(F-227) Glioblastoma culture in 3D hydrogel-based constructs highlights intertumoral heterogeneity and variable response to temozolomide

Introduction:: Glioblastoma (GBM) is a uniformly lethal cancer [1]. Conventional treatment, including maximal safe surgical resection followed by concurrent temozolomide (TMZ) and radiation therapy, fails to prevent a more than 90% recurrence rate. A plethora of studies theorize reasons for the dismal treatment response in GBM patients [2,3]. However, the role of intertumoral heterogeneity, wherein a similar dosage of chemotherapeutic drug fails to elicit a uniform response between GBM patients, is not fully known. Further, the attribute of heterogeneity in developing treatment resistance needs to be studied in a tumor microenvironment-mimetic model system. In this study, we demonstrate the phenotypic diversity and treatment-response variability in patient-derived GBM cell lines cultured in extracellular matrix-mimetic 3D hydrogel constructs. Using 4 GBM cell lines of distinct molecular mutations, we showcase the need for personalized regimen and possible targeted therapy for each GBM patient to prevent treatment resistance and achieve complete remission.


Materials and Methods:: Patient-derived glioblastoma (GBM) cell lines, GS54, GS304, GS187, and HK217 were cultured in DMEM/F12 with l-glutamine and 15 mm HEPES in 1× Gem21, 0.2% Normocin, 20 ng mL−1 human fibroblast growth factor-2, 50 ng mL−1 human epidermal growth factor, and 25 mg mL−1 heparin. Cells were passaged and seeded (600 000 cells/well) into individual wells of a 24-well AggreWell plate pre-coated with 5% Pluronic in 1× PBS solution. For 3D encapsulation, gliomaspheres (≈150 µm in diameter) were harvested from AggreWell plates and resuspended in 10 mg/ml thiolated hyaluronic acid-based (HA-SH) 3D hydrogel system [4].

Hydrogel precursor solution was prepared by dissolving HA-SH (0.5% w/v), 4-arm thiol terminated polyethylene glycol (PEG-SH), 8-arm norbornene terminated polyethylene glycol (PEG-Norb), 0.025% w/v lithium phenyl-2,4,6 trimethylben-zoylphosphinate (LAP) and 0.25 mM thiolated RGD peptides (GCGYGRGDSPG) in 20 mM HEPES buffer (pH = 7). The hydrogel precursor solution was cast into 8 mm diameter silicone rubber molds and irradiated with long-wave UV (365 nm, 4.2 mW/cm2) for 15 s [5].

Gliomasphere morphology and extent of migration was analyzed using ImageJ software. Cytotoxicity (IC50) and proliferation of temozolomide-treated (TMZ) GBM cells was determined using Cell Counting Kit-8 (CCK-8). For the latter, 2000 cells were encapsulated in hydrogels and treated with TMZ for three days, followed by a 3-day treatment-free period, and concluded by 3 days of treatment. CCK-8 readings were captured every 2 days. Statistical analysis was performed using ANOVA (one or two-way) followed up by Tukey post-hoc test.

Results, Conclusions, and Discussions:: Glioblastoma (GBM) cells derived from different patients exhibited distinct phenotypic properties. Notably, the rate of growth, migratory pattern in 3D hydrogels, and response to temozolomide (TMZ) treatment varied widely between cell lines. Based on the extent of cellular projection from gliomaspheres, it was noted that the relative migration of GS304 increased almost 6-times from Day 2 to Day 5, whereas that of GS187 remained less than 2-fold (Figure 1).

Further, upon assessing the cytotoxicity of TMZ on each cell line, the difference in response was more prominent. GS54, GS304, and HK217 demonstrated an IC50 around 50-70 μM, whereas the IC50 of GS187 was almost five times higher (Figure 2). In addition, while most cells responded positively to increasing concentrations of TMZ, the dose-response curve of GS304 indicated a pattern of acquired resistance.

A regimen of TMZ on and off treatment (Figure 3A) yielded interesting insights. Proliferation assay with each cell line's respective IC50 concentration showed a similar pattern of growth for all cells regardless of treatment (Figure 3B). However, upon closer observation, variable responses to treatment regimen are evident. Control and treated cells of GS54, GS304, and GS187 seem to follow a similar growth rate, while the TMZ-treated HK217 cells seem to outgrow their untreated counterpart by Day 8 (Figure 4). Nevertheless, in none of the cell lines do we note a complete response and possible remission when treated with TMZ.

These preliminary results indicate that GBM cell lines have a demonstrable heterogeneity in terms of phenotype and treatment response between patients. We also show how a 'one-dose-fits-all' approach may be ill-advised for GBM patients treated with TMZ. Furthermore, the accelerated growth of TMZ-treated HK217 cells implies that temozolomide treatment in some patients may lead to drug resistance and possible recurrence of GBM. To substantiate our findings, the study will also include comparison of molecular aberrations and signaling pathways between TMZ-treated lines using high-throughput Luminex platform. Overall, using a matrix-mimetic 3D hydrogel construct, our study demonstrates the need for personalized dosage regimen and potential targeted therapies in GBM patients.

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References (Optional): :
1. Ostrom, Q. T., Gittleman, H., Xu, J., Kromer, C., Wolinsky, Y., Kruchko, C., & Barnholtz-Sloan, J. S. (2016). CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2009-2013. Neuro-Oncology, 18(suppl_5), v1–v75. https://doi.org/10.1093/neuonc/now207

2. Auffinger, B., Spencer, D., Pytel, P., Ahmed, A. U., & Lesniak, M. S. (2015). The role of glioma stem cells in chemotherapy resistance and glioblastoma multiforme recurrence. Expert Review of Neurotherapeutics, 15(7), 741–752. https://doi.org/10.1586/14737175.2015.1051968

3. Wenger, K. J., Wagner, M., You, S.-J., Franz, K., Harter, P. N., Burger, M. C., Voss, M., Ronellenfitsch, M. W., Fokas, E., Steinbach, J. P., & Bähr, O. (2017). Bevacizumab as a last-line treatment for glioblastoma following failure of radiotherapy, temozolomide and lomustine. Oncology Letters, 14(1), 1141–1146. https://doi.org/10.3892/ol.2017.6251

4. Xiao, W., Zhang, R., Sohrabi, A., Ehsanipour, A., Sun, S., Liang, J., Walthers, C. M., Ta, L., Nathanson, D. A., & Seidlits, S. K. (2018). Brain-Mimetic 3D Culture Platforms Allow Investigation of Cooperative Effects of Extracellular Matrix Features on Therapeutic Resistance in Glioblastoma. Cancer Research, 78(5), 1358–1370. https://doi.org/10.1158/0008-5472.CAN-17-2429

5. Ghochani, Y., Muthukrishnan, S. D., Sohrabi, A., Kawaguchi, R., Condro, M. C., Bastola, S., Gao, F., Qin, Y., Mottahedeh, J., Iruela-Arispe, M. L., Rao, N., Laks, D. R., Liau, L. M., Mathern, G. W., Goldman, S. A., Carmichael, S. T., Nakano, I., Coppola, G., Seidlits, S. K., & Kornblum, H. I. (2022). A molecular interactome of the glioblastoma perivascular niche reveals integrin binding sialoprotein as a mediator of tumor cell migration. Cell Reports, 41(3), 111511. https://doi.org/10.1016/j.celrep.2022.111511