A Simple Three-Dimensional Hydrogel Platform Enables Ex Vivo Cell Culture of Patient and PDX Tumors for Assaying Their Response to Clinically Relevant Therapies

Author Manuscript Published OnlineFirst on February 12, 2019; DOI: 10.1158/1535-7163.MCT-18-0359 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Full Title: A simple three-dimensional hydrogel platform enables ex vivo cell culture of patient and PDX tumors for assaying their response to clinically relevant therapies

Running Title: 3D hydrogel culture platform for ex vivo compound testing

Kolin C. Hribar, PhD1, Chris Wheeler, PhD2, Alexey Bazarov, PhD1, Kunal Varshneya2,3, Ryosuke Yamada2, Padraig Buckley1, Chirag Patil, MD2 *

1 Cypre, Inc., 953 Indiana Street, San Francisco, CA, USA (www.cypre.co) 2 Department of Neurosurgery, Cedars-Sinai Hospital, Los Angeles, CA, USA 3 Department of Neurosurgery, Stanford University, Palo Alto, CA, USA

Funding: Precision Medicine Initiative for Brain Cancer Donor Grant

Conflict of Interest: KCH, AB, and PB were staff of Cypre Inc. during the preparation of this manuscript.

Statement of Significance: We propose a novel 3D hydrogel platform, VersaGel, to grow ex vivo tissue (patient, PDX) and assay therapeutic response using time-course image analysis.

Please send correspondence to: Chirag G. Patil, M.D., M.S. Center for Neurosurgical Outcomes Research, Maxine Dunitz Neurosurgical Institute Department of Neurosurgery, Cedars-Sinai Medical Center 127 S. San Vicente Blvd, Suite A6600

Downloaded from mct.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 12, 2019; DOI: 10.1158/1535-7163.MCT-18-0359 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Los Angeles, CA 90048 E-mail: [email protected]

ABSTRACT:

A cell culture platform that enables ex vivo tissue growth from patients or patient-derived xenograft (PDX) models and assesses sensitivity to approved therapies (e.g. Temozolomide, TMZ) in a clinically relevant timeframe would be very useful in translational research and personalized medicine. Here, we present a novel three-dimensional (3D) ECM hydrogel system, VersaGel, for assaying ex vivo growth and therapeutic response with standard image microscopy. Specifically, multicellular spheroids deriving from either five glioblastoma (GBM) patients or a renal cell carcinoma (RCC) PDX model were incorporated into VersaGel and treated with TMZ and several other therapies, guided by the most recent advances in GBM treatment. RCC ex vivo tissue displayed invasive phenotypes in conditioned media. For the GBM patient tumor testing, all 5 clinical responses were predicted by the results of our 3D-TMZ assay. In contrast, the MTT assay found no response to TMZ regardless of the clinical outcome, and moreover, basement membrane extract failed to predict the two patient responders. Finally, one patient was tested with repurposed drugs currently being administered in GBM clinical trials. Interestingly, IC50’s were lower than cmax for crizotinib and chloroquine, but higher for sorafenib. In conclusion, a novel hydrogel platform, VersaGel, enables ex vivo tumor growth of patient and PDX tissue and offers insight into patient response to clinically relevant therapies.

INTRODUCTION:

Ex vivo cell culture of resected tumors from patients and patient-derived xenografts (PDX) presents a unique opportunity to advance translational research and personalized medicine.1 Unfortunately, current in vitro techniques struggle to reliably grow ex vivo tissue and subsequently correlate drug responses with the clinical outcome. It has been shown that traditional two-dimensional (2D) cell culture systems may not be suitable models for investigating solid tumors and have shown inconsistencies with in vivo.2,3 Further, three- dimensional (3D) technologies such as spheroids or basement membrane extract do not provide the complete set of biomimetic microenvironments, such as appropriate scaffold architecture, or a defined, batch consistent extracellular matrix (ECM) for cell interactions, respectively.4,5 Basement membrane extract, a cell culture matrix derived from a mouse sarcoma, has known issues with growth-factor contamination and batch variability that may influence drug response and reproducibility.6 Spheroids in liquid suspension – that is, multi-cellular aggregates grown in hanging-drop or U-bottom plates – are cultured in a closely packed cell-only 3D environment, but lack external ECM binding modalities which strongly impacts various aspects of tumor progression (e.g. ECM-dependent growth, invasion, and cell migration), especially important for recapitulating solid tumors.7

