title: Lung cells support osteosarcoma cell migration and survival.
abstract: Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined.
  
  
  
  Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment.
  
  
  
  Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining.
  
  
  
  Lung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.
authors.affiliation: Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Shibing
authors.initials: S
authors.lastname: Yu
authors.affiliation: Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Mitchell Stephen
authors.initials: MS
authors.lastname: Fourman
authors.affiliation: School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Adel
authors.initials: A
authors.lastname: Mahjoub
authors.affiliation: Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Jonathan Brendan
authors.initials: JB
authors.lastname: Mandell
authors.affiliation: Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Jared Anthony
authors.initials: JA
authors.lastname: Crasto
authors.affiliation: Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Nicholas Giuseppe
authors.initials: NG
authors.lastname: Greco
authors.affiliation: Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA, USA. WeisKR@upmc.edu.|Cancer Stem Cell Laboratory, Department of Orthopaedic Surgery, 450 Technology Dr, Pittsburgh, PA, 15219, USA. WeisKR@upmc.edu.
authors.forename: Kurt Richard
authors.initials: KR
authors.lastname: Weiss
mesh_terms: D000080924:Aldehyde Dehydrogenase 1 Family; D000469:Alkaline Phosphatase / Q000235:genetics*; D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D018920:Coculture Techniques; D004221:Disulfiram / Q000008:administration & dosage; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008168:Lung / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009362:Neoplasm Metastasis; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D050697:Retinal Dehydrogenase / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D007527:Isoenzymes; D000080924:Aldehyde Dehydrogenase 1 Family; C510223:ALDH1A1 protein, human; C515028:ALDH1A1 protein, mouse; D050697:Retinal Dehydrogenase; D000469:Alkaline Phosphatase; D004221:Disulfiram
keywords: Aldehyde dehydrogenase; Disulfiram; Lung microenvironment; Metastasis; Osteosarcoma