Reovirus combined with a STING agonist enhances anti-tumor immunity in a mouse model of colorectal cancer

doi:10.21203/rs.3.rs-2251036/v1

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Reovirus combined with a STING agonist enhances anti-tumor immunity in a mouse model of colorectal cancer

https://doi.org/10.21203/rs.3.rs-2251036/v1

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Reovirus, a naturally occurring oncolytic virus, initiates the lysis of tumor cells while simultaneously releasing tumor antigens or proapoptotic cytokines in the tumor microenvironment to augment anticancer immunity. However, reovirus has developed a strategy to evade antiviral immunity via its inhibitory effect on interferon production, which negatively affects the induction of antitumor immune responses. The mammalian adaptor protein Stimulator of Interferon Genes (STING) was identified as a key regulator that orchestrates immune responses by sensing cytosolic DNA derived from pathogens or tumors, resulting in the production of type I interferon. Recent studies reported the role of STING in innate immune responses to RNA viruses leading to the restriction of RNA virus replication. In the current study, we found that reovirus had a reciprocal reaction with a STING agonist regarding type I interferon responses in vitro; however, we found that the combination of reovirus and STING agonist enhanced anti-tumor immunity by enhancing cytotoxic T cell trafficking into tumors, leading to significant tumor regression and survival benefit in a syngeneic colorectal cancer model. Our data indicate the combination of reovirus and a STING agonist to enhance inflammation in the tumor microenvironment might be a strategy to improve oncolytic reovirus immunotherapy.

Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer

Health sciences/Diseases/Cancer/Tumour immunology

reovirus

STING agonist

colorectal cancer

Human oncolytic reovirus is a non-enveloped double-stranded RNA virus composed of an outer and inner protein shell, which forms a 20-sided icosahedral capsid. Although reovirus infections are mild and sometimes subclinical, reovirus displays selective oncolytic activity against transformed and malignant cells^{1, 2}. Previous studies on the mechanisms involved in how reovirus kills tumor cells suggest Ras pathway activation is a key determinant of viral replication and subsequent oncolysis^{3, 4}. We investigated the efficacy of reovirus against gastrointestinal cancer and reported the strong antitumor activity of reovirus alone or in combination with anticancer drugs against gastric cancer cells⁵. Additionally, we found the antitumor efficacy of reovirus involved oncolytic effects of the reovirus as well as its immunostimulatory effects⁶. Multiple preclinical cancer models also demonstrated the functionality of reovirus as an immunotherapeutic agent^7–9 and the enhanced efficacy of reovirus in combination with immunotherapeutic agents including PD-1/PD-L1 blocking agents^10–13.

Reovirus has an intrinsic preference for propagation in KRAS-mutated cancer cells, and it has specific mechanisms to escape from type I interferon (IFN) antiviral responses¹⁴. A recent study reported that reovirus inhibited the production of type I IFNs by blocking IRF3 translocation into the nucleus¹⁵. Inhibition of type I IFNs by reovirus promoted its infection of cancer cells and its replication, which killed cancer cells, although it simultaneously suppressed type I IFN-induced antitumor immunity. Type I IFNs are key regulators that facilitate the antitumor immunity and activity of conventional cell death-inducing therapies by enhancing the activity of tumor infiltrating lymphocytes; therefore, the anti-tumor activity of reovirus might be partially opposed by its strategy for propagation in cancer cells.

Stimulator of interferon gene (STING) is a cytosolic DNA sensor involved in immune defense against pathogens such as bacteria and viruses. STING is activated by cyclic GMP-AMP (cGAMP) produced by the binding of cyclic GMP-AMP synthase (cGAS) to aberrant cytoplasmic double-stranded DNA (dsDNA) originating from intracellular pathogens, which induces the expression of type I IFNs. The cGAS-STING pathway is also stimulated by DNA leakage from cancer cells or by cytosolic fragments of DNA caused by irradiation or anticancer agents that break DNA strands. STING is a potent target for cancer immunotherapy and the compounds that modulate STING have been developed for cancer treatment^{16, 17}.

