for 1 year. Two patients are enrolled at the time of submission. Clinical trial information: NCT05101356.

Abstract A037: Mathematical modeling of the tumor-immune microenvironment in murine colon adenocarcinoma

Abstract Colon cancer is currently the fourth most common cancer in the United States, impacting over 100,000 people every year with increasingly younger patients affected by the malignancy. As a result, it is imperative to better understand the mechanisms that are involved in the development of colon cancer within the body. The intestine houses the largest compartment of the human body’s immune system, and the intestinal immune system has been implicated in the development of disease and malignancy of both the colon itself and other organ systems throughout the body. In particular, the colon contains high numbers of IgA antibodies (produced by B-cells) in conjunction with regulatory T cells (Treg). Because of the colon’s rich immune system involvement, we are interested in recapitulating several of the immune components and their roles in both targeting tumors and aiding tumors in their aggression. We strive to develop an accurate model of anti-tumor immune cells (CD8+ T-cells, CD4+ T-cells, Natural Killer Cells, Macrophages, Dendritic Cells, Neutrophils) and immunosuppressive cells (Treg, Myeloid-Derived Suppressor Cells) and their engagement with colon adenocarcinoma. The model is built in a nested fashion, beginning with the parametrization of a model of a monolayer from cell cycle and doubling time data. Growth parameters are subsequently obtained for spheroids with no immune cells, spheroids with immune cells, allograft studies in immunocompromised mice, and allograft studies in mice with intact immune systems. The model further incorporates cell cycle stages, quiescence, necrosis, VEGF and TNF-alpha production, and vascularization of the in vivo tumors. The data used to parametrize our model is obtained from literature centered around experiments with CT26 BALB/c murine colon adenocarcinoma both in vitro and in vivo. This cell line is of particular interest because of the large body of publications that utilize it and because the cells form tumors in both immunocompromised and syngeneic BALB/c mice thereby allowing for parametrization of the in vivo model both with and without immune interaction. There are additionally experimental studies through which spheroids were generated with various immune cell infiltrates, thereby enabling more accurate parametrization of each of the immune populations and their impacts on tumor development. We thus aim to develop a novel ODE model of colon adenocarcinoma and the various immune cells that engage in the tumor microenvironment to better evaluate the impacts of these immune populations and the outcomes of immunotherapy targeted at these cells. Citation Format: Frederika Rentzeperis, Maya Srinivasan, Dorothy Wallace. Mathematical modeling of the tumor-immune microenvironment in murine colon adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A037.

Solid lipid nanoparticles loaded with curcumin: development and in vitro toxicity against CT26 cells.

Aim: To develop a new curcumin carrier consisting of murumuru butter nanoparticles (SLN-Cs). Methods: A phase-inversion temperature method was used to produce SLN-Cs. The interaction of SLN-Cs with murine colon adenocarcinoma (CT26) cells in vitro was analyzed by confocal microscopy. Results: Stable SLN-Cs with a high curcumin-loading capacity were obtained. TheSLN-Cs were more toxicto CT26than free curcumin. Fluorescence microscopy images showed the SLN-Cs to be taken up by CT26 cells in vitro. Conclusion: These results indicate that SLN-Cs are suitable carriers of curcumin in aqueous media.

Abstract P003: Efficacy results of a novel vaccine composed of stimulated and haptenized tumors cells in Balbc mice grafted with murine colon adenocarcinoma CT26 cells

