Abstract 312: Profiling drug sensitivity and kinomic pathways utilizing a novel human tumor derived MicroTumor assay
Authors: Willey Christopher D. , Gilbert Ashley N. , Shevin Rachael , Langford Catherine P. , Singh Raj , Anderson Joshua C. , Gillespie G. Yancey 
Published: August 2015

Abstract
Introduction: At present, drug screening studies are commonly performed using monolayer or spheroid culture and xenograft models of tumor cell lines. However these do not fully replicate the primary tumor's microenvironment and fail to accurately predict clinical endpoints. Vivo Biosciences has developed a novel MicroTumor 3D matrix based assay system that emulates primary tumor multicellular growth and biology ex vivo, providing an advanced drug screening platform. We postulated that MicroTumors established from patient-derived xenograft (PDX) tumors will allow for accurate analysis of drug response and preserve molecular signaling of parent tumors. Glioblastoma multiforme (GBM), the most common primary brain malignancy, was used to test our hypothesis. MicroTumors were evaluated by comparing kinome activation profiles of GBM-MicroTumors with corresponding parental orthotopically implanted PDX; and determining single and combination treatment effects of small molecule kinase inhibitors (SMI) on GBM MicroTumors.
Methods: We investigated 6 GBM PDX tumor lines representing the 4 known molecular subtypes: Classical (JX10, X1016, X1046); Proneural (XD456); Mesenchymal (JX22P); and Neural (JX10). Four SMIs (primary kinase target indicated) were studied: WP1066 (JAK2), selumetinib (MEK1/2), crizotinib (c-MET, ALK), and cediranib (VEGFR, FLT-1, FLT-4, c-KIT, PDGFR). MTT assays and Calcein AM imaging were used for cytotoxicity assessment and PamStation 12 Kinomic analyses were performed (UAB Kinome Core).
Results: Kinomic analyses of GBM orthotopic PDX and GBM-MicroTumors revealed similar kinase signaling profiles based on comparison of commonly shared, most-variant phosphopeptides. Upstream kinase analyses identified these peptides as substrates of EGFR, AXL, ZAP70 and MERTK kinases. Initial drug response studies demonstrated dose dependency and PDX-specific responses for each drug used independently informing doses for ongoing combination studies. Interestingly, the least cytotoxic drug across all 6 MicroTumors, selumetinib, did impact MicroTumor morphology observed with Calcein AM imaging.
Conclusions: We identified kinomic alterations that may correlate MicroTumor and patient tumor biology and guide the use of molecularly targeted SMIs. SMI activities towards these targets highlighted that this novel 3D translational model for GBM can provide relevant drug sensitivity information. Future studies with in vivo PDX tumors will examine the most promising SMI combinations based on MicroTumor data. We believe this two-stage approach (Microtumor screening to predict PDX sensitivities) will improve preclinical drug screening in GBM and other cancers.
Citation Format: Christopher D. Willey, Ashley N. Gilbert, Rachael Shevin, Catherine P. Langford, Raj Singh, Joshua C. Anderson, G. Yancey Gillespie. Profiling drug sensitivity and kinomic pathways utilizing a novel human tumor derived MicroTumor assay. [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 312. doi:10.1158/1538-7445.AM2015-312