Direct visualization of the phenotype of hypoxic tumor cells at single cell resolution in vivo using a new hypoxia probe

ABSTRACT Tumor hypoxia is linked to tumor progression, metastasis, and therapy resistance. However, the underlying mechanisms behind this linkage are not fully understood. Here we present a novel fluorescent mCherry hypoxia-responsive marker that can be used in real time imaging to specifically and sensitively identify hypoxic cells in vivo at single cell resolution. Tumors derived from triple negative tumor cells expressing the hypoxia marker reveal that the hypoxic tumor cells congregate near flowing blood vessels. Using multiphoton microscopy, hypoxic MDA-MB-231 cells were directly visualized and showed a more persistent slow migration phenotype as compared to normoxic cells in the same field in vivo. Hypoxic tumor cells are enriched in the cell population that migrates toward human epithelial growth factor gradients in vivo, and has increased collagen degradation and intravasation activity, characteristics of dissemination and metastasis competent tumor cells. The hypoxia probe introduced in this study provides a specific reporter of hypoxic cell phenotypes in vivo which reveals new insights into the mechanisms by which hypoxia is linked to metastasis.

[1]  Yarong Wang,et al.  Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. , 2015, Cancer discovery.

[2]  Bojana Gligorijevic,et al.  Multiparametric Classification Links Tumor Microenvironments with Tumor Cell Phenotype , 2014, PLoS biology.

[3]  J. Yang,et al.  Invading one step at a time: the role of invadopodia in tumor metastasis , 2014, Oncogene.

[4]  S. McKeown,et al.  Defining normoxia, physoxia and hypoxia in tumours-implications for treatment response. , 2014, The British journal of radiology.

[5]  E. Puré,et al.  Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis. , 2013, Cancer discovery.

[6]  J. Condeelis,et al.  Tks5 and SHIP2 Regulate Invadopodium Maturation, but Not Initiation, in Breast Carcinoma Cells , 2013, Current Biology.

[7]  J. Bussink,et al.  The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response. , 2013, Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology.

[8]  S. Kiriakidis,et al.  Hypoxia-induced invadopodia formation: a role for β-PIX , 2013, Open Biology.

[9]  S. Higashiyama,et al.  Notch increases the shedding of HB-EGF by ADAM12 to potentiate invadopodia formation in hypoxia , 2013, The Journal of cell biology.

[10]  M. Clarke,et al.  Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors , 2013, Intravital.

[11]  Axel R. Pries,et al.  Angiogenesis: An Adaptive Dynamic Biological Patterning Problem , 2013, PLoS Comput. Biol..

[12]  G. Semenza,et al.  Procollagen Lysyl Hydroxylase 2 Is Essential for Hypoxia-Induced Breast Cancer Metastasis , 2013, Molecular Cancer Research.

[13]  J. Bussink,et al.  Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response , 2013, Breast Cancer Research.

[14]  E. Zelzer,et al.  HIF1α is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development , 2012, Development.

[15]  Yarong Wang,et al.  Selective gene-expression profiling of migratory tumor cells in vivo predicts clinical outcome in breast cancer patients , 2012, Breast Cancer Research.

[16]  M. Büchler,et al.  Hypoxia Induces EMT in Low and Highly Aggressive Pancreatic Tumor Cells but Only Cells with Cancer Stem Cell Characteristics Acquire Pronounced Migratory Potential , 2012, PloS one.

[17]  J. Myllyharju,et al.  Hypoxia-inducible Factor-1 (HIF-1) but Not HIF-2 Is Essential for Hypoxic Induction of Collagen Prolyl 4-Hydroxylases in Primary Newborn Mouse Epiphyseal Growth Plate Chondrocytes* , 2012, The Journal of Biological Chemistry.

[18]  G. Semenza,et al.  Hypoxia Regulates CD44 and Its Variant Isoforms through HIF-1α in Triple Negative Breast Cancer , 2012, PloS one.

[19]  R. Heeren,et al.  Localized hypoxia results in spatially heterogeneous metabolic signatures in breast tumor models. , 2012, Neoplasia.

