(P 293) GENERATION OF A SPONTANEOUS IMMUNOCOMPETENT MOUSE MODEL OF LIPOSARCOMA

Objective: Liposarcoma (LPS) is one of the most common types of soft tissue sarcoma. The most common subtypes are well-differentiated (WDLPS) and dedifferentiated (DDLPS) liposarcoma which are both characterized in over 90% of cases by genomic amplification of murine double minute 2 (MDM2), a negative regulator of the tumor suppressor p53, while 7% of human WDLPS/DDLPS instead have a TP53 mutation. Patients with metastatic disease have poor overall survival due to the low response rates to systemic chemotherapy. While immunotherapy has shown tremendous efficacy in solid tumors, research focusing on the immune biology of sarcoma has been slower than other tumor types. Genetically engineered mouse models (GEMMs) are needed in an effort to dependably mimic the human stroma and tumor microenvironment (TME) of liposarcoma. Our lab has set out to develop a spontaneous WDLPS/DDLPS GEMM along with syngeneic cell lines derived from these tumors in order to highly impact the sarcoma community by providing models useful in the pursuit of novel and effective immunotherapeutics for patients.

Methods: A viral construct with an adipocyte-specific promoter sequence driving an eGFP-Myc tag-Cre fusion molecule was used with adenovirus-associated virus (AAV8) vector which elicits low immune response. While PTEN mutations are not found in human LPS, ~80% of DDLPS have phosphorylated AKT. By knocking out Pten, the effect of constitutive phosphatidylinositol-3-kinase (PI3K)-AKT signaling is mimicked. We injected virus into flanks of Ptenf/f;Trp53f/f C57BL/6 mice, resulting in the development of spontaneous tumors. We performed H&E (n=24) and whole exome sequencing (WES) (n=6) on mouse tumors. Bulk RNA sequencing was done on 4 mouse and 9 human DDLPS with matched species-specific normal fat. Gene set enrichment analysis (GSEA) for the Molecular Signatures Database (MSigDb) Hallmark pathways of the differentially expressed genes (DEGs) in both mouse and human DDLPS were compared to respective normal fat. Tumor infiltrating lymphocytes (TILS) were analyzed by flow cytometry and multicolor immunohistochemistry (IHC).

Results: Spontaneous tumors were identified starting at 4 months from AAV8 injection. Histology confirmed all tumors had characteristics of human LPS with 60% DDLS, 10% WDLPS, and 30% mixed WD/DDLPS. Somatic copy number variation (CNV) calling from WES data revealed expected focal deletions of exon 5 in Pten (mean log2ratio = -5.6) and exons 2-5 in Trp53 (mean log2ratio = -5.9), and similar mutational load as published human LPS data. GSEA analysis revealed both mouse and human DDLPS, compared to respective normal fat, showed similar positive and negative pathway enrichment, supporting the significance of this mouse model to human disease (Fig. 1). Flow cytometry revealed 70% CD45+ TILS out of total live cells, of which 21% were CD3+ T cells with equal CD8:CD4 ratio, and 67% were CD11b+ myeloid cells with a small proportion of F4/80+ macrophages. Multicolor IHC confirmed a majority of myeloid cells, however, tumors with the highest number of TILs had areas with increased T cells and greater absolute number of CD8+ T cells compared to those with low TILs. Syngeneic cell lines were generated and consistently produced primary DDLPS tumors when injected orthotopically as well as lung metastasis via tail vein injection.

Conclusion: Primary mouse LPS generated with focally-delivered Cre recombinase and targeted gene deletion mimicked the biological behavior of human LPS and had similar histology, mutational burden, similarly altered pathways evaluated by GSEA, and immune cell subsets in the TME. Syngeneic mouse LPS cell lines can be used for both primary allografts and lung metastasis models in immune competent recipients. This model will be used to determine the key players in immune responses to LPS and preclinical testing of single agent and combination immuno-oncology therapies in order to inform the development of the critically needed effective therapies for liposarcoma patients.