Development of Genetically Flexible Mouse Models of Sarcoma Using RCAS-TVA Mediated Gene Delivery

<div><p>Sarcomas are a heterogeneous group of mesenchymal malignancies and unfortunately there are limited functional genomics platforms to assess the molecular pathways contributing to sarcomagenesis. Thus, novel model systems are needed to validate which genes should be targeted for therapeutic intervention. We hypothesized that delivery of oncogenes into mouse skeletal muscle using a retroviral (RCAS-TVA) system would result in sarcomagenesis. We also sought to determine if the cell type transformed (mesenchymal progenitors vs. terminally differentiated tissues) would influence sarcoma biology. Cells transduced with RCAS vectors directing the expression of oncoproteins Kras^G12D, c-Myc and/or Igf2 were injected into the hindlimbs of mice that expressed the retroviral TVA receptor in neural/mesenchymal progenitors, skeletal/cardiac muscle or ubiquitously (N-tva, AKE and BKE strains respectively). Disrupting the G1 checkpoint CDKN2 (<i>p16/p19^−/−</i>) resulted in sarcoma in 30% of <i>p16/p19^−/−</i>xN-tva mice with a median latency of 23 weeks (range 8–40 weeks). A similar incidence occurred in <i>p16/p19^−/−</i>xBKE mice (32%), however, a shorter median latency (10.4 weeks) was observed. <i>p16/p19^−/−</i>xAKE mice also developed sarcomas (24% incidence; median 9 weeks) yet 31% of mice also developed lung sarcomas. Gene-anchored PCR demonstrated retroviral DNA integration in 86% of N-tva, 93% of BKE and 88% of AKE tumors. Kras^G12D was the most frequent oncogene isolated. Oncogene delivery by the RCAS-TVA system can generate sarcomas in mice with a defective cell cycle checkpoint. Sarcoma biology differed between the different RCAS models we created, likely due to the cell population being transformed. This genetically flexible system will be a valuable tool for sarcoma research.</p></div

Introduction of RCAS vectors induces sarcoma in <i>p16/p19</i>-deficient <i>tva</i> mouse strains.

<p>A Kaplan-Meier analysis of mortality due to tumor formation at the RCAS injection site of <i>p16/p19^−/−</i>x AKE, <i>p16/p19^−/−</i>x BKE and <i>p16/p19^−/−</i>x N-<i>tva</i> mice. Mice were euthanized and tissues were removed for histopathologic assessment to confirm sarcoma formation, assess subtype and to confirm the presence of RCAS vector. Wild-type AKE and BKE mice did not develop RCAS-associated tumors.</p

Sarcoma development occurs at injection site in <i>p16/p19</i>-deficient <i>tva</i> mice following retrovirus oncogene delivery.

<p>Pulmonary sarcoma occurred in 12% of the affected mice.</p

Integration of RCAS DNA in the majority of RCAS-TVA induced sarcomas.

<p>Gene anchored PCR was performed to detect presence of RCAS DNA within harvested tumor specimens.</p><p>No RCAS DNA was detected in spontaneous tumors that arose in mice with <i>p16/p19-</i>deficiency only.</p

RCAS-TVA System is effective in gene delivery to neonatal hindlimbs.

<p>(<b>A</b>) In the RCAS-TVA system, DF1 chicken fibroblast cells are transfected with RCAS vector containing gene of interest (gene X). The virus produced is released into the culture medium where it infects remaining cells through endogenous TVA receptor uptake. Transgenic mice expressing the TVA receptor under a tissue-specific promoter allows for selection of which cell types are susceptible to RCAS infection and transformation. Infection of mouse cells with RCAS vector results in viral DNA integration and production of gene X's protein product. Mammalian cells do not translate the remaining viral proteins, which leaves the TVA receptor unoccupied allowing further infection of the mammalian cell by other RCAS vectors. (<b>B</b>) (i) Cells isolated from the hindlimbs of <i>p16/p19^−/−</i>xAKE mice are infected with RCAS-GFP in culture (100× magnification) and GFP expression was analysed by FACS. (ii) GFP positive cells are identified following injection of neonatal mice and subsequent harvest 7 days later. Cells were isolated and allowed to adhere to a 10 cm plate for 3 days at which time cells were imaged (100× magnification) and analysed for GFP expression. (<b>C</b>) Protein lysates from DF1 cells transfected (+) with (i) RCAS-Kras^G12D, (ii) RCAS-c-Myc, or (iii) RCAS-Igf2 or untransfected (-) were probed with antibodies to confirm protein expression of oncogenes of interest prior to retroviral gene delivery in neonatal hindlimbs.</p

The RCAS-TVA System Produces Multiple STS Immunophenotypes.

