The present study was carried out in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Kitasato University Medical Ethics Organization (approval no. KME B09-33). The patient agreed to participate in the study and provided written informed consent, as in a previous study by the authors (8).

The KU-Lu-MPPt3 MIP adenocarcinoma cell line was previously established in the authors' laboratory at Kitasato University (8). The KU-Lu-MPPt3 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Nacalai Tesque, Inc.) medium supplemented with 10% fetal bovine serum (FBS) on type I collagen-coated dishes (AGC Techno Glass Co., Ltd.) at 37°C with 5% CO₂. The cells initially exhibited adherent cell morphologies at their establishment. Some cells then formed tufty aggregates and floated away from the adherent cells. The aggregated floating cells were collected and separately cultured from the adherent cells in tissue culture dishes. The KU-Lu-MPPt3 adherent cells were classified as KU-Lu-MPPt3 adhesive (AD) cells, and the aggregated floating cells were classified as KU-Lu-MPPt3 clumpy and suspended (KU-Lu-MPPt3 CS) cells (Fig. 1A). The ratio of KU-Lu-MPPt3 AD to KU-Lu-MPPt3 CS cells was not clear. However, the number of KU-Lu-MPPt3 CS cells was less than that of the KU-Lu-MPPt3 AD cells when some cells formed tufty aggregates and floated away from the adherent cells and became KU-Lu-MPPt3 CS cells. Mycoplasma testing was conducted for these cell lines and confirmed to be negative for mycoplasma infection (data not shown).

Total RNA was isolated from the KU-Lu-MPPt3 AD and CS cells using the RNeasy Mini kit (Qiagen GmbH) according to the manufacturer's protocol. Quality checks analysis on the RNA samples was performed using the Human Clariom D array (cat. no. 902915; Thermo Fisher Scientific, Inc.) gene chip. All results were analyzed using the Transcriptome Analysis Console 4.0.2 software (Thermo Fisher Scientific, Inc.). Gene expression profiles between the KU-Lu-MPPt3 AD and CS cells were compared. The fold change in expression was defined as the ratio of expression in KU-Lu-MPPt3 AD cells to that in KU-Lu-MPPt3 CS cells. Genes with ratios ≥2 were considered to be significantly differentially expressed.

The DAVID 6.8 database (https://david.ncifcrf.gov/tools.jsp) was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The species was limited to Homo sapiens.

The following reagents were purchased commercially: Anti-CD36 D8L9T (14347; Cell Signaling Technology, Inc.), anti-aquaporin 1 (AQP1) B-11 (cat. no. sc-25287; Santa Cruz Biotechnology, Inc.), anti-phosphorylated (p-)Akt (Ser473; cat. no. 9271; Cell Signaling Technology, Inc.), anti-Akt 40D4 (cat. no. 2920; Cell Signaling Technology, Inc.), anti-focal adhesion kinase (FAK) (H-1; cat. no. sc-1688; Santa Cruz Biotechnology, Inc.), anti-p-FAK (Tyr397; cat. no. 44–624G; Thermo Fisher Scientific, Inc.), anti-actin I-19 (cat. no. sc-1616; Santa Cruz Biotechnology, Inc.), Alexa Fluor-phalloidin (cat. no. A12379; Thermo Fisher Scientific, Inc.), Akt inhibitor X (cat. no. sc-203811; Santa Cruz Biotechnology, Inc.) and FAK inhibitor (PF-00562271; AdooQ BioScience).

The KU-Lu-MPPt3 AD cells treated with TrypLE Express Enzyme (Thermo Fisher Scientific, Inc.) and the CS cells were collected, embedded in iPGell (GenoStaff Co., Ltd.), and fixed with 10% formaldehyde neutral buffer solution at 4°C overnight following the manufacturer's instructions. The lung cancer tissues were collected from the patient from whose tissue the KU-Lu-MPPt3 cells were established, and were paraffinized and cut into 4-µm-thick sections. All slides were subjected to autoclaving pretreatment for antigen retrieval using sodium citrate buffer (pH 6.0) and Tris-HCl buffer (pH 9.0). They were then blocked with Dako X0909 Protein Block, Serum-Free (Dako; Agilent Technologies, Inc.) at room temperature for 10 min, washed with PBS three times, and incubated with anti-CD36 (1:200 dilution), anti-AQP1 (1:100 dilution), anti-p-FAK (1:50 dilution) and anti-p-Akt (1:50 dilution) antibodies overnight at 4°C. Subsequently, the cells were washed with PBS three times and incubated with secondary antibodies (MAX-PO MULTI, cat. no. 424151; Nichirei Biosciences Inc.) for 30 min at room temperature. The slides were then washed with PBS three times and stained with stable DAB (Thermo Fisher Scientific, Inc.) at room temperature (anti-CD36, 6 min; anti-AQP1, 10 min; anti-p-FAK, 5 min; anti-p-Akt, 6 min). The slides were washed once again and counterstained with hematoxylin (cat. no. 115938; Sigma-Aldrich, Inc.) for nuclear staining at 4°C for 5 min. The slides were observed using an Olympus BX51 microscope (Olympus Corporation), and images were acquired using Olympus DP2-BSW software.

