Substrate stiffness regulates the recurrent glioblastoma cell morphology and aggressiveness.

Recurrent glioblastoma is highly aggressive with currently no specific treatment regime. Therefore, to identify novel therapeutic targets for recurrent GBM, we used a cellular model developed in our lab from commercially available cell line U87MG and patient-derived cultures that allows the comparison between radiation naïve (Parent) and recurrent GBM cells generated after parent cells are exposed to lethal dose of radiation. Total RNA-seq of parent and recurrent population revealed significant upregulation of cell-ECM interactions pathway in the recurrent population. These results led us to hypothesize that the physical microenvironment contributes to the aggressiveness of recurrent GBM. To verify this, we cultured parent and recurrent GBM cells on collagen-coated polyacrylamide gels mimicking the stiffness of normal brain (Young's modulus E = 0.5kPa) or tumorigenic brain (E = 10kPa) and tissue culture plastic dishes (E ∼ 1 GPa). We found that compared to parent cells, recurrent cells showed higher proliferation, invasion, migration, and resistance to EGFR inhibitor. Using orthotopic GBM mouse model and resection model, we demonstrate that recurrent cells cultured on 0.5kPa had higher in vivo tumorigenicity and recurrent disease progression than parent cells, whereas these differences were insignificant when parent and recurrent cells were cultured on plastic substrates. Furthermore, recurrent cells on 0.5kPa showed high expression of ECM proteins like Collagen, MMP2 and MMP9. These proteins were also significantly upregulated in recurrent patient biopsies. Additionally, the brain of mice injected with recurrent cells grown on 0.5kPa showed higher Young's moduli suggesting the ability of these cells to make the surrounding ECM stiffer. Total RNA-seq of parent and recurrent cells grown on plastic and 0.5kpa identified PLEKHA7 significantly upregulated specifically in recurrent cells grown on 0.5 kPa substrate. PLEKHA7 was also found to be high in recurrent GBM patient biopsies. Accordingly, PLEKHA7 knockdown reduced invasion and survival of recurrent GBM cells. Together, these data provide an in vitro model system that captures the observed in vivo and clinical behavior of recurrent GBM by mimicking mechanical microenvironment and identifies PLEKHA7 as a novel potential target for recurrent GBM.

Effect of Glucosamine Conjugate-Functionalized Liposomes on Glioma Cell and Healthy Brain: An Insight for Future Application in Brain Infusion.

Conjugation of D-glucosamine with lipophilic moiety can ease its application in surface modification of liposomes. Interestingly, although D-glucosamine is safe, studies have shed light on "toxic effect" of its conjugates on cancer cells and highlighted its application in targeting glioma. However, understanding the safety of such conjugates for local delivery to the brain is unavailable. Herein, after successful synthesis of D-glucosamine conjugate (GC), the toxicity of functionalized liposome was evaluated both in vitro and in vivo. The study revealed a significant effect on cytotoxicity and apoptosis in vitro as assessed on grade IV-resistant glioma cell lines, SF268, U87MG, using MTT assay and PI staining. Additionally, this effect was not observed on normal human erythrocytes in the hemolysis assay. Furthermore, we demonstrated that GC liposomes were non-toxic to the normal brain tissues of healthy Sprague-Dawley rats. Successful functionalization yielded liposome with uniform particle size, stability, and cellular uptake. With < 10% hemolysis, all the liposomal formulations demonstrated hemato-compatibility but led to high glioma cytotoxicity. The surface density of conjugate played an important role in tumor toxicity (0.5 < 1.0 ≤ 2.0% molar ratio). PI staining revealed that compared to control cell, functionalization led 26-fold increase in induction of apoptosis in glioma cells. Absence of histological and behavioral changes along with the absence of caspase-3 in brain tissue confirmed the suitability of the system for direct infusion in the brain. Thus, this study will aid the future development of clinically useful local chemotherapeutic without "add-in" side effects.