Although reovirus induces anti-tumor immunity in addition to direct tumor killing activity, it also represses type I IFN responses that modulate immune responses that eliminate cancer cells. Thus, we hypothesized that activation of the STING axis counteracts the suppression of type I IFN responses by reovirus, and that combination treatment with reovirus and a STING agonist promotes anticancer immunity by improving the tumor microenvironment. In this study, we investigated the effects of reovirus combined with a STING agonist on antitumor immunity.

Reagents

ADU-S100 (MIW815), a cyclic dinucleotide (CDN) agonist of STING, was purchased from CHEMIETEK (Indianapolis, IN), dissolved in double distilled water, and then stored at − 20°C until use. Reovirus was provided by the University of Calgary, Department of Molecular Biology and Oncology (Canada) and stored in the dark at neat concentrations in phosphate buffered saline (PBS) at − 80°C.

Cell culture

The murine colon cancer cell lines used for the experiments were CT26, MC38, and CMT93. CT26 and CMT93 cells were purchased from ATCC (Manassas, FL), and MC38 was purchased from Kerafast (Boston, MA). CT26, MC38, and CMT93 cells were cultured in low glucose Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and maintained at 37°C with 5% CO₂.

Cell proliferation assay

Cell proliferation was analyzed using the WST-8 cell proliferation assay. CT26 cells were seeded into 96-well culture plates at a concentration of 500 cells/100 µl/well and incubated for 8 hours. Cells were then treated with reovirus at a multiplicity of infection (MOI) of 3 or 10 on day 0. Cell proliferation was evaluated using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol, and the absorption at 450 nm was measured with a microplate spectrophotometer (SPECTRA MAX340, Molecular Devices, San Jose, CA). We determined the proliferative capacity by calculating the values from day 1 to day 3 with the number of cells on day 0 set to 1.

Quantitative reverse transcription–polymerase chain reaction

To investigate differences in gene expression induced by reovirus infection and ADU-S100 administration, reverse transcription–polymerase chain reaction (RT-PCR) was performed. We assessed the mRNA expression profiles of CT26 cells treated with reovirus at 10 MOI and 10 µM ADU-S100 for 1, 2, and 6 hours. Real-time quantitative RT-PCR analyses were performed using an ABI 7900 HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations.

Western blotting

Cells were washed with PBS 3 times and then added to 1 mL of cell lysis buffer (Cell Signaling Technology, Danvers, MA) containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO₄, and 1 µg/mL leupeptin. Cells were disrupted for 15 seconds on ice using a Bioruptor sonicator (Cosmo Bio, Tokyo, Japan) and centrifuged at 15 000 rpm for 20 minutes at 4°C. Each sample was normalized to an equal protein concentration using a Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Equal quantities of 2× sodium dodecyl sulphate–polyacrylamide gel electrophoresis sample buffer [0.5 M Tris-HCl (pH 7.2), 1% SDS, 100 mmol/L β-mercaptoethanol, and 0.01% bromophenol blue] were added to each sample, and samples were boiled for 5 minutes at 100°C. Aliquots of each sample were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis on a 10% gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in PBS for 1 hour at room temperature, followed by incubation with primary antibodies against IRF3 (ab68481, Abcam, Cambridge, UK), phosphorylated IRF-3 (Ser379, #79945, Cell Signaling Technology), Ser172, #5483,), cGAS (#31659, Cell Signaling Technology), STING (#13647, Cell Signaling Technology), and β-actin (Sigma-Aldrich, St. Louis, MO) overnight at 4°C. Membranes were washed with 0.05% Tween 20 in PBS three times at 5-minute intervals, incubated with the secondary antibody for 1 hour at room temperature, and washed again with 0.05% Tween 20 in PBS three times at 5-minute intervals. The membrane was incubated with western blotting detection reagent, ECL Prime (GE Healthcare, Buckinghamshire, UK) for 5 minutes at room temperature and filmed using ImageQuant LAS 4000 (GE Healthcare).

Detection of ICD Markers

CT26 cells seeded in 24-well plates (3×10⁴ cells/well) were treated with 10 MOI Reovirus, and cell supernatants collected 48h and 72h. Cell culture supernatants were centrifuged at 10000 rpm for 3 min at 4℃. HMGB1 released in culture supernatants was detected using an HMGB1 Detection ELISA kit (# 6010, Chondrex, Woodinville, WA). CT26 cells seeded in 96-well plates at 500 cells/well and incubated overnight were treated with 10 MOI Reovirus for 48h and 72h. Extracellular ATP was measured with a RealTime-Glo™ Extracellular ATP Assay (GA5010, Promega, Madison、WI).

Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, then permeabilized with 0.05% Triton X-100 for 10 minutes at room temperature. Immunostaining was performed with IRF3 and STING antibodies followed by fluorescence conjugated secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG) (Invitrogen, Carlsbad, CA). Images were taken with a FV3000 Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan).

Mouse tumor models

Female mice (BALB/cCr Slc), 6–8 weeks of age, with a body weight of 18–22 g, were obtained from Japan SLC, Inc. (Shizuoka, Japan). Syngeneic mouse models were established by subcutaneously implanting 5×10⁵ CT26 cells in 100 µl of PBS into the right flank or both flanks of mice. Mice were then treated with ADU-S100 and/or reovirus. Mice were divided into four groups; control, ADU-S100 IT injection, reovirus IT injection, and ADU-S100 and reovirus groups. Some mice were treated with one IT injection containing 10 µg/50 µL of ADU-S100. Other mice were treated with three IT injections of reovirus at a dose of 3×10⁸ plaque-forming units (pfu) per mouse in 50 µL on alternate days. The dose of reovirus was chosen based on our previous study^{5, 6}. The maximum tumor diameter (L) and the diameter at right angles to that axis (W) were measured twice a week using calipers, and the volume was calculated according to the formula, (L × W²)/2. Tumor size was measured and anti-tumor activity of reovirus or ADU-S100 was evaluated until the tumor volume reached 2 000 mm³. For histological and FACS analyses (see below), three days after the IT injection of anticancer agents, transplanted tumors were excised and fixed in formalin after euthanasia. No adverse effects, including weight loss or infection, were observed throughout the treatment period. The procedures in these experiments were approved by the Nagoya City University Center for Experimental Animal Science, and the mice were cared for according to the guidelines of the Nagoya City University for Animal Experiments.

Flow cytometry

Excised tumors were dissected into smaller fragments using scalpels and scissors, and further dissociated into single cell suspensions using the GentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, San Diego, CA). For each sample, 1×10⁶ cells were treated with mouse Fc block (#553142, BD Biosciences, Franklin Lakes, NJ) and then stained with Fixable Viability Stain 780 (#565388, BD Biosciences) and five different labeling antibodies. Anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-FoxP3 were purchased from BD Biosciences (San Jose, CA). Data were acquired on a BD FACSCanto II.

Immunohistochemistry

Tumors were fixed in 10% neutral buffered formalin for 24 hours. Formalin-fixed and paraffin-embedded sections (4 µm) were used for immunohistochemistry. Sections were stored dry until their use for histological analysis. After deparaffinization, slides submerged in 10 mM citrate buffer were heated in a microwave until boiling followed by 10 minutes at a sub-boiling temperature (95–98°C). Slides were cooled for 30 min. After pre-incubation with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity, sections were rinsed in PBS, and blocked with 100 µl of preferred 10% normal goat serum (Nichirei, Tokyo, Japan) for 10 minutes at room temperature. Primary antibodies were incubated overnight at 4°C. CD4, CD8, FoxP3, Ki67, Granzyme B, cleaved caspase-3, Arginase-I, and iNOS were visualized immunohistochemically by the streptavidin-biotin method using 1:500 rabbit anti-CD4 monoclonal antibody (ab183685, Abcam), 1:500 rabbit anti-CD8 monoclonal antibody (#98941, Cell Signaling Technology), 1:400 rabbit anti-FoxP3 monoclonal antibody (#12653, Cell Signaling Technology), 1:400 rabbit anti-Ki67 monoclonal antibody (#12202, Cell Signaling Technology), 1:100 rabbit anti-Granzyme B polyclonal antibody (ab4059, Abcam), 1:400 rabbit anti-cleaved caspase-3 monoclonal antibody (#9664, Cell Signaling Technology), 1:400 rabbit anti-Arginase-1 monoclonal antibody (#93668, Cell Signaling Technology), or 1:100 rabbit anti-iNOS polyclonal antibody (ab15323, Abcam) and the Histofine SAB-PO^® Kit (Nichirei). Sections were rinsed and incubated sequentially with secondary antibody (goat biotinylated anti-rabbit IgG antibody) and with the streptavidin-biotin-peroxidase complex. Sections were then washed with PBS, incubated in diaminobenzidine solution containing 0.006% hydrogen peroxide for 5 minutes. Sections were counterstained with hematoxylin and were then dehydrated through graded ethanol solutions (80–100%). For quantification, positive cells in a field approximately 70 000 µm², were counted randomly at 400× magnification. For objective cell counts, two independent researchers identified positive-stained cells in a blinded manner and observers blinded to the treatment condition, performed the cell counts. Mean counts from six fields were calculated. Observations were obtained from at least three independent experiments. To count the numbers of CD4, CD8, FoxP3, Ki67, Granzyme B, and cleaved caspase-3 positive cells in the sections at 200× magnification, we used ImageJ software (https://imagej.nih.gov/ij) with the immunohistochemistry profiler plugin as described previously by Shi et al¹⁸. Quantification of iNOS and Arginase-1 protein expression was performed using ImageJ software.

