Murine Colorectal Cancer Model Research Articles

Abstract PR011: Histone demethylase KDM5D drives sex-specific differences in colorectal cancer

Abstract Gender exerts a profound impact on cancer incidence, spectrum and outcomes, yet the molecular genetic bases of such sex differences are ill-defined and presumptively ascribed to X-chromosome genes and sex hormones. Such sex differences are particularly prominent in colorectal cancer (CRC) where men experience higher metastases and mortality. A murine CRC model, engineered with an inducible oncogenic Kras transgene (KRAS*) and conditional null alleles of Apc and Trp53 tumor suppressors (designated iKAP), revealed higher metastases and worse outcomes specifically in males with KRAS* CRC. Integrated cross-species molecular and transcriptomic analyses identified Y-chromosome gene histone demethylase KDM5D as a transcriptionally up-regulated gene driven by KRAS*-mediated activation of the STAT4 transcription factor. KDM5D-dependent chromatin mark and transcriptome changes showed repression of regulators of epithelial cell tight junction and MHC class I complex components. Deletion of KDM5D in iKAP cancer cells increased tight junction integrity, decreased cell invasiveness, and enhanced cancer cell killing by CD8+ T cells. Conversely, iAP mice engineered with a Kdm5d transgene to provide constitutive KDM5D expression specifically in iAP cancer cells exhibited an increased propensity for more invasive tumors in vivo. Thus, KRAS*-STAT4-mediated upregulation of Y chromosome KDM5D contributes significantly to the sex differences in KRAS* CRC via its disruption of cancer cell adhesion properties and tumor immunity, thus providing an actionable therapeutic strategy for metastasis risk reduction for men afflicted with KRAS* CRC. Citation Format: Jiexi Li, Zhengdao Lan, Wenting Liao, James W. Horner, Jielin Liu, Shan Jiang, Hong S. Shim, Max Slotnik, Kyle A. LaBella, Wen-Hao Hsu, Denise J. Spring, Y. Alan Wang, Ronald A. DePinho. Histone demethylase KDM5D drives sex-specific differences in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR011.

The Dual Blockade of the TIGIT and PD-1/PD-L1 Pathway as a New Hope for Ovarian Cancer Patients.

The prognosis for ovarian cancer (OC) patients is poor and the five-year survival rate is only 47%. Immune checkpoints (ICPs) appear to be the potential targets in up-and-coming OC treatment. However, the response of OC patients to immunotherapy based on programmed cell death pathway (PD-1/PD-L1) inhibitors totals only 6-15%. The promising approach is a combined therapy, including other ICPs such as the T-cell immunoglobulin and ITIM domain/CD155/DNAX accessory molecule-1 (TIGIT/CD155/DNAM-1) axis. Preclinical studies in a murine model of colorectal cancer showed that the dual blockade of PD-1/PD-L1 and TIGIT led to remission in the whole studied group vs. the regression of the tumors with the blockade of a single pathway. The approach stimulates the effector activity of T cells and NK cells, and redirects the immune system activity against the tumor. The understanding of the synergistic action of the TIGIT and PD-1/PD-L1 blockade is, however, poor. Thus, the aim of this review is to summarize the current knowledge about the mode of action of the dual TIGIT and PD-1/PD-L1 blockade and its potential benefits for OC patients. Considering the positive impact of this combined therapy in malignancies, including lung and colorectal cancer, it appears to be a promising approach in OC treatment.

WIN Symposium 2022 - Abstract Poster Presentations.

WIN Symposium 2022 - Abstract Poster Presentations.

A New Manganese Superoxide Dismutase Mimetic Improves Oxaliplatin-Induced Neuropathy and Global Tolerance in Mice.

