Regeneration Enhances Metastasis: A Novel Role for Neurovascular Signaling in Promoting Melanoma Brain Metastasis

Abstract

Neural repair after stroke involves initiation of a cellular proliferative program in the form of angiogenesis, neurogenesis, and molecular growth signals in the surrounding tissue elements. This cellular environment constitutes a niche in which regeneration of new blood vessels and new neurons leads to partial tissue repair after stroke. Cancer metastasis has similar proliferative cellular events in the brain and other organs. Do cancer and CNS tissue repair share similar cellular processes? In this study, we identify a novel role of the regenerative neurovascular niche induced by stroke in promoting brain melanoma metastasis through enhancing cellular interactions with surrounding niche components. Repair-mediated neurovascular signaling induces metastatic cells to express genes crucial to metastasis. Mimicking stroke-like conditions in vitro displays an enhancement of metastatic migration potential and allows for the determination of cell-specific signals produced by the regenerative neurovascular niche. Comparative analysis of both in vitro and in vivo expression profiles reveals a major contribution of endothelial cells in mediating melanoma metastasis. These results point to a previously undiscovered role of the regenerative neurovascular niche in shaping the tumor microenvironment and brain metastatic landscape.

Keywords: angiogenesis; astrocytosis; gliosis; neuroblast; stroke.

FIGURE 1

The regenerative neurovascular niche after stroke facilitates brain metastasis. (A) Representative brain images from control and stroke groups subjected to melanoma metastasis shown in the top panel. GFP⁺ cells identifies brain metastatic melanoma at high magnification in the bottom panel. (B) Schematics showing region from stroke brains and corresponding regions in control quantified. (C) Bar graphs of total number of metastatic cells in both control and stroke groups and (D) showing quantification and distribution of metastatic cells in different brain regions as mean ± SEM (n = 6–7, four sections per animal, ^∗∗p = 0.0082, ^∗∗∗p = 0.0012 Mann–Whitney, two-tailed t-test). PV-WM, periventricular white matter; ST, striatum; OB, olfactory bulb; SVZ, subventricular zone; RMS, rostral migratory stream; HF, hippocampus. (E) Representative images depicting vascular density in control and hypoxia + metastasis group. (F) Box and scatter plots with minimum and maximum percentage vascular density (surface area μm³) mean ± SEM (n = 6–8), (^∗∗p = 0.0090 unpaired, two-tailed t-test) (G) Stroke increases. brain metastasis significantly more than hypoxia mediated angiogenesis. Bar graphs showing number of metastatic foci in stroke and hypoxic groups shown as mean ± SEM (n = 6–8, four sections/animal, ^ap = 0.003, Mann–Whitney, two-tailed t-test). (H) Representative images from days 1 and 7 after stroke showing localization of fluorescent microspheres in green and vasculature (PECAM) in red. (I) Box and scatter plots with minimum and maximum percentage volume of fluorescent microspheres localized to the peri-infarct and contralateral regions at days 1 and 7 after stroke, mean ± SEM (n = 5–6, one-way ANOVA, Holm-Sidak for multiple comparisons p = 0.46, ns, non-significant). (J) Schematics of the method of quantification of fluorescent microspheres volume localized around the peri-infarct regions. (K,L) Representative immunohistochemical images and intensity of endogenous albumin extravasated from days 1 and 7 after stroke and contralateral regions, mean ± SEM (n = 5, three regions/animal, ^∗p = 0.0255, one-way ANOVA, Holm-Sidak for multiple comparisons).

FIGURE 2

Repair augments direct cellular interactions between the metastatic cell and neurovascular niche. (A) Immunohistochemical analysis of metastatic melanoma cells shown as GFP⁺ green, lectin-labeled vasculature in white and DCX⁺ neuroblasts in red. (B) Quantitative analysis of metastatic cell interaction with vessels associated with or without neuroblasts shown as scatter plot with mean ± SEM (n = 5–8, four sections/animal, ^aap < 0.0001; ^bp = 0.0354, Kruskal–Wallis, one-way ANOVA, Dunn’s for multiple comparisons). (C) Representative brain images of metastatic foci (GFP⁺) and neuroblasts (RFP⁺) over time (left). Control groups did not associate with neuroblasts in the corresponding brain region. Schematic representation of distance bins (25 μm intervals) used to analyze neuroblast distribution around metastatic foci. Bin closest to melanoma is shown in bright red and increasing distances showing in gradients of yellow and the farthest depicted in gray (right). (D) Box and scatter plot showing number of neuroblast contact surfaces interacting with metastatic foci. Mean ± SEM (n = 6–9, ^∗∗p = 0.0092, ^∗∗∗∗p = 0.0006, ^aap < 0.0001; one-way ANOVA, Holm-Sidak post hoc test used for multiple comparisons). (E) Bubble plot depicting density and distribution of neuroblasts around metastatic foci at 25 μm distances Mean ± SEM (n = 5–8, ^bp = 0.033, ^cp = 0.033, ^∗p < 0.0164; one-way ANOVA, Holm-Sidak post hoc test used for multiple comparisons). Bubble size represents number of neuroblasts and increasing distances are color coded as shown in schematic.

