Abstract 3236: Clonal heterogeneity in patient-derived xenografts: The SCLC model LG0904F1496M23 contains stable clones with epithelial or mesenchymal characteristics and differential drug sensitivities
Authors: Mullendore Michael , Stockwin Luke H. , Bonomi Carrie , Dougherty Kelly , Stotler Howard , Kimmel Adrienne , Das Biswajit , Datta Vivekananda , Lih Jason , Williams Mickie , Doroshow James , Hollingshead Melinda , Newton Dianne Lynn 
Published: August 2015

It is well established that cancers are heterogeneous diseases with biological, genetic, and histopathological differences among patients and within individual tumors. Consequently, it is not surprising that animal models based on established cell lines with their clonal nature and adaptations to defined media have limited preclinical value. Recent advances in the generation of patient-derived xenografts (PDX), where patient material is engrafted into immunosuppressed mice, suggests that this approach may provide a more representative surrogate for therapeutic development. In this study, a mixed cell culture derived from a SCLC PDX model was analyzed for clonal heterogeneity. Following two rounds of growth in soft agar, seven clones were derived; three were further characterized. The 1st clone, 7-7-2, similar in morphology to the parental culture was a mixture of cuboidal and spindle-shaped cells. FACs analysis confirmed two populations of cells; EpCAM+/CD90 low and EpCAM-/CD90 high population. The 2nd clone, 2-2-1, consisted of a single population of spindle-shaped EpCAM-/CD90+ cells; the 3rd clone, 7-7-1, contained a homogeneous population of cuboidal cells that were EpCAM+/CD90 low. DNA analysis confirmed all 3 clones were derived from the same patient. Furthermore, all 3 clones were tumorigenic in NSG mice.

To exclude fibroblast contamination, the clones were analyzed using a quantitative RT-PCR array capable of identifying human fibroblasts and other stromal constituents. None of the 3 clones had a classical fibroblast profile. Comparison of 2-2-1 relative to the other 2 clones revealed higher expression of VIM mRNA and lower KRT5, CDH1 and EpCAM. Results were confirmed by Western blot. Additionally, ITGAV and TWIST were up-regulated. These results suggest that clone 2-2-1 had undergone epithelial-mesenchymal transition (EMT). Further confirmation was provided by microarray and proteomic analysis where GNG11, MMP2, ZEB, DDR2, TESK2, DESM and MOES were elevated consistent with EMT.

Next, clones were tested for sensitivity against a panel of clinically relevant agents (trametinib, everolimus, temozolomide, ABT-888, carboplatin, MK 1775) either alone or in combination. Clone 2-2-1 proved to be more sensitive than parental cells or clones 7-7-1 or 7-7-2 to the majority of these drugs. Further studies showed the combination of temozolomide/ABT-888 was synergistic against each of the clones while the combination of MK 1775/carboplatin lacked synergism. In vivo, the parental tumor responded to the temozolomide/ABT-888 combination with tumor regressions.

In summary, these data serve to highlight 1) heterogeneity within this PDX model 2) profound biological differences between clones and 3) EMT is implicated and alters drug responses. Funded by NCI Contract No. HHSN261200800001E.

Citation Format: Michael Mullendore, Luke H. Stockwin, Carrie Bonomi, Kelly Dougherty, Howard Stotler, Adrienne Kimmel, Biswajit Das, Vivekananda Datta, Jason Lih, Mickie Williams, James Doroshow, Melinda Hollingshead, Dianne Lynn Newton. Clonal heterogeneity in patient-derived xenografts: The SCLC model LG0904F1496M23 contains stable clones with epithelial or mesenchymal characteristics and differential drug sensitivities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2015-3236