Female C57BL/6 mice (n=9; age, 6–8 weeks; weight, 18–22 g) were purchased from Anhui Medical University Laboratory Animal Center (Hefei, China) and were maintained in microisolator cages under pathogen-free conditions, at a temperature of 20–26°C under a 12-h light/dark cycle. Food and water was autoclaved before feeding and provided ad libitum. The mice were evenly divided into 3 groups; a PBS group, whole TCL group and TCL RACK1-/CTNNBL1-group. Experimental manipulation of the mice was undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (https://www.ncbi.nlm.nih.gov/books/NBK54050), with the approval of the Scientific Investigation Board of Science and Technology of Anhui Province and the animal experiments of the present study were also approved by the Ethics Committee of Wannan Medical College. The mouse Lewis lung cancer cell line was purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). The cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The present study was approved by the Ethics Committee of Wannan Medical College (Wuhu, China).

To prepare the TCL, cultured Lewis cells were lysed using a freeze-thaw cycle in PBS solution between −70°C and 37°C. After five cycles, the prepared TCL was stored at −70°C until further use. The TCL was observed under a microscope (Olympus Corporation, Tokyo, Japan) using trypan blue staining (Sigma-Aldrich; Merck KGaA) to ensure all of the cells were lysed.

To prepare the lysate, cultured type II alveolar epithelial cells were lysed using a freeze-thaw cycle in PBS solution between −70°C and 37°C. After five cycles, the prepared lysate was stored at −70°C until further use. The lysate was observed under a microscope (Olympus Corporation, Tokyo, Japan) using trypan blue staining (Sigma-Aldrich; Merck KGaA) to ensure all of the cells were lysed.

TCL was prepared from Lewis lung cancer cells or mouse type II alveolar epithelial cell using the above method. Protein concentration of TCL was determined using a bicinchoninic acid (BCA) assay using bovine serum albumin as standard (BCA Assay kit; Beyotime Institute of Biotechnology, Shanghai, China). Subsequently, 100 µg TCL protein was digested with trypsin (Promega Corporation, Madison, WI, USA) overnight at 37°C. Digested samples were labeled using the 8-plex iTRAQ kit (Applied Biosystems; Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. iTRAQ-labeled samples were diluted to 100 µl with 20 mM HCOONH₄, 2M NaOH (pH 10) prior to high-performance liquid chromatography on a Gemini-NX 3u C18 110A (150×2.00 mm) column (Phenomenex, Torrance, CA, USA). Peptides were separated using linear gradient elution; the mobile phases were comprised as follows: Mobile phase A, 20 mM HCOONH₄ and 2M NaOH; mobile phase B, 20 mM HCOONH₄, 2M NaOH and 80% acetonitrile (ACN). The gradient was increased from 5 to 40% mobile phase B in 30 min at a flow rate of 0.2 ml/min. The UV detector was set at 214/280 nm, and fractions were collected every 1 min. In total, 24 fractions were collected and dried by vacuum centrifuge in 12,000 × g overnight at 4°C. Acidified with 50% CF₃COOH, the peptides were separated using linear gradient elution; the mobile phases were comprised as follows: Mobile phase A, 5% ACN and 0.1% formic acid (FA); mobile phase B, 80% ACN and 0.1% FA. The gradient was increased from 5 to 90% mobile phase B in 50 min at a flow rate of 300 nl/min. The tandem mass spectrometry (MS/MS) analysis was performed using a Q Exative system (Thermo Fisher Scientific, Inc.) in Information-Dependent Mode. Briefly, MS spectra were acquired across the mass range of 350–1800 m/z in high resolution mode (>70,000 full width at half maximum) using a 40 msec accumulation time per spectrum. A maximum of 20 precursors per cycle were chosen for fragmentation from each MS spectrum with 60 msec minimum accumulation time for each precursor and dynamic exclusion for 20 sec. Tandem mass spectra were recorded in high sensitivity mode (resolution>17,500) with rolling collision energy on and iTRAQ reagent collision energy adjustment on. For protein identification, the MS/MS spectra were processed using ProteinPilot software version 5.0 (Sciex, Framingham, MA, USA). Differentially abundant proteins were examined using QuickGO (https://www.ebi.ac.uk/QuickGO/) for Gene Ontology annotation and enrichment analysis.

