1. IL-6 Secreted from Cancer-Associated Fibroblasts Mediates Chemoresistance in NSCLC by Increasing Epithelial-Mesenchymal Transition Signaling 
Yasushi Shintani, MD, PhD, Ayako Fujiwara, MD, Toru Kimura, MD, PhD, Tomohiro Kawamura, MD, PhD, Soichiro Funaki, MD, PhD, Masato Minami, MD, PhD, Meinoshin Okumura, MD, PhD 
Journal of Thoracic Oncology 
Volume 11, Issue 9, Pages (September 2016)
DOI: /j.jtho
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

2. Figure 1
The transforming growth factor-β (TGF-β)–interleukin-6 (IL-6) axis induces epithelial-to-mesenchymal transition and the acquisition of stemness in NSCLC cells and cancer tissue–originated spheroid (CTOS). A549 and NCI-H358 cells (A–C) or CTOS cells (D–F) that were established as described in Material and Methods were treated with Dulbecco's modified Eagle's medium (control), IL-6 (50 ng/mL), TGF-β1 (2 ng/mL), or IL-6 plus TGF-β1 for 2 days and were then analyzed as follows. (A and D) Phase contrast microscopy. Scale bar = 100 μm for A and 200 μm for D. (B and E) Western blotting for E-cadherin, N-cadherin, vimentin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (C and F). Real-time reverse-transcriptase polymerase chain reaction analysis of CD133 expression. Experiments were performed in triplicate and the results are expressed as means ± SD. In C, lane 3 versus lane 4, p = 0.12; lane 7 versus lane 8, p < In F, lane 3 versus lane 4, p < (G) CTOSs that were treated with TGF-β1 or IL-6 in addition to TGF-β1 for 2 days as already stated were fixed and stained for E-cadherin. Scale bar = 50 μm.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

3. Figure 2
Treatment with cisplatin increases transforming growth factor-β (TGF-β) production by NSCLC cells and cancer tissue–originated spheroid (CTOS). (A) Western blotting analysis of TGF-β and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in A549 cells, NCI-H358 cells, or CTOS that were treated with or without cisplatin (30 μM) for 2 days. CTOS1 and CTOS2 were isolated from different patients with NSCLC. (B) Enzyme-linked immunosorbent assay of the concentration of TGF-β in the conditioned medium (CM) of A549 and NCI-H358 cells that were treated with or without cisplatin (30 μM) for 4 days. The results were normalized to the cell number as described in Material and Methods. Lane 1 versus lane 2 (p < 0.05) and lane 3 versus lane 4 (p < 0.05). (C) Phase contrast microscopy of normal human lung fibroblasts (NFs) that were treated with Roswell Park Memorial Institute (RPMI) medium (control) or CM from A549 or NCI-H358 cells for 2 days. NFs were also treated with the TGF-β inhibitor SB (SB) in addition to CM from A549 cells. Scale bar = 100 μm. (D) Western blotting for α-smooth muscle actin (SMA) and control GAPDH of NFs that were treated with RPMI (control) or with CM from A549 or NCI-H358 cells with or without SB (10 μM) for 2 days. (E) Western blotting for α-SMA and control GAPDH of NFs that were treated with RPMI (control) or with CM from A549 cells treated with or without cisplatin (30 μM). SB (10 μM) was added to the CM as indicated. (F) Real-time reverse-transcriptase polymerase chain reaction analysis of interleukin-6 (IL-6) expression by NFs that were treated with RPMI (control) or with CM from A549 cells treated with or without cisplatin (30 μM). SB (10 μM) was added to the CM as indicated. The results are expressed as means ± SD. Lane 2 versus lane 4 (p < 0.05), lane 2 versus lane 3 (p < 0.05), and lane 4 versus lane 5 (p < 0.05). (G) Enzyme-linked immunosorbent assay of the concentration of IL-6 produced by NFs that were treated with RPMI (control) or with CM from A549 treated with or without cisplatin (30 μM) for 4 days. Subsequently, the NFs were incubated with fresh Dulbecco's modified Eagle's medium for 2 days, after which this CM was analyzed. SB (10 μM) was added to the CM as indicated. The results are expressed as means ± SD. Lane 2 versus lane 3 (p < 0.05) and lane 4 versus lane 5 (p < 0.05).
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

4. Figure 3
Interleukin-6 (IL-6) secreted from cancer-associated fibroblasts (CAFs) plays a role in development of the epithelial-to-mesenchymal transition factor phenotype and chemoresistance of NSCLC cells. (A) Western blotting analysis of smooth muscle actin (SMA) and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of fibroblasts that were isolated from surgically explanted lung tissue. Normal human lung fibroblasts (NFs) and CAFs were isolated from the resected tissues of patients with NSCLC (NF1 and CAF1, NF2 and CAF2, and NF3 and CAF3 were isolated from patients 1, 2 and 3, respectively.) (B) Enzyme-linked immunosorbent assay of the concentration of IL-6 in the conditioned medium (CM) from NF1-3 and CAF1-3 cells that were cultured with Dulbecco's modified Eagle's medium (DMEM) with 1% fetal bovine serum for 4 days. Lane 1 versus lane 2 (p < 0.05). (C and D) Western blot analysis of N-cadherin, E-cadherin, and control GAPDH of A549 cells, NCI-H358 cells, and cancer tissue–originated spheroid (CTOS) that were treated with DMEM (control) or with CM from NFs (NF-CM) or CAFs (CM-CAF) for 2 days. (E and F) Western blot analysis of N-cadherin, E-cadherin, and control GAPDH of A549 and NCI-H358 cells that were treated with DMEM (control) or with CM from NFs (NF-CM) or CAFs (CM-CAF) with or without IL-6 receptor neutralizing antibodies (Abs) (10 μg/mL IL-6 Abs) for 2 days. (G) The proliferation of A549 cells was quantified using a methyl thiazolyl tetrazolium (MTT) assay. The A549 cells were cultured in DMEM (as a control), DMEM including cisplatin (CDDP) (30 μM), CM from CAFs including CDDP (30 μM, CM + CDDP), or CM from CAFs including CDDP (30 μM) plus IL-6R Ab (10 μg/mL, CM + CDDP + IL6Ab) for 3 days (days 0–3 after treatment). A repeated measures analysis of variance was used to compare the results (*p < 0.05).
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

5. Figure 4
Interleukin-6 (IL-6) expression in cancer stroma correlates with epithelial-to-mesenchymal transition factor changes in NSCLC cells. (A) Immunohistochemical staining of the protein expression of E-cadherin, N-cadherin, transforming growth factor-β (TGF-β), and IL-6 in tumors after chemotherapy or chemoradiotherapy. All sections were scored in a semiquantitative manner as described in Materials and Methods. Scale bar = 50 μm. (B) Immunohistochemical staining of smooth muscle actin (SMA) that was used to analyze the distribution of cancer-associated fibroblasts (CAFs) after chemotherapy or chemoradiotherapy. The SMA staining pattern of tumors was classified as subepithelial or stromal as described in Materials and Methods. Scale bar = 50 μm.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions