Perfluorooctanesulfonic Acid and Perfluorooctanoic Acid Promote Migration of Three-Dimensional Colorectal Cancer Spheroids

Abstract

Perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are persistent environmental contaminants that are of increasing public concern worldwide. However, their relationship with colorectal cancer (CRC) is poorly understood. This study aims to comprehensively investigate the effect of PFOS and PFOA on the development and progression of CRC in vitro using a series of biological techniques and metabolic profiling. Herein, the migration of three-dimensional (3D) spheroids of two CRC cell lines, SW48 KRAS wide-type (WT) and SW48 KRAS G12A, were observed after exposure to PFOS and PFOA at 2 μM and 10 μM for 7 days. The time and dose-dependent migration phenotype induced by 10 μM PFOS and PFOA was further confirmed by wound healing and trans-well migration assays. To investigate the mechanism of action, derivatization-mass spectrometry-based metabolic profiles were examined from 3D spheroids of SW48 cell lines exposed to PFOS and PFOA (2 μM and 10 μM). Our findings revealed this exposure altered epithelial-mesenchymal transition related metabolic pathways, including fatty acid β-oxidation and synthesis of proteins, nucleotides, and lipids. Furthermore, this phenotype was confirmed by the downregulation of E-cadherin and upregulation of N-cadherin and vimentin. These findings show novel insight into the relationship between PFOS, PFOA, and CRC.

Graphical Abstract

INTRODUCTION

Per- and polyfluoroalkyl substances (PFAS) are a group of thousands of heterogeneous synthetic chemicals with a common carbon–fluorine bond in their structure, which determines their hydrophobicity, oleophobicity, resistance to degradation, and thermal stability.¹ These so-called “forever chemicals” have been broadly used in the manufacturing of industrial and household products, and some have been authorized by the Food and Drug Administration (FDA) for limited use in food contact applications.² In addition, individuals that have high occupational exposure to PFAS such as firefighters have a higher risk of cancer, including colon cancer.³ Since the late 1990s, PFAS that contain eight or more carbon atoms in length have raised increasing concerns about their safety on human and animal health.^4,5 Examples of these chemicals include perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). PFAS have been frequently detected in the environment (e.g., drinking water, indoor dust, cleaning products, coatings) and the human body⁶ and have been associated with the development of adverse health effects and diseases including cancer.⁷

Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer death in the world.⁸ It is widely known that sporadic CRC frequently arises upon activation of oncogenes such as KRAS (Kirsten rat sarcoma viral oncogene), whose constitutive activation causes aberrant cell growth and promotes cancer metastasis.⁹ The most frequent mutations in the KRAS gene are represented by point substitution in codons 12 and 13, which occur in approximately 35–45% of CRC.¹⁰ Beyond its genetic etiology, CRC has been widely associated with environmental risk factors including, but not limited to, diet, alcohol consumption, tobacco smoking, obesity, and interaction with pathogens.¹¹ The relationship between PFAS exposure and CRC has been investigated only by a few studies. Consequently, there is currently a lack of knowledge about the underlying biological mechanisms of how exposure to PFAS can affect CRC development and progression.

In this regard, metabolomics is gaining interest in PFAS research to identify metabolites of PFAS exposure or biological effect,¹² which could be linked to mechanisms underlying CRC development or progression. Previous studies have shown a differential metabolic profile of CRC based on its tumor localization and across CRC stages I–IV.¹³ Levels of specific amino acids (e.g., phenylalanine, valine), carboxylic acids (e.g., lactate), and lipids were shown to change from the early to the later and more aggressive stages of CRC with potential association with tumor metastases. Moreover, the presence of KRAS mutations in CRC was observed to be associated with suppressed expression of genes involved in immune pathways and suppression of iron-dependent cell death (i.e., ferroptosis).¹⁴ To date, no studies have provided detailed profiling of metabolic pathways in CRC after PFOS and PFOA exposure, which is necessary to generate hypotheses regarding their toxicity mechanisms in CRC development and progression.

In this study, we observed that exposure to PFOS and PFOA at a dosage of 10 μM can induce the migration of CRC spheroids of SW48 KRAS wild-type (WT) and KRAS G12A mutated cells. Initially, we evaluated the cytotoxic concentrations of PFOS and PFOA. Then, the migratory phenotype was analyzed through a wound healing and trans-well migration assay. Untargeted metabolomics was further performed, which revealed changes in fatty acids and amino acid metabolism in both SW48 KRAS WT and G12A cells after exposure to 10 μM PFOS and PFOA. Finally, the hypothesis that PFOS and PFOA contribute to CRC metastasis was verified through the analysis of the expression of epithelial-mesenchymal transition (EMT)-related markers. In conclusion, this work provides insights into the metabolic response of CRC to PFAS exposure and provides evidence of their metastatic potential (Figure 1).

Figure 1.

Overview of the study. a. PFOS and PFOA exposure to 3D CRC spheroids of both SW48 KRAS WT and SW48 KRAS G12A cell lines. b. Confirmation of migration phenotype by evaluating the cytotoxicity, wound healing, and trans-well assays, c. Untargeted metabolomics analysis of 3D CRC spheroid exposure to PFOS and PFOA at 2 or 10 μM. d. Validation of migration phenotype by evaluating the expression levels of E-cadherin, N-cadherin, and vimentin.

MATERIALS AND METHODS

Chemicals and Materials.

Metabolite standards were purchased from MetaSci (Toronto, Canada). Detailed information can be found in Table S1. p-Dimethylaminophenacyl (DmPA) bromine, dansyl chloride (DnsCl), 4-dimethylaminopyridine (DMAP), triethylamine (TEA), PFOS, and PFOA were purchased from Sigma-Aldrich (St. Louis, USA). The isotope reagents ¹³C₂-DmPA and ¹³C₂-DnsCl were purchased from TMIC (Edmonton, Canada). LC-MS grade formic acid (FA), water (H₂O), ethyl acetate (EA), and acetonitrile (ACN) were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA). The stock solutions of all standards were dissolved in LC-MS grade ACN or H₂O at a stock concentration of 1.0 mg/mL. ¹²C₂-DnsCl/¹³C₂-DnsCl and DMAP were at the concentrations of 20 mM in ACN, and TEA was prepared at the concentrations of 100 mM in ACN. All stock solutions were stored at −80 °C. All the primary and secondary antibodies were purchased from Cell Signaling Technology (Boston, U.S.A.).