Abstract BACKGROUND: Metastatic colorectal cancer (mCRC) is a major cause of death. Unmet medical need in immunotherapy is high for MSS patients and still present for MSI-H/dMMR patients. STC vaccine (Brenus Pharma) is composed of selected tumor cell lines, stimulated to overexpress TAA/TSA and neoantigens including resistant factors that are further haptenized to form immunogenic hapten-protein complex to educate the immune system to recognize and target the patient's tumor cells expressing the same resistance factors. We report results of two studies aiming to A) evaluate efficacy of a one cell line-based product (CT26) physical stimulated (S=irradiation and heat shock) and/or haptenized (H) w/o immunostimulant (IS=cyclophosphamide + mGM-CSF w/o BCG) and to B) investigate a potential increase of antitumoral effect of 3 cell lines vaccine (3CL-SH made of CT26, CMT-93, LTPA). STUDY DESIGN and METHODS: Female BALB/c mice were subcutaneously grafted with 5.104 CT26-WT cells. In study A, 9 groups (10 mice per group) are allocated to: G1) Control group, G2) IS, G3) CT26-S, G4) CT26-H, G5) CT26-SH, G6) CT26-S+IS, G7) CT26-H+IS, G8) CT26-SH+IS, G9) CT26-SH+IS+BCG. In study B, 5 groups (20 mice per group) are allocated to: G1) Control group, G2) CT26-SH + IS, G3) 3CL-SH, G4) 3CL-SH + IS once a week for 3 weeks and G5) 3CL-SH + IS twice a week for 4 weeks. Treatments were administered subcutaneously. Overall survival (OS) and tumor growth (TG) were recorded until 1000 mm3, safety endpoint or on D41 (study A) or D50 (study B). RESULTS: Stimulated cell-based treatments with IS (CT26-SH + IS) significantly increases OS compared to control group (Study A: G1/G8 p=0.0046 & study B: G1/G2 p=0.0023). Best mOS among groups is observed with 3CL-SH+IS (Study B: G1/G4 p<0.0001 38d vs 27d, Log-rank test). A direct comparison of 3CL-SH+IS and CT26-SH+IS confirmed a highly significant added benefit in favour of the 3 cell lines vaccine (Study B: G2/G4 p=0.0475) compared to the one cell line treatment. In addition, IS reinforced the effect of cell-based treatment, with CT26-SH (Study A: G1/G8, p<0.0001 at D20, Study B: G1/G2 p<0.0001 at D24) and with 3CL-SH (Study B: G4/G3 p=0.0004). Addition of BCG to CT26-SH+IS does not improve efficacy (Study A: G8/G9 p=NS). Results showed a significant impact on TG when cells were both physically stimulated then haptenized, such as CT26-SH (study A: G1/G5, p=0.003 at D20) or 3CL-SH (study B G1/G3 P<0.0001 at D24) compared to the control group. 3CL-SH+IS exhibits a significant efficacy on TG (Study B: G1/G4 p=0.0053 or G1/G5 p=0.0018) and OS whatever the administration schedule No side effect or inflammatory reaction towards the vaccines have been evidenced. CONCLUSION: Brenus STC vaccine based on physical stimulation and haptenization demonstrated a significant anticancer effect in mice with immunostimulant and confirmed a better efficacy of the 3 cell lines vaccine versus a single cell line vaccine. Further studies are ongoing to test the efficacy of STC vaccine in PD1 resistant preclinical model and in combination with SOC. Citation Format: Céline Gongora, Jacqueline Taleb, Benoît Pinteur, Lionel Chalus, Fanny De Luca, Paul Bravetti, François Ghiringhelli. Efficacy results of a novel vaccine composed of stimulated and haptenized tumors cells in Balbc mice grafted with murine colon adenocarcinoma CT26 cells [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 P003.

The combination therapy of oncolytic HSV-1 armed with anti-PD-1 antibody and IL-12 enhances anti-tumor efficacy

Cancer immunotherapy is a new therapeutic strategy for cancer treatment that targets tumors by improving or restoring immune system function. Therapies targeting immune checkpoint molecules have exerted potent anti-tumor effects and prolonged the overall survival rate of patients. However, only a small number of patients benefit from the treatment. Oncolytic viruses exert anti-tumor effects by regulating the tumor microenvironment and affecting multiple steps of tumor immune circulation. In this study, we engineered two oncolytic viruses that express mouse anti-PD-1 antibody (VT1093M) or mouse IL-12 (VT1092M). We found that both oncolytic viruses showed significant anti-tumor effects in a murine CT26 colon adenocarcinoma model. Importantly, the intratumoral combined injection with VT1092M and VT1093M inhibited growth of the primary tumor, prevented growth of the contralateral untreated tumor, produced a vaccine-like response, activated antigen-specific T cell responses and prolonged the overall survival rate of mice. These results indicate that combination therapy with the engineered oncolytic virus may represent a potent immunotherapy strategy for cancer patients, especially those resistant to PD-1/PD-L1 blockade therapy.

