Colorectal Cancer-bearing Mice Research Articles

Protective effect of electroacupuncture at ST36 against damage of intestinal mucosa, oxidative stress and apoptosis induced by 5-FU chemotherapy in mice with colon cancer.

To observe the effect of electroacupuncture (EA) at "Zusanli"(ST36) on intestinal mucosal damage, intestinal mucosal oxidative stress injury and apoptosis induced by 5-fluorouraeil (5-FU) chemotherapy in colorectal cancer-bearing mice. Thirty male BALB/c mice were randomly divided into normal control, colorectal cancer (CT26), 5-FU, non-acupoint and ST36 groups, with 6 mice in each group. Except for those of the normal control group, mice of the remaining 4 groups received subcutaneous implantation of colorectal CT26 cell suspension (0.1 mL) in the right armpit for establishing colorectal cancer model. Rats of the 5-FU group, non-acupoint group and ST36 group were given with 5 mg/mL 5-FU solution once every 3 days for a total of 21 days. For mice of the non-acupoint group and ST36 group, EA (2 Hz, 1-2 mA) was applied to bilateral ST36 or non-acupoints (the bilateral sunken spots about 3 mm to the midpoint between the tail root and the anus) for 5 min after each intraperitoneal infusion of 5-FU, once every 3 days, for a total of 21 days. After the intervention, the diarrhea index was assessed. The length of colon (from the endpoint of cecum to the anal orifice) was measured. Histopathological changes of colonic mucosa were observed by H.E. staining, and the length of colonic villi was measured. The content of malondialdehyde (MDA), and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of colonic tissue were detected by thibabituric acid, xanthine oxidase and colorimetric method, respectively. The rate of cell apoptosis in the colonic tissue was measured by TUNEL assay. The positive expressions of Bax and Bcl-2 in colonic tissue were determined by immunohistochemistry. The CT26 model group didn't show any significant changes in the diarrhea index, colon length, colon villus length, MDA content, SOD and GSH-Px activities, colonic cell apoptosis rate, and Bax and Bcl-2 expression levels when compared with the normal group. Compared with the CT26 group, the 5-FU group had a remarkable increase in the diarrhea index, MDA content, colonic cell apoptosis rate and Bax expression level (P<0.01, P<0.05), and a marked decrease in the colon length, colon villus length, SOD and GSH-Px activities and Bcl-2 expression level (P<0.01), suggesting the side effects of administration of 5-FU. Compared with the 5-FU group, the diarrhea index, MDA content, colonic cell apoptosis rate and Bax expression level were markedly decreased (P<0.05, P<0.01) and those of the colon length, colon villus length, SOD and GSH-Px activities and Bcl-2 expression level were obviously increased (P<0.01) in the ST36 group. Compared with the 5-FU group, the non-acupoint group also had an increase in the colon villus length, SOD and GSH-Px activities (P<0.01, P<0.05) and a decrease in the cell apoptosis rate (P<0.01). EA at ST36 has a positive effect in reducing intestinal mucosal damage induced by 5-FU chemotherapy in cancer-bearing mice, which may be related to its function in relieving oxidative stress injury and inhibiting apoptosis of colonic tissue.

Electroacupuncture at “Zusanli(ST36)” alleviates 5-fluorouracil-induced renal injury in colorectal cancer-bearing mice by exerting effects on oxidative stress, inflammatory responses, and cell apoptosis: 电针“足三里”减轻大肠癌荷瘤小鼠5-FU化疗后肾损伤及其氧化应激、炎症反应和细胞凋亡的研究