Evaluation of Cytotoxicity of PFOS and PFOA.

SW48 KRAS WT and SW48 KRAS G12A cells were obtained from Horizon Discovery Ltd. (Cambridge, U.K.). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL of streptomycin. Cells were maintained at 37 °C in a 5% CO₂ humidified incubator. The hanging-drop method was used to enable cellular spheroid formation which has been described in a previous method.¹⁵ Approximately 1,000 SW48 KRAS WT and SW48 KRAS G12A cells were in each drop (20 μL). The imaging of 3D CRC spheroids was taken on day 7 using a ZEISS Zen 3.3 (blue edition) microscope software. PFOS and PFOA were directly dissolved in DMEM medium, and their cytotoxicity was evaluated in both SW48 KRAS WT and SW48 KRAS G12A cells. Briefly, cells were seeded in the form of 3D CRC spheroids and treated with PFOS or PFOA at different concentrations of 0, 0.025, 0.2, 2, 10, 30, and 100 μM for 1, 2, and 7 days. Trypan blue was used to assess cell count and viability after PFOS and PFOA exposure, and normalization was performed in the reconstitution step for extract preparation based on cell count results.

Wound Healing and Trans-well Assays.

Wound-healing assays were first performed for the evaluation of the migration ability of SW48 KRAS WT and SW48 KRAS G12A cells after exposure to PFOS and PFOA. Cells (5 × 10⁵) were seeded onto 6-well dishes and incubated for 24 h; the monolayer was then scratched with a pipet tip (size 200 μL), and photographs were taken at 0, 24, and 48 h postscratch in a phase-contrast microscope (Leica DM IL LED, Wetzlar, Germany). Experiments were carried out in triplicate, and four fields of each point were recorded. The scratch area was calculated by measuring the gap enclosed by the cells using ImageJ software.¹⁶ The wound healing percentages were calculated according to the following formula:

$$\begin{gathered} \mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d}\mathrm{h}\mathrm{e}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} \\ =\left[\left({\mathrm{A}}_{\mathrm{t}=0\mathrm{h}}-{\mathrm{A}}_{\mathrm{t}=24\mathrm{o}\mathrm{r}48\mathrm{h}}\right)/{\mathrm{A}}_{\mathrm{t}=0\mathrm{h}}\right]\times 100\mathrm{\%} \end{gathered}$$

${\mathrm{A}}_{\mathrm{t}=0\mathrm{h}}$ and ${\mathrm{A}}_{\mathrm{t}=24\mathrm{o}\mathrm{r}48\mathrm{h}}$ represent the scratch areas measured at 0, 24, and 48 h, respectively.

Trans-well migration assays were performed to assess cancer cell migration upon treatments in trans-well chamber with the noncoated membrane (24-well insert, pore size: 8 mm, Corning, Life Sciences).¹⁷ Approximately 3 × 10⁴ cells of SW48 KRAS WT and SW48 KRAS G12A cell lines were dosed with PFOS or PFOA (2 and 10 μM) and plated in the top chamber. FBS-free medium was added to the upper chamber, and the medium containing 10% fetal bovine serum was added in the lower chamber. After 48 h, the cells were fixed in 10% neutral buffered formalin solution for 30 min and stained with 0.5% crystal violet solution for an additional 30 min. The cells that had successfully invaded through the pores and reached the lower surface of the insets were subsequently counted under an inverted microscope, in five independent microscopic fields.

Metabolomic Profiling.

3D spheroids are demonstrated to have improved accuracy for monitoring cell number, response to stimuli, and drug metabolism, compared to 2D models.¹⁸ The effect of environmental exposure on humans is usually long-term progress. Therefore, exposure of SW48 KRAS WT and SW48 KRAS G12A 3D CRC spheroids to PFOS or PFOA at 0, 2, and 10 μM for 7 days were used for metabolomics analysis (~6.0 × 10⁵ cells each sample). It has been reported that individuals living near contamination sites and those with occupational exposures have a serum concentration as high as 1.2 and 7.2 μM PFOA, respectively. Therefore, this concentration approximates the distributed concentrations reported in populations residing near these contamination sites or individuals with occupational exposure.¹⁹ Detailed interpretations can be found in Text S1. Liquid chromatographic separation was achieved on an Acquity UPLC BEH C18 column (100 × 2.1 mm i.d., 1.7 μm; Waters, Milford, USA). The injection volume was 2 μL, and triplicate measurements were performed for every experiment. The confirmation of all derivatization products was performed under full scan on Waters ACQUITY UPLC system-Xevo G2-XS hybrid quadrupole-time-of-flight mass spectrometer (Waters, Milford, MA, USA), equipped with an electrospray ionization source operating in positive ionization mode. Quality control (QC) samples were prepared in parallel by pooling an aliquont of each sample and treated in the same way as used for the study samples to assess the stability and accuracy of the analytical method. Detailed sample pretreatment and reaction conditions and LC-MS parameter setting are displayed in Text S2.

Western Blot.

Proteins of 3D spheroids exposed to PFOS and PFOA were quantified according to the BCA Protein Quantitation Specification (Thermo Fisher Scientific, Waltham, MA, USA); a standard curve was drawn, and the protein loading volume was calculated. Equal amounts of protein (10 μg) were separated by 10–15% SDS-PAGE and transferred onto the PVDF membrane, followed by 5% skim milk for blocking the membrane. Membranes were further incubated at 4 °C overnight with these antibodies including GAPDH (catalog no. 5174), E-cadherin (catalog no. 94386), N-cadherin (catalog no. 4061), and vimentin (catalog no. 46173). Subsequently, the membranes were washed with TBST and incubated with the corresponding HRP-linked secondary antibody at room temperature for 2 h, followed by visualization using the enhanced chemiluminescence system. Western blotting was quantified using Quantity One software (Bio-Rad). All the primary and secondary antibodies were purchased from Cell Signaling Technology (Boston, USA).