Here, we analyzed the ex vivo culture of a renal cell carcinoma PDX tumor as a proof of concept and, later, five patients with glioblastoma multiforme (GBM). GBM is the most prevalent and aggressive primary intracranial tumor. With an occurrence rate of 2 out of 100,000 people, GBMs account for over 82% of malignant gliomas and 5–20% of all primary intracranial tumors.8 Despite multimodal treatment, including radiation and chemotherapy following aggressive surgical resection, the prognosis remains poor with a median survival of

13-15 months.9,10 Younger age, good functional status (KPS), extent of resection, IDH-1 status and methylated O6-methylguanine DNA methyltransferase (MGMT) are the main predictors of a more positive prognosis.11,12

In this study, we sought to determine if a batch consistent, growth-factor free, 3D hydrogel culture system, VersaGel, enabled 3D ex vivo tumor growth and in vitro drug validation, compared to the liquid suspension MTT assay, MGMT promoter methylation and actual clinical responses to chemotherapy. Culturing tumor cells in 3D enables stronger physiological relevance to in vivo due, in part, to the increased cell-to-cell and cell-to-ECM interactions.13,14 More specifically, 3D scaffolds and ECM-mimicking hydrogels, may better simulate the native tumor microenvironment ECM and provide more accurate drug efficacy analyses.15 We propose a new scaffold-based 3D cell culture platform, VersaGel, that efficiently crosslinks to low intensity UV (365nm) and blue light (405nm). This type of light-crosslinking approach been widely used in tissue engineering applications, and moreover the low intensity has been shown to have little affect of cytotoxicity.16-18 The objective of this study is to evaluate the efficacy of VersaGel in growing ex vivo patient and PDX samples, retrospectively predicting treatment efficacy and outcomes for five GBM patients to Temozolomide (TMZ) – first line treatment in GBM – and prospectively testing known secondary therapeutics without clinical validation, as a proof of concept for personalizing therapies.

METHODS:

Therapeutic Compounds Temozolomide (TMZ) was purchased from Tocris Bioscience. Chloroquine, Crizotinib, and Sorafenib were all purchased from Selleckchem.

Patient Tumor Specimen Processing and Growing Neurospheres Tumor specimen diagnosed as glioblastoma as defined by World Health Organization criteria, were collected for patients undergoing tumor resection. Informed consents were obtained from

those patients, and the specimens were handled in accordance with Cedars-Sinai Medical Center Institutional Review Board (IRB). Within 3 hours of surgical resection, tumors were washed with PBS, mechanically minced with a scalpel and enzymatically dissociated into single cells1. Tumor cells were then isolated using gradient centrifugation.

Tumor cells were cultured in Neurosphere Media (NM), consisting of DMEM-based media with added recombinant bFGF (20ng/ml), EGF (50ng/ml), B27 supplement, heparin (160ng/ml), and penicillin-streptomycin. Ultra-low attachment (ULA) dishes and flasks (Nunc, ThermoFisher) were used as a culturing surface to induce cellular aggregation and neurosphere formation. Alternatively, Aggrewell-400 plates (Stemcell) could be used to grow more homogenously sized spheroids. Cells were cultured in neurosphere media for at least three days prior to use in VersaGel (Figure 1).

Clinically patients were categorized as non-responders to TMZ if they has a PFS of less than 6 months, partial responders if their PFS was between 6 months and 1 year and clinical responders if they had a PFS of more than 1 year.

VersaGel platform for 3D spheroid-hydrogel culture The VersaGel solution, 15%, was warmed to 37C for 10 minutes out of light. VersaGel was then mixed with spheroids and cell media in a 1:2 dilution for a final concentration of 5% (i.e. 200µL cell media : 100µL VersaGel) (Figure 1). To prevent mechanical disaggregation of the spheroid during the pipetting step, wide-bore tips and slow pipetting were employed during the process. The mixed VersaGel/cell media solution with spheroids was carefully pipetted into glass-bottom 24-well plates (Sensoplate, Greiner) and placed on a custom low-intensity light apparatus. The light was turned on for 30 seconds to expose the light inside the wells and chemically crosslink the VersaGel solution. After the light was turned off, the plate was removed from the apparatus and wells were washed twice with pre-warmed DPBS, followed by the addition of neurosphere

media, and finally cultured at 37°C and 5% CO2.

