Neurovascular signaling for repair enhances brain metastasis

Tue, 10/08/2019 - 09:02

High expression of GFAP (green) identifies astrocytes in rat cerebellum

By Jamshed Arslan, Pharm. D., PhD.

Stroke    is a leading cause of mortality and morbidity worldwide. Cellular players – neurons, astrocytes, endothelial and stromal cells – involved in post-stroke repair through angiogenesis   , neurogenesis, and tissue reorganization, are collectively referred to as the regenerative neurovascular niche. Interestingly, signaling in post-stroke repair is similar to that observed in the brain during metastasis. For example, both processes involve PTEN inhibition leading to axon regrowth, endothelial cell proliferation and migration induced by VEGF and angiopoietin, angiogenesis partly mediated by matrix metalloproteinases, and the TGF-beta-guided tissue reorganization. This made a group of researchers in Israel    and the USA    ask three pertinent questions:

  1. Can the regenerative neurovascular niche in stroke create a facilitative environment for brain metastasis?
  2. Does the similarity between repair and metastatic processes enhance the interactions between metastatic cells and the regenerative neurovascular niche?
  3. Can neurovascular signaling occurring during repair change the metastatic transcriptome to increase metastasis?

Using cell culture, immunodeficient mice, and human brain samples, the researchers found that the answer to all of these questions is affirmative. That is, reparative processes enhance brain metastasis.

Immunodetection of beta-III tubulin and GFAP serve as useful markers for the identification of brain cells in culture. Rat cortical neurons and astrocytes may be identified by immunostaining with primary antibodies specific for beta-III tubulin (red) and GFAP (green).Identification of cortical neurons and astrocytes: Rat cortical neurons were immunostained with beta III Tubulin: 5 µg/mL Mouse Anti-neuron-specific Mouse beta III Tubulin Monoclonal (clone TuJ 1) Antibody (MAB1195). Rat astrocytes were detected by immunostaining with GFAP: 10 µg/mL Sheep Anti-Human GFAP Antigen Affinity-purified Poly-clonal Antibody (AF2594). Fluorescent-conjugated secondary antibodies were used as follows: red-Northern-Lights 557-conjugated Anti-Mouse IgG Secondary Antibody (NL007) for beta III Tubulin and green-Northern-Lights 493-conjugated Anti-Sheep IgG Secondary Antibody (NL012) for GFAP.

Regenerative neurovascular niche facilitates brain melanoma metastasis

The researchers injected GFP-labeled melanoma cells into an immunocompromised mouse model of stroke to visualize brain melanoma metastasis. Unsurprisingly, the melanoma preferentially localized to the regenerative neurovascular niche. Genetically labeling neuroblasts with red fluorescent protein indicated that post-stroke neurogenesis, especially the neuroblast-associated vessels within the regenerative neurovascular niche, governs the preferential localization of melanoma to the regenerative neurovascular niche. This means that the regenerative neurovascular niche creates a facilitative environment for brain melanoma metastasis.

Melanoma cells are identified by the expression of Melan/MART-1 marker. Melanoma cells in human skin cancer/melanoma paraffin embedded tissue were immunostained for Melanoma marker MART-1 which shows a cytoplasmic and perinuclear distribution within melanoma cells but not stromal cells.Melanoma marker MART-1: Melanoma cells, in Human Skin Cancer/Melanoma Paraffin embedded tissue, were identified by immunocytochemistry in with Melan-A/MART-1 Antibody (A103) [NBP2-46603]. MART-1 was predominantly detected in the cytoplasm and around the nucleus.

Tissue repair-mediated direct interaction between metastatic cells and neurovascular niche

The team quantified neuroblasts as well as melanoma-neuroblast contact surfaces to determine direct interaction over time. They discovered two phases of melanoma-neuroblast association.

  1. Vascular phase: melanoma cells attach to neuroblast-associated vasculature near the injection site. By day 3, the number of melanoma-neuroblast contacts decreases with melanoma extravasation, leading to the next stage.
  2. Tissue phase: melanoma-neuroblast associations increase in the regenerative neurovascular niche until day 7.

To determine the cellular elements that interact with human brain tumors, the researchers immunostained metastatic melanoma samples. An increase in the association of metastatic cells with surrounding neuroblasts and astrocytes supported the idea that repair increases direct interaction between metastatic cells and the regenerative neurovascular niche.

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Metastatic transcriptome shaped by neurovascular signaling in repair

To determine the regenerative neurovascular niche-mediated gene expression changes in metastatic melanoma, the team sequenced total RNA from the FACS-isolated melanoma cells. They found 1,118 upregulated and 1,146 downregulated genes in metastatic melanoma within the regenerative neurovascular niche. The differentially expressed genes were related to axon guidance, phagosome formation and thrombin pathway   . Most importantly, neurovascular signaling affected the genes related to pathways governing melanoma metastasis, such as mTOR, angiopoietin pathways, ephrin receptor signaling, semaphorin signaling and PPAR/RXR-alpha. These results indicate that signaling in the regenerative neurovascular niche mediates a unique molecular profile in the metastatic melanoma cells.

Significance of understanding the metastatic transcriptome

Studying the regenerative neurovascular niche in brain melanoma metastasis led to the identification of unique gene expression profile that can have prognostic value. This research highlights certain molecular targets that can promote recovery after brain injury (stroke) or disease without evoking cancer-related mechanisms.

Jamshed Arslan Jamshed Arslan, Pharm D, PhD   
Dr. Arslan is an Assistant Professor at Dow University of Health Sciences, Pakistan,
where he teaches Pharmacology to future pharmacists.


Prakash, R., Izraely, S., Thareja, N. S., Lee, R. H., Rappaport, M., Kawaguchi, R., … Thomas Carmichael, S. (2019). Regeneration enhances metastasis: A novel role for neurovascular signaling in promoting melanoma brain metastasis. Frontiers in Neuroscience.


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