Statistical Analysis.

Statistical analysis between different groups was performed by Student’s t test (two groups) and analysis of variance (ANOVA) (multiple groups). All analyses were adjusted for multiple comparisons using Benjamini–Hochberg-based false discovery rates (FDRs) correction. Adjusted p-value < 0.05 and fold change > 1.5 (log₂ 1.5 = 0.585) or <0.67 (log₂ 0.67 = −0.578) were considered statistically significant. Overall precision was then evaluated by analysis of the total variance using principal component analysis (PCA). MetaboAnalyst 5.0 is used for pathway mapping and figure visualization.²⁰ The graphical abstract and Figure 1 and Figure 3 were created using BioRender, and bar graphs were created by GraphPad Prism 8.0.

Figure 3.

Migration of SW48 KRAS WT and SW48 KRAS G12A induced by 10 μM PFOS or PFOA exposure confirmed by wound healing and trans-well assays. a. Wound healing assays of SW48 KRAS WT cell exposure to PFOS and PFOA. Cells were plated in 6-well plates and grown to 70% confluence. After generating a scratch in the monolayer, cells were incubated with or without PFOS and PFOA. Images were captured at 0, 24, and 48 h after wounding (Scale bar: 100 μm). b. Wound healing assays of SW48 KRAS G12A cell exposure to PFOS and PFOA (Scale bar: 100 μm). c. Trans-well migration assays in SW48 KRAS WT cells with indicated treatments. Cells treated with or without PFOS and PFOA were seeded in the upper chamber. Following 24 h of incubation, cells migrated to the lower chamber and were fixed and stained, and images were captured for counting and representation (purple) (Scale bar: 200 μm). d. Trans-well migration assays in SW48 KRAS G12A cells with the indicated treatments. Cells were stained by crystal violet (Scale bar: 200 μm). (All assays were performed in triplicate and at least four fields of each point were recorded. ANOVA was performed. *p < 0.05, **p < 0.01, ***p < 0.001.)

RESULTS

Phenotypic Change of 3D CRC Spheroid Exposure to PFOS and PFOA.

3D CRC spheroids of SW48 KRAS WT and SW48 KRAS G12A were used in this study. The latter genetic mutation has been associated with a lower survival rate among all KRAS mutation types.²¹ At 2 μM PFOS exposure, cell spreading behavior was observed in both cell lines (Figure S1a). Concentrations of PFOS < 2 μM did not elicit cell spreading (Figure S1a); however, as PFOS exposure levels increased to 100 μM, the spreading phenotype was subsequently more pronounced. Similarly, cell spreading was also observed in both cell lines exposed to PFOA from 2 μM to 100 μM (Figure S1b), and not at doses < 2 μM. We speculated that such a phenotype was suggestive of migration of 3D CRC spheroids in the study.

Cytotoxicity of PFOS and PFOA to 3D CRC spheroids.

To determine optimal dosing concentrations for subsequent phenotypic confirmation and metabolomics analysis, we further evaluated the cell viability of 3D spheroids of both cell lines exposed to seven concentrations of PFOS and PFOA from 0 to 100 μM. The viability of each spheroid exposure to different levels of chemicals was >95%, which was confirmed by trypan blue stain. After exposure to the seven doses of 0 to 100 μM PFOS and PFOA for 24 and 48 h, the cell numbers of both cell lines did not change (Figure 2a,b). Conversely, after exposure to PFOS and PFOA for 7 days, the cell number of SW48 KRAS WT was decreased when the exposure level of PFOS was ≥30 μM and PFOA was ≥100 μM (p < 0.001) compared to controls (0 μM), suggesting that cell proliferation was inhibited by PFOS and PFOA (Figure 2c). Similarly, the cell numbers of SW48 KRAS G12A decreased when the exposure level of PFOS was ≥30 μM and PFOA was ≥100 μM. Overall, a short-time (24 and 48 h) exposure to PFOS and PFOA, regardless of the exposure levels, had no effect on cell proliferation. When exposed to PFOS and PFOA for a longer time (7 days), lower exposure levels had no effect on cell proliferation, while higher exposure (30 μM) levels inhibited cell proliferation to a certain extent. Taking the distribution levels of PFOS and PFOA in vivo into consideration, two low-cytotoxic concentrations of PFOS and PFOA, 2 and 10 μM, were used for subsequent experiments.

Figure 2.

Cell viability after exposure to different levels of PFOS and PFOA. a. 24 h. b. 48 h. c. 7-day exposure time periods. Triplicates were performed for each group (n = 3). ANOVA was conducted for the analysis of variance. *p < 0.05 compared to Ctr group, **p < 0.01. Abbreviation: Ctr, control.

Effect of PFOS and PFOA on the Migration Ability of SW48 Cells.

To confirm the cell spreading phenotype induced by PFOS or PFOA at concentrations ≥ 2 μM, the migration ability of both cell lines was assessed after 24 and 48 h exposure. Upon exposure to 2 μM of either chemical, the migration ability of SW48 KRAS WT cells slightly increased from 20% to 25% (PFOS) or 23% (PFOA) after 24 h, and from 34% to 39% (PFOS) or 35% (PFOA) after 48 h, but it was not statistically significant. Conversely, exposure to 10 μM PFOS and PFOA for 48 h significantly increased the migration ability of SW48 KRAS WT cells from 34% to 50% (PFOS, p < 0.05) or 46% (PFOA, p < 0.05) (Figure 3a), respectively. For SW48 KRAS G12A cells, the migration ability, after exposure to PFOS and PFOA at 2 or 10 μM, was comparable to that of SW48 KRAS WT cells (Figure 3b). No difference was found in SW48 KRAS G12A cell exposure to 2 μM of either compound for 24 and 48 h. However, when the cells were exposed to 10 μM PFOS and PFOA for 24 h, the migration ability of SW48 KRAS G12A cells significantly increased from 28% to 50% (PFOS, p < 0.01) and 42% (PFOA, p < 0.05), respectively. These results indicated that exposure to 10 μM PFOS and PFOA increased the migration ability of both cell lines.