Reactive oxygen species (ROS) are produced by every aerobic cell during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Superoxide Dismutases (SOD) are antioxidant proteins that convert superoxide anions (O2•-) to hydrogen peroxide (H2O2) and dioxygen. Using the differential in the level of oxidative stress between normal and cancer cells, SOD mimetics can show an antitumoral effect and prevent oxaliplatin-induced peripheral neuropathy. New Pt(IV) conjugate prodrugs (OxPt-x-Mn1C1A (x = 1, 1-OH, 2)), combining oxaliplatin and a Mn SOD mimic (MnSODm Mn1C1A) with a covalent link, were designed. Their stability in buffer and in the presence of sodium ascorbate was studied. In vitro, their antitumoral activity was assessed by the viability and ROS production of tumor cell lines (CT16, HCT 116, KC) and fibroblasts (primary culture and NIH 3T3). In vivo, a murine model of colorectal cancer was created with subcutaneous injection of CT26 cells in Balb/c mice. Tumor size and volume were measured weekly in four groups: vehicle, oxaliplatin, and oxaliplatin associated with MnSODm Mn1C1A and the bis-conjugate OxPt-2-Mn1C1A. Oxaliplatin-induced peripheral neuropathy (OIPN) was assessed using a Von Frey test reflecting chronic hypoalgesia. Tolerance to treatment was assessed with a clinical score including four items: weight loss, weariness, alopecia, and diarrhea. In vitro, Mn1C1A associated with oxaliplatin and Pt(IV) conjugates treatment induced significantly higher production of H2O2 in all cell lines and showed a significant improvement of the antitumoral efficacy compared to oxaliplatin alone. In vivo, the association of Mn1C1A to oxaliplatin did not decrease its antitumoral activity, while OxPt-2-Mn1C1A had lower antitumoral activity than oxaliplatin alone. Mn1C1A associated with oxaliplatin significantly decreased OIPN and also improved global clinical tolerance of oxaliplatin. A neuroprotective effect was observed, associated with a significantly improved tolerance to oxaliplatin without impairing its antitumoral activity.

TRPM8 indicates poor prognosis in colorectal cancer patients and its pharmacological targeting reduces tumour growth in mice by inhibiting Wnt/β-catenin signalling.

Transient receptor potential melastatin type-8 (TRPM8) is a cold-sensitive cation channel protein belonging to the TRP superfamily of ion channels. Here, we reveal the molecular mechanism of TRPM8 and its clinical relevance in colorectal cancer (CRC). TRPM8 expression and its correlation with the survival rate of CRC patients was analysed. To identify the key pathways and genes related to TRPM8 high expression, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted in CRC patients. TRPM8 functional role was assessed by using Trpm8-/- mice in models of sporadic and colitis-associated colon cancer. TRPM8 pharmacological targeting by WS12 was evaluated in murine models of CRC. TRPM8 is overexpressed in colon primary tumours and in CD326+ tumour cell fraction. TRPM8 high expression was related to lower survival rate of CRC patients, Wnt-Frizzled signalling hyperactivation and adenomatous polyposis coli down-regulation. In sporadic and colitis-associated models of colon cancer, either absence or pharmacological desensitization of TRPM8 reduced tumour development via inhibition of the oncogenic Wnt/β-catenin signalling. TRPM8 pharmacological blockade reduced tumour growth in CRC xenograft mice by reducing the transcription of Wnt signalling regulators and the activation of β-catenin and its target oncogenes such as C-Myc and Cyclin D1. Human data provide valuable insights to propose TRPM8 as a prognostic marker with a negative predictive value for CRC patient survival. Animal experiments demonstrate TRPM8 involvement in colon cancer pathophysiology and its potential as a drug target for CRC.

131I-C19 Iodide Radioisotope and Synthetic I-C19 Compounds as K-Ras4B-PDE6δ Inhibitors: A Novel Approach against Colorectal Cancer-Biological Characterization, Biokinetics and Dosimetry.

In 40–50% of colorectal cancer (CRC) cases, K-Ras gene mutations occur, which induce the expression of the K-Ras4B oncogenic isoform. K-Ras4B is transported by phosphodiesterase-6δ (PDE6δ) to the plasma membrane, where the K-Ras4B–PDE6δ complex dissociates and K-Ras4B, coupled to the plasma membrane, activates signaling pathways that favor cancer aggressiveness. Thus, the inhibition of the K-Ras4B–PDE6δ dissociation using specific small molecules could be a new strategy for the treatment of patients with CRC. This research aimed to perform a preclinical proof-of-concept and a therapeutic potential evaluation of the synthetic I-C19 and 131I-C19 compounds as inhibitors of the K-Ras4B–PDE6δ dissociation. Molecular docking and molecular dynamics simulations were performed to estimate the binding affinity and the anchorage sites of I-C19 in K-Ras4B–PDE6δ. K-Ras4B signaling pathways were assessed in HCT116, LoVo and SW620 colorectal cancer cells after I-C19 treatment. Two murine colorectal cancer models were used to evaluate the I-C19 therapeutic effect. The in vivo biokinetic profiles of I-C19 and 131I-C19 and the tumor radiation dose were also estimated. The K-Ras4B–PDE6δ stabilizer, 131I-C19, was highly selective and demonstrated a cytotoxic effect ten times greater than unlabeled I-C19. I-C19 prevented K-Ras4B activation and decreased its dependent signaling pathways. The in vivo administration of I-C19 (30 mg/kg) greatly reduced tumor growth in colorectal cancer. The biokinetic profile showed renal and hepatobiliary elimination, and the highest radiation absorbed dose was delivered to the tumor (52 Gy/74 MBq). The data support the idea that 131I-C19 is a novel K-Ras4B/PDE6δ stabilizer with two functionalities: as a K-Ras4B signaling inhibitor and as a compound with radiotherapeutic activity against colorectal tumors.

