A Trp53fl/flPtenfl/fl mouse model of undifferentiated pleomorphic sarcoma mediated by adeno-Cre injection and in vivo bioluminescence imaging

Abstract

Genetic mouse models of soft tissue sarcoma provide critical insights into disease pathophysiology, which are oftentimes unable to be extracted from human tumor samples or xenograft models. In this study we describe a mouse model of soft tissue sarcoma mediated by adenoviral-Cre recombinase injection into Trp53fl/fl/Ptenfl/fl lox-stop-lox luciferase mice. Injection of adenovirus expressing Cre recombinase, either subcutaneously or intramuscularly in two experimental groups, results in viral infection and gene recombination with 100% penetrance within the first 24 hours following injection. Luciferase expression measured by real-time bioluminescence imaging increases over time, with an initial robust increase following viral injection, followed by a steady rise over the next several weeks as primary tumors develop and grow. Intramuscular injections were more commonly associated with evidence of systemic viral distribution than subcutaneous injections. All mice developed soft tissue sarcomas at the primary injection site, with histological examination identifying 93% of tumors as invasive pleomorphic sarcomas based on microscopic morphology and immunohistochemical expression of sarcoma markers. A lymphocytic infiltrate was present in 64% of the sarcomas in this immunocompetent model and 71% of tumors expressed PD-L1. This is the first report of a viral-Cre mediated Trp53/Pten mouse model of undifferentiated pleomorphic sarcoma. The bioluminescence imaging feature, along with high penetrance of the model and its immunological characteristics, makes it suited for pre-clinical studies of soft tissue sarcoma.

Fig 1. Generation of Trp53/Pten mice associated with luciferase expression.

A) ROSA26 Lox-stop-Lox Luciferase, Pten^fl/fl male mice were crossed with Trp53^fl/fl female mice to generate offspring with ROSA26 LSL-Luc, Pten^fl/fl, and Trp53^fl/fl. Ad with a Cre recombinase expression vector was injected either subcutaneously over the right leg/flank or intramuscularly into the right quadriceps to promote somatic recombination. Somatic expression of Cre recombinase promoted expression of luciferase and homozygous knockout of Pten and Trp53. B) PCR from DNA isolated from either a tumor of Cre injected mice (Cre+) or from the tail of a non-injected mouse (Cre-) (Ctrl is a primer only control) to assay for the presence of recombination and floxed Pten or Trp53.

Fig 2. Trp53/Pten mice injected with Ad CMV-Cre demonstrate a longitudinal increase in BLI signal.

A) BLI of Trp53/Pten mice injected either SQ or IM with Ad CMV-Cre. Mice were injected with intraperitoneal D-luciferin and imaged using an Ami X imager. Shown are representative images of five mice from each cohort at 1 week, 9 weeks, and 17 weeks post-viral injection, demonstrating a steady increase in luciferase signal over time. B) Quantified whole-body in vivo BLI values as measured in photons/sec/cm²/sr are shown for individual mice for each injection group (n = 9), and compared between the two injection groups C), highlighting similar luciferase expression kinetics and values between SQ- and IM-injected animals. The difference between SQ and IM injection was statistically significant for day 1 post-injection (p = 0.045), but not thereafter. From the 2way ANOVA, overall variance due to injection method was statistically significant (p = 0.0019). In comparison to the first day post-injection, all values for both injection methods were significant (p<0.0001).

Fig 3. IM-injected Ad CMV-Cre results in increased systemic viral spread.

A) Quantified leg-only or abdomen-only BLI values are shown for individual Trp53^fl/flPten^fl/fl mice injected either SQ or IM with Ad CMV-Cre. B) Comparisons between injection cohorts demonstrate lower non-specific signal in the abdomen with SQ injection. There was no significant difference in luciferase signal between the SQ- or IM-injected legs; however, there is a significant difference between SQ and IM abdomen injections for week 1 and beyond (p<0.05). The difference in comparison to the first day post-injection was statistically significant for both SQ (p<0.002) and IM (p<0.0001) leg injections as well as IM abdominal BLI (p<0.02). No significant difference was found for SQ abdominal BLI in comparison to day 1. C) Ex vivo BLI images of livers from one SQ- and one IM-injected mouse, depicting multifocal low-level recombination and luciferase expression in the livers of mice with abdominal BLI signal, suggesting systemic viral spread and infection (left panels). Histological sections of liver demonstrated extramedullary hematopoiesis, noted with arrows, but no neoplasm was identified (right panel).

Fig 4. Trp53^fl/flPten^fl/fl mice injected with Ad CMV-Cre form soft tissue tumors with 100% penetrance.

A) Photographs of representative intact SQ and IM soft tissue tumors (*). B) Primary tumor dissections highlighting the superficial nature of tumors in the SQ-injected group compared to the deep, invasive tumors in the IM group, which develop within the native quadriceps muscle and adhere firmly to the surrounding femur and musculature. C) Palpable tumor latency as defined by time to earliest manual palpation of soft tissue tumor (p = 0.3201) D) Primary tumor mass and tumor volume with corresponding BLI measurements. Tumor mass (blue squares, right y-axis) measured after surgical excision and volume (red circles, left y-axis) measured by calipers are correlated with photon flux (X-axis); Spearman r = 0.857 and r = 0.733, respectively. Asterisk indicates one outlying tumor that was removed from the analysis.

Fig 5. Trp53^fl/flPten^fl/fl mice injected with Ad CMV-Cre form undifferentiated pleomorphic sarcoma.

A) Histology and immunophenotype of UPS arising after SQ injection of Ad CMV-Cre. H&E section reveals a proliferation of markedly atypical spindle cells with numerous mitotic figures (arrows) as well as tumor necrosis (*). No line of differentiation was identified by the neoplasm’s light microscopic appearance or immunohistochemical profile, supporting classification as UPS. B) Histology and immunophenotype of typical UPS arising in skeletal muscle. The histological appearance and immunophenotype is similar to the subcutaneous UPS present in (A). SMA is variably positive in different examples, similar to human UPS. The asterisk highlights skeletal muscle fibers in the H&E section as well as the desmin stain, where they serve as an internal control. C) Histology and immunophenotype of PRMS. H&E section reveals a proliferation of markedly atypical spindle cells admixed with rounded rhabdomyoblasts (arrow). Skeletal muscle differentiation is confirmed by the identification of various foci expressing myogenin and desmin. D) Phospho-AKT was variably present. In some sarcomas, such as the PRMS tumor and some of the UPS tumors, phospho-AKT could be identified more diffusely (left panel). In other UPS tumors, it was more focal (right panel).

Fig 6. Trp53/Pten undifferentiated pleomorphic sarcomas demonstrate immune tolerance.

A) Histology of UPS reveals an infiltrate of small mature lymphocytes (arrow), which is further confirmed by immunohistochemistry for CD45 (B, arrow). Immunohistochemistry was performed for PD-L1 (B7-H1), showing expression patterns ranging from focal/variable staining (C), to diffusely and strongly positive (D), suggesting that UPS in the Trp53/PTEN model are immunogenic and might evade immunosurveillance by PD-L1 upregulation.