The impact of PFOS or PFOA on the migration ability of SW48 cells was further evaluated using trans-well assays; spatial migration occurred from the upper to lower chamber. The findings revealed that exposure to 10 μM PFOS and PFOA for 24 h resulted in a significant increase in the number of migrated cells in SW48 KRAS WT cells, from 50 (controls) to 180 (PFOS, p < 0.001) or 172 (PFOA, p < 0.001), respectively (Figure 3c). Given that same number of SW48 KRAS WT and SW48 KRAS G12A cells were seeded, this indicated that cell migration of the untreated SW48 KRAS G12A (112 cells) was more than twice as high as that of SW48 KRAS WT (50 cells). Furthermore, exposure of SW48 KRAS G12A cells to 10 μM PFOS and PFOA for 24 h resulted in an increased number of migrated cells from 112 (control) to 268 (PFOS, p < 0.001) or 235 (PFOA, p < 0.001), respectively (Figure 3d). However, exposure to 2 μM PFOS or PFOA did not significantly alter the migration ability of either cell line. Overall, exposure to 10 μM PFOS or PFOA induced the migration of SW48 KRAS WT and SW48 KRAS G12A confirmed by wound healing and trans-well assays.

Metabolic Profiles of 3D CRC Spheroids in Response to PFOS and PFOA Exposure.

We further investigated the metabolic changes of SW48 KRAS WT and SW48 KRAS G12A in response to PFOS and PFOA exposure. Using multivariate analysis and principal component analysis (PCA), we observed a clear visualization of metabolic data and their clustering across the exposure conditions. A clear separation was observed between SW48 KRAS WT cells treated with the highest concentration (10 μM PFOS or PFOA). The PCA scores plot of SW48 KRAS WT cells indicated minimal residual technical errors with a R2X = 0.563 (Figure 4a). Similar results were also observed in SW48 KRAS G12A cells with a R2X = 0.599 (Figure 4b).

Figure 4.

Metabolic changes in 3D CRC spheroids with exposure to different concentrations of PFOS or PFOA. a. PCA scores plot of SW48 KRAS WT cell lines exposed to PFOS and PFOA at 2 or 10 μM (R2X = 0.563). b. PCA scores plot of SW48 KRAS G12A cell lines exposed to PFOS and PFOA at 2 or 10 μM (R2X = 0.599). c. Significantly changed metabolites between the controls of SW48 KRAS WT and SW48 KRAS G12A cells. Each group contains three replicates (n = 3). Student’s t test was conducted for the analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001.

Initially, we compared the metabolic profiles of controls of SW48 KRAS WT and SW48 KRAS G12A. Metabolites were identified using standards and three metrics: matching to MS, MS/MS, and retention time (Table S2, Figure S2). We observed downregulation of short chain fatty acids (SCFAs), propionic acid, and butyric acid in SW48 KRAS G12A cells compared to SW48 KRAS WT cells. Additionally, several amino acids (alanine, phenylalanine, proline), fatty acids, and monoglycerides (glycerol monooleate, glycerol monopalmitate) were significantly upregulated in SW48 KRAS G12A cells (Figure 4c, Table S3).

Next, subsequent metabolic analysis was focused on 10 μM PFOS or PFOA treatments due to a more distinct metabolic response observed by PCA. SW48 KRAS WT exposure to 2 μM PFOS and PFOA was clustered with controls (0 μM). Conversely, SW48 KRAS WT cells exposed to 10 μM PFOS or PFOA were clearly separated from the above groups. Compared to controls, 23 and 22 altered metabolites were observed in SW48 KRAS WT and SW48 KRAS G12A groups, respectively (Tables 1 and 2). The majority of differentially altered metabolites were fatty acids and amino acids (Figure S3).

Table 1.

Differences in Metabolite Levels between SW48 KRAS WT Control, PFOS, and PFOA-Treated Groups^a