Multiplex gene expression analysis

Total RNA was extracted from the tumor of the mice three days after treatment with reovirus, ADU-S100, or the combination of reovirus and ADU-S100 (RNeasy Lipid Tissue Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Samples with RNA integrity values > 7.0 (4200 TapeStation System, Agilent, Santa Clara, CA) were included for multiplex gene expression analysis. Data were collected on a NanoString nCounter digital analyzer using the nCounter Mouse PanCancer Immune Profiling Panel (Nanostring Technologies, Seattle, WA) according to the manufacturer´s instructions. Data analysis was performed by the nSolver analysis software (Nanostring Technologies) normalized to the internally provided control genes set.

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 8.4.2 (GraphPad Software, La Jolla, CA). The Student’s t-test was used when two independent groups were compared, and multiple comparisons between groups were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Survival data were analyzed by the log-rank (Mantel-Cox) test. P-values less than 0.05 were considered to indicate statistical significance.

Reovirus inhibits the growth of murine colorectal cancer cell lines.

We investigated the cytotoxic effect of reovirus against CT26, CMT93, and MC38 using cell viability assay. After treatment with reovirus for three days, proliferation was significantly decreased in all cell lines at various degrees (Fig. 1A). Next, we examined the expression of a cytosolic DNA sensor, cGAS and STING, in murine colorectal cancer cell lines including CT26, CMT93, and MC38. Western blot analysis demonstrated that cGAS was expressed in CT26 and MC38. STING showed varied expression in all cell lines examined (Fig. 1B). A STING agonist, ADU-S100, had no significant additive effect on cytotoxicity of the reovirus against all cell lines we examined in vitro (supplemental Fig. 1).

A STING agonist suppresses the inhibitory effect of reovirus against IFN responses.

We used CT26 cells to investigate the activation of the STING signaling pathway responsible for type I IFN regulation in CT26 cells. We examined the activation of STING signaling stimulated by reovirus alone or in combination with ADU-S100 by evaluating the phosphorylation of IRF-3, downstream factor of STING signaling, using western blot analysis. No activation of STING signals was detected in CT26 cells treated with reovirus. In contrast, STING agonist, ADU-S100 significantly induced the phosphorylation of IRF3; however, this effect was attenuated when ADU-S100 was administered with the reovirus (Fig. 2A, B). We performed immunofluorescence staining to examine the translocation of IRF3 in CT26 cells treated with reovirus. One hour after treatment with ADU-S100, IRF3 localized into the nucleus confirming the western blot analysis. Although reovirus alone did not induce the translocation of IRF3 into the nucleus, IRF3 was detected in the nucleus of cells treated with reovirus combined with ADU-S100 (Fig. 2C). Quantitative RT-PCR revealed that CT26 cells produced IFN-β in a concentration-dependent manner in response to ADU-S100 (supplemental Fig. 2). Reovirus significantly suppressed the expression of IFN-β enhanced by ADU-S100 in CT26 cells (Fig. 2D). STING agonist also suppresses the inhibitory effect of reovirus against IFN responses in MC 38 cells (supplemental Fig. 3). These results indicate the suppressive effect of reovirus against IFN-β production was partially canceled by a STING agonist in vitro.

