Murine Colon Carcinoma Research Articles

A Chinese Medicine, Huajie Xiaoliu Formula, Enhances the Anti-Colon Cancer Effect of Tegafur, Gimeracil, and Oteracil Potassium Capsules Through Bcl-2/Bax Signaling Pathway

Research Objective This study was aimed to determine the main ingredients of Huajie Xiaoliu Formula (HJF) and investigate the anti-colon cancer effect of its combination with Tegafur, Gimeracil, and Oteracil Potassium Capsules (S-1) in vivo as well as to preliminarily explore its possible mechanism. Materials and Methods The chemical composition of HJF was determined by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The murine colon carcinoma cell (CT 26.WT) subcutaneous xenograft model of colon cancer mice was established to evaluate the in vivo anti-colon cancer effect of HJF. Evaluation of pathological changes in tumor tissue was evaluated by hematoxylin–eosin staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expressions of interleukin-1β (IL-1β), inflammatory cytokines interleukin (IL-6), and tumor necrosis factor-α (TNF-α) in mice serum. Immunohistochemistry was used to evaluate B cell lymphoma-2 (Bcl-2), Bax, and Ki67 protein expression in tumor tissue. Results Twenty chemicals in HJF were identified by UPLC-QTOF-MS. In vivo experiments showed that HJF has obvious inhibitory effects on the subcutaneous xenograft model of colon cancer mice. Results of ELISA showed HJF is able to increase mice body weight, inhibit tumor growth, and reduce the serum levels of IL-1β and IL-6 along with TNF-α. Immunohistochemistry results demonstrated that HJF can significantly inhibit the levels of Bax, Bcl-2, and Ki67 in mice with colon cancer transplant tumors. Furthermore, the combination of HJF and S-1 demonstrated a more significant antitumor effect than any other group in vivo. Conclusions Huajie Xiaoliu Formula is effective in CT 26.WT subcutaneous xenograft model of colon cancer mice by inhibiting tumorigenesis, metastasis, and angiogenesis through the Bcl-2/Bax signaling pathway, resulting in anti-colorectal tumor effects. Huajie Xiaoliu Formula provides a strong theoretical basis for its clinical use and also provides a basis for further research.

Blockade of tumor-derived colony-stimulating factor 1 (CSF1) promotes an immune-permissive tumor microenvironment

The macrophage colony-stimulating factor 1 (CSF1) is a chemokine essential for the survival, proliferation, and differentiation of mononuclear phagocytes from hemopoietic stem cells. In addition to its essential physiological role in normal tissues, the CSF1/CSF1 receptor axis is known to be overexpressed in many tumor types and associated with poor prognosis. High levels of CSF1 within the tumor microenvironment have been shown to recruit and reeducate macrophages to produce factors that promote tumor invasiveness and accelerate metastasis. In this study, we demonstrate, for the first time, that treating established syngeneic murine colon and breast carcinoma tumors with a CSF1R-blocking antibody also promotes the expansion of neoepitope-specific T cells. To assess the role of tumor-derived CSF1 in these model systems, we generated and characterized CSF1 CRISPR-Cas9 knockouts. Eliminating tumor-derived CSF1 results in decreased tumor growth and enhanced immunity against tumor-associated neoepitopes, potentially promoting an immune permissive tumor microenvironment in tumor-bearing mice. The combination of neoepitope vaccine with anti-PDL1 in the MC38 CSF1−/− tumor model significantly decreased tumor growth in vivo. Moreover, anti-CSF1R therapy combined with the adeno-TWIST1 vaccine resulted in tumor control, decreased metastasis, and a synergistic increase in CD8 T cell infiltration in 4T1 mammary tumors. Analysis of the tumor microenvironment demonstrated greater CD8 T cell infiltration and a reduction in tumor-associated macrophages following CSF1R inhibition in both tumor models. Our findings thus add to the therapeutic potential of CSF1 targeting agents by employing combinations with vaccines to modulate anti-neoepitope responses in the tumor microenvironment.

