Cancer stem cells in solid tumors: elusive or illusive?

During the past years in vivo transplantation experiments and in vitro colony-forming assays indicated that tumors arise only from rare cells. These cells were shown to bear self-renewal capacities and the ability to recapitulate all cell types within an individual tumor. Due to their phenotypic resemblance to normal stem cells, the term "cancer stem cells" is used. However, some pieces of the puzzle are missing: (a) a stringent definition of cancer stem cells in solid tumors (b) specific markers that only target cells that meet the criteria for a cancer stem cell in a certain type of tumor. These missing parts started an ongoing debate about which is the best method to identify and characterize cancer stem cells, or even if their mere existence is just an artifact caused by the experimental procedures. Recent findings query the cancer stem cell hypothesis for solid tumors itself since it was shown in xenograft transplantation experiments that under appropriate conditions tumor-initiating cells are not rare

Identifizierung und Charakterisierung von Krebsstammzellen im kutanen malignen Melanom

Abbreviations viii List of figures x List of tables xii Abstract in English xiii Abstract in German xiv 1 Introduction 1 1.1 Human skin 1 1.1.1 Melanogenesis 2 1.2 Malignant melanoma 3 1.2.1 Epidemiology and etiology 3 1.2.2 Pathophysiology 5 1.2.3 Diagnosis, classification and staging of melanoma 6 1.2.4 Therapy of malignant melanoma 9 1.3 Cancer stem cells 10 1.3.1 History of the CSC theory 10 1.3.2 CSCs and the failure of conventional cancer therapies 11 1.3.3 Current standards of identification 13 1.4 Human embryonic and tissue stem cells 18 1.4.1 Characteristics 18 1.4.2 Importance of TGF-β signaling for hESC self-renewal 19 1.4.3 Pluripotency factor OCT4 20 1.5 Aims of this work 23 2 Materials and Methods 24 2.1 Materials 24 2.2 Cell culture 32 2.2.1 Establishment of primary cell cultures 32 2.2.2 Maintenance of primary cell cultures 33 2.2.3 Melanocyte culture 33 2.2.4 Cell culture with hESC medium 33 2.3 Cell sorting 34 2.3.1 MACS 34 2.3.2 FACS 34 2.4 RNA and DNA analyses 34 2.4.1 Isolation and quantification of genomic DNA 34 2.4.2 RNA isolation and quantification 35 2.4.3 Reverse transcription 35 2.4.4 Primer design 35 2.4.5 Standard polymerase chain reaction (PCR) 35 2.4.6 Real- time polymerase chain reaction (real-time PCR) 36 2.4.7 Gel electrophoresis 37 2.4.8 Restriction digest 37 2.4.9 Synthesis of biotin-labelled cRNA and Illumina Bead Chip hybridization 37 2.4.10 Gene expression and cluster analyses 38 2.4.11 Cell cycle analysis 38 2.5 Cycle Sequencing 39 2.6 Whole transcriptome mRNA sequencing 40 2.6.1 rRNA removal 40 2.6.2 Ethanol precipitation 40 2.6.3 Double-stranded cDNA synthesis 40 2.6.4 Sequencing data analysis 40 2.7 Plasmid works 41 2.7.1 phOCT4-EGFP-1 construct 41 2.7.2 Transformation and E. coli culture 42 2.7.3 Isolation of plasmid DNA 42 2.7.4 Plasmid transfection 43 2.7.5 Cultivation of stable clones 43 2.8 Protein analyses 43 2.8.1 Protein isolation and quantification 43 2.8.2 Fractionation of total protein lysates 43 2.8.3 SDS-PAGE gel electrophoresis 44 2.8.4 Western blotting 44 2.8.5 Immunocytochemistry 45 2.9 Functional assays 45 2.9.1 Dye exclusion assay 45 2.9.2 Drug resistance analyses 46 2.9.3 Anchorage-independent growth assay 46 2.9.4 Invasion and mobility assays 47 2.10 Xenotransplantation 48 3 Results 50 3.1 Identification of CSCs in malignant melanoma by established methods 50 3.1.1 Spheroid formation under ES cell culture conditions 50 3.1.2 Identification of a Hoechst SP in most melanoma cell lines 51 3.1.3 Expression of putative CSC markers in melanoma cell lines 52 3.2 Comparison of CD133+ and CD133- melanoma cells 57 3.2.1 CD133 related gene expression profiling 58 3.2.2 Activated FGF pathway in CD133+ melanoma cells 60 3.2.3 Whole transcriptome mRNA sequencing of CD133+ and CD133- melanoma cells 60 3.2.4 Is the CD133 expression epigenetically regulated? 61 3.2.5 Functional comparison of CD133+ and CD133- cells 63 3.3 Dynamic of CD133 expression 68 3.4 Analysis of OCT4A expression in melanoma cells 72 3.4.1 OCT4A is expressed in melanoma cells 72 3.4.2 Differential expression of OCT4A between CD133+ and CD133- melanoma cells 73 3.4.3 Sorting of OCT4-EGFP+ and OCT4-EGFP- melanoma cells 74 3.4.4 Comparison of OCT4-EGFP+ and OCT4-EGFP- melanoma cells 77 4 Discussion 81 4.1.1 Sphere formation assays 81 4.1.2 Dye exclusion assay 82 4.1.3 Expression of CSC markers 84 4.2 CD133+ melanoma cells comprise stem cell characteristics 88 4.2.1 Rare differential SNPs between CD133+ and CD133- cells on transcript level 90 4.2.2 Epigenetic regulation of CD133 90 4.3 Functional comparison of CD133+ and CD133- cells 92 4.3.1 BRAF mutations and targeted therapy for malignant melanoma 93 4.3.2 Drug resistance 95 4.4 Dynamic cell state transitions in tumors challenge cancer therapy concepts 95 4.5 Expression of the pluripotency factor OCT4A in rare melanoma cells 99 4.5.1 Data misconception 99 4.5.2 OCT4-EGFP+ melanoma cells overrepresent stem cell related pathways 100 4.6 Tumorigenic abilities of CSCs in in vivo experiments 103 4.7 Categorization of CSCs 106 5 Conclusion and perspectives 108 References 110 Appendix 125 Publications 125 Curriculum vitae 126 Supplementary tables 128Cutaneous malignant melanomas result from neoplastic growth of melanocytes and are the most severe type of skin cancer. To improve overall survival, a better understanding of melanoma tumorigenesis is needed. Recent findings suggest that within almost