		SW48 KRAS WT log2 fold change				SW48 KRAS WT adj. p-value
No.	Metabolites	PFOS 2 (μM/control)	PFOS 10 (μM/control)	PFOA 2 (μM/control)	PFOA 10 (μM/control)	PFOS 2 (μM/control)	PFOS 10 (μM/control)	PFOA 2 (μM/control)	PFOA 10 (μM/control)
1	2-Hydroxybutyric acid	−0.504	−1.185	−0.392	−0.615	3.65 × 10−02	7.94 × 10−04	7.08 × 10−02	4.24 × 10−03
2	Butyric acid	0.071	−0.901	0.099	−0.635	1.95 × 10−01	3.18 × 10−03	1.85 × 10−01	4.52 × 10−02
3	Valeric acid	−0.024	0.959	0.017	0.625	6.19 × 10−01	2.86 × 10−04	8.23 × 10−01	4.38 × 10−02
4	Caprylic acid	0.117	−1.000	0.098	−0.792	6.69 × 10−02	1.43 × 10−03	1.96 × 10−01	4.77 × 10−03
5	Undecanoic acid	−0.180	−1.344	−0.222	−0.891	2.84 × 10−03	3.20 × 10−03	6.05 × 10−02	1.19 × 10−05
6	Oleic acid	−0.028	0.766	−0.038	0.686	5.83 × 10−01	1.76 × 10−02	5.33 × 10−01	5.42 × 10−04
7	α-Linolenic acid	−0.801	−0.851	−0.304	−0.787	1.15 × 10−05	2.51 × 10−04	1.98 × 10−01	2.86 × 10−05
8	Adrenic acid	0.031	−0.939	−0.166	−0.075	3.90 × 10−01	1.13 × 10−03	1.14 × 10−01	1.02 × 10−01
9	5-HPETE	−0.090	0.600	−0.064	−0.067	3.99 × 10−02	1.08 × 10−02	1.13 × 10−01	4.82 × 10−02
10	Azelaic acid	−0.108	−0.966	−0.038	−0.594	5.54 × 10−01	6.77 × 10−03	8.40 × 10−01	2.28 × 10−02
11	Glycerol monomyristate	0.047	−1.078	−0.119	−0.648	2.93 × 10−01	3.18 × 10−04	1.25 × 10−01	5.80 × 10−04
12	Glycerol monooleate	−0.551	0.005	−0.656	−0.942	6.13 × 10−02	9.59 × 10−01	4.12 × 10−02	2.42 × 10−02
13	Alanine	−0.131	−0.851	−0.097	−1.436	6.08 × 10−01	4.73 × 10−02	8.12 × 10−01	4.38 × 10−02
14	Phenylalanine	−0.055	−1.481	−0.055	−0.691	8.22 × 10−01	3.61 × 10−04	8.18 × 10−01	2.62 × 10−03
15	Proline	−0.101	−0.663	0.256	−1.084	6.10 × 10−01	3.47 × 10−02	2.15 × 10−01	2.16 × 10−02
16	Valine	0.604	0.897	−0.392	0.913	3.65 × 10−02	1.13 × 10−03	1.08 × 10−01	3.86 × 10−03
17	N-Acetylglutamic acid	0.632	1.091	0.840	0.963	1.28 × 10−02	1.52 × 10−02	4.98 × 10−02	1.63 × 10−02
18	1-Dodecanol	−0.076	0.991	−0.040	1.489	2.68 × 10−01	2.02 × 10−04	6.37 × 10−01	2.07 × 10−05
19	1-Octanol	0.192	0.929	0.015	0.762	3.21 × 10−03	1.76 × 10−02	3.53 × 10−01	3.74 × 10−02
20	4-Hydroxybenzaldehyde	−0.182	−0.950	−0.060	0.065	5.11 × 10−02	2.10 × 10−04	2.03 × 10−01	1.06 × 10−01
21	Uridine	−0.915	−0.945	−0.599	−0.878	4.19 × 10−05	4.53 × 10−04	5.02 × 10−02	2.62 × 10−04
22	Creatinine	0.063	−0.012	−0.004	0.772	1.24 × 10−01	6.05 × 10−01	8.42 × 10−01	5.98 × 10−04
23	Guanidine	−0.014	0.119	−0.146	−0.755	3.36 × 10−01	3.50 × 10−01	7.17 × 10−01	4.90 × 10−02

^aTriplicates were performed for each group (n = 3). ANOVA was carried out and p-values were adjusted for FDR. Metabolites with adj. p < 0.05 and fold change >1.5 (log₂ 1.5 = 0.585) or <0.67 (log₂ 0.67 = −0.578) were considered as statistically significant. The p-values and fold changes that are significantly altered are displayed in bold. WT, wild type; PFOS, perfluorooctanesulfonic acid; PFOA, perfluorooctanoic acid; 5-HPETE, arachidonic acid 5-hydroperoxide; adj. p-value: p-value adjusted for false discovery rates (FDR).

Table 2.

Differences in Metabolite Levels between SW48 KRAS G12A Control, PFOS, and PFOA-Treated Groups^a

		SW48 KRAS G12A log2 fold change				SW48 KRAS G12A adj. p-value
No.	Metabolites	PFOS 2 μM/control	PFOS 10 μM/control	PFOA 2 μM/control	PFOA 10 μM/control	PFOS 2 μM/control	PFOS 10 μM/control	PFOA 2 μM/controll	PFOA 10 μM/control
1	2-Hydroxybutyric acid	0.011	−1.180	−0.338	−1.058	9.17 × 10−01	9.46 × 10−05	7.04 × 10−02	1.93 × 10−02
2	Butyric acid	0.007	−1.095	−0.013	−0.737	1.00 × 1000	2.14 × 10−03	9.34 × 10−01	2.75 × 10−03
3	Valeric acid	0.129	0.702	0.003	0.637	6.73 × 10−01	1.68 × 10−03	9.69 × 10−01	9.56 × 10−03
4	Oleic acid	−0.055	−1.147	−0.036	−0.875	1.94 × 10−01	1.09 × 10−02	4.52 × 10−01	9.90 × 10−03
5	α-Linolenic acid	0.038	−0.697	0.008	−0.622	2.24 × 10−01	1.23 × 10−04	7.93 × 10−01	1.65 × 10−02
6	Linoleic acid	0.001	−0.816	0.005	−0.604	1.00 × 1000	3.96 × 10−03	7.94 × 10−01	1.03 × 10−02
7	Adrenic acid	0.034	−0.608	−0.023	0.132	2.47 × 10−01	1.27 × 10−03	5.06 × 10−01	2.92 × 10−03
8	Arachidonic acid	0.019	0.328	0.196	0.960	1.00 × 1000	3.50 × 10−03	7.16 × 10−01	8.90 × 10−03
9	5-HPETE	0.024	−0.954	−0.032	−0.631	6.38 × 10−01	1.47 × 10−03	5.81 × 10−01	1.03 × 10−03
10	Azelaic acid	−0.431	−0.626	0.030	−0.617	3.72 × 10−03	2.81 × 10−02	5.24 × 10−01	2.74 × 10−03
11	Glycerol monomyristate	−0.099	−0.624	−0.284	−0.624	2.24 × 10−01	4.27 × 10−03	3.11 × 10−01	1.52 × 10−03
12	Glycerol monooleate	−0.603	−0.858	−0.110	0.045	3.20 × 10−02	3.42 × 10−03	7.14 × 10−01	6.07 × 10−01
13	Glycerol monopalmitate	−0.211	−0.569	−0.112	−0.605	2.36 × 10−01	1.71 × 10−02	5.01 × 10−01	1.24 × 10−02
14	Phenylalanine	−0.610	−1.071	−0.446	−0.951	3.06 × 10−03	1.36 × 10−05	2.14 × 10−04	3.61 × 10−04
15	Proline	−0.037	−0.679	−0.070	−0.051	8.76 × 10−01	3.67 × 10−03	5.46 × 10−01	5.24 × 10−01
16	N-Acetylglutamic acid	−0.076	0.609	−0.037	0.018	8.67 × 10−01	1.10 × 10−02	9.22 × 10−01	8.98 × 10−01
17	3-Hydroxybenzoic acid	0.134	0.941	0.096	0.975	2.59 × 10−01	1.95 × 10−02	4.95 × 10−01	2.82 × 10−02
18	1-Octanol	0.192	1.358	0.015	0.744	4.10 × 10−03	2.09 × 10−04	4.60 × 10−01	4.38 × 10−02
19	Uridine	−0.489	−0.915	−0.335	−0.938	5.68 × 10−02	4.00 × 10−05	3.75 × 10−03	7.55 × 10−05
20	Hypoxanthine	0.001	−1.046	0.063	−0.729	1.00	2.70 × 10−02	5.04 × 10−01	1.49 × 10−04
21	Creatinine	0.063	−0.012	0.235	0.741	2.04 × 10−01	5.79 × 10−01	9.68 × 10−03	6.27 × 10−04
22	Pipecolinic acid	0.097	−0.833	0.107	−0.780	2.42 × 10−02	1.09 × 10−02	4.87 × 10−01	6.16 × 10−04

