Mouse Model Of Colorectal Cancer Research Articles

Bioactive triterpenes of jujube in the prevention of colorectal cancer and their molecular mechanism research

BackgroundJujube, a popular fruit from the Rhamnaceae family, relieves colorectal inflammation caused by spleen deficiency and has been used in many formulas in clinical for decades to treat colorectal cancer (CRC). As of yet, the therapeutic substances and mechanism of their action are unknown. PurposeThe purpose of this study is to define the therapeutic substances of jujube and its mechanism of action in treating CRC. MethodsThe pharmacological effects of jujube extract and its fractions were evaluated in vivo using a CRC mouse model induced by AOM/DSS. The DAI value, colon length, mortality, tumor burden, and histological tumor size of the treated animals were compared. To explore the potential therapeutic substances, LC-MS analysis was conducted to characterize the serum migration components. A network pharmacology experiment was carried out for potential molecular targets. To verify the therapeutic substances as well as the molecular mechanism of jujube intervening CRC, cellular MTT assay of the serum migration components, Western blot and IHC tests were conducted. ResultsThe in vivo pharmacological studies showed that compared to AOM/DSS treated mice, the mortality and DAI value, tumor burden, and histological tumor size of jujube extract and its fat-soluble fraction (mainly contained triterpenes) treated mice were significantly reduced, and their colon lengths were obviously longer than AOM/DSS treated mice. The targeted-LC/MS analysis supposed triterpenes 3, 7, 9, 11, 12, 14, 17 – 21, and 25 – 28 to be the serum migration components, which might be the potential therapeutic substances. In the network pharmacology experiment, the GO annotation and enrichment analysis of the KEGG pathway indicated that PI3K-Akt pathway and inflammatory reaction were important factors for jujube inhibiting CRC. Cellular MTT assay of serum migration components indicated that the potential effective substances from fat-soluble fraction to be triterpenes 3, 7, 17, 19, 20, and 25. The Western blot and IHC assays implied that the jujube extract, its fat-soluble fraction, and triterpenes 7, 17, and 20 showed inhibition on the expression of PI3K/Akt/NF-κB signaling pathway-related proteins. Additionally, it was noted in the pharmacodynamic experiment that ZJL's effectiveness was more apparent than ZJH and SQL in tumor burden rate, colon length, and spleen weight, which indicated that the efficacy of ZJ is contributed from CD and SQ, and they may have a synergistic effect on anti-CRC. ConclusionThese results for the first time provide evidence that jujube triterpenes possess an anti-CRC effect, their mechanism was involving the control of the PI3K/Akt/NF-κB signaling pathway. What's more, the potential synergistic effect of the fat-soluble and water-soluble components found in this study provided a solid foundation for our deep understanding of how jujube can ameliorate CRC.

Establishment of a Selective Liver Lobe Tumor-Bearing Mouse Model of Colorectal Cancer.

Colorectal cancer is one of the most common causes of cancer-related death, and its main site of metastasis is the liver. The surgical method used for metastases of colorectal cancer in the liver varies according to the lobe affected, as does the prognosis. However, there is a lack of relevant basic research. Therefore, a good animal model is needed for basic studies of metastases from colorectal cancer to the different lobes of the liver. A CT26 colon cancer cell line transfected with a virus expressing green fluorescent protein was inoculated into BALB/C mice via the spleen. Tumor formation in the liver lobes was observed under a fluorescence microscope according to which portal vein branch was ligated and according to clamping time. The differential formation of metastatic lesions in the different lobes was then compared with physical anatomy. Serum samples were used to detect the changes in liver function postoperatively. Ligation and resection of the spleen 1 min after injection of the CT26 cells and release of the vessel clamp 1 min after splenectomy created an ideal tumor-bearing mouse model with little effect on liver function. Selective clamping of each portal vein branch and splenic injection of a CT26 cell line successfully established a selective liver lobe tumor-bearing model of colorectal cancer with distinct characteristics. This model provides an opportunity for investigation of the mechanisms of metastasis of colorectal cancer to different lobes of the liver and may provide a basis for clinical treatment.

Integrin-linked kinase expression in myeloid cells promotes colon tumorigenesis.

Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and treatment options for advanced CRC, which has a low 5-year survival rate, remain limited. Integrin-linked kinase (ILK), a multifunctional, scaffolding, pseudo-kinase regulating many integrin-mediated cellular processes, is highly expressed in many cancers. However, the role of ILK in cancer progression is yet to be fully understood. We have previously uncovered a pro-inflammatory role for myeloid-specific ILK in dextran sodium sulfate (DSS)-induced colitis. To establish a correlation between chronic intestinal inflammation and colorectal cancer (CRC), we investigated the role of myeloid-ILK in mouse models of CRC. When myeloid-ILK deficient mice along with the WT control mice were subjected to colitis-associated and APCmin/+-driven CRC, tumour burden was reduced by myeloid-ILK deficiency in both models. The tumour-promoting phenotype of macrophages, M2 polarization, in vitro was impaired by the ILK deficiency and the number of M2-specific marker CD206-expressing tumour-associated macrophages (TAMs) in vivo were significantly diminished in myeloid-ILK deficient mice. Myeloid-ILK deficient mice showed enhanced tumour infiltration of CD8+ T cells and reduced tumour infiltration of FOXP3+ T cells in colitis-associated and APCmin/+-driven CRC, respectively, with an overall elevated CD8+/FOXP3+ ratio suggesting an anti-tumour immune phenotypes. In patient CRC tissue microarrays we observed elevated ILK+ myeloid (ILK+ CD11b+) cells in tumour sections compared to adjacent normal tissues, suggesting a conserved role for myeloid-ILK in CRC development in both human and animal models. This study identifies myeloid-specific ILK expression as novel driver of CRC, which could be targeted as a potential therapeutic option for advanced disease.

Abstract P014: A novel role of mutant APC and n3-PUFA in reshaping cholesterol-dependent Wnt pathway-associated proteolipid nanocluster organization and signaling in colorectal cancer

Abstract Colorectal cancer (CRC) is the 3rd most common type of cancer in the U.S., and it is estimated it will account for an alarming 151,030 (8%) of new total cancer cases and 52,580 (9%) of total cancer deaths in 2022. Remarkably, dysregulation of the Wnt signaling pathway has been linked to ~90% of all colorectal cancer (CRC) cases. The vast majority (>80%) of sporadic CRC cases display mutations in Adenomatous Polyposis Coli (APC), a central Wnt signaling regulator. Loss of APC function causes aberrant stabilization of β-catenin (βcat), a crucial step in CRC initiation. Notably, attempts towards “drugging” this pathway still poses multiple hurdles due to poor tumor cell targeting, negative side effects associated with required long-term treatments and poorly understood mechanisms of action. Consequently, there is an urgent need to gather additional mechanistic insights associated with the Wnt pathway to develop novel therapeutic approaches. With respect to Wnt biology, two key plasma membrane receptors, LRP5/6 and Frizzled (Fzd) require lipid raft localization and nanoclustering for efficient signaling. Interestingly, various effectors that disrupt raft dynamics, alter LRP5/6-Fzd clustering, leading to reduced βcat stabilization. Thus, we examined the effect of mutant APC (mAPC) on plasma membrane lipid/protein-mediated interactions of Wnt-associated effectors in a CRC mouse model, human mAPC-expressing organoids and cell lines, and Drosophila. We show for the first time that mAPC significantly increased the levels of plasma membrane free cholesterol, a major component of lipid rafts known to selectively activate canonical Wnt signaling, in the intestinal epithelium including Lgr5+ colonic stem cells. Moreover, this change in plasma membrane cholesterol was associated with alterations in colonocyte membrane rigidity and raft organization. We subsequently examined the effect of mAPC on the interactions between Wnt signaling receptors/effectors in mAPC-expressing cell lines. mAPC significantly increased LRP6 and Fzd7 homo-clustering and LRP6-Fzd7 hetero-clustering as well as their interactions with key lipids, e.g., PIP2, resulting in the enhancement of Wnt signaling activation. Interestingly, the effects of mAPC were recapitulated by the addition of exogenous cholesterol to wild type colonocytes. Finally, we demonstrate that n-3 polyunsaturated fatty acids (n3-PUFA), i.e., DHA and EPA, reduces mAPC-driven colonic polyp formation in mice in part by reducing plasma membrane cholesterol, rigidity and LRP6-Fzd7 clustering in colonocytes. Collectively, these findings indicate that mAPC can directly perturb lipid homeostasis in the colon, thereby altering raft stability, LRP6-Fzd7 membrane nanoclusters and downstream Wnt signaling and supports the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane lipid composition, to reduce Wnt signaling and cancer risk. Citation Format: Alfredo Erazo-Oliveras, Mohamed Mlih, Mónica Muñoz-Vega, Eunjoo Kim, Venkat Thiriveedi, Michael L. Salinas, Xiaoli Wang, Jatin Roper, Jason Karpac, Robert S. Chapkin. A novel role of mutant APC and n3-PUFA in reshaping cholesterol-dependent Wnt pathway-associated proteolipid nanocluster organization and signaling in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P014.

Regulation of Fatty Acid Metabolism and Inhibition of Colorectal Cancer Progression by Erchen Decoction.

