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Selection of Tumorigenic Melanoma Cells Using ALDH

  • Author Footnotes
    8 These authors contributed equally to this work.
    Jim B. Boonyaratanakornkit
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, University of California, San Francisco, San Francisco, California, USA

    Dermatology Research, Veterans Affairs Medical Center, San Francisco, California, USA
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  • Author Footnotes
    8 These authors contributed equally to this work.
    Lili Yue
    Footnotes
    8 These authors contributed equally to this work.
    Affiliations
    Dermatology Research, Veterans Affairs Medical Center, San Francisco, California, USA
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  • Lauren R. Strachan
    Affiliations
    Department of Dermatology, University of California, San Francisco, San Francisco, California, USA

    Dermatology Research, Veterans Affairs Medical Center, San Francisco, California, USA
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  • Kenneth J. Scalapino
    Affiliations
    Division of Rheumatology, Department of Medicine, University of California, San Francisco and San Francisco Veterans Affairs Medical Center, San Francisco, California, USA
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  • Philip E. LeBoit
    Affiliations
    Departments of Pathology and Dermatopathology and Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA
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  • Ying Lu
    Affiliations
    Department of Radiology and Department of Epidemiology and Biostatistics, University of California, San Francisco, San Francisco, California, USA
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  • Stanley P. Leong
    Affiliations
    Department of Surgery, University of California, San Francisco, San Francisco, California, USA
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  • Janellen E. Smith
    Affiliations
    Department of Dermatology, University of California, Irvine, Irvine, California, USA
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  • Ruby Ghadially
    Correspondence
    Department of Dermatology, Institute for Regeneration Medicine, Veteran's Affairs Medical Center, University of California, 4150 Clement Street MC190, San Francisco, California 94121, USA
    Affiliations
    Department of Dermatology, University of California, San Francisco, San Francisco, California, USA

    Dermatology Research, Veterans Affairs Medical Center, San Francisco, California, USA
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  • Author Footnotes
    8 These authors contributed equally to this work.
      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.

      Abbreviations

      ALDH
      aldehyde dehydrogenase
      CI
      confidence interval
      MIC
      melanoma-initiating cell
      NOD/SCID
      non-obese diabetic/severe combined immunodeficiency
      SSC
      side scatter area
      UNF
      unfractionated

      Introduction

      According to the cancer stem cell hypothesis, most cancer cells are unable to proliferate extensively and only a small phenotypically distinct subset of cells can consistently proliferate and form new tumors upon transplantation (
      • Reya T.
      • Morrison S.J.
      • Clarke M.F.
      • et al.
      Stem cells, cancer, and cancer stem cells.
      ). The role of cancer stem cells in maintaining myeloid leukemia (
      • Lapidot T.
      • Sirard C.
      • Vormoor J.
      • et al.
      A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.
      ) and several solid cancers (
      • Hermann P.C.
      • Huber S.L.
      • Herrler T.
      • et al.
      Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.
      ;
      • O'Brien C.A.
      • Pollett A.
      • Gallinger S.
      • et al.
      A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.
      ;
      • Prince M.E.
      • Sivanandan R.
      • Kaczorowski A.
      • et al.
      Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.
      ;
      • Ricci-Vitiani L.
      • Lombardi D.G.
      • Pilozzi E.
      • et al.
      Identification and expansion of human colon-cancer-initiating cells.
      ) has been established through transplantation studies. Previous studies have also provided evidence for a phenotypically distinct tumor-initiating cell in melanoma. Melanoma cells propagated as non-adherent spheres expressed higher levels of CD20 than adherent cells, and were more tumorigenic than adherent cells when injected into mice (
      • Fang D.
      • Nguyen T.K.
      • Leishear K.
      • et al.
      A tumorigenic subpopulation with stem cell properties in melanomas.
      ).
      • Grichnik J.M.
      • Burch J.A.
      • Schulteis R.D.
      • et al.
      Melanoma, a tumor based on a mutant stem cell?.
      ) showed that small Hoescht dye-excluding melanoma cells shared many properties with normal stem cells, including slower proliferation and greater expansion in culture. Furthermore 100,000 CD133+ melanoma cells formed xenograft tumors, whereas 100,000 CD133 cells did not (
      • Monzani E.
      • Facchetti F.
      • Galmozzi E.
      • et al.
      Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential.
      ) and ABCB5 expressing melanoma cells were enriched for cells with tumorigenic capacity 6.8-fold over unfractionated (UNF) cells such that 1 in 158,170 cells was a melanoma-initiating cell (MIC) (
      • Schatton T.
      • Murphy G.F.
      • Frank N.Y.
      • et al.
      Identification of cells initiating human melanomas.
      ). Finally, using a murine melanoma model and injection into nude mice, melanoma formation occurred after every injection of individual CD34+p75 cell, but only rarely with CD34p75+ cells (
      • Held M.A.
      • Curley D.P.
      • Dankort D.
      • et al.
      Characterization of melanoma cells capable of propagating tumors from a single cell.
      ).
      Although the above work supports the existence of a phenotypically distinct MIC, when tumorigenic melanoma cells were examined in the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse, the tumorigenic cell was a frequent cell and no specific phenotype was found (
      • Quintana E.
      • Shackleton M.
      • Sabel M.S.
      • et al.
      Efficient tumour formation by single human melanoma cells.
      ).
      One approach to finding markers of tumorigenic cells is to focus on conserved progenitor cell functions (
      • Ginestier C.
      • Hur M.H.
      • Charafe-Jauffret E.
      • et al.
      ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.
      ). Using this approach we selected aldehyde dehydrogenase (ALDH) activity as a candidate marker of MICs. ALDH is involved in retinoic acid metabolism and confers a protective function against xenobiotics (
      • Vasiliou V.
      • Pappa A.
      • Petersen D.R.
      Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism.
      ). ALDH activity has been identified as a stem cell marker in normal human hematopoietic cells (
      • Hess D.A.
      • Meyerrose T.E.
      • Wirthlin L.
      • et al.
      Functional characterization of highly purified human hematopoietic repopulating cells isolated based on aldehyde dehydrogenase activity.
      ) and in multiple tumors (
      • Storms R.W.
      • Trujillo A.P.
      • Springer J.B.
      • et al.
      Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity.
      ;
      • Seigel G.M.
      • Campbell L.M.
      • Narayan M.
      • et al.
      Cancer stem cell characteristics in retinoblastoma.
      ;
      • Cheung A.M.
      • Wan T.S.
      • Leung J.C.
      • et al.
      Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential.
      ;
      • Ginestier C.
      • Hur M.H.
      • Charafe-Jauffret E.
      • et al.
      ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.
      ,
      • Ginestier C.
      • Korkaya H.
      • Dontu G.
      • et al.
      [The cancer stem cell: the breast cancer driver.].
      ).
      CD133 was also examined as a possible marker of MICs. CD133 is a cell surface glycoprotein (prominin-1), whose function has not been established. CD133 marks stem cells in normal (
      • Hess D.A.
      • Wirthlin L.
      • Craft T.P.
      • et al.
      Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells.
      ) and cancerous tissues (
      • Singh S.K.
      • Clarke I.D.
      • Terasaki M.
      • et al.
      Identification of a cancer stem cell in human brain tumors.
      ;
      • Hermann P.C.
      • Huber S.L.
      • Herrler T.
      • et al.
      Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.
      ;
      • O'Brien C.A.
      • Pollett A.
      • Gallinger S.
      • et al.
      A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.
      ;
      • Ricci-Vitiani L.
      • Lombardi D.G.
      • Pilozzi E.
      • et al.
      Identification and expansion of human colon-cancer-initiating cells.
      ).
      In this study, limiting dilution analyses in NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice revealed a 100-fold variation in MIC frequency in melanomas from different patients. In the NOD/SCID mouse, the ALDHhiSSClo population of human melanoma cells was enriched in MICs 16.8-fold over UNF cells. In the NSG mouse, ALDH+ cells were enriched in MICs 100-fold over the UNF cell population (such that one in four cells was a MIC) and tumors that developed from ALDH+ cells showed superior self-renewal in vitro over the occasional tumors that developed from ALDH cells. These studies provide evidence for a phenotypically distinct tumorigenic cell in melanoma that has superior self-renewal ability.