^aTriplicates were performed for each group (n = 3). ANOVA was carried out and p-values were adjusted for FDR. Metabolites with adj. p < 0.05 and fold change >1.5 (log₂ 1.5 = 0.585) or <0.67 (log₂ 0.67 = −0.578) were considered as statistically significant. The p-values and fold changes that are significantly altered are displayed in bold. PFOS, perfluorooctanesulfonic acid; PFOA, perfluorooctanoic acid; 5-HPETE, arachidonic acid 5-hydroperoxide; adj. p value: p value adjusted for false discovery rates (FDR).

Among the amino acids, 2-hydroxybutyric acid, derived from α-ketobutyrate, was also decreased in all 10 μM PFOS and PFOA treatments compared with controls, suggesting this is related to the decreased free amino acids due to increased protein synthesis. Other commonly identified amino acids, alanine, phenylalanine, and proline, were shown to be reduced in all treatments. Accordingly, phenylalanine metabolism was found to be a common metabolic pathway for all treatments, together with fatty acid metabolism. With this regard, fatty acids such as glycerol monomyristate and glycerol monooleate were reduced after exposure to 10 μM PFOS and PFOA in both the WT and G12A cells, suggesting a downregulation of monoglyceride synthesis in these cells. Moreover, elevated levels of intracellular fatty alcohols, such as 1-dodecanol and 1-octanol, were detected after PFOS and PFOA exposure in both cell types, which is indicative of the inhibition of cholesterol synthesis. Changes in nucleotides included reduced levels of the pyrimidine uridine.

Different metabolic compounds were also detected. After exposure to 10 μM PFOS and PFOA, aminoacyl-tRNA biosynthesis was altered in the SW48 KRAS WT cells (Figure S4), while biosynthesis of unsaturated fatty acids was altered in the SW48 KRAS G12A cells (Figure S5). On the one hand, decreased levels of alanine were observed in the SW KRAS WT cells exposed to PFOS and PFOA, while significant levels of valine increased upon both treatments. Additionally, the levels of free medium-chain fatty acids, including caprylic acid and undecanoic acid, were found to decrease in 10 μM PFOS and PFOA-treated SW48 KRAS WT cells. In SW48 KRAS G12A treated cells, fatty acids such as arachidonic acid, glycerol monopalmitate, and linoleic acid were detected, and their levels were decreased after treatment with PFOS and PFOA, except for arachidonic acids whose levels increased after exposure. Different metabolites were also specifically detected in the PFOS and PFOA treated samples. Decreased adrenic acid levels were found in both cell lines exposed to 10 μM PFOS, while increased creatinine levels were detected in both cell lines exposed to 10 μM PFOA.

Furthermore, we evaluated the metabolic impact of PFOS and PFOA at a lower concentration (2 μM), which is representative of exposure concentrations reported in individuals with high PFOS or PFOA exposure.¹⁹ The metabolic profiling of this concentration can provide a more accurate representation of the potential health implications of these contaminants in vivo. Our findings revealed that several metabolites, including α-linolenic acid, uridine, N-acetylglutamic acid, valine, glycerol monooleate, and phenylalanine, were significantly altered in 3D CRC spheroids after exposure to 2 μM PFOS and PFOA (Figure S6). In addition, as the exposure concentrations of PFOS and PFOA increased, the detected levels of these metabolites correspondingly increased or decreased in a dose-dependent manner.

In summary, the global metabolomics analysis of this study enabled the detection of similarities and differences in the metabolites of SW48 KRAS cells according to their genetic background (WT vs G12A mutated cells) and on the different treatment conditions (PFOS vs PFOA). Figure 5 summarizes the metabolic changes detected in this work and presents a putative metabolic map of the mechanism through which PFOS and PFOA can be related to metastasis development.

Figure 5.

Proposed migration related metabolic pathways induced by PFOS and PFOA exposure. Migration typically requires increased energy consumption. Cancer cells respond by enhancing the β-oxidation of fatty acids and TCA cycle function to produce more ATP. This leads to decreased levels of free fatty acids. The produced ATP was speculated to combine with amino acids through the aminoacyl-tRNA pathway to produce the proteins necessary for cell migration, resulting in reduced levels of free amino acids. As a result, the pathway for synthesizing monoglycerides was downregulated. Elevated levels of intracellular fatty alcohols were also detected, suggesting inhibition of cholesterol synthesis. Furthermore, the pathway for DNA synthesis was also upregulated in response to metastasis.

