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. 2026 Jul 20;481(9):4095–4106. doi: 10.1007/s11010-026-05663-2

Hypoxia and endothelial interaction cooperatively amplify free fatty acid receptor 1 (FFAR1)-mediated malignancy in highly invasive lung cancer cells

Shion Nagano 1, Yuka Kusumoto 1, Moemi Tamura 1, Nanami Shimomura 1, Hinata Amano 1, Mahiro Yanome 1, Nozomi Kano 1, Mizuki Ishida 1, Miwa Kawamoto 1, Toshifumi Tsujiuchi 1,✉
PMCID: PMC13638737  PMID: 42474976

Abstract

Free fatty acid receptor (FFAR)-mediated signaling plays a critical role in cancer pathogenesis. Within the tumor microenvironment (TME), stromal cells support cancer cell functions under hypoxic conditions, thereby promoting malignant progression. This study investigated whether FFAR1 contributes to stromal cell–induced malignancy in cancer cells under hypoxic conditions (1% O2), using highly invasive A549-M8 cells derived from lung cancer A549 cells. A549-M8 cells exhibit approximately eightfold higher invasiveness than A549 cells. Under 1% O2, FFAR1 expression was upregulated in A549-M8 cells but downregulated in A549 cells. A549-M8 cell invasion was markedly promoted by culturing at 1% O2, whereas A549 cell invasion decreased. TUG-770 (FFAR1 agonist) stimulated A549-M8 cell invasion, whereas GW1100 (FFAR1 antagonist) suppressed it. In contrast, A549-M8 cell invasion was inhibited by TUG-891 (FFAR4 agonist). Co-culture with mouse-derived endothelial F2 cells, which lack Ffar1 expression, further stimulated A549-M8 cell movement and motility relative to A549 cells. Under hypoxic co-culture conditions, A549-M8 cells exhibited significantly increased invasion, which was associated with elevated FFAR1 expression. These results suggest that FFAR1-mediated signaling is a key driver of tumor progression in highly invasive lung cancer cells under hypoxic conditions, with stromal cell interactions promoting FFAR1 induction and enhancing malignancy, whereas FFAR4 activation exerts a suppressive effect on invasive activity.

Keywords: FFAR1, Cell invasion, Hypoxia, Lung cancer cells, Endothelial cells

Introduction

G-protein-coupled free fatty acid (FFA) receptors, activated by FFAs, regulate diverse physiological functions including energy metabolism, gastrointestinal hormone secretions, lipid storage, and adipogenesis [1, 2]. FFA receptors (FFARs) are classified by their ligand specificity: FFAR1 (GPR40) and FFAR4 (GPR120) respond to medium- and long-chain fatty acids (C8-C22), whereas FFAR2 (GPR43) and FFAR3 (GPR41) are activated by short-chain fatty acids (C2-C4). Increasing evidence suggests that FFAR-mediated signaling is involved in tumor development and progression [1]. Altered FFAR4 expression has been reported in colon cancer cells compared to adjacent noncancerous regions [3]. Moreover, FFAR-mediated signaling modulates various aspects of cancer cell behavior. In colon cancer cells, FFAR4 activation enhances cell motility and angiogenic activity [3], whereas in pancreatic cancer cells, FFAR1 suppresses and FFAR4 promotes cell motility and invasion [4]. FFAR2 also enhances drug resistance and invasion but inhibits cell motility in osteosarcoma cells [5]. Conversely, previous research in lung cancer cells demonstrated that FFAR1 positively regulates malignant cellular behavior, whereas FFAR4 negatively regulates cancer progression, supporting the involvement of FFAR signaling in lung cancer progression [6]. Furthermore, FFAR4 activation was reported to suppress malignant behavior through regulation of mitochondrial metabolism in lung adenocarcinoma cells, suggesting that the functions of FFAR family members may differ depending on tumor type and cellular context [7]. Based on these findings, FFAR1 and FFAR4 were selected in the present study as candidate signaling molecules potentially involved in the enhanced malignant behavior of highly invasive A549-M8 cells. Previous studies have also indicated that lipid mediator-associated G-protein-coupled receptor signaling is involved in regulating malignant behavior of cancer cells under hypoxic conditions, suggesting potential crosstalk between hypoxia-related responses and lipid-mediated GPCR signaling pathways [8, 9].

Solid tumors consist of both cancer and stromal cells, forming a heterotypic tumor microenvironment (TME) [10, 11]. Irregular vasculature within tumors leads to inadequate oxygen supply, resulting in hypoxia (typically less than 1% O2) and nutrient deprivation. Despite these stresses, cancer cells adapt by activating genes that support cell survival, proliferation, and metastasis [10, 12]. Hypoxic conditions also drive resistance to cancer therapy and promote tumor progression [13, 14]. Additionally, stromal cells, such as fibroblasts, endothelial cells, and immune cells, contribute to the acquisition of malignant traits [15]. In this context, hypoxia and stromal cell interactions further enhance the malignant phenotype of cancer cells within the TME. However, it remains unclear whether FFAR signaling plays a role in this aggravating process.