Silver(I) complexes containing N-heterocyclic carbene azole drugs: Synthesis, characterization, cytotoxic activity, and their BSA interactions

Cancer is one of the main public health problems globally, there is a public demand for better drugs. Rational strategies or approaches are used to improve the success of drug discovery. Our strategy was to the repurposing of well-known antifungal agents as potential anticancer drugs, such as Clotrimazole (CTZ) and Ketoconazole (KTZ). We prepared the respective iodide imidazolium salt L1: (CTZ-Me)I and L2: (KTZ-Me)I to be the intermediates toward the synthesis of its respective NHC ligand and achieve the respective silver(I)-monoNHC and silver(I)-bisNHC derivatives: [Ag(L1)I] (1), [AgI(L2)] (2) [Ag(L1)2]I. (3), [Ag(L2)2]I. (4), as well as their corresponding coordination compounds [Ag(CTZ)2]NO3 (5) and [Ag(KTZ)2]NO3 (6) where these ligands (CTZ and KTZ) coordinate to silver through the N-imidazole atom. These compounds (L1, L2 and complexes 1–6) exhibited significant activity against the tested cancer cell lines (B16-F1, murine melanoma strains and CT26WT, murine colon carcinoma). The silver(I) complexes were more active than the free ligands, complexes 2 and 4 being the most selective in B16-F1 cancer cell line. Two possibles biological targets such as DNA and albumin were examined for the observed anticancer activity. Results show that DNA is not the main target, however, the interactions with albumin suggest it can transport/delivery the metal complexes.

Stimulatory effect of fluoxetine and desipramine, but not mirtazapine on C26 colon carcinoma hepatic metastases formation: association with cytokines.

Due to the high prevalence of depression among cancer patients, antidepressant medications are frequently administered as adjuvant treatment. However, the safety of such medications in the development of metastasis is unclear. In this study, we investigated the effects of fluoxetine, desipramine, and mirtazapine on the liver metastasis of murine C26 colon carcinoma (cc). Balb/c male mice were administered these antidepressants intraperitoneally (i.p.) for 14 days following intrasplenic injections of C26 colon carcinoma cells. Desipramine and fluoxetine, but not mirtazapine, significantly increased the number of tumor foci and total volume of the tumor in liver tissue. This effect was associated with a decrease in the ability of splenocytes to produce interleukin (IL)-1β and interferon (IFN)-γ and an increase in their ability to produce interleukin (IL)-10. Similar changes were observed in plasma IL-1β, IFN-γ, and IL-10 levels. The current study demonstrates that the stimulatory effect of desipramine and fluoxetine, but not mirtazapine, on experimental colon cancer liver metastasis is associated with a suppression of immune defenses against the tumor.

GSK-3 inhibitor elraglusib (9-ING-41) to enhance tumor-infiltrating immune cell activation in tumor biopsies and synergize with anti-PD-L1 in a murine model of colorectal cancer.