      Results

      The frequency of human melanoma initiating cells in NOD/SCID mice

      To determine the range in MIC frequency in melanomas from different patients, cell suspensions were xenografted subcutaneously into NOD/SCID mice at various doses and tumor growth was followed over time (Figure 1a). Only one patient sample failed to grow after transplantation. Xenografted tumors recapitulated the original patient tumor with similar histopathologic features (Figure 1b). The frequency of MICs in freshly obtained specimens from individual patients showed a 100-fold range, from 1 in 18,000 to 1 in 1,851,000 cells (Table 1). Thus, in all patients only a fraction of tumor cells initiated further tumor growth in NOD/SCID mice, and the size of the fraction varied greatly.
      Figure thumbnail gr1
      Figure 1Xenograft 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=10μm). (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).
      Table 1Characteristics of melanomas from patients with lymph node metastases (in NOD/SCID mice)
      Limiting dilution analysisGrowth in vivo
      Patient ID, age, sexNo. of cellsResponse ratioMIC frequency (95% CI)Time to metastasis (months)Xenograft latency (weeks)
      41, 60 years, F800,0002/21 in 18,000 (6,000–56,000)236
      400,0002/2
      100,0003/3
      25,0003/4
      28, 38 years, M1,600,0002/21 in 20,000 (6,000–64,000)1**5
      800,0004/4
      400,0004/4
      100,0006/6
      25,0002/3
      42, 63 years, F1,600,0003/31 in 61,000 (28,000–135,000)247
      800,0006/6
      400,0006/6
      100,0006/6
      25,0000/6
      29, 60 years, F1,600,0002/21 in 99,000 (39,000–249, 000)65
      800,0005/5
      400,0004/4
      100,0003/4
      25,0000/4
      36, 60 years, F1,600,0003/31 in 100,000 (47,000–214,000)1**5
      800,0006/6
      400,0006/6
      100,0003/6
      25,0002/6
      25, 63 years, M1,300,0001/11 in 113,000 (29,000–432,000)38
      800,0001/1
      500,0001/1
      400,0002/2
      100,0001/2
      23, 87 years, M1,600,0001/11 in 113,000 (62,000–204,000)46
      800,0008/8
      400,00013/14
      100,0007/9
      25,0000/2
      37, 54 years, M1,600,0003/31 in 161,000 (71,000–369,000)713
      800,0006/6
      400,0005/6
      100,0002/2
      25,0000/2
      24, 79 years, F1,700,0001/11 in 263,000 (134,000–517,000)1444
      800,0004/5
      400,0005/5
      100,0003/7
      25,0000/7
      52, 46 years, F400,0000/11 in 649,000 (80,000–5,300,000)30019
      100,0001/3
      22, 42 years, M3,200,0001/11 in 710,000 (368,000–1,370,000)3210
      800,0005/7
      400,0003/7
      100,0001/9
      25,0000/8
      19, 31 years, M1,700,0001/11 in 1,851,000 (484,000–7,072,000)6913
      400,0001/6
      100,0000/6
      25,0000/6
      6,2500/6
      Abbreviations: CI, confidence interval; F, female; M, male; MIC, melanoma-initiating cell; NOD/SCID, non-obese diabetic/severe combined immunodeficiency.
      **Metastases present at time of initial diagnosis.
      The time to first palpability was different in xenografts from different patients. In order to use data from all cell doses, a Cox regression model using cell doses as strata was used to associate time to palpability with MIC frequency. The hazards ratio for shortened time to palpability was 1.24 per 1 MIC increase in 150,000 cells (95% confidence interval (CI) 1.08 to 1.142, P=0.017). Thus, there was a significant association between increased MIC frequency and decreased time to first palpability (Figure 1c).
      For the 13 patients examined (Table 1, Supplementary Table S1 online), there was an inverse correlation between MIC frequency and metastasis-free survival (time between diagnosis of the primary melanoma and diagnosis of metastatic melanoma). Spearman's correlation coefficient was –0.6035 (P=0.038), such that the higher the MIC frequency, the shorter the time to metastasis (Supplementary Figure S1 online).
      In general, the melanoma samples effaced the lymph node and were sheets of melanoma cells microscopically. Cell counts under the microscope excluded lymphocytes (by size and morphology), which constituted 5–15% of cells. To examine whether it was likely that proportions of contaminating cells contributed to the results obtained above by affecting xenograft growth, we studied the number of CD45+ and CD31+ human cells in primary lesions and xenografts. For PM 41 (MIC frequency 1 in 18,000), PM 28 (1 in 20,000), PM 37 (1 in 161,000) PM 22 (1 in 710,000), and PM 47 (1 in >2,000,000), there was no evidence for a correlation between the number of contaminating cells (by immunoperoxidase for human-specific CD45 and CD31 and MIC frequency (Supplementary Figure S2 online). In the corresponding xenografts, neither CD45+ nor CD31+ human cells were found (Supplementary Figure S3 online). Thus, these data do not provide evidence that contaminating cells contributed significantly to the 100-fold difference in MIC frequency.