Potential of unglycosylated horseradish peroxidase variants for enzyme prodrug cancer therapy

Fighting cancer still relies on chemo- and radiation therapy, which is a trade-off between effective clearance of malignant cells and severe side effects on healthy tissue. Targeted cancer treatment on the other hand is a promising and refined strategy with less systemic interference. The enzyme horseradish peroxidase (HRP) exhibits cytotoxic effects on cancer cells in combination with indole-3-acetic acid (IAA). However, the plant-derived enzyme is out of bounds for medical purposes due to its foreign glycosylation pattern and resulting rapid clearance and immunogenicity. In this study, we generated recombinant, unglycosylated HRP variants in Escherichia coli using random mutagenesis and investigated their biochemical properties and suitability for cancer treatment. The cytotoxicity of the HRP-IAA enzyme prodrug system was assessed in vitro with HCT-116 human colon, FaDu human nasopharyngeal squamous cell carcinoma and murine colon adenocarcinoma cells (MC38). Extensive cytotoxicity was shown in all three cancer cell lines: the cell viability of HCT-116 and MC38 cells treated with HRP-IAA was below 1% after 24 h incubation and the surviving fraction of FaDu cells was ≤ 10% after 72 h. However, no cytotoxic effect was observed upon in vivo intratumoral application of HRP-IAA on a MC38 tumor model in C57BL/6J mice. However, we expect that targeting of HRP to the tumor by conjugation to specific antibodies or antibody fragments will reduce HRP clearance and thereby enhance therapy efficacy.

Ultrafine Jujube Powder Enhances the Infiltration of Immune Cells during Anti-PD-L1 Treatment against Murine Colon Adenocarcinoma.

Simple SummaryWhile modulating gut microbiota using dietary intervention with natural nutrients has proven to be effective in improving the response rate of immune checkpoint inhibitors (ICIs), the underpinning mechanism is poorly understood. This work demonstrates that the oral administration of ultrafine jujube powder (JP) let to a significant alteration of gut microbiota, an increased abundance of Clostridiales, including Ruminococcaceae and Lachnospiraceae, an elevated SCFA production, an intensified infiltration of CD8+ T cells to the tumor microenvironment, and a greatly improved response of anti-PD-L1 treatment against murine colon adenocarcinoma. Moreover, the size of the JP particles had a significant impact on the abovementioned attributes. The present study demonstrates that dietary intervention with nutrients is highly effective in modulating the gut microbiota for an improved immune checkpoint blockage therapy.Whereas dietary intervention with natural nutrients plays an important role in activating the immune response and holds unprecedented application potential, the underpinning mechanism is poorly understood. The present work was dedicated to comprehensively examine the effects of ultrafine jujube powder (JP) on the gut microbiota and, consequentially, the effects associated with the response rate to anti-PD-L1 treatment against murine colon adenocarcinoma. A murine colon adenocarcinoma model with anti-PD-L1 immunotherapy was established to evaluate how dietary interventions affect the microbiota. In vitro and in vivo experiments confirmed the role of SCFAs in the immune response. Oral administration of JP greatly improves the response of anti-PD-L1 treatment against murine colon adenocarcinoma. Such an improvement is associated with the alteration of gut microbiota which leads to an increased abundance of Clostridiales, including Ruminococcaceae and Lachnospiraceae, an elevated SCFA production, and an intensified infiltration of CD8+ T cells to the tumor microenvironment. This work demonstrates that JP is particularly effective in modulating the gut microbiota for an improved immune checkpoint blockage therapy by boosting cytotoxic CD8+ T cells in tumor-infiltrating lymphocytes. The experimental findings of the present study are helpful for the development of dietary intervention methods for cancer immunotherapy using natural nutrients.