Expression of EMT Markers in CRC Cells upon Exposure to PFOS/PFOA.

EMT has been considered as a critical mechanism for mediating the metastatic process in tumor cells.²² In addition, it has been reported that metabolic pathways which are involved in EMT include glycolysis, TCA cycle, and lipid and amino acid metabolism.²³ Therefore, we hypothesized that exposure to PFOS and PFOA (10 μM) would change the expression of important proteins in EMT, leading to these abnormal metabolites detected in 3D spheroids of SW48 KRAS WT and SW48 KRAS G12A.

To further understand the potential mechanisms of PFOS and PFOA-induced cell migration, we investigated the expression of several key proteins in EMT, including E-cadherin, N-cadherin, and vimentin, in controls and 10 μM PFOS or PFOA treatments (Figure 6a). Our results showed that exposure to 10 μM PFOS or PFOA resulted in downregulation of E-cadherin expression, indicating decreased cell adhesion and polarity in both SW48 KRAS WT and SW48 KRAS G12A cells.²⁴ In contrast, the expression levels of N-cadherin and vimentin were upregulated, suggesting a potential role for these chemicals in cell migration, invasion, and cytoskeletal remodeling (Figure 6b).²⁵ In addition, it was also found that there were decreased expression levels of E-cadherin and increased expression levels of vimentin in SW48 KRAS G12A compared to those of SW48 KRAS WT. This suggests that SW48 KRAS G12A cells have a stronger migration ability. Together these results suggested that the migration of 3D CRC spheroids induced by PFOS and PFOA is promoted through EMT.

Figure 6.

Potential mechanism study of migration induced by 10 μM PFOS and PFOA. a. Western blot of E-cadherin, N-cadherin, and vimentin in SW48 KRAS WT and SW48 KRAS G12A cells, b. Expression levels of E-cadherin, N-cadherin, and vimentin. (Experiments were carried out in triplicate. ANOVA was conducted for the analysis of variance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.)

DISCUSSION

Exposure to PFOS and PFOA has been shown to be associated with a various health outcomes.²⁶ Several studies have investigated their adverse effects on human health and in the development of cancer.²⁷ However, few studies have explored their cancerous and metastatic potential in CRC. Thus, it is currently unknown with which biological and biochemical mechanisms PFOS and PFOA might interfere during CRC progression and how they correlate to metastasis development.

In this work, we used human CRC cell lines, SW48, with a specific mutated form of the KRAS gene (G12A), as these cells are representative of an aggressive form of CRC of which no therapies are currently available.²⁸ Both SW48 KRAS WT and G12A mutated cell types were grown as spheroids because this represents a more sensitive in vitro model for toxicological assessments.²⁹ Both cell types were first employed in a phenotypic study to evaluate their response to PFOS and PFOA exposure through cell viability and migration assays, at two noncytotoxic concentrations, of 2 and 10 μM PFOS/PFOA for 24 and 48 h. Both concentrations promoted cell migration compared to the nontreated cells and were particularly effective after 48 h exposure in the mutated cells (G12A) compared to the WT cells. This suggested that the migration potential of PFOS and PFOA is dependent not only on concentration but also on the exposure time and genetic profile on cells. Overall, the phenotypic and molecular studies conducted on the SW48 KRAS WT and KRAS G12A cells confirmed that PFOS and PFOA determine CRC progression. We also investigated lower levels of PFOS and PFOA and observed that there was no effect on cell viability or cell spreading, suggesting that those who live near contaminated sites or who are occupationally exposed to the higher levels of these chemicals may be at increased susceptibility to the biological effects of these drugs. A recent report showed that occupations which have higher levels of exposure include firefighters and those working in fluorochemical manufacturing; environmental exposure can also occur to those who reside next to airports, military bases, landfills, and incinerators and also those that live near wastewater treatment plants and farms that use contaminated sewage sludge.³⁰

The mechanism of action of PFOS and PFOA was further investigated through MS-based untargeted metabolomics to generate a hypothesis as to their biological effects. Derivatization was performed to improve the coverage of detectable metabolites, including SCFAs. Recent studies have found that SCFAs can mitigate inflammation through the regulation of cytokine production (TNFα, IL2, IL6, and IL10), eicosanoids, and chemokines.³¹ In this study, SCFAs that were analyzed included propionic and butyric acids, which are known to be anti-inflammatory, antitumor, and antibacterial.³² Thus, the downregulation of these SCFAs in the KRAS mutated CRC cells (G12A) compared to the WT cells is in line with the hypothesis of activation of proinflammatory mechanisms in the mutated phenotype, ultimately leading to abnormal cell proliferation, invasion, and metastatic potential. According to this hypothesis, high levels of phenylalanine, a well-known amino acid associated with systemic inflammation in CRC,³³ were detected in both cell phenotypes.

