(F-217) Modeling Proliferation of Glioblastoma in Gelatin Maleimide Granular Hydrogels

Introduction:: Glioblastoma (GBM) is the most common primary malignant brain tumor. The prognosis is poor, with a median survival time of approximately one year and a five-year survival rate between 5% and 10%. Currently, there is a lack of preclinical models that can efficiently recapitulate tumor biology and tumor microenvironment. Previously, we have developed a bulk hydrogel in vitro model of the brain perivascular niche to modulate GBM cell invasion, proliferation, and track therapeutic response¹. We are now interested in adapting granular hydrogel approaches to take advantage of percolating pores to improve cell bioactivity as an alternative to bulk hydrogels. We are developing a jammed granular hydrogel model of GBM to mimic the tumor microenvironment and gain a better understanding of GBM invasion behavior. To date, granular materials have largely been realized as acellular hydrogel particles with cells cultured in the voids between particles. Efforts here are encapsulating GBM cells in hydrogel microdroplets that can be rapidly formed, have their matrix composition tailored for discrete cell populations, and be non-toxically degraded to eventually study multicellular interactions within the GBM microenvironment.




Materials and Methods::

42MGBA variants (WT and TMZ res) cells were provided by Dr. Rebecca Riggins (Georgetown University, Washington, DC). Briefly, a prepolymer solution of 4 wt% maleimide-functionalized gelatin (GelMAL) will be prepared to encapsulate 42MGBA cells at 4 x10⁶ cells mL^-1 density and crosslinked with DTT. GBM cells will be encapsulated in microdroplets using a flow-focusing microfluidic device, using an emulsion of DTT in light mineral oil/2% v/v SPAN80 to form droplets². Microgels containing GBM cells are loaded into the central channel of an AIM Biotech chip. TMZ will be added 24 hours after encapsulation at 8 serial conditions [0, 1, 3, 10, 30, 100, 300, and 1000 μM TMZ]. We will track their proliferation rates while exposed to TMZ dosage ranges using AlamarBlue at days 0, 3, 5, and 7. We will collect information regarding the cycling or proliferative state of the GBM cells by staining with cPARP and KI67. Analyzing the IC50 curves, we will decide on time frames before and after there is a large shift to inspect cPARP+/Ki67+ staining. We will fix using formalin, permeabilize using 0.5% Tween 20, and block with 5% donkey serum before staining with primary antibodies. Primary antibodies cleaved-PARP (1:400) and KI67 (1:500) and secondary antibodies Alexa Fluor 555 and 633 (1:500) will be used as we have previously done on bulk hydrogels. Antibodies will be applied overnight. Hoechst (1:2000) will be used as a nuclear marker. A comparison of Gel-MAL bulk hydrogels to granular hydrogels will be assessed to establish a size-scaled relationship.




Results, Conclusions, and Discussions::

We have demonstrated the ability to use a microfluidic emulsion method for repeatable and consistent microdroplet formation. 4 wt% acellular GelMAL microgels remain stable for a minimum of seven days (Fig. 1). GBM cells embedded into GelMAL microgels can be tracked with the non-toxic reagent CellTracker Green CMFDA (Figure 2). Microgels containing GBM cells were injected into an AIM Biotech chip that allowed us to continually track the same microgels for seven days. A decrease of CellTracker Green CMFDA at day three, no longer visible at day five, suggests cell proliferation. Results of ongoing experiments will reveal the proliferative status of encapsulated cells in response to single and metronomic doses of the frontline therapeutic TMZ as a means to validate a new modality to evaluate GBM cell activity within microgels in free suspension.






Granular hydrogels may possess the ability to better recapitulate the GBM microenvironment by allowing a higher diffusivity of nutrients to cells compared to bulk gels. This methodology will also mitigate cell migration and allow further insight into tumor-mediated restructuring of the microenvironment. We will establish a platform to examine drug response in vitro at a scaled-down version using our microdroplet system. Future studies will compare drug response of cells in bulk and granular hydrogels of the 42MGBA variants.










Acknowledgements (Optional): : This work was supported by the NIH (R01CA256481).

References (Optional): :
[1] Ngo et al. Adv. Sci. 2022; 220188 [2] Headen et al., Adv. Mater. 2014. 26(10): 3003-3008