Cell Penetrating Peptides Conjugated to Anti-Carcinoembryonic Antigen "Catch-and-Release" Monoclonal Antibodies Alter Plasma and Tissue Pharmacokinetics in Colorectal Cancer Xenograft Mice.

Cell penetrating peptides conjugated to delivery vehicles, such as nanoparticles or antibodies, can enhance the cytosolic delivery of macromolecules. The present study examines the effects of conjugation to cell penetrating and endosomal escape peptides (i.e., TAT, GALA, and H6CM18) on the pharmacokinetics and distribution of an anti-carcinoembryonic antigen "catch-and-release" monoclonal antibody, 10H6, in a murine model of colorectal cancer. GALA and TAT were conjugated to 10H6 using SoluLINK technology that allowed the evaluation of peptide-to-antibody ratio by ultraviolet spectroscopy. H6CM18 was conjugated to either NHS or maleimide-modified 10H6 using an azide-modified valine-citrulline linker and copper-free click chemistry. Unmodified and peptide-conjugated 10H6 preparations were administered intravenously at 6.67 nmol/kg to mice-bearing MC38CEA+ tumors. Unconjugated 10H6 demonstrated a clearance of 19.9 ± 1.36 mL/day/kg, with an apparent volume of distribution of 62.4 ± 7.78 mL/kg. All antibody-peptide conjugates exhibited significantly decreased plasma and tissue exposure, increased plasma clearance, and increased distribution volume. Examination of tissue-to-plasma exposure ratios showed an enhanced selectivity of 10H6-TAT for the GI tract (+25%), kidney (+24%), liver (+38%), muscle (+3%), and spleen (+33%). 10H6-GALA and 10H6-H6CM18 conjugates demonstrated decreased exposure in all tissues, relative to unmodified 10H6. All conjugates demonstrated decreased tumor exposure and selectivity; however, differences in tumor selectivity between 10H6 and 10H6-H6CM18 (maleimide) were not statistically significant. Relationships between the predicted peptide conjugate isoelectric point (pI) and pharmacokinetic parameters were bell-shaped, where pI values around 6.8-7 exhibit the slowest plasma clearance and smallest distribution volume. The data and analyses presented in this work may guide future efforts to develop immunoconjugates with cell penetrating and endosomal escape peptides.

Exceptional tumour responses to fasting-mimicking diet combined with standard anticancer therapies: A sub-analysis of the NCT03340935 trial

BackgroundCyclic fasting or calorie-restricted, low-carbohydrate, low-protein diets, collectively referred to as fasting-mimicking diets (FMDs), demonstrated additive or synergistic antitumour effects when combined with chemotherapy, targeted therapies, or immunotherapy in several preclinical in vivo models, including murine models of breast cancer, lung cancer, and colorectal cancer. However, no data on the antitumour efficacy of cyclic FMD in patients with cancer have been published so far. Here, we aim at reporting on patients with advanced cancer achieving complete and long-lasting tumour remissions with cyclic FMD in combination with standard anticancer therapies in the context of the phase Ib NCT03340935 trial. Patients and methodsThe NCT03340935 trial enrolled 101 patients with different tumour types, and it showed that a severely calorie-restricted FMD regimen is safe and feasible in patients with cancer receiving concomitant standard-of-care antineoplastic therapies. In addition, cyclic FMD resulted in positive metabolic and immunologic modifications, thus recapitulating the biological effects that in preclinical models were found to mediate the antitumour effects of fasting/FMD. ResultsOf the 101 patients enrolled in the NCT03340935 trial, we identified five patients with advanced, poor prognosis solid neoplasms (n = 1: extensive stage small cell lung cancer; n = 1: metastatic pancreatic adenocarcinoma; n = 1: metastatic colorectal cancer; n = 2: metastatic triple-negative breast cancer), who achieved complete and long-lasting tumour responses when treated with a combination of cyclic FMD and standard systemic treatments in the context of the NCT03340935 trial. ConclusionThese excellent responses prompt the initiation of clinical trials to investigate cyclic FMD in combination with standard antitumour therapies in specific clinical contexts.

