Murine Colorectal Cancer Model Research Articles

Human Colon Mucosal Biofilms and Murine Host Communicate via Altered mRNA and microRNA Expression during Cancer.

Disrupted interactions between host and intestinal bacteria are implicated in colorectal cancer (CRC) development. However, activities derived from these bacteria and their interplay with the host are unclear. Here, we examine this interplay by performing mouse and microbiota RNA sequencing on colon tissues and 16S and small RNA sequencing on stools from germfree (GF) and gnotobiotic ApcMin Δ 850/+ ;Il10-/- mice associated with microbes from biofilm-positive human CRC tumor (BF+T) and biofilm-negative healthy (BF-bx) tissues. The bacteria in BF+T mice differentially expressed (DE) >2,900 genes, including genes related to bacterial secretion, virulence, and biofilms but affected only 62 host genes. Small RNA sequencing of stools from these cohorts revealed eight significant DE host microRNAs (miRNAs) based on biofilm status and several miRNAs that correlated with bacterial taxon abundances. Additionally, computational predictions suggest that some miRNAs preferentially target bacterial genes while others primarily target mouse genes. 16S rRNA sequencing of mice that were reassociated with mucosa-associated communities from the initial association revealed a set of 13 bacterial genera associated with cancer that were maintained regardless of whether the reassociation inoculums were initially obtained from murine proximal or distal colon tissues. Our findings suggest that complex interactions within bacterial communities affect host-derived miRNA, bacterial composition, and CRC development.IMPORTANCE Bacteria and bacterial biofilms have been implicated in colorectal cancer (CRC), but it is still unclear what genes these microbial communities express and how they influence the host. MicroRNAs regulate host gene expression and have been explored as potential biomarkers for CRC. An emerging area of research is the ability of microRNAs to impact growth and gene expression of members of the intestinal microbiota. This study examined the bacteria and bacterial transcriptome associated with microbes derived from biofilm-positive human cancers that promoted tumorigenesis in a murine model of CRC. The murine response to different microbial communities (derived from CRC patients or healthy people) was evaluated through RNA and microRNA sequencing. We identified a complex interplay between biofilm-associated bacteria and the host during CRC in mice. These findings may lead to the development of new biomarkers and therapeutics for identifying and treating biofilm-associated CRCs.

C5aR1 is a master regulator in Colorectal Tumorigenesis via Immune modulation

Numerous factors have been claimed to play important roles in colorectal cancer (CRC) tumorigenesis, including myeloid-derived suppressor cells (MDSCs) and other immune cells, cytokines, and chemokines; however, the precise mechanisms of colorectal tumorigenesis remain elusive, and there is a lack of effective preventive treatments. Here, we investigated the role of complement system, a key regulator of immune surveillance and homeostasis, in colorectal tumorigenesis.Methods: The prototypical CRC model was induced by combined administration of azoxymethane (AOM)/ dextran sulfate sodium (DSS) in Wild-type (WT), C3-, C5-, C5ar1-, and C5ar2-deficient mice. Using flow cytometry, immunohistochemical staining and multiplex bead assay, we profiled the immune cells, cytokines and chemokines. Bone marrow transplantation was employed to determine the contribution of immune cells in colorectal tumorigenesis. Further, we used C5aR1 antagonist PMX205 to investigate the protective role in colorectal tumorigenesis.Results: Complement was extensively activated in inflamed tissues of AOM/DSS-induced murine CRC model, leading to multifaceted consequences. The deficiency of complement C5 or especially C5ar1, but not C3 almost completely prevented CRC tumorigenesis. C5a/C5aR1 signaling recruited MDSCs into the inflamed colorectum to impair CD8+ T cells, and modulated the production of critical cytokines and chemokines, thus initiating CRC. Moreover, the C5aR1 antagonist PMX205 strongly impeded colorectal tumorigenesis. Bone marrow transplantation further revealed that C5aR1 expression by immune cells was critical for colorectal tumorigenesis.Conclusion: Our study identifies C5a/C5aR1 signaling as a vital immunomodulatory program in CRC tumorigenesis and suggests a feasible preventive strategy.

Albumin Nano-Encapsulation of Piceatannol Enhances Its Anticancer Potential in Colon Cancer Via Downregulation of Nuclear p65 and HIF-1α.

