Isolation and molecular characterization of circulating melanoma cells - PubMed (original) (raw)

. 2014 May 8;7(3):645-53.

doi: 10.1016/j.celrep.2014.03.039. Epub 2014 Apr 18.

Devarati Mitra 2, Ryan J Sullivan 3, Ben S Wittner 4, Anya M Kimura 4, Shiwei Pan 4, Mai P Hoang 5, Brian W Brannigan 4, Donald P Lawrence 3, Keith T Flaherty 3, Lecia V Sequist 3, Martin McMahon 6, Marcus W Bosenberg 7, Shannon L Stott 8, David T Ting 3, Sridhar Ramaswamy 3, Mehmet Toner 9, David E Fisher 10, Shyamala Maheswaran 11, Daniel A Haber 12

Affiliations

Isolation and molecular characterization of circulating melanoma cells

Xi Luo et al. Cell Rep. 2014.

Abstract

Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice and rapidly declined after B-RAF inhibitor treatment. CTCs were shed early from localized tumors, and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled a comparison of RNA-sequencing profiles with matched primary tumors. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF-targeted therapy. Together, the capture and molecular characterization of CTCs provide insight into the hematogenous spread of melanoma.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Identification of CTCs in the B-RAFCA/+/PTENflox/flox mouse melanoma model

(A) Representative images of melanoma induced by focal tamoxifen injection. Arrowheads show tumor progression at the injection site from day 7 to day 24 (left) and cutaneous metastasis at day 56 after tumor induction (right). (B) Upper panel, representative image of a mouse melanoma CTC adjacent to a leukocyte. Lower panel, a cluster of four CTCs. DAPI, blue; CD45, green; S100, red; scale bars, 10μm. (C) Quantification of CTCs from a cohort of tumor-bearing (green, N=12) and control mice (red, genotype-matched tumor-free mice, N=8; blue, Tyr-CreER- mice received tamoxifen injection, N=5; open circles, syngeneic C57BL/6 mice, N=9). Solid lines, median CTC counts. Dashed line, threshold of ≥14 S100+ cells/ ml. Tam, tamoxifen. Concordant change in (D) tumor volume and (E) CTCs in tumor-bearing mice fed with control chow or PLX4720-containing chow are shown (PLX4720, blue; control, red) (Mean value, error bars represent standard deviation). (See also Figure S1)

Figure 2

Figure 2. Distant metastasis is associated with the presence of CTCs and is prevented by adjuvant B-RAF inhibition

(A) Tumor progression on 4-HT treated ear. (B) CTCs can be detected as early as day 7 after tumor induction and correspond to tumor progression. (C) CTC counts correlate with tumor thickness when thickness is greater than 0.1mm (N=12, R2=0.66). (D) Left panel, schematic diagram of the experimental procedures. Right panels, post-surgical administration of B-RAF inhibitor delays or prevents distant metastasis. After the affected ears were removed, the mice were fed with PLX4720-containing chow (N=17) or control chow (N=16) for 4 weeks. Subcutaneous metastases were observed in the control group (10 out of 16) but not in the PLX4720-fed group (0 out of 17). Tumors on the contralateral ear were also largely suppressed by PLX4720. (See also Figure S2)

Figure 3

Figure 3. Digital Gene Expression (DGE) analysis of mouse CTCs

(A) Schematic diagram of the DGE experiment. RNA was extracted from Chip-enriched cells, matched primary and metastatic tumors and subjected to Helicos DGE analysis. (B) Unsupervised clustering of transcripts enriched in CTCs, primary and metastatic tumors from the two mice (BP-55 and BP-73) demonstrating distinct expression profiles of CTCs from those of the primary tumor and metastasis. (C) Genes upregulated in CTC-enriched population compared with mock IgG-enriched cells. There were 1055 (left) and 275 (middle) upregulated genes in samples BP-55 and BP-73, respectively, with an overlap of 200 genes (right, “CTC-common”). Of these 200 genes, 132 were specifically overexpressed in CTCs compared with the primary tumor (D, left) and 125 were overexpressed in CTCs compared with the metastasis (D, middle), with 118 genes found to be in common (D, right). (E) Gene Set Enrichment Analysis (GSEA) on the 132 CTC-specific genes identified a number of gene sets related to cancer. Red bar, the 132 genes ranked by expression level from high (left, dark red) to low (right, light red); black vertical lines, overlap of the 132 genes with known gene sets. Poola breast, Poola invasive breast cancer genes (Poola et al., 2005); Hoshida liver, Hoshida liver carcinoma genes (Hoshida et al., 2009); Delys thyroid, Delys papillary thyroid cancer genes (Delys et al., 2007); Schuetz breast, Schuetz ductal invasive breast cancer genes (Schuetz et al., 2006); Onken melanoma, Onken aggressive uveal melanoma genes (Onken et al., 2006); Alonso melanoma, Alonso metastatic melanoma genes (Alonso et al., 2007). (F) The 132 CTC-specific gene signature is associated with increased motility in melanoma cell lines (Jeffs et al., 2009). (G) The signature also showed a progressive increase from benign or atypical nevus to primary and metastatic lesions. Benign, benign nevus; Atypica, atypical nevus; in situ, melanoma in situ; Vertical, melanoma vertical growth phase; Met, metastatic melanoma; Lymph, melanoma lymph node metastasis; Cult., metastatic melanoma short-term culture (Smith et al., 2005). (See also Figure S3)

Figure 4

Figure 4. Detection and characterization of circulating tumor cells in melanoma patients

(A) Representative images of CTCs from patients with metastatic melanoma. CTCs are defined as being “melanoma stain (MEL)”+ and CD45−. Upper panels, IF image of a MEL+CD45− melanoma CTC. Lower panels, bright-field (BF) image and merged image IF and BF of a CTC reveals intracellular “granules” marked with arrowheads. MEL, CSPG4/MCAM/TYRP-1/αSMA antibody cocktail (upper panels) or CSPG4 (lower panels). DAPI, blue; MEL, red; scale bar, 10μm. (B) CTC counts of 174 samples from metastatic patients, 21 pre-treatment samples and 11 healthy donors. Median MEL(C/M/T/S)+/CD45− cell count is 8 cells/2.5ml for all patient samples, 24 cells/2.5ml for pre-treatment samples and 1 cells/2.5ml for controls. Solid lines, median CTC counts. Dashed line, threshold of ≥ 3 cells/2.5ml. (C) CTCs display dynamic changes in patients and correlate with therapy responses. Radiographic tumor measurement, blue curve; CTC counts, red curve. Br. M, brain metastasis; SRS, Stereotactic Radiosurgery; Discont., treatment discontinued; B-RAFi/MEKi, dabrafenib/trametinib (Patient M1, M3 and M4) or LGX818/MEK163 (Patient M2). (See also Figure S4)

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