ATR-mediated CD47 and PD-L1 up-regulation restricts radiotherapy-induced immune priming and abscopal responses in colorectal cancer.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti-PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti-PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti-PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

Abstract 4166: Small-molecule inhibition of glycogen synthase kinase-3 (GSK-3) increases the efficacy of anti-PD-L1 therapy in a murine model of microsatellite stable colorectal cancer (CRC); Therapeutic response correlates with T cell ratios and serum cytokine profiles in mice

Abstract Immune checkpoint blockade (ICB) with αPD-1/PD-L1 has impressive efficacy in microsatellite unstable (MSI+) CRC but no significant activity in the 85% of CRC cases that are microsatellite stable (MSS). Our group previously demonstrated that 9-ING-41, a small-molecule inhibitor of GSK-3 in clinical development, stimulates both NK and T cell activity. This project characterizes the effects of 9-ING-41 in combination with ICB in vivo. In a syngeneic murine colon carcinoma BALB/c model using MSS cell line CT-26, we compared isotype, 9-ING-41 (70 mg/kg 2x/wk), αPD-1 (10 mg/kg 2x/wk), αPD-L1 (10 mg/kg 2x/wk), 9-ING-41 + αPD-1, and 9-ING-41 + αPD-L1 treatment groups. The median overall survival was 19 days (d) with 9-ING-41 (hazard ratio for death (HR) 0.12; 95% confidence interval (CI), 0.03-0.54; p=0.008), 16 d for 9-ING-41 + isotype (HR 0.40; 95% CI, 0.08-1.9; p=0.14), 20.5 d for αPD-1 (HR 0.15; 95% CI, 0.03-0.67; p=0.0185), 18 d for αPD-L1 (HR 0.21; 95% CI, 0.05-0.89; p=0.0559), 17 d for 9-ING-41 + αPD-1 (HR 0.37; 95% CI, 0.09-0.1.5; p=0.29), and 45.5 d for 9-ING-41 + αPD-L1 (HR 0.08; 95% CI, 0.02-0.36; p=0.0019), compared to 16 d for the isotype control group. Tumor response rates observed were 33.3% in the 9-ING-41 + αPD-L1 group, 16.6% in the αPD-1 group, and a 0% for all other treatment groups. We hypothesize that the 9-ING-41-mediated upregulation of PD-L1 in CRC cells (HCT-116, HT-29) observed via both flow cytometry (FC) and western blot analysis may contribute to the increased efficacy of combination therapy with αPD-L1, as compared to αPD-1. Significant differences between responders and non-responders in intratumoral and splenic natural killer (NK) and T cell subsets 14-days post-treatment initiation were shown by multi-color FC. Compared to non-responders, regardless of treatment group, responders had lower percentages of splenic CD4+ (p=0.0145) and CD8+ T cells (p=0.0001), increased percentages of splenic CD69+ activated T cells (p=0.0070) and FOXP3+ regulatory T cells (p=0.001), and increased percentages of tumor-infiltrating CD3+ (p=0.0006) and CD4+ T cells (p<0.0001). Responders had lower splenic CD8+/Treg (p=0.0007) and CD4+/Treg (p=0.001) ratios and higher intra-tumoral CD8+/Treg (p=0.0032) and CD4+/Treg (p=0.0001) ratios. Murine serum cytokine profiling showed that responders had lower concentrations of tumorigenic cytokines (BAFF, CCL7, CCL12, VEGF, VEGFR2, CCL21) and higher concentrations of immunomodulatory cytokines (CCL4, TWEAK, GM-CSF, CCL22, IL-12p70) compared to non-responders. These results demonstrate that small-molecule inhibition of GSK-3 with 9-ING-41 may increase the anti-tumor effects of ICB and improve response in patients with MSS CRC via modulation of anti-tumor immunity and cytokine signaling. Citation Format: Kelsey E. Huntington, Anna Louie, Lanlan Zhou, Benedito A. Carneiro, Wafik El-Deiry. Small-molecule inhibition of glycogen synthase kinase-3 (GSK-3) increases the efficacy of anti-PD-L1 therapy in a murine model of microsatellite stable colorectal cancer (CRC); Therapeutic response correlates with T cell ratios and serum cytokine profiles in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4166.