Ibudilast suppresses oxaliplatin-induced mechanical allodynia and neurodegeneration in rats

Oxaliplatin is a key drug used in the management of solid tumors, such as colorectal cancer; however, it causes peripheral neuropathy. In this study, we investigated the effect of ibudilast, a phosphodiesterase inhibitor, on oxaliplatin-induced mechanical allodynia and histological changes in rats. Ibudilast (7.5 mg/kg, i.p., 5 times per week) reduced mechanical allodynia and histological changes induced by oxaliplatin (4 mg/kg, i.p., twice a week). In contrast, ibudilast (0.01–10 μM) had no effect on oxaliplatin-induced tumor cytotoxicity in murine colon adenocarcinoma 26 cells. These findings suggest that ibudilast could be useful for preventing oxaliplatin-induced peripheral neuropathy in clinical settings.

Outcome of concurrent treatment with a-CTLA4 and metronidazole in murine model of colon adenocarcinoma.

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.

A Hepatitis B Virus-Derived Peptide Exerts an Anticancer Effect via TNF/iNOS-producing Dendritic Cells in Tumor-Bearing Mouse Model.

Simple SummaryRecently, it has been reported that a tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cell (Tip-DC) may play a pivotal role in the anticancer immune response by activating CD8+ T cells in tumor microenvironments. The development of a new immunotherapeutic agent that can enhance the oncolytic effect of Tip-DC has gained increasing attention in the cancer research field. In this study, we introduce a hepatitis B virus-derived peptide, Poly6, which elicited a strong anticancer immune response via enhanced Tip-DC activity. Our findings suggest that Poly6 could be a novel potential adjuvant/co-treatment partner in anticancer immunotherapy approaches.Recently, we reported a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, that exerts antiviral effects against human immunodeficiency virus type 1 (HIV-1). Here, we explored the immunotherapeutic potential of Poly6 via its administration into dendritic cells (DCs) in a mouse model. Our data revealed that Poly6 treatment led to enhanced production of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner via the induction of mitochondrial stress. Poly6 treatment in mice implanted with MC38 cells, a murine colon adenocarcinoma line, led to attenuated tumor formation, primarily due to direct cell death induced by Tip-DC mediated nitric oxide (NO) production and indirect killing by Tip-DC mediated cluster of differentiation 8 (CD8) cytotoxic T lymphocyte (CTL) activation via CD40 activation. Moreover, Poly6 treatment demonstrated an enhanced anticancer effect with one of the checkpoint inhibitors, the anti PD-L1 antibody. In conclusion, our data reveal that Poly6 treatment elicits an antitumor immune response in mice, possibly through NO-mediated oncolytic activity via Tip-DC activation and Tip-DC mediated CTL activation. This suggests that Poly6 represents a potential adjuvant for cancer immunotherapy by enhancing the anticancer effects of immune checkpoint inhibitors.

Downregulating testosterone levels enhance immunotherapy efficiency

ABSTRACT Low response rates to certain tumor types remain a major challenge for immune checkpoint blockade therapy. In this study, we first conducted an integrated biomarker evaluation of bladder cancer patients from confirmatory cohorts (IMvigor210) and found that no significant differences exist between sexes before acceptance of anti-PD-L1 treatment, whereas male patients showed a better response. Thus, we then focused on sex-related changes post anti-PD-L1 treatment and found no obvious impact on the gut microbiota in male mice but a significant decrease in the sex hormone levels. Further, castration dramatically enhanced the antitumor efficacy against murine colon adenocarcinoma in male mice. Moreover, a narrow-spectrum antibiotic, colistin was innovatively used for deregulation of testosterone levels to enhance the immunotherapy efficiency in male mice. These findings indicate that the impact on the sex hormone levels in males may contribute to the sexual dimorphism in response and provide a promising way to enhance immunotherapy efficiency.

High-fat diet-fed ovariectomized mice are susceptible to accelerated subcutaneous tumor growth potentially through adipose tissue inflammation, local insulin-like growth factor release, and tumor associated macrophages.

