Limits...
Efficient tumour formation by single human melanoma cells.

Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ - Nature (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Life Sciences Institute, Department of Internal Medicine, and Center for Stem Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.

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 (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.

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Only rare human melanoma cells form tumors in NOD/SCID micea, 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 melanoma40. 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).
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Figure 1: Only rare human melanoma cells form tumors in NOD/SCID micea, 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 melanoma40. 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).

Mentions: Melanoma-initiating (tumorigenic) cells were reported to be rare based on the observation that only 1 in 1,090,000 human metastatic melanoma cells formed tumors within 8 weeks of transplantation into NOD/SCID mice1. To assess this, we transplanted 102 to 107 freshly dissociated melanoma cells obtained directly from 7 patients subcutaneously into NOD/SCID mice (see Suppl. Table 1 for more information on tumors). Palpable tumors were evident in some mice eight weeks after injection of cells from four of seven melanomas (Fig 1a, b). Limiting dilution analysis20 indicated that the average frequency of cells that formed tumors within 8 weeks of transplantation into NOD/SCID mice was 1 in 837,000 (Fig. 1c), confirming the published estimate1. However, most tumors took more than 8 weeks to develop (Fig. 1a). On average, tumors first became palpable after 11.4±3.8 weeks (mean±s.d.), or 14.3±7.6 weeks for tumors that arose from less than 10,000 injected cells. Variability was high, but the average frequency of cells that formed tumors within 32 weeks was 1 in 111,000 (Fig. 1c; p<0.0001). The frequency of melanoma-initiating cells is therefore significantly underestimated when tumor formation is monitored for only 8 weeks.


Efficient tumour formation by single human melanoma cells.

Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ - Nature (2008)

Only rare human melanoma cells form tumors in NOD/SCID micea, 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 melanoma40. 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).
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Figure 1: Only rare human melanoma cells form tumors in NOD/SCID micea, 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 melanoma40. 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).
Mentions: Melanoma-initiating (tumorigenic) cells were reported to be rare based on the observation that only 1 in 1,090,000 human metastatic melanoma cells formed tumors within 8 weeks of transplantation into NOD/SCID mice1. To assess this, we transplanted 102 to 107 freshly dissociated melanoma cells obtained directly from 7 patients subcutaneously into NOD/SCID mice (see Suppl. Table 1 for more information on tumors). Palpable tumors were evident in some mice eight weeks after injection of cells from four of seven melanomas (Fig 1a, b). Limiting dilution analysis20 indicated that the average frequency of cells that formed tumors within 8 weeks of transplantation into NOD/SCID mice was 1 in 837,000 (Fig. 1c), confirming the published estimate1. However, most tumors took more than 8 weeks to develop (Fig. 1a). On average, tumors first became palpable after 11.4±3.8 weeks (mean±s.d.), or 14.3±7.6 weeks for tumors that arose from less than 10,000 injected cells. Variability was high, but the average frequency of cells that formed tumors within 32 weeks was 1 in 111,000 (Fig. 1c; p<0.0001). The frequency of melanoma-initiating cells is therefore significantly underestimated when tumor formation is monitored for only 8 weeks.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Life Sciences Institute, Department of Internal Medicine, and Center for Stem Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.

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 (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.

Show MeSH
Related in: MedlinePlus