GFP positive pericytes are found preferably within hypoxic regions of glioma.

<p>(A) GFP positive cells (arrows) are attracted more numerous within the penumbra zone around GLUT1 positive hypoxic regions close to the GL261 tumor border (dashed), scale bar is 200 μm. Normoxic and hypoxic parts of the tumor are marked with T_normox and T_hypox, respectively. (B) Few GFP positive pericytes (arrows) are found at normoxic regions within the tumor, scale bar as in A.</p

Pericytes are associated with laminin-expressing tumor satellites.

<p>(A) Overview image of GL261 tumor expressing high levels of laminin, scale bar is 1000 μm. (B) Higher magnification of laminin expression around the tumor border, scale bar is 200 μm. (C) GFP positive pericytes localize close to laminin-expressing tumor microsatellites outside the tumor (arrows), scale bar as in B. (D) However, they do not express laminin themselves. Scale bar is 500 μm in the low magnification image and 20 μm in the high magnification images.</p

A proportion of the pericytes proliferate intratumorally.

<p>(A) A subset of the GFP positive cells within GL261 tumors express the proliferation marker Ki67, (B) whereas a majority of the cells do not. Scale bar is 500 μm in the low magnification images and 20 μm in the high magnification images.</p

GFP positive cells become neither astrocytes nor microglia.

<p>None of the GFP positive cells express (A) S100B or (B) Iba1, ruling out the possibility that they become astrocytes or microglia. Scale bar is 500 μm in the low magnification images and 20 μm in the high magnification images.</p

Pericytes are activated around glioma.

<p>(A) Low magnification photomicrograph of a normal rgs5^GFP/+ mouse brain, scale bar is 500 μm. (B) Under normal conditions, quiescent GFP positive pericytes showed a flat morphology with a small cell body (arrows), scale bar is 20 μm. (C) However, in response to a GL261 glioma, the number of GFP positive pericytes within the cerebral cortex was significantly increased, both in the ipsilateral and contralateral hemisphere, compared to a normal mouse brain without tumor (n = 3, mean ± SEM, ***, p<0.001, ANOVA). (D) Low magnification photomicrograph of a representative GL261 tumor (dashed) in the rgs5^GFP/+ mouse brain, scale bar as in A. Pericytes showing a morphology consistent with activated pericytes were found in the cerebral cortex both in the (E) ipsilateral and (F) contralateral hemisphere, scale bars as in B. (G) Low magnification photomicrograph of a representative GL261 tumor (dashed) in the rgs5^GFP/+ mouse brain showing the SVZ (arrows), scale bar as in A. Activated pericytes are present (H) in the SVZ ipsilateral to the tumor but not (I) in the SVZ contralateral to the tumor, scale bar is 50 μm. (J) The morphology of the GFP positive pericytes inside the tumor was different compared to the pericytes in the cortex, with either (K) a flattened cell body with elongated processes (arrows) or (L) a prominent cell body with tuft-like processes (arrow). Scale bar in J as in H and scale bars in K-L as in B.</p

Pericytes within the tumor localize adjacent to vessels.

<p>The GFP positive cells within GL261 tumors are localized near cells expressing (A) CD31 and (B) VEGF-R, but do not express the markers themselves. Scale bar is 500 μm in the low magnification images and 20 μm in the high magnification images.</p

GFP positive cells express pericyte markers.

<p>(A) All GFP positive cells within the GL261 tumor are clearly positive for PDGFR-β. (B) Out of all PDGFR-β positive cells within the tumor, 57 ± 6.6% are host-derived GFP positive cells (n = 3, mean ± SEM). (C) A proportion of the cells are positive for the activation marker NG2. The majority of the GFP positive cells lack expression of the mesenchymal stromal cell marker CD13. (D) GFP positive cell at the tumor border expressing CD13. (E) The majority of the cells weakly express the pericyte marker α-SMA. Scale bar is 500 μm in the low magnification images and 20 μm in the high magnification images.</p

OGD produces a stronger gene regulatory response than hypoxia and glucose deprivation alone.

<p>(A) Normalised log2 transformed gene expression data was compared in a box plot to ensure that normalisation was equal between the samples of the bead-array. Error bars show standard deviation (SD). (B) PCA-plot of the top 500 probe sets p<0.05 comparisons to control to show that the replicates within the gene expression data group according to treatments. (C, E) Venn diagrams were constructed from the upregulated genes, (C) fold change (fc) >1.5 with a p-value <0.05 after 2hs or (E) 6 hs of: Control, Hypoxia, or oxygen and glucose deprivation (OGD). (D, F) Venn diagrams of down regulated genes fc<0.66, p-value <0.05 in (D) 2h or (F) 6 h treatments as in (C, E).</p

HIF1α BRGs 6h OGD.

<p>HIF1α BRGs 6h OGD.</p

STAT3 dominates the OGD responses but shows equal activity to HIF1α in the hypoxic responses in 6h treated pericytes.

<p>STAT3 or HIF1α bound regulated genes (BRGs) with binding site(s) 5Kbp of a TSS were identified by comparing ChIP-seq data to the pericytic Illumina gene expression data. (A) HIF1α alpha BRGs in the 6h hypoxia or (B) OGD condition (fc>1.5, p <0.05) was plotted in a heat map. Panel (C) shows STAT3 BRGs in the hypoxia and (D) OGD condition plotted in a heat map. (E) A Venn diagram was constructed to highlight the differences in STAT3 or HIF1α BRG distribution between 6 h hypoxic and OGD conditions.</p