Photodynamic Priming Overcomes Per- and Polyfluoroalkyl Substance (PFAS)-Induced Platinum Resistance in Ovarian Cancer

Brittany P Rickard; Xianming Tan; Suzanne E Fenton; Imran Rizvi

doi:10.1111/php.13728

. Author manuscript; available in PMC: 2024 Mar 1.

Published in final edited form as: Photochem Photobiol. 2022 Nov 7;99(2):793–813. doi: 10.1111/php.13728

Photodynamic Priming Overcomes Per- and Polyfluoroalkyl Substance (PFAS)-Induced Platinum Resistance in Ovarian Cancer^†

Brittany P Rickard ¹, Xianming Tan ^2,³, Suzanne E Fenton ^1,⁴, Imran Rizvi ^1,^3,^5,^6,^*

PMCID: PMC10033467 NIHMSID: NIHMS1838711 PMID: 36148678

Abstract

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants linked to adverse outcomes, including for female reproductive biology and related cancers. We recently reported, for the first time, that PFAS induce platinum resistance in ovarian cancer, potentially through altered mitochondrial function. Platinum resistance is a major barrier in the management of ovarian cancer, necessitating complementary therapeutic approaches. Photodynamic therapy (PDT) is a light-based treatment modality that reverses platinum resistance and synergizes with platinum-based chemotherapy. The present study is the first to demonstrate the ability of photodynamic priming (PDP), a low-dose, sub-cytotoxic variant of PDT, to overcome PFAS-induced platinum resistance. Comparative studies of PDP efficacy using either benzoporphyrin derivative (BPD) or 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) were conducted in two human ovarian cancer cell lines (NIH:OVCAR-3 and Caov-3). BPD and PpIX are clinically-approved photosensitizers that preferentially localize to, or are partly synthesized in, mitochondria. PDP overcomes carboplatin resistance in PFAS-exposed ovarian cancer cells, demonstrating the feasibility of this approach to target the deleterious effects of environmental contaminants. Decreased survival fraction in PDP + carboplatin treated cells was accompanied by decreased mitochondrial membrane potential, suggesting that PDP modulates the mitochondrial membrane, reducing membrane potential and re-sensitizing ovarian cancer cells to carboplatin.

Graphical Abstract

graphic file with name nihms-1838711-f0009.jpg

Select per- and polyfluoroalkyl substances (PFAS) have been shown to induce resistance to carboplatin, a platinum-based chemotherapeutic agent, in two ovarian cancer cell lines. A concomitant increase in mitochondrial membrane potential was observed. In the present study, photodynamic priming (PDP) to overcome PFAS-induced resistance was explored, using either benzoporphyrin derivative (BPD) or aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX). PDP in combination with low-dose carboplatin effectively reduced survival fraction in PFAS-exposed cells. A decrease in mitochondrial membrane potential following PDP was observed, suggesting that PDP modulates mitochondrial health to re-sensitize ovarian cancer cells to chemotherapy.

INTRODUCTION

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants that have been linked to adverse female reproductive health outcomes (1–4). More specifically, select PFAS have been linked to altered menstrual cyclicity and deleterious effects in ovarian tissue such as disrupted follicle growth and fertility as well as polycystic ovarian syndrome (PCOS) and PCOS-related infertility (2, 5–10). In the context of cancer, only one study has reported increased odds of ovarian cancer after exposure to very high levels of select PFAS (11); however, our recent study was the first to associate PFAS with platinum resistance in cancer (12). Specifically, we reported that select PFAS and PFAS mixtures induced resistance to carboplatin in two ovarian cancer cell lines, potentially through altered mitochondrial function as determined by an increase in mitochondrial membrane potential (ΔΨ_m) in both cell lines post-PFAS exposure alone as well as post-PFAS exposure and carboplatin treatment.

The current standard of care for advanced-stage ovarian cancer, the most lethal gynecologic malignancy, typically consists of surgical debulking along with a combination of platinum- and taxane-based chemotherapy (13–16). While this is initially effective, recurrence occurs in greater than 75% of patients with advanced-stage disease due, in large part, to the development of platinum-resistance (17, 18). The development of complementary therapeutic approaches to overcome platinum-resistance is critical for the effective treatment of ovarian cancer. Photodynamic therapy (PDT) is a photochemistry-based treatment modality that has been shown to overcome platinum resistance in ovarian cancer and to synergize with platinum-based chemotherapy (19–22). PDT involves the activation of a photosensitizer, or light-activable molecule, with a specific wavelength of visible light leading to the transient production of reactive molecular species and subsequent photodamage that is mechanistically distinct from conventional therapies (23–27). A study by Duska and colleagues (19) showed that a targeted variant of PDT re-sensitized cisplatin-resistant cancer cells to cisplatin. We and others have shown that PDT synergizes with low-dose chemotherapy and effectively targets ovarian tumors in 3D models and mouse models of ovarian carcinomatosis (20, 21, 24, 25, 28, 29). A study by Huang et al. (30) evaluated the effectiveness of photodynamic priming (PDP), or sub-tumoricidal PDT, as part of a rationally-designed combination with nanoliposomal irinotecan in an orthotopic xenograft model of pancreatic cancer. PDP significantly decreased the incidence of tumor relapse and metastasis, increased intratumoral drug accumulation and duration of drug exposure, and enhanced progression-free and 1-year disease-free survival, compared to the chemotherapy alone. Subsequent studies have demonstrated the efficacy of PDP as an enabling technology for durable low-dose chemotherapy (31) and tumor growth inhibition with no off-target toxicity (32). These studies suggest that in the context of ovarian cancer, combining sub-therapeutic or low-dose PDT with non-mechanistically overlapping therapeutic agents effectively reduces tumor burden and overcomes platinum resistance while reducing toxicity from conventional treatments (20–22, 24).

The present study describes the use of PDP to overcome PFAS-induced platinum resistance in ovarian cancer cells. Our recently published paper demonstrated, for the first time, that PFAS-induce platinum resistance following exposure of NIH:OVCAR-3 (OVCAR-3) and Caov-3 cells to select PFAS agents: perfluoroheptanoic acid (PFHpA), perfluoropentanoic acid (PFPA), and mixtures of these agents with or without perfluorooctanoic acid (PFOA) (12). In addition to PFAS-induced platinum resistance, our study also reported that PFAS exposure, with or without carboplatin treatment, increases ΔΨ_m, an indicator of cellular health. These findings suggest that PFAS-induced platinum resistance may be related to changes in mitochondrial function or health. Since mitochondrial disruption may underlie PFAS-induced platinum resistance, here we evaluate PDP efficacy in PFAS-exposed OVCAR-3 and Caov-3 cells by comparing two clinically-approved photosensitizers that have been shown to induce mitochondrial photodamage: Benzoporphyrin derivative (BPD), which preferentially localizes to the mitochondrion and endoplasmic reticulum (ER) (33, 34) or 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX or PpIX), a product of the heme biosynthesis pathway, that is partly synthesized in mitochondria (35–38). Optimal light doses for PDP, defined here as PDT at a dose with a < 10% decrease in survival fraction, were established based on light-dose-dependent curves for BPD-PDT and ALA-PpIX-PDT. Regardless of the photosensitizer used, in all instances where PFAS-induced platinum resistance was previously reported (12), platinum resistance was overcome by PDP. BPD-PDP more effectively enhanced carboplatin efficacy compared to ALA-PpIX-PDP, indicating that, in this study, BPD was a more potent photosensitizer for PDP in PFAS-exposed ovarian cancer cells than ALA-PpIX. Additionally, ΔΨ_m decreased following PDP in PFAS-exposed and carboplatin-treated cells, suggesting that a potential mechanism by which PDP synergizes with carboplatin to overcome PFAS-induced platinum resistance is by modulating mitochondrial membranes and possibly altering mitochondrial function. This study demonstrates, for the first time, the value of PDP in targeting resistance arising from exposure to environmental contaminants such as PFAS.

MATERIALS AND METHODS

Cell culture:

Human epithelial ovarian adenocarcinoma OVCAR-3 and Caov-3 cells were obtained from the The Physical Sciences-Oncology Network Bioresource Core Facility at American Type Culture Collection (ATCC). Both cell lines were cultured as previously described (12). Briefly, OVCAR-3 cells were grown in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS, Cytiva HyClone, Marlborough, MA, USA), 0.01 mg/mL recombinant human insulin (Gibco, Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Caov-3 cells were grown in Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM, Sigma-Aldrich) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Preparation of PFAS stocks:

PFAS stocks were prepared as described previously (12). Briefly, 10 mM stock solutions of PFOA (CAS#335–67-1, Synquest Laboratories, Alachua, FL, USA), PFHpA (CAS#375–85-9, Sigma-Aldrich), and PFPA (CAS#2706–90-3, TCI America, Portland, OR, USA) were prepared in 1.0 N potassium hydroxide in methanol (referred to as “methanol” or “vehicle control”, Lab Chem Inc., Zelienople, PA, USA). Stock solutions were kept at −20°C to enhance shelf-life.

Preparation of photosensitizer stocks:

A stock solution of 165.75 mM ALA hydrochloride (Sigma-Aldrich, Catalog #A7793–500MG) was prepared by dissolving 500 mg of ALA in 10 mL cell culture grade water (unbuffered). Stock solutions of 300 μM BPD (Sigma-Aldrich, Catalog #SML0534) were prepared by dissolving 5.39 mg BPD (MW = 718.79 g/mol) in 25 mL dimethyl sulfoxide (DMSO). 100 μL aliquots of BPD were prepared and stored at −20°C. BPD concentration was confirmed prior to each use using the QuickDrop (Molecular Devices, San Jose, CA, USA).

Evaluation of PDT efficacy at baseline:

OVCAR-3 and Caov-3 cells were seeded at densities of 5,000 cells/well and 10,000 cells/well, respectively, in white-walled, clear-bottom 96-well plates. Seeding densities were determined based on experiments designed for evaluating the linear dynamic range of the CellTiter-Glo Luminescent Cell Viability Assay (Catalog #C7572, Promega Corp., Madison, WI, USA) (12). For these experiments, the efficacy of two different photosensitizers, BPD and PpIX, a downstream product of ALA, were evaluated. To mimic the timeline used for PFAS experiments, cells were allowed to grow for 24 hours prior to replacing seeding medium with fresh medium for 48 hours. For BPD-PDT, 72 hours after seeding cells were incubated with 0.25 μM BPD for 90 minutes based on previous studies evaluating effective BPD doses and incubation times (20, 24, 39). Following the incubation period, BPD-containing medium was replaced with fresh medium prior to irradiation. Plates were irradiated using a 690 nm LED irradiation platform (LEDBox, BioLambda, Sao Paulo, SP, Brazil) at an irradiance of 20.12 mW/cm². For ALA-PpIX-PDT, 72 hours after seeding cells were incubated with 1 mM ALA for 4 hours based on previous studies evaluating effective ALA doses and incubation times (37, 40, 41). Plates were irradiated using a 630 nm LED irradiation platform (BioLambda) at an irradiance of 46.30 mW/cm². Irradiation times were automatically calculated to deliver pre-determined energy densities by a controller connected to the LED platform (BlackBox Smart, BioLambda). Survival fraction was evaluated using the CellTiter-Glo assay 48 hours following PDT. Briefly, 50 μL of medium was removed from each well prior to the addition of 50 μL CellTiter-Glo reagent. Plates were then shaken orbitally for 2 minutes using the SpectraMax iD3 plate reader (Molecular Devices). After orbital shaking, plates were removed from the plate reader, covered in foil, and the CellTiter-Glo signal was allowed to stabilize for 10 minutes. After 10 minutes, luminescence was read on the plate reader.

