Modulating the tumor microenvironment in a mouse model of colon cancer using a combination of HIF-1α inhibitors and Toll-Like Receptor 7 agonists

Abstract

The immunosuppressive tumor microenvironment (TME) plays a pivotal role in the response to various anticancer therapies, such as immune and chemotherapeutic agents. In this study, the synergistic effects of gene-targeting HIF-1α siRNA combined with Toll-Like Receptor 7 agonist on TME remodeling were investigated in a mouse model of colorectal cancer (CRC). A HIF-1α-specific siRNA duplex was formulated based on the ionic gelation of tripolyphosphate (TPP) with cationic chitosan (CH) as a nanoplex and evaluated in terms of size, charge, polydispersity index and gel retardation assay. MTT assay was conducted to assess the cytotoxicity of the specific siRNA duplex against CT26 cells. Hypoxic condition was generated to evaluate the gene and protein expression levels of HIF-1α, respectively. CT26 mouse model was established to assess the synergistic effect of silencing HIF-1α combined with oxaliplatin (OXA) and imiquimod (IMQ) on tumor growth. The mean diameter of the CH/siRNA nanoparticles was 243 ± 6 nm, as confirmed with Micrograph scanning electron microscope. There were no significant differences observed between the CT26 cells treated with nanoparticles alone and the untreated cells, indicating that these nanoparticles are safe and physiologically biocompatible (p ≥ 0.05). Triple combination therapy involving HIF-1α siRNA, OXA, and IMQ significantly retarded tumor growth and led to elevated levels of cytokines linked to cellular immunity (INF-γ and IL-12) compared with those in the other groups (P < 0.05). The positive correlation coefficient (r = 0.68) between tumor size and HIF-1α expression levels was statistically significant (P = 0.003). Compared with those in the control group, the expression levels of the anti-inflammatory cytokines IL-10 and IL-4 significantly decreased (P < 0.05). In conclusion, our findings suggest that inhibiting HIF-1α could serve as a rational strategy to enhance the antitumor response in the TME.

Keywords: Chemotherapy; Colorectal cancer; Combination therapy; Gene therapy; HIF-1α; Immunotherapy; TLR; Tumor microenvironment.

Fig. 1

Characterization of CH/HIF-1α siRNA nanoparticles: a Characterization of the size, zeta potential, and PDI of both CH nanoparticles and CH/HIF-1α siRNA nanoplexes. b Scanning electron microscopic (SEM) graph of CH/HIF-1α siRNA nanoplexes. c The gel retardation assay is presented here, evaluating CH/siRNA nanoparticles with varying N/P ratios. d The viability of CT26 cells was assessed following incubation with different concentrations of CH nanoparticles alone and CH-siRNA. e The efficiency of HIF-1α siRNA silencing by CH nanoplexes in CT26 cells was explored. Tumor cells were cultured and transfected with CH/HIF-1α siRNA nanoplexes containing different concentrations of HIF-1α siRNA (25, 50, 75, and 100 nM) at an N/P ratio of 60. After 48 h of incubation, HIF-1α mRNA levels were measured using qPCR. Beta-actin was used as a housekeeping gene for normalization. All the data are presented as the means ± SDs of three independent measurements

Fig. 2

Combination analysis of OXA, IMQ, and HsiRNA combination therapy on CT26 colon cancer cells. a Dose‒effect curve, b median-effect plot for single and triple combination therapy (TCT), c combination index plot, and d DRI plot for four combinations of OXA, IMQ & HsiRNA on CT26 colon cancer cells

Fig. 3

Effects of Combination Therapy on Tumor Weight and Volume in a Mouse Model of CRC. a Treatment protocol depicting the schedule for combination therapy designed to treat CT26 tumors. Treatments began 12–16 days after tumor implantation and were administered every two days. b Images of tumors harvested from untreated and treated mice. Resected tumors demonstrated that triple combination therapy resulted in a greater reduction in tumor size than dual therapy. c and e: Effects on tumor volume and weight. All combination therapies significantly reduced tumor volume and weight compared to those in the control group. d and f Double vs. triple therapy. These panels compare the effects of double and triple therapy. The largest reductions in both tumor volume and weight were observed in mice treated with the triple combination. g Tumor volume over time, indicating that triple combination therapy was more effective than dual therapy in suppressing tumor growth. Each line represents a specific treatment group and was plotted to show the primary tumor size relative to the days after tumor challenge

Fig. 4

Changes in HIF-1α and Apoptosis-Related Gene Expression during Combination Therapy. a to d The impact of CH/HIF-1α siRNA nanoplexes on HIF-1α, BAX, BAD and BCL2 expression in a mouse model of colorectal cancer (CRC). e & f Investigation of how combination therapy affects the mRNA and protein levels of HIF-1α and BAX. All the data are presented as the mean ± standard deviation (SD) of the gene expression levels

Fig. 5

This figure explores how combination therapy affects the expression of genes involved in cell proliferation. a and b depict the mRNA expression levels of vascular endothelial growth factor (VEGF). d and e show the mRNA expression levels of phosphorylated signal transducer and activator of p-STAT3. c The protein expression levels of p-STAT3. Beta-actin, a housekeeping gene, was used for data normalization. All the data are presented as the mean ± standard deviation (SD) of the gene expression levels

Fig. 6

This figure investigates how combination therapy influences the expression of genes associated with cytokines. a to c show the mRNA and protein expression levels of interferon-gamma (IFN-γ). d and e show the mRNA expression levels of interleukin-12 (IL-12). Beta-actin, a housekeeping gene, was used for data normalization. All the data are presented as the mean ± standard deviation (SD) of the gene expression levels. Within the triple therapy group, HIF-1α expression showed a positive correlation with VEGF (r = 0.8), although this correlation was not statistically significant (Fig. 6b). In contrast, negative correlations were observed between HIF-1α expression and the expression levels of both BCL2 (Fig. 6c) and IL-12 (Fig. 6d). The strongest correlation (p = 0.09) was found between HIF-1α and BCL2 expression

Fig. 7

Correlation of HIF-1α Expression with Tumor Size and Other Genes. a The correlation between HIF-1α gene expression and tumor size across all treatment and control groups was explored. A positive correlation was observed, indicating that higher HIF-1α mRNA expression is associated with larger tumors. b-d The correlation between HIF-1α expression and the expression of other genes in the triple combination therapy group was investigated. b The correlations with vascular endothelial growth factor (VEGF), c BCL2, and d interleukin-12 (IL-12) were investigated