Glioblastoma extracellular vesicles influence glial cell hyaluronic acid deposition to promote invasiveness

Introduction

Glioblastoma (GBM) is a highly aggressive and invasive brain tumour that inevitably recurs following standard-of-care treatments. GBM cells that infiltrate into healthy brain tissues beyond surgical margins are often the source of recurrent tumours¹. Prior studies have revealed that tumour cells migrate in an adhesion-dependent manner along blood vessels and white matter tracts. GBM cells also exhibit extensive crosstalk with cells present in their microenvironment: for example, they can form interconnected networks via tunneling nanotubes as well as bona fide excitatory synapses with surrounding neurons^2-5. Tumour cells can also influence neighbouring cells by secreting extracellular vesicles (EVs). For instance, tumour cell derived EVs have been demonstrated to promote an immune-suppressive environment to bolster tumour growth^6-9. To prevent recurrence and improve patient outcomes, there is an urgent need to investigate how crosstalk between GBM cells and their neighbours fosters GBM cell invasion.

In this study, the authors illustrate a pro-invasive signalling axis between GBM cells and neighbouring astrocytes. GBM cells bearing an oncogenic p53^R273H mutation release EVs containing podocalyxin into their microenvironment, which are picked up by astrocytes. In response, astrocytes increase their deposition of hyaluronic acid (HA)-rich extracellular matrix, providing a substrate for boosting GBM cell migration. Importantly, the authors demonstrate that genetic deletion of PODXL in GBM cells attenuates GBM cell invasion in vivo.

Key Findings

EVs from GBM cells influence astrocytes to deposit ECM

The authors used two patient-derived glioma stem-cell like lines (G7 and E2 cells) that exhibited different invasive characteristics in vivo. E2 cells infiltrate throughout the brain in a scattered manner, whereas G7 cells grow as a solid tumour mass with little invasion. The authors hypothesized that factors secreted by GBM cells could underlie their different migratory patterns through the ECM of the brain, which is primarily maintained by astrocytes. Therefore, they harvested EVs secreted by G7 and E2 cells, treated primary astrocyte cultures with EVs, then allowed astrocytes to deposit ECM. Interestingly, GBM cells that were plated onto ECM deposited by astrocytes treated with EVs from E2 cells migrated much more quickly than those treated with EVs from G7 cells, hinting that the composition of G7 versus E2-derived EVs differed in their ability to influence ECM deposition by astrocytes.

Deleting p53^R273H from GBM cells reduces ECM deposition from astrocytes and decreases GBM cell migration

Deep sequencing revealed that highly invasive E2 cells bear an oncogenic p53^R273H mutation. Previous studies showed that the p53^R273H mutation in carcinoma cells promotes cell migration by controlling the amount of podocalyxin (PODXL) in tumour-cell derived EVs¹⁰. After deleting p53^R273H in E2 cells using CRISPR-Cas9, the authors found PODXL levels within EVs were increased in p53^R273H KO E2 cells, and EVs derived from these cells had a decreased ability to promote ECM deposition from astrocytes. The authors hypothesized that PODXL levels within EVs must fall within a specific range to encourage astrocytes to deposit pro-migratory ECM. To verify this, they generated PODXL-overexpressing (OE) cells or deleted the PODXL gene in E2 cells (PODXL-KO). Astrocyte cultures treated with EVs from PODXL-OE or KO E2 cells did not deposit ECM in a way that supported GBM cell migration as observed via live cell imaging. Furthermore, pre-treatment of mouse brain slices with PODXL-OE or KO EVs decreased the migratory capacity of GBM cells seeded onto the slices, suggesting the level of PODXL within EVs must be finely tuned.

EVs promote GBM cell migration by tuning hyaluronic acid content of astrocyte-derived ECM

The ECM within the brain has a unique composition of proteo- and glycosamino-glycans which differs from fibrillar proteins that are typically found in the ECM of other organs¹¹. To determine how GBM-cell derived EVs influence the composition of ECM deposited by astrocytes, the authors performed a screen using a panel of lectins (proteins that bind carbohydrates) and other reagents that bind carbohydrate moieties. Notably, treating astrocytes with EVs from p53^R273H E2 cells increased the hyaluronic acid (HA) content in the ECM, as detected by hyaluronic acid binding protein (HABP) and immunofluorescence staining. HA levels were unaffected when astrocytes were treated with EVs from G7, p53^R273H-KO E2, or PODXL-KO E2 cells. To determine whether HA was responsible for boosting GBM cell migration, the authors treated astrocyte cultures with hyaluronidase (Hase), an enzyme that degrades HA. Hase treatment significantly decreased the migratory capacity of GBM cells, indicating that HA is a key ECM component that promotes the invasion of GBM cells.

PODXL drives mutant p53^R273H-driven infiltrative behaviour of GBM in vivo

To determine whether EV-mediated crosstalk between GBM cells and astrocytes promotes tumour cell invasion in vivo, the authors injected p53^R273H-KO E2, PODXL-KO E2 cells, or their control counterparts in the right forebrain of immunocompromised mice. p53^R273H-KO cells generated tumours that were too small to quantify invasive characteristics. In contrast, while PODXL-KO did not affect the overall proportion of proliferating (Ki67+) tumour cells, a much smaller proportion of GBM cells migrated to the left hemisphere of the brain compared to control, indicating their invasive capacity was compromised. Overall, these results suggest that targeting PODXL is an effective strategy for curbing GBM cell invasion.

Figure 1. A schematic outlining the EV-mediated crosstalk between GBM cells and astrocytes. GBM cells bearing the p53^R273H mutation secrete PODXL-containing EVs into their extracellular environment. Astrocytes respond to EVs by increasing HA deposition. Increased HA content in the ECM promotes GBM cell migration and infiltration into distant brain tissues. Schematic drawn by Reinier Prosée.

Why I chose this preprint

GBM is notoriously infiltrative, which contributes to its near-universal recurrence following surgery. I chose this preprint because it illustrates a complete signalling axis wherein GBM cells influence their neighbouring cell types into fuelling their invasion into healthy brain tissue. I am currently investigating the role of a specific protein in adhesion-dependent versus adhesion-independent migration in GBM. This preprint, along with other studies examining the crosstalk between tumour cells and their microenvironment, have inspired me to look beyond cell-autonomous factors that influence GBM cell invasion. Looking into microenvironmental relationships may be important for developing new therapies since GBM cells are highly heterogeneous.

Questions for the authors

1. Is the overall survival of the mice bearing PODXL-KO xenograft tumours prolonged compared to control?
2. How does EV-derived PODXL boost HA secretion in astrocytes?
3. Surprisingly, EVs derived from p53^R273H-KO E2 cells contained a greater amount of PODXL, yet their ability to encourage astrocytes to deposit pro-migratory ECM was reduced. How does p53^R273H tune the amount of PODXL present in EVs?
4. Can other cells in the GBM microenvironment (ex. microglia, neurons, oligodendrocytes) also perceive and respond to PODXL-containing EVs?

References

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Tags: astrocyte, extracellular matrix, glioblastoma, glioma, migration, vesicle

doi: https://doi.org/10.1242/prelights.34825

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