e15138 Background: Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase with key roles in myriad biological processes such as tumor progression, and inhibition of GSK-3 using a novel small-molecule elraglusib has shown promising preclinical antitumor activity in multiple tumor types. Methods: We characterize the effects of elraglusib in vitro on tumor and immune cells including cell killing and cytokine profiling , in vivo in combination with checkpoint inhibitors in a syngeneic murine colon carcinoma BALB/c model using MSS cell line CT-26, and in human tumor biopsies and plasma samples from patients with refractory solid tumors of multiple tissue origins enrolled in a Phase 1 clinical trial investigating elraglusib ( NCT03678883 ).We used human plasma samples from patients treated with elraglusib, paired pre- and post-treatment tumor biopsies and performed digital spatial proteomics. Results: Elraglusib promoted immune cell-mediated tumor cell killing, enhanced tumor cell pyroptosis, decreased tumor cell NF-κB-regulated survival protein expression, and increased immune cell effector molecule secretion. Synergy was observed between elraglusib and anti-PD-L1 in an immunocompetent murine model of colorectal cancer. Murine responders had more tumor-infiltrating T-cells, fewer tumor-infiltrating Tregs, lower tumorigenic circulating cytokine concentrations, and higher immunostimulatory circulating cytokine concentrations. Murine responders had lower serum concentrations of BAFF, CCL7, CCL12, VEGF, VEGFR2, and CCL21, and higher serum concentrations of CCL4, TWEAK, GM-CSF, CCL22, and IL-12p70 as compared to non-responders. We utilized human plasma samples from patients treated with elraglusib and correlated cytokine profiles with survival. Elevated baseline plasma levels of proteins such as IL-1 β and reduced levels of proteins such as VEGF correlated with improved PFS and OS. PFS was also found to be positively correlated with elevated plasma levels of immunostimulatory analytes such as Granzyme B, IFN-γ, and IL-2 at 24 hours post-treatment with elraglusib. Several of these secreted proteins correlated with results from the in vivo study where expression of proteins such as IL-1 β, CCL22, CCL4, and TWEAK was positively correlated with improved response to therapy while expression of proteins such as BAFF and VEGF negatively correlated with response to therapy. Using paired tumor biopsies, we found that CD45+ tumor-infiltrating immune cells had lower expression of inhibitory immune checkpoints and higher expression of T-cell activation markers in post-elraglusib patient biopsies. Conclusions: These results introduce several immunomodulatory mechanisms of GSK-3 inhibition using elraglusib, providing a rationale for the clinical evaluation of elraglusib in combination with immunotherapy.

Oncolytic therapy with recombinant vaccinia viruses targeting the interleukin-15 pathway elicits a synergistic response

We developed recombinant variants of oncolytic vaccinia virusLIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other invitro and invivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. Invitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. Invivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic Tcells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice.The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer.

Abstract 4899: Inhibition of murine tumor growth by decitabine is correlated with enhanced expression of the mouse mammary tumor virus

Abstract The purpose of this study is to clarify the mechanisms of the antineoplastic effect of decitabine (DAC), a DNA methyltransferase inhibitor. We previously reported that DAC inhibited the proliferation of the 4T1 mammary cancer cell line in vitro while inhibiting MMTV env and pol expression in the same cells. Here we present findings in vivo using two murine tumor models. We inoculated 4T1 cells into BALB/c mice from which the cell line was derived. In addition, we inoculated the murine colon carcinoma cell line, MC38, into C57BL/6 mice, the mouse strain of its origin. We treated the mice with DAC when tumors became palpable. DAC inhibited tumor growth in both tumor models and inhibited metastasis of 4T1 cells into the lungs. Quantitative PCR detected elevated expression of MMTV genes in the tumor tissues of DAC-treated mice in both models. The levels of MMTV env RNA in tumor tissues were negatively correlated with tumor mass in both models, so were the levels of the MMTV Env protein in 4T1 tumors. DAC also significantly mitigated tumor-induced splenomegaly. Hepatotoxicity of DAC was observed in C57BL/6 mice. Our data suggest the possibility that decitabine inhibits tumor growth in vivo and in vitro via different mechanisms. The findings also support the current understanding of decitabine upregulating endogenous retroviruses (ERVs) which subsequently activate an interferon response. The negative correlation between tumor mass and MMTV Env protein expression suggests a possible role of ERV proteins serving as tumor-associated antigens and decitabine enhancing adaptive immunity against cancer by stimulating ERV expression on tumor cells. Further studies of the interaction between decitabine and MMTV using immunodeficient mice may help us understand the possible involvement of adaptive immunity in the pharmacodynamics of decitabine as well as the role of ERVs in tumor development. Citation Format: Jieyu Zhang, Savannah Higgins, Riley Smith, Tania Reginald, Andrew Qi, Jiayi Li, Anna King, Yingguang Liu. Inhibition of murine tumor growth by decitabine is correlated with enhanced expression of the mouse mammary tumor virus. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4899.

