(P 106) CHARACTERIZATION OF AN IMMUNE COMPETENT, MURINE MODEL 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, more pre-clinical, immune competent models must be developed. Our laboratory has developed a new murine model of STS through lenti-viral cre-recombinase mediated gene knockouts of PTEN and p53, and activation of the KRAS oncogene (Trp53fl/fl; Ptenfl/fl; KrasG12D/+). Herein, we describe the histological characteristics and immune infiltrates of this novel STS in comparison to the previously published KP model of UPS.

Methods: The Trp53fl/fl; Ptenfl/fl; KrasG12D/+ mice were generated by breeding B6.129P2-
Trp53tm1Brn/J (Trp53fl), B6.129S4-Krastm4Tyj/J (KrasG12D), and B6.129S4-Ptentm1Hwu/J
mice. Once mice reached 6-8 weeks of age, 10μl of 1x10^8 pfu a CMV-driven cre-recombinase plasmid (lenti-cre) was administered in the deep margin on the tibialis anterior muscle of the right hindlimb following proximal-lateral tibia periosteal scratch with a 25-gauge needle to induce spontaneous tumor development. A female spontaneous primary STS tumor was processed to develop the cell line, KPP1053. Syngeneic tumors were developed through the orthotopic engraftment of 100,000 KPP1053 cells in the hindlimb (Fig. 1). As a control murine STS model for comparison, we will use Trp53fl/fl; KrasG12D/+ (KP) that our laboratory has previously characterized an immune competent murine model of STS through mutations to Trp53fl/fl; KrasG12D/+. The KP model is characterized as having an abundance of myeloid cells, with few tumor infiltrating lymphocytes, and poor response to immunotherapy. Spontaneous and cell line KP tumors are developed using the same strategy as above and used controls in comparison to spontaneous Trp53fl/fl; Ptenfl/fl; KrasG12D/+ and syngeneic KPP1053 tumors, respectively. Trp53fl/fl; Ptenfl/fl; KrasG12D/+, KPP1053, and KP spontaneous and syngeneic tumors were harvested for flow cytometry, histology, and mRNA sequencing at a tumor volume of 500-800mm^3. Assays to assess the systemic cytokine signatures of tumor bearing mice are currently taking place.

Results: Spontaneous tumors developed in 23/25 mice within 22-62 days of lenti-cre injection, which is significantly faster than lenti-cre induced KP tumors (p < 0.0001; Fig. 2A). Of mice that were orthotopically engrafted with KPP1053 cells, 100% developed primary STS tumors within 7 days of engraftment reaching humane endpoint by day 14, which is similar to KP cell line derived tumors (Fig. 2B). All spontaneous tumors retained similar histological features, with negative cytokeratin staining, focal or patchy desmin staining, and positive smooth muscle actin staining (Figs. 2C-E). In assessing immune infiltrates, flow cytometry revealed that macrophages and dendritic cells were similar across tumor models (Figs. 3A & B). However, KPP1053 syngeneic tumors appeared to have an elevated infiltration of neutrophils (Fig. 3C). Additionally, Trp53fl/fl; Ptenfl/fl; KrasG12D/+ spontaneous tumors had higher proportions of CD8+ T-cells compared to the KPP1053 syngeneic tumors (Fig. 3D), but no differences in CD4 T cells (Fig. 3E). However, in comparing KP syngeneic tumours to KPP1053 syngeneic tumors, there were no differences in lymphocytes, which was also shown for KP and Trp53fl/fl; Ptenfl/fl; KrasG12D/+ spontaneous tumors.

Conclusion: We have generated a new immune competent murine model of STS. This new model shows similar levels of myeloid cells and lymphocytes compared to the KP model of STS. Thus, this provides another murine STS model to assess pre-clinical treatments. This allows for more models to improve generalizability of study findings. Future experiments assessing the efficacy of immune checkpoint inhibitor immunotherapy and response to radiation therapy are ongoing.