Combined transcriptome and genome analysis of single micrometastatic cells

In human cancer, early systemic spread of tumor cells is recognized as a leading cause of death. Adjuvant therapies are administered to patients after complete resectioning of their primary tumors to eradicate the few residual and latent metastatic cells. These therapeutic regimens, however, are currently designed without direct information about the presence or nature of the latent cells. To address this problem, we developed a PCR-based technique to analyze the transcriptome of individual tumor cells isolated from the bone marrow of cancer patients. From the same cells, genomic aberrations were identified by comparative genomic hybridization. The utility of this approach for understanding the biology of occult disseminated cells and for the identification of new therapeutic targets is demonstrated here by the detection of frequent extracellular matrix metalloproteinase inducer (EMMPRIN; CD147) expression which was verified by immunostaining.

[1]  D. Barnes,et al.  Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents , 1992, Cell.

[2]  J. Haas,et al.  Tolerance to lipopolysaccharide involves mobilization of nuclear factor kappa B with predominance of p50 homodimers. , 1994, The Journal of biological chemistry.

[3]  A. Belyavsky,et al.  PCR-based cDNA library construction: general cDNA libraries at the level of a few cells. , 1989, Nucleic acids research.

[4]  J. Cooper,et al.  A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein , 1998, Nature.

[5]  G. Schlimok,et al.  Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients , 1992, The Lancet.

[6]  Fengzhi Li,et al.  Control of apoptosis and mitotic spindle checkpoint by survivin , 1998, Nature.

[7]  G. Schlimok,et al.  Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[8]  C. Gilles,et al.  Tumor Collagenase Stimulatory Factor (TCSF) Expression and Localization in Human Lung and Breast Cancers , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[9]  S. E. Lee,et al.  Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle , 1997, Molecular and cellular biology.

[10]  T. Dimpfl,et al.  Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. , 2000, The New England journal of medicine.

[11]  Yuri Lazebnik,et al.  Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells , 1997, The EMBO journal.

[12]  P. Rodríguez,et al.  Prothymosin alpha is a chromatin-remodelling protein in mammalian cells. , 1998, The Biochemical journal.

[13]  R. Cote,et al.  Detection and clinical importance of micrometastatic disease. , 1999, Journal of the National Cancer Institute.

[14]  S. Braun,et al.  ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients. , 2001, Cancer research.

[15]  H. Guo,et al.  The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. , 1995, Cancer research.

[16]  J. Davies,et al.  Molecular Biology of the Cell , 1983, Bristol Medico-Chirurgical Journal.

[17]  D. Brutlag,et al.  Addition of homopolymers to the 3'-ends of duplex DNA with terminal transferase. , 1979, Methods in enzymology.

[18]  G Brady,et al.  Construction of cDNA libraries from single cells. , 1993, Methods in enzymology.

[19]  M. Speicher,et al.  Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  S. Narumiya,et al.  Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. , 1999, Science.

[21]  T. Lindahl,et al.  Quality control by DNA repair. , 1999, Science.

[22]  C. Klein.  The biology and analysis of single disseminated tumour cells. , 2000, Trends in cell biology.

[23]  M. Duffy,et al.  The urokinase‐type plasminogen activator system in cancer metastasis: A review , 1997, International journal of cancer.

[24]  P. Steinlein,et al.  T cell activation-associated epitopes of CD147 in regulation of the T cell response, and their definition by antibody affinity and antigen density. , 1999, International immunology.

[25]  David Baltimore,et al.  NF-κB: Ten Years After , 1996, Cell.

[26]  Volker Herzog,et al.  Establishment of a human cell line (mono mac 6) with characteristics of mature monocytes , 1988, International journal of cancer.

[27]  C. Sherr.  Cancer Cell Cycles , 1996, Science.

[28]  W. J. Lucas,et al.  In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Matthew Tudor,et al.  Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation , 2001, Nature Genetics.

[30]  J. Warrington,et al.  A high-density probe array sample preparation method using 10- to 100-fold fewer cells , 1999, Nature Biotechnology.

[31]  R. Hill,et al.  Gene expression in individual cells: analysis using global single cell reverse transcription polymerase chain reaction (GSC RT-PCR). , 1999, Mutation research.

[32]  N. Tapon,et al.  Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton. , 1997, Current opinion in cell biology.

[33]  Robert C. Wolpert,et al.  A Review of the , 1985 .

[34]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[35]  F. Brasseur,et al.  Quantitative evaluation of the expression of MAGE genes in tumors by limiting dilution of cDNA libraries , 1999, International journal of cancer.

[36]  T. Hunter,et al.  A human peptidyl–prolyl isomerase essential for regulation of mitosis , 1996, Nature.

[37]  M. Jackson,et al.  Gene expression profiles of laser-captured adjacent neuronal subtypes , 1999, Nature Medicine.

[38]  G. Riethmüller,et al.  The epithelial cell surface antigen 17–1A, a target for antibody‐mediated tumor therapy: Its biochemical nature, tissue distribution and recognition by different monoclonal antibodies , 1986, International journal of cancer.

[39]  K. Pantel,et al.  Frequency and prognostic significance of isolated tumour cells in bone marrow of patients with non-small-cell lung cancer without overt metastases , 1996, The Lancet.

[40]  A. Montag,et al.  Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: a novel multimarker reverse transcription-polymerase chain reaction for diagnosis of micrometastatic disease. , 2002, Cancer research.