Prognostic impact of CXCL16 and CXCR6 in non-small cell lung cancer: combined high CXCL16 expression in tumor stroma and cancer cells yields improved survival

Patients surgically resected for stage I-IIIA NSCLC at the University Hospital of North Norway (UNN) and Nordland Hospital (NH) from 1990 through 2005 were included in this study. Of the 371 patients identified from the hospital databases, a total of 36 were excluded due to inadequate paraffin-embedded fixed tissue blocks (n = 13), other malignancy within 5 years prior to NSCLC diagnosis (n = 13), or radio-, or chemotherapy prior to surgery (n = 10). Thus, 335 patients were included in the study, 159 from UNN, and 176 from NH. Adjuvant chemotherapy had not been introduced in Norway during this period (1990–2004). This study includes follow up data as of January 2011. Patients were staged according to the revised 7th edition of UICC TNM classification of lung cancer [2]. The study was approved by The Norwegian Data Inspectorate and The Regional Committee for Medical and Health Research Ethics. Information about the study and subsequent written consent from patients was considered. However, as this was a retrospective study with more than half of patients deceased, with the rest of the patients having to be reminded about the death rate of the disease and the possible raising of unrealistic hope for the individual, The Norwegian Data Inspectorate, and The Regional Committee for Medical and Health Research Ethics waived the need for consent. All patient data were anonymized after collecting the clinicopathological variables for each patient and before doing the statistical analyses.

Two pathologists (S.Al-S and K.Al-S) reviewed the pathological specimens. Two representative areas of cancer cells (neoplastic epithelium) and two from the tumor surrounding stroma were marked on the paraffin donor blocks. Using a 0.6 mm-diameter stylet, one core from each marked area was transferred to a recipient block. Normal lung tissue localized distant from the tumor, in addition to samples from 20 patients without a diagnosis of cancer were used as controls. To include all tissue samples, a total of 9 TMAs were constructed. Multiple 4-μm-sections were cut with a Micron microtome (HM355S) before antibody staining for immunohistochemical analysis. The detailed methodology has been previously reported [29].

CXCR6 (goat polyclonal, ab125115, 1:100), and CXCL16 (rabbit polyclonal, ab101404, 1:100) antibodies from Abcam were used in the study. The antibodies were validated by the manufacturer for immunohistochemistry (IHC) on paraffin-embedded material. In addition, in-house validation by Western blot analysis was performed (Fig. 1). Cut sections were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was done by placing the sections in 0.01 M citrate buffer pH 6.0 before microwave heating for 20 minutes at 450 W. Endogenous peroxidase was blocked by incubation in 3 % H₂0₂ for 10 minutes. Sections were blocked in 5 % goat or rabbit serum for 1 hour before overnight incubation with the primary antibodies at 4 °C. The primary antibodies were visualized by adding a secondary biotin-conjugated antibody followed by an Avidin/Biodin/Peroxidase complex (Vectastain ABC Elite-kit, Vectastain), and substrate (Vector NovaRed, Vectastain). As negative staining controls, the primary antibodies were replaced with the primary antibody diluent. All slides were counterstained with haematoxylin to visualize the nuclei.

Sections were scored semi-quantitatively by two experienced pathologist (S.Al-S and E.R). The dominant staining intensity of stromal and cancer cells was scored as: 0 = negative, 1 = weak, 2 = intermediate, 3 = strong. For stromal CXCL16 and cancer cell CXCR6 density was also scored, in the following manner: 0 = no cells showing positivity, 1 = less than 25 % positivity, 2 = 25-50 % positivity, and 3 = 50-100 % positivity. CXCL16 displayed homogenous staining in cancer cells, while CXCR6 did not show positivity in stromal cells. For some patients, one, or both of the stromal or cancer tissue cores were missing. Favoring a conservative approach, we chose not to extrapolate scores from single cores. Consequently, all reported marker expressions are based on the evaluation of two separate tissue cores.