Combined therapy with methotrexate nanoconjugate and dendritic cells with downregulated IL-10R expression modulates the tumor microenvironment and enhances the systemic anti-tumor immune response in MC38 murine colon carcinoma.

Understanding the negative impact of the tumor microenvironment on the creation of an effective immune response has contributed to the development of new therapeutic anti-cancer strategies. One such solution is combined therapy consisting of chemotherapeutic administration followed by dendritic cell (DC)-based vaccines. The use of cytostatic leads to the elimination of cancer cells, but can also modulate the tumor milieu. Moreover, great efforts are being made to increase the therapeutic outcome of immunotherapy, e.g. by enhancing the ability of DCs to generate an efficient immune response, even in the presence of immunosuppressive cytokines such as IL-10. The study aimed to determine the effectiveness of combined therapy with chemotherapeutic with immunomodulatory potential - HES-MTX nanoconjugate (composed of methotrexate (MTX) and hydroxyethyl starch (HES)) and DCs with downregulated expression of IL-10 receptor stimulated with tumor antigens (DC/shIL-10R/TAg) applied in MC38 murine colon carcinoma model. With the use of lentiviral vectors the DCs with decreased expression of IL-10R were obtained and characterized. During in vivo studies MC38-tumor bearing mice received MTX or HES-MTX nanoconjugate as a sole treatment or combined with DC-based immunotherapy containing unmodified DCs or DCs transduced with shRNA against IL-10R (or control shRNA sequence). Tumor volume was monitored during the experiment. One week after the last injection of DC-based vaccines, tumor nodules and spleens were dissected for ex vivo analysis. The changes in the local and systemic anti-tumor immune response were estimated with the use of flow cytometry and ELISA methods. In vitro studies showed that the downregulation of IL-10R expression in DCs enhances their ability to activate the specific anti-tumor immune response. The use of HES-MTX nanoconjugate and DC/shIL-10R/TAg in the therapy of MC38-tumor bearing mice resulted in the greatest tumor growth inhibition. At the local anti-tumor immune response level a decrease in the infiltration of cells with suppressor activity and an increase in the influx of effector cells into MC38 tumor tissue was observed. These changes were crucial to enhance the effective specific immune response at the systemic level, which was revealed in the greatest cytotoxic activity of spleen cells against MC38 cells.

Macrophage STING signaling promotes NK cell to suppress colorectal cancer liver metastasis via 4-1BBL/4-1BB co-stimulation

Background and aimsMacrophage innate immune response plays an important role in tumorigenesis. However, the role and mechanism of macrophage STING signaling in modulating tumor microenvironment to suppress tumor growth at...

Effects of Iron on Efficacy of Photodynamic Therapy Using Photolon in a Mouse Model of CT26 Colon Cancer.

Photodynamic therapy (PDT) -a minimally invasive anti-cancer therapy-is undergoing experimental studies to increase its anti-cancer effects. This study investigated the influence of iron on the anti-cancer effects of PDT. PDT was performed in a cancer-bearing mouse model, which was created by using a murine colon carcinoma (CT26) cell line after administration of Photolon and iron. Tumor volume and the results of TdT-mediated dUTP-biotin nick end labeling (TUNEL), 8-OHdG, and TBARS assays were used to measure anti-cancer effect. On day 14, tumor volume had increased by 49% in the PDT group and decreased by 72% in the iron+PDT group. The percentage of TUNEL-positive cells in tumor tissues was 45% in the PDT group and 69% in the iron+PDT group, suggesting that the proportion of TUNEL-positive cells had increased in the iron+PDT group. The 8-OHdG content in tumor tissues was 33% higher in the iron+PDT group than in the PDT group. The TBARS content in tumor tissues was 46% higher in the iron+PDT group than in the PDT group. Iron enhances the anti-cancer effect of PDT using Photolon, most likely by increasing oxidative damage.

