Artigo Acesso aberto Revisado por pares

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity

2020; Springer Nature; Volume: 12; Issue: 9 Linguagem: Inglês

10.15252/emmm.201911908

ISSN

1757-4684

Autores

Claudia Koch, Andra Kuske, Simon A. Joosse, Gökhan Yigit, George Sflomos, Sonja Thaler, Daniel J. Smit, Stefan Werner, Kerstin Borgmann, Sebastian Gärtner, Parinaz Mossahebi Mohammadi, Laura Battista, Laure Cayrefourcq, Janine Altmüller, Gabriela Salinas-Riester, Kaamini Raithatha, Arne Zibat, Y. Goy, Leonie Ott, Kai Bartkowiak, Tuan Zea Tan, Qing Zhou, Michael R. Speicher, Volkmar Müller, Tobias M. Gorges, Manfred Jücker, Jean Paul Thiery, Cathrin Brisken, Sabine Riethdorf, Catherine Alix‐Panabières, Klaus Pantel,

Tópico(s)

Breast Cancer Treatment Studies

Resumo

Article15 July 2020Open Access Source DataTransparent process Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity Claudia Koch Claudia Koch Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Andra Kuske Andra Kuske Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Simon A Joosse Simon A Joosse orcid.org/0000-0002-4296-5615 Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Gökhan Yigit Gökhan Yigit Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author George Sflomos George Sflomos orcid.org/0000-0003-2972-0549 ISREC – Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Sonja Thaler Sonja Thaler European Centre for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Daniel J Smit Daniel J Smit orcid.org/0000-0002-3190-9511 Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Stefan Werner Stefan Werner Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Kerstin Borgmann Kerstin Borgmann Radiobiology& Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Sebastian Gärtner Sebastian Gärtner Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Parinaz Mossahebi Mohammadi Parinaz Mossahebi Mohammadi Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Laura Battista Laura Battista ISREC – Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Laure Cayrefourcq Laure Cayrefourcq Laboratory of Rare Human Circulating Cells (LCCRH), University Medical Centre, Montpellier, France Montpellier University, Montpellier, France Search for more papers by this author Janine Altmüller Janine Altmüller Cologne Center for Genomics, University of Cologne, Cologne, Germany Search for more papers by this author Gabriela Salinas-Riester Gabriela Salinas-Riester NGS Integrative Genomics Core Unit, Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Kaamini Raithatha Kaamini Raithatha NGS Integrative Genomics Core Unit, Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Arne Zibat Arne Zibat Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Yvonne Goy Yvonne Goy Radiobiology& Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Leonie Ott Leonie Ott Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Kai Bartkowiak Kai Bartkowiak Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Tuan Zea Tan Tuan Zea Tan orcid.org/0000-0001-6624-1593 Cancer Science Institute of Singapore, National University of Singapore, Singapore City, Singapore Search for more papers by this author Qing Zhou Qing Zhou orcid.org/0000-0001-7737-9643 Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria Search for more papers by this author Michael R Speicher Michael R Speicher orcid.org/0000-0003-0105-955X Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria Search for more papers by this author Volkmar Müller Volkmar Müller Department of Gynecology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Tobias M Gorges Tobias M Gorges Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Manfred Jücker Manfred Jücker Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Jean-Paul Thiery Jean-Paul Thiery orcid.org/0000-0003-0478-5020 INSERM Unit 1186, Comprehensive Cancer Center, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Cathrin Brisken Cathrin Brisken ISREC – Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Breast Cancer Now Research Centre, Institute of Cancer Research, London, UK Search for more papers by this author Sabine Riethdorf Sabine Riethdorf orcid.org/0000-0003-0028-5643 Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Catherine Alix-Panabières Catherine Alix-Panabières orcid.org/0000-0002-6401-2903 Laboratory of Rare Human Circulating Cells (LCCRH), University Medical Centre, Montpellier, France Montpellier University, Montpellier, France Search for more papers by this author Klaus Pantel Corresponding Author Klaus Pantel [email protected] orcid.org/0000-0001-5736-2772 Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Claudia