Erchen decoction (ECD) is a traditional Chinese prescription widely used in the treatment of various diseases such as obesity, fatty liver, diabetes, and hypertension. In this study, we investigated the effect of ECD on fatty acid metabolism in a colorectal cancer (CRC) mouse model fed a high-fat (HF) diet. The HF-CRC mouse model was established by azoxymethane (AOM)/dextran sulphate sodium (DSS) combined with a high-fat diet. Mice were then gavaged with ECD. Change in the body weight was recorded every two weeks for 26 weeks. Changes in blood glucose (GLU), total cholesterol (TC), total triglycerides (TG), and C-reactive protein (CRP) were measured. Colorectal tissues were collected to observe changes in colorectal length and tumorigenesis. Hematoxylin-eosin (HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and inflammatory markers. Fatty acids and the expression of related genes in colorectal tissues were also studied. ECD gavage inhibited HF-induced weight gain. CRC induction and HF diet intake resulted in increased GLU, TC, TG, and CRP, where ECD gavage reduced these elevated indicators. ECD gavage also increased colorectal length and inhibited tumorigenesis. HE staining revealed that ECD gavage suppressed inflammatory infiltration of colorectal tissues. ECD gavage suppressed the fatty acid metabolism abnormalities caused by HF-CRC in colorectal tissues. Consistently, ECD gavage lowered ACSL4, ACSL1, CPT1A, and FASN levels in colorectal tissues. Conclusions. ECD inhibited HF-CRC progression through the regulation of fatty acid metabolism.

Platelets promote CRC by activating the C5a/C5aR1 axis via PSGL-1/JNK/STAT1 signaling in tumor-associated macrophages.

Rationale: Platelets can influence the progression and prognosis of colorectal cancer (CRC) through multiple mechanisms, including crosstalk with tumor-associated macrophages (TAMs). However, the mechanisms underlying the crosstalk between platelets and TAMs remain unclear. The present study aimed to investigate the role of intratumoral platelets in regulating the function of TAMs and to identify the underlying mechanisms. Methods: The interaction of platelets with macrophages was assessed in the presence or absence of the indicated compounds in vivo. An azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC mouse model was used to investigate the role of platelets in controlling CRC development. Multiplexed immunofluorescence staining, fluorescence-activated cell sorting (FACS), and RNA sequence analysis were used to examine the changes in TAMs. TAMs and bone marrow-derived macrophages (BMDMs) were treated with the indicated compounds or siRNA against specific targets, and the expression levels of signal transducer and activator of transcription 1 (STAT1), c-Jun N-terminal kinase (JNK), and P-selectin glycoprotein ligand-1 (PSGL-1) were measured by Western blotting. The mRNA expression levels of complement 5 (C5), complement 5a receptor 1 (C5ar1), Arginase 1 (Arg1) and Il10 were measured by real-time RT-PCR, and the complement 5a (C5a) concentration was measured by ELISA. The dual-luciferase reporter assay and ChIP assay were performed to examine the potential regulatory mechanisms of platelet induction of C5 transcription in TAMs. Results: In our study, we found that an increase in platelets exacerbated CRC development, while inhibiting platelet adhesion attenuated tumor growth. Platelets signal TAMs through P-selectin (CD62P) binding to PSGL-1 expressed on TAMs and activating the JNK/STAT1 pathway to induce the transcription of C5 and the release of C5a, shifting TAMs toward a protumor phenotype. Inhibiting the C5a/C5aR1 axis or PSGL-1 significantly reduced CRC growth. Conclusions: An increase in intratumoral platelets promoted CRC growth and metastasis by CD62P binding to PSGL-1 expressed on TAMs, leading to JNK/STAT1 signaling activation, which promoted C5 transcription and activated the C5a/C5aR1 axis in TAMs. Our study examined the mechanism of the crosstalk between platelets and TAMs to exacerbate CRC development and proposed a potential therapeutic strategy for CRC patients.

Blocking Muscarinic Receptor 3 Attenuates Tumor Growth and Decreases Immunosuppressive and Cholinergic Markers in an Orthotopic Mouse Model of Colorectal Cancer

Tumor cells have evolved to express immunosuppressive molecules allowing their evasion from the host's immune system. These molecules include programmed death ligands 1 and 2 (PD-L1 and PD-L2). Cancer cells can also produce acetylcholine (ACh), which plays a role in tumor development. Moreover, tumor innervation can stimulate vascularization leading to tumor growth and metastasis. The effects of atropine and muscarinic receptor 3 (M3R) blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), on cancer growth and spread were evaluated in vitro using murine colon cancer cell line, CT-26, and in vivo in an orthotopic mouse model of colorectal cancer. In the in vitro model, atropine and 4-DAMP significantly inhibited CT-26 cell proliferation in a dose dependent manner and induced apoptosis. Atropine attenuated immunosuppressive markers and M3R via inhibition of EGFR/AKT/ERK signaling pathways. However, 4-DAMP showed no effect on the expression of PD-L1, PD-L2, and choline acetyltransferase (ChAT) on CT-26 cells but attenuated M3R by suppressing the phosphorylation of AKT and ERK. Blocking of M3R in vivo decreased tumor growth and expression of immunosuppressive, cholinergic, and angiogenic markers through inhibition of AKT and ERK, leading to an improved immune response against cancer. The expression of immunosuppressive and cholinergic markers may hold potential in determining prognosis and treatment regimens for colorectal cancer patients. This study's results demonstrate that blocking M3R has pronounced antitumor effects via several mechanisms, including inhibition of immunosuppressive molecules, enhancement of antitumor immune response, and suppression of tumor angiogenesis via suppression of the AKT/ERK signaling pathway. These findings suggest a crosstalk between the cholinergic and immune systems during cancer development. In addition, the cholinergic system influences cancer evasion from the host's immunity.