Evaluation of PDT efficacy post-PFAS exposure:

OVCAR-3 and Caov-3 cells were seeded as described above at 5,000 cells/well and 10,000 cells/well, respectively, and allowed to grow for 24 hours prior to PFAS exposure. PFAS were administered as described previously (12). Briefly, PFOA, PFHpA, PFPA, or mixtures of these agents were prepared in serum-free medium and 2X serum-containing medium containing HEPES to control for changes in pH resulting from either the addition of PFAS (acidic) or 1.0 N potassium hydroxide in methanol (basic). Changes in pH after the addition of ALA in the media used for OVCAR-3 cells (ALA + medium = 7.27, medium = 7.35) or Caov-3 cells (ALA + medium = 7.19, medium = 7.20) were negligible. Cells were exposed to 500 nM or 2 μM PFOA, PFHpA, and PFPA in 50 μL serum-free medium for 1 hour. After 1 hour, 50 μL 500 nM or 2 μM PFOA, PFHpA, and PFPA was added in 2X serum-containing medium for a final volume of 100 μL per well for 47 hours. For PFAS mixtures, PFOA + PFHpA, PFOA + PFPA, PFHpA + PFPA, or PFOA + PFHpA + PFPA were all formulated at 1:1 or 1:1:1 ratios at concentrations totaling 2 μM (1 μM + 1 μM) for 2 PFAS mixtures or 2.25 μM (750 nM + 750 nM + 750 nM) for the triple mix. PFAS mixtures were administered in serum-free medium for 1 hour followed by exposure in 2X serum-containing medium for 47 hours. For these experiments, serum-free or 2X serum-containing medium with 1% methanol was used as the vehicle control. After the 48-hour vehicle or PFAS exposure window, methanol- or PFAS-containing medium was removed and replaced with 100 μL medium containing 0.25 μM BPD or 1 mM ALA. Cells were exposed to BPD for 90 minutes or ALA for 4 hours prior to irradiation. After 90 minutes or 4 hours, photosensitizer-containing medium was removed and 50 μL fresh medium was added to each well. BPD-exposed cells were irradiated using a 690 nm LED at an irradiance of 20.12 mW/cm² at light doses ranging from 0.03 – 1 J/cm². ALA-exposed cells were irradiated using a 630 nm LED at an irradiance of 46.30 mW/cm² at light doses ranging from 0.1 – 5 J/cm² for OVCAR-3 cells or 1 – 25 J/cm² for Caov-3 cells. After irradiation, 50 μL of additional fresh medium was added to each well. Survival fraction was measured using the CellTiter-Glo assay 48-hours post-PDT. Plates were covered in foil during transport to protect the photosensitizer-exposed cells from additional light.

Evaluation of PDP in combination with carboplatin treatment:

OVCAR-3 and Caov-3 cells were seeded at 5,000 cells/well and 10,000 cells/well, respectively, and allowed to grow for 24 hours prior to PFAS exposure. After 24 hours, individual PFAS and PFAS mixtures were exposed as described above at concentrations of 500 nM or 2 μM for PFOA, PFHpA, and PFPA or 2 μM or 2.25 μM for PFAS mixtures. PFAS and PFAS mixtures were exposed for 1 hour in serum-free medium and 47 hours in serum-containing medium. To evaluate the effectiveness of PDP in combination with carboplatin treatment, sub-cytotoxic (< 10% decrease in survival fraction) light doses were chosen for each photosensitizer and for each cell line. For BPD-PDP, 0.03 J/cm² was selected for both OVCAR-3 and Caov-3 cells due to minimal observed toxicity in PDT experiments. For ALA-PpIX-PDP, 0.1 J/cm² was selected for OVCAR-3 cells and 1 J/cm² was selected for Caov-3 cells. The experiments follow the exposure timeline described above, since OVCAR-3 and Caov-3 cells were incubated with 0.25 μM BPD for 90 minutes or 1 mM ALA for 4 hours post-PFAS exposure. After incubation with the respective photosensitizer, photosensitizer-containing medium was removed and 50 μL of fresh medium was added to each well prior to irradiation. BPD-exposed OVCAR-3 and Caov-3 cells were irradiated using a 690 nm LED irradiation platform at an irradiance of 20.12 mW/cm² at a light dose of 0.03 J/cm² (~1 second). ALA-exposed OVCAR-3 and Caov-3 cells were irradiated using a 630 nm LED irradiation platform at an irradiance of 46.30 mW/cm² at a light dose of 0.1 J/cm² (~3 seconds) or 1 J/cm² (~21 seconds), respectively. After irradiation, cells were immediately treated with carboplatin (TCI America). Based on previous experiments (12), carboplatin doses ranging from 25 – 400 μM or 50 – 400 μM were used for OVCAR-3 and Caov-3 cells, respectively. Carboplatin working solutions were prepared by dissolving ~1.8563 mg carboplatin in 1 mL cell culture medium (5 mM stock). Since 50 μL of fresh medium was added to each well prior to irradiation, OVCAR-3 and Caov-3 cells were treated with 50 – 800 μM or 100 – 800 μM carboplatin, respectively, in 50 μL medium (total well volume = 100 μL). Cells were treated with carboplatin for 48 hours prior to measuring survival fraction. Plates were covered in foil during transport to protect the photosensitizer-exposed cells from additional light.

Evaluation of ΔΨ_m post-PFAS exposure, PDP, and/or carboplatin treatment:

To understand the effect of PDP on ΔΨ_m, OVCAR-3 and Caov-3 cells were seeded in black-walled, clear-bottom 96-well plates at a density of 40,000 cells/well (12). After allowing the cells to adhere and grow for 24 hours, OVCAR-3 and Caov-3 cells were concurrently exposed to PFAS (500 nM or 2μM) or PFAS mixtures (2 μM or 2.25 μM) and BPD (0.25 μM) for 90 minutes or ALA (1 mM) for 4 hours. Importantly, to provide adequate cell exposure to PFAS, both PFAS and the photosensitizers were prepared and exposed in serum-free medium. After 90 minutes or 4 hours, PFAS and photosensitizer-containing medium was removed and 50 μL fresh medium was added to each well. Plates were irradiated using a 690 nm LED irradiation platform (BPD-exposed cells) or 630 nm LED irradiation platform (ALA-exposed cells) at an irradiance of 20.12 mW/cm² or 46.30 mW/cm², respectively. PDP was performed at light doses of 0.03 J/cm² for BPD-exposed OVCAR-3 and Caov-3 cells and 0.1 J/cm² or 1 J/cm² for ALA-exposed OVCAR-3 and Caov-3 cells, respectively. After irradiation, 20 μg/mL JC-1 dye (Invitrogen, Thermo Fisher Scientific, Cat #T3168) was added in 50 μL to each well for 15 minutes. After 15 minutes, JC-1-containing medium was removed, and cells were washed with phosphate-buffered saline (PBS) prior to treatment with carboplatin. Cells were treated with 100 – 400 μM carboplatin in 100 μL complete cell culture medium for 1 hour prior to performing the JC-1 red:green aggregate ratio readout. This fluorescence readout was performed on the SpectraMax iD3 plate reader (green aggregate – excitation: 488 nm, emission: 529 nm; red aggregate – excitation: 488 nm, emission: 590 nm). Since JC-1 is easily photobleached, PDP was performed prior to incubating cells with JC-1 dye, and all dosing/readouts were performed in a minimally lighted room, and plates were covered with foil in lighted environments.

Statistical analysis:

To examine the effect of factors (e.g., PFAS concentration, PDT light dose, type of PDP, and carboplatin concentration) on outcomes of interest (e.g., survival fraction, mitochondrial membrane potential), unpaired t-tests and two-way ANOVAs were employed as appropriate, with control for potential plate effects. All tests are 2-sided at alpha level 0.05 unless otherwise specified. If a significant interaction effect is identified, the main effect of one factor (e.g., photosensitizer) is examined separately under each level of the other factor (e.g., carboplatin). All analyses were performed in R Statistical Software (v4.1.1; R Core Team 2021) (42) and Prism 9.0 software (GraphPad, San Diego, CA, USA). All supporting data and relevant analyses are available using the following link: (NIEHS CEBS link in progress; to be provided prior to publication).

RESULTS

PFAS exposure altered the efficacy of BPD-PDT or ALA-PpIX-PDT at select light doses in OVCAR-3 or Caov-3 cells

To examine the effects of PFAS exposure on the efficacy of BPD-PDT and ALA-PpIX-PDT in ovarian cancer cell lines, OVCAR-3 and Caov-3 cells were exposed to 500 nM or 2 μM PFHpA or PFPA for 48 hours prior to photosensitizer incubation and irradiation. These PFAS were selected based on our previous study demonstrating that PFHpA and PFPA, but not PFOA, induce platinum resistance in ovarian cancer cell lines (12). Since PFAS are known to bind proteins in human serum (43, 44), ovarian cancer cells were exposed to PFAS in serum-free medium for 1 hour (referred to as a serum-free pulse) followed by a 47-hour incubation with PFAS in complete medium. Absorbance spectra for each PFAS agent were also measured and were below detectable levels in the absorbance ranges of BPD and ALA (Figure S1). These data demonstrate that there is no absorbance by the chemicals at the wavelengths of light used for PDT. The effect of methanol on BPD-PDT and ALA-PpIX-PDT was also examined and data is shown in Figure S2.