Koch Claudia Koch Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Andra Kuske Andra Kuske Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Simon A Joosse Simon A Joosse orcid.org/0000-0002-4296-5615 Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Gökhan Yigit Gökhan Yigit Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author George Sflomos George Sflomos orcid.org/0000-0003-2972-0549 ISREC – Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Sonja Thaler Sonja Thaler European Centre for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany Search for more papers by this author Daniel J Smit Daniel J Smit orcid.org/0000-0002-3190-9511 Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Stefan Werner Stefan Werner Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Kerstin Borgmann Kerstin Borgmann Radiobiology& Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Sebastian Gärtner Sebastian Gärtner Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Parinaz Mossahebi Mohammadi Parinaz Mossahebi Mohammadi Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Laura Battista Laura Battista ISREC – Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Laure Cayrefourcq Laure Cayrefourcq Laboratory of Rare Human Circulating Cells (LCCRH), University Medical Centre, Montpellier, France Montpellier University, Montpellier, France Search for more papers by this author Janine Altmüller Janine Altmüller Cologne Center for Genomics, University of Cologne, Cologne, Germany Search for more papers by this author Gabriela Salinas-Riester Gabriela Salinas-Riester NGS Integrative Genomics Core Unit, Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Kaamini Raithatha Kaamini Raithatha NGS Integrative Genomics Core Unit, Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Arne Zibat Arne Zibat Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Yvonne Goy Yvonne Goy Radiobiology& Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Leonie Ott Leonie Ott Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Kai Bartkowiak Kai Bartkowiak Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Tuan Zea Tan Tuan Zea Tan orcid.org/0000-0001-6624-1593 Cancer Science Institute of Singapore, National University of Singapore, Singapore City, Singapore Search for more papers by this author Qing Zhou Qing Zhou orcid.org/0000-0001-7737-9643 Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria Search for more papers by this author Michael R Speicher Michael R Speicher orcid.org/0000-0003-0105-955X Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria Search for more papers by this author Volkmar Müller Volkmar Müller Department of Gynecology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Tobias M Gorges Tobias M Gorges Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Manfred Jücker Manfred Jücker Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Jean-Paul Thiery Jean-Paul Thiery orcid.org/0000-0003-0478-5020 INSERM Unit 1186, Comprehensive Cancer Center, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Cathrin Brisken Cathrin Brisken ISREC – Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Breast Cancer Now Research Centre, Institute of Cancer Research, London, UK Search for more papers by this author Sabine Riethdorf Sabine Riethdorf orcid.org/0000-0003-0028-5643 Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Catherine Alix-Panabières Catherine Alix-Panabières orcid.org/0000-0002-6401-2903 Laboratory of Rare Human Circulating Cells (LCCRH), University Medical Centre, Montpellier, France Montpellier University, Montpellier, France Search for more papers by this author Klaus Pantel Corresponding Author Klaus Pantel [email protected] orcid.org/0000-0001-5736-2772 Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Author Information Claudia Koch1,†, Andra Kuske1,†, Simon A Joosse1, Gökhan Yigit2, George Sflomos3, Sonja Thaler4, Daniel J Smit5, Stefan Werner1, Kerstin Borgmann6, Sebastian Gärtner1, Parinaz Mossahebi Mohammadi1, Laura Battista3, Laure Cayrefourcq7,8, Janine Altmüller9, Gabriela Salinas-Riester10, Kaamini Raithatha10, Arne Zibat2, Yvonne Goy6, Leonie Ott1, Kai Bartkowiak1, Tuan Zea Tan11, Qing Zhou12, Michael R Speicher12, Volkmar Müller13, Tobias M Gorges1, Manfred Jücker5, Jean-Paul Thiery14, Cathrin Brisken3,15,‡, Sabine Riethdorf1,‡, Catherine Alix-Panabières7,8,‡ and Klaus Pantel *,1 1Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany 3ISREC – Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 4European Centre for Angioscience (ECAS), Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany 5Institute