Background: The association between obesity and colorectal cancer (CRC) risk has been well established. This relationship appears to be more significant in men than in women, which may be attributable to sex hormones. However, controlled animal studies to substantiate these claims and the mechanisms involved are lacking.Materials and Methods: MC38 murine colon adenocarcinoma cells were injected subcutaneously into high-fat diet (HFD) fed male, female and ovariectomized (OVX) female C57BL/6 mice.Results: HFD increased tumor growth (main effect) that was consistent with metabolic perturbations (P < 0.01). HFD OVX mice exhibited the most significant tumor growth compared to HFD male and female mice (p < 0.05) and this was associated with increased subcutaneous adipose tissue (p < 0.05). Further, the subcutaneous adipose tissue depots within HFD OVX mice exhibited more severe macrophage associated inflammation compared to female (P < 0.01), but not male mice. Conditioned media from subcutaneous adipose tissue of HFD OVX contained higher IGF-1 levels compared to male (P < 0.01), but not female mice. Finally, HFD OVX mice had increased M2-like gene expression in their tumor-associated macrophages (TAMs) compared to female mice (P < 0.01).Conclusions: This work provides evidences suggesting adiposity, adipose specific IGF-1, macrophage associated adipose inflammation, and TAMs as potential mechanisms driving obesity-enhanced CRC in females lacking ovarian hormones.

Downregulation of Interferon-γ Receptor Expression Endows Resistance to Anti-Programmed Death Protein 1 Therapy in Colorectal Cancer.

Immune checkpoint inhibitors have emerged as a frontline treatment of a variety of malignancies. However, only a subset of patients respond to these therapies, and many initial responders eventually develop resistance, leading to tumor relapse. Programmed death protein 1 is one of the checkpoint inhibitors that is expressed on activated T cells and suppresses the antitumor immune response when binding to its ligand, programmed death ligand 1, on tumor cells. Previous studies indicated that loss-of-function mutations in the IFN-γ pathway could result in acquired resistance to immune checkpoint inhibitors in human patients with cancer. Here, we investigated the effects of the IFN-γ receptor downexpression on the response to an anti-PD-1 antibody (αPD1) in a murine colorectal cancer model and the underlying mechanisms of resistance. IFN-γ receptor (IFNGR) 1 was knocked down in MC38 cells, a murine colon adenocarcinoma cell line using IFNGR1 short hairpin RNA (shRNA) lentiviral particles. Then, MC38 IFNGR1 knockdown (KD) cells and negative control (SC) cells were used in this study. In the C57BL/6 xenograft model, the KD tumor demonstrated resistance to αPD1 in comparison with SC cells. The observed treatment resistance might be associated with reduced tumor-infiltrating immune cells (TILs). When mixed, the resistant (KD) and control cells (SC) grew in spatially separated tumor areas, and αPD1 did not impact this pattern of spatial distribution. Our findings have proved that downregulation of the IFNGR1 endowed resistance to αPD1 and provided the potential mechanisms involving the TILs. SIGNIFICANCE STATEMENT: Immunological checkpoint blockades have achieved substantial efficacy in a variety of tumors. However, only a subset of patients respond to these therapies, and innate and acquired resistance is widely present. Our study found that the downregulation of the IFN-γ receptor caused resistance to an anti-PD-1 antibody in a murine colorectal cancer model associated with the reduced tumor-infiltrating lymphocytes. Our findings have substantial implications for improving the efficacy of checkpoint blockades.

Tumor cell endogenous HIF-1α activity induces aberrant angiogenesis and interacts with TRAF6 pathway required for colorectal cancer development