[20]  J. Condeelis,et al.  N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors , 2012, Journal of Cell Science.

[21]  A. Sapino,et al.  HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast , 2012, BMC Cancer.

[22]  G. Semenza,et al.  HIF-1-dependent Expression of Angiopoietin-like 4 and L1CAM Mediates Vascular Metastasis of Hypoxic Breast Cancer Cells to the Lungs , 2011, Oncogene.

[23]  Karl-Erich Jaeger,et al.  Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor , 2012, BMC Biology.

[24]  K. Harper,et al.  Hypoxia-Induced Invadopodia Formation Involves Activation of NHE-1 by the p90 Ribosomal S6 Kinase (p90RSK) , 2011, PloS one.

[25]  R. Moon,et al.  Assessment of Hypoxia Inducible Factor Levels in Cancer Cell Lines upon Hypoxic Induction Using a Novel Reporter Construct , 2011, PloS one.

[26]  C. Yellowley,et al.  Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF‐1α , 2011, Cell biology international.

[27]  Jeffrey Wyckoff,et al.  Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging , 2011, Nature Protocols.

[28]  D. Lauffenburger,et al.  Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer , 2011, Journal of Cell Science.

[29]  P. Folk,et al.  Stress-induced expression of p53 target genes is insensitive to SNW1/SKIP downregulation , 2011, Cellular & Molecular Biology Letters.

[30]  J. Condeelis,et al.  An EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. , 2011, Cancer research.

[31]  J. Bussink,et al.  Metabolic markers in relation to hypoxia; staining patterns and colocalization of pimonidazole, HIF-1α, CAIX, LDH-5, GLUT-1, MCT1 and MCT4 , 2011, BMC Cancer.

[32]  Michihiko Sato,et al.  Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein. , 2010, American journal of physiology. Cell physiology.

[33]  I. Fidler,et al.  AACR centennial series: the biology of cancer metastasis: historical perspective. , 2010, Cancer research.

[34]  R. Huber,et al.  Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters , 2010, Applied and Environmental Microbiology.

[35]  U. Schaible,et al.  Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters , 2010, PloS one.

[36]  S. Lim,et al.  Hypoxic Tumor Cell Modulates Its Microenvironment to Enhance Angiogenic and Metastatic Potential by Secretion of Proteins and Exosomes* , 2010, Molecular & Cellular Proteomics.

[37]  S. Jeffrey,et al.  Circulating tumour cells demonstrate an altered response to hypoxia and an aggressive phenotype , 2010, British Journal of Cancer.

[38]  R. Johnson,et al.  Hypoxia and metastasis in breast cancer. , 2010, Current topics in microbiology and immunology.

[39]  Jeffrey Wyckoff,et al.  Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor. , 2009, Cancer research.

[40]  J. Condeelis,et al.  N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells. , 2009, Cell motility and the cytoskeleton.

[41]  G. Powis,et al.  HAF: The new player in oxygen-independent HIF-1α degradation , 2009 .

[42]  M. Celeste Simon,et al.  The impact of O2 availability on human cancer , 2008, Nature Reviews Cancer.

[43]  C. Ling,et al.  Noninvasive molecular imaging of hypoxia in human xenografts: comparing hypoxia-induced gene expression with endogenous and exogenous hypoxia markers. , 2008, Cancer research.

[44]  P. V. van Diest,et al.  Hypoxic regulation of metastasis via hypoxia-inducible factors. , 2008, Current molecular medicine.

[45]  F. Modugno,et al.  Identification of invasion specific splice variants of the cytoskeletal protein Mena present in mammary tumor cells during invasion in vivo , 2008, Clinical & Experimental Metastasis.

[46]  M. Hiraoka,et al.  The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts. , 2007, Biochemical and biophysical research communications.

[47]  J. Pouysségur,et al.  Hypoxia signalling controls metabolic demand. , 2007, Current opinion in cell biology.

[48]  María C Montoya,et al.  MT1‐MMP proinvasive activity is regulated by a novel Rab8‐dependent exocytic pathway , 2007, The EMBO journal.