VersaGel™-PDX fragment culture

Tumor tissues resected from the PDX mouse were supplied by Stanford University in cell media and mechanically dissociated using a razor blade in a petri dish with excess media. Small fragments were collected with a wide bore tip and mixed with the VersaGel solution in the same manner as described, prior, with spheroids.

RCC PDX spheroid culture Tissue fragments from the above PDX tissue were further processed via mechanical and enzymatic dissociation using the gentleMACS Dissociator (Miltenyi Biotech) and Multi-Tissue Dissociation Kit (Miltenyi Biotech). Single cells were cultured on ULA plates (Nunc, ThermoFisher) as previously noted to form spheroids. Alternatively, Aggrewell-400 plates (Stemcell) were used to produce more homogenously sized spheres.

RCC tumor growth conditions RCC cells, spheroids, or fragments were grown in either DMEM (Corning, supplemented with 10% FBS, 1% P/S) or EGM-2 media (Lonza).

3D Gel Generation VersaGel is a biocompatible, growth-factor free hydrogel for cell culture that forms interconnected 3D mesh networks when exposed to low intensity UV (365nm) or blue light (405 nm) for short durations (e.g. 30-60 seconds) and allows for cellular integrin binding and MMP degradation. Its light-activated chemical crosslinking mechanism provides enhanced matrix crosslinking and robustness, enabling long culture times (e.g. weeks to months). Its growth- factor free makeup is ideal for supporting serum-free conditions, e.g. neurospheres, though growth factors may be added to the media before mixing with VersaGel, or in the liquid culture for molecule diffusion through VersaGel’s micro-porous structure.

Additionally, it was empirically determined that crosslinked VersaGel attaches to glass bottom plates, providing an easier to use platform for media exchanges without damaging or accidentally lifting off the gel during pipetting.

Spheroid growth determination

Images of spheroids in each well were captured on a standard brightfield microscope and analyzed on ImageJ software. In ImageJ, the area around each sphere was traced and quantified for each time point and later compared to its day 0 area to determine percent (%) growth (Figure 2a and b). This practice normalized the data irrespective of neurosphere size variability (Figure 2c).

3D growth measurements and TMZ Drug Testing Brightfield images of neurospheres were captured using an EVOS microscope (Thermofisher) and individual spheres were tracked over time. Spheroid size was quantified on ImageJ software by taking the sphere area. Percent growth of each sphere was calculated by quantifying its current day size (i.e. day n) and relating to its size at day 0:

% growth = ( Areaday n – Areaday 0 )/ Areaday 0

In this way, each individual sphere was normalized to its initial day 0 size and average % growth could be determined for each timepoint and experimental condition.

Neurospheres in VersaGel were exposed to varying concentrations of TMZ (0, 5, 25, 125, 625µM) in 0.5% DMSO and neurosphere media. Drug was administered on day 0 and day 3, followed by standard neurosphere culture up to 14 days.

Cell viability Cytotoxicity was qualitatively assessed using the LIVE/DEAD assay (ThermoFisher), calcein AM (GFP-channel of the EVOS microscope) for living cells and ethidium homodimer (RFP- channel) for dead cells.

3D-IC50 determination 3D-IC50 curves were created using the Prism 7 software package (GraphPad). Neurosphere size averages taken at day 10 or 14 for each experimental drug condition were used. Growth values for each TMZ concentration were plotted in log base 10, and as such, the control group was estimated to be 0.5 µM for additional point of reference on the curve.

Liquid culture MTT assay Upon drug treatment, GSC viability with and without concentrations of predicted drugs was quantified according to Vybrant® Cell Proliferation Assay Kit instructions (ThermoFisher Scientific). Briefly, cells were seeded at 2000-5000 per well in a 96-well plate format, and cell the conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to insoluble formazan quantified per manufacturer instructions after solubilization in SDS. Formazan concentration was determined by optical density at 570nm. Cell number was determined by comparison to formazan levels in counted cell standards. Response or non-response determination was made by comparing test to published IC50. Bottom of upper quartile of published IC50s for individual drugs was used as a response/non-response threshold.

3D spheroid growth in Basement Membrane Extract (BME) Similar to VersaGel, neurospheres were mixed with basement membrane extract (BME) (Matrigel, Corning) in a 2:1 ratio, plated in 24-well plates and allowed to gel for 30 minutes at 37°C before adding neurosphere media. Spheroids in BME were imaged to assess 3D spheroid growth, invasion and drug response. Within an hour after plating, spheres began disaggregating and invading the matrix with projections, complicating growth curves of the original tumor, as evidenced in Supplementary Figure 2.

Imaging and Analysis Imaging was performed on an EVOS microscope (ThermoFisher) and neurosphere size quantification in ImageJ software (NIH).

Statistics Average neurosphere growth was quantified from at least three separate spheres for each experimental condition.

RESULTS:

VersaGel growth conditions for PDX ex vivo

VersaGel enabled growth and cultivation of a renal cell carcinoma (RCC) patient-derived xenograft (PDX) tissue. The authors explored several ways to showcase PDX tumor growth. The tumor tissue was first resected from the mouse and mechanically dissociated into smaller pieces capable of VersaGel embedding. Alternatively, the tissue was further enzymatically dissociated into single cells and grown into spheroids as previously described with GBM samples (Figure 3a). Spheroids were mixed with the VersaGel solution using conditioned media from the spheroid culture or with fresh media. Interestingly, VersaGel-embedded tumor fragments, and spheroids with conditioned RCC media, demonstrated invasive phenotypes, while VersaGel- embedded spheroids with fresh media showcased no such invasion, and instead grew as closely packed spheroids (Figure 3b). Secreted proteins in the media or original tumor tissue may have contributed to the invasion, though more experimental evidence is required to determine the exact cause. It can be appreciated, however, that this invasive phenotype was only possible in a scaffold-embedded system, and that conditioned media could modulate the invasiveness in growth-factor free VersaGel.

In practice, VersaGel can plausibly work for any spheroid culture. Spheroids from other human cancer cell lines, HepG2 and LN18, were embedded in VersaGel and imaged over time (Supplementary Figure 1). Interestingly, HepG2 spheroids grew as a tightly packed spheroid, while LN18 spheres grew and invaded VersaGel by day 22.

Patient Attributes for GBM testing Five GBM patients were included in this part of the study. Overall, median age of the cohort was 68 years at time of surgery, and 3 were female. All patients underwent standard of care GBM resection followed by temozolomide (TMZ) and radiation. Four out of five patient’s tissue samples were obtained during their initial surgery. One patient sample was from a 2nd repeat surgery following GBM recurrence after failure of TMZ and radiation. Cell cultures via polymerase chain reaction (PCR) indicated 2/5 patients had MGMT promoter methylation. Overall, median Karnofsky Performance Score (KPS) of the patient cohort was 80.