Piceatannol (PIC) is known to have anticancer activity, which has been attributed to its ability to block the proliferation of cancer cells via suppression of the NF-kB signaling pathway. However, its effect on hypoxia-inducible factor (HIF) is not well known in cancer. In this study, PIC was loaded into bovine serum albumin (BSA) by desolvation method as PIC–BSA nanoparticles (NPs). These PIC–BSA nanoparticles were assessed for in vitro cytotoxicity, migration, invasion, and colony formation studies and levels of p65 and HIF-1α. Our results indicate that PIC–BSA NPs were more effective in downregulating the expression of nuclear p65 and HIF-1α in colon cancer cells as compared to free PIC. We also observed a significant reduction in inflammation induced by chemical colitis in mice by PIC–BSA NPs. Furthermore, a significant reduction in tumor size and number of colon tumors was also observed in the murine model of colitis-associated colorectal cancer, when treated with PIC–BSA NPs as compared to free PIC. The overall results indicate that PIC, when formulated as PIC–BSA NPs, enhances its therapeutic potential. Our work could prompt further research in using natural anticancer agents as nanoparticels with possible human clinical trails. This could lead to the development of a new line of safe and effective therapeutics for cancer patients.

Dietary Fucoxanthin Induces Anoikis in Colorectal Adenocarcinoma by Suppressing Integrin Signaling in a Murine Colorectal Cancer Model.

Fucoxanthin (Fx), abundantly contained in edible brown algae, is a carotenoid with strong anti-cancer potential. Anoikis is an anchor-dependent apoptosis particularly related to integrin signaling, and a target for cancer preventive strategies. We recently demonstrated that Fx prevented colon cancer in azoxymethane-dextrane sodium sulfate (AOM/DSS) carcinogenic model mice, and that it increased anoikis-like integrin β1low/-/cleaved caspase-3high cells in colonic mucosal crypts. However, an induction mechanism of anoikis by Fx in adenocarcinoma tissue remains unresolved. Thus, we investigated anoikis in colonic adenocarcinoma in AOM/DSS mice. Fx administration (30 mg/kg body weight) significantly suppressed the incidence and multiplicity of colonic adenocarcinoma in AOM/DSS mice. A number of anoikis-like integrin β1low/-/cleaved caspase-3high cells in colonic adenocarcinoma and mucosal crypts were significantly increased, 8.3- and 3.5-fold in the Fx group compared with those of the control group, respectively. The results indicated the increase of anoikis-like cells occurred more strongly in colonic adenocarcinoma than in colonic mucosal crypts. In addition, integrin β1 expression, and pFAK (Tyr397) and pPaxillin (Tyr31) activation in mucosal tissue decreased 0.7-, 0.5- and 0.6-fold by Fx administration, respectively. The results suggest that Fx induces anoikis in colonic adenocarcinoma developed by AOM/DSS treatment through attenuation of integrin signaling.

Nanosecond Pulsed Electric Fields Induce Endoplasmic Reticulum Stress Accompanied by Immunogenic Cell Death in Murine Models of Lymphoma and Colorectal Cancer.

Depending on the initiating stimulus, cancer cell death can be immunogenic or non-immunogenic. Inducers of immunogenic cell death (ICD) rely on endoplasmic reticulum (ER) stress for the trafficking of danger signals such as calreticulin (CRT) and ATP. We found that nanosecond pulsed electric fields (nsPEF), an emerging new modality for tumor ablation, cause the activation of the ER-resident stress sensor PERK in both CT-26 colon carcinoma and EL-4 lymphoma cells. PERK activation correlates with sustained CRT exposure on the cell plasma membrane and apoptosis induction in both nsPEF-treated cell lines. Our results show that, in CT-26 cells, the activity of caspase-3/7 was increased fourteen-fold as compared with four-fold in EL-4 cells. Moreover, while nsPEF treatments induced the release of the ICD hallmark HMGB1 in both cell lines, extracellular ATP was detected only in CT-26. Finally, in vaccination assays, CT-26 cells treated with nsPEF or doxorubicin equally impaired the growth of tumors at challenge sites eliciting a protective anticancer immune response in 78% and 80% of the animals, respectively. As compared to CT-26, both nsPEF- and mitoxantrone-treated EL-4 cells had a less pronounced effect and protected 50% and 20% of the animals, respectively. These results support our conclusion that nsPEF induce ER stress, accompanied by bona fide ICD.