BPD-PDT:

Figure 1 shows that PFAS exposure altered survival fraction at specific light doses of BPD-PDT for both cell lines. In OVCAR-3 cells following BPD-PDT, increased survival fraction was noted in the 2 μM PFHpA exposure group at 0.25 J/cm² (0.524±0.326, Fig. 1a) compared to controls. Decreased survival fractions were observed in Caov-3 cells exposed to PFHpA and treated with BPD-PDT, in the no treatment (NT, 500 nM: 0.936±0.054) BPD only (500 nM: 0.926±0.057, 2 μΜ: 0.925±0.102), light only (hν: 1.0 J/cm²) (500 nM: 0.892±0.064, 2 μΜ: 0.907±0.097), and PDT only (hν: 0.03 J/cm²) (500 nM: 0.892±0.078, 2 μΜ: 0.882±0.095) groups, compared to controls (Fig. 1c). No significant changes in survival fraction were evident in OVCAR-3 or Caov-3 cells exposed to PFPA (Fig. 1b,d). Additionally, although PFHpA and PFPA were the focus of this study, the effects of PFOA on BPD-PDT were measured in OVCAR-3 and Caov-3 cells. In OVCAR-3 cells exposed to 500 nM PFOA, increased survival fraction was observed following BPD-PDT at a light dose of 0.5 J/cm² (0.140±0.135). Decreased survival fractions were observed in Caov-3 cells exposed to PFOA following BPD-PDT at 0.03 J/cm² (500 nM: 0.925±0.092, 2 μΜ: 0.908±0.085), 0.05 J/cm² (2 μΜ: 0.851±0.123), and 0.07 J/cm² (2 μΜ: 0.816±0.097) groups as well as the in the light only group (hν:1.0 J/cm²) (500 nM: 0.910±0.149). Among Caov-3 cells exposed to PFOA, increased survival fraction was only observed in the group that received BPD-PDT at a light dose of 0.25 J/cm² (500 nM: 0.373±0.277, 2 μΜ: 0.240±0.291) (Figure S3). While exposure to select PFAS led to significant changes in survival fraction compared to the vehicle control, the aim of PDP is to use the lowest light dose possible (e.g. a dose that is limited to a < 10% decrease in survival fraction). Since changes in survival fraction were limited to ~ 10% at the lowest light dose tested for BPD-PDT in OVCAR-3 and Caov-3 cells, 0.03 J/cm² was selected for BPD-PDP for the remainder of experiments.

ALA-PpIX-PDT:

Compared to BPD-PDT, PFAS-exposed cells treated with ALA-PpIX-PDT displayed more instances of increased survival fractions compared to vehicle-exposed cells. After PFHpA exposure and treatment with ALA-PpIX-PDT, OVCAR-3 survival fractions increased at 2.5 J/cm² (0.788±0.122) and 5.0 J/cm² (0.505±0.058, Fig. 1e). Survival fractions were also increased in the NT (2 μM: 1.073±0.062) and ALA only (2 μM: 1.134±0.128) groups. In PFHpA-exposed Caov-3 cells, survival fraction only increased in the light only group (hν: 25.0 J/cm²) (2 μM: 1.093±0.151, Fig. 1g) compared to controls. In PFPA-exposed cells, survival fraction was unchanged in Caov-3 cells (Fig. 1h) following ALA-PpIX-PDT; however, OVCAR-3 cell survival fraction was increased following ALA-PpIX-PDT at 0.10 J/cm² (500 nM: 1.298±0.302), 0.25 J/cm² (2 μM: 1.212±0.287), 0.50 J/cm² (2 μM: 1.168±0.228), 1.50 J/cm² (500 nM: 1.138±0.238) and 5.0 J/cm² (500 nM: 0.544±0.098, 2 μM: 0.498±0.071, Fig. 1f). The effects of PFOA on ALA-PpIX-PDT were also measured in OVCAR-3 and Caov-3 cells, and no significant changes in survival fraction, compared to controls, were observed at any light dose (Figure S3). Based on these data presented for ALA-PpIX-PDT in PFAS-exposed cells, light doses of 0.1 J/cm² and 1.0 J/cm² were selected for ALA-PpIX-PDP in OVCAR-3 and Caov-3 cells, respectively.

Exposure to mixtures of PFAS similarly affected the efficacy of BPD-PDT or ALA-PpIX-PDT at select light doses in OVCAR-3 and Caov-3 cells

Since PFAS mixtures are more relevant to human exposure than individual agents, the effects of PFAS mixtures on BPD-PDT and ALA-PpIX-PDT dose-response curves were examined. Because our previous study (12) demonstrated that 1 μM PFHpA + 1 μM PFPA and 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA induced platinum resistance in OVCAR-3 and Caov-3 cells, respectively, those mixtures were prioritized in these experiments. Generally, BPD-PDT was less sensitive than ALA-PpIX-PDT in both OVCAR-3 and Caov-3 exposed to PFAS mixtures (Fig. 2).

Figure 2. — Relative to the vehicle control, decreased survival fractions post-BPD-PDT were observed in OVCAR-3 cells exposed to (a) 1 μM PFHpA + 1 μM PFPA and (b) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA at 0.10 J/cm² and 0.25 J/cm². Decreased survival fraction in Caov-3 cells post-BPD-PDT was noted for (c) 1 μM PFHpA + 1 μM PFPA and (d) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA at 0.10 – 1.0 J/cm² and 0.07 – 1.0 J/cm², respectively. Increased survival fractions were noted in both cell lines after ALA-PpIX-PDT. OVCAR-3 cells exposed to (e) 1 μM PFHpA + 1 μM PFPA displayed increased survival fractions compared to controls at 0.25 J/cm², 0.50 J/cm², 1.00 J/cm², and 5.00 J/cm² while no significant changes in survival fraction were observed after (f) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA. Caov-3 survival fraction post-ALA-PpIX-PDT increased after exposure to (g) 1 μM PFHpA + 1 μM PFPA and (h) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA at light doses ranging from 5.0 – 25.0 J/cm². Data are expressed as mean ± SD and are represented as a percentage of the NT group for each respective medium condition (n= at least 3 independent experiments in duplicate). Significant differences between exposure groups at each light dose are denoted by * (p < 0.05).

BPD-PDT:

Similar to the effects reported after exposure to individual PFAS agents, PFAS mixtures influenced survival fractions only at select light doses in BPD-PDT-treated ovarian cancer cells. Interestingly, only decreased survival fractions were noted in both OVCAR-3 and Caov-3 cells, suggesting PFAS mixtures increase the sensitivity of ovarian cancer cells to BPD-PDT. In OVCAR-3 cells, survival fraction decreased in 1 μM PFHpA + 1 μM PFPA- (0.235±0.058, 0.094±0.051) and 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed cells (0.350±0.190, 0.091±0.042) treated with BPD-PDT at light doses of 0.10 J/cm² and 0.25 J/cm², compared to controls (Fig. 2a-b). Compared to OVCAR-3 cells, Caov-3 cells displayed more instances of decreased survival fraction following exposure to PFAS mixtures, suggesting their increased sensitivity to the effects of PFAS mixtures. In Caov-3 cells, survival fraction decreased after BPD-PDT at light doses of 0.10 J/cm² (0.318±0.247), 0.25 J/cm² (0.020±0.010), 0.5 J/cm² (0.010±0.009), and 1.0 J/cm² (0.015±0.012) in the 1 μM PFHpA + 1 μM PFPA exposure group (Fig. 2c) and at light doses of 0.07 J/cm² (0.584±0.314), 0.10 J/cm² (0.249±0.215), 0.25 J/cm² (0.014±0.010), 0.5 J/cm² (0.008±0.007), and 1.0 J/cm² (0.010±0.007) in the 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA exposure group (Fig. 2d) compared to controls. Similar to the effects observed in OVCAR-3 and Caov-3 cells after exposure to PFHpA + PFPA or PFOA + PFHpA + PFPA and treatment with BPD-PDT, survival fraction decreased in OVCAR-3 cells after BPD-PDT in 1 μM PFOA + 1 μM PFHpA or 1 μM PFOA + 1 μM PFPA exposure groups at light doses of 0.1 J/cm² or 0.1 J/cm² and 0.25 J/cm², respectively. (Figure S4a-b). Survival fraction decreased in Caov-3 cells treated with BPD-PDT at light doses of 0.1 J/cm², 0.25 J/cm², 0.5 J/cm², and 1.0 J/cm² in the 1 μM PFOA + 1 μM PFHpA and 1 μM PFOA + 1 μM PFPA exposure groups (Figure S4c-d), compared to non-PFAS exposed cells.

As observed with the individual PFAS agents in the previous section, differences in survival fraction of < 10% were observed in cells exposed to PFAS mixtures at the lowest light doses tested. While this may be a consequence of assay sensitivity, the lowest light dose tested for BPD-PDT (0.03 J/cm²) was selected for the remainder of the PDP experiments.

ALA-PpIX-PDT:

Compared to BPD-PDT, ALA-PpIX-PDT curves tended to shift upward (increased survival fraction) after exposure to PFAS mixtures, notably in Caov-3 cells. An upward shift of the curve indicated that light doses that were once sufficient at decreasing survival fraction were no longer as effective after exposure to PFAS mixtures. This effect differed from that observed in ovarian cancer cells exposed to PFAS mixtures then treated with BPD-PDT, since only decreased survival fractions were reported after BPD-PDT treatment. OVCAR-3 cells did not appear to be as sensitive to PFAS mixtures after ALA-PpIX-PDT as Caov-3 cells since survival fraction only increased at light doses of 0.25 J/cm² (1.167±0.126), 0.5 J/cm², 1.0 J/cm² (1.119±0.121), and 5.0 J/cm² in the 1 μM PFHpA + 1 μM PFPA exposure group compared to controls (Fig. 2e). Caov-3 cells were more sensitive to exposure to PFAS mixtures than OVCAR-3 cells, since survival fractions significantly increased following ALA-PpIX-PDT in cells exposed to 1 μM PFHpA + 1 μM PFPA (0.970±0.108 – 0.575±0.263) and 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA (0.951±0.117 – 0.609±0.257) at light doses ranging from 5.0 J/cm² – 25.0 J/cm² (Fig. 2g-h). Interestingly, significant rightward shifts (i.e. decreased response) of the ALA-PpIX-PDT curve were observed in Caov-3 cells exposed to 1 μM PFOA + 1 μM PFHpA and 1 μM PFOA + 1 μM PFPA as well (Figure S4g-h), possibly indicating that the PFAS mixtures tested may interfere with the heme biosynthesis pathway and PpIX production. Despite survival fractions increasing in OVCAR-3 and Caov-3 cells after exposure to PFAS mixture and ALA-PpIX-PDT, minimal changes in survival fraction (< 10%) were observed at the lowest light dose tested (0.1 J/cm² for OVCAR-3; 1 J/cm² for Caov-3), thus these doses were selected for PDP as well.

BPD-PDP was more effective than ALA-PpIX-PDP at reducing survival fraction in combination with carboplatin in PFAS-exposed ovarian cancer cell lines

One goal of PDP in this study was to decrease the carboplatin dose to ultimately mitigate off-target toxicity without compromising efficacy. In Fig. 3, the potencies of BPD-PDP and ALA-PpIX-PDP were compared in OVCAR-3 cells after exposure to PFHpA and PFPA + 50 – 200 μM carboplatin and Caov-3 cells after exposure to PFHpA and PFPA + 100 – 400 μM carboplatin treatment. These groups were compared to re-analyzed data from our previously published study (12) (grey bars, all treatment groups normalized to the vehicle control (no carboplatin) instead of vehicle control at each respective carboplatin dose) representing PFHpA or PFPA + carboplatin only (no PDP), to determine if there was a significant reduction in survival fraction at each respective carboplatin dose.

