Efficient tumour formation by single human melanoma cells

Abstract

A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells (0.1-0.0001%) form tumours when transplanted into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. However, the extent to which NOD/SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/SCID interleukin-2 receptor gamma chain null (Il2rg(-/-)) mice, can increase the detection of tumorigenic melanoma cells by several orders of magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumours under these more permissive conditions. In single-cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumours. Modifications to xenotransplantation assays can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.

Figure 1. Only rare human melanoma cells form tumors in NOD/SCID mice

a, Tumor development after subcutaneous injection of unfractionated primary melanoma cells directly from seven patients into NOD/SCID mice. Dots represent the times after injection at which individual tumors were first palpable and are colored according to cell dose. Crosses are injections that failed to form tumors. Dotted line indicates 8 weeks after injection. b, All tumors were diagnosed as metastatic melanoma by clinical pathology (see Suppl. Table 1 for more information). The tumors that formed in mice (i, arrow) became large, grew quickly once they were palpable, and were histologically similar to the patient tumors from which they were derived. Flow-cytometry demonstrated that the vast majority of tumor cells expressed human HLA (ii; dotted line represents unstained control). Some tumors were highly pigmented (iii) while others contained variable pigmentation (iv) or were amelanotic (scale bar=1cm). H&E stained sections through the same tumors showed pigmented cells (v, vi, see arrows, bars = 25μm). Cytospun cells contained melanin, as indicated by Fontana-Masson staining (vii, viii, arrows, bars = 25 μm), and showed widespread S100ß staining (ix, x), a marker used to diagnose melanoma. c, Limiting dilution analyses of the frequency of tumorigenic melanoma cells in Fig.1a at 8 weeks or 32 weeks after transplantation (*p<0.0001).

Figure 2. Modifications to the xenotransplantation assay reveal that many more human melanoma cells have tumorigenic potential than detected in NOD/SCID mice

a, Live human melanoma cells were isolated from xenografted tumors by flow-cytometry. After gating to eliminate debris and dead cells, additional gates were drawn to select human HLA+ cells and to exclude mouse hematopoietic (CD45 and TER119) and endothelial (CD31) cells (middle panel). The human HLA+ cells consistently formed tumors upon transplantation into immunocompromised mice while mouse hematopoietic and endothelial cells did not (data not shown). b, Tumor development after side-by-side subcutaneous injections of 4,000 human melanoma cells from a xenograft derived from patient 308 into NOD/SCID or NOD/SCID IL2Rγ^null mice. c, Tumor development after side-by-side injections of 400 human melanoma cells from a xenograft derived from patient 205 cells into NOD/SCID IL2Rγ^null mice, with or without Matrigel. Photographs in b, and c, show resulting tumors at the time of analysis (bars = 1 cm). Similar experiments conducted with xenografted and non-xenografted tumors from several patients demonstrated that a significantly higher frequency of human melanoma cells formed tumors when injected into NOD/SCID IL2Rγ^null mice (Suppl. Fig. 2) or with Matrigel (Suppl. Fig. 4b). The tumors were also palpable earlier and grew faster (Suppl. Figs. 1, 4a). d, Limiting dilution analyses of tumors that arose after 8 weeks in NOD/SCID or NOD/SCID IL2Rγ^null mice from the side-by-side transplantation of melanoma cells (derived from xenografts) mixed with vehicle or Matrigel, respectively. In all cases, melanoma-initiating cells were significantly (*p<0.05) more frequent in the modified assay and represented 1 in 5 to 1 in 15 cells. CI: confidence interval.

Figure 3. A high percentage of human melanoma cells are tumorigenic but normal human cells are not

a, Large doses of primary human melanocytes and/or mesenchymal stem cells were mixed with Matrigel and transplanted into NOD/SCID IL2Rγ^null mice. No tumors were palpable after 19 weeks. The frequency of melanoma-initiating cells in 12 human tumors obtained either from xenografts (b) or directly from patients (c) was calculated by limiting dilution analysis. Tumors from patients 498 and 509 were primary cutaneous lesions (cut), whereas other tumors were metastases (Suppl. Table 1). In each case, live human melanoma cells were isolated by flow-cytometry, mixed in Matrigel, and injected subcutaneously into NOD/SCID IL2Rγ^null mice. 11% of melanoma cells from xenografts and 25% of melanoma cells directly from patients formed tumors within 32 weeks of transplantation. Weeks to palpability (mean±s.d.) is indicated for the lowest dose of cells from each tumor. CI: confidence interval.

Figure 4. Efficient tumor development from the xenotransplantation of single human melanoma cells

a, Flow-cytometrically isolated human melanoma cells derived from xenografts from 4 patients were diluted into Terasaki microwells such that wells containing single cells could be identified by phase contrast microscopy. In control experiments, the presence of single cells was confirmed by the observation of single nuclei with Acridine Orange staining in 90 out of 90 cases. The single cells were mixed with Matrigel and injected into NOD/SCID IL2Rγ^null mice. Tumors arising from the injection of single cells were confirmed to be melanoma by H&E, S100, and HMB45 staining (right panels show sections from a tumor that arose from single cells obtained from patient 214) b, The percentage of single cell injections (69/254=27%) that formed tumors within 20 weeks of transplantation. CI: confidence interval. Weeks to first palpability (mean±s.d.) is indicated for each set of tumors.