Given the unclear role of FFAR signaling under hypoxic conditions in the TME, the present study investigated the involvement of FFAR1 in enhancing the malignant properties of cancer cells, particularly during co-culture with mouse-derived endothelial F2 cells under hypoxic conditions (1% O2), using highly invasive A549-M8 cells derived from lung cancer A549 cells. Our objective was to elucidate how interactions between highly invasive lung cancer cells and stromal cells under hypoxic conditions promote malignancy via FFAR1-mediated signaling and to provide new insights into the mechanisms underlying tumor progression.

Materials and methods

Cell culture

A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; FUJIFILM Wako Pure Chemical Corporation. Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) and cultured at 37 °C in a humidified atmosphere containing 5% CO2. In assays involving FFAR agonists and antagonists, 5% charcoal-stripped FBS was used to minimize the effects of endogenous fatty acids and other lipophilic ligands present in serum [4, 5]. For experiments under hypoxic conditions (1% O2), cells were cultured in gas-barrier packs with AnaeroPack Kenki (Sugiyama-Gen Co., Tokyo, Japan). Oxygen levels were monitored using the OXY-1 oxygen meter (JIKCO Co., Tokyo, Japan) [16].

Establishment of highly invasive cells

Highly invasive A549-M8 cells were generated from A549 cells as previously described [17]. Briefly, a Cell Culture Insert (8 μm pore size; BD Falcon, Franklin Lakes, NJ) was coated with Matrigel (12.5 µg/filter; BD Falcon) diluted in serum-free DMEM and allowed to dry. The filter was then placed in 24-well plates containing 10% FBS-DMEM. A549 cells (1 × 105) were suspended in serum-free DMEM and seeded into the filter, followed by incubation for 20 h. Migrated cells were collected using TrypLE™ Express (Thermo Fischer Scientific Inc., Waltham, MA), cultured in a 6-cm dish until reaching 90% confluence, and reseeded onto new Matrigel-coated insert. This selection process was repeated eight times to establish the A549-M8 cell line.

Cell proliferation assay

Cells were seeded at 3000 cells per well into 96-well plates and cultured in 10% FBS-DMEM. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The CCK-8 working solution was added to each well on days 0, 1 or 2, and the absorbance of the conditioned medium was measured at 450 nm. The CCK-8 assay detects metabolically active cells and is frequently used as an indirect indicator of cell growth, particularly in short-term experiments in which viable cell number reflects proliferative capacity. All experiments were independently performed three times [6, 18].

Cell invasion assay

Cell Culture Inserts (8 μm pore size) were coated with Matrigel (12.5 µg/filter) and air-dried for 1 day. The inserts were then placed in 24-well plates (lower chamber) containing 5% charcoal-stripped FBS-DMEM supplemented with either TUG-770 (1 µM) or TUG-891 (1 µM), both from Cayman Chemical Company (Ann Arbor, MI). Cells (1 × 105) were suspended in serum-free DMEM and seeded onto the filters (upper chamber). After incubation for 16 h, non-migrated cells were removed from the upper surface of the filter using cotton swabs. The cells on the lower surface were stained with Giemsa solution. Invaded cells were quantified by counting the number of stained cells in four randomly selected microscopic fields, and the values were compared with those of the control group. Some cells were pretreated with or without GW1100 (1 µM) (Cayman) for 30 min before TUG-770 treatment and the invasion assay. The concentration and treatment conditions used in this assay were selected based on previously established experimental protocols evaluating FFAR1-mediated signaling [6]. All assays were performed in triplicate [17].

Gene expression analysis

Total RNA was extracted using ISOGEN (NIPPON GENE CO., LTD., Toyama, Japan) and reverse-transcribed into cDNA using the PrimeScript RT Master Mix (Perfect Real Time) (TaKaRa Bio Inc., Shiga, Japan). Under hypoxic conditions, cells were cultured in 10% FBS-DMEM at 1% O2 for 16 h. For co-culture experiments, A549 or A549-M8 cells (2 × 105) were seeded in 6-well plates (lower chamber), and F2 cells (2 × 105) were seeded onto Cell Culture Inserts (0.4 μm pore size; BD Falcon) (upper chamber). Both cell types were maintained in 10% FBS-DMEM and incubated for 16 h before extracting RNA from A549 or A549-M8 cells. Quantitative real-time RT-PCR was conducted using TB Green Premix DimerEraser (TaKaRa Bio) with the Smart Cycler II System (TaKaRa Bio). Gene expression levels were normalized to GAPDH as an internal control. Threshold cycle (Ct) values were obtained from amplification curves, and relative mRNA expression was determined after normalization to GAPDH. The specificity of amplification was verified by melt-curve analysis showing a single peak. The primer sets used for quantitative real-time RT-PCR analysis are as follows: human FFAR1 (GenBank accession number; NM_005303) F: 5′- GCTGCTCTGCGTAGGACCCT-3′, R: 5′-ACACTCCAGGCACCCGTGAT-3′; human FFAR4 (GenBank accession number; NM_181745) F: 5′-AGGTGCAGTGGTTCATGCCT-3′, R: 5′-CGGGGGTCTCACCATGTTGG-3′; and human GAPDH (GenBank accession number; BC026907) F: 5′-AGTCCATGCCATCACTGCCA-3′, R: 5′-CAGGTCCACCACTGACACGT-3′.