of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 6Radiobiology& Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 7Laboratory of Rare Human Circulating Cells (LCCRH), University Medical Centre, Montpellier, France 8Montpellier University, Montpellier, France 9Cologne Center for Genomics, University of Cologne, Cologne, Germany 10NGS Integrative Genomics Core Unit, Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany 11Cancer Science Institute of Singapore, National University of Singapore, Singapore City, Singapore 12Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria 13Department of Gynecology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 14INSERM Unit 1186, Comprehensive Cancer Center, Institut Gustave Roussy, Villejuif, France 15Breast Cancer Now Research Centre, Institute of Cancer Research, London, UK † These authors contributed equally to this work as first authors ‡ These authors contributed equally to this work as senior authors *Corresponding author. Tel: +49 40 741053503; Fax: 49 40 7410-55379; E-mail: [email protected] EMBO Mol Med (2020)12:e11908https://doi.org/10.15252/emmm.201911908 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology. Synopsis Blood-born dissemination and subsequent outgrowth of tumor cells - a process called metastasis - is the leading cause of cancer-related death. Cell lines derived from circulating tumor cells (CTCs) in blood of cancer patients provide excellent models to study the largely unknown biology of CTCs. The cell line established from CTCs of an estrogen receptor (ER)-positive breast cancer patient mirrored the in situ CTCs and provided therefore a realistic model to investigate CTC biology. Xenograft experiments demonstrated a pattern of metastasis similar to ER-positive breast cancer patients involving bone, liver and lung as secondary organs. Growth pattern and protein analyses revealed subtle signs of epithelial-mesenchymal transition (EMT) with CTCs falling on the more epithelial end of the EMT spectrum. CTCs carried mutations in druggable genes relevant to cancer therapy (e.g., PIK3CA mutations). In vitro drug screening experiments indicated a cytostatic activity of the CDK4/6 inhibitor Palbociclib on CTCs. The paper explained Problem Metastases developing upon dissemination of tumor cells from the primary tumor, access to the blood circulation and outgrowth at distant organs are the most frequent causes of breast cancer-related death. The vast majority of breast cancer cases is driven by hormone receptors enabling endocrine treatment as promising therapeutic strategy. However, tumor cells can develop resistance to this therapy, which constitutes a significant clinical problem, especially in the metastatic stage of the disease. Analyzing circulating tumor cells (CTCs) might be helpful to identify new therapeutic targets, but functional studies giving insight into the biology of CTCs are limited due to their low frequency and the lack of appropriate models. Results This study describes the establishment and characterization of a novel CTC-derived estrogen receptor (ER)-positive breast cancer cell line from a patient with metastatic ER-positive breast cancer, designated CTC-ITB-01. Downstream ER signaling is constitutively active in CTC-ITB-01 independent of ligand availability. This ER-positive cell line is resistant to endocrine therapy; however, the CDK4/6 inhibitor Palbociclib strongly inhibits CTC-ITB-01 growth. Genomic analyses revealed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. Primary tumor and metastasis formation in an intraductal PDX mouse model reflected the clinical progression of ER-positive breast cancer. Impact Our work established and characterized a novel CTC cell line that mirrored in situ CTCs. This cell line allows first in-depth insights into the functional properties of CTCs in the most common ER+ breast cancer subtype and enables further experimental steps to uncover resistance mechanisms and to identify new therapeutic targets. Introduction Detection and characterization of circulating tumor cells (CTCs) have prognostic value in various tumor entities as demonstrated by several large clinical studies, e.g., for patients with breast and prostate cancer (Bidard et al, 2014; Goldkorn et al, 2014; Scher et al, 2015; Alix-Panabieres & Pantel, 2016). Moreover, these cells have the potential to be exploited as monitoring markers and might function as a blood-based biopsy guiding personalized treatment decisions (Keller & Pantel, 2019; Pantel & Alix-Panabieres, 2019). The perspective to accompany or even replace invasive tumor tissue biopsies in order to gain important