Anti-tumor effects of Auraptene through induction of apoptosis and oxidative stress in a mouse model of colorectal cancer

The main strategy of cancer cells for survival is uncontrolled cell division and escape from apoptosis. The use of anticancer agents inducing the production of reactive oxygen species (ROS) and controlling cell division might be a therapeutic approach to eradicate cancer cells. Herein, we examined the therapeutic effects of Auraptene on CT26 cells as well as on a mouse model of colorectal cancer (CRC). The spheroid assay was also conducted to analyze the anti-proliferative activity of Auraptene. We also assessed the in vitro analysis of ROS generation. The impact of Auraptene on oxidant/antioxidant markers, as well as the mRNA expression of Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin genes, was evaluated by qPCR in tumor samples. As a result, Auraptene significantly reduced the size of CT26 spheroids at a dose of 200 µM. After 12 h, ROS levels were significantly elevated in CT26 cells. The administration of Auraptene induced apoptosis and the cell cycle arrest by modulating Bax, Bcl-2, Nrf2, Cyclin D1, and Survivin mRNA levels. Furthermore, our results demonstrated that Auraptene suppressed CAT, GSH (reduced Glutathione), and FRAP while increasing MDA in tissue homogenates which in turn could raise oxidative stress and stimulate apoptosis. Therefore, Auraptene may act as a powerful adjuvant therapy in CRC since it triggers apoptosis and cell cycle.

Microenvironmental ammonia enhances T cell exhaustion in colorectal cancer

Effective therapies are lacking for patients with advanced colorectal cancer (CRC). The CRC tumor microenvironment has elevated metabolic waste products due to altered metabolism and proximity to the microbiota. The role of metabolite waste in tumor development, progression, and treatment resistance is unclear. We generated an autochthonous metastatic mouse model of CRC and used unbiased multi-omic analyses to reveal a robust accumulation of tumoral ammonia. The high ammonia levels induce Tcell metabolic reprogramming, increase exhaustion, and decrease proliferation. CRC patients have increased serum ammonia, and the ammonia-related gene signature correlates with altered Tcell response, adverse patient outcomes, and lack of response to immune checkpoint blockade. We demonstrate that enhancing ammonia clearance reactivates Tcells, decreases tumor growth, and extends survival. Moreover, decreasing tumor-associated ammonia enhances anti-PD-L1 efficacy. These findings indicate that enhancing ammonia detoxification can reactivate Tcells, highlighting a new approach to enhance the efficacy of immunotherapies.

Anti-Programmed Cell Death-1 Antibody and Dasatinib Combination Therapy Exhibits Efficacy in Metastatic Colorectal Cancer Mouse Models.

In this study, we investigated the in vivo metastasis suppression effects of the platelet-derived growth factor receptor inhibitor dasatinib, which targets cancer-associated fibroblasts (CAFs), in combination with an anti-programmed cell death-1 (PD-1) antibody. We classified clinical CRC cases as inflamed, excluded, or desert using immunohistochemical analysis and evaluated the tumor stroma. The excluded type was the most common, and cases with high-volume stroma in the primary lesions also had a high stromal volume in the liver metastatic lesions. Liver-metastasis mouse models with different stromal volumes were established and treatment-induced changes in the tumor immune microenvironment were evaluated. The anti-PD-1 antibody alone exhibited a therapeutic effect for the liver metastases with low stromal volumes but not for the liver metastases with high stromal volumes. In contrast, antitumor effects were observed with anti-PD-1 antibody/dasatinib combination therapy even in the liver metastases with high stromal volumes. Combination therapy reduced the stromal volume, promoted immune cell infiltration, induced antitumor cytotoxic T-cell responses, activated antitumor immunity, and promoted tumor regression. These results suggest that CAFs play an important role in the immune evasion of CRC and that anti-PD-1 antibody/dasatinib combination therapy has potential as a treatment option for patients with metastatic CRC for whom immunotherapy alone is ineffective.