Total RNA was extracted from mouse Lewis lung cancer cell whole TCL and reverse transcribed into cDNA using an RNAprep pure kit (Tiangen Biotech Co. Ltd., Beijing, China) and FastQuant RT kit (with gDNase; Tiangen Biotech Co. Ltd.), respectively. Oligo dT mixed with total RNA was and placed in a warm bath at 70°C for10 min, and then into ice-water mixture. dNTP\RTase\Rnase free H₂O was added to prepare the reverse transcription reaction system. The system was warmed at 42°C for 1 h, and then was heated to 70°C for10 min to produce cDNA. Subsequently, qPCR was performed to detect the mRNA expression levels of RACK1, CTNNBL1, Cullin 3, B-cell lymphoma 2-associated transcription factor 1 (BCLAF1), ribosomal protein S6 (RPS6), superoxide dismutase 1 (SOD1), high mobility group box 1 (HMGB1), inhibitor of growth family member 1 (ING1), ERCC excision repair 3, TFIIH core complex helicase subunit (ERCC3) and GAPDH in mouse Lewis lung cancer cell TCL using a SuperReal PreMix Plus kit with SYBR Green (Tiangen Biotech Co. Ltd.). Thermocycling conditions were 30 sec at 95°C, 2 step PCR at 95°C for 0.5 sec and at 60°C for 30 sec, for 40 cycles. Following dissociation was 1 cycle at 95°C for 15 sec, at 60°C for 30 sec and at 95°C for 15 sec successively. The qPCR data were analyzed with the ABI Step One PCR system (Applied Biosystems; Thermo Fisher Scientific Inc.). Relative gene expression was quantified using the 2^−ΔΔCq (13) and normalized to GAPDH (14). All qPCR primer sequences are presented in Table I.

Protein concentration of the TCL prepared from Lewis lung cancer cells was measured using the BCA protein assay kit. Subsequently, IP was conducted using a Pierce Classic IP kit (cat. no. 26146; Pierce; Thermo Fisher Scientific, Inc.). For 1 mg lysate, 80 µl Control Agarose Resin slurry was added to preclear the lysate. Subsequently, anti-RACK1 (cat. no. 5432; dilution, 1:50; Cell Signaling Technology, Inc., Danvers, MA, USA) was added to the precleared cell lysate in a microcentrifuge tube; the antibody/lysate solution was diluted to 300–600 µl with PBS. The solution was incubated overnight at 4°C to form the immune complex in the spin column. To capture the immune complex, the antibody/lysate sample was added to Protein A/G Plus Agarose in the spin column (supplied in the IP kit), and the column was incubated with gentle end-over-end shaking for 1 h at 4°C. Subsequently, the sample was centrifuged 100 × g for 1 min and the flow-through was saved. The spin column containing the resin was transferred into a new collection tube and 2X reducing sample buffer was added and incubated at 100°C for 5–10 min. The samples were further centrifuged to collect the eluate. Finally, the eluate and flow-through were separated by 10% SDS-PAGE and analyzed by western blotting. First round IP can remove RACK1 from the TCL. To remove CTNNBL1, the flow-through of first round IP was collected and then used to process second round of IP with anti-CTNNBL1 at 5 µg/mg of lysate (cat. no. ab95170; Abcam, Cambridge, MA, USA). Besides antibody, other steps of second round of IP is the same as the first round.

C57/BL6 mice (3 per group) were used for spleen isolation. The spleen was grinded, and depleted of red blood cells using a red blood cell lysis buffer (Tiangen Biotech Co. Ltd.). Then, the spleen cells were centrifuged at 500 × g for 5 min at room temperature, counted and cocultured at a density of 1×10⁶/ml with either TCL prepared from Lewis lung cancer cells (1×10⁶), TCL with RACK1 and CTNNBL1 removed or PBS for 48 h at 37°C. Subsequently, the cells were collected, washed and resuspended (1,000 cells) in PBS supplemented with 1% heat-inactivated FCS. Thereafter, the mouse spleen cells were stained at room temperature for 30 min with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis detection kit (cat. no. KGA107; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) to analyze apoptosis, or with FITC-labeled anti-cluster of differentiation (CD)69 (cat. no. 11–0691-81; eBioscience; Thermo Fisher Scientific, Inc.) to detect activation of spleen cells. The cells were then stored at 4°C for 30 min, washed with 1X PBS and analyzed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) using FlowJo version is 7.6 (FlowJo LLC, Ashland, OR, USA). For flow cytometric detection, lymphocytes and monocytes were gated, and ≤1×10⁶ splenocytes were acquired per test.

TCL was prepared from Lewis cells (1×10⁶) and was separated by 10% SDS-PAGE at 20 µg per lane. The proteins were then transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% evaporated milk for 1 h at room temperature and was then incubated overnight at 4°C with anti-RACK1 (cat. no. 5432; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-CTNNBL1 (cat. no. ab95170; 1:1,000; Abcam, Cambridge, MA, USA), anti-Cullin 3 (cat. no. ab108407; 1:1,000; Abcam) or anti-GAPDH monoclonal antibodies (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA). The membrane was then incubated for 1.5 h with IRDye 800CW goat anti-mouse secondary antibody (cat. no. P/N 925-32211; 1:10,000; LI-COR Biosciences, Lincoln, NE, USA) at room temperature. To visualize the protein on the membrane, the membrane was scanned and analyzed using the Odyssey fluorescent scanning system (LI-COR Biosciences).

Data are presented as the mean ± standard deviation of 3 independent experiments. Statistical significance was determined using χ² test. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS software version 22.0 (IBM Corp., Armonk, NY, USA).

