MULTISCALE BIOMATERIALS-ENABLED GLIOBLASTOMA STEM CELL-TARGETED THERAPY WITH microRNA TO OVERCOME CANCER RECURRENCE

Abstract

Glioblastoma (GBM) is one of the most lethal diseases in the world with a dismal ~7.2% 5-year survival rate and median survival time of ~15 months. More than 90% of GBM patients experience recurrence, which is primarily attributed to the presence of a rare subpopulation of cancer cells inside tumors called glioblastoma stem cells (GSCs). These GSCs are highly drug-resistant and tumorigenic. To address recurrence in GBM, it is crucial to isolate true GSCs, which possess two main abilities of all classical stem cells: self-renewal and multilineage differentiation. Current isolation technologies for GSCs, and all cancer stem cells (CSCs), predominantly include 1) sorting based on the expression of putative markers for CSCs, and 2) 3D suspension culture in defined, serum-free medium. However, these methods are unreliable because there is no definitive marker for CSCs and there is merging of cells in 3D suspension culture. Due to the deficiencies of these methods, no GSCs have been previously isolated that include the capability of both self-renewal and multi-lineage differentiation. Using our previously developed microfluidics-enabled 1-cell culture method, we demonstrated that true breast cancer stem cells with the ability for self-renewal and multi-lineage differentiation can be isolated. With the same method, we isolated patient GSCs that are capable of self-renewal and multi-lineage differentiation, which has never been demonstrated before. With microRNA (miR) sequencing, we identified miRs-10a/b as candidates that are differentially expressed in the GSCs from 1-cell culture compared to “GSCs” from conventional 3D suspension culture. These miRs were encapsulated inside nitric oxide (NO) precursor-laden, fucoidan-decorated nanoparticles. These nanoparticles generate NO gas and disassemble under low pH conditions, and are thus named “nanobombs” due to this explosive effect. The fucoidan is decorated on the nanoparticle surface for targeting of both activated blood vessels adjacent to GBM cells, and GBM cells directly. The NO-releasing, fucoidan-decorated nanobombs demonstrate efficient blood-brain barrier (BBB) crossing in vitro and endosomal escape into the cytosol of GBM cells. After treatment with miR-10a/b-laden, fucoidan-decorated nanobombs, GBM cells show diminished stemness marker expression and self-renewal capability. Moreover, miR-10a/b-laden, fucoidan-decorated nanobombs and temozolomide (TMZ) co-treatment eliminates recurrence in vitro. However, recurrence occurs in GBM neurospheres that are administered with all other control treatments, including miR-scrambled-laden, fucoidan-decorated nanobombs plus TMZ, miR-10a-, miR-10b-, and miR-scrambled-laden, fucoidan-decorated nanobombs, free TMZ, and DMSO. Using a multi-scale approach that includes 1) microfluidics-enabled GSC isolation, 2) fucoidan-decorated nanobombs which are NO gas-generating, BBB-penetrating and endosomal-escaping, and 3) miR target identification in GSCs, there is promise to target true GSCs and inhibit recurrence in GBM patients.