Neoadjuvant PD-1 inhibition prior to partial cryoablation of murine hepatocellular carcinoma modulates the tumor microenvironment towards favorable immunological profiles

PURPOSE: To evaluate the impact of neoadjuvant systemic PD-1 immune checkpoint inhibition on the local immune response in residual tumors following partial cryoablation in a TIB-75 murine HCC model. METHODS: 48 BALB/c mice (6-12 weeks) were orthotopically implanted with TIB-75 cells to induce a single lesion of HCC. Mice were randomized into 4 treatment groups: (a) control, (b) anti-PD-1, (c) partial cryoablation, and (d) anti-PD-1 and partial cryoablation. Anti-PD-1 was administered on days 7, 9 and 11 post-inoculation, followed by partial cryoablation on day 13 and tumor harvest on day 18. The presence of T-cell subsets (CD3(+), CD4(+), CD8(+)), tumor-associated macrophages (CD68(+), CD206(+)), PD-1, and PD-L1 were assessed by histopathological analysis of immunohistochemistry. The percentage of positively stained cells within the tumor was determined using QuPath. RESULTS: Mice treated with anti-PD-1 (n=12) had greater infiltration of CD3(+), CD4(+) and CD8(+) T-cells into residual tumors than control (CD3(+): median 22.4% vs. 5.5%; p=<0.001, CD4(+): median 19.8% vs. 5.1%; p<0.001, CD8(+): median 8.2% vs. 3.1%; p=0.007). Partial cryoablation alone (n=12) increased CD206(+) M2-like macrophages (median 36.6% vs. 14.7%; p=0.03). Partial cryoablation combined with neoadjuvant anti-PD-1 (n=12) showed significantly higher infiltration of CD3(+) T-cells (median 14.3% vs. 4.5%; p=0.048) than partial cryoablation alone (n=12) and significantly lower PD-1 expression than anti-PD-1 alone (median 2.9% vs. 7.3%; p=0.004). CONCLUSION: In a mouse model of HCC, neoadjuvant PD-1 immune checkpoint inhibition can modulate the immunosuppressive tumor microenvironment observed after cryoablation. This highlights the potential of a combination therapy to treat both early- and advanced-stage HCC.