Abstract 1943: Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning

Abstract Clinical benefit is observed in only a limited fraction of patients that undergo immunotherapy for a variety of cancer types. Therefore, the determination of robust biomarkers for accurate prediction of response to immunotherapeutic agents remains one of the grand challenges of 21st century. Our primary goal was to investigate if phenotypic differences due to treatment with ICI can be recognized with minimally perturbative molecular tools and guide personalization of immunotherapy. In this work, we provide first-in-class evidence that Raman spectroscopy (an optical method) and machine learning allows sensitive detection of early changes in biomolecular composition of tumors in response to immunotherapy with immune checkpoint inhibitors (ICI). By studying the widely investigated CT26 murine model of colorectal cancer treated with anti-CTLA-4 (n = 8) and anti-PD-L1 (n = 7) ICIs and controls (n = 10), we revealed new biological insights into the nature and degree of microenvironmental modifications induced by exposure to clinically relevant doses of ICI. Multivariate curve resolution-alternating least squares (MCR-ALS) decomposition of Raman spectral datasets revealed early changes in lipid, nucleic acid, and collagen content of the tumors following therapy. We trained supervised classification models - support vector machines and random forests on the spectral datasets and showed that the models provided excellent prediction accuracies for response to both ICIs and delineated spectral markers specific to each therapy, consistent with their differential mechanisms of action. On the basis of these findings, we sought to determine if the tumor microenvironment changes delineated by Raman spectroscopy correspond to proteomic alterations via quantitative mass spectrometry. Of the more than 6600 proteins identified, about 136 proteins were found to be significantly different in treated tumors relative to controls (p < 0.05 and log2 fold change of > 2). A subset of these differentially expressed proteins is known to regulate lipid metabolism and extracellular matrix composition, while others are known to control transcriptome dynamics or are associated with response to ICI therapy, thereby corroborating our Raman spectroscopic measurements. Our observation of biomolecular changes in the TME should catalyze detailed investigations for translating such markers and label-free Raman spectroscopy for clinical monitoring of immunotherapy response in cancer patients. Citation Format: Santosh K. Paidi, Joel R. Troncoso, Narasimhan Rajaram, Ishan Barman. Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1943.

Lactobacillus rhamnosus GG re-shapes gut microbiota and triggers STING-type I IFN-dependent antitumor immunity

Abstract Lactobacillus rhamnosus GG (LGG) is one of the most well characterized and widely used probiotics. The beneficial roles of LGG in modulating immune response and inflammatory state during cancer development have been well demonstrated. However the true therapeutic importance of its role in current radiotherapies and immunotherapies is still unclear. Using murine models of colorectal cancer and melanoma, we demonstrate that oral administration of live LGG augmented the antitumor activity of antiPD-1 immunotherapy and/or radiotherapy in either germ-free mice or specific pathogen-free mice. Whole genome shotgun metagenome sequencing analyses revealed that the combination treatment shifted the gut microbial community towards enrichment in Bacteroidetes, Actinobacteria, Proteobacteria, and Cyanobacteria. Notably, among the enriched species, Lactobacillus murinus and Bacteroides uniformis are known to increase DC activation and CD8+ T cell tumor recruitment. Immunologically, the synergistic antitumor effect of LGG and anti-PD1 relied on the increased tumor-infiltrating dendritic cells (DCs) and cytotoxic CD8+ T cells. Mechanistically, treatment with live LGG triggered cGAS/STING-dependent type I interferon (IFN) production in DCs, which resulted in enhanced cross-priming of tumor-specific CD8+ T cells. In DCs, LGG induced IFN-β production depended on cGAS/STING/TBK1/IRF7 axis, as further evidenced by the transcriptome sequencing. Overall, our results demonstrate that oral administration of LGG mediates antitumor immunity through activating DCs in a cGAS/STING-type I IFN-dependent manner. The study provides an alternative and effective strategy to advance cancer immunotherapies and radiotherapies.

Fecal DNA Virome Is Associated with the Development of Colorectal Neoplasia in a Murine Model of Colorectal Cancer.