Figure 3. — Relative to the vehicle control (dashed line), survival fraction significantly decreased in OVCAR-3 cells exposed to (a) 500 nM PFHpA and (b) 2 μM PFHpA after treatment with BPD-PDP (hν: 0.03 J/cm²) + 50 – 100 μM and 50 – 200 μM carboplatin, respectively. Treatment with BPD-PDP (hν: 0.03 J/cm²) + 100 – 400 μM carboplatin also reduced survival fraction in (c) 500 nM PFHpA and (d) 2 μM PFHpA-exposed Caov-3 cells. Survival fraction only decreased in OVCAR-3 cells exposed to (e) 500 nM PFPA after BPD-PDP (hν: 0.03 J/cm²) + 100 μM carboplatin but decreased significantly in (f) 2 μM PFPA-exposed cells after BPD-PDP (hν: 0.03 J/cm²) and ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 – 200 μM carboplatin treatment. Survival fraction also decreased in Caov-3 cells after (g) 500 nM PFPA and 2 μM PFPA exposure in BPD-PDP + 100 – 400 μM and ALA-PpIX-PDP (hν: 1.0 J/cm²) + 100 μM carboplatin groups. Grey bars represent previously published PFAS + carboplatin only (no PDP) data that has been re-analyzed (all treatment groups normalized to the vehicle control (no carboplatin) instead of vehicle control at each respective carboplatin dose). Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n= at least 3 independent experiments in duplicate). BPD-PDP was performed using 690 nm light at an energy density of 0.03 J/cm²; ALA-PpIX-PDP was performed using 630 nm light at an energy density of 0.1 J/cm² for OVCAR-3 cells and 1.0 J/cm² for Caov-3 cells. Significant differences between PDP groups and the PFAS + carboplatin only group (grey bar) at each carboplatin dose are denoted by * (p < 0.05).

BPD-PDP:

After PFHpA or PFPA exposure in ovarian cancer cells, treatment with BPD-PDP + carboplatin significantly decreased survival fraction compared to PFAS + carboplatin only (no PDP) controls (Fig. 3, grey bars). A significant reduction in survival fraction was observed in OVCAR-3 cells exposed to PFHpA followed by BPD-PDP (hν: 0.03 J/cm²) then treatment with 50 – 200 μM carboplatin at 500 nM PFHpA (50 μM carboplatin: 0.387±0.213, 100 μM carboplatin: 0.166±0.088) or 2 μM PFHpA (50 μM carboplatin: 0.378±0.172, 100 μM carboplatin: 0.167±0.092, 200 μM carboplatin: 0.077±0.030) (Fig. 3a-b). BPD-PDP (hν: 0.03 J/cm²) in combination with 25 μM carboplatin was also investigated in OVCAR-3 cells, and the data is shown in Figure S5. Similar to OVCAR-3 cells, survival fraction decreased in Caov-3 cells after BPD-PDP (hν: 0.03 J/cm²) + 100 – 400 μM carboplatin treatment following exposure to 500 nM PFHpA (100 μM carboplatin: 0.527±0.124, 200 μM carboplatin: 0.207±0.065, 400 μM carboplatin: 0.049±0.025) and 2 μM PFHpA (100 μM carboplatin: 0.448±0.078, 200 μM carboplatin: 0.165±0.045, 400 μM carboplatin: 0.046±0.019) (Fig. 3c-d). Decreased survival fraction was also observed in PFOA-exposed OVCAR-3 and Caov-3 cell after BPD-PDP (hν: 0.03 J/cm²) + 50 – 400 μM carboplatin (Figure S5e-h).

The efficacy of BPD-PDP + carboplatin in reducing survival fraction compared to PFAS + carboplatin only (no PDP) treated cells was also compared after exposure to PFAS mixtures. Interestingly, in OVCAR-3 cells, BPD-PDP (hν: 0.03 J/cm²) + 100 – 400 μM carboplatin had no effect on survival fraction in 1 μM PFHpA + 1 μM PFPA-exposed cells compared to the PFAS + carboplatin only (no PDP) group (Fig. 4a). Survival fraction decreased in 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed OVCAR-3 cells after treatment with BPD-PDP (hν: 0.03 J/cm²) + 100 – 200 μM carboplatin (0.102±0.024 – 0.061±0.008, Fig. 4b) and in the 1 μM PFOA + 1 μM PFPA group after BPD-PDP (hν: 0.03 J/cm²) + 200 μM carboplatin (Figure S6b). BPD-PDP (hν: 0.03 J/cm²) + 100 – 400 μM carboplatin significantly reduced survival fraction compared to the PFAS + carboplatin only (no PDP) group in Caov-3 cells exposed to 1 μM PFHpA + 1 μM PFPA (200 μM: 0.192±0.118, Fig. 4c), 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA (100 μM: 0.386±0.122, 200 μM: 0.191±0.101, 400 μM: 0.072±0.059, Fig. 4d), and 1 μM PFOA + 1 μM PFPA (100 μM: 0.404±0.144, 200 μM: 0.200±0.101, Figure S6d) as well.

Figure 4. — Relative to the vehicle control (dashed line), survival fraction did not significantly change in OVCAR-3 cells exposed to (a) 1 μM PFHpA + 1 μM PFPA + BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 0.10 J/cm²) + carboplatin at any dose while survival fraction significantly decreased in (b) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed cells after treatment with BPD-PDP (hν: 0.03 J/cm²) + 100 – 200 μM carboplatin and ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 μM carboplatin. Caov-3 cell survival fraction significantly decreased after BPD-PDP (hν: 0.03 J/cm²) + 200 μM carboplatin in (c) 1 μM PFHpA + 1 μM PFPA-exposed cells, and significantly decreased after BPD-PDP (hν: 0.03 J/cm²) + 100 – 400 μM carboplatin in (d) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed cells. Grey bars represent previously published PFAS + carboplatin only (no PDP) data that has been re-analyzed (all treatment groups normalized to the vehicle control (no carboplatin) instead of vehicle control at each respective carboplatin dose). Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n= at least 4 independent experiments in duplicate). BPD-PDP was performed using 690 nm light at an energy density of 0.03 J/cm²; ALA-PpIX-PDP was performed using 630 nm light at an energy density of 0.1 J/cm² for OVCAR-3 cells and 1.0 J/cm² for Caov-3 cells. Significant differences between PDP groups and the PFAS + carboplatin only group (grey bar) at each carboplatin dose are denoted by * (p < 0.05).

ALA-PpIX-PDP:

Compared to BPD, ALA-PpIX was a less potent photosensitizer for PDP in ovarian cancer cells exposed to select PFAS. In most cases, survival fraction in ovarian cancer cells post-PFAS exposure and ALA-PpIX-PDP + carboplatin treatment did not significantly differ from the PFAS + carboplatin only (no PDP) control (Figs. 3–4, grey bars). Specifically, in OVCAR-3 cells, survival fraction was unchanged after ALA-PpIX-PDP (hν: 0.10 J/cm²) + carboplatin in PFHpA-exposed cells (Fig. 3a-b), while survival fraction decreased in 2 μM PFPA-exposed cells after ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 – 200 μM carboplatin (100 μM: 0.179±0.032, 200 μM: 0.085±0.029, Fig. 3f) and in PFOA-exposed cells after ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 μM carboplatin (0.164±0.016, Figure S5e). In Caov-3 cells, only two instances of decreased survival fraction were noted compared to the PFAS + carboplatin only (no PDP) control in the PFPA-exposure group after ALA-PpIX-PDP (hν: 1.0 J/cm²) + 100 μM carboplatin treatment (500 nM: 0.605±0.064, 2 μM: 0.589±0.044, Fig. 3g-h). Interestingly, more instances of increased survival fraction (5) were noted in Caov-3 cells after ALA-PpIX-PDP (hν: 1.0 J/cm²) + carboplatin in cells exposed to 500 nM (0.210±0.070) or 2 μM PFHpA (0.173±0.048) + 400 μM carboplatin, 500 nM PFPA + 400 μM carboplatin (0.146±0.059), and 500 nM (0.762±0.093, 0.180±0.051) or 2 μM PFOA (0.760±0.067, 0.217±0.111) + 100 μM and 400 μM carboplatin (Fig. 3c,d,g and Figure S5g-h).

In the context of PFAS mixtures, only one instance of significant reduction in survival fraction was observed with ALA-PpIX-PDP followed by carboplatin. This significant reduction was observed in OVCAR-3 cells exposed to 750 nM PFOA + 750 nM PFHpA + 750 nM, subjected to ALA-PpIX-PDP (hν: 0.10 J/cm²) and then treated with 100 μM carboplatin: 0.116± 0.020, Fig. 4b. No other significant changes in survival fraction post- exposure to PFAS mixtures followed by ALA-PpIX-PDP (hν: 0.10 J/cm² for OVCAR-3 or 1.0 J/cm² for Caov-3) + carboplatin treatment were noted (Fig. 4, Figure S6).

Together, these findings suggest that BPD is a more potent photosensitizer than ALA-PpIX for PDP in ovarian cancer cells exposed to select PFAS. Even when statistical significance was not achieved, BPD-PDP typically decreased survival fraction to a greater extent than ALA-PpIX-PDP, especially in Caov-3 cells. Additionally, significant decreases in survival fraction for BPD-PDP were observed at lower doses of carboplatin (50 – 200 μM rather than 400 μM), while survival fractions in ALA-PpIX-PDP + carboplatin treated groups actually increased in some cases. Interestingly, in both OVCAR-3 and Caov-3 cells, significant interactions between BPD-PDP + 100 μM carboplatin or ALA-PpIX-PDP + 100 μM carboplatin (indicative of the priming effects), were only observed in exposure groups where PFAS-induced platinum resistance was not observed (Fig. 5; OVCAR-3: 2 μM PFOA [p = 0.013]; Caov-3: 500 nM PFOA [p = 0.032], 2 μM PFHpA [p = 0.024], 1 μM PFOA + 1 μM PFHpA [p = 0.022], 1 μM @PFOA + 1 μM PFPA [p = 0.008]). Among the groups in which PFAS-induced platinum resistance was observed, no significant interactions between BPD-PDP + 100 μM carboplatin or ALA-PpIX-PDP + 100 μM carboplatin were observed, indicating that both photosensitizers were equally effective at overcoming resistance (Figures S7-8). Photosensitizer-dependent priming efficacy was only compared at 100 μM carboplatin to determine whether BPD or ALA-PpIX was more effective at potentiating the effects of a low dose of carboplatin.

