Differential Immune Microenvironments and Response to Immune Checkpoint Blockade among Molecular Subtypes of Murine Medulloblastoma

Abstract

Purpose: Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma, the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and group 3 medulloblastoma for preclinical evaluation in immunocompetent C57BL/6 mice.

Experimental design and results: Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid-derived suppressor cells, and tumor-associated macrophages in murine SHH model tumors compared with group 3 tumors. However, murine group 3 tumors had higher percentages of CD8(+) PD-1(+) T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial group 3 tumors compared with SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1(+) peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3(+) T cells within the tumor microenvironment.

Conclusions: This is the first immunologic characterization of preclinical models of molecular subtypes of medulloblastoma and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment.

©2015 American Association for Cancer Research.

Figures

Figure 1. Characterization of markers of activation and antigen presentation in Ptch1 MB and NSC MB

Freshly dissociated tissue from moribund tumor-bearing animals and cerebella from normal animals was measured for markers of immune activation and antigen presentation by multi-color flow cytometry. Representative flow data with gating strategy are shown for each marker (left). Histograms of all samples for each tissue type are also shown (right). Percentages of CD11c+ cells costained with: (A) CD80, (B) CD40 and (C) I-A/I-E (MHC Class II). Percentages of CD11c, CD80+ were not significantly different between the two subtypes (p=0.17) but percentages of CD11c, CD40+ and CD11c, I-A/I-E+ cells were significantly higher in the Ptch1 MB subtype compared to the NSC MB subtype (p=0.04 and 0.03 by unpaired t-test, respectively).

Figure 2. Characterization of infiltrating CD4 and CD8 T-cells in Ptch1 MB and NSC MB

T-cell infiltration in freshly dissociated tissue from moribund tumor-bearing animals and cerebella from normal animals was measured by multi-color flow cytometry. Representative flow data with gating strategy are shown for each marker (left). Histograms of all samples for each tissue type are also shown (right). (A) Overall CD4+ and (B) CD8+ frequencies were significantly higher in Ptch1 MB tumors (p=0.0088 and 0.0034 by unpaired t-test, respectively). (C) Proportions of CD4+CTLA-4+ were not significantly different between the two subtypes (p=0.64), but (D) CD8+PD-1+ of CD3 T cells was significantly higher in NSC MB tumor bearing animals (p=0.0049 by unpaired t-test).

Figure 3. Evaluation of PD-L1 expression in Ptch1 MB and NSC MB

Freshly dissociated tumor from moribund tumor-bearing animals was stained for PD-L1 expression. Representative histograms NSC MB (left) and Ptch1 MB (right). (A) Percentage of PD-L1 expression on total cells was significantly higher in the Ptch1 MB subtype compared to the NSC MB subtype (p=0.04 by unpaired t-test). (B) PD-L1 co-expression on infiltrating myeloid cells was also significantly higher in the Ptch1 MB subtype (p=0.03 by unpaired t-test). (C) Myeloid derived suppressor cells and (D) tumor associated macrophages infiltration frequencies were significantly higher in the Ptch1 MB subtype compared to the NSC MB subtype (p=0.01 and 0.03 by unpaired t-test, respectively).

Figure 4. Immunologic differences within tumor microenvironment do not affect immunogenicity of total tumor antigens of both MB subtypes

Animals were implanted with the minimum tumorigenic dose of either NSC MB or Ptch1 MB tumor cells. (A) Splenocytes from tumor-bearing animals were harvested at Day 16 (NSC MB) or Day 21 (Ptch1 MB) and expanded with primary dendritic cells pulsed with total tumor RNA (ttRNA). (B) Splenocytes expanded with DCs pulsed with Ptch1 MB ttRNA or (C) NSC MB ttRNA showed specific Th1 responses upon restimulation with tumor targets.

Figure 5. Anti-PD-1 blockade confers superior antitumor treatment effect in NSC MB tumor bearing animals

(A) Animals were implanted with the minimum tumorigenic dose of either NSC MB or Ptch1 MB tumor cells and administered CTLA-4 and PD-1 blocking antibodies, both alone and in combination. (B) Ptch1 MB tumor bearing animals did not respond to either CTLA-4 or PD-1 blockade whereas (C) NSC MB tumor bearing animals treated with anti-PD-1 alone and in combination with CTLA-4 blockade showed significant extension in median survival (p=0.02 and 0.009, respectively, by log-rank test, N=7 per group). (D) Explanted NSC MB and Ptch1 MB tumors from untreated and anti-PD-1 treated animals were stained for PD-1 and PD-L1 IHC. (E) Explanted NSC MB and Ptch1 MB tumors from untreated and anti-PD-1 treated animals were evaluated for differences in Ki67 and cleaved caspase-3 staining by IHC, with a significant decrease in Ki67 positive cells in anti-PD-1 treated NSC MB tumor bearing mice (top right, p=0.02 by unpaired t-test), but no significant differences in caspase-3 reactivity in both MB subtypes (bottom right).

Figure 6. Anti-PD-1 blockade acts systemically and results in increased population of CD3 T cells within the tumor microenvironment

(A) Representative flow cytometric analyses of anti-PD-1 mAb staining via mIgG1 detection on CD3+ cells within the tumor, spleen, cervical lymph node (CLN) and inguinal lymph node (ILN). A rat anti-mouse IgG1 antibody was used to detect the anti-PD-1 mAb because it is a mouse IgG1 isotype. (B) Quantification of anti-PD-1 mAb positive CD3 T cells within the tumor and lymphoid organs of symptomatic tumor-bearing NSC and Ptch1 MB untreated controls and anti-PD-1 treated animals. (C) PD-1 blockade in both Ptch1 MB and NSC MB treated animals resulted in significant increases in CD3 infiltration (p=0.002 and 0.01 by unpaired t-test, respectively). (D) Representative flow cytometric analyses (left) and histogram (right) of significantly increased CD3+ PD-1- infiltration in anti-PD-1 treated animals in both the Ptch1 and NSC MB subtypes (p=0.01 and 0.02 by unpaired t-test, respectively). (E) PD-1 blockade in both Ptch1 MB and NSC MB treated animals result in significant increases in CD8 infiltration (p=0.004 and 0.01 by unpaired t-test, respectively). (F) PD-1 blockade in both Ptch1 MB and NSC MB treated animals resulted in increases in CD4 infiltration, with a significant change in the NSC MB subtype (p=0.009 unpaired t-test).