VersaGel retrospective TMZ drug testing

Patient neurospheres embedded in VersaGel were exposed to Temozolomide (TMZ) of varying concentrations (0, 5, 25, 125, 625µM) on day 0 and day 3 and allowed to grow through day 14. Previous research has indicated this range as acceptable for GBM cell lines (eg U87), tho interestingly the TMZ cmax value – the maximum plasma concentration in the human – has been reported to be ~ 25µM.15,16 Image analysis revealed varying growth patterns and 3D-IC50 values in Patients 1 thru 5, correlating with the degree of clinical response and time to tumor progression (Table 1). Patient 1 and 2 were considered clinical Non-Responders with a time to progression below 6 months. In VersaGel, these patients exhibited no statistical difference between the control groups and all TMZ concentrations except 625µM, with 3D-IC50 values >125µM (Figure 4a). Patients 3 had already failed TMZ and radiation with a PFS of 5.8 months. Patient 3 tissue sample was obtained from the second surgery and hence, clinically Patient 3 was a non-responder. In VersaGel, this tumor exhibited no statistical difference between the control groups and all TMZ concentrations except 625µM, with a 3D-IC50 value of 128µM. Patient 4 was clinically considered a partial responder with time to progression above 6 months and below 1 year (8.7 months). In VersaGel, this patient’s tumor exhibited growth patterns that statistically varied in the higher 125µM and 625µM TMZ groups but showed no statistical difference with lower TMZ groups (Figure 4b) and a 3D-IC50 value of 49.8 µM. Hence, Patient 4 was classified as a partial responder per VersaGel 3D culture testing. Patient 5, the sole Responder from a clinical standpoint with PFS of 16 plus months, demonstrated statically slower growth in all TMZ concentration groups compared to the control (Figure 4c), with a 3D-IC50 value of 0.16 µM. Zero growth was reported in the 625µM TMZ group in all patients however cells remained viable, suggesting the cells underwent senescence at this upper limit of the experimental design (Figure 4d). Patient 4 showed zero growth in the higher concentrations of TMZ (125 and 625µM) during treatment through day 7. After switching to neurosphere media for the latter 7 days of culture, the neurospheres in the 125µM-treated group began to grow again, suggesting an incomplete response at that concentration (Figure 4e). Interestingly, this patient was a partial responder with time to progression of 8.7 months (compared to non-responders of < 6 month recurrence). And, Patient 5 was the only patient tumor to display an invasive phenotype in the control compared to TMZ treated group (Figure 4f), while in TMZ, the invasiveness was limited completely, suggesting that treatment was effective in vitro, and interestingly, the patient has been progression free for more than 16 months.

Liquid Suspension Culture and BME Culture MTT assay results on cells cultured in liquid neurosphere media correctly correlated to the clinical outcome for only three out of the five patients (Patients 1, 2, and 3, the Non-Responders) (Table 1, Figure 4). Indeed, all Patients (1 through 5) were universally resistant to TMZ under these conditions, with none reaching IC50. This low correlation may be due to phenotypic changes associated with growth of suspended cells or neurospheres in liquid culture as opposed to an ECM-mimicking scaffold like VersaGel, to intrinsic limitations of the MTT assay itself, or to both.

Basement membrane extract (BME) likewise failed to report the correct response for Patient 4 (the partial responder) and Patient 5 (the responder) (Supplementary Figure 2). BME’s rapid digestion by the tumor cells made measurements of individual neurosphere growth difficult to quantify. More importantly, as an example, neurospheres from Patients 4 and 5 showed invasive phenotypes in both 125 µM TMZ and control groups in Matrigel, which opposed the clinical outcome, which may be due to the batch variability and growth factor-enriched nature of BME.

VersaGel prospective drug testing for Patient 3 Nuerospheres derived from Patient 3 (Table 1) were grown in VersaGel and subjected to varying concentrations of Sorafenib, Crizotinib, and Chloroquine (Figure 5). Sorafenib is a kinase inhibitor that affects tumor proliferation and is currently subject to Phase I clinical trials as a combination therapy with TMZ and radiation.21 Crizotinib is an ALK and cROS-1 inhibitor that is FDA approved to treat non-small lung cancer and is currently being explored as glioblastoma treatment.22 Finally, Chloroquine is a medication used to treat for malaria and is currently undergoing Phase III trials for GBM treatment.23 The use of these therapies demonstrates the desire and willingness by the clinical community to repurpose existing drugs.24 Neurospheres in VersaGel were dosed appropriately with each drug and a 3D-IC50 value based on sphere growth was determined similar to previous experiments. Interestingly, Sorafenib’s 3D-IC50 was 27.11 µM, exceedingly higher than the reported cmax value of 6.7 µM. Crizotinb’s 3D-IC50 was 0.49 µM with a reported cmax of 0.93 µM.25 And, chloroquine’s 3D-IC50 was 4.71 µM compared to a cmax of 4.8 µM.

Discussion:

In translational and clinical cancer research, there is need for a simple to use, rapid in vitro system with the ability to grow ex vivo to tissue and correlate the clinical outcome. Current cell culture technologies have limited capacity to recapitulate the microenvironment, such as plastic plates, liquid suspension cultures of neurospheres, or growth factor enriched basement membrane extract (BME). Spheroids in liquid culture also fail to provide a suitable biomimetic environment in which cells may attach, grow and invade (all hallmarks of tumor progression), and additional assays like CellTiterGlo are required to assess drug response.