      The population of human melanoma cells with high ALDH activity is enriched in MICs in NOD/SCID mice

      We next determined whether cells marked by high ALDH activity (ALDHhiSSClo) were tumorigenic cells. Freshly obtained melanoma cells were sorted for ALDH activity using Aldefluor (Stemco Biomedical, Durham, NC) (Figure 2a). Diethylaminobenzaldehyde, an ALDH inhibitor, was the negative control. The selected ALDHhiSSClo population was 2% of live cells (Figure 2a). Dead cells were excluded using 7-aminoactinomycin D. Lymphocytes formed a smaller, less complex population than the melanoma cells and could be excluded on the forward scatter/side scatter (FSC/SSC) plot. Remaining hematopoietic cells were excluded using CD45 (Figure 2a), such that the ALDH+ population was found to be over 99.5% CD45CD31, and the ALDH population was over 95% CD45CD31 (not shown). ALDHhiSSClo cells from a melanoma cell line (WM-266-4) were enriched for MICs 5.7-fold over UNF cells so that 1 in 65 cells (95% CI 1 in 43 to 1 in 97 cells) versus 1 in 370 UNF cells (95% CI 1 in 190 to 1 in 710) was a MIC (Table 2a). ALDHhiSSClo cells from freshly collected patient melanomas (n=3) or human melanomas grown for one passage in the NOD/SCID mouse (n=6) were also enriched in MICs 16.8-fold, such that 1 in 21,000 cells (95% CI 1 in 14,000 to 1 in 30,000) versus 1 in 353,000 UNF cells (95% CI 1 in 246,000 to 1 in 507,000) was a MIC (Table 2b). Xenograft melanomas that developed from ALDHhiSSClo cells contained both ALDHhi and ALDH cells by FACS (Figure 2b). Furthermore, 5,000 ALDHhiSSClo cells secondarily transplanted into NOD/SCID mice could once again produce a xenograft melanoma. These studies provide evidence that the ALDHhiSSClo population contains the tumorigenic cells, can self-renew, and produce a range of progeny in the NOD/SCID mouse.
      Figure thumbnail gr2
      Figure 2Expression of aldehyde dehydrogenase (ALDH) and CD133 in human melanoma. (a) Cells from individual patients were sorted by flow cytometry. ALDH high (ALDHhi), non-complex (SSClo) 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 ALDHhiSSClo 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.
      Table 2The population of human melanoma cells with high ALDH activity is enriched in MICs
      (a) ALDH: WM-2664 cell line
      Number of tumors/number of injections Cells per injectionTumorigenic cell frequency (95% Cl)
      10,0001,936968242100307.53
      ALDHhiSSClo8/812/4710/360/81 in 65 (43–97)
      ALDH0/72/164/81/161 in 16,000 (6,880–35,000)
      UNF9/381 in 370 (190–710)
      (b) ALDH: patient (n=3) and xenograft (n=6) melanomas
      Number of tumors/number of injections Cells per injectionTumorigenic cell frequency (95% Cl)
      800,000400,000200,000100,00025,0005,000
      Xe12/41/4
      Xe23/41/4
      Xe34/62/6
      Xe41/11/1
      ALDHhiSSCloXe52/21/2
      Xe63/43/4
      PM11/30/3
      PM20/40/4
      PM34/53/6
      All20/3312/341 in 21,000 (14,000–30,000)
      Xe30/5
      Xe41/1
      Xe50/1
      ALDHXe60/21/6
      PM20/6
      PM32/4
      All1/62/121/71 in 588,000 (217,000–1,593,000)
      Xe11/13/30/40/4
      Xe22/24/40/40/4
      Xe30/20/2
      Xe42/22/21/21/2
      Xe51/11/11/3
      UNFXe62/22/20/4
      PM11/22/42/4
      PM22/23/42/40/4
      PM31/1
      All11/1418/236/251/141 in 353,000 (246,000–507,000)
      (c) CD133: patient melanomas (n=5)
      Number of tumors/number of injections Cells per injectionTumorigenic cell frequency (95% Cl)
      1,600,000800,000400,000100,00025,0005,000
      CD133+6/131/121 in 43,000 (20,000–90,000)
      CD13312/12
      UNF8/87/86/81 in 124,000 (61,000–252,000)
      Abbreviations: ALDH, aldehyde dehydrogenase; CI, confidence interval; MIC, melanoma-initiating cell; SSC, side scatter area; UNF, unfractionated.