all solid tumors in anology to hematopoetic tumors a small subpopulation of tumor cells exculsively have tumor initiating and propagating capacity. These so called cancer stem cells (CSCs) might also be the reason for the high rate of therapy failure, tumor relapse and metastasis. This study was aimed at establishing useful methods and identifying markers or combinations of markers to characterize CSCs in low-passage melanoma cells. After establishing primary melanoma cell cultures I identified subpopulations of melanoma cells that express CD133, which is correlated with asymmetric cell division and downregulated upon cell differentiation and OCT4A the master regulator of pluripotency. I enriched CD133+ as well as OCT4A+ cells from the bulk and found that crucial regulatory pathways related to oncogenesis and stemness such as Wnt, Hedgehog and Notch signaling are significantly overexpressed in the respective positive populations. I validated these results for the TGF-β signaling pathway, which is crucial to maintain the undifferentiated state in human embryonic stem cells (hESCs) on the protein level. I found this cascade exclusively activated in CD133+ melanoma cells. In addition, an overlap of both putative CSC populations was indicated by the overexpression of OCT4A in the CD133+ subpopulation compared to CD133- melanoma cells and vice versa. In vivo experiments showed enhanced tumor growth capabilities of CD133+ and OCT4A+ melanoma cells compared to their negative counterparts. Thus, CD133+ and particularly OCT4A+ melanoma cells comprise both the characteristics of stem-like cells and malignant tumors and provide strict criteria for self-renewal and asymmetric cell division to hold up the term of CSCs in solid tumors. Targeting these cells may lead to a more successful tumor intervention in the future. The dynamic conversion of tumor cell phenotypes that I have observed in my work could substantially hamper a successful therapeutic intervention. Until recently it was believed that tumor heterogeneity was due to a strict hierarchical order where only dedicated CSCs could fuel tumor-development and -fate in an unidirectional manner. My results indicate that a dynamic plasticity between both CSC and bulk tumor populations exists, which would explain tumor survival and rapid adaptation to unfavorable microenvironments like chemo- and radiotherapy. Further characterization of melanoma cancer stem cells and elucidation of the interconversion between tumor cell populations will be an important prerequisite for a lasting prevention of tumor recurrence and metastasis and may pave the way for better understanding tumor biology.Das kutane maligne Melanom, eine bösartige Entartung der Melanozyten, stellt die gefährlichste Form von Hautkrebs dar. Um die Gesamtüberlebenszeit der Melanom¬patienten zu verlängern, ist ein besseres Verständnis der Mechanismen der Melanom-Tumorigenese notwendig. Studien legen nahe, dass fast alle Tumore eine kleine Subpopulation von Tumorzellen enthalten, die ausschließlich die Fähigkeit zur Tumorinitiierung und Selbsterneuerung besitzen. Diese sogenannten Krebsstammzellen stellen vermutlich auch die Ursache für das häufige Therapieversagen, sowie das Auftreten von Tumorrezidiven und Metastasen dar. Ziel dieser Arbeit war die Etablierung geeigneter Methoden und Identifizierung eines geeigneten Markers oder einer Markerkombination für die Charakterisierung von Krebsstammzellen in primären, niedrig-passagigen Melanomzellen. Nach Etablierung primärer Melanom-Zellkulturen habe ich Subpopulationen von Melanomzellen identifiziert, die CD133 exprimieren, einem Marker, der mit asymmetrischer Zellteilung korreliert und während der Zelldifferenzierung herunterreguliert wird, sowie OCT4A, den wichtigsten Regulator der Pluripotenz. Ich habe CD133+ und OCT4A+ Zellen aus den Melanomkulturen isoliert und herausgefunden, dass wichtige regulatorische Entwicklungs- sowie krebsbezogene Signalwege signifikant in diesen Marker- positiven Populationen hochreguliert sind. Für den TGF-β-Signalweg, der in humanen embryonalen Stammzellen essenziell ist, um den undifferenzierten Zustand zu erhalten, habe ich diese Ergebnisse auf Proteinebene validiert. Die TGF-β-Kaskade war ausschließlich in CD133+ Melanomzellen aktiviert. Eine Überschneidung von beiden putativen Krebsstammzell-Populationen wurde außerdem durch Überexpression von OCT4A in der CD133+ Subpopulation im Vergleich zur CD133- Fraktion und vice versa angezeigt. In vivo Experimente zeigten schließlich, dass CD133+ und OCT4A+ Melanomzellen stärkere Tumor-initiierende und -propagierende Eigenschaften besitzen als CD133- und OCT4A- Zellen. Somit weisen CD133+ und OCT4A+ Melanomzellen sowohl die Eigenschaften von Stammzellen als auch malignen Tumorzellen auf und bieten strenge Kriterien für Selbsterneuerung und asymmetrische Zellteilung, um dem Begriff Krebsstammzellen in soliden Tumoren gerecht zu werden. Auf diese Zellen gerichtete Therapien könnten in der Zukunft effizientere Strategien zur Heilung von Melanompatienten darstellen. Die dynamische Konversion zwischen Krebsstammzell- und nicht-stammzellartigen Populationen, die ich während meiner Doktorarbeit beobachtet habe, könnte eine erfolgreiche therapeutische Intervention jedoch erschweren, weil sie das Tumorüberleben und eine rasche Adaption an Veränderungen im Tumorumfeld wie bei Chemo- und Strahlentherapie ermöglicht. Eine weitere Aufklärung der gegenseitigen Umwandlung von Tumorzellpopulationen wird die Voraussetzung für eine langanhaltende Verhinderung von Tumorrezidiven und Metastasen sein