diagnostically and therapeutically relevant information makes CTCs an essential contribution to non-invasive "real-time liquid biopsies" (Pantel & Alix-Panabieres, 2010; Bardelli & Pantel, 2017). In spite of an enormous progress in the development of approaches for the detection and molecular characterization of CTCs up to the single cell level (Joosse et al, 2012; Alix-Panabieres & Pantel, 2014a,b; Pantel & Alix-Panabieres, 2019), information on the functional properties of CTCs is still limited due to the very low concentrations of these cells in the peripheral blood of patients with cancer (Alix-Panabieres & Pantel, 2014a). Not all CTCs possess the potential to extravasate at distant sites and grow out to form a novel metastatic lesion (Wicha & Hayes, 2011). A plethora of different factors play into the survival of these CTCs in the blood stream and their capacity to extravasate and metastasize (Strilic & Offermanns, 2017; Giuliano et al, 2018), including the hemodynamic forces within the circulation (Follain et al, 2018) and genomic make-up of the tumor cells (Joosse & Pantel, 2016; Gkountela et al, 2019). Experimental models indicate that only few tumor cells are viable, survive shear forces within the blood flow, evade the immune system as well as systemic therapies, reach distant organs, and eventually have the potential to form an overt metastasis (Chambers et al, 2002, Y. Kang & Pantel, 2013). Adaptation to a new microenvironment and proliferation of a single tumor cell or a CTC cluster in a distant site requires highly specialized traits, most of which are largely unknown. In order to understand these underlying mechanisms of the metastatic cascade, functional characterization of viable CTCs capable of forming distant metastasis is required. A prerequisite for these analyses were therefore the recent advances in the ability to culture CTCs in vitro (Zhang et al, 2013; Yu et al, 2014; Cayrefourcq et al, 2015) or expand the CTC pool in vivo using xenografts (Baccelli et al, 2013; Hodgkinson et al, 2014; Carter et al, 2017). However, to our knowledge, none of these studies compared the characteristics of the original CTCs captured from the patients with cancer directly to the CTC line. Besides unraveling the biology of CTCs in patients with cancer, these studies allow testing of the unknown activity of cancer drugs against CTCs. In this context, the low number of estrogen receptor-positive (ER+) breast cancer cell lines presently available is disturbing, since 70–80% of patients with breast cancer harbor ER+ tumors and ER is the primary target of endocrine therapies in breast cancer (Pan et al, 2017). Here, we report the establishment and in-depth characterization of an ER+ breast CTC line with unique properties. Comparison of CTCs in situ before cell culture with the CTC line indicates that it mirrors the situation in ER+ breast cancer patients and therefore provides novel insights into the biology and drug response of patient-derived CTCs in the most common breast cancer subtype. Results Patient characteristics The patient with MBC the CTC-ITB-01 cell line derived from presented with a bilateral mammary carcinoma, lymph node (LN) metastases, and bone metastases (BM) at age 75, 2 years prior to blood collection for CTC analysis and begin of cultivation (Fig 1A). Histopathological analysis of biopsies performed for both breast tumors revealed a well-differentiated (G1) invasive lobular carcinoma (ILC) of the left breast and a well-differentiated (G1) invasive ductal carcinoma (IDC) of the right breast (Table EV1). E-cadherin re-staining of the tissue biopsies from both tumors revealed that the tumor described as lobular showed a sub-fraction of E-cadherin+ (Fig EV1A) besides E-cadherin− tumor cells (Fig EV1B). This could point toward a ducto-lobular histopathology. Both the primary right ductal tumor (Fig EV1C) and the vaginal metastasis (Fig EV1D) contained strongly E-cadherin+ tumor cells. Figure 1. CTC cell line establishment from peripheral blood of an mBCa patient A. Scheme of the breast cancer patient's clinical status and therapies. Course of disease progression (blue) and treatment scheme (green) of the patient giving rise to CTC-ITB-01 are indicated. Timeline of progression and treatment indicated in years and months (mo). Drugs were administered at standard dosage according to indicated pattern. The red star represents the time point of blood sample collection. More detailed information is available the in Appendix Supplementary Methods. B. Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45 negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 μm in diameter, 1, 2) and large size (larger than 10 μm in diameter, 3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of more than 4 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (7, 8). C. Bright field images of CTC-ITB-01 cells growing adherently. D. Bright field images of CTC-ITB-01 cells growing non-adherently (relation between adherent and non-adherent cells: 80/20%). Data information: White scale bars represent 10 μm. Black scale bars represent 40 μm. Source data are available online for this figure. Source Data for Figure 1 [emmm201911908-sup-0007-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. E-cadherin re-immunostaining of biopsies taken from the primary tumors and from the vaginal metastasisFormalin-fixed paraffin-embedded (FFPE) tissue of both primary tumors and of the vaginal metastasis was analyzed by immunohistochemistry for E-cadherin protein expression (brown). Nuclear visualization was performed using Mayer's hemalum solution. Figures show exemplary IHC images of E-cadherin staining. The scale bars correspond to 20 μm. A. Area of the primary left tumor (primarily diagnosed as lobular carcinoma) with strongly E-cadherin-positive tumor cells (red arrow). B. Area of the primary lobular left tumor with negative or very weakly E-cadherin-positive tumor cells (red arrow). The black arrow shows strong E-cadherin staining in normal mammary ductal epithelium. C. Strongly E-cadherin-positive tumor cells (red arrow) of the primary ductal (right) tumor. D. Strongly E-cadherin-positive tumor cells (red arrow) of the vaginal metastasis. E-cadherin staining in normal vulvar squamous epithelium is shown by a black arrow. Source data are available online for this figure. Download figure Download PowerPoint Both primary tumors were classified as estrogen receptor-positive (ER+ in ≥ 80% of nuclei), progesterone receptor positive (PR+ in ≥ 80% of nuclei), ERBB2 negative (ERBB2−), and presented with a low Ki67+ cell fraction of 5%. At time of blood collection, additional metastases in the spleen, liver, and vagina had been diagnosed. An overview of the disease progression and treatment scheme of this patient can be found in Fig 1A and in Appendix Supplementary Methods. Establishment of a CTC-derived breast cancer cell line The peripheral blood sample of this patient with metastatic breast cancer (MBC) was screened for CTCs using the CellSearch® System, resulting in a tumor cell count of 1,547 CTCs per ml of blood (total of 7.5 ml). In parallel, blood of the same patient was processed for cell culture. This led to the establishment of a permanent cancer cell line, designated CTC-ITB-01. The original CTCs at blood draw were comprised of single cells of various shapes and diameters (Fig 1B, e.g., panels 1–3), as well as approximately 700 small cell clusters of CTCs (Fig 1B, panels 5–6). CTCs showed negative or very weak (Fig 1B, panels 4 and 8) immunostaining for ERBB2. CTC-ITB-01 has now been successfully cultured for more than 4 years and cells grow in a mixed epithelial–mesenchymal morphology (Fig 1C) as well as in adherent (Fig 1C) and non-adherent fractions (Fig 1D). Genomic characteristics of CTC-ITB-01 cells Remarkably, a considerable number of the CTC cell line cells carried multiple and lobed nuclei, probably due to abnormal cytokinesis (Fig EV2A). Those cells were also detectable in the original CellSearch analysis (Fig 1B, panels 7–8), indicating that this is not an artificial effect originating in cell culture. Giant cancer cells carrying multiple nuclei have recently been associated with metastasis and disease relapse (Mirzayans et al, 2018). Karyotyping of the cell line showed a broad range of chromosomes per cell (32–110), resulting in a mean of 70.7 (s = 17.66) chromosomes (Fig EV2B and C). Click here to expand this figure. Figure EV2. CTC-ITB-01 nuclei and karyotype A. Representative ICC images of large CTC-ITB-01 cells containing multiple or lobed nuclei. Cells were stained with pan-keratin (green) or E-cadherin (orange). Nuclear visualization was performed with DAPI (blue). Gray scale bars represent 20 μm. B. Histogram showing the chromosome distribution across 66 measured CTC-ITB-01 cells. A mean chromosome count of 73.7 (SD = 12.9) was calculated, representing triploidy. C. Representative bright field images of Giemsa staining used for karyotyping (purple). Examples of 32 (upper image) or 75 chromosomes (lower image). Source data are available online for this figure. Download figure Download PowerPoint Whole-genome next-generation sequencing of single cells of the CTC-ITB-01 line

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