Isolation of tumorigenic circulating melanoma cells

https://doi.org/10.1016/j.bbrc.2010.10.091Get rights and content

Abstract

Circulating tumor cells (CTC) have been identified in several human malignancies, including malignant melanoma. However, whether melanoma CTC are tumorigenic and cause metastatic progression is currently unknown. Here, we isolate for the first time viable tumorigenic melanoma CTC and demonstrate that this cell population is capable of metastasis formation in human-to-mouse xenotransplantation experiments. The presence of CTC among peripheral blood mononuclear cells (PBMC) of murine recipients of subcutaneous (s.c.) human melanoma xenografts could be detected based on mRNA expression for human GAPDH and/or ATP-binding cassette subfamily B member 5 (ABCB5), a marker of malignant melanoma-initiating cells previously shown to be associated with metastatic disease progression in human patients. ABCB5 expression could also be detected in PBMC preparations from human stage IV melanoma patients but not healthy controls. The detection of melanoma CTC in human-to-mouse s.c. tumor xenotransplantation models correlated significantly with pulmonary metastasis formation. Moreover, prospectively isolated CTC from murine recipients of s.c. melanoma xenografts were capable of primary tumor initiation and caused metastasis formation upon xenotransplantation to secondary murine NOD-scid IL2Rγnull recipients. Our results provide initial evidence that melanoma CTC are tumorigenic and demonstrate that CTC are capable of causing metastatic tumor progression. These findings suggest a need for CTC eradication to inhibit metastatic progression and provide a rationale for assessment of therapeutic responses of this tumorigenic cell population to promising emerging melanoma treatment modalities.

Research highlights

► This study isolates for the first time viable tumorigenic circulating melanoma cells. ► Results provide evidence that circulating melanoma cells cause metastasis formation. ► Circulating melanoma cell detection correlates significantly with metastatic disease.

Introduction

Human melanoma is the most malignant skin cancer, with increasing incident rates worldwide. Although melanoma can be cured by surgical resection before the cancer has spread, metastatic melanoma is one of the most aggressive and drug-resistant cancers with very poor overall survival [1], [2], [3]. A more detailed understanding of the cellular mechanisms that drive metastatic melanoma progression would improve the development and assessment of more effective new or emerging treatment modalities that target disseminated disease.

In metastatic dissemination, primary tumor cells invade basement membranes, intravasate into blood or lymphatic vessels, reach secondary sites through the circulation, and extravasate and colonize again [4]. In recent years, circulating tumor cells (CTC) have been actively investigated as potential prognostic and therapeutic biomarkers [5]. In melanoma patients, CTC were first detected based on expression of tyrosinase RNA in the peripheral circulation [6]. Melanoma CTC have been found to correlate significantly with tumor stage and patient survival [7], and can serve as a biomarker for predicting anti-tumor responses in patients undergoing systemic therapies [8].

Different strategies have been employed to detect and characterize melanoma CTC, and several genes have been investigated as melanoma CTC biomarkers, including genes involved in melanin biosynthesis or genes encoding melanoma-associated antigens [9]. However, isolation of viable CTC, a prerequisite for functional studies in tumor initiation and metastasis of this cell population, has not been achieved to date. While observations of prognostic relevance of melanoma CTC have suggested that this malignant subpopulation might indeed be tumorigenic and responsible for the development of distant metastases, this hypothesis has not been experimentally proven to date.

Melanoma-initiating cells [10], [11] are minority subpopulations in which clinical virulence resides as a consequence of unlimited self-renewal capacity, resulting in inexorable tumor progression [1], [10], [11]. Our laboratory recently showed human malignant melanoma-initiating cells (MMIC) to express the targetable biomarker [10], [12], [13], [14] and multidrug resistance mediator [14], [15], ATP-binding cassette subfamily B member 5 (ABCB5) [10]. In this study, proof of principle of immune-mediated MMIC destruction and consequent inhibition of tumor growth was demonstrated [10]. More recently, we have shown that MMIC employ mechanisms to thwart endogenous anti-tumor immunity [16], which could account for the observed relative enrichment of MMIC in patient lymph node (LN) metastases compared to visceral metastases [10]. Whether ABCB5 might also be expressed by melanoma CTC hypothesized to exert important roles in metastatic progression, has not been evaluated to date.

Here, we provide initial evidence for the existence of circulating melanoma cells that are capable of causing tumor initiation and metastatic disease progression, considerably strengthening the rationale for ongoing clinical evaluations of melanoma CTC as a biomarker for disease progression, prognosis and outcome. Furthermore, we find that CTC can be comprised of heterogeneous cancer populations, including ABCB5-positive subsets previously associated with clinical metastatic melanoma progression. This additional result provides a rationale to examine in future studies whether specific CTC subsets might contribute differentially to tumor initiation and metastatic disease progression, of relevance to the selection of appropriate CTC markers for diagnostic, prognostic or therapeutic purposes.

Section snippets

Melanoma specimens

Clinical melanoma cells were derived from surgical specimens according to an Institutional Review Board-approved research protocol (University of Würzburg, Germany). Metastatic human C8161 melanoma cells [17] were provided by Dr. Mary Hendrix (Children’s Memorial Research Center, Chicago, IL) and metastatic human LOX and FEMX1 melanoma cells [18] were provided by Dr. Udo Schumacher (University Hospital Hamburg-Eppendorf, Hamburg, Germany). Melanoma cell lines were cultured as described [15].

Isolation of tumorigenic circulating melanoma cells

In order to demonstrate tumorigenic potential of human melanoma CTC, we developed a novel metastatic melanoma xenotransplantation model whereby fluorescent transgene-expressing metastatic human melanoma cells are xenografted s.c. to immunodeficient mice and, upon primary tumor formation and systemic spreading, human melanoma cells are isolated from the murine circulation for use in further tumorigenicity experiments (Fig. 1A). In order to maximize tumor take resulting from xenotransplantation

Discussion

CTC are thought to be important mediators of tumor dissemination and metastatic disease progression in human patients. They represent unique malignant subpopulations that emigrate from the cellular microenvironment and extracellular matrix support of the primary tumor site, survive in a fluid dynamic environment in the context of immunocompetent PBMC, and possess the capacity to eventually home to and establish tumorigenic growth in secondary sites of metastasis. Not all CTC might necessarily

Acknowledgments

This work was supported by the NIH/NCI (grants 1RO1CA113796 and 1R01CA138231 to M.H. Frank and grant 2P50CA093683 (Specialized Program of Research Excellence in Skin Cancer) to M.H. Frank and G.F. Murphy.

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