The Nerve Growth Factor Receptor CD271 Is Crucial to Maintain Tumorigenicity and Stem-Like Properties of Melanoma Cells

<div><p>Background</p><p>Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance. In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133.</p><p>Results</p><p>Here we confirmed that the nerve growth factor receptor (CD271) is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity and plasticity in melanoma cells. Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity. A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down. In a meta-analysis we have determined a shared set of 271 differentially regulated genes, linking CD271 to SOX10, a marker that specifies the neural crest. To dissect the connection of CD271 and CD133 we have analyzed 10 patient-derived melanoma-cell strains for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271⁺ cells in the majority of cell strains analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell strains harbored a CD133⁺ sub-set that in addition comprised a fraction of cells of a CD271⁺/CD133⁺ phenotype. Those cells were found in the label-retaining fraction and <i>in vitro</i> deduced from CD271⁺ but not CD271 knock-down cells.</p><p>Conclusions</p><p>Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes. Further, our data strongly suggest that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity.</p></div

CD271⁺ and CD133⁺ melanoma cells hold comparable tumor-initiating capacities.

<p>(<b>A</b>) Flow cytometry of enriched CD271⁺ cells show a high content of CD271 expressing cells before transplantation. (<b>B</b>) Immunofluorescence analysis and immunohistochemistry on paraffin sections (2 µm) of tumors derived from 1×10⁵ of either CD271⁺, CD133⁻, CD133⁺ or unsorted cells for CD133 (upper row) and HMB45 (center row), respectively. A representative tumor out of n = 3 is shown. H&E shows histological morphology of tumors (lower row). Haematoxylin served as counter stain for HMB45. Bar size is 50 µm. (<b>C</b>) Immunofluorescence analysis for CD271 and CD133 on sections of tumors described in (A) showing co-localization and discrete expression of both markers. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). (<b>D</b>) Growth of tumors following injection of 1×10⁵ of either CD271⁺, CD133⁻, CD133⁺ or unsorted cells was monitored for 57 days. Tumor volumes are shown as mean values ± SD of biological triplicates. Growth of 10⁶ of CD271⁺ cells is not shown. (<b>E</b>) Comparison of mRNA expression levels of either CD271⁺ cells with respective xenograft tumors derived from injection of CD271⁺ cells at different cell numbers (10⁵, 10⁶; left chart) or comparison of CD133⁺ cells with tumors as pointed (right chart). Expression in CD133⁺ cells was compared to xenograft tumors shown in (B) derived from injection of 10⁵ of CD133⁻, CD133⁺ or unsorted cells. mRNA expression levels of CD271, CD133, SOX10, MITF, MART-1 and TYR were determined by qPCR as indicated. Expression levels reveal the independence of the <i>in vivo</i> differentiation capacity of melanoma cells from the initial cell number or the cells phenotype. Shown are ΔCT values normalized to β-actin as mean value ± SD of biological triplicates. *p≤0.05; **p≤0.01 (t-test).</p