      The population of human melanoma cells that expresses CD133 is enriched in MICs in NOD/SCID mice

      The CD133-positive population was examined for MIC enrichment. By flow cytometry, CD133+ cells constituted 0.01–0.2% of cells (Figure 2c) and 2.9–29% of CD133+ were ALDHhi. AutoMACS was used to isolate CD133+ melanoma cells for limiting dilution analysis from three fresh melanoma samples. CD133+ cells were enriched for MICs 2.9-fold over UNF cells, such that 1 in 43,000 cells (95% CI 1 in 20,000 to 1 in 90,000) was a MIC (Table 2c). CD133 cells in large doses (800,000) contained MICs in 12 out of 12 samples, suggesting that there may be a distinct population of MICs that is CD133. This is reminiscent of findings in glioblastoma (
      • Beier D.
      • Hau P.
      • Proescholdt M.
      • et al.
      CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles.
      ). Given the lesser enrichment with CD133 versus ALDH, we did not pursue this marker further.

      Serial transplantation and MIC frequency in NOD/SCID mice

      To determine whether serial transplantation altered MIC frequency, we transplanted tumors with a range of MIC frequencies through six serial transplantations. After 2–3 transplantations, tumors characterized by low MIC frequency achieved high MIC frequency and had shorter time to first palpability (Figure 2d). Tumors characterized by high MIC frequency maintained high MIC frequency through serial transplantations, and exhibited decreased time to palpability after 2–3 serial transplantations (Figure 2d).

      The population of human melanoma cells that expresses ALDH is enriched for MICs in NSG mice

