Abstract No. 6 Treatment of HCC by Multimodal In Situ Vaccination Using Cryoablation and a Plant Virus Immunostimulant

Abstract

To test the hypothesis that cryoablation combined with intratumoral immunomodulating nanoparticles from cowpea mosaic virus (CPMV) as an in situ vaccination (ISV) approach induces systemic antitumoral immunity in a murine model of hepatocellular carcinoma (HCC). A bilateral, subcutaneous HCC tumor model was established using RIL-175 cells in male C57BL/6 mice. Contralateral, untreated tumors were monitored for an abscopal effect. Mice were randomized into one of four groups: phosphate-buffered saline injected (Control), cryoablation only (Cryo), CPMV-treated only (CPMV), and cryoablation plus CPMV-treated (Cryo+CPMV), (N = 10-12/group). Ten days after tumor inoculation, CPMV was administered intratumorally (day 0) and then on days 3, 6 and 9. Cryoablation was performed on day 3. Tumor growth was measured with digital calipers. Systemic chemokine/cytokine concentrations were measured from tail bleed serum samples. Two mice from each group were sacrificed at 1 week post treatment initiation, and tumors and spleens were harvested for immunohistochemistry and flow cytometry, using innate and adaptive immune cell panels. One-way ANOVA was performed for all statistical comparisons among treatment groups. A P value of < 0.05 served as the threshold for statistical significance. At 2 weeks post treatment, Cryo+CPMV outperformed solo treatment arms and Control. For the treated tumor, Cryo+CPMV significantly delayed and inhibited tumor progression compared with tumors in the Control group (1.6- ± 0.9-fold vs 6.3- ± 0.5-fold; P< 0.0001). For the untreated tumor, only the Cryo+CPMV group showed statistically significant tumor growth reduction compared with Control (9.2- ± 0.9-fold vs 17.8- ± 2.1-fold; P = 0.01). This was associated with a transient increase in IL-10 and a significantly lower concentration of CXCL1 in the Cryo+CPMV group. Flow cytometry revealed an enrichment of natural killer (NK) cells, activated CD4+ and CD8+ T cells, and an increase in PD-1 expression on both CD4+ and CD8+ T cells in untreated tumors and/or spleens of the Cryo+CPMV group. Immunohistochemistry demonstrated a robust increase in tumor-infiltrating lymphocytes in the Cryo+CPMV group, especially into the center of the tumors. Cryoablation combined with intratumoral CPMV as a multimodal ISV approach had potent efficacy against this aggressive HCC tumor model, both to the treated tumor and to the contralateral untreated tumor, indicating an abscopal effect.