[49]  Yarong Wang,et al.  Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. , 2007, Cancer research.

[50]  M. T. Santini,et al.  Three‐dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG‐63 spheroids using two‐photon excitation microscopy , 2007, FEBS letters.

[51]  R. Johnson,et al.  Hypoxia-Inducible Factor-1α Is a Key Regulator of Metastasis in a Transgenic Model of Cancer Initiation and Progression , 2007 .

[52]  Arvind P Pathak,et al.  Characterizing vascular parameters in hypoxic regions: a combined magnetic resonance and optical imaging study of a human prostate cancer model. , 2006, Cancer research.

[53]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[54]  M. Dewhirst,et al.  Observation of incipient tumor angiogenesis that is independent of hypoxia and hypoxia inducible factor-1 activation. , 2005, Cancer research.

[55]  M. Ogura,et al.  Real-time imaging of hypoxia-inducible factor-1 activity in tumor xenografts. , 2005, Journal of radiation research.

[56]  J. Pollard,et al.  A Paracrine Loop between Tumor Cells and Macrophages Is Required for Tumor Cell Migration in Mammary Tumors , 2004, Cancer Research.

[57]  A. Giaccia,et al.  Hypoxic gene expression and metastasis , 2004, Cancer and Metastasis Reviews.

[58]  M. Dewhirst,et al.  Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. , 2004, Cancer cell.

[59]  M. Lacroix,et al.  Relevance of Breast Cancer Cell Lines as Models for Breast Tumours: An Update , 2004, Breast Cancer Research and Treatment.

[60]  F. Gorin,et al.  Perinecrotic glioma proliferation and metabolic profile within an intracerebral tumor xenograft , 2004, Acta Neuropathologica.

[61]  F. Brosius,et al.  Implications of glucose transporter protein type 1 (GLUT1)-haplodeficiency in embryonic stem cells for their survival in response to hypoxic stress. , 2003, The American journal of pathology.

[62]  G. Semenza.  Targeting HIF-1 for cancer therapy , 2003, Nature Reviews Cancer.

[63]  H. Moch,et al.  Chemokine receptor CXCR4 downregulated by von Hippel–Lindau tumour suppressor pVHL , 2003, Nature.

[64]  P. Ratcliffe,et al.  Regulation of angiogenesis by hypoxia: role of the HIF system , 2003, Nature Medicine.

[65]  M. Lerman,et al.  Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 alpha stabilization: a role for phosphatidylinositol 3'-kinase. , 2002, Cancer research.

[66]  C. Kanthou,et al.  Limitations of the reporter green fluorescent protein under simulated tumor conditions. , 2001, Cancer research.

[67]  D. Vordermark,et al.  Green fluorescent protein is a suitable reporter of tumor hypoxia despite an oxygen requirement for chromophore formation. , 2001, Neoplasia.

[68]  J. Segall,et al.  The collection of the motile population of cells from a living tumor. , 2000, Cancer research.

[69]  A. Giaccia,et al.  Development of a hypoxia-responsive vector for tumor-specific gene therapy , 2000, Gene Therapy.

[70]  A. Koong,et al.  Loss of PTEN facilitates HIF-1-mediated gene expression. , 2000, Genes & development.

[71]  F. Ismail-Beigi,et al.  Stimulation of Glucose Transport by Hypoxia: Signals and Mechanisms. , 1999, News in physiological sciences : an international journal of physiology produced jointly by the International Union of Physiological Sciences and the American Physiological Society.

[72]  T. Fojo,et al.  p53 Inhibits Hypoxia-inducible Factor-stimulated Transcription* , 1998, The Journal of Biological Chemistry.

[73]  V. Nehls,et al.  Guided migration as a novel mechanism of capillary network remodeling is regulated by basic fibroblast growth factor , 1998, Histochemistry and Cell Biology.

[74]  B. Reid,et al.  Chromophore formation in green fluorescent protein. , 1997, Biochemistry.

[75]  Rakesh K. Jain,et al.  Interstitial pH and pO2 gradients in solid tumors in vivo: High-resolution measurements reveal a lack of correlation , 1997, Nature Medicine.