      We then examined whether ALDH-expressing cells were the tumorigenic cells in NSG mice. Although previous hematopoietic studies used ALDHhiSSClo populations, more recent studies in solid tumors used ALDH+ cells. We examined both 2% ALDHhiSSClo cells and ALDH+ cells (ALDH+ cells comprised 0.46–5.8% of melanoma cells) and no difference in either MIC frequency or in time to first palpability in the NSG mouse was detected (Supplementary Tables S2 and S3 online). Accordingly, in subsequent studies, we used the entire ALDH+ cell population. ALDH+ human melanoma cells were enriched in MICs such that 1 in 4 (95% CI 3–5) ALDH+ cells versus 1 in 418 (95% CI 302–579) UNF cells (P=0.0001), or 1 in 601 (95% CI 463–779) ALDH cells (P=0.0001) was capable of tumorigenesis (Figure 3a). Median time to first palpability was significantly shorter for tumors derived from ALDH+ cells versus tumors from equal numbers of ALDH or UNF cells (Figure 3b and Supplementary Figure S4 online). These studies demonstrate that ALDH+ cells were enriched in tumorigenic cells compared with ALDH or UNF cells and that tumors from ALDH+ cells had shorter time to first palpability than tumors from UNF or ALDH cells.
      Figure thumbnail gr3
      Figure 3The population of human melanoma cells with high aldehyde dehydrogenase (ALDH) activity is enriched in melanoma-initiating cells (MICs) in non-obese diabetic Cg-Prkdcscid Il2rgtm1Wjl/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+, ALDHhiSSClo, 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+, ALDHhiSSClo, 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.
      An increased MIC frequency might be found if all cells were equally tumorigenic, but the proliferation of the cells in one tumor versus another was more rapid. To address this possibility, we studied tumors from Figure 3 and assessed the frequency of ALDH+ cells by FACS, MIC frequency, mitotic index, Ki-67 expression, and the weeks to first palpability (an in vivo reflection of doubling time) (Figure 4). In these cases, the tumor with the highest proportion of ALDH+ cells in the tumor by FACS had the highest MIC frequency. There was no significant difference in mitotic index between the tumors, or in Ki-67 staining. For UNF cells, the time to first palpability was longest for the tumor with the lowest percentage of ALDH+ cells. However, when ALDH+ cells only were injected, no statistical difference was detected in the median weeks to first palpability. Thus, at least for the tumors examined, no correlation was found between the mitotic index and MIC frequency.
      Figure thumbnail gr4
      Figure 4Melanoma-initiating cell (MIC) frequency and tumor proliferation. (a, b) The tumors from 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=10μm). ALDH, aldehyde dehydrogenase.
      To determine whether ALDH+ melanoma cells were capable of ongoing self-renewal, whereas ALDH cells were not, serial transplantation in vitro was performed. Xenografts that developed from ALDH+ or ALDHhiSSClo melanoma cells contained similar proportions of ALDH+ cells to the tumors that developed from UNF cell injections (Figure 3c), whereas tumors that developed from ALDH cells contained small numbers of ALDH+ cells. Xenografts (from Figure 3) were transplanted in vitro to examine self-renewal ability (20,000 cells per well, in 24-well plates). Cells from xenografts that developed from ALDH+, ALDHhiSSClo, and UNF cells grew well in culture, whereas the xenografts that developed from ALDH cells did not grow or grew poorly (Figure 3d and Supplementary Figure S5 online). Thus, xenografts from ALDH+ melanoma cells displayed superior self-renewal to xenografts from ALDH cells.

      FACS sorting for ALDH cell populations with Aldefluor

      ALDH+ cells were present in the xenografts that developed from ALDH cells, consistent with either, ALDH+ cells present in the ALDH cells selected by FACS, or ALDH cells producing or becoming ALDH+ cells (see Figure 3c). To determine if ALDH+ cells persist in FACS-sorted ALDH populations, we performed double and triple sorts of FACS-sorted ALDH cells. When ALDH cells were double-sorted 0.2–1% of cells that were in the ALDH gate on the first sort were in the ALDH+ gate on the second sort, and after triple sorting, 0–0.4% of previously ALDH cells were found in the ALDH+ gate (n=4). FACS sorting produces highly purified populations of cells, yet is clearly unable to eliminate all ALDH+ cells.

      Immunostaining for ALDH

      We then performed immunoperoxidase studies to detect differences in ALDH expression in fresh human melanomas (Supplementary Figure S6 online). Eleven of 13 primary melanomas and 11 of 14 metastatic lesions had 0–10% ALDH-positive cells, whereas 2 of 13 primaries and 3 of 14 metastastic lesions had 50–100% positive cells. In tumors with small numbers of ALDH+ cells (0–10%), some ALDH+ cells had melanoma cell morphology (large aberrant nucleus) and a nested location, whereas other cells were infiltrating macrophages, making tumors with small numbers of ALDH-positive cells difficult to assess. In three paired primary/metastasis samples, we found that each of the primaries and each of the metastases had 0–10% positive cells, and thus there was no evidence for expansion of the ALDH+ population in metastases over corresponding primary lesions.