Gelatin zymography

Cells were plated at 7 × 105 cells per 6-cm dish and maintained in 10% FBS-DMEM. After 8 h, cells were gently washed twice with PBS and cultured in serum-free DMEM with or without TUG-770 (1 µM) or TUG-891 (1 µM) under 21% or 1% O2 for 16 h. Culture supernatants were loaded onto 7.5% acrylamide gel containing 0.1% gelatin. Gels were washed twice in wash buffer (pH 7.5; 2.5% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl2 and 1µM ZnCl2) for 30 min, then incubated in incubation buffer (pH 7.5; 1% Triton X-100, 50 mM Tris-Cl, 5 mM CaCl2, 0.02% NaN3 and 1µM ZnCl2) at 37 °C for 20 h. Gels were stained with 0.5% CBB R250 (FUJIFILM Wako) for 30 min. Band intensities were quantitated using NIH Image software (Bethesda, MD).

Cell motility assay in co-culture with F2 cells

F2 cells were seeded at 5 × 104 cells per well in 24-well plates (lower chamber) and maintained in 5% charcoal-stripped FBS-DMEM for 16 h. Cell Culture Inserts (8 μm pore) were placed in the wells, and A549 or A549-M8 cells were seeded at 1 × 104 cells into the filters (upper chamber) with serum-free DMEM containing TUG-770 (1 µM). After 16 h of incubation, cells that had migrated to the lower surface were stained with Giemsa solution and counted. During co-culture experiments, both cancer cells and F2 cells were exposed to TUG-770 and GW1100 through the shared culture medium.

Wound healing assay

A549 or A549-M8 cells (7 × 105) were seeded into the left well of a Culture-Insert 2 Well in a µ-Dish (35 mm; Ibidi GmbH, Gräfelfing, Germany), while F2 cells were plated into the right well at the same density. After 20 h, the inserts were removed, and cells were incubated in 5% charcoal-stripped FBS-DMEM containing TUG-770 (1 µM) under 21% or 1% O2 for 16 h. Some cells were pretreated with or without GW1100 (1 µM) for 30 min before TUG-770 treatment and the wound healing assay. The wound healing assay under TUG-770-treated conditions was performed to visualize and confirm FFAR1-associated cell movement observed in the quantitative motility assay. Under wound healing co-culture conditions, agonist and antagonist treatments were applied to the shared culture environment containing both cancer cells and F2 cells. The cell-free area was measured using NIH ImageJ software (Bethesda, MD). Assays were performed in triplicate.

Statistical analysis

Statistical analyses were performed using analysis of variance (ANOVA) followed by a post hoc test to assess differences among multiple groups, and comparisons between two groups were evaluated using Student’s t-test. A p-value of less than 0.01 was considered statistically significant. Data are presented as the mean ± SD from at least three independent biological replicates.

Results

Reduced FFAR1 and FFAR4 expression is associated with enhanced invasiveness in A549-M8 cells

A549 and A549-M8 cells exhibited similar morphology, without noticeable differences (Fig. 1A). To establish a baseline for comparison, FFAR1 and FFAR4 expression, cell growth, and invasive activity were first analyzed in A549 and A549-M8 cells. Previous study demonstrated FFAR1 and FFAR4 expression in A549 cells [6], which was also confirmed in the present study (Fig. 1B). A549-M8 cells showed increased cell proliferation compared to A549 cells (Fig. 1C). Representative images of the cell invasion assay are shown in Fig. 1D. The invasiveness of A549-M8 cells was approximately 8-fold higher than that of A549 cells (p < 0.001) (Fig. 1E). FFAR1 and FFAR4 expression levels were reduced in A549-M8 cells relative to A549 cells (Fig. 1F). These findings indicate that A549-M8 cells have enhanced invasive potential, which may be associated with reduced FFAR1 and FFAR4 expression levels.

Fig. 1.

Fig. 1

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Characteristics of highly invasive A549-M8 cells derived from A549 cells. A Morphology of A549 and A549-M8 cells in 10% FBS-DMEM. Scale bars, 100 μm. B FFAR1 and FFAR4 expression pattern in A549 cells by semi-quantitative RT-PCR analysis. C Cell proliferation assay. Data are presented as the percentage of cell number on day 0. *; p < 0.01 vs. A549 cells. D Representative images from the cell invasion assay. Migrated cells were stained using Giemsa staining. Scale bars, 100 μm. Arrow, migrated cell. Arrowhead, pore of the filter. E Cell invasion assay using Matrigel-coated Cell Culture Inserts in 10% FBS-DMEM. Columns indicate the mean of three studies; bars indicate SD. **; p < 0.001 vs. A549 cells. F FFAR1 and FFAR4 expression levels assessed by quantitative real-time RT-PCR analysis. *; p < 0.01 vs. A549 cells