Alteration of the gut virome has been associated with colorectal cancer (CRC); however, when and how the alteration takes place has not been studied. Here, we employ a longitudinal study in mice to characterize the gut virome alteration in azoxymethane (AOM)-induced colorectal neoplasia and identify important viruses associated with tumor growth. The number and size of the tumors increased as the mice aged in the AOM treated group, as compared to the control group. Tumors were first observed in the AOM group at week 12. We observed a significantly lower alpha diversity and shift in viral profile when tumors first appeared. In addition, we identified novel viruses from the genera Brunovirus, Hpunavirus that are positively associated with tumor growth and enriched at a late time point in AOM group, whereas members from Lubbockvirus show a negative correlation with tumor growth. Moreover, network analysis revealed two clusters of viruses in the AOM virome, a group that is positively correlated with tumor growth and another that is negatively correlated with tumor growth, all of which are bacteriophages. Our findings suggest that the gut virome changes along with tumor formation and provides strong evidence of a potential role for bacteriophage in the development of colorectal neoplasia.

New Class of Drug Modalities: Directed Evolution of a De Novo Designed Helix-Loop-Helix Peptide to Bind VEGF for Tumor Growth Inhibition.

As a small affinity molecule to serve as an alternative to antibodies, we have developed a conformationally constrained peptide with a de novo designed helix-loop-helix (HLH) scaffold. To evaluate its potential for biomedical applications, we performed directed evolution of HLH peptides to obtain an inhibitor for vascular endothelial growth factor-A (VEGF). A phage-displayed library of HLH peptides was constructed and screened against VEGF, giving the peptide VS42 that inhibits the VEGF/VEGF receptor-2 interaction (IC50 = 210 nM), which was further improved by in vitro affinity maturation using a yeast-displayed library. An identified HLH peptide, VS42-LR3, exhibited improved inhibitory activity (IC50 = 37 nM), high thermal stability, and excellent resistance against chemical denaturation. In biological activity tests, the HLH peptide was found to block VEGF-induced proliferation of human umbilical vein endothelial cells and suppress tumor growth in a murine xenograft model of human colorectal cancer.

Three-dimensional morphological characterization of colorectal pits from label-free microscopy images.

The prognosis of colorectal cancer (CRC), one of the most prevalent pathologies worldwide, is linked to early detection. Kudo's pit pattern classification states morphological pit patterns of the Lieberkühn crypts by analyzing the superficial mucosa, predicting the histology of colorectal lesions. Its use as a highly accurate two-dimensional diagnostic criterion has increased, mostly involving expert endoscopists' judgment. The processing of autofluorescence images could allow the diagnostic, bypassing staining techniques and decreasing the biopsies, resources and times involved in the inspection. That criterion could be extended by data of the pit three-dimensional (3D) morphology. Thus, this work was aimed at obtaining 3D morphological information by quantifying geometrical and shape descriptors through software processing and analysis of widefield autofluorescence microscopy image stacks acquired by fresh colon tissue samples from a murine model of CRC. Statistical analyses included pits from control mice and from the second (2nd), fourth (4th), and eighth (8th) weeks of treatment. Statistically significant differences were found for almost all parameters between the pits from control and from the 4th treated week, stating that the major morphological changes begin after the 2nd week. In particular, pits from control or initial treatment time points were more tubular, straighter and less rough than the ones from later treatment points. Therefore, they may be more associated to normal or non-neoplastic crypt lumens than linked to adenomas or even cancer crypts. These preliminary outcomes could be considered an advance in 3D pit morphology characterization.

Dendritic cell immunotherapy with miR-155 enriched tumor-derived exosome suppressed cancer growth and induced antitumor immune responses in murine model of colorectal cancer induced by CT26 cell line