Induction of immunogenic cell death in CT26 cells by reovirus

We measured the concentration of ATP in culture media of CT26 cells treated with reovirus and the HGMB-1 released from CT26 cells after exposure to reovirus to evaluate the immunogenic cell death induced by reovirus in CT26 cells. The concentration of extracellular ATP in culture media of CT26 treated with reovirus was significantly higher than that in culture media of CT26 without reovirus treatment (Fig. 2D). A higher concentration of HGMB-1 was detected in the culture media congaing CT26 treated reovirus (Fig. 2E). These results demonstrate that reovirus induces immunogenic cell death in CT26.

Efficacy of reovirus in combination with a STING agonist on the growth of CT26 cells in vivo.

To investigate whether reovirus and ADU-S100 synergistically induced anti-tumor efficacy against colorectal cancer (CRC) in immunogenic settings, we used a syngeneic murine model of CRC. To analyze the anti-tumor immune response, CT26 cells were subcutaneously injected into BALB/c mice. The tumor size was measured, and the tumor volume was calculated after the intratumoral (IT) injection of reovirus, ADU-S100, or reovirus and ADU-S100. Reovirus IT injection significantly inhibited tumor growth compared with controls. IT treatment with ADU-S100 also inhibited tumor growth, and the combination of reovirus and ADU-S100 demonstrated the strongest anti-tumor activity, and ADU-S100 showed the significant additive effect on anti-tumor activity of the reovirus (Fig. 3A). Additionally, the combination treatment significantly improved the survival of tumor-bearing mice compared with controls (supplemental Fig. 4). In the combination treatment group, four of eight mice achieved complete regression of the tumor. Histopathological analyses (supplemental data 5) of extracted tumors from mice three days after IT injection of anticancer agents revealed the number of Ki-67-positive cells was significantly decreased in the implanted tumor treated with the IT injection of reovirus, either alone or in combination with ADU-S100, compared with controls (Fig. 3B). Furthermore, the number of cleaved caspase-3-positive cells was increased in the combination reovirus and ADU-S100 group compared with controls (Fig. 3C). We also detected a significant increase in the number of granzyme B positive cells in tumors treated with the combination treatment compared with controls (Fig. 3D). These results suggest that reovirus synergizes with ADU-S100 to promote an anti-tumor effect in tumor-bearing mice and that the efficacy of the combination treatment might be derived by enhanced anti-tumor immunity.

Combined reovirus and ADU-S100 control tumor growth in a dual flank tumor model.

To investigate the role of immune responses in primary tumor growth inhibition, we performed a dual flank study using an immunocompetent syngeneic model. In the dual flank syngeneic mouse tumor model, CT26 cells were implanted to both sides of a mouse and then one tumor was injected with reovirus, ADU-S100, or reovirus and ADU-S100, while the other tumor remained untreated. The combination reovirus and ADU-S100 treatment induced the significant regression of the treated tumor and the untreated distant tumor, suggesting a systemic immune response was activated by the combination treatment in a dual flunk tumor model (Fig. 4A). In contrast, IT injection of reovirus or ADU-S100 could not significantly delayed tumor growth in the untreated distal tumor. The combination treatment also significantly prolonged the survival of dual tumor-bearing mice (Fig. 4B). Furthermore, we found the survival of tumor-bearing mice was determined by the growth of the untreated tumor in this model. These results demonstrate the abscopal effect mediated by tumor immunity induced by the focal IT injection of reovirus in combination with ADU-S100, and that control of the untreated tumor is essential for achieving long-term survival.

Microenvironment of tumors treated with reovirus and a STING agonist.

To investigate whether the STING agonist restricted the propagation of reovirus in vivo, we performed immunofluorescence staining of reovirus protein in tumor samples extracted from mice. Three days after reovirus IT injection, reovirus protein was detected in the reovirus IT injected tumors but not in untreated distal tumor grafts (Fig. 5A). In addition to that, in tumors treated with the IT injection of reovirus followed by ADU-S100, the number of reovirus protein positive cells were almost same as those in tumors injected with reovirus alone (Fig. 5B). These results indicate that pretreatment with a STING agonist did not restrict the propagation of reovirus in the tumor despite the activation of STING signaling, and that reovirus infection had not spread to the untreated distal tumor within 3 days after reovirus IT injection. Immunohistochemical staining demonstrated that combination reovirus and ADU-S100 treatment induced significant apoptotic cell death (Fig. 5C, D). Enhanced granzyme B staining and upregulation of IFN-β mRNA expression in untreated tumors of the combination group indicated the combination IT injection boosted cytotoxic immune responses (Fig. 5E, F). These results suggest the combination reovirus and ADU-S100 treatment enhanced antitumoral immune responses via type I IFNs without reovirus elimination.

