Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Aug 29;7(1):9834.
doi: 10.1038/s41598-017-10129-z.

CD271 determines migratory properties of melanoma cells

Josefine Radke  1   2   3   4 , Florian Roßner  5   3 , Torben Redmer  6   7   8   9
Affiliations

CD271 determines migratory properties of melanoma cells

Josefine Radke et al. Sci Rep. .

Abstract

Melanoma cell expression of the nerve growth factor receptor CD271 is associated with stem-like properties. However, the contributing role of the receptor in melanoma cell migration is elusive. Here, we explored extracranial (skin, soft tissue, lymph node and liver, n = 13) and matched brain metastases (BM, n = 12) and observed a heterogeneous distribution of phenotypically distinct subsets of CD271+ cells. In addition, we observed that CD271 expression gradually rises along with melanoma progression and metastasis by exploration of publicly available expression data of nevi, primary melanoma (n = 31) and melanoma metastases (n = 54). Furthermore, we observed highest levels of CD271 in BM. Sub-clustering identified 99 genes differentially expressed among CD271high and CD271low (p < 0.05) BM-subgroups. Comparative analysis of subsets revealed increased ( ≥ 1.5fold, log2) expression of migration-associated genes and enrichment of CD271-responsible genes involved in DNA-repair and stemness. Live cell-imaging based scratch-wound assays of melanoma cells with stable knock-down of CD271 revealed a significantly reduced cell migration (3.9fold, p = 1.2E-04) and a reduced expression of FGF13, CSPG4, HMGA2 and AKT3 major candidate regulatory genes of melanoma cell migration. In summary, we provide new insights in melanoma cell migration and suggest that CD271 serves as a candidate regulator, sufficient to determine cellular properties of melanoma brain metastatic cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
CD271 expressing cells are phenotypically distinct in extracranial and brain metastases. (A) Immunohistochemistry of three representative matched pairs of melanoma extracranial (EM) and corresponding brain metastases (BM, Patient 1 and 2), or soft tissue metastases (MET) and corresponding BM (Patient 3) for presence of CD271 and MART1 or hematoxylin/eosin (H&E). Scale bars indicate 50 µm. (B) Left panel: Box-plots show the number of CD271+ cells (%) of BM (n = 19), primary tumors (PM; n = 2) and EM (n = 14) as determined by counting of 50 visual fields at 200x magnification, p = 0.081. Right panel: Visualization of CD271/NGFR expression levels (log2) of skin cancer subtypes (GSE7553) including MET (n = 34), PM (n = 14), melanoma in-situ (MIS; n = 2), melanocytes (Mel.; n = 1), basal cell carcinoma (BCC; n = 15), squamous cell carcinoma (SCC; n = 11) and normal skin (NS). Significant difference in expression levels among MET/PM (p = 0.032) and PM/BCC (p = 0.034) is shown. Black filled circles indicate number of samples. If not stated otherwise, p-values were calculated by Wilcoxon rank-sum test.
Figure 2
Figure 2
CD271 expression characterizes melanoma metastases. (A) Left panel: Visualization of CD271/NGFR expression levels (log2) at different melanoma progression stages (GSE46517): MET (n = 54), PM (n = 31), melanocytic nevus (Nevus; n = 9), NS (n = 7) and melanocytes (Mel.; n = 1). Right panel: CD271/NGFR expression levels (log2) of brain metastases (Brain; n = 29, GSE50493) as well as extracranial metastases, lymph node (LN; n = 12), soft tissue (ST; n = 10), small intestine (S. int.; n = 4), lung (Lung; n = 6), Bowel (n = 1), Spleen (n = 4), Adrenal (n = 1), Bone (n = 3), Liver (n = 2). (B) Supervised clustering of brain metastases with high expression of CD271/NGFR (red, patients 15, 37, 42, 51 and 55) and very low expression (green, n = 13) of 99 genes, among them 62 genes with increased (≥1.5fold, log2; p < 0.05) and 37 genes with decreased (≤1.5fold, log2; p < 0.05) expression. (C) Left and right panel: Gene-set enrichment analysis (GSEA) of CD271high (BM-CD271high) and CD271low (BM-CD271low) brain metastases (study GSE50493) shows enrichment of CD271-responsive genes (n = 156; NES = normalized enrichment score; FDR = false discovery rate). Venn-diagram depicts the number of CD271-responsible genes, distinctly or commonly up-regulated in CD271high brain metastases in two independent studies (GSE50493, GSE44660; FClog2 ≥ 1.1, p ≤ 0.05). (D) Left panel: Representation of the migratory capacity of A375GFP and A375CD271/NGFR cells indicated by the wound width [µm], 24 hours after wounding. Right panel: Enrichment of genes involved in focal adhesion formation (Broad institute’s MSigDB) in A375CD271/NGFR cells as determined by GSEA (E) Confocal microscopy-based serial imaging of mouse brain slices, 48 h after injection of 10 000 A375GFP or A375CD271/NGFR cells. Left panels: GFP expression of image sections, Z0/0, Z7/14, Z14/14 (inset) as well as CD271 expression (red) for Z14/14 of slices injected with A375GFP are shown. Right panels: image sections Z0/7and Z7/7 of slices injected with A375CD271/NGFR are shown. Areas depicting migrating CD271+ cells are magnified. DAPI served as nuclear stain, scale bars indicate 50 µm. White dotted lines indicate initial tumor borders/non-migrated cells.
Figure 3
Figure 3