FFAR1 promotes, whereas FFAR4 suppresses, invasion without altering extracellular MMP-2 activity

We next evaluated the impact of FFAR1 and FFAR4 activation on cell invasion and MMP activity in A549-M8 cells.　A549-M8 cell invasion was stimulated by TUG-770 (FFAR1 agonist), whereas it was suppressed by TUG-891 (FFAR4 agonist) (Fig. 2A). Matrix metalloproteinase-2 (MMP-2) activity was markedly higher in A549-M8 cells than in A549 cells, while MMP-9 activity was not detected in either cell type. TUG-770 and TUG-891 had no impact on MMP-2 activity (Fig. 2B, C). Because gelatin zymography evaluates extracellular protease activity in conditioned media, the absence of changes in MMP-2 activity after TUG-770 or TUG-891 treatment does not exclude possible effects of FFAR signaling on MMP-2 expression or regulation. Under the present experimental conditions, however, extracellular MMP-2 activity in A549-M8 cells was not markedly altered by FFAR1 or FFAR4 activation. The present experiments examined acute ligand stimulation; therefore, indirect or long-term effects of FFAR-associated signaling on MMP-2 regulation cannot be excluded. Accordingly, FFAR1-mediated stimulation and FFAR4-mediated suppression of cell invasion in A549-M8 cells likely occur through mechanisms not directly associated with changes in extracellular MMP-2 activity.

Fig. 2.

Fig. 2

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Roles of FFAR1 and FFAR4 in cell invasion and MMP activation in highly invasive A549-M8 cells. A Cell invasion assay using Matrigel-coated Cell Culture Inserts in 5% charcoal-stripped FBS-DMEM with or without TUG-770 (1 µM) or TUG-891 (1 µM) for 16 h. Columns indicate the mean of three studies, bars indicate SD. *; p < 0.01 vs. control. control, TUG-770- or TUG-891-untreated A549-M8 cells. B Gelatin zymography. Cells were incubated in serum-free DMEM with or without TUG-770 (1 µM) or TUG-891 (1 µM) for 16 h, and culture supernatants were subjected to gelatin zymography. C Densitometric analysis of MMP-2 activity. Band intensities were quantitated using NIH Image. *; p < 0.01 vs. TUG-770- or TUG-891-untreated A549 cells. †; p < 0.01 vs. TUG-770- or TUG-891-treated A549 cells

Hypoxia selectively upregulates FFAR1 in A549-M8 cells

To explore the effects of hypoxia, we examined cell behavior and receptor expression under 1% O2. Under 1% O2 conditions, A549-M8 cell growth increased compared to A549 cells (Fig. 3A). Similarly, A549-M8 cells showed markedly higher invasive activity than A549 cells (17.2-fold, p < 0.001) (Fig. 3B). In A549 cells, FFAR1 and FFAR4 expression levels decreased under 1% O2. Conversely, FFAR1 expression was significantly elevated in A549-M8 cells under the same conditions, while FFAR4 expression remained unchanged (Fig. 3C). These observations suggest that FFAR1 is selectively upregulated in highly invasive cells under hypoxia, potentially contributing to their aggressive behavior.

Fig. 3.

Fig. 3

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Cell invasion and FFAR1 and FFAR4 expression in highly invasive A549-M8 cells under 1% O2. A Cell proliferation assay under 1% O2 for 2 days. Data are expressed as the percentage of cell number on day 0. *; p < 0.01 vs. A549 cells. B Cell invasion assay in 10% FBS-DMEM under 1% O2 for 16 h. Columns indicate the mean of three studies; bars indicate SD. **; p < 0.001 vs. A549 cells. C FFAR1 and FFAR4 expression levels determined by quantitative real-time RT-PCR analysis. *; p < 0.01 vs. 21% O2. 21% O2, cells cultured at 21% O2. 1% O2, cells cultured at 1% O2

Hypoxia enhances FFAR1-dependent invasion without directly increasing extracellular MMP-2 activity

Since TUG-770 enhanced A549-M8 cell invasion, we focused on FFAR1-mediated signaling to further analyze its role in invasion and MMP-2 activity under hypoxic conditions. In A549 cells, cell invasion decreased under 1% O2 relative to 21% O2. In contrast, invasion of A549-M8 cells was further enhanced under 1% O2. TUG-770 increased the invasiveness of both A549 and A549-M8 cells under normoxic and hypoxic conditions (Fig. 4A). The enhanced cell invasion of A549-M8 cells by TUG-770 was significantly suppressed by GW1100 (FFAR1 antagonist) (Fig. 4B). GW1100 alone showed little effect on basal invasion activity, whereas pretreatment with GW1100 suppressed the TUG-770-induced increase in invasion, supporting the involvement of FFAR1-mediated signaling in the enhanced invasive phenotype of A549-M8 cells. MMP-2 activity remained markedly higher in A549-M8 cells under both 21% and 1% O2 than in A549 cells. Under 1% O2, MMP-2 activity decreased in A549 cells, whereas A549-M8 cells exhibited an increase in MMP-2 activity. No marked changes in MMP-2 activity were observed following TUG-770 treatment in either A549 or A549-M8 cells under normoxic or hypoxic conditions (Fig. 4C, D). Although the increase in MMP-2 activity in A549-M8 cells under hypoxic conditions was relatively modest, A549-M8 cells simultaneously exhibited enhanced invasive activity under 1% O2, whereas parental A549 cells showed reduced invasion under the same conditions. These findings suggest that hypoxia-dependent changes in MMP-2 activity may be associated, at least in part, with the differential invasive behavior observed between A549 and A549-M8 cells. Therefore, the enhanced invasion of A549-M8 cells under hypoxic conditions may not be directly explained by changes in extracellular MMP-2 activity induced by FFAR1 activation alone. At present, the relationship between FFAR1 upregulation and increased MMP-2 activity under hypoxic conditions should therefore be interpreted as an association rather than direct mechanistic evidence of FFAR1-dependent MMP-2 regulation.