Figure 5. — The interaction between BPD-PDP (hν: 0.03 J/cm²) + 100 μM carboplatin and ALA-PpIX-PDP (hν: 0.10 J/cm² for OVCAR-3 and 1.0 J/cm² for Caov-3) + 100 μM carboplatin was significant in OVCAR-3 cells in (a) 2 μM PFOA-exposed cells [5] and Caov-3 cells in (b) 500 nM PFOA- [p = 0.032], (c) 2 μM PFHpA- [p = 0.024], (d) 1 μM PFOA + 1 μM PFHpA- [p = 0.022], and (e) 1 μM PFOA + 1 μM PFPA-exposed cells [p = 0.008].

Since an interaction indicates that one photosensitizer (BPD or ALA-PpIX) is more potent than the other, further analyses were performed to determine whether BPD-PDP was more effective than ALA-PpIX-PDP in the absence and presence of 100 μM carboplatin (Tables 1 and S1, analyses illustrating p values for BPD-PDP potency without carboplatin and + 100 μM carboplatin can be found at [insert CEBS data sharing link] below each graph in the row labeled “PhotosensitizerBPD-PDP). Analyses revealed that, out of the groups where platinum resistance was observed, BPD-PDP was more potent than ALA-PpIX-PDP in the absence of 100 μM carboplatin in OVCAR-3 cells exposed to 500 nM PFHpA and 1 μM PFHpA + 1 μM PFPA and in Caov-3 cells exposed to 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA (Table 1). BPD-PDP + 100 μM carboplatin was also more potent than ALA-PpIX-PDP + 100 μM carboplatin in Caov-3 cells exposed to 2 μM PFPA. Among all groups that received PDP alone (no carboplatin), BPD-PDP was more potent than ALA-PpIX-PDP in 12 exposure groups in both OVCAR-3 and Caov-3 cells, while BPD-PDP + 100 μM carboplatin was more potent than ALA-PpIX-PDP + 100 μM carboplatin in 7 exposure groups across the two cell lines (Table S1). These findings suggest that BPD-PDP alone and in combination with carboplatin more effectively reduced survival fraction than ALA-PpIX-PDP, and was more effective than ALA-PpIX-PDP at overcoming a platinum resistance in ovarian cancer cells exposed to select PFAS.

Table 1.

Summary of results. Data illustrating platinum resistance, PDP overcoming PFAS-induced platinum resistance, BPD-PDP (hν: 0.03 J/cm²) potency with or without carboplatin, and ΔΨ_m post-BPD-PDP (hν: 0.03 J/cm²) and ALA-PpIX-PDP (hν: 0.10 J/cm² for OVCAR-3 and 1 J/cm² for Caov-3) are shown in Figure 6, Figures 3, 4, and 6, Figures 5, S7, and S8, and Figures 7, 8, S13, and S14, respectively.

	Platinum Resistance	PDP Overcomes Resistance	Potency of BPD-PDP > ALA-PpIX-PDP	Potency of BPD-PDP + 100 μM Carboplatin > ALA-PpIX-PDP + 100 μM Carboplatin	ΔΨ_m Post- BPD-PDP	ΔΨ_m Post- ALA-PpIX-PDP
OVCAR-3
500 nM PFHpA	Yes	Yes	Yes	No	↓	↓
2 μM PFHpA	Yes	Yes	No	No	↓	↓
2 μM PFPA	Yes	Yes	No	No	↓	↓
1 μM PFHpA + 1 μM PFPA	Yes	Yes	Yes	No	↓	↓
Caov-3
2 μM PFPA	Yes	Yes	No	Yes	↓	No Change
750 nM PFOA + 750 nM PFHpA + 750 nM PFPA	Yes	Yes	Yes	No	↓	No Change

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Combination of BPD-PDP or ALA-PpIX-PDP with carboplatin significantly decreased survival fraction and overcame PFAS-induced platinum resistance in OVCAR-3 and Caov-3 cells

Our previous study (12) demonstrated that select PFAS, including PFHpA and PFPA, and PFAS mixtures increased survival fraction in ovarian cancer cells following treatment with carboplatin, indicative of platinum resistance (Fig. 6, grey bars). Since PDT has been shown to synergize with platinum-based chemotherapy, and to reverse platinum resistance in ovarian cancer (19, 20), the ability of BPD-PDP and ALA-PpIX-PDP to overcome PFAS-induced platinum resistance was examined. Importantly, to determine whether light affected the survival fraction of ovarian cancer cells, OVCAR-3 and Caov-3 cells exposed to PFAS- and PFAS mixtures were treated with 630 nm (hν: 0.10 J/cm² for OVCAR-3 or 1.0 J/cm² for Caov-3) or 690 nm (hν: 0.03 J/cm²) light (no photosensitizer) in combination with carboplatin (Figures S9-12). Of the 200- treatment groups evaluated, significant differences in survival fraction were observed in three groups (two increases and one decrease), relative to no light controls (Figure S9). It is important to note that while an increase survival fraction was observed in OVCAR-3 cells exposed to 2 μM PFPA and treated with 630 nm light (hν: 0.10 J/cm²), irradiation with light alone was likely not responsible for the observed increase, since a similar survival fraction was observed in cells exposed to 2 μM PFPA alone. An increase in survival fraction was also observed in OVCAR-3 cells exposed to 500 nM PFHpA then treated with 690 nm light (hν: 0.03 J/cm²) and 400 μM carboplatin; however, this result is very similar to our previously published observation showing that survival fraction increased in 500 nM PFHpA-exposed OVCAR-3 cells treated with 400 μM carboplatin. As a result, it is likely that this effect is an artifact of PFAS exposure rather than treatment with light. Lastly, a significant decrease in survival fraction was observed in OVCAR-3 cells exposed to 2 μM PFOA and treated with 690 nm light (hν: 0.03 J/cm²) followed by 200 μM carboplatin, but the substantial variability in this group is noteworthy.

Figure 6. — PDP prior to carboplatin reduced survival fraction to levels that are similar to vehicle controls (dashed line, no PFAS) in OVCAR-3 and Caov-3 cells exposed to PFAS, or mixtures of PFAS, based on the following groups: (a) 2 μM PFHpA + BPD-PDP (hν: 0.03 J/cm²) + 50 μM carboplatin, (b) 2 μM PFPA + BPD-PDP (hν: 0.03 J/cm²) + 50 μM carboplatin, (c) 2 μM PFPA + BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 μM carboplatin, (d) 2 μM PFPA + BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 0.10 J/cm²) + 200 μM carboplatin, (e) 500 nM PFHpA + ALA-PpIX-PDP (hν: 0.10 J/cm²) + 400 μM carboplatin, (f) 2 μM PFHpA + ALA-PpIX-PDP (hν: 0.10 J/cm²) + 400 μM carboplatin, (g) 1 μM PFHpA + 1 μM PFPA + BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 0.10 J/cm²) + 400 μM carboplatin, (h) 2 μM PFPA + BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 1.0 J/cm²) + 400 μM carboplatin, and (i) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA + BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 1.0 J/cm²) + 400 μM carboplatin. Grey bars represent previously published PFAS + carboplatin only (no PDP) data. Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n= at least 3 independent experiments in duplicate). BPD-PDP was performed using 690 nm light at an energy density of 0.03 J/cm²; ALA-PpIX-PDP was performed using 630 nm light at an energy density of 0.1 J/cm² for OVCAR-3 cells and 1.0 J/cm² for Caov-3 cells. Significant differences between exposure groups and the vehicle control are denoted by * (p < 0.05). Significant differences between the PFAS + carboplatin only group (no PDP, grey bars) and the PFAS + PDP + carboplatin groups are denoted by # (p < 0.05).

BPD-PDP:

In all scenarios of PFAS exposure + carboplatin treatment where platinum resistance was previously observed (12), BPD-PDP (hν: 0.03 J/cm²) in combination with carboplatin overcame PFAS-induced resistance (Fig. 6). Although platinum resistance in OVCAR-3 cells exposed to individual PFAS agents was previously reported by us at doses of carboplatin as high as 400 μM carboplatin, in the present study only doses from 25 to 200 μM carboplatin were examined in combination with BPD-PDP since a goal of PDP is to use lower doses of each of the individual modalities. In OVCAR-3 cells, platinum resistance was previously observed in cells exposed to 2 μM PFHpA then treated with 50 μM carboplatin and in cells exposed to 2 μM PFPA then treated with 50 – 200 μM carboplatin (Fig. 6a-d, grey bars). Importantly, when PFAS-exposed OVCAR-3 cells were treated with BPD-PDP (hν: 0.03 J/cm²) prior to carboplatin, survival fractions were significantly lower, relative to the PFAS + carboplatin only groups, and did not significantly differ from the vehicle control in 2 μM PFHpA + 50 μM carboplatin (0.871±0.132) or 2 μM PFPA + 50 – 200 μM carboplatin-exposed (0.922±0.110, 0.897±0.094, 0.988±0.151) cells (Fig. 6a-d).

Since Caov-3 cells are less sensitive than OVCAR-3 cells to carboplatin (12), BPD-PDP (hν: 0.03 J/cm²) was administered in combination with 100 – 400 μM carboplatin treatment. Our previous study (12) reported that in Caov-3 cells, only 2 μM PFPA induced carboplatin resistance at a dose of 400 μM (Fig. 6h, grey bar). In the present study, BPD-PDP (hν: 0.03 J/cm²) in combination with 400 μM carboplatin (0.966±0.153) successfully overcame the previously observed platinum resistance in Caov-3 cells exposed to 2 μM PFPA. Survival fraction decreased significantly compared to the PFAS + carboplatin only group and did not differ from the vehicle control (Fig. 6h).

In addition to individual PFAS agents inducing carboplatin resistance in ovarian cancer cell lines, select PFAS mixtures also induced platinum resistance in cells treated with 400 μM carboplatin (12). Specifically, resistance was observed in OVCAR-3 cells exposed to 1 μM PFHpA + 1 μM PFPA and Caov-3 cells exposed to 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA (Fig. 6g,i, grey bars). Platinum resistance induced by PFAS mixtures was also overcome by BPD-PDP in combination with carboplatin. Survival fractions of 1 μM PFHpA + 1 μM PFPA-exposed OVCAR-3 cells (0.893±0.105) and 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed Caov-3 cells (0.840±0.182) treated with BPD-PDP (hν: 0.03 J/cm²) + 400 μM carboplatin were significantly lower than the comparable groups without PDP (PFAS mixture + carboplatin only group), and not significantly different from the vehicle control. These findings demonstrate that BPD-PDP in combination with carboplatin can overcome PFAS- and PFAS mixture-induced resistance in OVCAR-3 and Caov-3 cells.