Nowadays, various strategies are considered to prime Dendritic cells (DCs) with tumor antigens. The tumor cell-derived exosomes are recognized as one of the most efficient strategies for achieving this purpose. In this regard, MicroRNA 155 (miR-155) is employed as one of the most prominent miRNAs, which play substantial roles in DCs maturation and IL-12 production. This study investigates the tumor growth suppression and antitumor effects of DCs primed with miR-155-enriched exosome on the BALB/c murine model of colorectal cancer induced by CT-26 cell lines.Therefore, a holistic framework is proposed for the analysis procedure. In the first stage, miRNA-155 was electroporated into texosomes. In the second stage, bonemarrow-derived DCs were treated with miRNA-155 enriched texosomes. Then, antitumor properties of manipulated DC have been evaluated in the BALB/c mice model of colorectal cancer. After DC immunotherapy, several features have been assessed for each animal, including survival, body weight, tumor volume/size, histopathology, and serum cytokine levels. Also, flow cytometric evaluation has been performed for the spleen and the tumor tissue T-cell subsets.The findings demonstrated that the primed DCs could significantly increase IL-12p70 and IFN-γ in serum and accelerate the differentiation, proliferation, and cytotoxicity effects on the Th and CTL cells. Also, the treatment also increased the infiltration of Th and CTL cells into the tumor microenvironment while decreasing Tregs. This situation causes tumor growth control, and survival improvement.Therefore, DC immunotherapywith miR-155-enriched texosomes can be employed as a the desired approach for inducing antitumor immune responses, controlling tumor growth, and improving survival in mice with colorectal cancer. However, it is essential to perform more investigations to confirm the clinical application of this approach in humans and other types of tumors.

Abstract P009: Correlation between immune modulation of macrophage recruitment and new blood vessel formation in a subcutaneous murine mouse model of colorectal cancer

Abstract Purpose: Colorectal cancer is the fourth most common cancer in the United States, accounting for more than 50,000 deaths annually1. The standard treatment is 5-fluorouracil (5-FU)-based chemotherapy, but emerging methods of immunotherapy have gained the attention of investigators and clinicians. A specific cytokine-based strategy for immunotherapy is the blockade of CCL2/MCP-1 (monocyte chemoattractant protein-1)2. CCL2 is a chemotactic cytokine that recruits circulating monocytes to the tumor microenvironment2. These monocytes differentiate into tumor-associated macrophages (TAMs), which promote pro-tumor functions including tumor growth, immunosuppression, and angiogenesis. CCL2 blockade in certain mouse models of cancer has been shown to reduce tumor burden but had not been studied in colorectal cancer. In this study, we examine how cytokine-targeted anti-CCL2 immunotherapy affects tumor-associated macrophage recruitment as well as the promotion of angiogenesis as a standalone and in combination with 5-FU. Methods: Nine-week-old Balb/c mice (n=13) were acclimated for a week in the Small Animal Facility at the University of Arkansas. CT26 cells, a murine colon carcinoma cell line, were cultured in RPMI-1640 media, supplemented with 10% bovine serum, 0.2% amphotericin B/gentamicin, and 1% antibiotic antimycotic solution. 1 × 106 CT26 cells were subcutaneously injected into the left flank of Balb/c mice (10 weeks old). The tumors grew to 75 mm3, upon which Balb/c mice were randomly divided into saline control, immunotherapy, chemotherapy, and combination (immunotherapy + chemotherapy) groups. The immunotherapy and chemotherapy groups were intraperitoneally injected with a 28G insulin syringe with anti-CCL2 and 5-FU at a concentration of 5.0 mg/kg/dose and 15mg/kg/dose, respectively. The control group was injected with an equal volume of sterile saline on days while the combination group received a dose of anti-CCL2 and 5-FU. Tumor-associated macrophages were quantified via immunohistochemistry using anti-CD68 and tumor vasculature was quantified using anti-CD31. Correlation plots were also created to determine a relationship between macrophage recruitment and new blood vessel formation. Results: There was a decrease in the total number of tumor-associated macrophages from Day 3 to 7 (33% decrease) when tumors were treated with anti-CCL2 and 5-FU. The area of vasculature in the combination group showed a slight decrease from Day 3 to Day 7 (29% decrease). There was a slight positive correlation between total macrophage recruitment and new blood vessel formation (R2 = 0.3041). Conclusion: Overall, we have demonstrated that anti-CCL2 in combination with 5-FU has modestly slowed tumor growth, reduced the area of vasculature, and decreased the number of recruited tumor-associated macrophages in a murine model of colorectal cancer. Acknowledgments: This material is based on work supported by the National Institutes of Health (1R15CA202662- 01), National Science Foundation CAREER award (CBET 1751554), and the Arkansas Biosciences Institute. Citation Format: Shelby N. Bess, Timothy J. Muldoon. Correlation between immune modulation of macrophage recruitment and new blood vessel formation in a subcutaneous murine mouse model of colorectal cancer [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 P009.