Fig. 4.

Fig. 4

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Effects of hypoxic conditions on cell invasion and MMP activation in highly invasive A549-M8 cells. A Cell invasion assay in 5% charcoal-stripped FBS-DMEM with or without TUG-770 (1 µM) under 21% or 1% O2 for 16 h. Columns indicate the mean of three studies; bars indicate SD. *; p < 0.01 vs. 21% O2. 21% O2, cells cultured at 21% O2. 1% O2, cells cultured at 1% O2. B Effects of GW1100 on A549-M8 cell invasion under 1% O2 for 16 h. Cells were pretreated with or without GW1100 (1 µM) for 30 min before TUG-770 stimulation. *; p < 0.01 vs. control. control, untreted cells. #: p < 0.01 between the indicated groups. C Gelatin zymography. Cells were incubated in serum-free DMEM with or without TUG-770 (1 µM) under 21% or 1% O2 for 16 h, and culture supernatants were subjected to gelatin zymography. D Densitometric analysis of MMP-2 activity. Band intensities were quantitated using NIH Image. *; p < 0.01 vs. TUG-770-untreated A549 cells cultured at 21% O2. †; p < 0.01 vs. TUG-770-treated A549 cells cultured at 21% O2. #: p < 0.01 between the indicated groups

Endothelial cell co-culture increases FFAR1 expression and enhances migration in A549-M8 cells

Since FFAR1 enhanced the cellular response to hypoxic conditions in A549-M8 cells, we next investigated how interaction with endothelial cells influences cancer cell migration and FFAR1-mediated signaling. When A549 cells were cocultured with F2 cells, FFAR1 expression increased 4.2-fold (p < 0.01). In addition, A549-M8 cells cocultured with F2 cells exhibited a pronounced elevation in FFAR1 expression, reaching 16-fold increase (p < 0.001) (Fig. 5A). Ffar1 expression was undetectable in F2 cells by semi-quantitative RT-PCR analysis (Fig. 5B). Therefore, the effects of TUG-770 and GW1100 observed in the co-culture experiments were interpreted primarily in relation to FFAR1-associated signaling in A549 and A549-M8 cells. The motility of A549-M8 cells was markedly enhanced by co-culture with F2 cells compared with A549 cells (6.2-fold, p < 0.001). To evaluate the effect of FFAR1 activation on cell motility, untreated and TUG-770-treated conditions were compared in the motility assay. Under co-culture conditions, TUG-770 increased the motility of A549 and A549-M8 cells by approximately 1.2- and 1.5-fold, respectively (Fig. 5C). In the presence of TUG-770, co-culture with F2 cells elevated the motility of both A549 and A549-M8 cells relative to each cell type cultured alone (Fig. 5D). Because the motility assay in Fig. 5C demonstrated that TUG-770 enhanced cell motility, the wound healing assay shown in Fig. 5E, F was performed under TUG-770-treated conditions to confirm and visualize FFAR1-associated cell movement during co-culture with F2 cells. The cell movement of A549-M8 and F2 cells was faster than that of A549 and F2 cells in the presence of TUG-770 (Fig. 5E, F). These results suggest that interaction with endothelial cells enhances FFAR1-mediated motility, particularly in highly invasive cancer cells.

Fig. 5.

Fig. 5

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Cell movement and motility of highly invasive A549-M8 cells co-cultured with F2 cells. A FFAR1 expression measured by quantitative real-time RT-PCR analysis after co-culture with F2 cells for 16 h. *; p < 0.01 vs. A549 cells. **; p < 0.001 vs. A549 cells. #: p < 0.01 between the indicated groups. ##: p < 0.001 between the indicated groups. A549 + F2, A549 cells co-cultured with F2 cells. A549-M8 + F2, A549-M8 cells co-cultured with F2 cells. B Ffar1 expression in F2 cells by semi-quantitative RT-PCR analysis. C Cell motility assay during co-culture with F2 cells in the presence or absence of TUG-770 for 16 h. *; p < 0.01 vs. TUG-770-untreated A549 + F2. **; p < 0.001 vs. TUG-770-untreated A549 + F2. ††; p < 0.001 vs. TUG-770-treated A549 + F2. #: p < 0.01 between the indicated groups. D Effects of F2 cell co-culture on A549 and A549-M8 cell motility in the presence of TUG-770. *; p < 0.01 vs. A549 or A549-M8. E Wound healing assay of A549 or A549-M8 cells co-cultured with F2 cells in the presence of TUG-770 for 16 h. Scale bar: 200 μm. F Remaining cell-free area in the wound healing assay. Data are presented as the percentage of cell-free area at 0 h. Columns represent the mean of three experiments; bars indicate SD. #: p < 0.01 between the indicated groups

