title: Fibroblasts weaken the anti-tumor effect of gefitinib on co-cultured non-small cell lung cancer cells.
abstract: BACKGROUND
  Non-small cell lung cancer (NSCLC) is the most common lung malignancy worldwide. The metastatic potential of NSCLC cells has been shown to be associated with the tumor microenvironment, which consists of tumor cells, stroma, blood vessels, immune infiltrates and the extracellular matrix. Fibroblasts can produce numerous extracellular matrix molecules and growth factors. Gefitinib has been evaluated as a first-line treatment in selected patients, and it has shown favorable efficacy especially in NSCLC, but it is not effective for everyone.
  
  
  METHODS
  In this study, we examined the antitumor activity of gefitinib on lung fibroblasts co-cultured of lung cancer cells. A series of co-culture experiments that employed cell counting kit-8 (CCK8), transwells, real-time polymerase chain reaction (RT-PCR) and Western blotting with HFL-1 fibroblasts and A549 human lung carcinoma cells were performed to learn more about tumor cell proliferation, migration and invasion; and to determine any change of epithelial mesenchymal transition (EMT)-associated tumor markers vimentin, matrix metallopro-teinase 2 (MMP2) and chemotaxis cytokines receptor 4 (CXCR4) mRNA levels.
  
  
  RESULTS
  A549 cell proliferation in the presence of HFL-1 cells was not significantly increased compared with A549 cells alone, but A549 cell spheroid body formation was increased after co-culture, and treatment with gefitinib increased further. Our study also revealed that fibroblasts attenuated the lung cancer cell inhibition ratio of migration and invasion after gefitinib treatment in vitro. To further study this mechanism, RT-PCR analysis showed that vimentin, MMP2 and CXCR4 mRNA levels were more highly expressed in the lung cancer cells after co-culture, but did not obviously decrease compared with the control cells following gefitinib treatment. This suggests the mechanism by which fibroblasts attenuate gefitinib-induced expression of EMT-associated tumor markers. Finally, our results demonstrated that co-culture with A549 lung cancer cells does not alter the cell cycle distribution of HFL-1 fibroblasts. Furthermore, HFL-1 fibroblasts had no effect on the cell cycle distribution of HFL-1 cells treated with gefitinib.
  
  
  CONCLUSIONS
  Gefitinib has lower anti-tumor activity on A549 lung cancer cells when co-cultured with HFL-1 fibroblasts.
authors.affiliation: Department of Pneumology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yong
authors.affiliation: Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Peiqin
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Emergency, Affiliated Hospital of Medical College Qingdao University, Qingdao, Shandong 266000, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Department of Pneumology, Affiliated Hospital of Medical College Qingdao University, Qingdao, Shandong 266000, China.
authors.forename: Wenchen
authors.initials: W
authors.lastname: Yu
authors.affiliation: Department of Pneumology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Shang
authors.affiliation: Department of Pneumology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Han
authors.affiliation: Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China. Email: sky110319@163.com.
authors.forename: Pingping
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Department of Pneumology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. Email: liqressh@hotmail.com.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002289:Carcinoma, Non-Small-Cell Lung; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects*; D000077156:Gefitinib; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D011799:Quinazolines / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D011799:Quinazolines; D000077156:Gefitinib