The KU-Lu-MPPt3 CS cells were cultured on round coverslips coated with 1.8 mg/ml type I collagen (Koken Co., Ltd.) for 9 days, fixed in 10% formaldehyde neutral buffer solution for 10 min, washed with PBS three times, and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min at room temperature. After rinsing with PBS three times, the cells were blocked with 5% BSA at room temperature for 1 h. The cells were then incubated with anti-p-FAK (1:50 dilution) antibody overnight at 4°C. The cells were then washed with PBS three times and incubated with secondary antibodies (cat. no. A11035; Thermo Fisher Scientific, Inc.) (1:200 dilution) and phalloidin (1:500 dilution) for 1 h at room temperature. After washing with PBS once again, the cells were labeled with 4,6-diamidino-2-phenylindole (DAPI) (cat. no. D9542; Sigma-Aldrich, Inc.) for nuclear staining (1:1,000 dilution). A laser scanning confocal imaging system (LSM700; Carl Zeiss AG) was used to acquire fluorescence images. Three dimensional images were reconstituted using IMARIS software (Bitplane AG).

The cells were lysed in RIPA buffer (16488; Nacalai Tesque, Inc.) supplemented with a protease inhibitor cocktail (25955; Nacalai Tesque, Inc.) and phosphatase inhibitor cocktail solution II (FUJIFILM Wako Pure Chemical Corporation), to prepare whole-cell lysates. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 15 µg protein lysate was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% SDS gel (cat. no. 414954; Cosmo Bio Co., Ltd.). The separated proteins were transferred onto a PVDF membrane and the membrane was blocked for 1 h at room temperature using blocking buffer (05999-84; Nacalai Tesque, Inc.), and incubated overnight at 4°C with primary antibodies against p-Akt (1:1,000 dilution), p-FAK (1:1,000 dilution), Akt (1:1,000 dilution), FAK (1:200 dilution) and actin (1:200 dilution). The membrane was then washed with PBST three times and incubated with secondary antibodies (anti-rabbit IgG: cat. no. 1706515; Bio-Rad Laboratories, Inc., anti-mouse IgG: cat. no. 1706516; Bio-Rad Laboratories, Inc., anti-goat: cat. no. A16005; Thermo Fisher Scientific, Inc.) (1:10,000 dilution) for 1 h at room temperature. Proteins were visualized and analyzed using an Image Quant LAS 4000 system with Image Quant TL7.0 analysis software imaging system (Cytiva). All western blot bands were semi-quantified using ImageJ 1.53i software (National Institutes of Health).

The KU-Lu-MPPt3 CS cells were cultured on collagen-coated 96-well plates (AGC Techno Glass Co., Ltd.) with or without inhibitors (Akt inhibitor X and FAK inhibitor PF-00562271) and incubated for 4 days at 37°C. The cells were then incubated with WST-8 (CCK-8; Dojindo Molecular Technologies, Inc.) at a final concentration of 10% (v/v), for 150 min at 37°C. The absorbances of the culture supernatants were measured at 450 nm using a SpectraMax M2 multifunctional microplate reader (Molecular Devices, LLC).

The KU-Lu-MPPt3 CS cells were plated onto collagen-coated 96-well plates with or without inhibitors (Akt inhibitor X and FAK inhibitor PF-00562271) and cultured for 3 days at 37°C in RPMI medium with 10% FBS. In preparation for the measurement of the numbers of attached cells, the wells were washed twice with PBS and filled with fresh medium containing 10% (v/v) WST-8. In preparation for the measurement of the total viable cell numbers, WST-8 was added without washing to the wells at a final concentration of 10% (v/v). Plates were then incubated for 150 min at 37°C, and the absorbances of the culture supernatants were measured at 450 nm using a SpectraMax M2 multifunctional microplate reader. The proportions of adherent cells were defined as the attached cell number/total cell number.