To obtain an overview of the immune cell profile within the tumor microenvironment with each treatment, the expression of cancer immunology-related genes of the tumors was analyzed using the NanoString nCounter PanCancer Immune Profiling. Gene clusters involved in immune cell functions, including adaptive immune cells, T cell function, and interferon responses, were found to be upregulated in the distal tumors of mice treated with reovirus combined with ADU-S100 compared to those in control (Fig. 6A). In contrast, a remarkable upregulation of cluster genes related to immune functions could not be detected in the distal tumors in the mice with reovirus or ADU-S100 single treatment. We also identified tumor-infiltrating lymphocytes using the NanoString nCounter data analysis and found that cytotoxic cells and CD8 cells scores were upregulated in the distal tumor in mice treated with the combination of reovirus and ADU-S100 compared to the distal tumor in mice treated with reovirus or ADU-S100 alone (Fig. 6B). These results imply that the enhanced anti-tumor activity in the tumor with a combination treatment reovirus and ADU-S100 was induced by accumulation of cytotoxic immune cells within tumor microenvironment.

To examine the profile of immune cells in the tumor microenvironment that might promote enhanced anti-tumor immunity in the combination group, we performed immunohistochemical staining of tumors treated with reovirus alone or in combination with ADU-S100. The proportions of M1 and M2 tumor-associated macrophages (TAMs) infiltrated in tumors were evaluated by determining M1 marker inducible nitric oxide synthesis (iNOS) and the M2 marker arginase-I with immunohistochemical staining. The expression of iNOS was not significantly upregulated in the tumor tissue of mice treated with the reovirus alone or in combination with ADU-S100 (Fig. 7A). The expression of arginase I was significantly downregulated in all treatment groups as compared with controls, and we detect the significant difference in the expression of arginase I between the mice treated with the reovirus alone and the mice treated with reovirus in combination with ADU-S100. (Fig. 7B). From these results, the polarization of TAMs seems to be partially involved in the anti-tumor immunity enhanced by the combination treatment. To further verify the immunotherapeutic effect of the T cell infiltration in tumor tissues of mice receiving the combination treatment, we also performed the tumor-infiltrating lymphocytes profiling in the tumor tissues of mice with the treatment. The number of CD8⁺ T cells was significantly increased in the treated and untreated tumors in all treatment groups compared with controls (Fig. 7D). By contrast, decreased numbers of CD4⁺ T cells were observed in the treated and untreated tumors of all treated groups (Fig. 7C). Next, we investigated the profile of immune cells in the tumor microenvironment using fluorescence-activated cell sorting (FACS). Tumor samples were collected and dissociated 3 days after IT injection of reovirus with or without pretreatment with ADU-S100, and subjected to FACS analysis. Significantly lower CD4:CD8 ratios of T cells were observed in the treated and untreated tumors from CT26 tumor-bearing mice treated with the combination reovirus and ADU-S100 IT injection compared with untreated controls, whereas no significant differences in the CD4:CD8 ratio were observed between the control and other treatment groups (Fig. 7E). We detect a significant reduction in the ratio of Foxp3⁺CD45⁺CD4⁺CD3⁺ regulatory T cells in tumors treated with reovirus alone or the combination reovirus and AUD-S100 compared to the control (Fig. 7F). Taken together, local treatment with reovirus in combination with a STING agonist increased the levels of infiltrating CD8⁺ T cells in the treated tumor and untreated distal tumor. The significant tumor regression and survival benefit observed in combination treated mice were mediated by an inflamed tumor microenvironment associated with the increased numbers of infiltrating cytotoxic T cells.