Lactate oxidase/catalase-displaying nanoparticles efficiently consume lactate in the tumor microenvironment to effectively suppress tumor growth

The aggressive proliferation of tumor cells often requires increased glucose uptake and excessive anaerobic glycolysis, leading to the massive production and secretion of lactate to form a unique tumor microenvironment (TME). Therefore, regulating appropriate lactate levels in the TME would be a promising approach to control tumor cell proliferation and immune suppression. To effectively consume lactate in the TME, lactate oxidase (LOX) and catalase (CAT) were displayed onto Aquifex aeolicus lumazine synthase protein nanoparticles (AaLS) to form either AaLS/LOX or AaLS/LOX/CAT. These complexes successfully consumed lactate produced by CT26 murine colon carcinoma cells under both normoxic and hypoxic conditions. Specifically, AaLS/LOX generated a large amount of H2O2 with complete lactate consumption to induce drastic necrotic cell death regardless of culture condition. However, AaLS/LOX/CAT generated residual H2O2, leading to necrotic cell death only under hypoxic condition similar to the TME. While the local administration of AaLS/LOX to the tumor site resulted in mice death, that of AaLS/LOX/CAT significantly suppressed tumor growth without any severe side effects. AaLS/LOX/CAT effectively consumed lactate to produce adequate amounts of H2O2 which sufficiently suppress tumor growth and adequately modulate the TME, transforming environments that are favorable to tumor suppressive neutrophils but adverse to tumor-supportive tumor-associated macrophages. Collectively, these findings showed that the modular functionalization of protein nanoparticles with multiple metabolic enzymes may offer the opportunity to develop new enzyme complex-based therapeutic tools that can modulate the TME by controlling cancer metabolism.Graphical

Bioprocessed Black Rice Bran Potentiates the Growth Inhibitory Activity of an Immune Checkpoint Inhibitor against Murine Colon Carcinoma

Bioprocessed Black Rice Bran Potentiates the Growth Inhibitory Activity of an Immune Checkpoint Inhibitor against Murine Colon Carcinoma

IGF1R and HIF-1α Gene Silencing Inhibits Cancer Cell Growth

Background: According to several studies, hypoxia-inducible factor-1α (HIF-1α) and insulin-like growth factor 1 receptor (IGF1R) promote cancer progression and drug resistance. The overexpression of IGF1R and HIF-1 α is observed in various cancers, including breast and colon cancers. Thus, we tried to inhibit the progression of tumor cells by blocking IGF1R and HIF-1α. Methods: This study used Lipofectamine to transfect small interfering RNAs (siRNAs) into tumor cells. We treated murine mammary carcinoma (4T1) and murine colon carcinoma (CT26) cells with anti-IGF1R and anti-HIF-1 siRNAs. The real-time polymerase chain reaction (PCR) assay studied the impact of siRNA transfection on the mRNA levels of affected factors. Moreover, the viability of cancer cells was investigated by the MTT assay. Results: The investigations demonstrated that the mRNA levels of IGF1R and HIF-1α strongly reduced in tumor cell lines following siRNA transfection. Moreover, silencing IGF1R and HIF-1 additively downregulated cell viability in cancer cell lines. Conclusion: These findings imply that cancer combination therapy based on IGF1R and HIF-1α targeting may be a novel promising approach for the immunotherapy of breast and colorectal cancers; however, more study is required.

