Subpopulation Of Melanoma Cells Research Articles

Phenotypic Heterogeneity among Tumorigenic Melanoma Cells from Patients that Is Reversible and Not Hierarchically Organized

We investigated whether melanoma is hierarchically organized into phenotypically distinct subpopulations of tumorigenic and nontumorigenic cells or whether most melanoma cells retain tumorigenic capacity, irrespective of their phenotype. We found 28% of single melanoma cells obtained directly from patients formed tumors in NOD/SCID IL2Rγ(null) mice. All stage II, III, and IV melanomas obtained directly from patients had common tumorigenic cells. All tumorigenic cells appeared to have unlimited tumorigenic capacity on serial transplantation. We were unable to find any large subpopulation of melanoma cells that lacked tumorigenic potential. None of 22 heterogeneously expressed markers, including CD271 and ABCB5, enriched tumorigenic cells. Some melanomas metastasized in mice, irrespective of whether they arose from CD271(-) or CD271(+) cells. Many markers appeared to be reversibly expressed by tumorigenic melanoma cells.

Investigation of structure–activity relationships for mitochondrial toxicity

Inactivation of the tumor suppressor neurofibromin 1 (NF1) presents a newly characterized melanoma subtype, for which currently no targeted therapies are clinically available. Preclinical studies suggest that extracellular signal-regulated kinase (ERK) inhibitors are likely to provide benefit, albeit with limited efficacy as a single agent; therefore, there is a need for rationally designed combination therapies. Here, we evaluate the combination of the ERK inhibitor SCH772984 and the biguanide phenformin. A combination of both compounds showed potent synergy in cell viability assays and cooperatively induced apoptosis. Treatment with both drugs was required to fully suppress mechanistic target of rapamycin signaling, a known effector of NF1 loss. Mechanistically, SCH772984 increased the oxygen consumption rate, indicating that these cells relied more on oxidative phosphorylation upon treatment. Consistently, SCH772984 increased expression of the mitochondrial transcriptional coactivator peroxisome proliferator-activated receptor gamma, coactivator 1-α. In contrast, cotreatment with phenformin, an inhibitor of complex I of the respiratory chain, decreased the oxygen consumption rate. SCH772984 also promoted the expansion of the H3K4 demethylase KDM5B (also known as JARID1B)-positive subpopulation of melanoma cells, which are slow-cycling and treatment-resistant. Importantly, phenformin suppressed this KDM5B-positive population, which reduced the emergence of SCH772984-resistant clones in long-term cultures. Our results warrant the clinical investigation of this combination therapy in patients with NF1 mutant melanoma.

A Temporarily Distinct Subpopulation of Slow-Cycling Melanoma Cells Is Required for Continuous Tumor Growth

Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.

Cancer/testis antigens can be immunological targets in clonogenic CD133+ melanoma cells.

"Cancer stem cells" that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133(+) melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133(+) clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8(+) T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma.

Stemming the flood

The cancer stem cell debate over thelast 10 months has been an excitingtime for melanoma research. At theoutset of 2008, Markus Frank’s labora-tory published evidence that only atiny subpopulation of melanoma cells(approximately one cell in a million) hasthe capacity to maintain tumour pro-gression (Schatton et al., 2008). By thestandards of the day, it was an extre-mely important work, linking togethercancer stem cells, disease progressionand therapeutic resistance, and itseemed that a great leap forward hadbeen made in the fight against meta-static disease. However, at the closeof the year Sean Morrison’s groupraised the standards and presented evi-dence opposing the idea of a tiny sub-population of melanoma cells which arecritical for tumour progression, findinginstead that as much as a quarter of allmelanoma cells within any given lesionare tumorigenic (Quintana et al., 2008).The cancer stem cell (CSC) hypothe-sis rests on the premise that there area small number of specialized cancercells which combine the stem cell-likeproperties of limitless renewal andasymmetric division with the capacityto invade and populate regions distalfrom the original transformative event.The attractions of this hypothesisinclude that these cells may explainboth the cellular heterogeneity oftumours and resistance of metastaticdisease to current therapeuticapproaches. That melanoma metasta-ses are composed of different popula-tions of malignant cells is now wellaccepted; however, the explanation forthis heterogeneity has not yet beenestablished. Asymmetric division ofmelanoma stem cells to yield differentlycommitted progeny is a good model forpersistent heterogeneity throughoutdisease progression. Likewise, resis-tance to therapy may occur becausetumour cells are targeted via character-istics which, although widely expressedin melanoma tissue, may not beexpressed by a truly tumorigenic sub-population. The possibility that mela-noma stem cells underlie heterogeneityand therapeutic failure is plausible andthe seminal work by Schatton andco-workers appeared to bear this out.Challenges to the CSC hypothesis,include concerns about whether or notthe models used to evaluate CSCs arevalid. Xenograft models take advantageof compromised immune mechanismsto provide an essentially ‘black box’environment in which cancer cellproliferation may be experimentallymonitored. A typical CSC experimentseparates cells into fractions character-ized by surface markers known to beexpressed by normal stem cells, subcu-taneously injects them into immuno-compromised mice, and evaluates theircapacity to form tumours. It has beensuggested that xenografting underesti-mates the number of cells truly capableof driving tumourigenesis, because non-human environments may proliferativelyselect, in a non-informative fashion,some cancer cells over others. In theirstudy, Quintana et al. compared xeno-graft viability in the NOD⁄SCID mouseused by Schatton et al. with that of amore severely immunocompromisedmodel lacking natural-killer cell activity(NOD⁄SCID Il2rg