VersaGel provides a scaffold-based platform that recapitulates ECM cues for the tumor microenvironment in a growth factor free manner. To demonstrate its feasibility with ex vivo tissue, a renal cell carcinoma PDX tumor directly following resection was cultured and grown in VersaGel. Interestingly, there was phenotypic variance between different pre-culture conditions. When tumor fragments (directly from resection) or spheroids that were grown following tissue dissociation and pre-cultivation on ULA plates, were mixed with VersaGel and conditioned media – that is, media which came from the ULA plates – the tumor aggregates or spheroids in VersaGel displayed an invasive phenotype. Spheroids that were first washed with fresh media and then embedded in VersaGel grew as spherical shape with no invasive protrusions. This phenotypic alteration suggests that the secreted proteins in the conditioned media or original tumor tissue are critical for maintaining the invasive kidney carcinoma phenotype and could be an important consideration in future research with PDX models.

Clinically, TMZ response is predicted by MGMT methylation status. Unfortunately MGMT does not correctly predict response to TMZ in about 30% of cases.12 Therefore, cell assays offer a promising tool to verify TMZ response prior to treatment, however technologies to date have failed to correlate response. In our own testing, liquid suspension cell culture using the MTT assay predicted the correct TMZ response in only 3/5 patients, and neurospheres in BME were hindered by rapid invasion into the matrix surroundings, irrespective of TMZ culture. It has become increasingly accepted that unsupported liquid cell culture has limited capacity in

recapitulating the tumor microenvironment, and thus confers phenotypic and genetic alterations that affect drug response.2-5 BME contains varying amounts of growth factors that influence cell behavior, suggesting it may be a less reliable platform in this instance. As evidenced in Patient 5, a clinical Responder to TMZ, the neurospheres in BME failed to demonstrate any response and instead reported an invasive phenotype similar to the control group.

VersaGel, on the other hand, correctly demonstrated response to TMZ in all five patients, offering insight into the patient response in a rapid, in vitro setting. Interestingly, patient response in VersaGel, as noted by the degree of the reported 3D-IC50, correlated to the degree of response and time to progression in the clinical setting. As a proof of concept, VersaGel was utilized with one GBM patient (Patient 3) in the prospective testing of several compounds. It is interesting to note that IC50 values could be determined for all. While the data is early, the utility of repurposed drugs suggests a shifting trend with clinicians looking to find off-indication treatments for individual patients, especially after first-line treatment failure.26

Though our tests were performed in standard 24-well plates, the platform is amenable to higher throughput and to high content analysis, making VersaGel a potential candidate for oncology drug screening and personalized diagnostics. This study provides a stepwise protocol for groups to explore various PDX and patient tumor types in VersaGel. Further patient testing should be considered before the assay is considered clinically validated and used in the clinical decision making process. The appropriate laboratory or University with access to patient samples may find this type of testing compelling when building a database of retrospective and prospective therapeutic responders. It is the hope of the authors that a synergy between 3D ex vivo tumor testing in VersaGel and other robust technologies like next-generation sequencing may assist translational oncology researchers and clinical decision makers to advance personalized medicine for oncology patients.

Conclusion:

VersaGel enabled ex vivo growth of 5 patient tumors and one PDX tumor. In all five patients, the assay correctly correlated the clinical outcome to TMZ, showing that the 3D-IC50 values related to time of progression in these patients. Interestingly, for prospective testing of Patient 3, 3D-IC50’s were lower than cmax for crizotinib and chloroquine, but higher for sorafenib. Expanded studies, both retrospective and prospective, will be necessary to broaden clinical validation for advancing personalized medicine and clinician decision making, however this tool could be readily explored for many cancers in oncology drug screening and translational research.

Acknowledgements: The authors graciously thank funding from the Precision Medicine Initiative for Brain Cancer Donor Grant (C. Patil). We also thank Professor Donna Peehl, PhD, and her lab staff of Stanford University for access to renal cell carcinoma PDX tissue and Robert Bell, PhD, at Telo Therapeutics for access to their HepG2 and LN18 human cancer cell lines.