CRC is the third most diagnosed cancer worldwide according to the database of GLOBOCAN 2018, and in Japan, it is a leading cause of cancer death. Although the outcome of inoperable CRC has improved after the development of biological therapies, it is still difficult to control CRC. In addition to the limited efficacy of conventional treatment for CRC, numbers of elderly CRC patients, who are intolerant to chemotherapy toxicity, are increasing. Under these circumstances, cancer immunotherapy has emerged as a promising next-generation cancer therapy to overcome obstacles related to conventional cancer treatment. Although cancer immunotherapy for microsatellite instability (MSI)-high CRC using immune checkpoint inhibitors was recently approved¹⁹, the proportion of MSI-high CRC is approximately 15–16% of all CRC cases^{20, 21}. Therefore, the development of novel cancer immunotherapies effective for CRC irrespective of MSI status is indispensable to improving the outcome of CRC patients.

The oncolytic effect of reovirus against human CRC cell lines was previously reported²² and another study demonstrated that CRC is a viable target for reovirus therapy mediated by direct and innate immune killing²³. As reported previously, reovirus induces immunogenic cell death²⁴ which might confer the utility of reovirus as a potential anti-tumor immune agent, and we also found that reovirus induced immunogenic cell death in CT26 (Fig. 2D, E). Preclinical studies using genetically modified reovirus^{25, 26} and combination treatment of reovirus with other anticancer agents^{5, 27, 28} reported enhanced anti-tumor activity and tumor targeting of reovirus. Based on these studies, multiple phase I/II clinical trials of reovirus alone or in combination with chemotherapy against CRC have been conducted²⁹. Of note, a phase I study of reovirus against metastatic CRC investigated tumor immune responses, and the analysis of blood and tumor samples from patients revealed that reovirus had an immunomodulatory effect that enhanced the destruction of tumor cells in addition to its oncolytic effect against tumor cells³⁰.

In this study, we used STING expressing murine colorectal CT26 cancer cells that are sensitive to reovirus (Fig. 1A). CT26 cells are used as a non-hypermutated/MSS model without mutations in mismatch repair genes³¹. Evaluation of the tumor-immune profile of murine syngeneic tumor models demonstrated CT26 cells are moderately immunogenic, which may explain why CT26 cells are not sensitive to PD-1 based immunotherapy³². Indeed, our study demonstrated a low level of CD8⁺ T cell infiltration within the non-treated CT26 tumor microenvironment (Fig. 6B, 7D, E). Regarding the immunogenic profile of tumors, the CT26 syngeneic mouse model is appropriate for evaluating our strategy. Our findings confirm the rationale for the combination reovirus and STING agonist treatment of CRC without an inflamed tumor microenvironment (Fig. 4A, B).

Mammalian cells express cellular pattern recognition receptors, which detect the presence of pathogens or molecules released by damaged cells. dsRNA viruses including reovirus are recognized by pattern recognition receptors, retinoic acid inducible gene-I, melanoma differentiation-associated protein 5, Toll-like receptors, and the dsRNA-activated protein kinase R, senses viral RNAs, which induce type I IFN responses that restrict virus replication and dissemination³³. Reovirus has developed several mechanisms to inhibit type I IFN responses allowing it to evade the innate immune system¹⁴. In this study, we demonstrated no significant upregulation of IFNβ mRNA expression, or phosphorylation and nuclear translocation of IRF3 in CT26 cells infected with reovirus (Fig. 2B, C). However, IFN-β mRNA expression was moderately upregulated in injected and distal tumors treated with the combination of reovirus and the STING agonist (Fig. 5E) despite the inhibitory effect of the virus on IFN-β. In the syngeneic mouse model, our results indicated the combination treatment utilized antitumor immunity mediated by the STING agonist instead of the induction of reovirus restriction by STING activity.

In the current study, we selected ADU-S100 as a STING agonist, because it is stable and activates all five human STING alleles¹⁶. ADU-S100 induced the phosphorylation of IRF3 and its translocation into the nucleus of CT26 cells that express STING (Fig. 2A, B). It also strongly enhanced IFN-β mRNA expression in CT26 cells, which was concentration dependent (supple mental Fig. 2).

