title: Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation.
abstract: BACKGROUND
  The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment.
  
  
  METHODS
  4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5 bar, 100% O₂, à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis.
  
  
  RESULTS
  The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors.
  
  
  CONCLUSIONS
  The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway. An anti-angiogenic effect after intermittent HBO was observed, and reflected in the gene expression profile.
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway. ingrid.moen@biomed.uib.no
authors.forename: Ingrid
authors.initials: I
authors.lastname: Moen
authors.forename: Charlotte
authors.initials: C
authors.lastname: Jevne
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.forename: Karl-Henning
authors.initials: KH
authors.lastname: Kalland
authors.forename: Martha
authors.initials: M
authors.lastname: Chekenya
authors.forename: Lars A
authors.initials: LA
authors.lastname: Akslen
authors.forename: Linda
authors.initials: L
authors.lastname: Sleire
authors.forename: Per Ø
authors.initials: PØ
authors.lastname: Enger
authors.forename: Rolf K
authors.initials: RK
authors.lastname: Reed
authors.forename: Anne M
authors.initials: AM
authors.lastname: Øyan
authors.forename: Linda E B
authors.initials: LE
authors.lastname: Stuhr
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005109:Extracellular Matrix / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006931:Hyperbaric Oxygenation; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020782:Matrix Metalloproteinases / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID
chemical_list: D049452:Green Fluorescent Proteins; D020782:Matrix Metalloproteinases