Neutralizing tumor-promoting inflammation with polypeptide-dexamethasone conjugate for microenvironment modulation and colorectal cancer therapy

Tumor is known as “a wound that does not heal”. Tumor-promoting inflammation plays a crucial role in carcinogenesis, tumor progression, tumor metastasis, as well as chemotherapy resistance. Therefore, reducing tumor-promoting inflammation may be a key aspect in targeting the tumor microenvironment for cancer therapy. Dexamethasone (DEX), a commercial drug in the treatment of many different inflammatory diseases, can effectively inhibit the release of substances causing inflammation. However, as a corticosteroid medication, direct use of DEX results in many severe side effects. In this study, a redox and pH dual sensitive polypeptide-DEX conjugate (L–SS–DEX) was synthesized, and the L–SS–DEX dramatically increased the tumoral accumulation of DEX in murine colorectal cancer model (CT26) compared to free DEX. Importantly, at equal dose (10 mg/kg), L–SS–DEX showed superior antitumor activity over free DEX: 86% tumor suppression rate of L–SS–DEX treatment group compared to 49% of free DEX treatment group. Further analysis of the tumor tissues showed that cyclooxygenase-2 (COX-2) and α-smooth muscle actin (α-SMA) were significantly reduced after the L–SS–DEX treatment compared with control groups. In addition, the immunosuppressive microenvironment of the CT26 tumor was effectively relieved after L–SS–DEX treatment, characterized by increased CD8+ T cell infiltration, increased ratio of M1 over M2 macrophages, as well as markedly decrease in regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The above results suggest that anti-inflammatory drugs hold great potential in modulating the tumor microenvironment when delivered properly, and can also result in significant tumor inhibition effects. Since dramatic amounts of anti-inflammatory drugs have been used in clinic, our results may provide improved tumor therapy options of using anti-inflammatory drugs for cancer therapy.

Chemotherapy-induced mucositis development in a murine model of colitis-associated colorectal cancer

Objectives: Ulcerative colitis is an incurable inflammatory bowel disease that increases the risk of colorectal cancer (CRC). 5-Fluorouracil (5-FU) is the predominant chemotherapy for CRC patients; however, undesirable side-effects, including mucositis, are common. This study utilised 5-FU-treatment in a model of colitis-associated CRC to develop a pre-clinical setting of intestinal mucositis coincident with manifestation of CRC.Materials/methods: On day 0, female C57BL/6 mice (n = 10/group); (1) saline control, (2) AOM/DSS control, or (3) AOM/DSS + 5-FU were injected with saline or AOM (i.p; 7.4 mg/kg). Groups 2 and 3 underwent cycles of seven days 2%w/v DSS followed by 14 days plain water. After three cycles, 5-FU was administered weekly (i.p; 75 mg/kg) to group 3 for five weeks. Clinical indicators were measured daily and colonoscopy performed at four time-points. Mice were euthanized at 13 weeks (day 91). Intestinal sections were collected for histological and biochemical analyses. p < .05 was considered significant.Results: AOM/DSS resulted in bodyweight loss, increased disease activity index, colitis-severity and tumour number compared to saline controls (p < .05). 5-FU-treatment in AOM/DSS mice decreased bodyweight and disease activity index at selected time-points compared to AOM/DSS controls (p < .05). 5-FU did not impact colitis-severity or overall tumour burden; although, resulted in fewer small tumours compared to AOM/DSS controls (<2mm; p < .05). AOM/DSS increased histological severity scores in intestinal sections (p < .05), however, 5-FU-treatment did not further increase histologically-assessed disease severity (p > .05).Conclusion: Weekly 5-FU administration at a dose of 75 mg/kg was insufficient to reduce overall tumour burden or induce intestinal mucositis in the AOM/DSS mouse model.

Bufalin, a Traditional Chinese Medicine Compound, Prevents Tumor Formation in Two Murine Models of Colorectal Cancer

Chemoprevention is cost-effective for colorectal cancer when targeted at intermediate- or high-risk populations. Bufalin is a cardiac glycoside extracted from the traditional Chinese medicine (TCM) "Chan Su," which has been used as an anticancer agent. On the basis of the relative safety of bufalin, we investigated whether bufalin could act as a chemoprophylactic agent to prevent colon tumorigenesis in two murine models, namely colitis-associated colorectal cancer and Apc germline mutation-developed colorectal cancer. Our results revealed that long-term (12-16 weeks) administration of low-dose bufalin (0.5 mg/kg) effectively suppressed tumorigenesis in both colorectal cancer models, accompanied by attenuated epithelial cell proliferation (reduced bromodeoxyuridine incorporation, lower levels of cyclin A, cyclin D1, cyclin E, and cyclin-dependent kinases-2/4, and higher levels of p21 and p27) and promoted apoptosis (increased TUNEL positivity and caspase-3/9 cleavages, reduced levels of Bcl-2, Bcl-xL and survivin, and increased levels of Bax and Bak). Bufalin also suppressed the expression of proinflammatory mediators [reduced levels of cyclooxygenase-2, tumor TNFα, IL1β, IL6, C-X-C motif chemokine ligand (CXCL)-1, CXCL-2, and CXCL-5] in the colitis-associated colorectal cancer model. These effects were associated with the inhibition of oncogenic NF-κB and PI3K/Akt pathways. Our findings unveil a novel chemoprophylactic action of bufalin in colorectal cancer in vivo and provided efficacy data and mechanistic evidence for further clinical evaluation of this TCM compound for colorectal cancer chemoprevention in individuals at risk of colorectal cancer.

