Increased perfluorooctanoic acid accumulation facilitates the migration and invasion of lung cancer cells via remodeling cell mechanics

Abstract

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are widely used in industrial and household products, raising serious concerns due to their environmental persistence and mobility. Epidemiological studies have reported potential carcinogenic risks of PFAS based on their widespread occurrence and population exposure. In this study, we observed that perfluorooctanoic acid (PFOA), a common PFAS, functions as a mechanical regulator in lung cancer cells. PFOA exposure reduces cell stiffness, thereby decreasing cell adhesion and enhancing immune evasion, ultimately exacerbating tumor metastasis. In various lung cancer models, more aggressive tumor metastases have been observed in the PFOA exposure group. Additionally, serum PFOA levels in patients with advanced lung adenocarcinoma were significantly higher than those in patients with early-stage disease. Mechanistically, the interaction between PFOA and transmembrane integrins in cancer cells triggers changes in cellular mechanical properties, leading to the reorganization of the cytoskeleton, and activation of the intracellular FAK-PI3K-Akt signaling pathway. Our findings demonstrate that in individuals with lung adenocarcinoma, PFOA can increase the risk of cancer metastasis even at daily exposure levels.

Keywords: PFOA accumulation; PI3K-Akt pathway; cell mechanics re-modulation; lung adenocarcinoma.

Fig. 1.

Increased PFAS levels in patients with lung adenocarcinoma. (A) Schematic illustration of detecting PFAS concentration in serum or tumor tissue from patients with lung adenocarcinoma and healthy individuals. (B) Composition profile of each PFAS subtype in the Normal and LA groups. (C) Comparison of concentrations of top ten PFAS subtypes. (Normal group, n = 150; LA group, n =120). (D) Composition profile of top ten PFAS subtype in LA tumor tissues. (E) Comparison of PFOA concentration in patients with different stages (Stage I group, n = 64; Stage II–IV group, n = 56). (F) Comparison of PFOA concentration in patients with different metastatic characteristics. (Nonmetastasis group (N0M0), n = 74; Metastasis group (NxMx), n = 46). LA, lung adenocarcinoma; SPE, solid-phase extraction; PFOA, perfluorooctanoic acid; PFOS, perfluorooctane sulfonates; 6:2Cl-PFESA, 6:2chlorinatedpolyfluorinatedethersulfonates; PFBA, perfluorobutanoic acid; PFHxA, perfluorohexanoic acid; PFHpA, perfluoroheptanoic acid; PFNA, perfluorononanoic acid; PFDA, perfluorodecanoic acid; PFUdA, perfluoroundecanoic acid; PFDoA, perfluorododecanoic acid; PFTrA, perfluorotridecanoic acid; PFTeDA, perfluorotetradecanoic acid; PFHxDA, perfluorohexadecanoic acid; PFODA, perfluorooctadecanoic acid; PFBS, perfluorobutanesulfonic acid; PFHxS, perfluorohexanesulfonic acid; PFHpS, perfluoroheptanesulfonic acid; PFDS, perfluorodecanesulfonic acid; 8:2Cl-PFESA, chlorinatedpolyfluorinatedethersulfonates; 4:2FTS, 4:2 fluorotelomer sulfonic acid; 6:2FTS, 6:2 fluorotelomer sulfonic acid; 8:2FTS, 8:2 fluorotelomer sulfonic acid; 10:2FTS, 10:2 fluorotelomer sulfonic acid; NaDONA, sodium dodecafluoro-3H-4,8-dioxanonanoate; PFPeS, perfluoropentanesulfonic acid; PFNS, perfluorononanesulfonate; PFDoS, perfluorodecane sulfonic acid; PFPeA, perfluoropentanoic acid; N0M0, no lymph node involvement and no distant metastasis; NxMx, lymph node or distant metastasis involvement. Data are presented as bar plots, shown as means ± SEM. Two-tailed unpaired t tests are employed for significance testing.

Fig. 2.

Oral PFOA exposure promotes the metastasis of lung cancer. (A) Schematic illustration of oral PFOA exposure and the orthotopic lung cancer mouse model. (B) Small animal imaging of lung cancer metastasis in the PFOA treatment groups. (C) Representative H&E staining images of intrapulmonary metastases. The left lung is denoted by a dashed gray line. In the lung images, the primary tumors are circled with dashed red lines, while the intrapulmonary nodules are indicated by red arrows in the histological images. Representative images and H&E-stained sections of the chest wall metastatic nodules in the different PFOA-treated groups are also displayed. In these images, the metastatic nodules are marked with a dashed red line, while red arrows indicate the metastases in the H&E images. (D) Enumeration of intrapulmonary nodules in the indicated PFOA-treated groups (n = 3). (E) Enumeration of chest wall metastatic nodules in the indicated PFOA-treated groups (n = 6). (F) Comparison of PFOA concentration in tumor samples (n = 4). (G) Proportion of total PFOA accumulation in the indicated tissues and serum at day 28. The data are presented as violin plots with two-tailed unpaired t tests.

Fig. 3.