Supplementation of quinoa peptides alleviates colorectal cancer and restores gut microbiota in AOM/DSS-treated mice

Quinoa protein hydrolysate has been previously reported to exert anti-cancer effects in cultured colon cancer cells. Here, we investigated the effect of quinoa protein and its hydrolysate on an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced mouse model of colorectal cancer (CRC) and examined its underlying mechanism using gut microbiota analysis and short chain fatty acids (SCFAs) production analysis. Our results showed that quinoa protein or its hydrolysate mitigated the clinical symptoms of CRC and increased SCFAs contents in colon tissues. Moreover, administration of quinoa protein or its hydrolysate partially alleviated gut microbiota dysbiosis in CRC mice by decreasing the abundance of pathogenic bacteria and increasing the abundance of probiotics. Additionally, PICRUSt analysis revealed that the functional profile of gut microbiota in the quinoa protein treated groups was more similar to that of the control group. These findings indicated that the modulation of gut microbiota by quinoa protein diet intervention may ameliorate AOM/DSS-induced CRC.

Dietary Supplementation with Black Raspberries Altered the Gut Microbiome Composition in a Mouse Model of Colitis-Associated Colorectal Cancer, although with Differing Effects for a Healthy versus a Western Basal Diet.

Black raspberries (BRB) are rich in anthocyanins with purported anti-inflammatory properties. However, it is not known whether dietary supplementation would ameliorate Western-diet enhanced gut inflammation and colon tumorigenesis. We employed a mouse model of colitis-associated colorectal cancer (CAC) to determine the effects of dietary supplementation with 5 to 10% (w/w) whole, freeze-dried BRB in male C57BL/6J mice fed either a standard healthy diet (AIN93G) or the total Western diet (TWD). In a pilot study, BRB suppressed colitis and colon tumorigenesis while also shifting the composition of the fecal microbiome in favor of taxa with purported health benefits, including Bifidobacterium pseudolongum. In a follow-up experiment using a 2 × 2 factorial design with AIN and TWD basal diets with and without 10% (w/w) BRB, supplementation with BRB reduced tumor multiplicity and increased colon length, irrespective of the basal diet, but it did not apparently affect colitis symptoms, colon inflammation or mucosal injury based on histopathological findings. However, BRB intake increased alpha diversity, altered beta diversity and changed the relative abundance of Erysipelotrichaceae, Bifidobacteriaceae, Streptococcaceae, Rikenellaceae, Ruminococcaceae and Akkermansiaceae, among others, of the fecal microbiome. Notably, changes in microbiome profiles were inconsistent with respect to the basal diet consumed. Overall, these studies provide equivocal evidence for in vivo anti-inflammatory effects of BRB on colitis and colon tumorigenesis; yet, BRB supplementation led to dynamic changes in the fecal microbiome composition over the course of disease development.

Nanoliposome-Loaded Phenolics from Nasturtium officinale Improves Health Parameters in a Colorectal Cancer Mouse Model.

Nasturtium officinale contains high amounts of phytochemical compounds that work against oxidative damages leading to improved health conditions in animals as well as humans. The study was performed to investigate the health benefits of nonencapsulated and nanoliposome-encapsulated phenolic rich fractions obtained from Nasturtium officinale on mice induced colorectal cancer. The experiment focused on encapsulation efficiency in improving the effectiveness of plant bioactive compounds. Phenolic rich fractions (PRF) were successfully loaded in the nanoliposome structure, a nanometer in size, of spherical shape and with homogeneous dispersion. Induction of colorectal cancer in mice impaired weight gain and feed intake, liver function and structural characteristics of ileum, while the dietary administration of nanoliposome-encapsulated PRF regulated the expression of Caspase 3, Bax, Bcl2, iNOS and SOD genes in the tumor tissue. The addition of nonencapsulated PRF and nanoliposome encapsulated PRF at the concentration of 100 mg TPC/kg BW/day improved the genes expression, although the nanoliposome-encapsulated PRF revealed better health outcomes compared to nonencapsulated PRF. Furthermore, both PRF improved intestinal morphology when the mice were challenged with colorectal cancer. The higher health promoting activity of nanoliposome-encapsulated PRF could be associated with its enhanced intestinal absorption, bioavailability, bioaccessibility and bioactivity. Consequently, the nanoliposome-encapsulated PRF could be considered as a promising anticancer agent against colorectal cancer.