<p>(<b>A</b>) Immunohistochemistry in a <i>p16/p19^−/−</i>xAKE leg mass has positive tumor cell staining for desmin, SMA and MyoD1 consistent with rhabdomyosarcomatous differentiation. (<b>B</b>) A leg sarcoma in a <i>p16/p19^−/−</i>xAKE mouse is SMA positive, consistent with myofibroblastic differentiation. (<b>C</b>) The corresponding lung sarcoma from (<b>B</b>). (<b>D</b>) The sarcoma seen in a <i>p16/p19^−/−</i>xBKE limb has negative IHC and fascicular arrangements of cells consistent with fibrosarcomatous differentiation. (<b>E</b>) H&E stains of four human sarcomas demonstrates similar morphology to RCAS-TVA induced murine tumors. All images at ×200 magnification, except (<b>A</b>) MyoD1 insert x400. (<b>F</b>) In <i>p16/p19^−/−</i>xAKE mice, myofibrosarcoma was the predominant STS seen in tumors that arose under the α-actin promoter. A high proportion of fibrosarcoma was seen in <i>p16/p19^−/−</i>xBKE and <i>p16/p19^−/−</i>xN-<i>tva</i> mice. Myofibro = Myofibrosaroma, RMS = rhabdomyosarcoma and Fibro = fibrosarcoma. Other included round cell tumour.</p

shRNA MGAT1 suppresses N-glycan branching.

<p><b>A</b>) HeLa cells were infected with lentiviral vectors targeting MGAT1(shRNA1 or shRNA2) or the control shRNA sequences. Stable cell populations were selected by the addition of puromycin (1 µg/mL). <b>A</b>)MGAT1 mRNA were measured by qRT-PCR, <b>B</b>) MGAT1 enzyme activity, <b>C</b>) L-PHA reactive surface N-glycans by Array scan microscope, <b>D</b>) Proliferation over 4 days Data represent the mean ± SD relative expression of mRNA relative to control sequence (n = 3 independent experiments performed in triplicate).</p

MGAT1 knockdown decreases tumor migration, and invasion in prostate cancer cells.

<p>PC-3-Yellow cells with MGAT1-shRNA2 or the control shRNA sequences were assessed for <b>A</b>) MGAT1 mRNA levels by qRT-PCR. <b>B</b>)MGAT1 enzyme activity, <b>C</b>) L-PHA reactive surface N-glycans by Arrayscan microscope, <b>D</b>) invasion through a Matrigel barrier and migration (<b>E</b>) using the xCELLigence Real-Time Cell Analyzer. Data represents mean ±SD cell index (3 independent experiments with quadruplicates). (<b>F</b>) Tumor size and (<b>G</b>) metastasis in mice injected with PC-3-Yellow cells with MGAT1-shRNA2 or the control shRNA sequences. The mice were injected orthotopically, with 0.5×10⁶ cell per mouse, into the prostate of the sub-lethally irradiated SCID mice. Four weeks after injection, mice were sacrificed and their organs were imaged using a fluorescent microscope. The numbers of metastatic nodules in all five lobes of the lungs were quantified using image analysis software. Each point represents one mouse.</p

MGAT1 shRNA inhibits HeLa cell migration and invasion <i>in vitro</i>.

<p><b>A</b>) HeLa cells were plated into chambers with 8-µm pores and 10% FBS was used as a chemoattractant. <b>B</b>) HeLa cells were plated into invasion chambers with Matrigel. After 48 h, cells that had migrated through the pores were fixed, stained and counted automatically. C) migration and D) invasion assays with or without pretreatment for 72 h with 2 µM swainsonine (SW) for 72 hours prior. The mean number of migrated cells ± SD of 3 independent experiments performed in triplicate were graphed. (<b>E</b>) Cell morphology by staining with phospho-paxillin and TRITC-conjugated phalloidin for F-actin, shown as a merged image.</p