1231P - Master checkpoint Cbl-b inhibition: Anti-tumour efficacy in a murine colorectal cancer model following siRNA-based cell therapy

BackgroundThe E3 ubiquitin ligase Cbl-b is a key master immune checkpoint regulator that limits immune activation critical for anti-tumor immunity. We first reported that Cbl-b deficiency in mice confers spontaneous in vivo rejection of tumor cells. This checkpoint role in anti-tumor immunity was confirmed in multiple mouse and human studies. Further discovery efforts led to the advancement of APN401, an ex vivo human Cbl-b siRNA-based autologous cellular therapy, currently in clinical development in patients with advanced solid tumors. APN401 has been target validated and its preclinical efficacy was established in various mouse syngeneic tumor models. We present here the potent anti-tumor efficacy of APN401 immunotherapy of Cbl-b silenced murine T cells in a syngeneic MC38 colorectal tumor model. MethodsT cells isolated from MC38 tumor-bearing donor C57Bl/6 mice were silenced ex vivo with APN401 murine Cbl-b specific siRNA vs control siRNA, adoptively transferred into MC38 tumor-bearing recipient mice, and tumor growth was monitored using an in vivo imaging system. ResultsMurine APN401 treatment resulted in a significant MC38 tumor growth inhibition of 63% (p=0.008) vs controls after just a single dose application. Profound anti-tumor efficacy induced by Cbl-b-silenced T cells strongly correlated with enhanced production of TH1 cytokines IL-2 and IFN-γ. Furthermore, in vivo tracking of fluorescently labeled and silenced T cells revealed migration to relevant lymphoid organs and local tumor sites. Murine APN401 treatment was safe and well-tolerated similar to recent human APN401 clinical phase Ia study results in patients with advanced solid tumors. ConclusionsIn a model of murine colon cancer, Cbl-b-silencing induced vigorous in vivo anti-tumorigenic immune responses. The treatment modality was safe and well tolerated. These data provide direct preclinical proof of concept that siRNA-based silencing of Cbl-b provides a novel, effective and tunable approach as cellular immunotherapy. Targeting Cbl-b through human APN401 cell therapy is a promising novel therapeutic approach for the treatment of solid cancers, also highlighted by our fast-tracked global APN401 clinical development. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Neutrophils Deficient in Innate Suppressor IRAK-M Enhances Anti-tumor Immune Responses.

Tumor-associated immune-suppressive neutrophils are prevalent in various cancers, including colorectal cancer. However, mechanisms of immune-suppressive neutrophils are not well understood. We report that a key innate suppressor, IRAK-M (interleukin-1 receptor-associated kinase M), is critically involved in the establishment of immune-suppressive neutrophils. In contrast to the wild-type (WT) neutrophils exhibiting immune-suppressive signatures of CD11bhighPD-L1highCD80low, IRAK-M-deficient neutrophils are rewired with reduced levels of inhibitory molecules PD-L1 and CD11b, as well as enhanced expression of stimulatory molecules CD80 and CD40. The reprogramming of IRAK-M-deficient neutrophils is mediated by reduced activation of STAT1/3 and enhanced activation of STAT5. As a consequence, IRAK-M-deficient neutrophils demonstrate enhanced capability to promote, instead of suppress, the proliferation and activation of effector Tcells both invitro and invivo. Functionally, we observed that the transfusion of IRAK-M-/- neutrophils can potently render an enhanced anti-tumor immune response in the murine inflammation-induced colorectal cancer model. Collectively, our study defines IRAK-M as an innate suppressor for neutrophil function and reveals IRAK-M as a promising target for rewiring neutrophils in anti-cancer immunotherapy.

Modulation of Nanostructure-Based Lipopolysaccharide Active Immunotherapy in Cancer: Size and Composition Determine Short- and Long-Term Tolerability.