Hypoxia and inflammation are key factors for colorectal cancer tumorigenesis. The colonic epithelium belongs to the tissues with the lowest partial pressure of oxygen in the body, and chronic inflammation is associated with an increased chance to develop colon cancer. How the colonic epithelium responds to hypoxia and inflammation during tumorigenesis remains to be elucidated. Here we show, that murine colon adenocarcinoma cells with attenuated response to hypoxia, due to a knock-down (KD) of HIF-1α, produce smaller and less hypoxic tumors in an orthotopic mouse model when compared to tumors induced with control cells. HIF-1α-KD tumors showed more functional perfused vasculature associated with increased levels of vessel-stabilizing factors and reduced levels of proangiogenic factors, including extracellular matrix protein Cyr61/CCN1. Intratumoral injection of Cyr61 in HIF-1α-KD tumors revealed an in increased vessel permeability and tumor hypoxia. Further bioinformatics analysis identified a possible interaction between HIF-1α and TRAF6, an upstream effector of the NF-κB pathway that was confirmed by coimmunoprecipitation in MC-38 and CT26 colon adenocarcinoma cells and in situ by proximity ligation assay. Down-regulation of TRAF6 resulted in virtual abrogation of orthotopic tumor growth. Subcutaneous TRAF6-KD tumors were smaller and contained reduced vessel size and differently polarized macrophages. These data demonstrate that the tumor cell response to increased hypoxia in the colon leads to promotion of nonfunctional angiogenesis, regulated by both hypoxia and TRAF6 pathways.

Anticancer drug-loaded mesenchymal stem cells for targeted cancer therapy

Mesenchymal stem cells (MSCs) have a tumor-homing ability—they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.

FUT9-Driven Programming of Colon Cancer Cells towards a Stem Cell-Like State

Simple SummaryAberrant glycosylation, for instance heightened expression of fucosylated structures, is a frequent feature observed in tumor cells. Our paper outlines the role of aberrant fucosylation by the Fucosyltransferase 9 (FUT9) as a potent reprogramming factor marking the acquisition of a stem-like state both by murine and human colon cancer cells. Importantly, our study reinforces the implication of aberrant fucosylation in promoting tumor growth and resistance to chemotherapy in the context of colon cancer.Cancer stem cells (CSCs) are located in dedicated niches, where they remain inert to chemotherapeutic drugs and drive metastasis. Although plasticity in the CSC pool is well appreciated, the molecular mechanisms implicated in the regulation of cancer stemness are still elusive. Here, we define a fucosylation-dependent reprogramming of colon cancer cells towards a stem cell-like phenotype and function. De novo transcriptional activation of Fut9 in the murine colon adenocarcinoma cell line, MC38, followed by RNA seq-based regulon analysis, revealed major gene regulatory networks related to stemness. Lewisx, Sox2, ALDH and CD44 expression, tumorsphere formation, resistance to 5-FU treatment and in vivo tumor growth were increased in FUT9-expressing MC38 cells compared to the control cells. Likewise, human CRC cell lines highly expressing FUT9 displayed phenotypic features of CSCs, which were significantly impaired upon FUT9 knock-out. Finally, in primary CRC FUT9+ tumor cells pathways related to cancer stemness were enriched, providing a clinically meaningful annotation of the complicity of FUT9 in stemness regulation and may open new avenues for therapeutic intervention.

Mouse Model for Efficient Simultaneous Targeting of Glycolysis, Glutaminolysis, and De Novo Synthesis of Fatty Acids in Colon Cancer.

Colon cancer is a highly anabolic entity with upregulation of glycolysis, glutaminolysis, and de novo synthesis of fatty acids, which also induces a hypercatabolic state in the patient. The blockade of either cancer anabolism or host catabolism has been previously proven to be a successful anticancer experimental treatment. However, it is still unclear whether the simultaneous blockade of both metabolic counterparts can limit malignant survival and the energetic consequences of such an approach. In this chapter, byusing the CT26.WT murine colon adenocarcinoma cell line as a model of study, we provide a method to simultaneouslyperformapharmacological blockade of tumor anabolism and host catabolism, as a feasible therapeutic approach to treat cancer, and to limit its energetic supply.

Nanoparticles Presenting Potent TLR7/8 Agonists Enhance Anti-PD-L1 Immunotherapy in Cancer Treatment.