After exposure to PFOS and PFOA at 2 and 10 μM, a clear separation among the metabolic features of treated and nontreated cells was observed for all tested conditions, especially at the highest concentration (10 μM). Shared classes of metabolites were represented mainly by amino acids and fatty acids, indicating that they have a high impact in determining the observed metabolic differences. Among the amino acids, reduced levels of proline were found in both KRAS WT and KRAS G12A mutated SW48 cells exposed to 10 μM PFOS and PFOA. This association between low levels of proline and increased proliferative and migration potential of CRC cells is in contrast with other studies where downregulation of proline biosynthesis was found to be antiproliferative and to inhibit EMT in CRC cells.³⁴ Regarding lipid metabolism, common lipids altered among both cell phenotypes and exposure conditions included butyric acid and valeric acid, and long-chain fatty acids (oleic acid, α-linolenic acid, glycerol mono myristate, and glycerol monooleate). Butyric acid, as mentioned prior, is known to have critical inhibitory effects on glucose metabolism supporting cancer proliferation,³⁵ which is also supported by the findings of this work showing significantly low levels of butyric acid in both WT and G12A mutated cells after PFAS treatment. Oleic and α-linolenic acids are known to have a protective effect against CRC.³⁶ Low levels of both fatty acids in the KRAS G12A cells exposed to PFOS and PFOA further support the potential of PFAS in enhancing CRC cell progression. Moreover, reduced levels of fatty acids can be a sign of rewired lipid metabolism in CRC from fatty acid synthesis to their oxidation, in order to support the high ATP requirement for proliferation and metastasis formation.³⁷ In line with this assumption, other studies have demonstrated that PFAS activates the peroxisome proliferator-activated receptor alpha (PPARα),³⁸ which is mainly expressed in tissues with a high rate of fatty acid oxidation. PPARα positively regulates mitochondria β-oxidation especially for polyunsaturated fatty acids (PUFA), which have a high binding affinity to PPARs.³⁹

Beyond amino acids and lipid metabolism, nucleotide (pyrimidine) metabolism was also detected to be relevant for SW48 KRAS WT and G12A cells after exposure to PFOS and PFOA. The decreased levels of uridine in all exposure conditions can be indicative of the disrupted pathways for the synthesis of nucleotides which are used for DNA and RNA synthesis. Creatinine was found to be altered in all conditions, and its levels were significantly upregulated in both WT and G12A mutated cell exposure to PFOA but not PFOS. Recently, low and high serum creatinine levels have been associated with poor overall survival of CRC patients,⁴⁰ indicating that its metabolism is critical in determining the aggressiveness of CRC.

Different classes of metabolites were also detected for each different exposure condition. For example, both WT and G12A mutated cells treated with PFOS, but not PFOA, were enriched in docosanoid (C22) and eicosanoid (C20) compounds. These included adrenic acid and 5-hydroperoxyeicosatetraenoic (5-HPETE), known as intermediates of arachidonic metabolism, which regulates tumor immunity⁴¹ and has been observed to serve as an antitumorigenic agent in CRC cells.⁴² Thus, the reduced levels of both adrenic acid and 5-HPETE observed in the mutated cells after PFOS exposure may indicate their increased aggressiveness. On the other hand, specifically representative of the WT phenotype were phenols (4-hydroxybenzaldehyde) that can have a protective effect on CRC. In the SW48 KRAS G12A cells alteration in choline metabolism was detected, which is in line with other studies where disruption of choline metabolism was associated with KRAS mutations.⁴³

In general, the metabolomics study of SW48 KRAS (WT and G12A) cells showed similarities and differences in the metabolic features of the two cellular phenotypes before and after exposure to PFOS and PFOA. This was correlated to a different response of cells to the two pollutants. In particular, the SW48 KRAS G12A cell type exposure to either PFOS or PFOA presented a metabolic content associated with a more aggressive and migrated phenotype, which is in line with the phenotypic and molecular analyses initially described in this work.

A further confirmation of the PFAS metastatic potential in CRC cells was carried out by the investigation of the expression of EMT markers such as E-cadherin, N-cadherin, and vimentin. E-cadherin is a cell adhesion and tumor suppressor protein that inhibits uncontrolled cellular proliferation and differentiation toward a malignant phenotype.⁴⁴ Thus, the loss of this protein is associated with cancer invasion and progression. N-cadherin is a transmembrane protein similar to E-cadherin, but it is required for cancer proliferation, invasion, and metastasis; thus its high expression is associated with malignant cancer progression.⁴⁵ The switched expression between E-cadherin (low) and N-cadherin (high) and the high levels of vimentin were detected mainly in the KRAS G12A mutated cells upon treatment with either PFOS or PFOA. This indicates that mutation in the KRAS gene leads to a more aggressive phenotype and increased susceptibility to cancerous and metastatic environmental pollutants like PFOS and PFOA. The metastatic progression of cancer requires metabolic changes to support cancer proliferation and migration. For example, previous studies have shown that the activation of the transforming growth factor beta 1 (TGFβ-1) leads to increased glycolysis and fatty acid oxidation in cancer cells by regulating the expression of certain enzymes involved in these pathways (e.g., FASN, CPT1).⁴⁶ Accordingly, our findings present increased levels of metabolites of fatty acid oxidation upon exposure to PFAS. Moreover, it has been demonstrated that branched-chain amino acids (BCAAs) play a potential role in regulating the EMT program. This further aligns with our findings of higher levels of the BCAA valine in the SW48 KRAS WT cells after treatment with PFOS and PFOA. Thus, there are specific metabolic changes that accompany EMT, which can be indicators of cancer cells gaining metastatic potential.

Our findings indicate that exposure to 10 μM PFOS and PFOA increase the migration potential of 3D CRC spheroids. Metabolic profiling analysis revealed that such exposure led to decreased free fatty acids and amino acids in SW48 KRAS WT and SW48 KRAS G12A cells, likely due to an increase in glycolysis, TCA cycle, and β-oxidation of fatty acids. The EMT mechanism was further indicated by the observed decreased expression level of E-cadherin and the increased expression level in N-cadherin and vimentin. These results provide valuable insights into the potential impacts of PFOS and PFOA exposure on CRC progression, highlighting the importance of monitoring these environmental chemicals to reduce harmful effects on human health. These results are also important for vulnerable populations that may be occupationally exposed to high levels of PFOS and PFOA including firefighters and those who work in fluorochemical manufacturing.

Supplementary Material

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.3c04844.

Sample pretreatment, chemical isotope labeling and LC-MS conditions; information of identified significantly changed metabolites; significantly changed metabolites between control groups; 3D CRC spheroids and cell viability after PFOS or PFOA exposure; matching of MS/MS of metabolites in cell extracts and standards; distribution of the chemical structures of significantly changed metabolites; metabolic pathway analysis by MetaboAnalyst 5.0 (PDF)