Theoretically, all the proteins expressed in the tumor cells should exist in the TCL prepared with from these cells. To determine protein variety and quantitation in TCL, a TCL was prepared from Lewis lung cancer cells and was analyzed using iTRAQ (Fig. 1). Using the iTRAQ detection method, 4,444 confidence proteins were detected in the TCL (Fig. 1C). A confidence protein is one that can be searched in the currently available protein information databases, with high quantitation and structural integrity. Among these proteins, 768 proteins were upregulated in Lewis lung cancer TCL compared with mouse type II alveolar epithelial cell lysate. The Lewis lung cancer TCL/type II alveolar epithelial cell lysate ratio was >0.67 (Fig. 1B). A total of 769 proteins were downregulated in the Lewis lung cancer TCL compared with the mouse type II alveolar epithelial cell lysate with a ratio of >1.5 (Fig. 1B). These proteins could be classified based on molecular function, cellular component or biological process (Fig. 2), including cell growth, cell division, cell death, cell differentiation, cell proliferation and cell cycle. Among the detected proteins that were differentially expressed between the TCL and the alveolar epithelial cell lysate, 4.56% were associated with cell growth, 7.42% were associated with cell division, 10.16% were associated with cell death, 14.32% were associated with cell differentiation, 8.72% were associated with cell proliferation and 15.23% were associated with cell cycle (Fig. 2). These proteins were selected as the target proteins in the present study, since they serve an important role in cell proliferation and apoptosis.

Using the iTRAQ method, a large number of proteins were detected in the TCL that are correlated with cell proliferation. To determine the proteins that have the most influence on cell proliferation and apoptosis, the functions of these proteins were determined using protein databases and previously published literature, and the ratio of protein expression in Lewis lung cancer cell TCL/type II alveolar epithelial cell lysate was analyzed. Subsequently, the following proteins were selected as targets in the present study: RACK1, CTNNBL1, Cullin 3, BCLAF1, RPS6, SOD1, HMGB1, ING1 and ERCC3. These proteins have been revealed to promote cell proliferation and inhibit apoptosis in numerous protein databases (Panther, http://pantherdb.org/; UniProt, http://www.uniprot.org/; and NCBI Protein, https://www.ncbi.nlm.nih.gov/protein) and in the literature (8,9,15–21). In addition, these proteins exist in ≥2 signaling pathways that are associated with cell proliferation or apoptosis, and the value of type II alveolar epithelial cell lysate/Lewis lung cancer cell TCL calculated by iTRAQ was <0.3 for these proteins. Western blot analysis and RT-qPCR were used to detect the expression levels of these proteins in TCL. The results demonstrated that RACK1, CTNNBL1 and Cullin 3 can be detected by WB, and the expression levels of RACK1 and CTNNBL1 were higher than Cullin 3 in TCL (Fig. 3A). However, BCLAF1, RPS6, SOD1, HMGB1, ING1 and ERCC3 can only be detected by RT-qPCR, which is known to be more sensitive (Fig. 3B). In addition, protein expression was increased in whole TCL compared with the TCL supernatant as determined by western blotting (Fig. 3A). This may be caused by the accumulation of proteins in the lysis cells debris; therefore, the whole TCL was used to investigate its function in subsequent experiments.

As determined by western blot analysis and RT-qPCR, RACK1 and CTNNBL1 were selected as targets to investigate the function of TCL on mouse splenocytes. The present study used a novel protein extraction method based on IP. To remove both proteins, the method was repeated two times. In the first round, a RACK1 antibody was used to remove RACK1 from the TCL. Following IP, western blotting demonstrated that RACK1 was removed from TCL; however, CTNNBL1 was still detected in the flow-through (Fig. 4A). Similarly, in the second round, CTNNBL1 was removed from the processed TCL after the first round using an anti-CTNNBL1 antibody (Fig. 4B). This resulted in the removal of CTNNBL1 from TCL but not GAPDH; the flow-through of the second round of IP did not contain RACK1 or CTNNBL1; however, other proteins, including GAPDH, were still present. Using two rounds of IP, RACK1 and CTNNBL1 were successfully removed from TCL. The subsequent experiments were performed using TCL that was devoid of RACK1 and CTNNBL1.

To investigate the function of RACK1 and CTNNBL1 in splenocyte activation and apoptosis, mouse splenocytes were incubated with TCL lacking RACK1 and CTNNBL1, and were then analyzed by flow cytometry. The results demonstrated that the expression of the early apoptosis marker Annexin V was higher in mouse splenocytes stimulated with whole TCL compared with in the control splenocytes stimulated with PBS; however, the result was not statistically significant (P>0.05; Fig. 5A). In addition, the early activation marker CD69 was significantly upregulated in splenocytes stimulated with whole TCL (P<0.05; Fig. 5B). Conversely, CD69 expression in mouse splenocytes stimulated with TCL lacking RACK1 and CTNNBL1 was downregulated (P<0.05; Fig. 5B), whereas Annexin V expression was upregulated in these cells (P>0.05; Fig. 5A). These results indicated that RACK1 and CTNNBL1 may be the main elements in TCL that affect activation and apoptosis of mouse splenocytes.