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SHORT ABSTRACT Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis. LONG ABSTRACT The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Glioblastoma multiforme (GBM), the most common form of primary malignant brain cancer in adults, is a devastating disease for which effective treatment has remained elusive for over 75 years. One reason for the minimal progress during this time is the lack of accurate preclinical models to represent the patient’s tumor’s in vivo environment, causing a disconnect in drug therapy effectiveness between the laboratory and clinic. While patient-derived xenografts (PDX’s or xenolines) are excellent human tumor representations, they are not amenable to high throughput testing. Therefore, we developed a miniaturized xenoline system (microtumors) for drug testing. Nineteen GBM xenolines were profiled for global kinase (kinomic) activity revealing actionable kinase targets associated with intracranial tumor growth rate. Kinase inhibitors for these targets (WP1066, selumetinib, crizotinib, and cediranib) were selected for single and combination therapy using a fully human-derived three-dimensional (3D) microtumor model of GBM xenoline cells embedded in HuBiogel for subsequent molecular and phenotype assays. GBM microtumors closely resembled orthotopically-implanted tumors based on immunohistochemical analysis and displayed kinomic and morphological diversity. Drug response testing could be reproducibly performed in a 96-well format identifying several synergistic combinations. Our findings indicate that 3D microtumors can provide a suitable high-throughput model for combination drug testing.

Accurate patient-derived models of cancer are needed for profiling the disease and for testing therapeutics. These models must not only be accurate, but also suitable for high-throughput screening and analysis. Here we compare two derivative cancer models, microtumors and spheroids, to the gold standard model of patient-derived orthotopic xenografts (PDX) in glioblastoma multiforme (GBM). To compare these models, we constructed a custom NanoString panel of 350 genes relevant to GBM biology. This custom assay includes 16 GBM-specific gene signatures including a novel GBM subtyping signature. We profiled 11 GBM-PDX with matched orthotopic cells, derived microtumors, and derived spheroids using the custom NanoString assay. In parallel, these derivative models underwent drug sensitivity screening. We found that expression of certain genes were dependent on the cancer model while others were model-independent. These model-independent genes can be used in profiling tumor-specific biology and in gauging therapeutic response. It remains to be seen whether or not cancer model-specific genes may be directly or indirectly, through changes to tumor microenvironment, manipulated to improve the concordance of in vitro derivative models with in vivo models yielding better prediction of therapeutic response.

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