Brn-2 Represses Microphthalmia-Associated Transcription Factor Expression and Marks a Distinct Subpopulation of Microphthalmia-Associated Transcription Factor–Negative Melanoma Cells

The origin of tumor heterogeneity is poorly understood, yet it represents a major barrier to effective therapy. In melanoma and in melanocyte development, the microphthalmia-associated transcription factor (Mitf) controls survival, differentiation, proliferation, and migration/metastasis. The Brn-2 (N-Oct-3, POU3F2) transcription factor also regulates melanoma proliferation and is up-regulated by BRAF and beta-catenin, two key melanoma-associated signaling molecules. Here, we show that Brn-2 also regulates invasiveness and directly represses Mitf expression. Remarkably, in melanoma biopsies, Mitf and Brn-2 each mark a distinct subpopulation of melanoma cells, providing a striking illustration of melanoma tumor heterogeneity with implications for melanoma therapy.

Distribution of muscarinic receptor subtype M3 in melanomas and their metastases

Muscarinic acetylcholine receptors (mR) are involved in the regulation of cancer cell motility and cancer progression. mR have been shown in melanoma cell lines and cryostat sections of melanomas. To substantiate the experimental data, here the correlation of mR-expression with invasive growth was studied on the cellular level by comparison with HMB-45 immunoreactivity. mR were detected by a M3 subtype-specific polyclonal antibody in normal skin, benign compound nevi, primary melanomas [nodular type, nodular malignant melanoma (NMM)] and metastases, and were compared with HMB-45 staining in parallel paraffin sections. The general staining pattern of anti-M3 and HMB-45 was similar with accentuation of zones with infiltrative growth. On the cellular level, only a subpopulation of the HMB-45 positive melanoma cells expressed mR. Immunoreactivity was encountered in 3 of 15 nevi, in 9 of 14 NMM and in 10 of 14 melanoma metastases. Polymorphonuclear granulocytes also exhibited strong reactivity for anti-M3. mR-expression is associated with invasive migration of melanomas.

Sanguinarine cytotoxicity on mouse melanoma K1735-M2 cells—Nuclear vs. mitochondrial effects

Sanguinarine (SANG) is an alkaloid recognized to have anti-proliferative activity against various human tumour cell lines. No data is available on the susceptibility of advanced malignant melanoma to SANG, although this disease has a very poor prognosis if not detected in time due to the resistance to conventional chemotherapy. The present work was designed to study the nuclear and mitochondrial involvement in the pro-apoptotic effect of SANG in an invasive mouse melanoma cell line. The results obtained show that SANG is primarily accumulated by the cell nuclei, causing inhibition of cell proliferation and inducing cell death, as confirmed by an increase in sub-G1 peaks. At low concentrations, SANG induces mitochondrial depolarization in a sub-population of melanoma cells, which also generally displayed strong nuclear labelling of phosphorylated histone H2AX. Western blotting revealed an increase in p53, but not Bax protein, in both whole-cell extracts and in mitochondrial fractions. Isolated hepatic mitochondrial fractions revealed that SANG affects the mitochondrial respiratory chain, and has dual effects on mitochondrial calcium loading capacity. We suggest that SANG is able to induce apoptosis in metastatic melanoma cells. The knowledge of mitochondrial vs. nuclear effects of SANG is important in the development of this promising compound for clinical use against aggressive melanoma.