Total RNA was extracted from the KU-Lu-MPPt3 AD and CS cells using Sepasol-RNA I super G (Nacalai Tesque, Inc.) and subjected to reverse transcription to synthesize cDNA using the iScript™ cDNA Synthesis Kit (cat. no. 1708890; Bio-Rad Laboratories, Inc.). The reverse transcription conditions were as follows: 25°C for 5 min, 42°C for 30 min, 85°C for 5 sec, holding at 4°C. Thereafter, qPCR was performed using SYBR-Green (cat. no. 1725271; Bio-Rad Laboratories, Inc.) on a CFX96 Touch Real-time PCR Detection System (Bio-Rad Laboratories, Inc.) and β-actin was used as the internal reference gene. The PCR cycles were performed as follows: Initial denaturation at 95°C for 30 sec and then 40 cycles of 95°C for 10 sec and 60°C for 30 sec. A calibration curve (standard curve) was created and analyzed using the analysis was conducted with the 2^−ΔΔCq quantification method (9). The following primers were used: CD36 forward, 5′-TGGGACCATTGGTGATGAG-3′ and reverse, 5′-GCAACAAACATCACCACACC-3′; AQP1 forward, 5′-TTTCTGTTTCCTGGCCTCAG-3′ and reverse, 5′-TCCACAACTTCAAGGGAGTG-3′; 3-phosphoinositide-dependent protein kinase-1 gene (PDK1) forward, 5′-GAAGATGAGTGACCGAGGAGG-3′ and reverse, 5′-GTAAAGACGTGATATGGGCAATC-3′; Janus kinase 1 gene (Jak1) forward, 5′-GGTCAGCATTAACAAGCAGGACAA-3′ and reverse, 5′-AGCCATCTACCAGGGACACAAAG-3′; TIMP metallopeptidase inhibitor 1 (TIMP1) forward, 5′-TATCCATCCCCTGCAAACTG-3′ and reverse, 5′-TTTTCAGAGCCTTGGAGGAG-3′; Paxillin forward, 5′-ACAGTCGCCAAAGGAGTC-3′ and reverse, 5′-TGGTAGTGCACCTCACAGTA-3′; and β-actin forward, 5′-TCACCCACACTGTGCCCATCTACGA-3′ and reverse, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′.

All values are presented as the mean ± standard error, and all error bars represent the standard error of the mean. Statistical analysis was performed using JMP Pro 16 software (SAS Institute, Inc.). All results were analyzed using the unpaired Student's t-test and the one-way ANOVA followed by Dunnett's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

The KU-Lu-MPPt3 cell line was recently established by the authors as the first established MIP adenocarcinoma cell line, at least to the best of our knowledge (8). Although the cells adhere to and grow on collagen-coated dishes, some cells aggregate and float away from adherent cells during culture (Fig. 1A). To compare the characteristics of the KU-Lu-MPPt3 CS and AD cells, their gene expression profiles were examined, using DNA microarray analysis. In total, 8,054 genes were differentially expressed >2-fold, of which 3,882 and 4,172 genes were upregulated and downregulated, respectively (Table SI), in the KU-Lu-MPPt3 CS cells in comparison with the KU-Lu-MPPt3 AD cells. The most prominently differentially expressed genes are shown in Fig. 1B.

Among these genes, CD36, which encodes a fatty acid receptor (10), and AQP1, which has been reported to encode a water channel protein involved in the progression, invasion and metastasis of lung adenocarcinoma tumors (11), were highly expressed in the KU-Lu-MPPt3 CS cells (Fig. 1C). The CD36 and AQP1 differential expression levels at the protein level were then confirmed using immunohistochemistry. Their expression patterns were also compared with those of histopathological specimens from the patient with MIP adenocarcinoma. It was revealed that the KU-Lu-MPPt3 CS cells were positive for both CD36 and AQP1 proteins; however, the KU-Lu-MPPt3 AD cells were not (Fig. 2E, F, H and I). Several MIP component cells of the alveolar spaces in MIP adenocarcinoma specimens were positive for these proteins (Fig. 2D and G). Therefore, the KU-Lu-MPPt3 CS cells may retain properties similar to those of the characteristic tumor cells discovered in the alveolar spaces of patients with MIP adenocarcinoma. The tumor cells detected in the alveolar spaces have often been discovered in only a section of MIP adenocarcinoma. Fewer KU-Lu-MPPt3 CS than KU-Lu-MPPt3 AD cells existing when certain cells are being separated from adherent cells and transforming into KU-Lu-MPPt3 CS cells is consistent with this clinical feature.