The Novel IGF-1R Inhibitor PB-020 Acts Synergistically with Anti-PD-1 and Mebendazole against Colorectal Cancer

CRC is one of the leading causes of cancer mortality worldwide. Chemotherapy is widely used for the treatment of CRC, but its efficacy remains unsatisfactory, mainly due to drug resistance. Therefore, it is urgent to develop new strategies to overcome drug resistance. Combination therapy that aims to achieve additive or synergistic therapeutic effects is an effective approach to tackle the development of drug resistance. Given its established roles in tumor development, progression and metastasis, IGF-1R is a promising drug target for combination therapy against CRC. In this study, we revealed that the novel IGF-1R inhibitor PB-020 can act synergistically with mebendazole (MBZ) to reduce the viability of CRC cells and block xenograft CRC progression. Moreover, the PB-020/anti-PD-1 combination synergistically blocked CRC propagation in the MC38 murine colon carcinoma model. Both combination therapies potently suppressed the PI3K/AKT signaling pathway genes in CRC that may be associated with the development of drug resistance. Our findings establish a preclinical proof-of-concept for combating CRC using combined multi-target treatment with PB-020 and clinical anticancer drugs, which may provide useful clues for clinical trials to evaluate the efficacy and safety of these drug combinations in CRC patients.

WIN Symposium 2022 - Abstract Poster Presentations.

WIN Symposium 2022 - Abstract Poster Presentations.

Evaluation of a Sumoylation Inhibitor as an Intrinsic and Extrinsic Radiosensitizer

<h3>Purpose/Objective(s)</h3> Radiation therapy (RT) reduces tumor burden through both intrinsic initiation of tumor cell death and enhancement of anti-tumor immunity. However, radiation treatments are not universally efficacious due to variation in radiosensitivity and anti-tumor immunity. Low proliferative rate, hypoxia, and T cell effector dysfunction can significantly degrade response to RT. Here we evaluate a rational combination therapy pairing radiosensitization and immune activation in conjunction with standard of care RT. Recent studies demonstrated the sumoylation inhibitor, TAK-981, has intrinsic capacity to reduce the proliferation of tumor cells <i>in vitro</i> as a monotherapy and decrease tumor volumes in syngeneic murine colon carcinoma models (CT26 and MC38) in combination with PD-1 or CTLA-4 checkpoint blockade. In these models a dendritic cell dependent type I interferon pathway was implicated to mediate the anti-tumor immune response and enhanced in vivo tumor control in TAK-981 and checkpoint blockade combination therapy, similar to previously elucidated mechanisms of immune activation following hypofractionated radiotherapy. Additionally, multiple intrinsic and extrinsic protein mediators (Myc, IRF3, p21) of tumor cell response to radiation are regulated by the post-translational modification of sumoylation. Consequently, we evaluated whether combining RT with TAK-981 resulted in enhanced tumor radiosensitivity in vitro and improved local tumor control in vivo, consistent with enhanced anti-tumor immune response in murine tumor models. <h3>Materials/Methods</h3> We utilized a longitudinal real time microscopic proliferation assay to evaluate RT response. Titrations of TAK-981 were added to established in vitro tumor cell cultures 4 hours prior to increasing doses of single fraction photon RT. Impacts of RT, TAK-981 or the combination treatment were assessed based upon cell proliferation for 72 hours. We assessed efficacy of this novel combination in vivo using the MC38 colorectal carcinoma model. C57BL6 mice with established tumors (> 50mm2) received TAK981 (15mg/kg) alone three times weekly or in combination with single fraction 8 Gy RT and compared to RT alone to evaluate local tumor control and impacts on overall survival. <h3>Results</h3> Our data demonstrates a synergistic and dose dependent reduction in MC38 colon carcinoma in vitro proliferation in response to combination RT and TAK-981. In vivo MC38 tumor models demonstrate no effect of TAK-981 alone compared to vehicle treated mice while co-administration with a single 8 Gy fraction of radiotherapy synergistically enhanced tumor control and overall survival compared to RT alone. <h3>Conclusion</h3> Our preliminary data support potential dual synergistic roles for the sumoylation inhibitor TAK-981 with radiotherapy, functioning as both an intrinsic radiosensitizer and immunomodulator.

