Downregulation of the neonatal Fc receptor expression in non-small cell lung cancer tissue is associated with a poor prognosis

Lung cancer is the leading cause of cancer-related death worldwide. Although the recommended tumor, node and metastasis (TNM) classification and stage determination are important to select therapeutic options for patients with non-small cell lung carcinoma (NSCLC), additional molecular markers are required to indicate the prognosis, in particular within a specific stage, and help with the management of patients. Because neonatal Fc receptor (FcRn) has recently been involved in colon cancer immunosurveillance, we measured its expression in non-cancerous and NSCLC lung tissues and evaluated its prognostic value in overall survival for patient with NSCLC. FcRn expression was determined at both mRNA and protein levels on cancerous and adjacent non-cancerous tissues from 80 NSCLC patients. In NSCLC, FcRn was mainly found in resident and tumor infiltrating immune cells. The corresponding mRNA and protein were significantly less abundant in lung tumor than non-cancerous tissue. Moreover, analysis of our cohort and datasets from the public data bases show that FCGRT mRNA down-regulation is a robust and independent, unfavorable predictive factor of NSCLC patient survival. We conclude that FCGRT mRNA expression may be a useful additional marker for immunoscoring, reflecting tumor immune system, and help in the decision-making process for NSCLC patients.


Total RNA isolation and cDNA synthesis NSCLC and non tumoral adjacent tissues
Frozen lung tissues were embedded in Tissue-Tek OCT (Sakura Finetek Europe) and 8μm sections were cut on a cryotome (Thermo SCIENTIFIC). Sections (up to 50 mg) were immediately placed in lysis buffer (RLT Plus, Qiagen) containing 143 μM β-mercaptoethanol and vortexed thoroughly for 2 minutes. The lysates were then centrifuged in a microfuge for 1 min at full speed. Aliquots of each supernatant were collected and stored at -80°C. RNA was purified on a QIAsymphony SP workstation using the RNA CT 400 protocol (QIAsymphony RNA kit, Qiagen), including digestion of genomic DNA with DNAse I. Total RNA was eluted RNAse-free water and quantified using a NanoDrop 2000c spectrophotometer (Thermo SCIENTIFIC). RNA integrity was assessed using an Agilent 2100 bioanalyzer. Only samples with an RNA integrity number (RIN) > 6 were considered for RT-qPCR analysis (80 patients matched for tumoral and non tumoral adjacent tissue).
Single-strand cDNA was synthesized from 2μg total RNA from each sample with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to the manufacturer's instructions.

CRC and non tumoral adjacent tissues
All tissue samples were maintained at 180°C (liquid nitrogen) until RNA extraction and were weighed before homogenization. Tissue samples were then disrupted directly into a lysis buffer using Mixer Mill MM 300 (Qiagen). Total RNA was isolated from tissue lysates using the RNeasy Mini Kit (Qiagen), and additionalDNAsedigestion was performed on all samples during the extraction process (RNase-Free DNase Set Protocol for DNase treatment on RNeasy Mini Spin Columns; Qiagen). After each extraction, a small fraction of the total RNA preparation was taken to determine the quality of the sample and the yield of total RNA. Controls analyses were performed by UV spectroscopy and analysis of total RNA profile using the Agilent RNA 6000 Nano LabChip Kit with the Agilent 2100 Bioanalyser (Agilent Technologies) to determine RNA purity, quantity, and integrity.

Quantitative real-time PCR
The FCGRT mRNA expression in lung samples was determined by quantitative real-time PCR in a LightCycler 480 (Roche Diagnostics GmbH). The concentration of FCGRT mRNA was normalized to the geometric mean of mRNAs of two reference genes. TATA-binding protein (TBP) and hypoxanthine phosphoribosyltransferase 1 (HPRT1). This gives more reliable results than using a single reference gene (Vandesompele et al. 2002). Gene-specific primer pairs were designed according published mRNA sequences so that the amplicons generated spanned two exons. The sequences of the primers used in this study are:

Gene
Forward Reverse PCR reactions were carried out using 20ng cDNA as template, 0.2μM each of forward and reverse primer and 1x SYBR Premix Ex Taq (Takara Bio Inc). Each reaction was performed in triplicate. The thermal protocol consisted of an initial denaturation step at 95°C for 30 sec followed by 40 cycles of denaturation at 95°C for 5 sec and primer annealing and extension at 60°C for 20 sec. Melting curves were generated for each amplified cDNA to check the specificity of the reactions. In the standard protocol, fluorescence was read at 60°C during the annealing and extension step. However, we included an additional step (heating at 82°C for 15sec) to record the fluorescence. This increased the specificity of FCGRT measurement by eliminating all non-specific signals. Each PCR run included a no-template control and a calibrator AV090211 (a pool of cDNA from many lung tumors) to evaluate inter-assay variability.
Serial dilutions of the AV090211 calibrator (a pool of 10 cDNA from tumoral and non tumoral samples) (80ng cDNA to 0.625ng cDNA) were used to create a standard curve for each gene. These curves were constructed by plotting the crossing point (Cp) values against the initial quantity of the AV090211 calibrator. The Cp of a sample, defined as the point where the fluorescence curve of the sample was above the background fluorescence, was calculated according to the second derivative maximum method by the LightCycler 480 software. The efficiencies of PCR for the target and reference genes were calculated from the formula E = 10 -1/slope ; they were close to 100% (i.e. efficiency E = 2): FCGRT: E = 1.986; TBP: E = 2.041; HPRT1: E = 1.979. Thus, the software converted Cp data from the samples into a concentration for each gene using the PCR efficiency and the initial amount of AV090211 calibrator. Finally, the results for each sample were normalized by dividing the FCGRT value by the geometric mean of HPRT1 and TBP reference genes.

Western blot
Protein extracts (40μg) of a pool of 10 cancerous tissues and 10 matched non-cancerous tissues from NSCLC patients, chosen blindly and human recombinant FcRn protein were separated on a NUPAGE 4-12% Bis-Tris gel (Life Technologies) and then transferred onto PVDF (polyvinyldenedifluoride) membranes by electroblotting. Membranes were blocked and then incubated with anti-FcRn (Novus Biologicals) or antialpha tubulin as a loading control antibody (Abcam) under the conditions recommended by the manufacturers. After incubation with the appropriate conjugated-HRP secondary antibody, membranes were developed using an enhanced chemiluminescence western blotting detection reagent (Amersham Biosciences). The FcRn human recombinant protein used as a positive control is homologous over its entire region to the amino acid sequence PAKS, from there it is deleted on the entire Ct final region and the original sequence was replaced by a synthetic sequence which corresponds to the sequence factor X, followed by the V5 epitope and a polyhistidine (10). The construction has been obtained by a 2-step cloning 1) a deletion from PAKS (since the beginning of exon 6) then 2) an insertion of the synthetic sequence at BamHI-NotI site.

Immunohistochemical analysis
10μm-thick tissue sections from both NSCLC and non-cancerous tissues were deparaffinized, rehydrated and subjected to heat antigen retrieval in a citrate buffer (pH 6.0). Samples were blocked for endogenous peroxidase activity in 3% hydrogen peroxide-methanol. The VECTASTAIN Elite ABC Kit (Goat IgG,VECTOR Laboratories) was used for immunostaining following the manufacturer's instructions. Tissue sections were incubated with anti-FcRn polyclonal antibody (Novus Biologicals) overnight at 4°C. A standard avidinbiotinimmunoperoxidase method and diaminobenzine as chromogen (DakoCytomation) were used for visualization. Rabbit IgG, whole molecule (Jackson ImmunoResearch), was used as a negative control.