MDR1 expression identifies human melanoma stem cells

ABC transporters are often found to be inherently expressed in a wide variety of stem cells, where they provide improved protection from toxins. We found a subpopulation of human melanoma cells expressing multidrug-resistance gene product 1 (MDR1). This fraction co-expresses the ABC transporters, ABCB5 and ABCC2 in addition to the stem cell markers, nanog and human telomerase reverse transcriptase (hTERT). The clonogenicity and self-renewal capacity of MDR1 + melanoma cells were investigated in single cell settings using the limiting dilution assay. We found that the MDR1 + cells, isolated by FACS sorting, demonstrated a higher self-renewal capacity than the MDR1 − fraction, a key stem cell feature. Moreover, MDR1 + cells had higher ability to form spheres in low attachment conditions, a hallmark of cancer. In conclusion, these novel findings imply that the MDR1 + cells represent melanoma stem cells and thus should be considered as a unique cellular target for future anti-melanoma therapies.

Reprogramming metastatic melanoma cells to assume a neural crest cell-like phenotype in an embryonic microenvironment

Human metastatic melanoma cells express a dedifferentiated, plastic phenotype, which may serve as a selective advantage, because melanoma cells invade various microenvironments. Over the last three decades, there has been an increased focus on the role of the tumor microenvironment in cancer progression, with the goal of reversing the metastatic phenotype. Here, using an embryonic chick model, we explore the possibility of reverting the metastatic melanoma phenotype to its cell type of origin, the neural-crest-derived melanocyte. GFP-labeled adult human metastatic melanoma cells were transplanted in ovo adjacent to host chick premigratory neural crest cells and analyzed 48 and 96 h after egg reincubation. Interestingly, the transplanted melanoma cells do not form tumors. Instead, we find that transplanted melanoma cells invade surrounding chick tissues in a programmed manner, distributing along host neural-crest-cell migratory pathways. The invading melanoma cells display neural-crest-cell-like morphologies and populate host peripheral structures, including the branchial arches, dorsal root and sympathetic ganglia. Analysis of a melanocyte-specific phenotype marker (MART-1) and a neuronal marker (Tuj1) revealed a subpopulation of melanoma cells that invade the chick periphery and express MART-1 and Tuj1. Our results demonstrate the ability of adult human metastatic melanoma cells to respond to chick embryonic environmental cues, a subset of which may undergo a reprogramming of their metastatic phenotype. This model has the potential to provide insights into the regulation of tumor cell plasticity by an embryonic milieu, which may hold significant therapeutic promise.

Rodent models of brain metastasis in melanoma

Metastasis of melanoma to the central nervous system (CNS) remains one of the major barriers to successful treatment of this disease. Available treatment modalities are of limited clinical efficacy. This problem is compounded by the presence of the blood-brain barrier (BBB), an important consideration in the development of new therapeutic agents. Only in animal models can the dual properties of experimental tumours and the BBB be explored in one system. A variety of rodent models have been developed, utilizing both murine and human melanoma cell lines. These models have highlighted the complex biology of cerebral metastasis, involving apparent disease progression through the selection of subclones at each stage, eventually leading to disease in the brain. As demonstrated in a number of animal studies, different subpopulations of metastatic melanoma cells are likely to be responsible for parenchymal and leptomeningeal CNS disease. In addition, these animal systems have been used to demonstrate the potential efficacy of new chemotherapeutic drugs, radiation treatments and immunotherapeutic approaches for the treatment of melanoma brain metastasis. Key biological questions remain to be answered. In particular, the molecular and cellular mechanisms responsible for establishing cerebral melanoma must be clearly delineated. Several molecules, including vascular endothelial growth factor (VEGF) and integrins, appear to play important, but not definitive, roles. Other, as yet undefined, molecules appear to be critical. The identification of these factors in experimental models, with confirmatory studies in humans, will expand our understanding of cerebral melanoma and provide valuable new therapeutic targets for intervention in this difficult clinical problem.