Despite holding promise for cancer immunotherapy, the strong pro-inflammatory properties of lipopolysaccharide (LPS) also account for severe localized and systemic side effects, restricting its administrable dosage and the possibility of chronic dosing. Herein, we exploited the surface-active properties of LPS molecules to develop pathogen-mimicking LPS-decorated nanostructures with different compositions (lipid nanoemulsion vs polymeric nanospheres) and sizes (volumetric mean diameters of 100 nm vs 700 nm). The formulations were tested in cell culture for their immunostimulatory properties and in vivo against a murine subcutaneous colorectal cancer model. While all nanostructures resulted in similar levels of apoptotic cell death in tumor cells cultured with splenocytes, both the size and the composition of the nanostructures were found to govern the short- and long-term tolerability of LPS-based immunotherapy in vivo. The toxicity-related end point of the animal trials was decided upon in the case of a body condition score (BCS) of 1 and poor hair coat, or more than 15% loss of the original body weight, while in the absence of long-term intolerability, the experiments were terminated in the case of full remission or once the tumor surpassed a volume of 1000 mm3. Size was an important determinant of short-term tolerability, with larger particles being associated with higher incidence and extent of localized necrosis (3-6% necrotic surface area). Nanostructure composition, on the other hand, predominantly governed the long-term systemic tolerability. Within this context, the higher affinity of LPS molecules to the triglyceride core of the nanoemulsion compared to the polymeric matrix significantly improved the tolerability of the former over time. In fact, the mean survival estimate of the animals treated with small LPS nanoemulsion (LPS-NE (small)) was at least 42 days longer than that of the LPS and the LPS-decorated polymeric nanoparticle (LPS-NP) groups. Unlike other treatment groups, the experiments on 80% of the animals in LPS-NE (small) were terminated due to complete remission or tumor volume >1000 mm3. While a better understanding of these findings requires a larger scale, mechanistic-oriented trial on larger animal models, they indicate the role of nanostructures as beyond the carriers of the incorporated immunotherapeutic cargos. This highlights the importance of a wise selection of nanoparticle composition and a purposeful tuning of their physicochemical properties to enhance the safety profile and improve the eventual immunotherapeutic outcome.

Colon Cancer: Epithelial Notch Signaling Recruits Neutrophils to Drive Metastasis

In this issue of Cancer Cell, Jackstadt etal. report that pathological Notch signaling in colorectal cancer (CRC) cells can initiate a neutrophil-dependent, TGF-β-mediated cascade that leads to metastatic disease. Inhibition of this pathway with clinically relevant compounds blocks metastatic spread in a developed murine model of CRC.

Mapping insoluble indole metabolites in the gastrointestinal environment of a murine colorectal cancer model using desorption/ionisation on porous silicon imaging

Indole derivatives are a structurally diverse group of compounds found in food, toxins, medicines, and produced by commensal microbiota. On contact with acidic stomach conditions, indoles undergo condensation to generate metabolites that vary in solubility, activity and toxicity as they move through the gut. Here, using halogenated ions, we map promising chemo-preventative indoles, i) 6-bromoisatin (6Br), ii) the mixed indole natural extract (NE) 6Br is found in, and iii) the highly insoluble metabolites formed in vivo using desorption/ionisation on porous silicon-mass spectrometry imaging (DIOS-MSI). The functionalised porous silicon architecture allowed insoluble metabolites to be detected that would otherwise evade most analytical platforms, providing direct evidence for identifying the therapeutic component, 6Br, from the mixed indole NE. As a therapeutic lead, 0.025 mg/g 6Br acts as a chemo-preventative compound in a 12 week genotoxic mouse model; at this dose 6Br significantly reduces epithelial cell proliferation, tumour precursors (aberrant crypt foci; ACF); and tumour numbers while having minimal effects on liver, blood biochemistry and weight parameters compared to controls. The same could not be said for the NE where 6Br originates, which significantly increased liver damage markers. DIOS-MSI revealed a large range of previously unknown insoluble metabolites that could contribute to reduced efficacy and increased toxicity.

Abstract 343: A potent and selective cfms inhibitor EI-1071 inhibits CSF1R signaling and regulates the tumor-associated macrophages

Abstract We have found that tumor-associated macrophages (TAMs) isolated from metastatic tumors displayed a predominant M2 phenotype. Preclinical studies show that M2-polarized TAMs forge an immunosuppressive microenvironment at metastatic niches to facilitate colonization. Colony-stimulating factor 1 receptor (CSF-1R) signaling is known to regulate the survival and maintenance of M2 TAMs, suggesting targeting CSF-1R might have therapeutic potential in halting cancer progression. To this end, we developed an orally active and selective small-molecule CSF-1R inhibitor EI-1071. Experimental procedures: EI-1071 was evaluated in kinase and cell-based bioassays, including macrophage proliferation and osteoclast differentiation. In vivo activity was verified using murine syngeneic models of colorectal cancer and breast cancer. EI-1071 was administered individually or in combination with an anti-PD1 antibody. Tumor infiltrating leucocytes were profiled using flow cytometric analysis. Results: EI-1071 exhibited nanomolar potency for inhibition of CSF-1R but not other 453 kinases tested. In cell-based bioassays, EI-1071 inhibited CSF-1R activity in human monocytes/macrophages, AML-5 leukemia, MNFS-60 leukemia, and human osteoclasts. In preclinical studies, EI-1071 displayed a favorable ADME properties, including hepatocyte stability, high Caco-2 permeability, and pharmacokinetic profiles. Oral administration of EI-1071 reduced TAM infiltration in the MC38 colorectal cancer and EMT6 breast cancer models. Moreover, EI-1071 dosed with an anti-PD-1 antibody showed further enhanced antitumor activity in vivo . Conclusions: Preclinical characterization of EI-1071 demonstrated that it is a potent and selective CSF-1R inhibitor and has a favorable drug like properties for further clinical evaluation. Therefore, EI-1071 is undergoing IND-enabling activities for the First-In-Human (FIH) Phase 1 study. Citation Format: Hung-Kai Chen, Huey-Wen Hsiao, Li-Ming Lu, Chih-Lun Hsiao, Yin-Ping Wang, Jing-Yi Huang, Chu-Bin Liao, Shao-Zheng Peng, Daw-Tsun Shih. A potent and selective cfms inhibitor EI-1071 inhibits CSF1R signaling and regulates the tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 343.

Abstract 558: MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys

Abstract MERTK, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is a pleiotropic immune modulator that controls efferocytosis. Engagement of MERTK with its ligand GAS6, found anchored to phosphatidylserine exposed on the outer membrane of apoptotic cells, triggers MERTK phosphorylation and signaling events that culminate in the removal of apoptotic debris. Recent studies have highlighted the expression of MERTK on tumor-associated macrophages, and Mertk-deficient mice show reduced tumor cell growth accompanied by inflammatory cytokine production and alterations in macrophage activation. Thus, MERTK has emerged as a promising therapeutic target for augmenting innate antitumor immune responses. MERTK is also expressed in retinal pigmented epithelium (RPE) cells of the eye where it mediates phagocytosis of photoreceptor outer segment fragments. Mutations in MERTK that disrupt its expression or kinase activity lead to marked retinal degeneration and blindness in mice, rats, and humans. Due to known differences in blood-retinal permeability, we explored whether therapeutic antibodies targeting MERTK could inhibit macrophage-mediated efferocytosis and promote antitumor activity while sparing RPE toxicity. A diverse panel of high-affinity antibodies was developed to explore MERTK blockade in vitro and in vivo. Multiple antibodies disrupted MERTK-GAS6 binding and blocked human and murine macrophage-mediated efferocytosis. Two antibodies targeting distinct GAS6 binding epitopes were selected for further characterization. Both antibodies demonstrated antitumor activity in murine CT26 and MC38 syngeneic colorectal cancer models and led to alterations in immune cell-related gene expression. To investigate potential effects on RPE biology with MERTK antibodies, a multi-dose, 4-week cynomolgus monkey study with several in-life and post-mortem ophthalmologic endpoints was designed. While no abnormal ophthalmic or electroretinography (ERG) findings were detected, all animals treated with either MERTK antibody at all doses showed histological abnormalities of the retina, including vacuolation of the outer segments of photoreceptors, displacement of RPE cells, and single cell necrosis of the outer nuclear layer. These data suggest that inhibition of efferocytosis by antibody-mediated blockade of MERTK can promote immune activation and inhibit tumor growth in vivo; however, retinal toxicity consistent with histological observations made in Mertk mutant animals is an on-target effect. As several therapeutics that block MERTK function are currently in preclinical development, a thorough evaluation of retinal toxicity is warranted. Citation Format: Kerry F. White, Matthew Rausch, Jing Hua, Katherine H. Walsh, Christine E. Miller, Christopher C. Wells, Devapregasan Moodley, Benjamin H. Lee, Scott C. Chappel, Pamela M. Holland, Jonathan A. Hill. MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 558.

