Fig 1.
Mutation of Trp53 and Kras in C57Bl/6 mice results in rapid tumour formation.
(A-B) Sub-periosteal lenti-cre injection into the tibialis anterior muscle of C57Bl/6 mice results localized tumour growth involving the entire hindlimb. (C) Tumours develop in greater than 90% of injected mice with a short latency of 2–3 months from lenti-virus injection until tumours are palpable. (D-E)10x and 200x magnification photomicrographs of UPS tumours showing a malignant, highly pleomorphic spindle cell sarcoma. (F-G) Immunohistochemical studies show strong actin staining and patchy Myogenin expression, consistent with a diagnosis of UPS. White arrows delineate the tumour margin on both gross necropsy and low magnification microscopy.
Fig 2.
Transcriptomic analyses show murine sarcomas resemble human UPS.
(A) Unsupervised hierarchical clustering of murine sarcoma RNAseq data with that of muscle lineage human soft tissue sarcoma shows murine sarcomas group with human UPS. (B-C) Additional gene set enrichment analyses the top differentially expressed genes of spontaneous murine tumours sarcomas is most similar to human UPS.
Fig 3.
Cell lines derived from spontaneous UPS tumours are easily engrafted in the hindlimb and lung of naïve, immunologically competent C57Bl/6 mice.
(A-B) Following injection of 100,000 UPS cells into the hindlimb of C57Bl/6 mice, tumours are detectable via bioluminescence imaging and result in large palpable tumours by three weeks. (C-E) Routine histology and immunohistochemistry of cell line derived tumours is consistent with UPS and similar to spontaneous UPS tumours. (F-G) Tail vein injections result in bioluminescence detectable tumours in the lung with multiple tumour lung nodules on gross examination of lung tissue. (H-J) Routine histology and immunohistochemistry of cell line derived lung tumours is consistent with UPS and similar to spontaneous UPS tumours.
Fig 4.
Spontaneous and cell line derived murine UPS tumours are macrophage rich and lymphocyte poor.
(A) Using FACS analyses, macrophages are the dominant intra-tumoural immune population (14%), whereas lymphocyte numbers are low (<2%). (B) In a separate analysis comparing spontaneous UPS, cell lined derived (transplanted) UPS and the inflamed M3-9-M ERMS model, CD4 and CD8 lymphocyres are low (1–4%) and significantly less than the infamed M3-9-M-Female model (6–9%). The cell line UPS model is more inflamed and contains more lymphocytes than the spontaneous model, althogh these differences did not reach stastistical significance. (C-D) NanoString® immune profiling of UPS tumours also demonstrates high macrophage scores, and negative lymphocyte, NK cell, dendritic cell and adaptive immune pathway scores compared to an inflamed sarcoma model.
Fig 5.
Murine UPS tumour growth is unaffected by lymphocyte deficiency and resistant to immunological checkpoint blockade therapy.
(A-B) Survival and growth kinetics of UPS cells engrafted into lymphocyte deficient Rag2 KO mice were no different from control C57Bl/6 mice. (C) Despite 45–55% of CD4 and CD8 TILs expressing PD1, UPS bearing mice were resistant to anti-PD1 and anti-CTLA-4 therapy, whereas survival was 66% in the inflamed M3-9-M model similarly treated with anti-PD1 (D-F).