The identification of chromosome abnormalities associated with the invasive phenotype of uveal melanoma in vitro

Tumour cell cultures are often highly heterogeneous, containing sub-populations of cells with differing characteristics. To identify chromosome abnormalities that are associated with the invasive phenotype, we isolated highly invasive uveal melanoma cell populations using the Transwell assay. Using this invasion assay, invasive sub-populations of primary uveal melanoma short-term cultures, and an established cell line, were specifically isolated. A series of sequential assays were undertaken to enrich the invasive population, and the enhanced invasive ability was confirmed by Transwell invasion assay. Chromosome abnormalities in invasive and parental cells were identified by karyotyping and confirmed by comparative genome hybridisation. Invasive sub-populations of uveal melanoma cells were isolated from 3 uveal melanoma short term cultures and a uveal melanoma cell line. In all cases, invasive sub-populations had either acquired additional chromosome abnormalities to those present in the parental cell line, or other abnormalities present in the parental lines were lost. In the established cell line (SOM 157), invasive cells were characterised by widespread chromosomal instability, frequent telomere associations and additional copies of chromosome 20. The invasive phenotype of SOM 196 associated with the presence of a derivative chromosome 5, der(5)t(5;11)(q35;q12) whilst a translocation t(17;20)(q12;q13) was predominant amongst non-invasive cells. In two additional cultures, deletions on chromosome 6q were associated with reduced invasive ability. In conclusion, highly invasive populations of uveal melanoma cells demonstrate chromosomal abnormalities that differ from non-invasive cells. These include chromosome instability and abnormalities of chromosome 20, observations echoing those seen in metastatic uveal melanoma.

The effect of Taurolidine on adherent and floating subpopulations of melanoma cells.

The annual incidence of malignant melanoma is estimated at 10-12 per 100000 inhabitants in countries of Central Europe and the US, with more recent estimates showing a dramatic upward trend. Taurolidine (Carter/Wallace, Cranberry, NJ) is a novel, potentially effective, antitumor chemotherapeutic agent. We hypothesized that Taurolidine could inhibit the growth, induce apoptosis, affect the cell cycle and change morphology of melanoma cells. We expected this process to be different in adherent and floating subpopulations that may be reflective of solid tumors and their metastases. Analysis of MNT-1 human and B16F10 murine melanoma cells showed that at 72 h the IC(50) of Taurolidine was 25.4+/-3.3 microM for MNT-1 human melanoma cells and 30.9+/-3.6 microM for B16F10 murine melanoma cells. Taurolidine induced DNA fragmentation of melanoma cells in a dose-dependent manner. Taurolidine (75 and 100 microM) induced 52-97% Annexin-V binding (apoptosis), respectively. Evaluation of cell cycle after 72 h exposure to Taurolidine (0-100 microM) revealed that the percentage of melanoma cells in S phase increased from 27 to 40% in the adherent subpopulation and from 33 to 49% in the floating subpopulation. Phase contrast microscopy revealed a marked swelling of melanoma cells and decreasing cell numbers in adherent subpopulation starting at 24 h with 25 microM Taurolidine. Shrinkage of cells dominated at 75-100 microM Taurolidine. Using Cytospin assay in the floating population, we observed swelling of melanoma cells induced by 25-100 micro Taurolidine and appearance of giant (multinuclear) forms resulting from exposure to 75-100 micro Taurolidine. Some floating cells with normal morphology were observed with low concentrations of Taurolidine (0-25 microM). These data show that effects of Taurolidine may be different in adherent and floating subpopulations of melanoma cells. More importantly, floating subpopulations that may contain some viable melanoma cells, may be reflective of potential metastasis after treatment of solid tumors in vivo.

Ribosomal 18S RNA Prevails over Glyceraldehyde-3-Phosphate Dehydrogenase and β-Actin Genes as Internal Standard for Quantitative Comparison of mRNA Levels in Invasive and Noninvasive Human Melanoma Cell Subpopulations

The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and β-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and β-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.