ObjectiveTo observe the effect of electroacupuncture at “Zusanli (ST36)” on 5-fluorouracil (5-FU)-induced renal injury in colorectal cancer-bearing mice, and to further investigate its effects on oxidative stress, inflammatory responses, and cell apoptosis in mouse renal tissue. MethodsThirty-five male BALB/c mice were randomly divided into five groups of seven mice each, namely the control, CT26, 5-FU, sham point (SP), and ST36 (which received EA at the “ST36”) groups. With the exception of the control group, each group was subjected to establishment of a subcutaneous implantation tumor model using the murine CT26 colorectal cancer cell line. Once the models were successfully established, the 5-FU, SP, and ST36 groups received 5-FU injection solution intraperitoneally at a dose of 5 mg/mL once every three days over a 21-day period. Mice in the SP and ST36 groups additionally received an EA intervention after each intraperitoneal 5-FU injection. EA were performed on mice of the SP group at bilateral sham acupoints and on mice of the ST36 group at the bilateral “ST36” using the continuous wave mode at a frequency of 2 Hz for a duration of 5 min, intervention was administered once every two days for a duration of 21 days. Samples were collected from the mice at the end of the experiment. The pathological morphology of the renal tissue was observed using hematoxylin and eosin (HE) staining; the contents of creatine (Cre), blood urea nitrogen (BUN) and malondialdehyde (MDA), and superoxide dismutase (SOD) activity were measured using biochemical assays; the expression and nuclear translocation of nuclear factor kappa B p65 subunit (NF-κB p65) were measured by immunofluorescence; the expression levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in serum were measured by ELISA; cell apoptosis in renal tissue was detected using the TUNEL assay; and the expression levels of the anti-B-cell lymphoma/leukemia 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, cleaved caspase-9, and cytochrome C (cyt c) in renal tissue were measured by Western blotting. ResultsCompared with the control group, mice of the CT26 group showed a significant increase in serum Cre content (P<0.01), but the difference in BUN content was not statistically significant (P>0.05). HE staining of renal tissue revealed clear structures with normal glomerular and renal tubular morphology. SOD activity was decreased (P<0.01); MDA content was increased, but the increase was not statistically significant (P>0.05). Differences in NF-κB p65 protein expression in the cytoplasm of renal tissue and serum levels of TNF-α, IL-6, and IL-1β were not statistically significant (all P>0.05). Results of immunofluorescent TUNEL staining indicated an absence of significant cell apoptosis. In the renal tissue, Bcl-2 protein expression was slightly increased (P<0.05), and the expression levels of Bax (P<0.01), cleaved caspase-3 (P>0.05), cleaved caspase-9 (P<0.01), and cyt c (P>0.05) were increased. Compared with the CT26 group, mice of the 5-FU group exhibited an increase in Cre (P<0.01) and BUN (P<0.05) content. HE staining of renal tissue revealed the presence of glomerular atrophy and dilated Bowman's capsular spaces. SOD activity was significantly decreased (P<0.01), while the increase of MDA content was not significant (P>0.05). The expression of NF-κB p65 in the nucleus and serum TNF-α, IL-6, and IL-1β levels showed significant increases (P<0.05). The cell apoptosis level was significantly increased. The protein expression of Bcl-2 (P<0.05), Bax (P<0.01), cleaved caspase-9 (P<0.01), and cyt c (P<0.05) was significantly increased. Compared with the 5-FU group, the ST36 group showed decreased serum Cre and BUN levels (both P<0.01). HE staining of renal tissue showed less renal tissue injury and less dilation of renal capsular cavities. SOD activity was significantly higher (P<0.01), while MDA content was lower (P<0.05). Nuclear expression of NF-κB p65 and serum TNF-α (P<0.05), IL-6 (P<0.05), and IL-1β (P<0.01) levels were lower. The cell apoptosis level was decreased relative to the 5-FU group. Bcl-2 protein expression was significantly increased (P<0.01), while the protein expression levels of Bax (P<0.01), cleaved caspase-3 (P<0.01), cleaved caspase-9 (P<0.01), and cyt c (P<0.05) also were reduced. ConclusionEA at “ST36” attenuated 5-FU-induced renal injury in colorectal cancer-bearing mice. The mechanism may be related to the regulation of renal oxidative stress, alleviation of inflammatory responses, and inhibition of cell apoptosis.