REFERENCES:

1. Friedman AA, Letai A, Fisher DE, Flaherty KT. Precision medicine for cancer with next- generation functional diagnostics. Nat Rev Cancer. 2015;15:746-756.

2. Mehta G, Hsiao AY, Ingram M, Luker GD, Takayama S. Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J Control Release. 2012;164(2):192-204. 3. Sinek J, Frieboes H, Zheng X, Cristini V. Two-dimensional chemotherapy simulations demonstrate fundamental transport and tumor response limitations involving nanoparticles. Biomed Microdevices. 2004;6(4):297-309. 4. Katt ME, Placone AL, Wong AD, Xu ZS, Searson PC. In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform. Front Bioeng Biotechnol. 2016;4:12. 5. Hoffmann OI, Ilmberger C, Magosch S, Joka M, Jauch KW, Mayer B. Impact of the spheroid model complexity on drug response. J Biotechnol. 2015;205:14-23. 6. Patel R, Alahmad AJ. Growth-factor reduced Matrigel source influences stem cell derived brain microvascular endothelial cell barrier properties. Fluids Barriers CNS. 2016;13:6. 7. Vukicevic S, Kleinman HK, Luyten FP, Roberts AB, Roche NS, Reddi AH. Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res. 1992;202(1):1-8. 8. Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987-996. 9. DeAngelis LM. Brain tumors. N Engl J Med. 2001;344(2):114-123. 10. Hegi ME, Liu L, Herman JG, et al. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J Clin Oncol. 2008;26(25):4189-4199. 11. Chaichana KL, Chaichana KK, Olivi A, et al. Surgical outcomes for older patients with glioblastoma multiforme: preoperative factors associated with decreased survival. Clinical article. J Neurosurg. 2011;114(3):587-594. 12. Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med. 2005;352(10):997-1003. 13. Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol. 2014;12(4):207-218. 14. Herrmann D, Conway JR, Vennin C, et al. Three-dimensional cancer models mimic cell- matrix interactions in the tumour microenvironment. Carcinogenesis. 2014;35(8):1671- 1679. 15. Song HH, Park KM, Gerecht S. Hydrogels to model 3D in vitro microenvironment of tumor vascularization. Adv Drug Deliv Rev. 2014;79-80:19-29. 16. Mironi-Harpaz I, Wang DY, Venkatraman S, Seliktar D. Photopolymerization of cell- encapsulating hydrogels: crosslinking efficiency versus cytotoxicity. Acta Biomater. 2012;8(5):1838-1848. 17. Liu VA, Bhatia SN. Three-Dimensional Photopatterning of Hydrogels Containing Living Cells. Biomed Microdevices. 2002;4(4):257–266. 18. Ifkovits JL, Burdick JA. Photopolymerizable and Degradable Biomaterials for Tissue Engineering Applications. Tissue Eng. 2007;13(10):2369-2385.

19. Hammond LA, Eckardt JR, Baker SD, et al. Phase I and pharmacokinetic study of temozolomide on a daily-for-5-days schedule in patients with advanced solid malignancies. J Clin Oncol. 1999;17(8):2604-2613. 20. Portnow J, Badie B, Chen M, Liu A, Blanchard S, Synold TW. The neuropharmacokinetics of temozolomide in patients with resectable brain tumors: potential implications for the current approach to chemoradiation. Clin Cancer Res. 2009;15(22):7092-7098. 21. Hottinger AF, Aissa AB,1 Espeli V, et al. Phase I study of sorafenib combined with radiation therapy and temozolomide as first-line treatment of high-grade glioma. Br J Cancer. 2014;110(11):2655-2661. 22. Junca A, Villalva C, Tachon G, et al. Crizotinib targets in glioblastoma stem cells. Cancer Med. 2017;6(11):2625–2634. 23: Briceño E, Reyes S, Sotelo J. Therapy of glioblastoma multiforme improved by the antimutagenic chloroquine. Neurosurg Focus. 2003;14(2):e3. 25: Orbach RC, Zineh I. NDA 202570 Clinical Pharmacology Review - Crizotinib. 2011. Application #: 202570Orig1s000. 26. Abbruzzese C, Matteoni S, Signore M. Drug repurposing for the treatment of glioblastoma multiforme. J Exp Clin Cancer Res. 2017;36:169.