Hypoxia and endothelial co-culture synergistically enhance FFAR1-dependent migration in A549-M8 cells

Finally, we examined how hypoxia and endothelial cell interaction cooperatively affect cell migration. Under hypoxic conditions, FFAR1 expression was elevated in A549 cells co-cultured with F2 cells compared with cells cultured at 21% O2. Moreover, FFAR1 expression was markedly higher in A549-M8 cells co-cultured with F2 cells maintained at 1% O2 (Fig. 6A). In the presence of TUG-770, the motility of A549 and A549-M8 cells co-cultured with F2 cells at 1% O2 increased approximately 3- and 6-fold, respectively, compared to 21% O2 (Fig. 6B). The cell movement was markedly faster in the co-culture of A549-M8 cells and F2 cells than in the co-culture of A549 cells and F2 cells under 1% O2 conditions (Fig. 6C, D). GW1100 inhibited the cell movement of A549-M8 cells in the co-culture setting under 1% O2 (Fig. 6E, F). These results demonstrate that hypoxia and endothelial co-culture synergistically enhance FFAR1-driven migration in highly invasive cancer cells.

Fig. 6.

Fig. 6

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Cell motility and movement of highly invasive A549-M8 cells co-cultured with F2 cells under hypoxia. A FFAR1 expression by quantitative real-time RT-PCR analysis after co-culture under 21% or 1% O2 for 16 h. *; p < 0.01 vs. A549 + F2 or A549-M8 + F2 cultured at 21% O2. #: p < 0.01 between the indicated groups. A549 + F2, A549 cells co-cultured with F2 cells. A549-M8 + F2, A549-M8 cells co-cultured with F2 cells. B Cell motility assay during co-culture with F2 cells in the presence of TUG-770 under 21% or 1% O2 for 16 h. *; p < 0.01 vs. A549 + F2 (21% O2). **; p < 0.001 vs. A549 + F2 (21% O2). ††; p < 0.001 vs. A549 + F2 (1% O2). ##: p < 0.001 between the indicated groups. C Wound healing assay of A549 or A549-M8 cells co-cultured with F2 cells in the presence of TUG-770 under 1% O2 for 16 h. Scale bar: 200 μm. D Remaining cell-free area. Data are expressed as the percentage of the cell-free area at 0 h. Columns represent the mean of three experiments; bars indicate SD. #: p < 0.01 between the indicated groups. E Effects of GW1100 on cell movement of A549-M8 and F2 cells under 1% O2. Cells were pretreated with or without GW1100 (1 µM) for 30 min before TUG-770 stimulation. F Remaining cell-free area in the wound healing assay. #: p < 0.01 between the indicated groups

Discussion

Tumor invasion, a key early step in metastasis, is strongly enhanced by hypoxia [14, 19]. Highly invasive cancer cells serve as a useful model for evaluating the mechanisms underlying tumor progression [17]. In the present study, we investigated whether A549-M8 cells exhibit increased aggressiveness under hypoxic conditions relative to parental A549 cells and examined the involvement of FFAR1-mediated signaling in this process. Consistent with their aggressive phenotype, A549-M8 cells exhibited markedly elevated invasiveness and increased MMP-2 activity relative to A549 cells (Fig. 2B, C). MMP-2 is widely recognized as a key mediator of extracellular matrix degradation and tumor cell invasion [20], and its upregulation is frequently associated with metastatic progression in lung cancer [21]. These results suggest that A549-M8 cells are a suitable model to investigate mechanisms driving invasive behavior and that MMP-2 activation may support the enhanced invasion observed in these cells.