The Sesquiterpene Lactone-Rich Fraction of Inula helenium L. Enhances the Antitumor Effect of Anti-PD-1 Antibody in Colorectal Cancer: Integrative Phytochemical, Transcriptomic, and Experimental Analyses.

Treatment strategies combining immune checkpoint inhibitors with sesquiterpene lactones have attracted much attention as a promising approach for cancer treatment. We systemically analyzed gene expression profiles of cells in response to two major sesquiterpene lactones, alantolactone and isoalantolactone, and determined whether the sesquiterpene lactone-rich fraction of Inula helenium L. (SFIH) enhances the antitumor effect of anti-PD-1 antibody in MC38 colorectal cancer-bearing mice. Gene expression and pathway analysis using RNA sequencing data were used to identify the SFIH-driven combined activity with anti-PD-1 antibody. The results showed that SFIH significantly enhanced the antitumor effect of anti-PD-1 antibody by reducing tumor growth and increasing the survival time of mice. Specifically, SFIH exhibited antitumor activity when combined with anti-PD-1 antibody, and the effects were further enhanced compared with monotherapy. An analysis of immune cells indicated that combination treatment with SFIH and anti-PD-1 antibody significantly increased the proportion of CD8+ T cells. Moreover, combination treatment enhanced antitumor immunity by decreasing the population of myeloid-derived suppressor cells and increasing the number of M1-like macrophages. Pathway enrichment analysis revealed that combination therapy activated immune-related pathways to a greater extent than monotherapy. In conclusion, our integrative analysis demonstrates that SFIH enhances the response of murine tumors to anti-PD-1 antibody. These findings provide insight into developing integrative therapeutics and molecular data for the use of natural products as an adjunct treatment for colorectal cancer.

Self-assembled peptide-paclitaxel nanoparticles for enhancing therapeutic efficacy in colorectal cancer.

Chemotherapy is one of the main treatments for colorectal cancer, but systemic toxicity severely limits its clinical use. Packaging hydrophobic chemotherapeutic drugs in targeted nanoparticles greatly improve their efficacy and reduce side effects. We previously identified a novel colorectal cancer specific binding peptide P-LPK (LPKTVSSDMSLN) from phage display peptide library. Here we designed a self-assembled paclitaxel (PTX)-loaded nanoparticle (LPK-PTX NPs). LPK-PTX NPs displayed a superior intracellular internalization and improved tumor cytotoxicity in vitro. Cy5.5-labeled LPK-PTX NPs showed much higher tumor accumulation in colorectal cancer-bearing mice. Furthermore, LPK-PTX NPs exhibit enhanced antitumor activity and decreased systemic toxicity in colorectal cancer patient-derived xenografts (PDX) model. The excellent in vitro and in vivo antitumor efficacy proves the improved targeting drug delivery, suggesting that peptide P-LPK has potential to provide a novel approach for enhanced drug delivery with negligible systemic toxicity.

Anti-tumor effects of PEGylated-nanoliposomes containing ginger extract in colorectal cancer-bearing mice.

Objective(s):This study aimed to develop a nanoliposomal formulation containing ginger ethanolic extract with a higher therapeutic effect for cancer treatment. Materials and Methods:The present study aimed to prepare PEGylated nanoliposomal ginger through the thin film hydration method plus extrusion. Physicochemical characteristics were evaluated, and the toxicity of the prepared liposomes was assessed using the MTT assay. In addition, tumor size was monitored in colorectal cancer-bearing mice. Also, the anticancer effects of liposomal ginger were evaluated by gene expression assay of Bax and Bcl-2 and cytokines including TNF-α, TGF-β, and IFN-γ by Real-time PCR. Also, cytotoxic T lymphocytes (CTLs) and regulatory T lymphocytes (Treg cells) were counted in spleen and tumor tissue by flow cytometry assay.Results:The nanoliposomes’ particle size and polydispersity index (PDI) were 94.95 nm and 0.246 nm, respectively. High encapsulation capacity (80 %) confirmed the technique’s efficiency, and the release rate of the extract was 85% at pH 6.5. In addition, this study showed that liposomal ginger at 100 mg/kg/day enhanced the expression of Bax (P<0.05) and IFN-γ (P<0.01) compared with ginger extract in the mouse model. Also, the number of tumor-infiltrating lymphocytes (TILs) and CTLs cell count in tumor tissue showed a significant increase in the LipGin group compared with the Gin group (P<0.05).Conclusion:Results indicated that the liposomal ginger enhanced the antitumor activity; therefore, the prepared liposomal ginger can be used in future clinical trials.