ALA-PpIX-PDP:

Similar to BPD-PDP, ALA-PpIX-PDP in combination with carboplatin successfully overcame PFAS-induced resistance in all exposure groups where platinum resistance was previously reported (12) (Fig. 6, grey bars). Since ALA-PpIX was expected to be less potent than BPD, based on the dose response curves shown in Figs. 1 and 2, ALA-PpIX-PDP treated OVCAR-3 (hν: 0.10 J/cm²) and Caov-3 (hν: 1.0 J/cm²) cells received 100 – 400 μM carboplatin. As outlined above, platinum resistance was observed in OVCAR-3 cells exposed to 2 μM PFHpA then treated with 50 μM carboplatin and in cells exposed to 2 μM PFPA then treated with 50 – 200 μM carboplatin (Fig. 6a-d, grey bars). Additionally, platinum resistance was observed in both 500 nM and 2 μM PFHpA-exposed OVCAR-3 cells after treatment with 400 μM carboplatin (Fig. 6e-f, grey bars). Treatment with ALA-PpIX-PDP (hν: 0.10 J/cm²) in combination with 100 – 200 μM carboplatin overcame PFAS-induced resistance in 2 μM PFPA-exposed cells (0.919±0.313, 0.755±0.170) and 400 μM carboplatin overcame PFAS-induced resistance in 500 nM (1.132±0.359) and 2 μM PFHpA-exposed (1.088±0.339) OVCAR-3 cells (Fig. 6c-f). Additionally, while platinum resistance was observed in Caov-3 after exposure to 2 μM PFPA and treatment with 400 μM carboplatin (Fig. 6h, grey bar), resistance was overcome by ALA-PpIX-PDP (hν: 1.0 J/cm²) + 400 μM carboplatin (0.949±0.178).

As described above, platinum resistance was also observed in OVCAR-3 cells exposed to 1 μM PFHpA + 1 μM PFPA and Caov-3 cells exposed to 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA then treated with 400 μM carboplatin (Fig. 6g,i, grey bars). ALA-PpIX-PDP in combination with 400 μM carboplatin overcame resistance in both OVCAR-3 (hν: 0.10 J/cm², 0.938±0.120) and Caov-3 (hν: 1.0 J/cm², 0.938±0.073) cells (Fig. 6g,i). These findings collectively demonstrate that ALA-PpIX-PDP in combination with carboplatin could be used to overcome PFAS-induced platinum resistance in OVCAR-3 and Caov-3 cells. Importantly, while ALA-PpIX-PDP + carboplatin overcame PFAS-induced platinum resistance in both ovarian cancer cell lines, BPD-PDP was statistically more effective, both alone and in combination with 100 μM carboplatin (Tables 1, S1), than ALA-PpIX-PDP, suggesting BPD is a more potent photosensitizer for overcoming PFAS-induced resistance than ALA-PpIX.

Mitochondrial membrane potential (ΔΨ_m) decreased post-PDP + carboplatin in PFAS-exposed OVCAR-3 and Caov-3 cells

Our recent study (12) demonstrated that ΔΨ_m increased in OVCAR-3 and Caov-3 cells post-PFAS exposure and post-PFAS exposure + carboplatin treatment. These findings were suggestive of improved cellular health post-PFAS exposure and warrant further investigation into mitochondrial mechanisms underlying PFAS-induced platinum resistance in ovarian cancer. To evaluate changes in ΔΨ_m following PFAS exposure and after PDP + carboplatin treatment, OVCAR-3 and Caov-3 cells were exposed to PFAS and BPD or ALA concurrently for 90 minutes or 4 hours in serum-free medium, respectively. After the incubation period, cells were irradiated using 690 nm (BPD) or 630 nm (ALA-PpIX) light at the lowest light dose evaluated per group (OVCAR-3 and Caov-3 BPD-PDP = 0.03 J/cm², OVCAR-3 ALA-PpIX-PDP = 0.1 J/cm², and Caov-3 ALA-PpIX-PDP = 1.0 J/cm²). Cells were then treated with carboplatin for 1 hour prior to performing the JC-1 readout. Consistent with our previous findings (12), ΔΨ_m increased in OVCAR-3 and Caov-3 cells exposed to PFHpA, PFPA, or PFOA then treated with 400 μM carboplatin (Fig. 7, Figure S13).

Figure 7. — Relative to the PFAS + 400 μM group (no PDP), ΔΨ_m decreased post-BPD-PDP (hν: 0.03 J/cm²) and carboplatin treatment in (a) 500 nM and 2 μM PFHpA- and (b) 500 nM (100 – 200 μM carboplatin only) and 2 μM PFPA-exposed OVCAR-3 cells along with (c) 2 μM PFHpA- and (d) 500 nM and 2 μM PFPA-exposed Caov-3 cells. ΔΨ_m also decreased post ALA-PpIX-PDP (hν: 0.10 J/cm²) and carboplatin treatment in (e) 500nM (100 and 400 μM carboplatin only) and 2 μM PFHpA- (100 – 200 μM carboplatin only) and (f) 2 μM PFPA-exposed OVCAR-3 cells. No significant differences were observed in (g) 500 nM or 2 μM PFHpA-exposed Caov-3 cells after ALA-PpIX-PDP (hν: 1.0 J/cm²) and carboplatin treatment, but ΔΨ_m decreased in (h) 500 nM PFPA-exposed cells. Data are expressed as mean ± SD and are represented as either a percentage of the vehicle control (PFAS + 400μM carboplatin, no PDP group) or as a percentage of the PFAS + 400μM carboplatin (no PDP) group for each respective medium condition (n=2 in duplicate for grey bars, n=3 independent experiments in duplicate for colored bars). Significant differences between the vehicle control and PFAS-exposed cells are denoted by * (p < 0.05), while significant differences between PDP-exposed groups and the PFAS + 400μM carboplatin (no PDP) group are denoted by # (p < 0.05).

BPD-PDP:

After treatment with BPD-PDP (hν: 0.03 J/cm²) + carboplatin, ΔΨ_m decreased significantly in OVCAR-3 and Caov-3 cells in all PFAS exposure groups where platinum resistance was previously observed (Fig. 7). In OVCAR-3 cells, ΔΨ_m decreased after BPD-PDP (hν: 0.03 J/cm²) + carboplatin compared to the PFAS + carboplatin only (no PDP) control in the 500 nM PFHpA (100 μM: 0.812±0.061, 200 μM: 0.830±0.078, 400 μM: 0.914±0.095), 2 μM PFHpA (100 μM: 0.865±0.085, 200 μM: 0.863±0.129, 400 μM: 0.877±0.193), and 2 μM PFPA exposure groups (100 μM: 1.026±0.135, 200 μM: 0.980±0.085, 400 μM: 0.943±0.121, Fig. 7a-b). Similarly, ΔΨ_m decreased in Caov-3 cells exposed to 2 μM PFPA (100 μM: 0.959±0.110, 200 μM: 0.918±0.099, 400 μM: 0.925±0.145, Fig. 7d) then treated with BPD-PDP (hν: 0.03 J/cm²) + carboplatin at all doses tested. Decreases in ΔΨ_m were also observed in OVCAR-3 and Caov-3 cells even in PFAS exposure groups that did not induce platinum resistance, including 500 nM PFPA and 2 μM PFOA for OVCAR-3 cells and 2 μM PFHpA, 500 nM PFPA, and 500 nM PFOA for Caov-3 cells (Fig. 7, Figure S13).

To evaluate the effects of PFAS mixtures on ΔΨ_m, OVCAR-3 and Caov-3 cells exposed to PFAS mixtures were treated with BPD-PDP (hν: 0.03 J/cm²) + carboplatin prior to performing the JC-1 readout. In our previous study (12), we reported that ΔΨ_m increased in OVCAR-3 and Caov-3 exposed to PFAS mixtures alone and in combination with carboplatin. In accordance with the previous report, in the present study ΔΨ_m increased in OVCAR-3 and Caov-3 exposed to 1 μM PFHpA + 1 μM PFPA or 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA then treated with 400 μM carboplatin, compared to controls (Fig. 8). The same was observed in most cases for 1 μM PFOA + 1 μM PFHpA and 1 μM PFOA + 1 μM PFPA in both cell lines (Figure S14). In OVCAR-3 and Caov-3 cells exposed to PFAS mixtures, carboplatin resistance was only observed in the 400 μM carboplatin treatment group after exposure to 1 μM PFHpA + 1 μM PFPA or 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA, respectively. In OVCAR-3 cells, ΔΨ_m decreased following BPD-PDP (hν: 0.03 J/cm²) then carboplatin at all carboplatin doses tested in cells exposed to 1 μM PFHpA + 1 μM PFPA (100 μM: 0.886±0.058, 200 μM: 0.886±0.078, 400 μM: 0.967±0.098, Fig. 8a). Interestingly, while platinum resistance was not observed after exposure to any other PFAS mixture in OVCAR-3 cells, ΔΨ_m decreased after BPD-PDP (hν: 0.03 J/cm²) + carboplatin at all doses tested in the 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA and 1 μM PFOA + 1 μM PFPA exposure groups as well (Fig. 8b, Figure S14b). In Caov-3 cells, where platinum resistance was observed in the 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA exposure group, ΔΨ_m also decreased after BPD-PDP (hν: 0.03 J/cm²) + carboplatin at all carboplatin doses tested (100 μM: 0.938±0.050, 200 μM: 0.857±0.092, 400 μM: 1.017±0.077, Fig. 8d). Although platinum resistance was not observed in other PFAS mixture groups, ΔΨ_m also decreased after BPD-PDP (hν: 0.03 J/cm²) + carboplatin treatment in Caov-3 cells exposed to 1 μM PFHpA + 1 μM PFPA or 1 μM PFOA + 1 μM PFPA (Fig. 8c, Figure S14d).

Figure 8. — Relative to the PFAS mixture + 400μM group (no PDP), ΔΨ_m decreased post-BPD-PDP (hν: 0.03 J/cm²) and carboplatin treatment in (a) 1 μM PFHpA + 1 μM PFPA- and (b) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed OVCAR-3 cells along with (c) 1 μM PFHpA + 1 μM PFPA- (100 – 200 μM carboplatin only) and (d) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed Caov-3 cells. ΔΨ_m also decreased post ALA-PpIX-PDP (hν: 0.10 J/cm² for OVCAR-3 and 1 J/cm² for Caov-3) and carboplatin treatment in (e,g) 1 μM PFHpA + 1 μM PFPA-exposed OVCAR-3 and Caov-3 cells and (h) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed Caov-3 cells. Data are expressed as mean ± SD and are represented as either a percentage of the vehicle control (PFAS + 400μM carboplatin, no PDP group) or as a percentage of the PFAS + 400μM carboplatin (no PDP) group for each respective medium condition (n=2 in duplicate for grey bars, n=3 independent experiments in duplicate for colored bars). Significant differences between the vehicle control and PFAS-exposed cells are denoted by * (p < 0.05), while significant differences between PDP-exposed groups and the PFAS + 400μM carboplatin (no PDP) group are denoted by # (p < 0.05).

