Distinct Tumor Microenvironments Are a Defining Feature of Strain-Specific CRISPR/Cas9-Induced MPNSTs

Abstract

The tumor microenvironment plays important roles in cancer biology, but genetic backgrounds of mouse models can complicate interpretation of tumor phenotypes. A deeper understanding of strain-dependent influences on the tumor microenvironment of genetically-identical tumors is critical to exploring genotype-phenotype relationships, but these interactions can be difficult to identify using traditional Cre/loxP approaches. Here, we use somatic CRISPR/Cas9 tumorigenesis approaches to determine the impact of mouse background on the biology of genetically-identical malignant peripheral nerve sheath tumors (MPNSTs) in four commonly-used inbred strains. To our knowledge, this is the first study to systematically evaluate the impact of host strain on CRISPR/Cas9-generated mouse models. Our data identify multiple strain-dependent phenotypes, including changes in tumor onset and the immune microenvironment. While BALB/c mice develop MPNSTs earlier than other strains, similar tumor onset is observed in C57BL/6, 129X1 and 129/SvJae mice. Indel pattern analysis demonstrates that indel frequency, type and size are similar across all genetic backgrounds. Gene expression and IHC analysis identify multiple strain-dependent differences in CD4+ T cell infiltration and myeloid cell populations, including M2 macrophages and mast cells. These data highlight important strain-specific phenotypes of genomically-matched MPNSTs that have implications for the design of future studies using similar in vivo gene editing approaches.

Keywords: CRISPR/Cas9; MPNST; mouse models; sarcoma; tumor microenvironment.

Figure 1

Host strain determines tumor onset but does not alter tumor growth kinetics. (A) Kaplan–Meyer curve of tumor-free survival. Formation of Nf1/p53-deleted malignant peripheral nerve sheath tumors (MPNSTs) is accelerated in BALB/c mice. Tumor initiation occurs within a similar timeframe in mice from 129/SvJae, C57BL/6, and 129X1 backgrounds. (B) Growth kinetics are similar across all background strains for genetically-identical MPNSTs (n= 6–8 tumors per strain). Growth rates are calculated as the number of days required for tumors to double from an initial volume of 150 mm³. 129X1 (red circles), C57BL/6 (black triangles), BABL/c (white squares), and 129/SvJae (blue triangles). (C) Representative images of MPNSTs from different host strains stained for H&E (20×), S100 (20×), and Ki67 (20×). (D) Quantification of Ki67 confirms that background strain does not alter the rate of tumor proliferation (n= 5 tumors per strain). (B,D) analyzed by one-way ANOVA with Tukey’s multiple comparison test.

Figure 2

CRISPR/Cas9-induced insertions and deletions detected in Nf1 and p53 in MPNST-derived cell lines from different genetic backgrounds. Indel pattern analysis of the sgRNA-targeted regions of Nf1 (A) and p53 (B) demonstrates disruption of genomic targets in all tumors. The majority of indels detected in both Nf1 and p53 are frameshift mutations that result in inactivation of targeted proteins.

Figure 3

The MPNST immune landscape is determined by genetic background. (A) Levels of CD8+ T cells in terminally-harvested MPNSTs are similar across all host strains. (B) Infiltration of CD4+ T cells are significantly lower in tumors from C57BL/6 mice compared to MPNSTs in mice from 129X1, BALB/c, and 129/SvJae backgrounds. (C) Foxp3+ Tregs are detected at higher levels in tumors from 129X1 mice compared to C57BL/6 and BALB/c mice. (D) MPNSTs from BALB/c mice have significantly higher levels of infiltrating F4/80+ macrophages compared to C57BL/6 mice. (E) Mast cell infiltration is higher in tumors from BALB/c mice compared to 129/SvJae, C57BL/6, and 129X1 mice. Mast cell levels are lowest in MPNSTs from C57BL/6 mice. 129X1 (red circles), C57BL/6 (black triangles), BABL/c (white squares), and 129/SvJae (blue triangles). Analyzed by one-way ANOVA with Tukey’s multiple comparison test. A p-value of less than 0.05 is considered statistically significant and is denoted by “*” (n = 5 tumors per strain).

Figure 4

Expression of key genes in the MPNST microenvironment. (A) RT-qPCR analysis of markers for innate immunity, adaptive immunity, angiogenesis, and cytokine signaling in terminally-harvested tumors shows a large degree of heterogeneity between host strains and individual tumors. Samples are normalized to a single C57BL/6 tumor, shown as reference (n = 5 tumors per strain). (B) Expression analysis determines that MPNSTs from BALB/c mice express significantly higher levels of Arg-1 mRNA, a marker of immunosuppressive M2 macrophages, when compared to tumors from 129/SvJae, C57BL/6, and 129X1 mice. (C) In contrast, levels of Nos2 mRNA, a marker of M1 macrophages, is similar in tumors from all background strains. 129X1 (red circles), C57BL/6 (black triangles), BABL/c (white squares), and 129/SvJae (blue triangles). Analyzed by one-way ANOVA with Tukey’s multiple comparison test. A p-value of less than 0.05 is considered statistically significant and is denoted by “*”.