Gene Expression Signatures of a Preclinical Mouse Model during Colorectal Cancer Progression under Low-Dose Metronomic Chemotherapy.

Simple SummaryColorectal cancer is one of the most frequent types of cancer world-wide, leading to over 500,000 cancer-related deaths each year. Although many primary colorectal cancer patients can be cured by surgery, up to 60% will develop metastases. Chemotherapeutic strategies are well-established, but finally often lead to chemo-resistance and tumor relapse. A specific chemotherapeutic approach is low dose metronomic (LDM) therapy, which is based on a constant administration of low doses of a chemotherapeutic compound instead of high-dose pulses, which are often a huge burden for patients and also may induce rapid resistance. However, the molecular mechanism of LDM chemotherapy is not fully understood. Our study therefore aims at identifying gene signatures of colorectal cancer progression under LDM chemotherapy which eventually provides new potential biomarkers for therapeutic interventions.Understanding the molecular signatures of colorectal cancer progression under chemotherapeutic treatment will be crucial for the success of future therapy improvements. Here, we used a xenograft-based mouse model to investigate, how whole transcriptome signatures change during metastatic colorectal cancer progression and how such signatures are affected by LDM chemotherapy using RNA sequencing. We characterized mRNAs as well as non-coding RNAs such as microRNAs, long non-coding RNAs and circular RNAs in colorectal-cancer bearing mice with or without LDM chemotherapy. Furthermore, we found that circZNF609 functions as oncogene, since over-expression studies lead to an increased tumor growth while specific knock down results in smaller tumors. Our data represent novel insights into the relevance of non-coding and circRNAs in colorectal cancer and provide a comprehensive resource of gene expression changes in primary tumors and metastases. In addition, we present candidate genes that could be important modulators for successful LDM chemotherapy.

Inhibition of tumor growth in CT-26 colorectal cancer-bearing mice with alcoholic extracts of Curcuma longa and Rosmarinus officinalis

Herbal medicinal products for cancer therapy is one of the complementary approaches with few side effects. In this study, Rosmarinus officinalis (Rosemary) and Curcuma longa (Turmeric) extracts were selected to assess the anti-cancer effects of their mixture on colorectal cancer both in vitro and in vivo. Rosemary and Turmeric extractions were performed by a Soxhlet apparatus. The MTT assay was carried out for the measurement of cell cytotoxicity. Apoptosis was evaluated using the Annexin-V assay. In vitro and in vivo gene expression of Bax, Bcl-2, and caspase-3 was measured by real-time PCR. Inhibition of tumor growth and infiltration of cytotoxic T lymphocytes (CTLs) and regulatory T lymphocytes (Tregs) were then investigated in the treated colorectal cancer-bearing mice via flow cytometry. The MTT assay showed a concentration-dependent increase in mixture group cytotoxicity in CT-26 cells (P < 0.05). Moreover, treatment of cells with the half-maximal inhibitory concentration (IC50) of Rosemary, Turmeric, and mixture induced apoptosis (P < 0.05). In vivo studies revealed that mixture treatment inhibited tumor growth in mice transplanted with CT-26 cells (P < 0.05), without any side effects, such as weight loss or changes in kidney and liver functions. Moreover, mice treated with a mixture of Rosemary and Turmeric showed a significant increase in the cytotoxic T lymphocyte (CTL)/Treg cell ratio (P < 0.05).The results of the present study showed that the combination of Rosemary and Turmeric had the potential to inhibit cancer cell growth in the CT-26 cell line and reduce tumor growth in colorectal cancer-bearing mice.

