(P 327) CHARACTERIZATION OF TWO NOVEL IMMUNE COMPETENT, MURINE MODELS OF SOFT TISSUE SARCOMA

Objective: Soft tissue sarcomas (STS) are rare and aggressive tumors that are notoriously difficult to treat as they rarely respond to conventional systemic therapies and require extensive operations. To achieve a stronger understanding of STS biology and to investigate new therapeutic interventions for this disease, pre-clinical mouse models are essential. Previous studies have developed various transgenic mouse models of bone sarcomas and STS. However, these models are limited by immune deficiency, long tumor latency, multiple tumors in diverse anatomical locations, and mixed genetic backgrounds. Our laboratory has developed two novel murine models of STS. Herein, we describe the histological characteristics and immune phenotypes of these two new models.

Methods: We developed two novel mouse models for this study; 1) Trp53fl/fl; Ptenfl/fl; KrasG12D/+ mice, and 2) Trp53fl/fl; Ptenfl/fl mice. Additionally, we used the Trp53fl/fl; KrasG12D/+ mouse model previously published by our group as a reference tumor model. This model is characterized as having an abundance of myeloid cells, few tumor infiltrating lymphocytes, and poor response to immunotherapy. Once mice from each group reached 6-8 weeks of age, we performed 10-20 scratches to the periosteal area of the proximal-lateral tibia. Next, 10μl of 1x10^8 pfu a CMV-driven cre-recombinase plasmid (lenti-cre) was administered in the deep margin on the tibialis anterior muscle to induce tumor development. One female inducible primary STS tumor from each group was processed to develop a cell line for syngeneic tumor experiments. Syngeneic tumors were developed by engrafting 100,000 STS cells into the right hindlimb or tail vein of C57Bl/6 mice. Inducible and syngeneic tumors were harvested at a volume of 500-800mm^3 to be assessed by flow cytometry, histology, and bulk RNA sequencing.

Results: Inducible tumors developed in the Trp53fl/fl; Ptenfl/fl; KrasG12D/+ mice ~30-40-days, whereas Trp53fl/fl; Ptenfl/fl tumors developed between ~100-350 days (Figure 1A). When Trp53fl/fl; Ptenfl/fl; KrasG12D/+ derived cells were engrafted into the hindlimb of C57Bl/6 mice, tumors reached experimental endpoint in ~2-weeks, whereas Trp53fl/fl; Ptenfl/fl derived cells in the hindlimb produced tumors that reached experimental endpoint by ~3-weeks (Figure 1B). While no evidence of metastasis was found in any of the models, injecting STS cell lines into the tail vein of C57Bl/6 mice yielded Trp53fl/fl; Ptenfl/fl; KrasG12D/+ lung tumors (Figure 1C). However, no Trp53fl/fl; Ptenfl/fl lung tumors have yet developed. Interestingly, both inducible and cell line tumors from both novel models resembled undifferentiated pleomorphic sarcoma (UPS) or myxoid UPS based on histological evaluation by sarcoma pathologists (Figure 2A). However, assessing sarcoma subtype using bulk RNA sequencing yielded more heterogeneous results, but showed that the Trp53fl/fl; Ptenfl/fl; KrasG12D/+ model aligns closely with human leiomyosarcoma (Figure 2B). In assessing immune infiltrates, flow cytometry revealed that myeloid cells and macrophages are the most prevalent immune cells across all models, with a small proportion of lymphocytes (Figures 3A & 3B). We compared our bulk RNA sequencing data to the “sarcoma immune classes” established by Petitprez et al (Nature, 2020). We found that the tumors largely clustered in class A, which is known to be an immune suppressive phenotype (Figure 3C). Lastly, the syngeneic models did not respond to anti-PD1 immune checkpoint blockade (Figure 4).

Conclusion: There is a need for new mouse models of STS that are inducible, transplantable and immune competent. We have generated two novel murine models of STS that meet these needs. These models appear to be UPS/myxoid UPS based on histological characteristics but show a more heterogeneous transcriptional signature in comparison to human STS subtypes. These models appear to be an immune suppressed phenotype. These provide models to study UPS biology and novel ways to sensitize immune suppressed UPS tumors to immunotherapy, essential for improving treatment options for UPS patients.