FIGURE 3

Repair enhances direct cellular interactions between the metastatic cell and astrocytes and increases neovascularization. (A) Representative images showing metastatic melanoma interaction with reactive astrocytes during melanoma extravasation. (B) Quantitative analysis of % volume of metastatic cell extravasated. Mean ± SEM [^∗∗∗p = 0.0082, ^aap < 0.0001, two-way ANOVA, Holm-Sidak post hoc test used for multiple comparisons (n = 5–8)]. (C) Astrocyte coverage at corresponding time points around each metastatic foci. Mean ± SEM [^aap < 0.0001, ^ap = 0.0006, two-way ANOVA, Holm-Sidak post hoc test used for multiple comparisons (n = 5–8)]. (D) Methodology of analysis of neovascularization and infection point analysis. (E) Changes in neovascularization response over time mean ± SEM, (n = 5–8), (^∗p = 0.0424, ^∗∗p = 0.0349, ^ap = 0.0454, ^bp = 0.0015, ^cp = 0.0163, ^dp = 0.0474, ^ep = 0.0009, two-way ANOVA, Fisher LSD). (F) Bar graphs showing temporal changes in inflection point ratios [^∗p = 0.0032, ^∗∗p = 0.0215; ^∗∗∗p = 0.0012; ^aap < 0.0001; two-way ANOVA, Holm-Sidak post hoc test used for multiple comparisons, (n = 5–8)].

FIGURE 4

Human brain metastatic melanoma interact with neuroblasts and astrocytes. (A) Representative image depicting characterization of antibodies on human brain samples. Human hippocampus section showing staining for DCX, PSA-NCAM, GFAP, and Tuc-4. DCX positive neuroblasts colocalize with Tuc-4. (B) Schematics showing human brain tumor margin immunostained for neuroblasts and astrocyte association. (C) Immunohistochemical representative images of brain metastatic melanoma show novel interactions with neuroblasts excised from human patients (4/15 show close neuroblast–melanoma interactions). (D) Representative images of brain metastatic melanoma from human patients show interactions with astrocytes.

FIGURE 5

(A) Schematic representation of tissue processing and FACS sort methodology performed. (B) Gating areas used during FACS and the GFP and MCSP-PE double positive cells were isolated for RNA sequencing. MCSP, melanoma-associated chondroitin sulfate proteoglycan.

FIGURE 6

Molecular signaling systems in regeneration-potentiated brain metastasis. (A) Schematic representation of flow of transcription profiling and regenerative neurovascular niche components interacting with metastatic cells. Significant and differentially expressed genes between regeneration-potentiated and contralateral metastatic cells as determined by log₂ fold change > 0.5 and p < 0.05 (Stroke-MET). (B) Top differentially regulated canonical pathways with largest negative logP shown in metastatic melanoma cells from the regenerative neurovascular niche compared to contralateral tissue. The dashed line represents the ratio of the number of molecules in the dataset over the number of molecules in the pathway. (C) Volcano plot showing p-values correlated with log₂ fold change in metastatic cells isolated from regenerative neurovascular niche vs. contralateral side. Genes systematically identified from top pathways from (B), are marked in blue. (D,E) GSEA of hallmark genes up and downregulated in the Stroke-MET transcriptome. Estimated probability of FDR < 25% represent 3/4 chances of a gene set being valid.

FIGURE 7

OGD alters chemokine/cytokine responses in the neurovascular niche and enhances migration of brain metastatic cells. (A) Schematics showing the interaction of human astrocytes (h-Astro) and human endothelial cells (h-EC) with metastatic melanoma cells. (B) Schematics of method of oxygen glucose deprivation (OGD) conditioned media exposed to metastatic melanoma cells. (C) Migratory response of metastatic melanoma cells exposed to OGD conditioned media from cells are shown as mean ± SD, ^∗P < 0.05. (D,E) Heatmap of relative expression of secreted proteins profiled from the medium of h-Astro (E) and h-EC (D). Functionally grouped enriched pathways are shown in one color. Enrichment/depletion tests performed with ClueGO application on cytoscape, κ score ≥ 0.4.

FIGURE 8

Oxygen-glucose deprivation (OGD) affects viability of human-endothelial cells (h-EC), astrocytes (h-Astro) and adhesion of metastatic melanoma cells. (A,B) Percentage viability of h-EC (A) and h-Astro (B) subjected to OGD for 4 h followed by a 4, 20, and 44 h reperfusion phase. Cell viability as measured by XTT assay. The bars represent the average % viability of OGD exposed cells, normalized to control cells subjected to normal oxygen and glucose levels, mean ± SD, ^∗p < 0.05. The different time points indicate the hours in reperfusion after OGD/control conditions. (C) h-EC were subjected to OGD for 4 h followed by a 20 h reperfusion phase. m-Cherry expressing macro- and micro-metastatic melanoma cells were seeded on top of the h-EC monolayer and incubated for 30 min, to allow adhesion to occur. The fluorescence signal of labeled cells was measured before and after removal of non-adherent cells. The bars represent the % adherent cells in the wells. The graph represents an average of three independent experiments + SD. ^∗P < 0.05. (D) m-Cherry expressing metastatic melanoma cells were seeded on top of the OGD or control h-EC monolayers and incubated for 30 min with 5 μg/mL VCAM-1 blocking Ab, an isotype control (mIgG1), or without IgG. The fluorescence signal of labeled cells was measured before and after removal of non-adherent cells. The bars represent % adherent melanoma cells seeded with anti-VCAM-1 blocking Ab or isotype control normalized to % adherent melanoma cells seeded without Ab. The graph represents an average of two independent experiments + SD. ^∗p < 0.05.

FIGURE 9

Overlapping transcription landscapes from regenerative NV niche after stroke and melanoma brain metastasis. (A,B) Common canonical signaling pathways between the in vivo Stroke-MET and in vitro OGD-_ECMET/OGD-_AstroMET transcriptome. (C,D) Rank–rank hypergeometric overlap (RRHO) analysis of common genes expression spread between Stroke-MET and OGD-_ECMET/OGD-_AstroMET transcriptome. RRHO analysis of common genes expression spread between Stroke-MET and OGD-_AstroMET datasets. Red and blue pixels on the heatmap depict a high and low number of overlapping genes, respectively. Stroke-MET and OGD_EC-MET display stronger hypergeometric overlap those with OGD_Astro-MET transcriptome. (E,F) Overlapping up and downregulated genes between in vivo Stroke-MET and in vitro OGD-_ECMET/OGD-_AstroMET transcriptome are depicted in the Venn diagram. (G–J) Heatmap of log₂ FC of overlapping genes between Stroke-MET and OGD-_ECMET/OGD-_AstroMET transcriptome. The up and down regulated overlapping genes are shown in red and blue a change in gradients of these color correspond to the level of log₂ FC as shown on the scale bars. Genes classified based on the cellular location and the color coded as shown in the key at the bottom of the heatmaps : Blue: extracellular, green: plasma membrane, orange: cytoplasm, violet: nucleus, and black: other.

FIGURE 10

Gene expression changes in OGD exposed metastatic melanoma cells. (A) The effect of OGD exposed astrocyte secreted factors on melanoma gene expression. Astrocytes were subjected to OGD or control conditions for 4 h followed by a 20 h reperfusion phase. Metastatic melanoma cells were treated for 24 h with the OGD or control conditioned media collected from the reperfusion phase. Total RNA was extracted and gene expression was determined by RT-qPCR. mRNA expression levels were normalized to the expression of RS9. Bars represent gene expression in cells treated with conditioned medium of “stroked” astrocytes normalized to gene expression in cells treated with control astrocyte conditioned medium mean ± SD of three independent experiments, ^∗p < 0.05, ^∗∗p < 0.01. (B) Primer sequences used for the qPCR analysis shown in (A,B).

FIGURE 11

Astrocyte–metastatic cell and endothelial cell–metastatic cell interactome. Extracellular and plasma membrane bound proteins were chosen to determine putative gene interactions between metastatic melanoma cell with two cell types in the regenerative neurovascular niche–astrocyte/endothelial cells. Astrocyte–metastatic cell interactome display differentially expressed proteins in blue shows OGD exposed astrocyte secretome (blue panel) and their interactions with Genes in the metastatic melanoma cells from the Stroke-MET transcriptome colored in green (green panel). Endothelial cell–metastatic cell interactome differentially expressed proteins in red shows OGD exposed astrocyte secretome (red panel) and their interactions with genes in the metastatic melanoma cells from the Stroke-MET transcriptome colored in green (green panel). Yellow panels depicts molecules in the extracellular space. Molecules in yellow: secretome, red: OGD exposed endothelial cells, blue: OGD exposed astrocytes, green: stroke responsive melanoma Stroke-MET.

FIGURE 12

Gene network analysis of highly repetitive classes of coordinated diseases and functions from the Stroke-MET transcriptome. Network of gene interactions representing genes associated with “cancer” (A), “Neurological disease and injury” (B), “Development” (C). Red: upregulated genes, blue: downregulated genes, and larger nodes: central and highly connected genes. Nodes shown in gradients of red and blue are up- and down-regulated, respectively. Centrality values are provided in the Table 2. Central genes with higher connectivity with neighboring genes are shown as larger nodes in each category.

FIGURE 13

Unique molecular signaling systems in brain metastasis. (A) Schematic representation of brain and distant sites of metastasis compared. The significant and differentially expressed genes between Brain metastasis (stroke responsive metastatic cells) and distant metastasis (liver metastasis) were compared. (B) Unique extracellular and plasma membrane genes that interact with the surrounding niche in each of these two transcriptomes were identified at FPKM values > 4. (C) Top canonical pathways differentially regulated in brain metastatic melanoma over a distant metastatic site (liver) with the largest negative logP values are shown. The dashed line represents the ratio of the number of molecules in the dataset over the number of molecules in the pathway. (D,E) GSEA of hallmark genes up and downregulated in the differentially expressed genes of brain metastatic vs. liver metastasis.