      Discussion

      Isolating tumorigenic populations of cancer cells is a first step in identifying cells that may constitute cancer stem cells, based on the ability for long-term self-renewal and on a differentially expressed and stable stem cell lineage marker/phenotype.
      There was a 100-fold range in the frequency of human melanoma cells capable of tumorigenesis in NOD/SCID mice. Previous studies in breast cancer also showed that while a few tumors have extensive ALDH activity (essentially 100% ALDH-positive cells), most tumors have only 0–10% ALDH-positive cells by immunohistochemistry (
      • Ginestier C.
      • Hur M.H.
      • Charafe-Jauffret E.
      • et al.
      ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.
      ). These findings are consistent with the low frequency of tumor-initiating cells by NOD/SCID xenotransplantation in most tumors, and the wide range of frequency between tumors.
      We, similar to others, found that melanoma cells that can produce tumor in a NOD/SCID mouse are rare (
      • Schatton T.
      • Murphy G.F.
      • Frank N.Y.
      • et al.
      Identification of cells initiating human melanomas.
      ). The frequency of MICs found in NSG mice in the studies presented here is somewhat lower than that found previously (
      • Quintana E.
      • Shackleton M.
      • Sabel M.S.
      • et al.
      Efficient tumour formation by single human melanoma cells.
      ). However, tumors appeared with similar time to first palpability. The patient samples are different and the methods may have been somewhat different. For example, the type of Matrigel used is not stated by
      • Quintana E.
      • Shackleton M.
      • Sabel M.S.
      • et al.
      Efficient tumour formation by single human melanoma cells.
      ), so that they may have used the High Concentration Matrigel No. 354248 (whereas we used the regular Matrigel Basement Membrane Matrix No. 354234 (BD Biosciences, San Jose, CA).
      The fact that tumors develop from ALDH cells suggests three possibilities: ALDH+ cells are present in the ALDH population as a result of technical limitations of FACS, ALDH cells produce or become ALDH+ cells, or, tumors form from ALDH cells that are part of a distinct MIC population. We believe that the limitation of FACS-sorting is the most likely cause of tumors that develop from ALDH cells because; (1) ALDH+ cells were present in the ALDH cell-derived xenografts, (2) ALDH+ cells persisted in FACS-sorted populations of ALDH cells, and (3) while ALDH+ cells produce tumors more rapidly than equivalent numbers of ALDH cells, the time to first palpability for 5,000 ALDH cells is much longer for ALDH cells from the primary tumor containing the least ALDH+ cells (there are presumably less contaminating ALDH+ cells in this ALDH sample, and therefore slower tumor growth). However, this does not eliminate the possibility that ALDH cells produce or become ALDH+ cells. Extensive molecular studies will be needed to determine whether ALDH can be used for enrichment for melanoma-initiating activity without necessarily supporting a cancer stem cell theory (ALDH is not a stable phenotype), or whether ALDH cells represent true cancer stem cells with a stable phenotype.
      Serial transplantation has been shown to lead to the in vivo selection of cells that recapitulate tumors with increased aggressiveness (
      • Clark E.A.
      • Golub T.R.
      • Lander E.S.
      • et al.
      Genomic analysis of metastasis reveals an essential role for RhoC.
      ). In the studies presented here, serial transplantation of tumors resulted in an increased frequency of MICs as well as decreased time to first palpability. This may be related to the altered environment, with the niche and immune system of the NOD/SCID mouse perhaps doing less to inhibit tumorigenesis than the original human host.
      Growth of tumors from freshly isolated cells could be affected by the presence of non-melanoma cells. Lymphocytes were easily excluded from cell counts based on size. Immunoperoxidase studies showed that CD45 and CD31 expressing human cells did not survive in the xenograft, and staining of the human samples did not reveal a correlation between the MIC frequency and the degree of infiltrating human non-melanoma cells. However, the loss/absence of non-melanoma cells could be a contributory factor to the increased MIC frequency observed with serial transplants, and the presence of non-melanoma cells could affect whether or not a tumor develops.
      In summary, human melanomas show a 100-fold variation in the number of cells with tumor-initiating ability in the NOD/SCID mouse, and tumors with high MIC frequency have shorter time to first palpability in a xenograft model than melanomas with low MIC frequency. Furthermore, the population of human melanoma cells that expresses ALDH is enriched for MICs in both the NOD/SCID and NSG mouse models and has stem cell characteristics of self-renewal and ability to generate heterogeneous descendants.

