Research ArticleMelanoma

Human Melanoma Metastasis in NSG Mice Correlates with Clinical Outcome in Patients

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Science Translational Medicine  07 Nov 2012:
Vol. 4, Issue 159, pp. 159ra149
DOI: 10.1126/scitranslmed.3004599
  • Fig. 1

    Human melanomas exhibited intrinsic differences in their metastatic efficiency in NSG mice. (A) Twenty-seven melanomas from 25 patients were transplanted subcutaneously into NSG mice in doses between 1 and 50,000 cells per injection. Mice with subcutaneous tumors were monitored until the tumors grew to 1.4 ± 0.9 cm (mean ± SD) in diameter 21.8 ± 10.2 weeks after transplantation. Upon necropsy, organs were visually examined for the presence of metastases. “AJCC clinical stage” is the American Joint Committee on Cancer stage of the patient at the time of melanoma removal. “Tumor site” reflects the location of the tumor in the patient. Melanomas 633/634 and 498/499 (boxed) were pairs of tumors obtained from different locations in the same patients at the same time. The metastatic behavior of each melanoma was examined in 2 to 22 independent experiments with 6 to 136 mice that formed subcutaneous tumors per melanoma. The rate of metastasis (%) is the percentage of mice with subcutaneous tumors that developed macrometastases. (B) Tumors that formed at the injection site are shown alongside macrometastases in mice transplanted with cells from four patients. Metastases were confirmed as melanoma by a dermatopathologist after staining sections with hematoxylin and eosin (H&E) and S100.

  • Fig. 2

    BLI confirmed intrinsic differences in metastatic efficiency among human melanomas in NSG mice. Luciferase-GFP+ melanoma cells from different patients exhibited intrinsic differences in metastatic efficiency revealed by BLI in NSG mice. (A and B) BLI of an NSG mouse 2 days (A) and 45 days (B) after subcutaneous transplantation of 100 luciferase-GFP+ cells from melanoma 205, a heavily pigmented melanoma. (C) BLI of an NSG mouse 13 weeks after subcutaneous transplantation of 100 luciferase-GFP+ cells from melanoma 487. (D to J) BLI of individual organs dissected from the mouse in (C) revealed metastases in the lungs (D), stomach (F), pancreas (G), kidneys, adrenal glands, and ovaries (H), but not in the brain (E), liver (I), or spleen (J). The maximum luminescence shown in red in (C) and (D) is 31 × 106 photons s−1 cm−2 sr−1 and in (E) to (J) is 6.2 × 106 photons s−1 cm−2 sr−1. (K) Summary of the percentage of mice with subcutaneous tumors that developed metastases detected by BLI for each melanoma. Melanomas 608, 499, and 597 did not undergo BLI.

  • Fig. 3

    Metastatic efficiency in NSG mice correlated with disease progression in patients. (A) Side-by-side comparison of distant metastasis in patients versus metastasis in mice for all 22 patients from whom follow-up clinical data were available after tumor banking. Most of the melanomas were lymph node metastases from stage IIIB/C patients at the time of banking. The melanomas were ranked by clinicians in terms of the aggressiveness of disease in patients after banking, based primarily on the rate of metastasis, from 514 at the top (most aggressive) to 498/499 at the bottom (least aggressive). The latest AJCC stage (or stage at time of death) and the time (in months) of survival after tumor banking are indicated in the “Most recent survival and staging data” column. The patients with melanomas 514 to 492 formed distant metastases (stage IV disease) and died 3.4 to 40.6 months after melanoma banking. The patient who donated melanoma 600 remained alive at last follow-up, 33.2 months after melanoma banking, but had progressed to stage IV disease with brain metastasis. All of the patients who progressed to stage IV disease did so within 22 months of tumor banking. The patients with melanomas 651 to 498/499 did not form distant metastases or progress to stage IV disease. Patient 528 died 40.7 months after melanoma banking with lung cancer and advanced chronic obstructive pulmonary disease but no evidence of melanoma at the time of death. (B) Side-by-side comparison of brain metastasis in patients and in NSG mice injected subcutaneously with the same melanomas. (C) Kaplan-Meier survival curves for all patients with at least 30 months of follow-up after tumor banking. Patients with melanomas that did not metastasize widely in mice lived significantly longer than all patients or than patients whose melanomas metastasized widely in mice (log-rank test, P < 0.05).

  • Fig. 4

    The frequency of circulating melanoma cells in the blood of xenografted NSG mice correlated with metastatic efficiency. Blood was collected from untransplanted control or melanoma-transplanted experimental NSG mice by cardiac puncture. Live nucleated blood cells were analyzed for HLA+ melanoma cells by flow cytometry. (A and B) The blood samples shown were obtained from mice bearing an efficiently metastasizing melanoma (A; melanoma 405) and an inefficiently metastasizing melanoma (B; melanoma 528). HLA+ cells were not detected in the blood of untransplanted control mice [(A) and (B), <0.01% background staining] or in the blood of mice bearing inefficiently metastasizing melanomas [(B), <0.01% background staining] but were detected in the blood of mice bearing efficiently metastasizing melanomas [(A), 0.02 to 0.51%]. Note that melanoma 528 (B) was analyzed side by side with melanoma 405 (A), such that the same negative control [shown in the left frame in (A) and (B)] applied to both analyses. (C) Frequency of HLA+ melanoma cells in the blood of all mice. The difference between mice bearing efficiently metastasizing (n = 15 mice transplanted with melanomas 405, 481, and 633) and inefficiently metastasizing melanomas (n = 7 mice transplanted with melanomas 528 and 651) was statistically significant [P = 0.0023 by analysis of variance (ANOVA) comparing efficient metastasizers to both other treatments; P = 0.0004 by post hoc pairwise Mann-Whitney t tests]. (D) Several of the mice with inefficiently metastasizing melanomas had large tumor burdens. (E) NSG mice were injected subcutaneously with 100 HLA+/GFP+ cells from the blood of NSG mice with subcutaneous melanomas. Palpable subcutaneous tumors developed within 2 months after injection. Mice were analyzed by BLI for metastases when the subcutaneous tumor reached 2 cm in diameter. Widespread metastasis was observed, just as in the primary melanomas from which the circulating melanoma cells derived.

Additional Files

  • Supplementary Materials for:

    Human Melanoma Metastasis in NSG Mice Correlates with Clinical Outcome in Patients

    Elsa Quintana, Elena Piskounova, Mark Shackleton, Daniel Weinberg, Ugur Eskiocak, Douglas R. Fullen, Timothy M. Johnson, Sean J. Morrison*

    *To whom correspondence should be addressed. E-mail: Sean.Morrison{at}UTSouthwestern.edu

    Published 7 November 2012, Sci. Transl. Med. 4, 159ra149 (2012)
    DOI: 10.1126/scitranslmed.3004599

    This PDF file includes:

    • Table S1. Clinical data are presented on the 25 patients in this study, including therapy before and after melanoma banking.
    • Table S2. Human melanomas exhibited intrinsic differences in their metastatic efficiency in NSG mice.
    • Table S3. Differences in the metastatic efficiency of human melanomas in NSG mice were consistently observed in multiple independent experiments.
    • Table S4. Sites of metastasis in tumor-bearing NSG mice were detected by macroscopic evaluation at necropsy and by BLI for each melanoma.
    • Fig. S1. The rate of metastasis was not significantly correlated with subcutaneous tumor growth by linear regression analysis.

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