Functional Investigation of Fas Ligand Expressions in Human Non-Small Cell Lung Cancer Cells and Its Clinical Implications  Yidan Lin, MD, PhD, Lunxu Liu, MD, PhD, Ting Zhang, Jin Liu, PhD  The Annals of Thoracic Surgery  Volume 95, Issue 2, Pages 412-418 (February 2013) DOI: 10.1016/j.athoracsur.2012.08.012 Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Expressions of Fas and Fas ligand (FasL) in primary lung cancer cells (PLCCs) (A), cell lines (B), and autologous tumor-infiltrating lymphocytes (TILs) (C). (A) PLCCs were derived from surgical samples of 5 patients suffering pathologically confirmed non-small cell lung cancer (NSCLC). Each of the 5 serial numbers represents 1 single patient. The whole-cell protein lysates were prepared from each of the 5 PLCCs and subjected to Western blot analysis. As a result, Fas and FasL were both identified in all of the 5 PLCCs. Moreover, the expressions of Fas seemed to be in the lower levels than that of FasL in 4 of the 5 samples (Nos.1, 2, 4, and 5). (B) Jurkat cell is a human T cell leukemia cell line and A549, H460, Calu-6 are human NSCLC cell lines, which are all commercially available and stored in our lab. Western blot analysis was adopted to detect the expressions of Fas and FasL in these cell lines. As expected, both Fas and FasL were identified in Jurkat cells. And, as similar as in PLCCs, Fas and FasL were both expressed by all of the 3 NSCLC cell lines and Fas was again in the lower levels than FasL in 2 of the 3 lung cancer cell lines (A549 and Calu-6). (C) Autologous TILs were derived from the same 5 tumor samples. To induce the resting TILs to become activated and specific to PLCCs, the viable TILs were stimulated in a T-cell receptor dependent manner by interleukin (IL)-2, IL-17, and irradiated PLCCs weekly for 3 weeks. And the resting Jurkat cells were stimulated in a T-cell receptor-independent manner by a co-stimulation of PMA and ionomycin for 16 hours. Cell protein lysates were prepared from both resting cells and activated cells. As expected, Western blot analysis identified Fas and FasL expressions in TILs and Jurkat cells. Interestingly, the expressions of Fas were in the lower levels than that of FasL and would be elevated after activations of TILs or Jurkat cells. The Annals of Thoracic Surgery 2013 95, 412-418DOI: (10.1016/j.athoracsur.2012.08.012) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Gene silencing of cancer-expressed FasL attenuates the counterattack against Jurkat Cells. Genetically modified A549 cells were the effector cells and the activated Jurkat cells were the target cells. A549 cells were cultured in 6-well plates and the next day transfected with control (Ctrl) or FasL small interfering RNA. Twenty-four hours after the transfection, these cells were reseeded and labeled and on the second day subjected to the preparation of whole cell lysates for Western blot analysis (A) and to the co-culture with the activated Jurkat cells (B). (A) Western blot analysis proved that cancer-expressed FasL had been effectively downregulated by gene silencing method; β-actin was used as a control. (B) PKH-26 labeled A549-Ctrli or A549-FasLi cells were incubated in triplicate at the indicated E/T ratios with activated Jurkat cells. The percentage of PKH-26 negative/annexin V positive cells (dying target Jurkat cells) was determined by flow cytometry. (bars = SDs; FasL = fas ligand; Points = means of 3 replicate determinations; siRNA = small interfering RNAs.) The Annals of Thoracic Surgery 2013 95, 412-418DOI: (10.1016/j.athoracsur.2012.08.012) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Specific blockade of cancer-expressed Fas ligand (FasL) enhances the cytotoxicity of autologous tumor-infiltrating lymphocytes (TILs). (A) Similar cell densities of the primary lung cancer cells (PLCCs) or TILs were treated with 1.5 μg/mL NOK-1 overnight, then harvested by trypsinization and subjected to detect FasL using immunofluorescent staining measured by a flow cytometry. This figure shows 1 of the 5 samples as a representation. The mean fluorescent intensity (MFI) for immunoglobulin G (IgG) staining in tumor cells was 2.3, for FasL was 24.8 prior to NOK-1 treatment, and was 10.5 after NOK-1 treatment (upper panel of A). The MFI for IgG staining in TILs was 6.9, for FasL was 62.3 prior to NOK-1 treatment, and was 8.9 after NOK-1 treatment (lower panel of A). (B) Cytotoxicity of TILs tested by a classic chromium release assay (E/T = 100:1) when NOK-1 was directly added into the co-culture at a dose up to 1.5 μg/mL. Mild inhibitions of TIL's cytotoxicity were observed in 4 samples (Nos.1, 2, 4, 5), prominent inhibition was noticed in 1 sample (No.3). (C) The cytotoxicities of TILs in all 5 samples were dramatically enhanced when target tumor cells were pretreated with NOK-1 (pretrNOK-1). (Ctrl = control). The Annals of Thoracic Surgery 2013 95, 412-418DOI: (10.1016/j.athoracsur.2012.08.012) Copyright © 2013 The Society of Thoracic Surgeons Terms and Conditions