A cancer cell membrane coated, doxorubicin and microRNA co-encapsulated nanoplatform for colorectal cancer theranostics

Endogenous microRNAs (miRNA) in tumors are currently under exhaustive investigation as potential therapeutic agents for cancer treatment. Nevertheless, RNase degradation, inefficient and untargeted delivery, limited biological effect, and currently unclear side effects remain unsettled issues that frustrate clinical application. To address this, a versatile targeted delivery system for multiple therapeutic and diagnostic agents should be adapted for miRNA. In this study, we developed membrane-coated PLGA-b-PEG DC-chol nanoparticles (m-PPDCNPs) co-encapsulating doxorubicin (Dox) and miRNA-190-Cy7. Such a system showed low biotoxicity, high loading efficiency, and superior targeting ability. Systematic delivery of m-PPDCNPs in mouse models showed exceptionally specific tumor accumulation. Sustained release of miR-190 inhibited tumor angiogenesis, tumor growth, and migration by regulating a large group of angiogenic effectors. Moreover, m-PPDCNPs also enhanced the sensitivity of Dox by suppressing TGF-β signal in colorectal cancer cell lines and mouse models. Together, our results demonstrate a stimulating and promising m-PPDCNPs nanoplatform for colorectal cancer theranostics.

Combination of immune checkpoint inhibitors with radiation therapy in cancer: A hammer breaking the wall of resistance.

Immuno-oncology is an emerging field in the treatment of oncological diseases, that is based on recruitment of the host immune system to attack the tumor. Radiation exposure may help to unlock the potential of the immune activating agents by enhancing the antigen release and presentation, attraction of immunocompetent cells to the inflammation site, and eliminating the tumor cells by phagocytosis, thereby leading to an overall enhancement of the immune response. Numerous preclinical studies in mouse models of glioma, murine melanoma, extracranial cancer, or colorectal cancer have contributed to determination of the optimal radiotherapy fractionation, as well as the radio- and immunotherapy sequencing strategies for maximizing the antitumor activity of the treatment regimen. At the same time, efficacy of combined radio- and immunotherapy has been actively investigated in clinical trials of metastatic melanoma, non-small-cell lung cancer and renal cell carcinoma. The present review summarizes the current advancements and challenges related to the aforementioned treatment approach.

MgIG Attenuates Oxaliplatin-induced Hepatotoxicity through Suppression of Connexin 43 in Hepatic Stellate Cells.

Oxaliplatin is widely used in cancer chemotherapy with adverse effects such as liver toxicity. Magnesium isoglycyrrhizinate (MgIG) has hepatoprotective effects, but the underlying mechanism remains elusive. The study's aim was to investigate the mechanism underlying the hepatoprotective effects of MgIG against oxaliplatin-induced liver injury. A xenografted colorectal cancer mouse model was established with MC38 cells. Mice were given oxaliplatin (6 mg/kg/week) for 5 weeks to mimic oxaliplatin-induced liver injury in vivo. LX-2 human hepatic stellate cell s(HSCs) were employed for in vitro studies. Serological tests, hematoxylin and eosin staining, oil red O staining and transmission electron microscopy were used for histopathological examinations. Real-time PCR, western blotting, immunofluorescence and immunohistochemical staining were used to determine Cx43 mRNA or protein levels. Flow cytometry was used to assay reactive oxygen species (ROS) and mitochondrial membrane. Short hairpin RNA targeting Cx43 was lentivirally transduced in LX-2 cells. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to determine MgIG and metabolite concentration. MgIG (40 mg/kg/day) treatment significantly reduced serum aspartate transaminase (AST) and alanine transaminase (ALT) levels in the mouse model, and alleviated liver pathological changes, including necrosis, sinusoidal expansion, mitochondrial damage, and fibrosis. MgIG reduced the abnormal expression of Cx43 in the mitochondria and nuclei of HSCs. MgIG inhibited the activation of HSCs via reducing ROS generation, mitochondrial dysfunction, and N-cadherin transcription. MgIG's inhibition of HSCs activation was abolished after knockdown of Cx43 in LX-2 cells. Cx43 mediated MgIG's hepatoprotective effects against oxaliplatin-induced toxicity.

Abstract A020: Exploring the effects of resveratrol on the gut microbiome in high fat diet-fed BRAFV600E mutant mice

