title: A co-culture model with brain tumor-specific bioluminescence demonstrates astrocyte-induced drug resistance in glioblastoma.
abstract: BACKGROUND
  Although several studies suggest that stromal fibroblasts mediate treatment resistance in several cancer types, little is known about how tumor-associated astrocytes modulate the treatment response in brain tumors. Since traditionally used metabolic assays do not distinguish metabolic activity between stromal and tumor cells, and since 2-dimensional co-culture system does not recreate the formidable complexity of the microenvironment within 3-dimensional structures such as solid tumor tissue, we instead established a glioblastoma (GBM) cell-specific bioluminescent assay for direct measurements of tumor cell viability in the treatment of clinical relevant drugs.
  
  
  METHODS
  Using lentiviral transfection, we established a panel of human GBM cell lines constitutively expressing a fusion transgene encoding luciferase and the enhanced green fluorescence protein (eGFP). We then initiated co-cultures with immortalized astrocytes, TNC-1, and the eGFP/Luc GBM cell lines. Next, we treated all eGFP/Luc GBM cell lines with Temozolomide (TMZ) or Doxorubicin, comparing co-cultures of glioblastoma (GBM) cells and TNC-1 astrocytes with mono-cultures of eGFP/Luc GBM cells. Cell viability was quantitated by measuring the luciferase expression.
  
  
  RESULTS
  Titration experiments demonstrated that luciferase expression was proportional to the number of eGFP/Luc GBM cells, whereas it was not influenced by the number of TNC-1 cells present. Notably, the presence of TNC-1 astrocytes mediated significantly higher cell survival after TMZ treatment in the U251, C6, A172 cell lines as well as the in vivo propagated primary GBM tumor cell line (P3). Moreover, TNC-1 astrocytes mediated significantly higher survival after Doxorubicin treatment in the U251, and LN18 glioma cell lines.
  
  
  CONCLUSIONS
  Glioma cell-specific bioluminescent assay is a reliable tool for assessment of cell viability in the brain tumor cell compartment following drug treatment. Moreover, we have applied this assay to demonstrate that astrocytes can modulate chemo sensitivity of GBM tumor cells. These effects varied both with the cell line and cytotoxic drug that were used, suggesting that several mechanisms may be involved.
authors.affiliation: Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, China. yangning2103@hotmail.com.|Department of Biomedicine, University of Bergen, Bergen, Norway. yangning2103@hotmail.com.|Brain Science Research Institute, Shandong University, Jinan, China. yangning2103@hotmail.com.
authors.forename: Ning
authors.initials: N
authors.lastname: Yang
authors.affiliation: Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, China. dryantao@126.com.|Department of Biomedicine, University of Bergen, Bergen, Norway. dryantao@126.com.|Brain Science Research Institute, Shandong University, Jinan, China. dryantao@126.com.
authors.forename: Tao
authors.initials: T
authors.lastname: Yan
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. Huaiyang.Zhu@biomed.uib.no.
authors.forename: Huaiyang
authors.initials: H
authors.lastname: Zhu
authors.affiliation: The Gade Laboratory of Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway. Xiao.Liang@k1.uib.no.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liang
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. Lina.Leiss@biomed.uib.no.|Neuro Clinic, Haukeland University Hospital, Bergen, Norway. Lina.Leiss@biomed.uib.no.
authors.forename: Lina
authors.initials: L
authors.lastname: Leiss
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. Per.Sakariassen@biomed.uib.no.
authors.forename: Per Øystein
authors.initials: PØ
authors.lastname: Sakariassen
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. kaioveskaftnesmo@gmail.com.
authors.forename: Kai Ove
authors.initials: KO
authors.lastname: Skaftnesmo
authors.affiliation: Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, China. huangbin_sdu@qq.com.|Brain Science Research Institute, Shandong University, Jinan, China. huangbin_sdu@qq.com.
authors.forename: Bin
authors.initials: B
authors.lastname: Huang
authors.affiliation: The Gade Laboratory of Pathology, Department of Clinical Medicine, University of Bergen, Bergen, Norway. Daniela.Costea@k1.uib.no.
authors.forename: Daniela Elena
authors.initials: DE
authors.lastname: Costea
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. Per.Enger@biomed.uib.no.|Department of Neurosurgery, Haukeland University Hospital, Bergen, Norway. Per.Enger@biomed.uib.no.
authors.forename: Per Øyvind
authors.initials: PØ
authors.lastname: Enger
authors.affiliation: Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, China. Lixg@sdu.edu.cn.|Brain Science Research Institute, Shandong University, Jinan, China. Lixg@sdu.edu.cn.
authors.forename: Xingang
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biomedicine, University of Bergen, Bergen, Norway. jian.wang@biomed.uib.no.
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
mesh_terms: D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D001932:Brain Neoplasms / Q000188:drug therapy / Q000473:pathology*; D002451:Cell Compartmentation; D002452:Cell Count; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D018920:Coculture Techniques / Q000379:methods*; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D005909:Glioblastoma / Q000188:drug therapy / Q000473:pathology*; D006801:Humans; D049449:Luminescence; D008954:Models, Biological; D015203:Reproducibility of Results; D018874:Spheroids, Cellular / Q000473:pathology; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism; D000077204:Temozolomide
chemical_list: D003606:Dacarbazine; D004317:Doxorubicin; D000077204:Temozolomide