Since FFAR1 and FFAR4 are activated by long- and medium-chain fatty acids [1, 2], we assessed their roles in regulating A549-M8 cell functions. FFAR1 and FFAR4 expression levels were lower in A549-M8 cells than in A549 cells (Fig. 1F). A549-M8 cell invasion was stimulated by the FFAR1 agonist TUG-770 and inhibited by the FFAR4 agonist TUG-891 (Fig. 2A) [22, 23]. These results indicate that FFAR1 positively regulates, whereas FFAR4 negatively regulates, A549-M8 cell invasion. Taken together, reduced FFAR4 expression may contribute to the intrinsically high invasive potential of A549-M8 cells. A previous study demonstrated that A549 cell motility and invasion were enhanced by FFAR1 and suppressed by FFAR4 [6]. In contrast, FFAR1 reduced, whereas FFAR4 promoted, cell motility and invasion in pancreatic cancer cells [4]. These findings suggest that the functional effects of FFAR1 and FFAR4 are cell type-dependent. Although MMP-2 activity is elevated in A549-M8 cells, pharmacological activation of FFAR1 or FFAR4 did not alter MMP-2 activity (Fig. 2B, C), indicating that MMP-2 is not directly regulated by FFAR-mediated signaling in these cells. This suggests that MMP-2 may contribute to the intrinsically high invasive capacity of A549-M8 cells; however, FFAR1-dependent enhancement of invasion occurs through MMP-2-independent mechanisms. Thus, elevated MMP-2 activity may contribute to the intrinsically high invasive capacity of A549-M8 cells, whereas FFAR expression and signaling responsiveness appear to represent additional determinants regulating the invasive phenotype. In our previous study, prolonged incubation with supernatant from FFAR1 knockdown cells reduced MMP-2 activity [6]. Therefore, although acute FFAR1 activation did not markedly alter extracellular MMP-2 activity in the present experiments, indirect or chronic FFAR1-associated regulation of MMP-2 cannot be excluded. The discrepancy between the present and previous findings may reflect differences in experimental design, particularly incubation duration and indirect paracrine effects, rather than direct acute regulation of MMP-2 by FFAR1.

Based on these observations, we focused on FFAR1 to elucidate its contribution to FFAR-mediated signaling and the malignant phenotype of A549-M8 cells. Under 1% O2, A549 cell invasion decreased in the presence of TUG-770, whereas A549-M8 cell invasion increased (Fig. 4A). Furthermore, the elevated invasive activity of A549-M8 cells under 1% O2 was suppressed by the FFAR1 antagonist GW1100 (Fig. 4B) [24]. Because GW1100 was administered before TUG-770 stimulation, the observed suppression likely reflects inhibition of FFAR1-mediated signaling activated under these experimental conditions. FFAR1 expression was downregulated in A549 cells but upregulated in A549-M8 cells under 1% O2 (Fig. 3C), suggesting an association between FFAR1 expression and invasive potential under hypoxic conditions. However, the present findings do not demonstrate direct transcriptional regulation of FFAR1 or FFAR4 by hypoxia itself, and the underlying mechanisms responsible for the altered receptor expression patterns under hypoxic conditions remain to be clarified. Hypoxia regulates gene expression primarily through stabilization of hypoxia-inducible factor (HIF) transcription factors, which bind to hypoxia-responsive elements in target genes and reprogram metabolic and signaling pathways [25]. Consistent with this concept, FFAR family genes were reported to be upregulated under 1% O2 in fibrosarcoma cells [26]. Although hypoxia-mediated regulation of FFAR expression has been described, direct evidence linking FFAR1 induction to enhanced invasive behavior under hypoxic stress has been limited. Our findings therefore provide functional evidence that hypoxia-induced FFAR1 upregulation is associated with acquisition of a more aggressive phenotype.

The differential involvement of MMP-2 under normoxic and hypoxic conditions represents an important mechanistic insight. Hypoxia promotes MMP-2 transcription and activation through HIF-dependent pathways that contribute to remodeling of the TME [19]. In our study, FFAR1 expression and MMP-2 activity increased in A549-M8 cells under 1% O2 but decreased in A549 cells (Fig. 4C, D), indicating that hypoxic regulation of proteolysis is cell context-dependent. Under hypoxia, basal MMP-2 activity was elevated, and invasion became sensitive to FFAR1 modulation, supporting the interpretation that hypoxic conditions establish a proteolytic background in which FFAR1 signaling enhances invasive capacity. Importantly, the present findings do not demonstrate that FFAR1 directly induces MMP-2 expression or activation, because TUG-770 treatment itself did not markedly alter extracellular MMP-2 activity under either normoxic or hypoxic conditions. Instead, these findings suggest that FFAR1-mediated signaling promotes invasive behavior under hypoxia in a cellular environment already associated with elevated MMP-2 activity. Accordingly, the observed relationship between FFAR1 upregulation and increased MMP-2 activity under hypoxic conditions should be interpreted cautiously as a functional association rather than direct evidence of FFAR1-dependent MMP-2 regulation. Collectively, these observations are consistent with the interpretation that A549-M8 cells acquire a hypoxia-adaptive invasive phenotype characterized by coordinated FFAR1 upregulation and increased proteolytic activity. These results suggest that hypoxia may promote malignant progression in cancer cells capable of upregulating and responding to FFAR1-mediated signaling. Consequently, FFAR1 expression under hypoxic conditions may represent a potential indicator of aggressive behavior in invasive lung cancer subpopulations.

