CHARACTERIZATION OF TWO IMMUNE-COMPETENT MURINE MODELS OF SARCOMA

Soft tissue sarcomas (STS) are rare and aggressive tumours 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 two novel murine models of STS through lenti-viral cre-recombinase mediated gene deletions of PTEN, p53, and activation of the KRAS oncogene. Herein, we describe the histological characteristics and immune infiltrates of these novel STS in comparison to the previously published KP model of UPS. C57Bl/6 mice with the following genetic mutations were used in this study: 1) Trp53fl/fl; Ptenfl/fl; KrasG12D/+ (KPP), 2) Ptenfl/fl; KrasG12D/+ (PTEN), and Trp53fl/fl; KrasG12D/+ (KP) mice. Once mice reached 6-8 weeks of age, 10μl of 1x108 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 tumour development. A female STS tumour from each group was processed to develop cell lines. These cell lines were also used to generate transplantable tumours by engrafting 105 STS cells in the hindlimb (Fig. 1). The KP tumour models (lenti-viral and cell line derived) have been previously established, and used as a control model for the KPP and PTEN models. All lenti-viral and cell lined tumours were harvested for flow cytometry, histology, and mRNA sequencing at a tumour volume of 500-800mm3. Some tumours formed bone but were not assessed by flow cytometry. Tumor Diagnostics: Thus far, only one inducible tumor for each KPP and PTEN models have been assessed by a pathologist. KPP was deemed poorly differentiated high-grade sarcoma infiltrating skeletal muscle. There are areas of osteoid formation which make this a high-grade osteosarcoma. PTEN was deemed poorly differentiated high-grade sarcoma involving skeletal muscle. Focal areas were reminiscent of osteoid formation, but there is not enough evidence to be an osteosarcoma. Thus, these models produce both bone and soft tissue sarcomas. Immune infiltrates: F4/80+ and CD11c+ myeloid cells were highly abundant in all tumours. There were no differences in F4/80+ or CD11c+ myeloid cells in inducible tumours. There are significantly fewer F4/80+ myeloid cells in transplantable KPP. However, tumour macrophages can express CD11c, which may confound this data. Ly6G+ myeloid cells were a smaller proportion of tumour immune infiltrates. Lymphocytes represented a smaller proportion of immune cells within the tumour microenvironment. In inducible tumours, KPP tumours had higher CD8+ lymphocytes than KP tumours. In transplantable tumours, PTEN tumors had the most CD8+ lymphocytes. CD19+ lymphocytes accounted for < 1 % of CD45+ cells. CD19+ lymphocytes were higher in transplantable KPP than KP tumours. We were able to develop two inducible and transplantable immune competent mouse models of sarcoma. These models have mutations that are among the common mutations in human sarcomas. These models appeared to have elevated lymphocyte infiltration, suggesting more immunogenic tumour models compared to KP models. Having multiple models with varying immunogenicity allows for improved ability to test preclinical immunotherapies (e.g., immune checkpoint blockade and cGAS-STING activation). For any figures or tables, please contact the authors directly.