The specificity of the CXCL16 and CXCR6 antibodies was investigated by Western blotting. Cell lysates were incubated with NuPAGE LDS Sample Buffer (Life Technologies, USA) for 5 minutes at 85 °C, sonicated briefly, and run on a NuPAGE® 4-12 % Bis Tris Gel (cat#NP0322, Life Technologies, USA). Blotting was performed on a Hybond nitrocellulose membrane (cat# RPN2020D, GE Healthcare) using the NuPAGE blotting system (Life Technologies, USA). The membrane was incubated with Odyssey blocking buffer (cat# 927–40000, LI-COR Biosciences, Germany) for 1 hour at room temperature. Primary and secondary antibodies were diluted in the blocking buffer. Anti-CXCL16 antibody (cat# 101404) was used in the dilution of 1:500, anti-CXCR6 in the dilution 1:500 (cat# 125115) and anti-actin (cat#A2066, Sigma) 1:2000. IRDye CW secondary antibodies (cat# 926–32213 and 926–68073, LI-COR, Germany) were used in dilution 1:10000. Molecular weight markers used were SeeBlue Plus 2 (cat# LC5925, Life Technologies, USA), and Magic Mark XP (cat# LC5602, Life Technologies, USA). Images were acquired on the ODYSSEY Sa Infrared Imaging System (LI-COR, Germany).

NCI-H460 cells (ATCC#HTB-177) were grown in RPMI-1640 media (21875–034, Gibco), A549 cells (ATCC# CCL-185) were grown in Ham’s F-12 K (Kaighn’s) media (21127–022, Gibco). Media for all cell lines contained 10 % fetal calf serum (Biochrome, Berlin, Germany), and 1 % of mixed 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma, cat#P0781, Sigma-Aldrich, St. Louis, MO).

Cells were transfected either in 6-well plates (for antibody specificity validation) or in E-plates 16 (Roche, cat# 05469830001) with human CXCL16 Silencer Select siRNA (Ambion, s33807), or CXCR6 Silencer Select siRNA (Ambion, s20963) using Lipofectamine 2000 (Invitrogen, cat# 11668–027, Carlsbad, CA) according to the manufacturer’s recommendations. A scrambled negative control siRNA was included in all experiments (Silencer Negative Control #2 siRNA, Ambion, Austin, TX). BLOCK-iT™ Fluorescent Oligo Reagent (cat# 2013, Invitrogen) was used for transfection efficiency control. Cells were harvested 24 hours after siRNA transfection.

NCI-H460 and A549 cells were trypsinized briefly until detached. Cells were resuspended in complete growth media and counted. Optimal cell number per well (5000) was determined in initial titration experiments. According to xCELLigence (Roche) manufacturer’s instructions, cells were seeded in duplicates into E-plates 16 (Roche, cat# 05469830001) after baseline measurement. Plates were incubated for 1 hour at room temperature, and then placed into the RTCA DP instrument (Roche, cat# 05469759001) located in an incubator preserving same temperature and CO₂ concentration as were used for routine cultivation of the cells. siRNA transfection mix was added to the cells 6 hours after seeding, and left there for 4 hours. After that, transfection mix was replaced with regular growth media. Cell index (arbitrary unit reflecting the cell-sensor impedance) was measured every 15 minutes during the first 4 hours for better resolution at attachment and spreading phase. Further measurements were taken every 30 minutes. Doubling times were calculated with RTCA software 1.2 (Roche). At least three independent experiments were performed.

We used Western blotting to verify the specificity of the CXCL16 and CXCR6 antibodies (Fig. 1) as described. The molecular weight of the detected protein (strongest bands) corresponded well with the predicted weight and data provided by the manufacturers. The weaker extra bands may represent chemically modified or degraded proteins, or products of alternative splicing. siRNAs targeted against CXCL16 and CXCR6 caused marked decrease in the intensity of the bands, compared to scrambled control siRNA. This confirms the specificity of the used antibodies.