CD271 controls expression of mediators of cell migration. (A) Left panels: Absence of CD271 in melanoma cells (T20/02) stably transfected with a CD271-targeting shRNA (shCD271/sh#3) but high expression in control cells (shCtl.) as determined by immunofluorescence and a directly labeled (PE, red) CD271 antibody. Right panels: A changed morphology of CD271 knock-down cells is indicated by phalloidin (red) and bright field images (PH), as well as a decreased number of BrdU+ (red) but not Ki67+ (green) cells as compared to shCtl. cells. (B) Left panel: Live cell-imaging based scratch-wound assay of cells described in (A) at 0, 24, 48 and 72 hours after wounding. Initially, 30 000 cells/well (96-well plate) were seeded, reproducible wounds were scratched using the wound maker tool (Essen bioscience). Scale bars indicate 200 µm, the initial wound is indicated by white dotted lines. Right panel: Quantification of the scratch-wound assay, the wound width [µm] was determined every 3 hours. Shown are mean values ± sdv of n = 8 replicates, p = 3.1E-05 (shCtl.1 vs. sh#3) or 9.6E-07 (shCtl.2 vs. sh#3), ttest. (C) Left and right panel: CD271-responsible regulation of genes associated with formation of cell projections as determined by GSEA of T20/02 cells engineered for stable down-regulation (T20/02k.d.) or over expression (T20/02CD271/NGFR) of CD271. (D) Representation of genes found either up-regulated or down-regulated by knock-down or over expression, respectively. A common set of 20 genes followed a CD271-responsible regulation among them FGF13 and CSPG4. (E) Left panel: Determination of expression levels of potential migration-associated genes CSPG4, FGF13, HMGA2 and AKT3 by qPCR of T20/02 cells either stably transfected with control shRNA (shCtl) or effective CD271-targeting shRNAs (sh#3, sh#4; sh#2, not effective). Relative expression levels are shown as mean values ± sdv of biological triplicates, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, ttest. Right panel: Immunofluorescence microscopy for levels of CSPG4, FGF13 and HMGA2 in shCtl. and CD271 knock-down cells (sh#3). DAPI served as nuclear stain, scale bars indicate 50 µm.
Figure 4
Figure 4
Exploration of multiple brain metastases reveal a phenotypical concordance of CD271 expression. (A) Schematic representation of plausible mechanisms of primary tumor (PM) or extracranial metastasis (EM) seeding to the brain suggesting either a multiple or single seeding of PM/EM or a combination of both leading to high phenotypic heterogeneity (left and right scheme) or phenotypic homogeneity (center scheme). Brain schemes were obtained from Servier (http://servier.com/Powerpoint-image-bank) as per Servier’s usage license (https://creativecommons.org/licenses/by/3.0/legalcode) and modified. (B) Immunohistochemistry of autopsied multiple brain metastases. In patient T1, all tumors (n = 3) located in the cerebellum and frontal cortex showed strong expression of CD271. (C) Areas indicating CD271+ cell migration /invasion into adjacent brain parenchyma of tumors described in (B), are shown. Insets show distant CD271+ cells. (D) Exploration of brain micrometastases located in the hippocampus and cortex of patient T3 for presence of CD271+ cells (black arrows).
Figure 5
Figure 5
CD271 high brain metastases lack expression of MITF. (A) Co-immunohistochemistry of a solitary BM of patient 1 (two different tumor areas) as well as multiple BM of patient T1 for CD271 (membrane, red) and MITF (nuclear, brown). (B) Upper and center panels: Staining of patient 1 BM for CD271 and Ki67. Lower panel, quantification of Ki67+, CD271+ and double positive cells of BM (n = 5) and EM (n = 6). (C) MITF expression levels (log2) of CD271high and CD271low BM of two independent studies (GSE50493, GSE44660) are shown. (D,E) GSEA of BM subsets analyzed in (C) and T20/02 cells with over expression of CD271 (T20/02CD271/NGFR) for enrichment of MITF-target genes as provided by TCGA, compared to untransfected cells (Mock) is shown. In addition, GSEA revealed enrichment of melanoma aggressiveness-associated genes in A375 cells (CD271/NGFR vs. GFP) and BM. In (B) and (C) p-values were calculated by Wilcoxon rank-sum test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Balch CM, et al. Final version of 2009 AJCC melanoma staging and classification. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2009;27:6199–6206. doi: 10.1200/JCO.2009.23.4799. - DOI - PMC - PubMed
    1. Eyles J, et al. Tumor cells disseminate early, but immunosurveillance limits metastatic outgrowth, in a mouse model of melanoma. The Journal of clinical investigation. 2010;120:2030–2039. doi: 10.1172/JCI42002. - DOI - PMC - PubMed
    1. Bonaventure J, Domingues MJ, Larue L. Cellular and molecular mechanisms controlling the migration of melanocytes and melanoma cells. Pigment cell & melanoma research. 2013;26:316–325. doi: 10.1111/pcmr.12080. - DOI - PubMed
    1. Damsky WE, Theodosakis N, Bosenberg M. Melanoma metastasis: new concepts and evolving paradigms. Oncogene. 2014;33:2413–2422. doi: 10.1038/onc.2013.194. - DOI - PubMed
    1. Flanigan JC, et al. Melanoma brain metastases: is it time to reassess the bias? Curr Probl Cancer. 2011;35:200–210. doi: 10.1016/j.currproblcancer.2011.07.003. - DOI - PMC - PubMed

Publication types

Substances