      Materials and Methods

      Melanoma cell preparation

      Metastatic melanomas were obtained intraoperatively from patients at the UCSF Helen Diller Cancer Center, with written, informed patient consent, adherence to the declaration of Helsinki principle guidelines and Committee on Human Research approval. As in most human melanoma studies (
      • Monzani E.
      • Facchetti F.
      • Galmozzi E.
      • et al.
      Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential.
      ;
      • Quintana E.
      • Shackleton M.
      • Sabel M.S.
      • et al.
      Efficient tumour formation by single human melanoma cells.
      ;
      • Schatton T.
      • Murphy G.F.
      • Frank N.Y.
      • et al.
      Identification of cells initiating human melanomas.
      ), metastatic melanomas were used, as obtaining fresh primary melanomas is difficult due to the requirement of the entire lesion for pathological evaluation. Extraneous tissue was trimmed and the tumor was minced and placed in 1000 collagen digestion Uml−1 of Collagenase, 1300Uml−1 of Hyaluronidase, and 200 Kunitz Uml−1 DNase I (Sigma, St Louis, MO) in Hank's solution (Cambrex, Walkersville, MD) with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). After 90–120minutes at 37°C, the cell suspension was filtered (100μm strainer, Becton Dickinson, Mountain View, CA). Cell counts and viability were determined using a Neubauer hemacytometer (Hausser Scientific, Horsham, PA). Dead cells were excluded using 0.4% Trypan Blue (Sigma), and lymphocytes by size and morphology.

      Cell culture

      Patient melanoma cells and the cell line WM-266-4 (from a human metastatic melanoma in lymph node) were cultured in Eagle's minimum essential medium (ATCC, Manassas, VA), 10% fetal bovine serum, and 1% antibiotic/antimycotic (Invitrogen).

      Subcutaneous injection of melanoma cells

      The NOD/SCID mouse and the NSG mouse (Jackson Laboratory, Bar Harbor, ME) models were utilized. Melanoma cells were injected subcutaneously on the dorsal surface. Cell viability was ∼90%. Tumors were followed for up to 24 (NOD/SCID) or 36 (NSG) weeks. After killing, injection sites were excised, and histological examination determined the presence of tumor. Tumors were excised for serial transplantations at 1cm diameter.

      FACS

      Melanoma cells were incubated with 1μM BODIPY aminoacetaldehyde (Aldefluor), a fluorescent substrate to ALDH, according to the manufacturer's protocol. Cells were also incubated with phycoerythrin-conjugated anti-human CD133/1(AC133) antibody (Miltenyi, Bergisch Gladbach, Germany), according to the manufacturer's protocol. Allophycocyanin-conjugated anti-human CD45 antibody was used to exclude bone marrow-derived cells. Sorting was conducted on a FACSAria (Becton Dickinson) utilizing FACSDiva software and a purity prioritization algorithm. FlowJo software (Treestar, Ashland, OR) was used for data analysis. CD133+ melanoma cells were separated by an AutoMACS Separator (Miltenyi Biotec, Auburn, CA). The CD133+ population was confirmed by FACS.

      Immunostaining

      Paraffin-embedded sections were deparaffinized, rehydrated, and antigen enhanced with Reaction Buffer (Ventana Medical Systems, Oro Valley, AZ). ALDH1 antibody (ab52492, Abcam, Cambridge, MA) was used according to the manufacturer's instructions. Human-specific mAb anti-CD45 and anti-CD31 antibodies (Cell Marque, Rocklin, CA) and mAb anti Ki-67 antibody (Ventana Medical Systems) were used.

      Statistical analysis

      Injection sites were scored as positive or negative for tumor, based on gross observation and subsequent histological confirmation. The ratio of positive results/total injections for each dose of cells injected was determined. Using this data, limiting dilution analysis of the frequency of MICs for each cell population was performed using L-Calc (Stemsoft, Vancouver, Canada) statistical software. The Spearman correlation coefficient was used to assess the association between variables. A Stratified Cox regression model was used to assess differences in time to first palpability.

      ACKNOWLEDGMENTS

      This work was supported by an NIH AR01 grant (RG), the Department of Veterans Affairs (RG), as well as gifts from D Gregory, JC McIntosh, and S Reeves. We thank C Largman, A Charruyer, HJ Lawrence, T Jenkins, M Florero, and L-C Liu for invaluable advice and support. We thank S Fong for his outstanding technical support. Dr Scalapino's work is supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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