ALA-PpIX-PDP:

Compared to BPD-PDP, ALA-PpIX-PDP was less effective at reducing ΔΨ_m in ovarian cancer cells, specifically Caov-3 (Fig. 7). Platinum resistance was previously observed in 500 nM PFHpA, 2 μM PFHpA, and 2 μM PFPA-exposed OVCAR-3 cells, and in each of these groups, ALA-PpIX-PDP (hν: 0.10 J/cm²) + carboplatin decreased ΔΨ_m in at least one carboplatin treatment group. Specifically, ΔΨ_m decreased after ALA-PpIX-PDP (hν: 0.10 J/cm²) + carboplatin treatment in OVCAR-3 cells exposed to 500 nM PFHpA (100 μM: 1.054±0.063, 400 μM: 1.053±0.166), 2 μM PFHpA (100 μM: 1.056±0.095, 200 μM: 1.036±0.134), and 2μM PFPA (100 μM: 0.936±0.042, 200 μM: 0.972±0.092, 400 μM: 1.087±0.208, (Fig. 7e-f). Interestingly, while ΔΨ_m decreased in OVCAR-3 cells after ALA-PpIX-PDP (hν: 0.10 J/cm²) + carboplatin in the groups in which PFAS-induced platinum resistance was previously observed, ΔΨ_m was unaffected in Caov-3 cells exposed to 2 μM PFPA (Fig. 7h). Decreased ΔΨ_m in Caov-3 cells was observed in the 500 nM PFPA exposure group, though, in which platinum resistance was not previously observed (Fig. 7h). In Caov-3 cells exposed to PFOA, no significant decrease in ΔΨ_m was observed following ALA-PpIX-PDP (hν: 1.0 J/cm²) + carboplatin (Figure S13d). A significant decrease in ΔΨ_m was observed in OVCAR-3 cells exposed to 2 μM PFOA; however, an increase in ΔΨ_m was observed in the same cells exposed to 500nM PFOA followed by ALA-PpIX-PDP (hν: 0.10 J/cm²) then 400 μM carboplatin (Figure S13b).

Relative to individual agents, ALA-PpIX-PDP + carboplatin was equally as effective as BPD-PDP + carboplatin in reducing ΔΨ_m in Caov-3 cells exposed to PFAS mixtures, but slightly less effective in OVCAR-3 cells. For example, ΔΨ_m decreased compared to the PFAS + carboplatin only (no PDP) group in OVCAR-3 cells exposed to 1 μM PFHpA + 1 μM PFPA + BPD-PDP (hν: 0.03 J/cm²) + carboplatin treatment at all carboplatin doses tested (100 μM: 1.058±0.114, 200 μM: 1.040±0.079, 400 μM: 1.006±0.179) and Caov-3 cells exposed to 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA + BPD-PDP (hν: 0.03 J/cm²) + carboplatin treatment at all platin doses tested (100 μM: 0.982±0.132, 200 μM: 0.943±0.130, 400 μM: 0.976±0.159) (Fig. 8e,h). Significant decreases in ΔΨ_m were also measured in Caov-3 cells exposed to 1 μM PFHpA + 1 μM PFPA and 1 μM PFOA + 1 μM PFPA, despite no platinum resistance being reported in either exposure group for the respective cell line (Fig. 8g, Figure S14h).

Together, these findings demonstrate that, in all exposure groups where platinum resistance was observed in OVCAR-3 and Caov-3 cells, BPD-PDP (hν: 0.03 J/cm²) + carboplatin significantly decreased ΔΨ_m compared to the PFAS + carboplatin only (no PDP) control. While ALA-PpIX-PDP (hν: 0.10 J/cm² for OVCAR-3 and 1.0 J/cm² for Caov-3) + carboplatin effectively reduced ΔΨ_m in OVCAR-3 cells, ΔΨ_m did not decrease significantly in all the PFAS exposure groups in which Caov-3 cells demonstrated platinum resistance. Additionally, in groups where platinum resistance was not observed, BPD-PDP continued to decrease ΔΨ_m in many scenarios, whereas ALA-PpIX-PDP was not always effective. These findings suggest that BPD-PDP was more effective than ALA-PpIX-PDP at altering ΔΨ_m.

DISCUSSION

PFAS are ubiquitous environmental contaminants that are known to adversely affect various bodily systems, including the female reproductive system (2, 5–9, 45, 46, 10). PFAS have been associated with adverse health effects in uterine and ovarian tissue, as well as increased risk of reproductive disorders such as PCOS and even cancer (5–7, 9, 11, 47–49). Our recent study (12) linked exposure to select PFAS with platinum resistance in ovarian cancer. Specifically, it was shown that PFHpA, PFPA, and mixtures of the agents, as well as a mixture of PFOA, PFHpA, and PFPA induce resistance to carboplatin in OVCAR-3 and/or Caov-3 cells (summarized in Table 1). Since platinum resistance is a major barrier to the effective treatment of ovarian cancer, and our prior study was the first linking environmental contaminants with platinum resistance in ovarian cancer, we sought to determine whether a mechanistically-distinct therapeutic approach, such as PDP, could overcome platinum resistance induced by environmental contaminants.

The most common route of exposure to PFAS is through consumption of contaminated drinking water sources and food supplies (1, 50, 51). Although this is the main route of exposure, PFAS are present in a variety of everyday items including food packaging, non-stick cookware, and cosmetics (50–52). PFAS are also commonly used in the apparel, automotive, electronics, textile, and medical industries (52). Medical products such as dental floss, contact lenses, microbubble-based ultrasound contrast agents, and dialysis membranes each utilize PFAS (52). In these items, PFAS are generally included for their useful chemical properties and thermal stability; however, it is possible that any exposure to PFAS, including from medical sources, can induce deleterious health effects.

It is also important to note that the concentrations of PFAS used in this study may be human relevant in contaminated communities. Since the Environmental Protection Agency recently released new health advisory levels for select PFAS (53), the estimated number of Americans living in contaminated communities and exposed to PFAS in their drinking water supply has increased from an estimated 6 million (50) to over 200 million (54). Although PFAS exposure levels differ due to many sociodemographic factors, the concentrations used in this study are within range, or within an order of magnitude, of those reported in contaminated communities (12, 55–57). Additionally, while epidemiologic studies evaluating PFAS exposure levels in ovarian cancer patients are lacking, many ovarian cancer patients present with disease later in life and may therefore be subject to increased PFAS levels as a result of a lifetime of bioaccumulation.

Prior to the current study, PDT had not been examined in the context of environmental contaminants. Thus, light dose-response curves in PFAS-exposed ovarian cancer cell lines were established for both BPD-PDT and ALA-PpIX-PDT in order to determine the appropriate light dose for PDP (< 10% decrease in survival fraction). For OVCAR-3 and Caov-3 cells, the lowest light dose used for both BPD-PDT (0.03 J/cm²) and ALA-PpIX-PDT (0.1 J/cm² or 1.0 J/cm²) were selected because survival fraction was minimally affected at these doses compared to the vehicle control. It is important to note that it remains unclear whether a lack of observed effects from PFAS exposure at these low light doses were due to limitations in assay sensitivity, since PFAS exposure had an impact on survival fraction at higher light doses. To investigate the impact of PFAS mixtures, OVCAR-3 and Caov-3 cells were exposed to mixtures of PFHpA, PFPA, or PFOA then treated with BPD-PDT or ALA-PpIX-PDT. For both cell lines, the survival fraction following treatment with BPD-PDT or ALA-PpIX-PDT at the lowest light dose used was unchanged compared to controls, confirming that the doses previously selected were appropriate for PDP after PFAS exposure. Interestingly, PFAS mixtures significantly shifted the light dose-response curve for ALA-PpIX-PDT upwards, indicating decreased sensitivity to ALA-PpIX-PDT in OVCAR-3 and Caov-3 cells post-PFAS exposure.

Whether these observations are a result of PFAS interference with the heme biosynthesis pathway remains unclear and warrants further investigation. Relative to normal cells, PpIX synthesis is often enhanced in cancer cells due to altered metabolism (58). This metabolic differential leads to the accumulation of PpIX in malignant tissues that can be further enhanced by administering exogenous ALA for fluorescence guided resection or PDP/PDT (37, 58, 38). In the heme biosynthesis pathway, there are a variety of enzymes and transporters required for the production of PpIX (35), thus interference by PFAS at any of these steps could lead to decreased PpIX production and therefore less effective treatment with ALA-PpIX-PDP/PDT. In a healthy cell, ALA is converted to uroporphyrinogen III and then coporphyrinogen III by uroporphyrinogen III synthase and uroporphyrinogen decarboxylase, respectively. Coporphyrinogen III then enters the mitochondrion via the ATP-binding cassette (ABC) B6 transporter where it becomes protoporphyrinogen IX and finally PpIX via coporphyrinogen oxidase and protoporphyrin oxidase, respectively (35). Disruptions by PFAS at any of these steps could affect PpIX production. Although no studies have examined interactions between PFAS and the heme biosynthesis pathway, PFAS are known to bind serum albumin in the blood (43, 44), which is a key carrier of heme (59). Additionally, while studies examining associations between PFAS and altered ABC transporter activity are lacking, reports have demonstrated the ability of PFAS, specifically GenX, to inhibit P-glycoprotein and breast cancer resistance protein transport activity (60). Limited studies have also shown that ABC transporters may also play key roles in PFAS transport within the body (61–63). Since endocrine-disrupting chemicals, such as PFAS, often interfere with the normal activity of transporters and enzymes, exploring PFAS-induced effects within the heme biosynthesis pathway is needed to better understand the upward shift of the ALA-PpIX-PDT dose-response curve post-PFAS mixture exposure in ovarian cancer cells.

In our recently published study (12), we reported that select PFAS and PFAS mixtures induced platinum resistance in OVCAR-3 and Caov-3 cells. Specifically, 500 nM and 2 μM PFHpA, 2 μM PFPA, 1 μM PFHpA + 1 μM PFPA, and 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA induced resistance to at least one carboplatin dose ranging from 50 – 400 μM in OVCAR-3 or Caov-3 cells, or both. Although not directly examined, it is unlikely that PFAS-induced platinum resistance is related to cross-reactivity between PFAS and carboplatin or inactivation of carboplatin by PFAS. The carbon-fluorine bonds present in PFAS are extremely strong and resistant to degradation (64, 65). Additionally, when PFAS are fully fluorinated (perfluorinated compounds), they don’t readily cross-react with other molecules. PFAS can lose their fully fluorinated status if they breakdown, which occurs very slowly at ambient temperatures, or requires the compounds to be heated to high temperatures to change the rate of degradation. A recent study by Trang et al. (66) showed that a subset of perfluorocarboxylic acids, including both PFHpA and PFPA, could be degraded through low-temperature mineralization. The temperatures typically ranged from 40 – 120° Celsius, which were higher than those in our present study. Because the compounds (PFHpA, PFPA, and PFOA) evaluated in the present study, and in our previous publication are fully fluorinated, were not heated substantially during experiments, and are not known to breakdown into shorter-chain PFAS, the potential for these chemicals to cross-react with, or to inactivate carboplatin, is unlikely. In the current study, we found that BPD-PDP or ALA-PpIX-PDP in combination with carboplatin decreased ovarian cancer cell survival fraction post-PFAS exposure, indicating that the combination of PDP + carboplatin was effective in overcoming PFAS-induced resistance (summarized in Tables 1, S1). In all instances where PFAS-induced resistance had previously been observed, treatment with BPD-PDP and/or ALA-PpIX-PDP + carboplatin decreased survival fraction in PFAS-exposed ovarian cancer cells to levels comparable to (i.e. not significantly different from) the vehicle control at each respective carboplatin dose.

