Characterization of melanoma cells capable of propagating tumors from a single cell

Abstract

Questions persist about the nature and number of cells with tumor-propagating capability in different types of cancer, including melanoma. In part, this is because identification and characterization of purified tumorigenic subsets of cancer cells has not been achieved to date. Here, we report tumor formation after injection of single purified melanoma cells derived from three novel mouse models. Tumor formation occurred after every injection of individual CD34+p75- melanoma cells, with intermediate rates using CD34-p75- cells, and rarely with CD34-p75+ cells. These findings suggest that tumorigenic melanoma cells may be more common than previously thought and establish that multiple distinct populations of melanoma-propagating cells (MPC) can exist within a single tumor. Interestingly, individual CD34-p75- MPCs could regenerate cellular heterogeneity after tumor formation in mice or multiple passages in vitro, whereas CD34+p75- MPCs underwent self-renewal only, showing that reestablishment of tumor heterogeneity is not always a characteristic of individual cells capable of forming tumors. Functionally, single purified MPCs were more resistant to chemotherapy than non-MPCs. We anticipate that purification of these MPCs may allow a more comprehensive evaluation of the molecular features that define tumor-forming capability and chemotherapeutic resistance in melanoma.

Figure 1

Mouse melanomas contain putative MPCs based on single-cell FACS. A, Kaplan-Meier survival graph of 4-OHT–treated mouse cohorts. Median tumor-free survival times (T_m) between the two tumor-bearing cohorts were not significantly different (P = 0.17). B, top, representative pictures of a mouse melanoma (1118) arising on the ear and low-power view of histologic section from ear melanoma; bottom, mouse melanoma (3297) from the flank with mixed pigmentation. H&E staining reveals melanin pigment and spindled and epithelioid cells with atypical mitoses. C, dual-color flow cytometry with CD34 and p75 showed three major subsets in each cultured melanoma. D, CD34⁺ and CD34⁻p75⁻ sorted fractions in all three melanomas exhibited high and moderate colony formation frequencies, respectively, whereas p75⁺ cells only rarely formed colonies. **, P < 0.001, ANOVA, for CD34⁺ versus CD34⁻p75⁻ groups; ***, P < 0.0001, ANOVA, for CD34⁺ versus p75⁺ groups; ****, P < 0.0001, ANOVA, for CD34⁻p75⁻ versus p75⁺ groups.

Figure 2

Purified single-cell s.c. injections into nude mice reveal multiple MPC subsets. A, representative photographs of nude mice bearing tumors of culture 1445 grown from one CD34⁺ cell (left), one CD34⁻p75⁻ cell (middle), and a mouse without tumors from p75⁺ cell injections (right). B, tumor growth over time revealed that CD34⁺ cells propagated tumors significantly faster than CD34⁻p75⁻ tumors in all three melanomas. Two tumors that formed from p75⁺ single-cell inoculations in culture 1118 grew significantly more slowly compared with all other subsets. *, P < 0.05, unpaired Student’s t test, CD34⁺ versus CD34⁻p75⁻; **, P < 0.005, unpaired Student’s t test, CD34⁺ versus CD34⁻p75⁻; #, P < 0.05, unpaired Student’s t test, CD34⁻p75⁻ versus p75⁺.

Figure 3

Evaluation of MPC subsets in cultured and uncultured melanomas using the Braf/Pten mouse model. A, left, flow cytometry on Braf/Pten cultured tumor 2697; right, 2697 single-cell FACS confirmed high, intermediate, and low colony-forming rates with CD34⁺, CD34⁻p75⁻, and p75⁺ single cells, respectively, indicating that MPC subsets are the same across all three mouse melanoma models. Columns, mean (n = 3); bars, SE. **, P < 0.001, unpaired Student’s t test; ***, P < 0.0001, unpaired Student’s t test, relative to CD34⁺. B, left, fresh Braf/Pten tumors 3297 and 3474 were dissociated and stained with propidium iodide and sorted to deplete dead/apoptotic cells. Live cells were then stained with CD34 and p75 antibodies for subset sorts followed by single-cell inoculations of live cells into nude mice. Right, marker profiles of parental tumors 3297 and 3474. Numbers in blue within quadrants represent tumors grown per total sites injected for that subset.

Figure 4

Evaluation of the reestablishment of cellular heterogeneity in the progeny of individual melanoma cell–derived colonies. A, colonies grown from CD34⁺ single cells mainly produced CD34⁺ progeny. B, CD34⁻p75⁻ clones gave rise to progeny with variable cell surface marker expression. C, infrequent colonies from p75⁺ single cells in cultures 1445 and 1111 predominantly gave rise to p75⁺ cells; however, p75⁺ colonies formed in culture 1118 generated both p75⁺ and CD34⁻p75⁻ progeny. D, left, serial single-cell FACS on cell progeny from colonies was analyzed for colony-forming ability. Numbers above data columns indicate the number of clones analyzed. Columns, mean; bars, SE. Right, representative flow cytometry on melanoma 1118 serial colonies formed from a CD34⁺ cell (top), CD34⁻p75⁻ cell (middle), and a p75⁺ cell (bottom) each sorted from a CD34⁻p75⁻ cell–derived colony.

Figure 5

Analysis of the reestablishment of cellular heterogeneity in grafted tumors. A, engrafted tumors were dissociated and analyzed by flow cytometry. Tumors from single cells displayed similar marker phenotypes compared with in vitro colonies grown from single cells. Representative flow analyses are displayed for tumors grown from one CD34⁺ cell (top) and one CD34⁻p75⁻ cell (middle) from culture 1445 and one p75⁺ cell (bottom) from culture 1118. CD34⁺ single cells predominantly self-renewed in vivo in contrast with CD34⁻p75⁻ cells. B, single-cell CD34⁻p75⁻–derived tumors from short-term melanoma cultures showed variability in their propensity to reestablish heterogeneity, similar to observations seen in culture. C, individual CD34⁺ cells injected directly from freshly dissociated tumors self-renewed to form homogeneous CD34⁺ tumors, whereas those derived from individual CD34⁻p75⁻ reestablished heterogeneity, consistent with short-term culture engraftments.

Figure 6

Multiple subsets of melanoma MPCs exhibit chemoresistance. A, treatment of parental melanoma cultures 1445 and 1118 with 500 μmol/L temozolomide or 0.5 μg/mL cisplatin (IC₉₀ doses) yields marked reduction of the p75⁺ populations and persistence of the CD34⁺ subsets. Numbers highlighted in red indicate a loss in subset percentage, whereas green indicates percentage gain compared with vehicle controls (left). B, single-cell FACS of subsets sorted directly into temozolomide- or cisplatin-spiked medium showed that some CD34⁺ and CD34⁻p75⁻ cells were capable of recovering from each chemotherapy treatment, whereas p75⁺ cells were not. Percent survival shown was normalized to untreated controls. Columns, mean (n = 3); bars, SE.