Abstract 93: Anti-LAP-TGFb antibodies inhibit tumor growth in a CT26 syngeneic tumor model in combination with radiation therapy

Abstract Anti-LAP-TGFβ antibodies inhibit tumor growth in a CT26 syngeneic tumor model in combination with radiation therapy. TGFβ is a major immunosuppressive cytokine that acts within the TME and is implicated in resistance to checkpoint inhibitors. TGFβ is synthesized as a pro-protein complex in which the mature cytokine is caged within LAP, the latency associated peptide of TGFβ1, and serves to hold TGFβ1 in a latent state. Anti-LAP-TGFβ antibodies have efficacy in mouse models of cancer (Gabriely et al., 2017) and offer a promising new treatment approach in oncology. Radiation is standard of care in many cancer indications and is a known potent inducer of TGFβ. TGFβ is also implicated in resistance to radiation treatment. Thus, combination treatment with anti-LAP antibodies and radiation may yield a novel approach for enhancing therapeutic responses in a population with significant unmet medical need. We have evaluated the anti-tumor effects of targeting TGFβ via an anti-LAP antibody in combination with radiation therapy in a murine syngeneic CT26 colorectal cancer model. We identified a unique class of anti-LAP antibodies, TLS-01, that specifically target LAP on the surface of cells, but do not bind to LAP-TGFβ in the extracellular matrix. We assessed the effects of combination of TLS-01 and radiation on both tumor efficacy and modulation of immune cell subsets within the TME. CT26 tumor bearing mice (tumor size ~300 mm3) were treated with either TLS-01, an isotype control antibody, 12 Gy or 20 Gy of targeted radiation, or a combination of TLS-01 with 12 Gy or 20 Gy of radiation. Seven days after treatment, 3 animals per group were analyzed for tumor infiltrating immune cell subsets and the remaining animals were followed for tumor growth and survival for up to 19 days. Radiation inhibited tumor growth in a dose dependent fashion. Combination treatment with TLS-01 and radiation inhibited tumor growth to a greater extent than was seen with TLS-01 or radiation treatment alone. Radiation treatment dramatically increased the number of CD8+ T cells in the TME and concomitantly increased both the number and immunosuppressive properties of inhibitory cell populations in the TME, including increases in CD73 expression on M-MDSCs and M2 macrophages. Treatment of irradiated mice with TLS-01 reduced Tregs, increased the CD8 / Treg ratio and reduced CD73 expression on immune suppressive cells. Combination of TLS-01 with radiation resulted in enhanced efficacy when compared to either agent alone. Our data provides mechanistic insights underpinning this enhanced efficacy. TLS-01 moderated immunosuppression in the TME by radiation via modulation of multiple immunosuppressive cell types. These data support the therapeutic utility of combination treatment of TLS-01 and radiation therapy in the treatment of solid tumors. Citation Format: Kenneth J. Simon, Stavros Kopsiaftis, Randall Burton, Patricia E. Rao, Jessie M. English, Barbara S. Fox. Anti-LAP-TGFb antibodies inhibit tumor growth in a CT26 syngeneic tumor model in combination with radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 93.

Abstract 2011: Potassium usnate inhibits invasion and metastasis in colorectal cancer: Implication of clinical application of a water-soluble form of usnic acid in anticancer therapy

Abstract Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity in vitro, whereas its effect in vivo is limited. Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy. Citation Format: Yi Yang, Woo Kyun Bae, Kyung Hwa Lee, Kyung-Sub Moon, So-Yeon Park, Rui Zhou, Isa Taş, Chathurika Gamage, Sueun Lee, Chaeyong Jung, Kyung Keun Kim, Hyung-Ho Ha, Hangun Kim. Potassium usnate inhibits invasion and metastasis in colorectal cancer: Implication of clinical application of a water-soluble form of usnic acid in anticancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2011.

MORPHOLOGICAL CHARACTERIZATION OF COLORECTAL PITS USING AUTOFLUORESCENCE MICROSCOPY IMAGES.

Colorectal cancer is one of the most prevalent pathologies. Its prognosis is linked to the early detection and treatment. Currently diagnosis is performed by histological analysis from polyp biopsies, followed by morphological classification. Kudo's pit pattern classification is frequently used for the differentiation of neoplastic colorectal lesions using hematoxylin-eosin stained samples. Few articles have reported this classification with image software processing, using exogenous markers over the samples. The processing of autofluorescence images is an alternative that could allow the characterization of the pits from the crypts of Lieberkühn, bypassing staining techniques. Processing and analysis of widefield autofluorescence microscopy images obtained by fresh colon tissue samples from a murine model of colorectal cancer in order to quantify and characterize the pits morphology by measuring morphology parameters and shape descriptors. Adult male BALB/cCmedc strain mice (n=27), ranging from 20 to 30 g, were randomly assigned to four and five groups of treated and control animals. Colon samples were collected at day zero and at fourth, eighth, sixteenth and twentieth weeks after treatmentwith azoxymethane. Two-dimensional (2D) segmentation, quantification and morphological characterization of pits by image processing applied using macro programming from FIJI. Type I is the pit morphology prevailing between 53 and 81% in control group weeks. III-L and III-S types were detected in reduced percentages. Between the 33 and 56% of type I was stated as the prevailing morphology for the 4th, 8th and 20th weeks of treated groups, followed by III-L type. For the 16th week, the 39% of the pits was characterized as III-L type, followed by type I. Further, pattern types as IV, III-S and II were also found mainly in that order for almost all of the treated weeks. These preliminaries outcomes could be considered an advance in two-dimensional pit characterization as the whole image processing, comparing to the conventional procedure, takes a few seconds to quantify and characterize non-pathological colon pits as well as to estimate early pathological stages of colorectal cancer.