Abstract The polyphenol resveratrol, widely known for its cancer protective activities has been shown to protect against the pro-tumorigenic effects induced by high-fat diets (HFD) in mouse models of colorectal cancer (CRC). Although, the mechanisms of actions of this compound in humans still remain elusive, recent studies suggest its effects may be mediated partly through its interaction with the gut microbiota. Here we sought to determine the changes induced by HFD on the intestinal microbiome and the impact of resveratrol supplementation on these alterations in a mouse model of BRAFV600E mutant CRC. BRAFV600E; BRAFWT (wild-type) mice were randomised into 8 groups and fed either a standard diet or HFD supplemented with low or high-dose of resveratrol (0.7mg/kg/day and 14mg/kg/day, respectively) for 6-weeks. The mouse faecal microbial profile was then assessed by targeting the V3-V4 area of the bacterial 16S rRNA gene. Consumption of HFD was associated with significant alternations in the gut microbiome composition in both BRAFV600E and BRAFWT based on beta-diversity analysis (PADONIS<0.05). HFD increased the Firmicutes and Clostridia abundance in BRAFWT compared to animals on a standard diet (P<0.01, P<0.008 respectively), while in BRAFV600E mice, alterations were observed in lower taxonomic levels including the enrichment of Faecalibaculum (P<0.02) and growth inhibition of Lactobacillus johnsonhii (P<0.05). High-dose resveratrol resulted in significant differences in the microbial composition compared to HFD-treated group in the BRAFV600E mice (PADONIS<0.05) but not in BRAFWT . Resveratrol administration did not counteract the HF-induced taxonomic changes but high-dose resveratrol changed Muribaculum, and UBA181 genera abundances in BRAFV600E mice (P<0.001 and P<0.05 respectively) compared to HF-treated mice. Low dose resveratrol had no significant effects on bacterial abundance in both BRAFV600E and BRAFWT mice. It was also observed that BRAFV600E mutation influenced the gut microbiome independently of diet based on beta and alpha-diversity (PADONIS<0.05, PChao1<0.05 respectively). Resveratrol administration modified the gut microbiota composition of BRAFV600E mice but did not completely reverse the HFD-induced changes indicating that alternative mechanisms may contribute to the protective effects of resveratrol in this mouse model. Citation Format: Despoina Theofanous, Hong Cai, Karen Brown. Exploring the effects of resveratrol on the gut microbiome in high fat diet-fed BRAFV600E mutant mice [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A020.

Evaluation of Innate Lymphoid Cells (ILCs) Population in the Mouse Model of Colorectal Cancer.

Innate Lymphoid Cells (ILCs) promote tissue homeostasis, contribute to the immune defense mechanisms, and play important roles in the initiation of immune responses and chronic inflammation. To understand the roles of innate lymphoid cells in the pathophysiology of colorectal cancer (CRC) in the mouse model. CRC was induced using azoxymethane (AOM) and dextran sulfate sodium (DSS) in Balb/c mice (the chemically induced group=18 mice), or orthotopic injection of CT-26 cell line into the colon of another set of Balb/c mice (the orthotopic group=14mice). Normal saline was injected into 18 mice, as the sham group. After 80 days, the chemically induced group was divided into two subgroups, dysplasia (8 mice) and reparative change (10 mice), based on pathological examinations. The frequencies of ILC1, 2, and 3 were then measured in colon tissues using flow cytometry by four markers including an anti-mouse lineage cocktail (FITC anti-mCD3/FITC anti-mGr-1/FITC anti-mCD11b/ FITC anti-mCD45R (B220)/FITC anti-mTer-119), PE/Cy7 anti-mouse CD45, PE anti-mouse CD117 (c-kit), and APC anti-mouse IL-33 Rα (ST2). The total ILC population was significantly higher in the chemically induced reparative change compared with the sham group. ILC1 percentage in the chemically induced reparative change was significantly higher compared to those in the other three groups (Sham, chemically induced dysplasia and orthotopic dysplasia). The orthotopic dysplasia group showed more ILC3 percentage than the other groups. ILC1 and ILC3 subgroups increased significantly in reparative and dysplastic experimental CRC respectively. Thus ILC1 may have an inhibitory effect on tumor growth whereas ILC3 promotes tumor progression.

Abstract B013: Activin’s influence on the tumor microenvironment in colon cancer

Abstract Background and aims: Activin A (activin) is a key molecule that regulates cell specific and context dependent signaling in tumor growth and metastasis as well as local and systemic inflammatory responses. Understanding the link between tumor-promotive and inflammatory effects may provide a novel opportunity for targeted therapy in colorectal cancer (CRC). We have previously shown that activin signaling promotes epithelial to mesenchymal transition, tumor migration, and that serum activin correlates with advanced stage in CRC patients. Recently, activin has been implicated in enhancing CD4+ to CD8+ communications to promote cytotoxic elimination of tumor cells. We now hypothesize that activin exerts cell-specific effects in the tumor microenvironment (TME) to both promote anti-tumoral activity of immune cells and pro-metastatic behavior of tumor cells in a context-dependent manner. Methods: Western blot and transwell migration assays with and without activin were performed in human epithelial colon cancer cells with varying expression levels of ACVR2 and in human colonic fibroblasts. The influence of the canonical Smad4 pathway was elucidated in a Ts4-Cre;Apcflox;Smad4flox mouse model for CRC. We analyzed a TMA of 1055 stage II and III CRC patients from the QUASAR2 cohort to correlate activin and CD4+ expression with outcome. The TMAs were also analyzed via Digital Spatial Profiling (DSP, NanoString) to determine the immune cell heterogeneity and cell signaling patterns within the tumor microenvironment relative to activin. Results: The non-canonical pAkt pathway is activated in ACVR2-restored but not ACVR2-mutated HCT116+chr2 colon cancer cells, which leads to tumor cell migration in a PI3K-dependent manner. In vivo, ablation of downstream canonical SMAD signaling is associated with elevated activin, alpha-SMA and pAkt and increases in tissue dysplasia, intestinal stromal disorganization, and animal mortality. While there are direct pro-metastatic effects of activin on tumor cells, activin expression in the TME of stage II or III CRC patients is associated with elevated CD4 and survival benefit (High activin/high CD4: 1.665 hazard ratio, 95% Cl of ratio 1.379 to 2.010). DSP analysis confirmed co-localization of activin with immune cells in the TME which influences cell signaling patterns found in these regions. Conclusion: In colon cancer cells, activin leads to preferential prometastatic PI3K/Akt pathway activation. Within the TME, high levels of activin in the presence of CD4+ T-cells is associated with better outcomes in CRC patients. This work lays the foundation to further study context-dependent activin signaling as an adjunct or target in CRC. Citation Format: Mark B. Wiley, Jessica Bauer, Kunaal Mehrotra, David N. Church, Rachel S. Kerr, David J. Kerr, Paul Grippo, Barbara Jung. Activin’s influence on the tumor microenvironment in colon 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 B013.

