title: Fluorescence detection and depletion of T47D breast cancer cells from human mononuclear cell-enriched blood preparations by photodynamic treatment: Basic in vitro experiments towards the removal of circulating tumor cells.
abstract: OBJECTIVE
  A major obstacle for permanent cancer eradication is the persistence of circulating tumor cells (CTCs) in blood, which often escape radio- or chemotherapy. Currently no efficient strategy to remove CTCs from peripheral blood in order to lower the risk of metastases or tumor recurrence exists. Photodynamic treatment (PDT) using aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) as photosensitizer offers an innovative approach to overcome this problem. This study aims at providing basic evidence towards fluorescence detection and photodynamic depletion of scattered cancer cells from blood preparations.
  
  
  METHODS
  The breast cancer cell line T47D, endothelial GP8 cells, red blood cells (RBCs) and peripheral blood mononuclear cells (MNCs) have been tested for ALA-induced formation kinetics of PPIX by flow cytometry and microplate fluorescence analysis. The influence of the presence of RBCs on the PPIX-accumulation in cancer cells was evaluated by flow cytometry; the efficacy of PDT on cancer cells and MNCs has been tested by resazurin assay. Mixtures of T47D and GP8 cells and MNCs spiked with cancer cells were tested to determine the limit of fluorescence detection by flow cytometry and antibody co-staining.
  
  
  RESULTS
  T47D cells accumulated significantly higher PPIX-amounts after ALA-incubation than any other cell type tested. The presence of RBCs had no impact on PPIX-formation in T47D cells. Experiments towards the fluorescence detection of cancer cells in blood revealed that the sensitivity of this method is yet limited. Viability testing after PDT showed that cancer cells where almost completely eradicated after illumination whereas MNCs were almost spared.
  
  
  CONCLUSIONS
  We clearly demonstrate in vitro tumor cell selectivity of PPIX-accumulation over endothelial cells, MNCs and RBCs. Breast cancer cells are efficiently killed by PDT with minor depletion of MNCs. Our findings provide a basis for the PDT of blood samples for a future depletion of CTCs.
authors: Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
authors: Verena G
authors: VG
authors: Ziegler
authors: Julia
authors: J
authors: Knaup
authors: Dorothea
authors: D
authors: Stahl
authors: Barbara
authors: B
authors: Krammer
authors: Kristjan
authors: K
authors: Plaetzer
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy; D000622:Aminolevulinic Acid / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy*; D002460:Cell Line; D045744:Cell Line, Tumor; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000378:metabolism; D009360:Neoplastic Cells, Circulating / Q000187:drug effects* / Q000378:metabolism; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D011524:Protoporphyrins / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D051381:Rats
chemical_list: D017319:Photosensitizing Agents; D011524:Protoporphyrins; D000622:Aminolevulinic Acid; C028025:protoporphyrin IX