PFOA remodels tumor cell stemness and facilitates the migration of lung cancer cells in vitro. A549 cells were exposed to PFOA at 150 μM for three weeks. (A) Representative SEM images of cells with or without PFOA treatment. (B) AFM images of cell morphology and height differences. Cell heights of adherent A549 cells are presented as violin plots comprising the data from 20 cells in the PBS group and 16 cells in the PFOA group. (C) Cell adhesion status is determined by a scattering assay (n = 3). (D) Relative ability of A549 cells to adhere to collagen I and gelatin (n = 4). (E) Representative images from the wound healing assay. (F) Representative transwell assay images, along with a comparison of the cell count from a field of view (n = 5). (G) Bubble plot depicting the enriched pathways in PFOA-treated cells. The data are represented as the mean ± SEM, and the data are analyzed by the two-tailed t test and one-way ANOVA, ***P < 0.001.

Fig. 4.

PFOA induces cytoskeleton remodeling and cell softening of lung cancer cells, leading to tumor escape. (A) Representative immunofluorescence images of cytoskeleton and focal adhesion morphology of A549 cells treated with or without PFOA and jasplakinolide (Jas). In the PFOA group, A549 cells are exposed to PFOA at 150 μM for three weeks. In the Jas group, A549 cells are treated with 50 nM Jas for 12 h. In the PFOA+Jas group, A549 cells are pre-exposed to PFOA at 150 μM for three weeks, followed by 50 nM Jas treatment for 12 h. (B) Relative ability of A549 to adhere to collagen I and gelatin with or without PFOA and Jas treatment. (n = 6) (C) Cell scattering status comparison, with or without PFOA and Jas treatment. (D) Schematic illustration of AFM measurements (Upper panel) and representative images of cells on the AFM stage (Lower panel). (E) Representative force curves from AFM experiments. (F) Elastic modulus of A549 cells presented as violin plots. Each data point is the average of at least 30 force curve measurements of a single cell. At least 20 cells are measured per group. (G) Schematic illustration of the optical tweezer experiment to measure the stiffness of A549 cells. (H) Representative force curves of A549 cells treated with PBS or PFOA from the optical tweezer experiment. (I) Stiffness of A549 cells with or without PFOA treatment, as measured by optical tweezers (at least seven individual cells). (J) Percentage of apoptotic A549 cells after 48 h of coculture with macrophages (n = 3). In the A549 + Macrophage group, A549 is cocultured with macrophage for 48 h. In the PFOA A549 + Macrophage group, A549 cells are exposed to 100 or 150 μM PFOA for three weeks and then cocultured with macrophages for 48 h. During the coculture process, PFOA is removed from the medium. (K) Representative live-cell imaging of A549 cells cocultured with macrophages, from Movie S1. (L) Relative elapsed times for macrophage-triggered A549 death. The data are presented as a violin plot from five experiments from each group. The bar plots are represented as the mean ± SEM, and the data are analyzed by the two-tailed t test and one-way ANOVA.

Fig. 5.

PFOA stimulates the FAK-PI3K-Akt signaling pathway to remodel cell mechanics. (A) GSEA enrichment plots of relevant KEGG pathways from the transcriptome of PFOA-treated A549 cells. (B) Protein expression of AKT/p-AKT, FAK/p-FAK, PTEN/p-PTEN, and PI3K/p-PI3K in A549 cells exposed to 150 μM PFOA for three weeks and in the orthotopic lung cancer model, as determined by western blot analysis. (C) Protein expression in the PI3K-Akt pathway with or without PFOA and LY294002 treatment. Cells are treated with LY294002 at 1 mM for 24 h after 150 μM PFOA exposure for three weeks. (D) Protein expression in the PI3K-Akt pathway with or without PFOA and PF-566271 treatment. Cells are treated with PF-562271 at 10 μM for 24 h after 150 μM PFOA exposure for three weeks. (E) Protein–protein interaction network illustrating the interactions between relevant genes and signaling pathways. (F) Molecular docking simulations of the binding of PFOA to integrins, with the binding energy of PFOA, and core integrins marked in pink. (G) Affinities of PFOA with integrin proteins are determined by microscale thermophoresis.

Fig. 6.

High PFOA levels are associated with the expression of integrin proteins in patients with lung adenocarcinoma. (A) Representative images indicating tumor tissues isolated from patients with lung adenocarcinoma stained with FITC-phalloidin, DAPI, anti-Integrin α2 antibody, anti-Integrin α5 antibody, or anti-Integrin α10 antibody. The carcinoma areas are denoted by dashed white lines. Representative areas of carcinoma and adjacent lung tissues are shown. (Scale bar, 50 μm.) (B–D) The mean fluorescence intensity (MFI) and positive area proportion of integrin α2, α5, and α10 are presented as violin plots with two-tailed unpaired t tests (PFOA-low group, n = 22; PFOA-high group, n = 23). (E–G) Scatter plots indicate the correlation between the concentration of PFOA and MFI of integrin α2, α5, and α10 in lung adenocarcinoma tissues (n = 45). The Spearman test is used to assess the correlation.

Fig. 7.

Schematic diagram of a working model of PFOA stimulation of lung cancer metastasis. PFOA is significantly accumulated in lung adenocarcinoma patients, particularly in those with metastasis. PFOA primarily enters the body through dietary intake. This chemical is then transported systemically and accumulates in the lungs. PFOA directly influences tumor cells, facilitating the metastasis of lung cancer cells by regulating cytoskeletal dynamics and cellular stiffness. The softening of tumor cells contributes to the reduced adhesion to the extracellular matrix (ECM), enhanced immune evasion, and increased stemness, collectively driving tumor metastasis. Integrins on the cell membrane are potential targets of PFOA that activate the downstream FAK-PI3K-Akt signaling pathway to promote lung cancer development and metastasis. (Created in BioRender. BioRender.com.)