Abstract B04: Preclinical activity and safety profile or JANX008, a novel EGFR-targeting tumor-activated T cell engager for treatment of solid tumors

Abstract Introduction: Epidermal growth receptor (EGFR) is the most expressed membrane oncogenic protein in human cancers. KRAS and BRAF mutations are significant drivers of resistance to EGFR-targeted therapies. Unlike other treatments, EGFR-targeting, CD3 bispecific T cell engagers (TCEs) can potentially retain activity against tumors bearing resistance mutations. However, cytokine release syndrome (CRS), on-target off-tumor toxicities, and poor pharmacokinetics (PK) properties present significant clinical limitations for these potent immunomodulators. To overcome these challenges, Janux has developed JANX008, an EGFR- and CD3-targeted tumor-activated T cell engager (TRACTr). JANX008 is a humanized tri-specific protein that contains EGFR- and CD3-binding domains, an albumin binding domain to extend circulating half-life, and two different peptide masks fused to the molecule through tumor protease cleavable linkers. One peptide mask inhibits EGFR engagement on target cells, and the other inhibits CD3 engagement on T cells. Once the cleavage sequences undergo proteolysis by tumor proteases, the EGFR and CD3 masks are released, and the resulting active molecule can bind EGFR and CD3 on target cells. Methods: Peptide masks against EGFR- and CD3-binding domains were identified via phage display. The efficiency of the masks was evaluated using human EGFR and CD3 ELISAs. JANX008-induced cleavage-dependent T cell killing was evaluated in human PBMC/tumor cell co-culture assays. Anti-tumor efficacy of JANX008 was tested in multiple preclinical models, including EGFR antibody-resistant KRAS- and PIK3CA-mutant mouse colon cancer model (HCT116) and a fully human primary colorectal cancer (CRC) tumoroid system. The pharmacokinetic and safety profile of JANX008 was evaluated in non-human primate studies. Results: JANX008 target engagement was cleavage-dependent where masking reduced EGFR and CD3 binding by >300x and >1,000x, respectively. JANX008 exhibited potent cleavage- and dose-dependent activity in multiple preclinical models, including EGFR antibody-resistant tumor and T cell co-culture assays, humanized mouse CRC model, and a human primary CRC tumoroids with an intact tumor microenvironment. JANX008 showed a significantly enhanced safety profile in NHPs compared to non-masked EGFR-TCE, including decreased CRS-associated cytokines and healthy tissue toxicities at high exposures. Clinical chemistry, hematology, and pathology measurements all supported No-Observed-Adverse-Effect-Level ≥ 0.6 mg/kg/dose. Finally, the cleavable albumin-binding domain extended the half-life of JANX008 to ~94h, relative to the ~1.3h half-life of non-masked TCE, supporting its weekly clinical dosing. Conclusions: Preclinical data demonstrate key characteristics of JANX008, including cleavage-dependent activity, half-life extended PK, the potential for superior safety, and manufacturability properties that could mitigate significant limitations of TCEs and support JANX008 clinical development. Citation Format: Thomas R DiRaimondo, Natalija Budimir, Lina Ma, Simon Shenhav, Vanessa Cicchini, Hu Wu, Renee Jocic, Fabrece Roup, Calvin Campbell, Carolina Caffaro, Hans Aerni, Ugur Eskiocak, Wayne Godfrey, Charles Winter, Marc Nasoff, Neil Gibson, David Campbell, shahram Salek-Ardakani. Preclinical activity and safety profile or JANX008, a novel EGFR-targeting tumor-activated T cell engager for treatment of solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B04.