All differentially expressed genes identified in the microarray analysis were subjected to KEGG pathway enrichment analysis. Cell adhesion molecules (CAMs) and the Akt signaling pathway were among the enriched pathways. Among the genes identified using the microarray analysis, genes related to CAMs and the Akt signaling pathway were examined. CD36, PDK1 and Jak1, which were highly expressed in the KU-Lu-MPPt3 CS cells and have been associated with the activation of the Akt pathway (10,12–16), and paxillin and TIMP1, which were highly expressed in the KU-Lu-MPPt3 AD cells and are associated with the activation of the FAK pathway (17–20), were detected (Fig. 1B). Therefore, these genes were confirmed to be differentially expressed between the two cell types using RT-qPCR (Fig. 1C).

It was then examined whether Akt and FAK were differentially activated between the two cell types by performing western blot analysis with phospho-specific antibodies. It was demonstrated that the expression of total FAK was decreased in the KU-Lu-MPPt3 CS cells (Fig. 3A). Apart from this difference in total FAK levels, the p-FAK levels normalized to the total FAK levels were significantly decreased in the KU-Lu-MPPt3 CS cells (Fig. 3B). By contrast, whereas the total Akt levels were almost comparable between the KU-Lu-MPPt3 AD and CS cells (Fig. 3A), the normalized p-Akt levels were significantly increased in the KU-Lu-MPPt3 CS cells (Fig. 3B). Thus, the KU-Lu-MPPt3 CS cells exhibited a decreased and increased activity of FAK and Akt, respectively (Fig. 3A and B).

In the histopathological specimens, p-Akt was detected primarily in the MIP component cells in the alveolar space, being consistent with the results obtained for the KU-Lu-MPPt3 CS cells (Fig. 3C-d and -f). By contrast, p-FAK was detected in all tumor cells in the histopathological specimens, regardless of location (Fig. 3C-e). Since p-FAK was detected in both the KU-Lu-MPPt3 AD and CS cells at comparable levels (Fig. 3C-a and -c), the immunostaining performed herein may have failed to accurately quantify the levels of p-FAK. Furthermore, in addition to CD36, the expression of stem cell markers, such as SOX2, CD44, C-X-C chemokine receptor type 4 (CXCR4) and aldehyde dehydrogenase 1 family, member A1 (ALDH1a) was higher in the KU-Lu-MPPt3 CS cells than in the KU-Lu-MPPt3 AD cells in the microarray analysis, and similar results were obtained using RT-qPCR (unpublished data). The microarray data were uploaded in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database; GEO accession number GSE184883 (https://www.ncbi.nlm.nih.Gov/geo/query/acc.cgi?acc=GSE184883).

The KU-Lu-MPPt3 CS cells slowly proliferated in suspension when cultured in tissue culture dishes; however, when cultured on type I collagen, some cells changed their morphologies, resembling KU-Lu-MPPt3 AD cells within 9 days (Fig. 4A). Subsequently, several cells formed tufty aggregates with rising edges, of which specific cells detached and reverted to the KU-Lu-MPPt3 CS cell morphology (data not shown).

Thus, the Akt and FAK phosphorylation states between the KU-Lu-MPPt3 CS and transformed AD cells (hereafter referred to as glued KU-Lu-MPPt3 CS cells) were compared, following incubation on type I collagen-coated dishes at 37°C for 9 days. Consistent with the cell morphology, the p-Akt and p-FAK levels were decreased and increased, respectively, in the glued KU-Lu-MPPt3 CS cells (Fig. 4B and C). Confocal microscopic analysis revealed that these cells formed lamellipodia-like protrusions with accumulated p-FAK (Fig. 4D). These findings suggest that the adhesiveness of the KU-Lu-MPPt3 cells was associated with their Akt and FAK activities.

It was further confirmed using western blot analysis that Akt and FAK inhibitors respectively decreased the p-Akt and p-FAK levels significantly in the KU-Lu-MPPt3 CS cells (Fig. 5A and B). Of note, it was also demonstrated that the Akt inhibitor decreased the p-FAK levels, whereas the FAK inhibitor increased the p-Akt levels (Fig. 5A and B), suggesting a possible signaling crosstalk between Akt and FAK in the KU-Lu-MPPt3 cells. In order to investigate the involvement of Akt and FAK activities in the proliferation and adhesion of the KU-Lu-MPPt3 CS cells, the effects of Akt and FAK inhibitors on their proliferation and adhesion to type I collagen-coated plates were evaluated using the WST-8 assay. Although the untreated cells proliferated in type I collagen-coated plates, they failed to proliferate in the presence of either Akt or FAK inhibitors, presenting a significant decrease in cell numbers (Fig. 5C). Furthermore, both inhibitors markedly inhibited cell adhesion (Fig. 5D). The formation of lamellipodia-like protrusions with accumulated p-FAK was inhibited by the Akt inhibitor (Fig. 5E). These results suggested that the activities of both Akt and FAK are required for the adhesion and proliferation of KU-Lu-MPPt3 CS cells.