Selection of tumorigenic melanoma cells using ALDH

Abstract

Despite increasing knowledge regarding melanoma-initiating cells (MICs), questions persist regarding the number and phenotypic nature of cells with tumor-generating capability. Evidence for a phenotypically distinct human MIC has been found in NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice. However, a phenotypically distinct human MIC was not found in the NOD/SCIDIl2rg(-)/(-) (NSG) mouse model. The demonstration of a distinct population of human melanoma cells responsible for tumorigenesis and tumor cell self-renewal would provide an important target for new melanoma therapies. In this study, we show a 100-fold range in MIC frequency in human melanoma (1 in 18,000 to 1 in 1,851,000 cells) in the NOD/SCID mouse. In this model, human melanoma cells with high aldehyde dehydrogenase (ALDH) activity were enriched 16.8-fold in tumorigenic cells over unfractionated (UNF) cells, such that 1 in 21,000 cells was a MIC. In the NSG mouse, the ALDH expressing cell population was enriched 100-fold in tumorigenic cells over UNF cells, such that one in four cells was a MIC. Xenograft melanomas that developed from ALDH(+) cells displayed robust self-renewal, whereas those from ALDH(-) cells showed minimal self-renewal in vitro. Thus, ALDH(+) melanoma cells have enhanced tumorigenicity over ALDH(-) cells and superior self-renewal ability.

Figure 1. Xenograft growth in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice

Cells from fresh human metastatic melanoma were injected subcutaneously into NOD/SCID mice. (a) Tumors were excised for pathological confirmation of melanoma and serial transplantation. (b) Primary melanoma in patient (above) and xenograft in mouse (below) (ID 42) showing similar histopathological features (bar=10 μm). (c) Time to first palpability of the xenograft is significantly longer for tumors with low melanoma-initiating cell (MIC) frequency. Linear regression was used to plot a trend line (P=0.017).

Figure 2. Expression of aldehyde dehydrogenase (ALDH) and CD133 in human melanoma

(a) Cells from individual patients were sorted by flow cytometry. ALDH high (ALDH^hi), non-complex (SSC^lo) cells, comprising 2% of the total live population, were isolated. CD45⁺ hematopoietic cells were excluded. (b) Flow cytometry of a non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft tumor derived from 5,000 ALDH^hiSSC^lo cells, showing cellular heterogeneity. (c) CD133⁺ cells comprise 0.01–0.2% of the total population in our samples. (d) Serially transplanted tumors. Melanoma-initiating cell (MIC) frequency for tumors with lower initial MIC frequency increased with successive transplantations and approached that of tumors with high initial MIC frequency by the third serial transplant. With successive serial transplants, tumors were palpable earlier. *MIC frequency at first passage <1 in 1 million. SSC-A, side scatter area.

Figure 3. The population of human melanoma cells with high aldehyde dehydrogenase (ALDH) activity is enriched in melanoma-initiating cells (MICs) in non-obese diabetic Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice

(a) Flow cytometry was used to select ALDH⁺, ALDH⁻, and unfractionated (UNF) cells from human melanoma xenografts. CD45, CD31, and Ter119 were used to exclude murine cells. 7-Aminoactinomycin D was used to exclude dead cells. A range of cell doses was injected into NSG mice. After 32 weeks, limiting dilution analysis was used to assess MIC frequency. (b) The median weeks until tumors were first palpable was recorded for xenografts derived from ALDH⁺, ALDH⁻, and UNF cell populations. (c) Xenograft melanomas that had developed from UNF, ALDH⁺, ALDH^hiSSC^lo, or ALDH⁻ cell populations were harvested, and FACS analysis was used to determine the frequency of ALDH⁺ cells. (d) Xenograft melanomas that developed from UNF, ALDH⁺, ALDH^hiSSC^lo, or ALDH⁻ cell populations were harvested, dissociated, and plated at 20,000 cells per well into 24-well plates (2cm²) for 14 days, after which wells were stained with toluidine blue. NG, no growth.

Figure 4. Melanoma-initiating cell (MIC) frequency and tumor proliferation

(a, b) The tumors from Figure 3a were assessed for the percentage of ALDH⁺ cells by FACS, MIC frequency, mitotic rate, Ki-67 expression (cells in active cell cycle), and time (weeks) to first palpability for the xenograft (bar=10 μm). ALDH, aldehyde dehydrogenase.