Application of the Tumor Site Recognizable and Dual-Responsive Nanoparticles for Combinational Treatment of the Drug-Resistant Colorectal Cancer.

Combination of PCI and chemotherapy represents a promising strategy for combating drug resistance of cancer. However, poor solubility of photosensitizers and unselectively released drugs at unwanted sites significantly impaired the treatment efficacy. Therefore, in the present study, we aimed to develop a nano-platform which could efficiently co-entrapping photosensitizers and chemotherapeutics for active targeting therapy of drug resistant cancers. Two pro-drugs were respectively developed by covalently linking the Ce6 with each other via the GSH-sensitive linkage and the PTX with mPEG-PLA-COOH through the ROS sensitive-linker. The dual-responsive nanoparticles (PNP-Ce6) was developed by emulsion/solvent evaporation method and further modified with tLyp-1 peptides. Physicochemical properties of nanoparticles were determined by the TEM and DLC. Cellular uptake assay was investigated with the Ce6 acting as the fluorescent probe and cell growth was studied by the MTT experiment. In vivo tumor targeting and anti-tumor assay was investigated on the colorectal cancer-bearing mice. The developed tPNP-Ce6 were stable enough under the normal physiological conditions. However, free Ce6 and PTX were completely released when exposed the tPNP-Ce6 to the redox environment. Excellent tumor-targeting drug delivery was achieved by the tPNP-Ce6, which in turn resulted in satisfactory anit-tumor effect. Of great importance, super inhibition effect on tumor progress was achieved by the combination therapy when compared with the group only received with chemotherapy.. The results obtained in the present study indicated that the developed tPNP-Ce6 may have great potential in enhancing the therapeutic efficacy of drug-resistant colorectal cancer. Graphical Abstract Left: Targeting delivery of drug to tumor site by the tumor recognizable and dual-responsive nanoparticles and penetrating into tumor inner via the mediation of irradiation. Right: Nanoparticle distribution within tumor tissues with green represents the blood vessels stained with CD31, blue signal represents the cell nuclei stained with DAPI and red shows fluorescence of Ce6 as the indicator of the nanoparticles.

Fecal Microbiota Transplantation Prevents Intestinal Injury, Upregulation of Toll-Like Receptors, and 5-Fluorouracil/Oxaliplatin-Induced Toxicity in Colorectal Cancer.

FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin), a 5-fluorouracil (5-FU)-based chemotherapy regimen, is one of most common therapeutic regimens for colorectal cancer. However, intestinal mucositis is a common adverse effect for which no effective preventive strategies exist. Moreover, the efficacy and the safety of fecal microbiota transplants (FMT) in cancer patients treated with anti-neoplastic agents are still scant. We investigated the effect of FMT on FOLFOX-induced mucosal injury. BALB/c mice implanted with syngeneic CT26 colorectal adenocarcinoma cells were orally administered FMT daily during and two days after five-day injection of FOLFOX regimen for seven days. Administration of FOLFOX significantly induced marked levels of diarrhea and intestinal injury. FMT reduced the severity of diarrhea and intestinal mucositis. Additionally, the number of goblet cells and zonula occludens-1 decreased, while apoptotic and NF-κB-positive cells increased following FOLFOX treatment. The expression of toll-like receptors (TLRs), MyD88, and serum IL-6 were upregulated following FOLFOX treatment. These responses were attenuated following FMT. The disrupted fecal gut microbiota composition was also restored by FMT after FOLFOX treatment. Importantly, FMT did not cause bacteremia and safely alleviated FOLFOX-induced intestinal mucositis in colorectal cancer-bearing mice. The putative mechanism may involve the gut microbiota TLR-MyD88-NF-κB signaling pathway in mice with implanted colorectal carcinoma cells.