Immune targeting of three independent suppressive pathways (TIGIT, PD-L1, TGFβ) provides significant antitumor efficacy in immune checkpoint resistant models

ABSTRACT Immune checkpoint blockade (ICB) therapy, while groundbreaking, must be improved to promote enhanced durable responses and to prevent the development of treatment-refractory disease. Cancer therapies that engage, enable, and expand the antitumor immune response will likely require rationally designed combination strategies. Targeting multiple immunosuppressive pathways simultaneously may provide additional therapeutic benefit over singular targeting. We therefore hypothesized that the use of two molecules which inhibit three independent, but overlapping, pathways (TIGIT:CD155, PD-1/PD-L1, and TGFβ) would provide significant antitumor efficacy in the syngeneic ICB resistant colorectal tumor model MC38 expressing human carcinoembryonic antigen (CEA) in CEA transgenic mice. This novel combination treatment strategy has significant antitumor activity and survival benefit in two models of murine carcinomas, MC38-CEA (CRC) and TC1 (HPV+ lung carcinoma). MC38-CEA mice that responded to αTIGIT and bintrafusp alfa combination therapy generated memory responses and were protected from rechallenge. These effects were dependent on CD4+ and CD8+ T cells, as well as increased immune infiltration into the TME. This combination induced production of tumor-specific CD8+ T cells, and an increase in activation and cytotoxicity resulting in an overall activated immune landscape in the tumor. Data presented herein demonstrate the αTIGIT and bintrafusp alfa combination has efficacy across multiple tumor models, including the checkpoint-resistant model of murine colon carcinoma, MC38-CEA and the HPV+ model TC-1.

The Antitumor and Toxicity Effects of Ruthenium(II) Complexes on Heterotopic Murine Colon Carcinoma Model

Abstract The aim of the present study was to examine the antitumor and toxicity effects of ruthenium(II) complexes, [Ru(Cltpy)(en)Cl][Cl] (Ru-1) and [Ru(Cl-tpy)(dach)Cl][Cl] (Ru-2) on heterotopic murine colon carcinoma model. For tumor induction, 1×106 CT26 cells suspended in 100 μl of DMEM were injected subcutaneously into flank of male BALB/c mice. Treatment groups were as follows: Ru-1, Ru-2, oxaliplatin and control (saline). The intraperitoneal administration of the tested complexes began on 6th day after CT26 cells inoculation. Each complex was administered at dose of 5 mg/kg, twice weekly, four doses in total. To assess toxicity, serum values of urea, creatinine, AST and ALT were determined and histopathological analysis of organs and tumor were performed. In order to assess the effects of Ru(II) complexes on markers of oxidative stress and antioxidant defense system, we determined the TBARS, GSH, SOD and CAT in the homogenate of tumor, heart, liver, lungs and kidney tissues. The findings indicate that Ru-1 and Ru-2 exerts equal or better antitumor activity in comparison with oxaliplatin, but with pronounced toxic effects such as reduced survival rate, cardiotoxicity, nephrotoxicity and hepatotoxicity. The increased index of lipid peroxidation in the tissues of the kidneys and heart, but decreased in tumor tissue, after Ru(II) complexes administration, indicates the importance of the induction of oxidative stress as a possible mechanism of nephrotoxicity and cardiotoxicity, but not the mechanism by which they realize antitumor activity. Additional studies are needed to elucidate the mechanism of antitumor activity and toxicity of the Ru(II) complexes.

Chidamide plus Tyrosine Kinase Inhibitor Remodel the Tumor Immune Microenvironment and Reduce Tumor Progression When Combined with Immune Checkpoint Inhibitor in Naïve and Anti-PD-1 Resistant CT26-Bearing Mice.

Combined inhibition of vascular endothelial growth factor receptor (VEGFR) and the programmed cell death protein 1 (PD-1) pathways has shown efficacy in multiple cancers; however, the clinical outcomes show limited benefits and the unmet clinical needs still remain and require improvement in efficacy. Using murine colon carcinoma (CT26) allograft models, we examined the efficacy and elucidated novel tumor microenvironment (TME) remodeling mechanisms underlying the combination of chidamide (a benzamide-based class l histone deacetylase inhibitor; brand name in Taiwan, Kepida®) with VEGF receptor tyrosine kinase inhibitor (TKIs; cabozantinib/regorafenib, etc.) and immune checkpoint inhibitors (ICIs; anti-PD-1/anti-PD-L1/anti-CTLA-4 antibodies). The TME was assessed using flow cytometry and RNA-sequencing to determine the novel mechanisms and their correlation with therapeutic effects in mice with significant treatment response. Compared with ICI alone or cabozantinib/regorafenib + ICI, combination of chidamide + cabozantinib/regorafenib + ICI increased the tumor response and survival benefits. In particular, treatment of CT26-bearing mice with chidamide + regorafenib + anti-PD-1 antibody showed a better objective response rate (ORR) and overall survival (OS). Similar results were observed in anti-PD-1 treatment-resistant mice. After treatment with this optimal combination, in the TME, RNA-sequencing revealed that downregulated mRNAs were correlated with leukocyte migration, cell chemotaxis, and macrophage gene sets, and flow cytometry analysis showed that the cell numbers of myeloid-derived polymorphonuclear suppressor cells and tumor-associated macrophages were decreased. Accordingly, chidamide + regorafenib + anti-PD-1 antibody combination therapy could trigger a novel TME remodeling mechanism by attenuating immunosuppressive cells, and restoring T-cell activation to enhance ORR and OS. Our studies also showed that the addition of Chidamide to the regorafenib + anti-PD-1 Ab combination could induce a durable tumor-specific response by attenuating immune suppression in the TME. In addition, this result suggests that TME remodeling, mediated by epigenetic immunomodulator combined with TKI and ICI, would be more advantageous for achieving a high objective response rate, when compared to TKI plus ICI or ICI alone, and maintaining long-lasting antitumor activity.

CGX, a standardized herbal syrup, inhibits colon-liver metastasis by regulating the hepatic microenvironments in a splenic injection mouse model.

Background: Colon-liver metastasis is observed in approximately 50% of patients with colorectal cancer and is a critical risk factor for a low survival rate. Several clinical studies have reported that colon-liver metastasis is accelerated by pathological hepatic microenvironments such as hepatic steatosis or fibrosis. Chunggan syrup (CGX), a standardized 13-herbal mixture, has been prescribed to patients with chronic liver diseases, including fatty liver, inflammation and fibrotic change, based on preclinical and clinical evidence. Aim of the study: In the present study, we investigated anti-liver metastatic the effects of CGX in a murine colon carcinoma (MC38)-splenic injection mouse model. Materials and methods: C57BL/6N mice were administered with CGX (100, 200 or 400 mg/kg) for 14 days before or after MC38-splenic injection under normal and high-fat diet (HFD) fed conditions. Also, above experiment was repeated without MC38-splenic injection to explore underlying mechanism. Results: The number of tumor nodules and liver weight with tumors were sup-pressed by preadministration of CGX in both normal and HFD fed mice. Regarding its mechanisms, we found that CGX administration significantly activated epithelial-cadherin (E-cadherin), but decreased vascular endothelial-cadherin (VE-cadherin) in hepatic tissues under MC38-free conditions. In addition, CGX administration significantly reduced hepatic steatosis, via modulation of lipolytic and lipogenic molecules, including activated adenosine monophosphate activated protein kinase (AMPK) and peroxisome proliferator activated receptor-alpha (PPARα). Conclusion: The present data indicate that CGX exerts an anti-colon-liver metastatic property via modulation of hepatic lipid related microenvironments.