Clinical Months Clinical MGMT MGMT MTT MTT VersaGel VersaGel to Recurrance Outcome Methylation Prediction IC50 (µM) Prediction IC50 (µM) Prediction Patient 1 2.7 NR – NR NA NR >125µM NR Patient 2 5.0 NR – NR NA NR >125µM NR Patient 3 5.8 NR – NR NA NR 128 NR Patient 4 8.7 PR + R NA NR 49.8 PR Patient 5 >16 R + R NA NR 0.16 R Table 1: 3D-VersaGel IC50 as compared to clinical data, MGMT methylation status, and liquid suspension MTT assay for 5 GBM patients. MTT values did not reach IC50 therefore were written as NA (not available). NR = Non-responder; PR = Partial-Responder; R = Responder.

FIGURE LEGENDS

Figure 1: This figure describes the process workflow of culturing ex vivo (patient, animal- derived) spheroids in VersaGel: 1) resect the tumor from the patient or animal; 2) dissociate the tissue fragments to single cells and subsequent culture in liquid on ultra low attachment (ULA) plates to generate multicellular spheroids; 3) mix spheroids with VersaGel solution and pipette in standard multi-well glass-bottom plates; 4) solidify VersaGel with low intensity UV light; 5) culture in standard media (eg. Neurosphere media for GBM spheroids) with titrated drug concentrations to assess 3D growth of spheroids and 3D-IC50.

Figure 2: Quantification of spheres, showing original image of spheroids embedded in VersaGel (A), magnified image of one spheroid and its area circled in analysis software (B), and growth analysis resulting from multiple timepoints of individual spheres. Because VersaGel remains attached to the glass bottom plate and spheroids grow in the same position, time-course analysis using a coordinate system is feasible and adaptable to high content analysis. Scale bars in A and B are 300 µm and 50µm, respectively.

Figure 3: PDX tumor growth in VersaGel. A) illustration depicting the two approaches explored with a renal cell carcinoma (RCC) ex vivo PDX tissue: spheroid approach (conditioned media or fresh media) and tissue fragment approach. B) representative images of the live cultures on day 3 after VersaGel embedding, noting invasiveness in the fragment and conditioned media spheroid approaches and non-invasion in the spheroid, fresh media group.

Figure 4: Retrospective TMZ response using VersaGel and comparison with MTT analysis. A-C) Liquid suspension culture using MTT (IC50) versus 3D VersaGel culture (Growth, IC50) for Non-Responder Patient 1 (A), Partial Responder Patient 4 (B), Responder Patient 5 (C) according to different TMZ concentrations (0, 5, 25, 125, 625 µM); D) representative viability

image for Patient 4 taken at day 14 at concentration 625 µM TMZ, highlighting the viability of the tumor cells despite the TMZ concentration well above the cmax of 25µM; E) Patient 4 (partial responder) neurospheres at day 0, 7, 14 showcasing regrowth at 25 µM TMZ concentration. Scale bar = 200µm; F) Patient 5 neurospheres at Day 0 and Day 18 with day 18 magnified to show their growth and invasiveness in VersaGel of Control (DMSO) spheres compared to 25µM and 125µM TMZ conditions. Scale bar = 200µm.

Figure 5: personalized, prospective testing of GBM Patient 3, as listed in Table 1. A) images of neurospheres embedded in VersaGel for various concentrations (µM) of Sorafenib, Crizotinib, and Chloroquine, with IC50 values labeled on the right. Scale bar = 100 µm. B) resulting 3D growth curves from which IC50 values were found.

A simple three-dimensional hydrogel platform enables ex vivo cell culture of patient and PDX tumors for assaying their response to clinically relevant therapies

Kolin C Hribar, Christopher J Wheeler, Alexey Bazarov, et al.

Mol Cancer Ther Published OnlineFirst February 12, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-18-0359

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2019/02/12/1535-7163.MCT-18-0359.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/early/2019/02/12/1535-7163.MCT-18-0359. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.