Electrophoretic Heterogeneity of Pigmented Hamster Melanoma Cells

Pigmented hamster melanoma tumors growing in situ contain two subpopulations of melanoma cells that have different electrophoretic mobilities (EPM). A mild neuraminidase treatment, which removes sialic acid residues from the cell surface glycoproteins, reduces the EPM of both groups of melanoma cells yielding an electrophoretically uniform population. This shows that the differences in the EPM between the subpopulations of pigmented melanoma cells stem from the different content of sialic acid residues on the cell surface. The relationship between the different EPM melanoma cell subpopulations was, therefore, examined during tumor growth, development, and formation of metastases. The relative content of cells having high electrophoretic mobility, the "fast moving" cells, increases as the tumors grow larger. However, tumors of the same diameter contain nearly the same fraction of "fast moving" cells despite their age. The proportion of the "fast moving" cells is significantly higher in the central part than in the outermost layer of pigmented melanoma tumors. These data suggest that the development of "fast moving" cells is promoted by some size-dependent changes in the intratumor environment. In vivo selection of melanoma cells for their ability to colonize lungs renders tumors that reveal elevated metastatic potential and contain a significantly higher fraction of cells possessing high electrophoretic mobility than the parent tumor. Moreover, the metastatic nodules contain a remarkably elevated fraction of the "fast moving" cells. The reported correlation between the "fast moving" cell fraction and the metastatic potential suggests that the relative content of cells having high electrophoretic mobility may determine the metastaticity of pigmented hamster melanoma.

αIIbβ3 Integrin expression and function in subpopulations of murine tumors

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G 2 M phase of the cell cycle. Both B16a and 3LL cells express α IIb β 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of α IIb β 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with α IIb β 3 -specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that α IIb β 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the α IIb β 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.

Increased expression of alpha IIb beta 3 integrin in subpopulations of murine melanoma cells with high lung-colonizing ability.

Four subpopulations of B16 amelanotic melanoma cells, possessing different abilities to induce platelet aggregation (TCIPA) and to form lung colonies, were isolated by centrifugal elutriation. The expression of alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 integrins was examined in the 4 subpopulations in order to determine the relationship between integrin receptor expression and tumor-cell metastatic potential. The mRNA of alpha IIb, alpha 5, beta 1 and beta 3 was detectable in the 4 subpopulations by Northern blotting. A gradual increase in mRNAs and cell-surface immunoreactivity of the alpha IIb beta 3 receptor, but not in their gene copies, was observed from the low to the high metastatic subpopulations. The ability of tumor cells to adhere to fibronectin and subendothelial matrix (SEM) increased in parallel. In the high metastatic cells, the alpha IIb beta 3 receptors, but not the alpha 5 beta 1 receptors, were localized to focal adhesion plaques. Incubation of the high metastatic cells with alpha IIb beta 3-specific antibodies reduced their matrix adhesion, TCIPA and lung-colonizing abilities. In contrast, in the low met- astatic cells, SEM adhesion and lung-colony formation were not affected by anti-alpha IIb beta 3 antibody treatment. Incubation of either the low or the high metastatic subpopulation with an alpha 5 beta 1-specific antibody had no effect in vitro and showed a slight inhibition of lung colonization in vivo. Our results suggest that several phenotypic characteristics of the enhanced metastatic potential of B16a subpopulations may be mediated by increased expression of alpha IIb beta 3 receptors and that expression of these receptors may be regulated at the transcriptional level.

Purification of human melanocytes by monoclonal antibody combined with Percoll TM gradients

Cultures of human melanocytes obtained by differential plating of human epidermal cells in 12-myristate 13-acetate (PMA) were purified using a monoclonal antibody R 24 which detects a restricted glycolipid antigen present on melanoma cells and melanocytes. Melanocytes were rosetted using protein A-conjugated human red blood cells and separated from non-rosetted ftbroblasts on discontinuous Percoll ™ gradients. 100% pure cultures of melanocytes obtained in this fashion were then successfully grown in tissue culture in the presence of PMA and cholera toxin for at least thirty passages (corresponding to approx. sixty cell doublings). We conclude that: 1. 1. Pure cultures of melanocytes are a prerequisite for the establishment of long-term cultures. 2. 2. Since most human melanomas express substantial levels of R 21 antigen, this method can be applied to purification of melanomas and can be easily adapted to separation of subpopulations of melanocytes and melanoma cells recognized by specific monoclonal antibodies.