title: Quantitative analysis of chemotherapeutic effects in tumors using in vivo staining and correlative histology.
abstract: OBJECTIVE
  To microscopically analyze the chemotherapeutic response of tumors using in vivo staining based on an annexinV-Cy5.5 probe and independently asses their apoptotic count using quantitative histological analysis.
  
  
  METHODS
  Lewis Lung Carcinomas cells, that are sensitive (CS-LLC) and resistant (CR-LLC) to chemotherapy were implanted in nude mice and grown to tumours. Mice were treated with cyclophosphamide and injected with a Cy5.5-annexinV fluorescent probe. In vivo imaging was performed using Fluorescence Molecular Tomography. Subsequently tumours were excised and prepared for histology. The histological tumour sections were stained for apoptosis using a terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. A minimum of ten tissue sections were analyzed per tumour for apoptosis quantification by TUNEL staining and corresponding Cy5.5 distribution.
  
  
  RESULTS
  We detected higher levels of apoptosis and corresponding higher levels of Cy5.5 fluorescence in the CS-LLC vs. the CR-LLC tumours. The cell count rate on CS-LLC sections over CR-LLC was found to be approximately 2 :1 where the corresponding area observed on Cy5.5 distribution measurements revealed a approximately 1.7 :1 ratio of CS-LLC over CR-LLC. These observations are consistent with the higher apoptotic index expected from the CS-LLC cell line.
  
  
  CONCLUSIONS
  Quantitative analysis of histological slices revealed higher fluorescence and higher apoptotic count in the CS-LLC tumour images compared to the CR-LLC tumour images. These observations demonstrate that the annexinV-Cy5.5 probe sensed the chemotherapeutic effect of cyclophospamide and further confirmed in vivo FMT measurements.
authors.affiliation: Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA. hkchoi@mitl.inje.ac.kr
authors.forename: Heung Kook
authors.initials: HK
authors.lastname: Choi
authors.forename: Doreen
authors.initials: D
authors.lastname: Yessayan
authors.forename: Hyun Ju
authors.initials: HJ
authors.lastname: Choi
authors.forename: Eyk
authors.initials: E
authors.lastname: Schellenberger
authors.forename: Alex
authors.initials: A
authors.lastname: Bogdanov
authors.forename: Lee
authors.initials: L
authors.lastname: Josephson
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
authors.forename: Vasilis
authors.initials: V
authors.lastname: Ntziachristos
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000008:administration & dosage / Q000031:analogs & derivatives*; D017302:Annexins; D017209:Apoptosis / Q000187:drug effects; D002232:Carbocyanines / Q000008:administration & dosage*; D018827:Carcinoma, Lewis Lung / Q000188:drug therapy / Q000235:genetics / Q000473:pathology*; D002452:Cell Count; D045744:Cell Line, Tumor; D003520:Cyclophosphamide / Q000008:administration & dosage / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005240:Feasibility Studies; D005260:Female; D005456:Fluorescent Dyes / Q000008:administration & dosage*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D041622:Tomography, Optical / Q000379:methods*
chemical_list: D017304:Annexin A5; D017302:Annexins; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; C506056:Cy5.5-annexin V conjugate; D005456:Fluorescent Dyes; C089187:cyanine dye 3; D003520:Cyclophosphamide