Q27 - Single-cell transcriptomics reveal overexpression of PTPRN2 promotes glioblastoma cell migration in a tumor-brain hydrogel model

Introduction: : Glioblastoma (GBM) is a highly aggressive primary brain tumor with poor prognosis. Primary GBM tumors are highly heterogeneous with cells that are known to invade from the tumor edge diffusively into adjacent brain tissue, making these cells impossible to resect and difficult to treat. These poorly characterized invasive cells are thought to cause tumor recurrence seen in nearly all patients. The difference in transcriptomic expression between migratory and non-migratory cells is not well understood. Recent studies have shown a mechanical difference between the tumor bulk and peritumoral brain tissue; however, the extent to which mechanical changes can influence the migratory behavior of GBM cells is not clear. Utilizing heritable DNA barcodes and single-cell transcriptomics we investigated the transcriptomic differences between the migratory and non-migratory cells in a tumor-brain hydrogel interface model. We identified various genes associated with a distinct migratory subpopulation, including tyrosine phosphatase receptor type N2 (PTPRN2) which we chose to further study for its role in GBM migration.

Materials and

Methods: : Patient-derived primary GBM cells were transduced with ClonMapper cell barcodes. These heritable 20-bp mRNA-expressed DNA barcodes allow for the tracking of clonal lineages within our cell population. To investigate migration at the tumor-brain interface, we have developed an interface hydrogel platform to mimic the mechanical change from the tumor edge to peritumoral tissue. Hydrogels consist of thiolated-hyaluronic acid photocrosslinked with PEG-norbornene, which allows us to adjust the crosslinking density to match the stiffness of tumor and brain. HA concentration, RGD integrin binding sites, and diffusion rates were held constant, so that the only effect on migration was stiffness. Barcoded GBM cells were encapsulated into a hydrogel with tumor-like stiffness and allowed to migrate into a hydrogel with brain-like stiffness. Cells were cultured for two weeks then cells from each hydrogel were isolated and submitted for single-cell RNA sequencing. Based on cell counts between conditions in the sequencing data, we used a binomial test to infer if pre-existing subpopulations associated with higher migratory activity were present. Differentially expressed genes between migratory and non-migratory subpopulations were identified based on a >0.5-logfold change and a p-value of < 5*10^(-6) .
To confirm the over expression of PTPRN2 influences GBM migration, knockdown studies were conducted using lentiviral transduction of PTPRN2 shRNA. Cells were encapsulated into interface hydrogels and migration was monitored using live cell imaging over a 10-day period. Invasion rates were calculated by measuring the new confluence area in the migratory gel over time.

Results, Conclusions, and Discussions:: Evaluating differential gene expression between the migratory and non-migratory cells revealed that the most downregulated genes in the migrating cells were related to the matrisome and ECM production while many of the highly upregulated genes were associated with motility and poor survival. We identified 5 lineages with statistically significant migratory activity and 10 lineages with statistically significant non-migratory tendencies. We conducted differential gene expression on these pre-existing lineages using their expression in a control soft gel to investigate the genes and pathways which may indicate a higher tendency for a cell to be migratory. Enriched pathways in the migratory lineages include transportation and regulation of secretory cargo and regulation of the microtubule and actin cytoskeleton.
We chose to further investigate protein PTPRN2 for its role in GBM migration. PTPRN2 is a transmembrane protein which is proposed to influence cell migration and vesicle formation by regulating plasma membrane signaling protein PI(4,5)P2. First, we verified that when separated, the migratory cells had significantly higher expression of PTPRN2 compared to the non-migratory cells, as evident through immunofluorescence and seen in Figure 1a. We then investigated the effect of gene knockdown using lentiviral transduction of PTPRN2 shRNA. When cells were encapsulated in an interface hydrogel, we observed significantly less cell invasion into the soft brain-like hydrogel in the knockdown condition compared to the control group seen in Figure 1b. These results suggest that over expression of PTPRN2 is related to a more migratory phenotype as identified in our single cell sequencing data and knockdown study.
Our hydrogel interface model is a relevant model of the mechanical transition seen at the tumor-brain interface. We found that pre-existing migratory subpopulations exist within the tumor which are better adapted for a migratory phenotype. We investigated the expression of PTPRN2 using gene knockdown and found cells migrated significantly less than control cells indicating that PTPRN2 plays a role in GBM cell migration at the tumor edge.

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