A previous study reported that the IT injection of ADU-S100 induced antitumor immune responses by inflaming the tumor microenvironment³⁴. Sivick et al. optimized the dose of ADU-S100 IT required to induce the local activation and systemic expansion of tumor-specific CD8⁺ T cells³⁵. In that study, a high dose of ADS-S100 ablated the IT injected tumor but diminished systemic tumor-specific T cell responses that are required for durable anti-tumor immunity. Therefore, we used a low dose, 10 mM, of ADU-S100, which enhanced systemic antitumor immunity. The IT injection of ADU-S100 demonstrated anti-tumor efficacy in CT26-bearing mice regardless of the single or dual flank model (Figs. 3A, 4A). However, the low dose IT injection of ADU-S100 did not significantly prolong survival in double tumor-bearing mice compared with controls (Figs. 4B). Consistent with a previous study³⁵, our data suggest the efficacy of the IT injection of ADU-S100 alone might be limited as an inducer of systemic antitumor immunity.

In this study, we IT injected reovirus combined with a STING agonist to CT26-bearing mice. Therefore, the interaction between reovirus and the STING agonist occurred within the tumor microenvironment. Currently, how the STING axis is involved in immune responses induced by RNA viruses including reovirus is unknown. STING is a key DNA sensing molecule that induces type I IFN production in response to DNA viruses and aberrant cytosolic double-stranded DNA originating from cancer cells^{36, 37}. Sensing of RNA viruses also activates type I IFN responses that depend on retinoic acid inducible gene-I, melanoma differentiation-associated protein 5, and protein kinase R. Previous studies demonstrated that the downstream signaling components of the RNA and DNA sensing pathways are physically and functionally interconnected^{38, 39}. Recent studies also reported the role of STING in innate immune responses to RNA viruses, where it was required to restrict the replication of diverse RNA viruses including reovirus⁴⁰. In our mouse model, we did not observe a difference in the expression of reovirus protein between tumors treated with an IT injection of reovirus alone or reovirus combined with ADU-S100 (Fig. 5A, B) although our observation period of reovirus protein expression within the tumor was limited. We found that reovirus and ADU-S100 cooperatively enhanced greater anti-tumor immunity compared with reovirus or ADU-S100 alone, indicating pretreatment with ADU-S100 does not restrict reovirus propagation within tumors. The antiviral effect of STING might depend on the timing and the order of the administration of agents; therefore, the efficacy of the combination treatment of reovirus and STING agonist might be improved further.

In conclusion, we demonstrated oncolytic reovirus-induced tumor regression in a CT26 syngeneic tumor model, and that antitumor immunity was enhanced by combination treatment of reovirus and a STING agonist. This boosted antitumor immunity was partially mediated by IFN-induced T cell activation. Although further investigation is required to clarify the mechanism underlying STING agonist synergy with reovirus to provoke immune responses in the context of anti-tumor activity, our strategy using the combination treatment of reovirus and a STING agonist might have potential for immunotherapy against CRC.

Acknowledgements

We would like to thank Mrs. Yukimi Ito from Nagoya City University for her technical assistance. We thank J. Ludovic Croxford, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript.

Data availability statement

The data supporting this study's findings are available from the corresponding author (E.K.) upon reasonable request.

Ethics declarations

Conflicts of interest

All authors state that they have no conflicts of interest.

Author contributions

S.N., E.K., Y.M., and H.K. were involved with the conception and performance of experiments, statistical analysis, and writing the manuscript. M.T., T.S., and S.T. assisted with some of the experiments. R.N.J. and M.A. were involved with the conception and design of experiments. All authors reviewed and edited the manuscript.

Supplementary Material

Supplemental data for the article can be accessed on the publisher’s website.

Funding

This work was supported by Grants-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant Number 18K07951and 22K16002.

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There is NO conflict of interest to disclose.

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Reovirus combined with a STING agonist enhances anti-tumor immunity in a mouse model of colorectal cancer

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Abstract

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Introduction

Materials And Methods

Reagents

Cell culture

Cell proliferation assay

Quantitative reverse transcription–polymerase chain reaction

Western blotting

Detection of ICD Markers

Immunofluorescence

Mouse tumor models

Flow cytometry

Immunohistochemistry

Multiplex gene expression analysis

Statistical analysis

Results

Induction of immunogenic cell death in CT26 cells by reovirus

Discussion

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References

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Supplementary Files

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