The demographic and clinical characteristics of the patients are presented in Table 1. The median patient age was 67 (range 28–85) and the majority were male (76 %). Nearly all patients (96 %) were present or previous smokers. The median follow-up was 105 months (range 73 – 234). There were 191 squamous cell carcinomas (SCC), 113 adenocarcinomas (AC) and 31 large-cell carcinomas (LCC).

The expression of CXCL16 was predominantly cytoplasmic, while CXCR6 exhibited membranous staining (Fig. 2). CXCL16 was expressed in both stromal and cancer cells, whereas CXCR6 was only expressed in cancer cells (table 2). The stromal cells displaying positivity for CXCL16 were fibroblasts, endothelial cells, macrophages, and plasma cells. Of the controls (20 patients without a diagnosis of cancer), 50 % showed varying degrees of positivity for CXCR6 while 100 % showed strong positivity for CXCL16. There were no significant correlations between CXCR6 or CXCL16 and adaptive immunological (CD4, CD8, CD20), innate immunological (CD68, CD56, CD1A), or angiogenic (vascular endothelial growth factors and receptors, platelet derived growth factors, and receptors and fibroblast growth factor-2 and receptor-1) markers (data not shown). For both CXCR6 and CXCL16 the correlations with clinicopathological factors were only weak or not significant (r < 0.2). A correlation was observed between stromal and cancer cell CXCL16 expression (r = 0.368, P < 0.01), however no significant correlation was observed between CXCR6 and CXCL16 in cancer cells.

The prognostic impacts of clinicopathologic variables on DSS are given in Table 1. WHO Performance status (P = 0.016), histology (P = 0.028), differentiation (P < 0.001), surgical procedure (P = 0.007), pathological stage (P < 0.001), tumor stage (P < 0.001), nodal stage (P < 0.001) and vascular infiltration (P <0.001) were significant prognosticators.

The influence of marker expression on survival is presented in Table 2. High CXCL16 expression in stromal cells was significantly associated with an improved DSS (P = 0.016, Fig. 3a), with similar trends observed for each hospital separately (NH, P = 0.045; UNN, P = 0.137). The combination of high stromal and high cancer cell CXCL16 was also significantly associated with an improved DSS (P = 0.016, Fig. 3b). Cancer cell CXCR6 and CXCL16 did not have significant impact on survival in univariate analyses.

Significant clinicopathologic variables were entered into multivariate analysis in two separate models, one with stromal CXCL16 expression variable (model 1) and one with the co-expression variable of cancer and stromal cell CXCL16 (model 2).

Tumor stage, nodal stage, tumor differentiation, performance status, vascular infiltration, and histology were independent prognostic variables in both models (Table 3).

In model 1, high expression of stromal CXCL16 was an independent positive prognostic factor (HR: 0.55; 95 % CI: 0.35 – 0.87, P = 0.011). The combined high expression of CXCL16 in stromal and cancer cells was an independent positive prognostic factor for DSS in model 2 (HR: 0.42; 95 % CI: 0.20 – 0.88, P = 0.022) when compared to the combined low expression.

The increased survival seen in patients with combined high CXCL16 expression in cancer and stromal cells led us to investigate the effect of CXCL16 on cell proliferation. We employed the xCELLigence platform (Roche) which facilitates studying of cell proliferation in real time. This is a micro electric assay based on changing impedance of bottom electrodes in presence of the cells. Attachment and initial spreading of the cells typically took 3–6 hours, after which cells were transfected with siRNAs. We repeatedly observed that knockdown of CXCL16 with siRNA caused activation of proliferation compared to the negative scrambled control (P < 0.001, Fig. 4). This was evident both from the growth curves and from the doubling time calculations. The same effect was observed in two different NSCLC cell lines: A549 and NCI-H460.