Abstract 4843: TP53-stabilization with APR-246 enhances antitumor effects of immune checkpoint blockade in preclinical models

Abstract An emerging body of literature suggests a role of TP53 pathway in antitumor immunity including antigen presentation and T cell proliferation. Stabilization of TP53 could therefore alter the immune tumor microenvironment, enabling the immune system to target tumor cells more effectively. APR-246 covalently modifies the core domain of cellular TP53 through the alkylation of thiol groups, leading to reactivation of endogenous TP53 activity. To further study the effect of APR-246 on antitumor immunity we used a B16 pre-clinical melanoma mouse model. In vitro treatment of B16 cells with APR-246 caused intracellular accumulation of TP53, leading to increased apoptosis. However, treatment of B16-melanoma bearing mice with APR-246 monotherapy did not result in a statistically significant change in tumor growth or survival. Analyses of the tumor immune microenvironment showed increased immune potentiating M1 polarized tumor-associated macrophages, and Granzyme B activity in CD8+ T cells, suggesting enhanced anti-tumor immunity. Concomitantly, there was increased PD-L1 expression in the macrophages, PD-1 expression on CD8+ T cells, and Foxp3+ Tregs in tumors from APR-246 treated animals. Therefore, we decided to combine anti-PD-1 antibody (RMP-1) to APR-246 treatment in tumor-bearing mice. The combination led to a significant delay in tumor growth (P &amp;lt; 0.001) and improved survival of B16-bearing mice compared to anti-PD1 or APR-246 monotherapies (P &amp;lt; 0.01). Improved responses were also seen in MC38 colorectal cancer-bearing mice (P &amp;lt; 0.01). The anti-tumor effects of APR-246 and anti-PD1 were T cell dependent and abrogated in hosts lacking T cells. Analyses of the tumor immune microenvironment showed that the combination decreases proliferation but increases cytolytic activity of CD8+ T cells compared to anti-PD1 alone. We next studied the effect of combining APR-246 with anti-PD1+ anti-CTLA-4 (9B9). Combination of APR-246 with dual immune checkpoint blockade using anti-PD1 and anti-CTLA-4 showed further tumor growth inhibition in B16-melanoma compared to monotherapies (P &amp;lt; 0.001). Our studies support a role for restoring TP53 in the tumor microenvironment and provide evidence that reactivation of TP53 by APR-246 can enhance anti-tumor immune responses and inhibit tumor growth in preclinical models. Citation Format: Arnab Ghosh, Judith Michel, Lauren Dong, Nathan Suek, Hong Zhong, Sadna Budhu, Olivier de Henau, Jedd Wolchok, Taha Merghoub. TP53-stabilization with APR-246 enhances antitumor effects of immune checkpoint blockade in preclinical models [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 4843.

Lactobacillus casei Variety rhamnosus Probiotic Preventively Attenuates 5-Fluorouracil/Oxaliplatin-Induced Intestinal Injury in a Syngeneic Colorectal Cancer Model.

