Ashley N. Gilbert

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing.

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.

Kinomic Profiling of Electromagnetic Navigational Bronchoscopy Specimens: A New Approach for Personalized Medicine

Purpose Researchers are currently seeking relevant lung cancer biomarkers in order to make informed decisions regarding therapeutic selection for patients in so-called “precision medicine.” However, there are challenges to obtaining adequate lung cancer tissue for molecular analyses. Furthermore, current molecular testing of tumors at the genomic or transcriptomic level are very indirect measures of biological response to a drug, particularly for small molecule inhibitors that target kinases. Kinase activity profiling is therefore theorized to be more reflective of in vivo biology than many current molecular analysis techniques. As a result, this study seeks to prove the feasibility of combining a novel minimally invasive biopsy technique that expands the number of lesions amenable for biopsy with subsequent ex vivo kinase activity analysis. Methods Eight patients with lung lesions of varying location and size were biopsied using the novel electromagnetic navigational bronchoscopy (ENB) technique. Basal kinase activity (kinomic) profiles and ex vivo interrogation of samples in combination with tyrosine kinase inhibitors erlotinib, crizotinib, and lapatinib were performed by PamStation 12 microarray analysis. Results Kinomic profiling qualitatively identified patient specific kinase activity profiles as well as patient and drug specific changes in kinase activity profiles following exposure to inhibitor. Thus, the study has verified the feasibility of ENB as a method for obtaining tissue in adequate quantities for kinomic analysis and has demonstrated the possible use of this tissue acquisition and analysis technique as a method for future study of lung cancer biomarkers. Conclusions We demonstrate the feasibility of using ENB-derived biopsies to perform kinase activity assessment in lung cancer patients.

Modeling Physiologic Microenvironments in Three-Dimensional Microtumors Maintains Brain Tumor Initiating Cells

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity, including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy, because BTICs are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using the human-derived biomatrix HuBiogel™ and maintained with physiologically relevant microenvironments. BITC percentages were determined using cell surface marker expression, label retention, and neurosphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient-restricted microtumors significantly increased the percentage of both CD133+ and CFSEhigh cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintained neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions

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.

Abstract 1925: Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting. Citation Format: Ashley Gilbert, Kiera Walker, Anh Tran, Yancey Gillespie, Raj Singh, Anita B. Hjelmeland. Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2017-1925

Abstract A16: Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions

Introduction: Drug testing of Glioblastoma multiforme (GBM) has revealed that many promising preclinical therapies fail when translated to the clinic. Serious limitations in preclinical models (often monolayer, serum-treated immortalized cell models) likely contribute to this translational failure. Although patient-derived xenografts (PDX), in which patient tumors are serially passaged in immunocompromised mice, are potentially superior model systems of human disease, high throughput drug studies in GBM PDX tumors are not feasible due to time, cost and throughput. To overcome these challenges, we have developed a GBM PDX MicroTumor system in which GBM PDX cells are grown in a novel 3D extracellular matrix material (HuBiogel™, Vivo Biosciences Inc., Birmingham, AL). We hypothesized that this 3D MicroTumor system would better emulate in vivo PDX tumor growth while providing a high throughput assay system for drug combination studies that can be performed in a more rapid timeframe. Methods: Six GBM PDX tumor “xenolines” were selected from the UAB Brain Tumor Animal Model Core to represent the 4 known molecular subtypes of GBM: Classical (JX10, X1016, X1046); Proneural (XD456); Neural (JX10) and Mesenchymal (JX22P). Tumor cells were prepared as single cell suspensions from subcutaneously passaged tumors and embedded in HuBiogel™ beads to form MicroTumors using Neuro-basal media devoid of serum. Single and combinatorial drug cytotoxicity studies were performed using MTT mitochondrial viability assays and Calcein AM imaging. Small molecule inhibitors selected for drug testing were: 1) selumetinib-targeting MEK1/2; 2) crizotinib-targeting c-MET and ALK; 3) cediranib-targeting VEGFR, FLT-1, FLT-4, c-KIT, and PDGFR; and 4) WP1066-targeting JAK2/STAT3. Combinatorial interactions were determined by Chou-Talalay combination index (CI) calculation using Calcusyn software. Global kinase signaling (kinomic profiling) was assessed using paired total cell lysates of GBM-PDX from orthotopic tumors grown in athymic nu/nu mice and as 3D MicroTumors. Testing was performed on the PamStation12 high content peptide microarray platform within the UAB Kinome Core. Results: Following optimization of model system conditions, 4 drugs were tested at multiple concentrations to identify effective dose ranges. While crizotinib, cediranib and WP1066 demonstrated significant cytotoxicity in at least 1 of the GBM-PDX MicroTumors tested, selumetinib was ineffective in producing cytotoxicity as a monotherapy. However, in combinatorial drug testing, selumetinib was effective in promoting synergistic activity (CI Conclusions: The GBM-PDX 3D MicroTumor provides a relatively high throughput drug screening model system that displays similar kinase signaling pathway activation as compared to their in vivo intracranial counterpart. Specific synergistic drug combinations were identified through MicroTumor testing. Ongoing drug combination testing of intracranially implanted GBM-PDX will determine how robustly the MicroTumor system predicts in vivo efficacy. Citation Format: Christopher D. Willey, Ashley N. Gilbert, Rachael Shevin, Catherine P. Langford, Christine W. Duarte, Raj Singh, Joshua C. Anderson, G. Yancey Gillespie. Combinatorial drug testing using 3D microtumors derived from GBM PDX reveals cytotoxic synergisms in pharmacokinomics-informed pathway interactions. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A16.

Abstract 312: Profiling drug sensitivity and kinomic pathways utilizing a novel human tumor derived MicroTumor assay

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 tumor9s 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