The TME of solid tumors comprises cancer cells and various stromal components [10, 11]. Endothelial cells facilitate tumor proliferation, invasion, and metastasis [27], and tumor-associated endothelial cells further support progression through enhanced proliferative and immunosuppressive functions [28]. Because hypoxic conditions within the TME altered FFAR1 expression in A549-M8 cells (Fig. 3C), we further investigated whether stromal cell interactions could modulate FFAR1-associated malignant behavior under microenvironment-like conditions. To assess the role of FFAR1 in endothelial cell-induced cancer cell functions, A549-M8 cells were co-cultured with F2 cells, which lack Ffar1 expression (Fig. 5B). Co-culture with F2 cells markedly enhanced A549-M8 cell motility and movement in the presence of TUG-770 (Fig. 5C–F), accompanied by increased FFAR1 expression (Fig. 5A). These findings indicate that endothelial interaction further elevates FFAR1 expression in cancer cells and augments FFAR1-dependent migration. Because F2 cells are mouse-derived endothelial cells, potential species differences in FFAR1-associated signaling should be considered when interpreting the co-culture experiments. Nevertheless, Ffar1 expression was undetectable in F2 cells under the present experimental conditions, supporting the interpretation that the observed pharmacological responses primarily reflected FFAR1-associated signaling in A549 and A549-M8 cells. The specific endothelial-derived factors responsible for induction of FFAR1 expression were not identified in the present study. Endothelial cells produce various soluble mediators and microenvironment-associated signals that may influence receptor expression and cancer cell behavior. Further studies will be necessary to clarify the molecular mechanisms underlying endothelial cell-associated enhancement of FFAR1 signaling. Endothelial-derived signals therefore appear to prime highly invasive cancer cells for increased responsiveness to FFAR1 activation. We next examined whether hypoxia intensifies these endothelial-mediated effects. Under co-culture conditions at 1% O2, A549-M8 cell motility and movement were more pronounced than in A549 cells (Fig. 6B–D), indicating that endothelial stimuli impose a stronger migratory phenotype than hypoxia alone. Furthermore, GW1100 inhibited A549-M8 cell movement under hypoxic conditions (Fig. 6E, F), demonstrating that FFAR1 is required for this enhanced responsiveness. Although the wound healing assay was performed under a representative experimental condition using TUG-770 treatment, these findings were consistent with the quantitative motility assay results and supported the involvement of FFAR1 signaling in hypoxia- and co-culture-associated cell movement. Collectively, these results support a model in which endothelial signals and hypoxia act cooperatively to amplify FFAR1-mediated pathways (Fig. 6A), thereby reinforcing malignant traits in highly invasive lung cancer cells. The TME thus functions not only as a structural scaffold but also as an active regulator of receptor-dependent signaling programs that drive invasive behavior. In this context, FFAR1 is a key determinant of the heightened sensitivity of A549-M8 cells to endothelial interaction under hypoxic conditions.

In conclusion, cancer metastasis occurs when tumor cells invade surrounding tissue, enter the bloodstream, and colonize distant organs. Among these steps, cancer cell invasion is a critical determinant of metastatic progression [19, 29]. A deeper understanding of the mechanisms modulating invasion is essential for developing effective anti-metastatic therapies. Our findings demonstrate that highly invasive A549-M8 cells exhibit enhanced malignant behavior under hypoxic conditions and during co-culture with F2 cells, relative to parental A549 cells. Collectively, these findings suggest that hypoxia and endothelial cell interaction enhance malignant behavior in A549-M8 cells in association with FFAR1-related signaling, indicating that FFAR1-mediated pathways may represent potential therapeutic targets for limiting tumor progression. However, several limitations should be acknowledged. FFAR1 and FFAR4 expression was primarily assessed at the mRNA level, and protein-level validation together with downstream signaling analyses will be necessary to confirm functional receptor activation. FFAR1 is known to couple with multiple G proteins, including Gq, Gi, and Gs, resulting in activation of several intracellular signaling pathways such as regulation of cAMP signaling and PLC/IP3/DAG-associated signaling cascades [1, 2]. Therefore, multiple downstream signaling mechanisms may contribute to the enhanced invasive and migratory behavior observed in A549-M8 cells under hypoxic and co-culture conditions. The present study did not directly investigate intracellular signaling pathways associated with FFAR1 activation, and further mechanistic analyses will be necessary to clarify how FFAR1-mediated signaling promotes malignant behavior under hypoxic conditions. In the present study, Western blot analysis using several commercially available antibodies did not yield reproducible or specific detection of FFAR1 or FFAR4 proteins, highlighting the technical difficulty of reliable protein-level evaluation of these receptors in the examined cell lines. Therefore, additional approaches such as validated immunodetection methods or receptor localization analyses will be important subjects for future investigation. In addition, the present study was conducted exclusively using in vitro cell culture systems, including hypoxic and endothelial co-culture models, and no in vivo experiments were performed. Although these systems provide mechanistic insight into FFAR1-associated invasion and migration within a tumor microenvironment-like setting, further studies using xenograft or metastasis models will be necessary to determine the physiological relevance and translational significance of FFAR-mediated signaling in lung cancer progression in vivo.

Author contributions

S.N. performed all experiments and analyzed data. Y.K., M.T., N.S., H.A., M.Y., N.K., M.I. and M.K. assisted in cell culture experiments. T.T. designed this study and edited the manuscript. All authors reviewed and approved the manuscript.

Funding

This work was supported by Japan Society for the Promotion of Science (Grant Numbers 21K07109).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Footnotes

Publisher’s Note

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Data Availability Statement

No datasets were generated or analysed during the current study.

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