Cancer immunotherapy can be augmented with toll-like receptor agonist (TLRa) adjuvants, which interact with immune cells to elicit potent immune activation. Despite their potential, use of many TLRa compounds has been limited clinically due to their extreme potency and lack of pharmacokinetic control, causing systemic toxicity from unregulated systemic cytokine release. Herein, we overcome these shortcomings by generating poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) nanoparticles (NPs) presenting potent TLR7/8a moieties on their surface. The NP platform allows precise control of TLR7/8a valency and resulting surface presentation through self-assembly using nanoprecipitation. We hypothesize that the pharmacokinetic profile of the NPs minimizes systemic toxicity, localizing TLR7/8a presentation to the tumor bed and tumor-draining lymph nodes. In conjunction with antiprogrammed death-ligand 1 (anti-PD-L1) checkpoint blockade, peritumoral injection of TLR7/8a NPs slows tumor growth, extends survival, and decreases systemic toxicity in comparison to the free TLR7/8a in a murine colon adenocarcinoma model. These NPs constitute a modular platform for controlling pharmacokinetics of immunostimulatory molecules, resulting in increased potency and decreased toxicity.

Abstract 4583: Generation of peptide vaccines targeting labyrinthin, a pan-adenocarcinoma biomarker

Abstract We have previously shown that labyrinthin is expressed on the surface of essentially all adenocarcinomas, including lung, breast, prostate, and colorectal cancer, but not in normal cells. The present work includes further experiments that demonstrate the selective cancer cell surface localization of labyrinthin by FACS analysis of multiple normal, adenocarcinoma, and other (non-adeno) cancer cell lines. In addition, single-cell analysis showed mouse monoclonal anti-labyrinthin antibody MCA 44-3A6 bound to the surface of A549 human lung adenocarcinoma cells, but not to normal WI38 lung fibroblasts. Because available data collectively indicate that labyrinthin is a promising therapeutic target for adenocarcinomas, regardless of tissue origins, several therapeutic strategies targeting labyrinthin are under development, including vaccines against labyrinthin. In the first-in-human clinical trial we demonstrated no toxicity and seroconversion to the vaccine in 5 patients with metastatic lung adenocarcinoma. Using the cloned sequence and computer simulation modeling, a 2nd generation synthetic peptide vaccine was generated, LabVax 3(22)-23 (LabVax), that is comprised of four synthetic peptides to labyrinthin. LabVax incorporates the 1st generation vaccine epitopes with additional labyrinthin-based amino acids to potentiate not only B-cell, but also T-cell responses. LabVax safety was determined in BALB/c mice (male:female 50:50; N=20 per treatment group). A separate study in female C57/BL6 transgenic mice expressing human PD-1/PD-L1 implanted with the murine colon adenocarcinoma cell line MC-38-huPD-L1 also tested for safety, immune responses and anti-tumor effects [LabVax ± mGM-CSF adjuvant, ± pembrolizumab]. Control tumor weights on day 15 were 138 ± 31 mg (x¯ ± SD) and steadily rose across all data collection time points (days 22, 29, 36, 43, 48) up to 1597 ± 577mg by day 48. As expected, pembrolizumab eliminated all tumors by day 29. LabVax + mGM-CSF eliminated the tumor in one mouse by day 29, and significantly (p&amp;lt;0.05) reduced tumor growth in the remaining mice at each time point (day 48 = 884 ± 503mg; N=6). Pembrolizumab increased CD3, CD4, and CD4 cell counts (end of study, day 48), but not LabVax. LabVax alone or in combination with pembrolizumab treatments were well tolerated (no morbidity, mortality, weight loss). The data indicate LabVax is a prototype pan-adenocarcinoma vaccine. A phase I study evaluating the safety of LabVax (± adjuvant) alone or in combination with a PD-1 inhibitor in patients with refractory, metastatic adenocarcinomas is to be conducted at UC Davis Comprehensive Cancer Center. We will also determine the safety of LabVax and the ability of patients to induce an appropriate immune response (e.g., antibody production, T-cell response) against the vaccine. Citation Format: Michael Babich, James A. Radosevich, Tianhong Li. Generation of peptide vaccines targeting labyrinthin, a pan-adenocarcinoma biomarker [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4583.

Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail.

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.