Multimodal reprogramming of the tumor microenvironment by MMR and dual checkpoint blockade in hepatocellular carcinoma models

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide, thus, there is an urgent need to develop more effective therapeutic options for this dismal condition. Tumor-infiltrating lymphocytes (TILs) are associated with improved response to immune checkpoint blockade in HCC, but their low abundance in most cases limits their therapeutic efficacy. Here, we demonstrate, in mice, that low-dose intratumoral immunovirotherapy with the trivalent measles, mumps, and rubella vaccine (MMR) induces superior tumor-growth delay and extended host survival compared to individually administered vaccines for measles, mumps, or rubella viruses. Further, our results show that MMR therapy synergizes with PD-1 and CTLA-4 blockade to reprogram the tumor microenvironment, resulting in increased CD8+ TIL infiltration and reduced PD-1 expression on TILs, among other effects. These changes in the immunological landscape translated into greater survival and more durable tumor-specific and memory immune responses for hosts. Comprehensive toxicology analysis revealed no evidence of MMR-induced liver or kidney toxicity after intrahepatic administration. This work reinforces an unrecognized role of MMR plus ICB in reprogramming the immune landscape in HCC through multimodal immune activation, providing a strong rationale for further development of MMR-based therapies for HCC.

Keywords: MMR vaccine; hepatocellular carcinoma; immune checkpoint blockade; innate and adaptive immunity modulation; tumor microenvironment.

Figure 1

Trivalent measles, mumps, and rubella vaccine (MMR) shows no systemic toxicity. (A) To assess MMR-related toxicities, naïve non-tumor-bearing mice were treated with an intrahepatic injection of MMR (1 × 10² TCID₅₀) or PBS. Plasma was collected at 24 h to assess markers of liver toxicity, nephrotoxicity, and electrolytes. (B) At 24 h post-treatment, body weights were not altered by intrahepatic MMR. Plasma markers of organ toxicity—albumin (C, ALB), alkaline phosphatase (D, ALP), alanine aminotransferase (E, ALT), amylase (F, AMY), total bilirubin (G, TBIL), blood urea nitrogen (H, BUN), calcium (I, CA+), Potassium (J, K+), and creatinine (K, CRE)—were not altered by intrahepatic MMR.

Figure 2

Trivalent measles, mumps, and rubella vaccine (MMR) did not affect blood cell counts and did not damage tissues. To assess MMR-related toxicities, naïve non-tumor-bearing mice were treated with an intrahepatic injection of MMR (1 × 10² TCID₅₀) or PBS. Blood cell counts were obtained at baseline (i.e., before intrahepatic injection; day 0) and on days 1, 7, and 21. Brain and liver tissues were harvested at the end of the experiment (21 days post-injection) and were stained and prepared for pathology analysis. (A-G).

Figure 3

Anti-tumor activity of vaccines for individual measles (MeV), mumps (MuV), and rubella (RuV) viruses and of the trivalent vaccine (MMR) in murine HCC Hepa 1–6 model. (A) Hepa 1–6 tumors treated with 3 intratumoral doses (1 weekly dose for 3 weeks) of MeV, MuV, RuV, MMR (1 × 10² TCID₅₀ per mouse), or PBS (n = 7/group). (B) Survival (shown as Kaplan-Meier curves) was monitored. (C-H) Hepa1–6 cells grown in culture were treated with MMR or individual viruses (MeV, MuV, or RuV) for 48 h before RNA extraction and qPCR amplification to characterize MHC class I and β-2-microglobulin (B2M) expression. (C, D) Expression of classical murine MHC class I (H2Kb, H2Db), (E) B2M, and (F-H) transporter associated with antigen-processing (TAP 1/2 and Tapbp) complex.

Figure 4

Trivalent measles, mumps, and rubella vaccine (MMR) combined with anti-PD-1 and anti-CTLA-4 prolongs survival in the murine R1LWT HCC subcutaneous (SQ) model. We investigated the anti-tumor effects of combining MMR with immune checkpoint blockade (ICB) in an immunocompetent HCC SQ mouse model (R1LWT). (A) We treated tumor-bearing female C57BL/6J mice with a weekly intratumoral injection of MMR (1 × 10² TCID₅₀) with or without ICB (intraperitoneal injections of anti-PD-1 and anti-CTLA4 antibodies; twice per week) for 3 weeks. (B) Survival was monitored. (C) shows a rechallenge result where all R1LWT-cured mice and naïve control mice were (re)challenged with SQ injection of R1LWT cells.

Figure 5

Trivalent measles, mumps, and rubella vaccine (MMR) in combination with antibodies that target PD-1 and CTLA-4 signaling enhances infiltration of cytotoxic T lymphocytes in R1LWT tumors. (A) Female C57BL6/J mice were implanted with R1LWT cells (n = 7/group). When the average tumor volume reached 80−120 mm³, PBS or MMR (1 × 10² TCID₅₀) was injected intratumorally on days 0, 7, and 14 with or without addition of immune checkpoint blockade (intraperitoneal injection of anti-PD-1 and anti-CTLA4 antibodies; twice per week, for 3 weeks). (B) Tumor volume was recorded weekly. Tumors were harvested at the end of the study for downstream analysis. (C) We used flow cytometry to analyze the effects on immune cell infiltration into the tumor microenvironment. (D-I) show levels of tumor immune infiltration (such as CD8+, PD-1+ CD44+) in ICB and the combined treatment groups.

Figure 6

Immunotherapy treatments affect macrophage polarization differently, shifting the balance between M1 and M2 phenotypes. Female C57BL6/J mice were implanted with R1LWT cells (n = 7/group). (A) When the average tumor volume reached 80−120 mm³, mice were administered intratumoral injections of PBS or MMR (1 × 10² TCID₅₀) on days 0, 7, and 14 with or without addition of immune checkpoint blockade (intraperitoneal injection of anti-PD-1 and anti-CTLA4 antibodies; twice per week, for 3 weeks). (B-H) We harvested tumors and used flow cytometry to analyze the effects on macrophage polarization in the tumor microenvironment.

Figure 7

Systemic administration of MMR in combination with anti-PD-1 and anti-CTLA-4 resulted in enhanced survival in a metastatic murine R2LWT HCC model. (A) A metastatic HCC orthotopic mouse model was established with 5 × 10⁵ luciferase-expressing R2LWT cells surgically implanted into the livers of female C57BL/6J mice (n = 7/group). When tumors reached 4−5 mm in diameter (approximately 7 days post-implantation), mice were randomly assigned to different study groups (PBS, MMR, αPD-1+αCTLA-4 antibodies, and MMR+αPD-1+αCTLA-4 antibodies) based on IVIS imaging. For 3 consecutive weeks (on days 0, 7, and 14) mice were intraperitoneally injected with PBS or MMR (1 × 10² TCID₅₀). (B) IVIS imaging shows tumor burden. (C) Survival was monitored. (D) A subset of cured mice was rechallenged with R2LWT injected subcutaneously and compared with naïve control mice that were challenged with R2LWT; tumor volumes were monitored.