Adjuvant 5-fluorouracil (5-FU)-based chemotherapy, including FOLFOX (5-FU, leucovorin, and oxaliplatin), is recommended for colorectal cancer. However, intestinal mucositis remains a common adverse effect for which no effective preventive strategies are available. To develop a convenient and novel way to alleviate mucositis, we investigated the effect of Lactobacillus casei variety rhamnosus (Lcr35) on FOLFOX-induced mucosal injury. BALB/c mice subcutaneously injected with syngeneic CT26 colorectal adenocarcinoma cells were orally administered Lcr35 daily before, during, and after 5-day injection of FOLFOX regimen, for 14 days. The following methods were used: diarrhea score for toxicity, ELISA for cytokine production, histopathology for intestinal injury, immunohistochemistry for apoptosis/proliferation and regulatory proteins, RT-PCR for cytokine mRNA expression, and DNA sequencing for fecal gut microbiota. FOLFOX administration to colorectal cancer-bearing mice significantly inhibited tumor growth and the accompanying marked diarrhea and intestinal injury histologically characterized by the shortening of villi and destruction of intestinal crypts. Preventive administration of Lcr35 dose-dependently reduced the severity of diarrhea and intestinal mucositis without affecting the anti-tumor effect of FOLFOX. The numbers of apoptotic, NF-κB-, and BAX-activated cells increased after FOLFOX, and these responses were mitigated by Lcr35. TNF-α and IL-6 upregulation by FOLFOX treatment was attenuated by Lcr35. The fecal gut microbiota composition of Firmicutes and Bacteroidetes disturbed by FOLFOX was significantly reversed by Lcr35 toward a preferential profile. In conclusion, the oral probiotic Lcr35 prevented FOLFOX-induced intestinal mucositis in colorectal cancer-bearing mice. The putative mechanism might involve modulation of gut microbiota and proinflammatory responses with suppression of intrinsic apoptosis in intestinal injury.

Low-dose decitabine enhances the effect of PD-1 blockade in colorectal cancer with microsatellite stability by re-modulating the tumor microenvironment

PD-1 blockade has demonstrated impressive clinical outcomes in colorectal cancers that have high microsatellite instability. However, the therapeutic efficacy for patients with tumors with low microsatellite instability or stable microsatellites needs further improvement. Here, we have demonstrated that low-dose decitabine could increase the expression of immune-related genes such as major histocompatibility complex genes and cytokine-related genes as well as the number of lymphocytes at the tumor site in CT26 colorectal cancer-bearing mice. A more significant inhibition of tumor growth and a prolongation of survival were observed in the CT26 mouse model after treatment with a combination of PD-1 blockade and decitabine than in mice treated with decitabine or PD-1 blockade alone. The anti-tumor effect of the PD-1 blockade was enhanced by low-dose decitabine. The results of RNA sequencing and whole-genome bisulfite sequencing of decitabine-treated CT26 cells and tumor samples with microsatellite stability from the patient tumor-derived xenograft model have shown that many immune-related genes, including antigen-processing and antigen-presenting genes, were upregulated, whereas the promoter demethylation was downregulated after decitabine exposure. Therefore, decitabine-based tumor microenvironment re-modulation could improve the effect of the PD-1 blockade. The application of decitabine in PD-1 blockade-based immunotherapy may elicit more potent immune responses, which can provide clinical benefits to the colorectal cancer patients with low microsatellite instability or stable microsatellites.

Inhibitory Effect of Tetrandrine on Pulmonary Metastases in CT26 Colorectal Adenocarcinoma–Bearing BALB/c Mice

Tumor metastasis is a major cause of mortality in cancer patients. The anti-metastatic effect of tetrandrine, an alkaloid isolated from Stephania tetrandrae S. Moore, was investigated in a pulmonary metastatic model of colorectal cancer-bearing mice. Tetrandrine decreased the viability of murine colorectal adenocarcinoma CT26 cells in a time- and dose-dependent manner. CT26 cells were injected into BALB/c mice via a tail vein to establish pulmonary metastases. After this, the mice were given intraperitoneal injections of tetrandrine (10 mg/kg/day), 5-fluorouracil (5-FU) at the same dose, or vehicle for 5 consecutive days. Mice treated with tetrandrine had 40.3% fewer metastases than vehicle-treated mice, and those treated with 5-FU had 36.9% fewer metastases than controls. Both tetrandrine- and 5-FU-treated mice survived longer than mice in the untreated control group. There was no acute toxicity or obvious changes in body weight in any of the mice. These results suggest that tetrandrine may be a useful anti-metastatic agent.