CD271 is a predominant marker of malignant melanoma.

<p>(<b>A</b>) Immunohistochemistry (IHC) of CD271 and melanoma differentiation markers TYR, HMB45 and MITF of a representative melanoma metastasis (skin). The mutually exclusive expression of CD271 is marked by the red dashed line. Haematoxylin was used as a counter stain in IHC and H&E discriminates differentiation marker positive cells with large nuclei from negative cells with small nuclei. Scale bars indicate 50 µm. (<b>B</b>) Immunofluorescence microscopy of serial sections of a hepatic metastasis (patient T20/15) shows expression of CD271 and absence of CD133. (<b>C</b>) Co-expression and membranous localization of CD133 and the melanoma antigen MART-1 of a representative out of 16 tumors. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). (<b>D</b>) Illustration of patient-derived melanoma metastases (PM) derivatives: PMX, PM derived xenografted tumors; PMC, PM derived established cell strains; PMCX, PMC derived xenograft tumors. (<b>E</b>) Roundup of qPCR results of PM, PMX and PMC. Shown are the expression levels of <i>CD271</i>, <i>CD133</i>, <i>ABCB5</i>, <i>SOX10</i>, <i>SOX2</i>, <i>NES</i>, <i>TYR</i>, <i>MART-1</i> and <i>MITF</i> (<i>MITF-M</i>) (n = 6). The color code indicates high (red) or median (yellow) expression or low/absence (green). A375, MeWo and human melanocytes served as controls (metastases of SKIN  =  cutaneous; PUL  =  pulmonary/lung and LN  =  lymph node).</p

Asymmetrically dividing cells are CD133⁺ or double positive.

<p>(<b>A</b>) Flow cytometry for CD271 and CD133 in CD133⁺ enriched cells. Results depict the presence of single and double positive cells. The experiment was carried out in independent biological triplicates, a representative example is shown. (<b>B</b>) Distribution of CD271 and CD133 in symmetrically and asymmetrically dividing CD133⁺ cells detected by immunofluorescence microscopy for both markers. (<b>C</b>) Asymmetric cell division in unsorted cells is illustrated by the asymmetrical retention of PKH26. (<b>D</b>) Symmetric cell division is demonstrated by the symmetric orientation of microtubules indicated by α-Tubulin staining (red) and symmetrical distribution of CD133 (green). Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue).</p

The label-retaining melanoma cell-fraction comprises CD271/CD133 double positive cells.

<p>(<b>A</b>) Co-expression of CD271 and CD133 in melanoma cell cultures shown by flow cytometry. The double positive fractions are highlighted and indicated as % ± SD of biological triplicates. (<b>B</b>) Immunofluorescence microscopy for co-expression of CD271 and CD133 in cell cultures shows a discrete or simultaneous expression of these markers. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). Representatives of n = 5 are shown. (<b>C–D</b>) Isolation of highly fluorescent cells that retained the lipophilic dye PKH26 of two melanoma cell strains (Mel9-1 and Mel4-7, left panels) by FACS, 7 days after labeling. PKH26^high fractions are indicated as % ± SD of n = 3 independent FACS experiments. Confirmation of isolated, dye retaining fractions for presence of CD271⁺ and CD133⁺ cells by co-labeling and flow cytometry (right panels). Analysis shows the presence of cells with discrete and co-expression of markers. Representative plots indicate fractions as % ± SD of n = 3 independent experiments. (<b>E</b>) Analysis of isolated dye-retaining cells 7 days after sorting supports the co-localization of CD271 (green) and PKH26 (red, white arrows, first panel) or co-expression of CD271 (red) and CD133 (green) (panels 2–4). (<b>F</b>) Co-localization of CD271 (green) and PKH26 (red, white arrows, first panel) and low expression of CD133 in Mel4-7 cells. Scale bars indicate 50 µm. Nuclei were stained with DAPI (blue). Representatives of n = 3 are shown.</p