The anti-tumor effects of CT-26 derived exosomes enriched by MicroRNA-34a on murine model of colorectal cancer

AimsAs conventional therapeutics failed to provide satisfied outcomes against one of the most prevalent cancers, colorectal cancer (CRC), we purposed to implicate MicroRNA (miR)-34a, as a major tumor suppressor, to be delivered by tumor-derived exosomes (TEXs) and investigated its anti-tumor functions in-vivo. Main methodsTEXs were isolated from CT-26 cell line and loaded with miR-34a mimic. Then, mice bearing CRC were treated with miR-34a-enriched TEX (TEX-miR-34a) and then examined for the relative tumor-suppressive impacts of the TEX as well as its potential in promoting an anti-tumor immune response. Key findingsTEX-miR-34a significantly reduced tumor size and prolonged survival of mice bearing CRC. TEX-miR-34a was able to diminish gene expressions related to invasion, angiogenesis and immune evasion. It was also capable of inducing T cell polarization toward CD8+ T subsets among tumor-infiltrating lymphocytes, draining lymph nodes (DLNs) and spleen cells. Moreover, cytotoxic T cells were professionally induced in mice receiving TEX-miR-34a and the secretion of interleukin (IL)-6, IL-17A and tumor necrosis factor (TGF)-β was reduced in DLNs. However, the enhanced levels of interferon-γ were evaluated in DLN and spleen displaying the polarization of anti-tumor immune responses. Interestingly, mice receiving TEX alone showed a noticeable reduction in certain oncogenic gene expressions as well as IL-17A secretion in DLNs. SignificanceTEX-miR-34a demonstrated the potential to induce beneficial anti-tumor immune responses and TEXs, aside from the delivery function of miRNA, revealed certain anti-tumor beneficial characteristics which could introduce TEX-miR-34a as a promising approach in CRC combination therapies.

Lactobacillus gallinarum modulates the gut microbiota and produces anti-cancer metabolites to protect against colorectal tumourigenesis

ObjectiveUsing faecal shotgun metagenomic sequencing, we identified the depletion of Lactobacillus gallinarum in patients with colorectal cancer (CRC). We aimed to determine the potential antitumourigenic role of L. gallinarum in...

Tumor-immune profiling of CT-26 and Colon 26 syngeneic mouse models reveals mechanism of anti-PD-1 response

BackgroundImmune checkpoint blockade (ICB) therapies have changed the paradigm of cancer therapies. However, anti-tumor response of the ICB is insufficient for many patients and limited to specific tumor types. Despite many preclinical and clinical studies to understand the mechanism of anti-tumor efficacy of ICB, the mechanism is not completely understood. Harnessing preclinical tumor models is one way to understand the mechanism of treatment response.MethodsIn order to delineate the mechanisms of anti-tumor activity of ICB in preclinical syngeneic tumor models, we selected two syngeneic murine colorectal cancer models based on in vivo screening for sensitivity with anti-PD-1 therapy. We performed tumor-immune profiling of the two models to identify the potential mechanism for anti-PD-1 response.ResultsWe performed in vivo screening for anti-PD-1 therapy across 23 syngeneic tumor models and found that CT-26 and Colon 26, which are murine colorectal carcinoma derived from BALB/c mice, showed different sensitivity to anti-PD-1. CT-26 tumor mice were more sensitive to the anti-PD-1 antibody than Colon 26, while both models show similarly sensitivity to anti-CTLA4 antibody. Immune-profiling showed that CT-26 tumor tissue was infiltrated with more immune cells than Colon 26. Genomic/transcriptomic analyses highlighted thatWnt pathway was one of the potential differences between CT-26 and Colon 26, showing Wnt activity was higher in Colon 26 than CT-26. .ConclusionsCT-26 and Colon 26 syngeneic tumor models showed different sensitivity to anti-PD-1 therapy, although both tumor cells are murine colorectal carcinoma cell lines from BALB/c strain. By characterizing the mouse cells lines and tumor-immune context in the tumor tissues with comprehensive analysis approaches, we found that CT-26 showed “hot tumor” profile with more infiltrated immune cells than Colon 26. Further pathway analyses enable us to propose a hypothesis that Wnt pathway could be one of the major factors to differentiate CT-26 from Colon 26 model and link to anti-PD-1 response. Our approach to focus on preclinical tumor models with similar genetic background but different sensitivity to anti-PD-1 therapy would contribute to illustrating the potential mechanism of anti-PD-1 response and to generating a novel concept to synergize current anti-PD-1 therapies for cancer patients.