Strategies of Mesenchymal Invasion of Patient-derived Brain Tumors: Microenvironmental Adaptation

Abstract

The high mortality in glioblastoma multiforme (GBM) patients is primarily caused by extensive infiltration into adjacent tissue and subsequent rapid recurrence. There are no clear therapeutic strategies that target the infiltrative subpopulation of GBM mass. Using mesenchymal mode of invasion, the GBM is known to widely infiltrate by interacting with various unique components within brain microenvironment such as hyaluronic acid (HA)-rich matrix and white matter tracts. However, it is unclear how these GBM microenvironments influence the strategies of mesenchymal invasion. We hypothesize that GBM has different strategies to facilitate such invasion through adaptation to their local microenvironment. Using our in vitro biomimetic microenvironment platform for three-dimensional GBM tumorspheres (TSs), we found that the strategies of GBM invasion were predominantly regulated by the HA-rich ECM microenvironment, showing marked phenotypic changes in the presence of HA, which were mainly mediated by HA synthase (HAS). Interestingly, after inhibition of the HAS gene, GBM switched their invasion strategies to a focal adhesion (FA)-mediated invasion. These results demonstrate that the microenvironmental adaptation allowed a flexible invasion strategy for GBM. Using our model, we suggest a new inhibitory pathway for targeting infiltrative GBM and propose an importance of multi-target therapy for GBM, which underwent microenvironmental adaptation.

Figure 1. Pro-invasive factors within brain tumor microenvironment.

(A) Schematic illustration of brain tumor-favorable microenvironment. Hyaluronic acid (HA)-rich extracellular matrix (ECM) and pre-existing brain anatomy (e.g. white matter tract). (B) Illustration of three major components in HA metabolism. Transcriptomic analysis of Gene Expression Omnibus (GEO) data for three major HA metabolic genes between normal brain tissue and GBM tissue: HA receptors (CD44, RHAMM), HA Synthases (HAS1, HAS2, and HAS3), and Hyaluronidase (HYAL1, HYAL2, HYAL3, and HYAL4). The normalized gene expression ranging from 0 (no expression) and 1 (fully expression). Red bar indicates the statistical significance between normal and GBM tissue.

Figure 2. Features of GBM within Hyaluronic acid (HA)-rich ECM environment.

HA can be interpenetrating with a fibrillary, crosslinked collagen (COL) hydrogel, forming hyaluronic acid-collagen semi-interpenetrating polymer network (HA-COL semi-IPN). The patient-derived GBM cells were isolated with CD133 markers using flow cytometry, then labelled with green fluorescent protein (GFP). (A) Structural scheme for HA-COL semi-IPN as HA-rich ECM environment.(B) Scanning electron images of COL and HA-COL semi-IPN matrices. Scale bar: 10 μm. (C) The culture process of three-dimensional GBM tumorsphere (TS) in ECM matrix. (D) Increased relative expression of HA receptors, CD44 and RHAMM in GBM, cultured within non-HA (COL only) and HA-rich (HA-COL semi-IPN) environment. (n = 5~6; Asterisks indicate a significant difference by student’s t-test, *p < 0.05). (E) Normalized proliferation of GBM cultured in non-HA (COL only) and HA-rich (HA-COL semi-IPN) environment (red line: non-HA, yellow line: HA-rich) in a fold-change. (F) Representative fluorescent images of invaded GBM TS within non-HA and HA-rich environment according to the time-variant. The invasiveness of GBM TS within HA-rich ECM environment was higher than one of GBM TS within non-HA environment.

Figure 3. Features of GBM in in vitro biomimetic microenvironment platform.

(A) The culture process of three-dimensional GBM tumorsphere (TS) in in vitro biomimetic microenvironment platform. (B) Morphological characteristics of GBM TSs within the in vitro biomimetic microenvironment platform. Fluorescent images of TSs along the electrospun fiber. (Blue: nucleus, Green: cytosol, Red: F-actin). (C) Relative expression of HA metabolic genes: synthetic genes (HAS) and enzymatic genes (HYAL) (n = 5~6; Asterisks indicate a significant difference by student’s t-test, **p < 0.001; no sign for Non-significant difference).

Figure 4. Inhibition of HAS as a new therapeutic suggestion.

(A) The inhibition of HAS genes in 4-methylumbelliferone (4-MU) treated GBM TSs within biomimetic environment after 72 hr invasion. 4-MU was treated after 12 hr invasion. Inset: 4-MU treated GBM TSs at 0 h. (B) Normalized proliferation of GBM in presence of 4-MU treatment in a fold-change. (A) Magnified immunofluorescent images for invaded GBM TSs in presence of 4-MU treatment. The 4-MU was treated at 12-hour after GBM invasion. Scale bar: 50 μm. (C) Molecular expression profiles in presence of 4-MU. (n = 5~6; Asterisks indicate a significant difference statistically by student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001).