Novel murine glioblastoma models that reflect the immunotherapy resistance profile of a human disease

Abstract

Background: The lack of murine glioblastoma models that mimic the immunobiology of human disease has impeded basic and translational immunology research. We, therefore, developed murine glioblastoma stem cell lines derived from Nestin-CreERT2QkL/L; Trp53L/L; PtenL/L (QPP) mice driven by clinically relevant genetic mutations common in human glioblastoma. This study aims to determine the immune sensitivities of these QPP lines in immunocompetent hosts and their underlying mechanisms.

Methods: The differential responsiveness of QPP lines was assessed in the brain and flank in untreated, anti-PD-1, or anti-CTLA-4 treated mice. The impact of genomic landscape on the responsiveness of each tumor was measured through whole exome sequencing. The immune microenvironments of sensitive (QPP7) versus resistant (QPP8) lines were compared in the brain using flow cytometry. Drivers of flank sensitivity versus brain resistance were also measured for QPP8.

Results: QPP lines are syngeneic to C57BL/6J mice and demonstrate varied sensitivities to T cell immune checkpoint blockade ranging from curative responses to complete resistance. Infiltrating tumor immune analysis of QPP8 reveals improved T cell fitness and augmented effector-to-suppressor ratios when implanted subcutaneously (sensitive), which are absent on implantation in the brain (resistant). Upregulation of PD-L1 across the myeloid stroma acts to establish this state of immune privilege in the brain. In contrast, QPP7 responds to checkpoint immunotherapy even in the brain likely resulting from its elevated neoantigen burden.

Conclusions: These syngeneic QPP models of glioblastoma demonstrate clinically relevant profiles of immunotherapeutic sensitivity and potential utility for both mechanistic discovery and evaluation of immune therapies.

Keywords: 1; 4; Anti; CTLA; PD; anti; glioblastoma; immunotherapy.

Figure 1.

Four QPP lines grow in both immune-deficient and immunocompetent mice. 1 × 10⁶ cells of each QPP cell line were subcutaneously implanted in (A) C57BL/6J (n = 4) and (B) B6 Rag1 knockout (RagKO) mice (n = 2) and their growth was measured with calipers. Each curve and error bar represents the mean and SEM of each QPP line. A comparison of growth kinetics between C57BL/6J and B6 Rag1 knockout is also demonstrated (C).

Figure 2.

T cell checkpoint blockade sensitivity of flank-implanted QPP cell lines. 1 × 10⁶ cells were subcutaneously implanted on day 0 and mice were treated i.p.with anti-PD-1 (29F.1A12 or RMP1-14) or anti-CTLA-4 (9H10) antibody or PBS on days 7, 10, and 13. Tumors were measured with calipers, and a tumor measuring more than 800 mm³ is considered a death event. Each curve represents the survival curve from 2 experiments of 5 mice per group.

Figure 3.

QPP lines engraft orthotopically and are differentially sensitive to checkpoint blockade. 1 × 10⁵ (QPP7), 2 × 10⁵ (QPP4 and 5) and 3 × 10⁵ (QPP8) cells were stereotactically implanted into the right striatum of C57BL/6J mice. On days 7, 10, and 13, mice were treated i.p. with anti-PD-1 (29F.1A12 or RMP1-14) or anti-CTLA-4 (9H10) antibodies or PBS. Survival and significance are shown based on the Log-Rank (Mantel-Cox) test. Two (QPP4 and 5) to 3 (QPP7 and 8) experiments were performed with 4–5 mice per group.

Figure 4.

Comparative infiltrates analysis of flank versus brain implanted QPP8. 1 × 10⁶ cells and 5 × 10⁵ cells of QPP8 were implanted in the flank or brain respectively. Mice received 3 doses of i.p. anti-CTLA-4 (9H10) or anti-PD-1 (RMP1-14) antibody or PBS every 3 days and were sacrificed 2 days after the last treatment. Tumor-infiltrating immune cells were extracted, enriched, and stained with phenotypic and functional markers before analysis by flow cytometry. Immune infiltrate density fold changes compared to the control group (A) and T cell subset frequencies (B) in response to T cell checkpoint blockade in both flank and brain tumors are shown as mean ± SEM. Fold changes of CD8 T cells over suppressive subset ratios (C) and functional marker MFI fold changes compared to the matched control group for each immune subset in the indicated site were calculated and presented on a log scale (D). Data reflect 3 experiments with 3–8 mice per group. (*P < .05 by Student’s t-test).

Figure 5.

QPP7 remains checkpoint sensitive in the CNS despite adaptive upregulation of Arginase. 1 × 10⁵ QPP7 cells or 5 × 10⁵ QPP8 cells were implanted in the right striatum. Mice received 3 doses of i.p. anti-CTLA-4 (9H10), anti-PD-1 (RMP1-14) antibody, or PBS every 3 days and sacrificed 2 days after the last treatment. Tumor-infiltrating immune cells were extracted, enriched, and stained for phenotypic and functional markers before analysis by flow cytometry. Immune infiltrate density fold changes compared to the control group (A) and T cell subset frequencies (B) in response to T cell checkpoint blockade in both QPP7 and QPP8 brain tumors are shown as mean ± SEM. Fold changes of CD8 T cells over suppressive subset ratios (C) and functional marker MFI fold changes compared to the matched control group for each immune subset in the indicated site were calculated and presented on a log scale (D). Data reflect 3 experiments with 3–8 mice per group (*P < .05 by Student’s t-test). 6 × 10⁵ QPP7 and QPP8 cells (dash line) were seeded in 10 mL of media either with or without arginine and were incubated for 6 days before image analysis by fluorescent and light microscopy (E) and viable cell number quantified (F). Graph shows mean and SEM of the ratios of the cell numbers incubated with and without arginine from 3 independent experiments (*P < .05 by Student’s t-test). (^†Separate evaluation of PD-1 expression in Supplementary Figure S7).

Figure 6.

Genomic profile of QPP cell lines. DNA extracted from the cultured QPP lines was sent for whole exome sequencing (Novogene). Tumor mutational burden in each QPP line is presented as the number of single-nucleotide variants (SNV) in coding regions and non-synonymous SNV (A); as well as the number of small insertion and deletion (Indels) and frameshifts by Indels (B). Neoantigens were predicted MuTect and NetMHC program and the numbers of the predicted neoantigens are presented (C). DNA extracted from the CD4 T cells and CD8 T cells isolated from QPP7 and 8 brain tumors were sent for TCR sequencing (ImmunoSeq). TCR richness (D) and Simpson clonality (E) of the intratumoral CD4 and CD8 T cells were calculated and are presented (*P < .05 by Student’s t-test).