Impact of the Total Western Diet for Rodents on Colon Mucosal Gene Expression in a Multigenerational Murine Model of Colitis-associated Colorectal Cancer (OR04-03-19)

ObjectivesA Western type dietary pattern is a major risk factor for colitis-associated colorectal cancer (CAC). Observations from transgenerational studies suggest that epimutations may be inherited, resulting in persistent aberrant gene expression in offspring. Previously, our group reported that ancestral exposure to the total Western diet (TWD) markedly increased colon cancer incidence and disease severity in F3 offspring that were not fed this diet directly. Moreover, exposure to TWD over multiple generations markedly exacerbated the disease in F3 offspring as compared to those fed TWD directly. For the present work, we hypothesized that ancestral or multiple generation exposure to the TWD may result in differential expression of cancer critical genes in such a way that explains the greater tumor abundance and burden observed in these mice. MethodsC57BL/6 J mice were bred for three generations, during which they were fed a standard diet (AIN93G) for all generations or the total Western diet for rodents (TWD) during only the F0 generation (ancestral), the F0 through F3 generations (multi-generation), or only the F3 generation (direct). The azoxymethane and dextran sodium sulfate (AOM/DSS) model of CAC was employed in F3 offspring, from which colon mucosa RNA was isolated and used for Illumina RNAseq with EdgeR for differential expression analysis. ResultsAbout 700 to 4500 differentially expressed genes (DEGs) were identified for colon mucosa from AOM/DSS-initiated offspring compared to their sham controls. For AOM/DSS-initiated mice, >100 DEGs were identified comparing multi-generation TWD-fed mice to their AIN93G-fed counterparts, and 36 genes were different from those direct TWD-fed. Of note, in sham-initiated mice, 101 DEGs were identified comparing direct TWD-fed mice to multi-generation TWD-exposed offspring. Interestingly, these DEGs were associated with defense response, immune response, and response to interferon biological process ontology terms. ConclusionsExposure to the TWD over multiple generations caused significant changes in genes involved in immune response in third generation offspring. Assessment of genome-wide patterns of DNA methylation in these colon tissue samples is ongoing. Funding SourcesUSDA NIFA AFRI Grant No. 2014-67017-21755; Utah Agricultural Experiment Station Project UTA-1178.

A Tumor-Peptide-Based Nanoparticle Vaccine Elicits Efficient Tumor Growth Control in Antitumor Immunotherapy.

Recognition of immunoactive oligonucleotides by the immune system, such as Toll-like receptor ligand CpG, leads to increased antibody and T-cell responses. Systemic application often results in unwanted generalized nonantigen-specific activation of the immune system. Nanoparticles are ideal carriers for small and large molecules. Recently, we have demonstrated that calcium phosphate (CaP) nanoparticles functionalized with CpG, and viral antigens are able to induce specific T-cell immunity that protects mice against viral infection and efficiently reactivates the exhausted CD8+ T-cell compartment during chronic retroviral infection. Therefore, CaP nanoparticles are promising vaccine vehicles for therapeutic applications. In this study, we investigated the therapeutic potential use of these nanoparticles in a murine xenograft colorectal cancer model. Therapeutic vaccination with CaP nanoparticles functionalized with CpG and tumor model antigens increased the frequencies of cytotoxic CD8+ T cells in the tumor in a type I interferon-dependent manner. This was accompanied with significantly repressed tumor growth in contrast to the systemic administration of soluble CpG and antigens. Combination therapy of CaP nanoparticles and immune checkpoint blocker against PD-L1 further enhanced the cytotoxic CD8+ T-cell response and eradicated the tumors. Strikingly, vaccination with CaP nanoparticles functionalized with CpG and a primary tumor cell lysate was also sufficient to control the tumor growth. In conclusion, our results represent a translational approach for the use of CaP nanoparticles as a potent cancer vaccine vehicle.