Although this is the first study evaluating the ability of PDP + carboplatin to overcome environmental contaminant-induced platinum resistance in the context of any cancer, other studies have demonstrated the ability of PDT, and targeted variants, to enhance platinum efficacy and to overcome platinum resistance in ovarian cancer (19, 20, 28, 67). A study by Duska et al. (19) reported that photoimmunotherapy, a variant of PDT using antibody-based conjugates, reverses platinum resistance in ovarian cancer cell lines and primary ovarian cancer cultures. Specifically, when photoimmunotherapy was combined with cisplatin, cytotoxicity increased by 12.9-fold in platinum-resistant cells, compared to those treated with cisplatin alone. A study in a 3D model for ovarian cancer (20) demonstrated the ability of BPD-PDT to synergize with low-dose carboplatin to reduce residual tumor volume. More recently, photoimmunotherapy was shown to significantly reduce tumor burden, with decreased toxicity and fewer chemotherapy cycles than chemotherapy alone, in a mouse model for disseminated ovarian cancer (28, 67). These studies highlight that PDT in combination with chemotherapy can sensitize, or re-sensitize, ovarian cancer cells to traditional therapies. Since platinum resistance is a main contributor to the high lethality rate of ovarian cancer (17, 18) and PFAS are found in the serum of nearly all residents of developed countries (1, 68–70), further studies exploring the ability of PDP to sensitize PFAS-induced platinum resistant 3D ovarian cultures and mouse models of ovarian carcinomatosis should be explored to promote clinical integration of PDP for PFAS-exposed, platinum resistant ovarian cancer patients.

PDT dose parameters (such as photosensitizer concentration, fluence, and irradiance) have been shown to significantly impact cytotoxic efficacy (23, 27, 28, 39, 71–75). An additional consideration is the photosensitizer itself, which can be selected based on the preferential localization of the molecule or its potency. This study compares the PDP efficacy of two clinically-relevant photosensitizers that have been shown to induce mitochondrial photodamage: BPD and ALA-PpIX. BPD passively diffuses across the cell membrane and preferentially localizes to mitochondria among other organelles such as the ER (24, 33, 34). Activation of BPD leads to targeted photodamage in the corresponding subcellular organelles as well as dark toxicity and photodamage to the anti-apoptotic protein Bcl-2 (76). PpIX is the penultimate molecule in the heme biosynthesis pathway, which occurs, in part, in the mitochondria (35, 36). Heme synthesis requires the addition of iron to PpIX, by a rate-limiting enzyme, ferrochelatase, which is often mutated in cancer cells (35, 58). The administration of exogeneous ALA further increases the accumulation of PpIX in target cells and in subcellular organelles such as the mitochondria (37, 38). While the mitochondrion is, in part, the site of PpIX synthesis and is among the sites of preferential localization of BPD, a comparative analysis of PDP efficacy using these photosensitizers in the context of PFAS-induced platinum resistance is critical to developing rational combinations that enhance efficacy and minimize toxicity.

In this study, while BPD-PDP and ALA-PpIX-PDP in combination with carboplatin demonstrated comparable efficacy in OVCAR-3 cells (as evaluated by reduction in survival fraction), Caov-3 cells were more sensitive to BPD-PDP + carboplatin than ALA-PpIX-PDP + carboplatin. To better understand the role of PFAS in photosensitizer efficacy, we evaluated whether significant interactions were evident between BPD-PDP + carboplatin and ALA-PpIX-PDP + carboplatin in both OVCAR-3 and Caov-3 cells under each exposure condition. Interestingly, for both cell lines, significant differences in interactions between BPD-PDP + carboplatin and ALA-PpIX-PDP + carboplatin were only observed in PFAS exposure groups where platinum resistance was not observed. Where platinum resistance was observed in OVCAR-3 and Caov-3 cells, both BPD-PDP + carboplatin and ALA-PpIX-PDP + carboplatin were equally effective at overcoming resistance. This suggests that the mechanism by which PFAS exposure induces platinum resistance may also render cells more susceptible to PDP + carboplatin treatment and warrants further investigation. Additional analyses evaluating BPD-PDP vs. ALA-PpIX-PDP potency alone and in the presence of the lowest dose of carboplatin tested in both cell lines (100 μM) revealed that in many cases, BPD-PDP was more potent than ALA-PpIX-PDP (summarized in Table 1). Additionally, while PFOA alone, or in certain mixtures, did not induce platinum resistance in either cell line, BPD-PDP proved more effective than ALA-PpIX-PDP alone and in combination with 100 μM carboplatin in both cell lines in several of these exposure groups (Table 1). This suggests that even though PFOA does not induce platinum resistance in OVCAR-3 or Caov-3 cells at the concentrations used, PFOA may alter ovarian cancer cell biology in a way that differentially affects susceptibility to BPD-PDP versus ALA-PpIX-PDP, warranting further investigation. These findings demonstrate that BPD-PDP + carboplatin is more effective at reducing survival fraction than ALA-PpIX-PDP in Caov-3 cells and after various PFAS exposures. Whether this is due to interference by PFAS of enzymes or transporters involved in the heme biosynthesis pathway remains to be explored.

As a mechanistic basis for the observed efficacy of PDP in combination with carboplatin in PFAS-induced platinum-resistant ovarian cancer cells, ΔΨ_m was explored using the JC-1 probe in PFAS-exposed OVCAR-3 and Caov-3 cells treated with BPD-PDP or ALA-PpIX-PDP + carboplatin. In the context of PFAS-induced, platinum-resistant ovarian cancer, our previous study (12) showed that after PFAS exposure alone or PFAS exposure + carboplatin treatment, the JC-1 red:green aggregate ratio increased compared to controls, suggestive of improved cellular health. Similar findings are reported in this study, based on increased ΔΨ_m in OVCAR-3 and Caov-3 cells exposed to PFAS and PFAS mixtures then treated with 400 μM carboplatin. Interestingly, ΔΨ_m decreased even after low-dose treatment with BPD-PDP or ALA-PpIX-PDP + carboplatin compared to PFAS + 400 μM carboplatin-treated cells (no PDP, summarized in Table 1). More instances of reduction in ΔΨ_m were observed following BPD-PDP + carboplatin than were observed following ALA-PpIX-PDP + carboplatin in both ovarian cancer cell lines exposed to PFAS, but the difference in PDP-mediated ΔΨ_m reduction was especially evident in Caov-3 cells. These findings further highlight the enhanced efficacy of BPD-PDP + carboplatin in overcoming PFAS-induced resistance. It is important to note that while ΔΨ_m decreased in PFAS + PDP + carboplatin exposed cells compared to those exposed to PFAS then treated with carboplatin alone, the JC-1 aggregate ratio in OVCAR-3 and Caov-3 cells remains around 1.0 or even slightly higher. Generally, a JC-1 aggregate ratio below 1.0 is indicative of decreased cellular health and a shift towards apoptosis (77), which was reported in our previous study in OVCAR-3 and Caov-3 cells treated with carboplatin alone (without prior PFAS exposure) (12). While the JC-1 aggregate ratios in PFAS-exposed OVCAR-3 and Caov-3 cells post-PDP + carboplatin treatment remain around 1.0 (at the low doses tested), the decrease in ΔΨ_m resulting from PDP may be enough to enhance responsiveness of PFAS-exposed ovarian cancer cells to carboplatin treatment.

Our findings demonstrating that ΔΨ_m decreased following PDP are similar to those reported by others (78–85). For example, Singh et al. (84) reported that the fluorescence of Rhodamine-123, a dye that localizes to viable mitochondrial membranes, delocalized and filled the cell cytoplasm following PDT with Photofrin II as a photosensitizer, indicating that mitochondria are among the targets of Photofrin II-mediated PDT. A study by Kessel (85) also reported that following BPD-PDT, ΔΨ_m, as measured by Mitotracker orange staining, decreased with increased PDT dose. Interestingly, even when cell viability decreased significantly post-PDT, the decrease in ΔΨ_m was reversible, suggesting that the initial, transient, change in ΔΨ_m following PDT can be indicative of photokilling (85).

While many limitations accompany the use of ΔΨ_m dyes (86), there is one limitation inherent to the JC-1 dye that is particularly relevant to the current study. The JC-1 probe is easily photobleached (86), therefore it is important to optimize the assay in such a way that the JC-1 probe is not present during PDP. In this study, PFAS-exposed cells were treated with PDP prior to the administration of the JC-1 probe, informed by optimization experiments illustrating that the JC-1 readout was comparable when the dye was administered pre- and post-PDP (Figure S15). The JC-1 readout was also not affected by incubation with the photosensitizer alone or irradiation without the photosensitizer (Figure S15). While it is known that the JC-1 probe is easily photobleached, the lack of effect after irradiation may be due to the low light doses used, since irradiation time ranged from 1–21 seconds. Other studies evaluating PDT have also used the JC-1 probe to estimate ΔΨ_m and, consistent with the present study, JC-1 dye was administered after irradiation (79, 87–90). It is worth noting that administration of JC-1 dye prior to PDT has been reported (78).

Here, we show for the first time that BPD-PDP and ALA-PpIX-PDP in combination with carboplatin can be used to overcome PFAS-induced platinum resistance in OVCAR-3 and Caov-3 cells. Interestingly, while individual PFAS affected BPD- or ALA-PpIX-PDT curves only at select light doses in Caov-3 cells, PFAS mixtures reduced the efficacy of ALA-PpIX-PDT, suggesting that PFAS mixtures may interfere with the production of PpIX. Thus, further investigation is warranted into the interaction of PFAS mixtures with key enzymes and transporters involved in PpIX production. Comparing the potencies of BPD and ALA-PpIX, we observed that BPD-PDP in combination with carboplatin was more effective in decreasing survival fraction in Caov-3 cells, whereas the effectiveness of BPD-PDP versus ALA-PpIX-PDP in combination with carboplatin was largely comparable in OVCAR-3 cells. As a potential mechanism underlying the decreased survival fraction observed post-PDP + carboplatin, we report that ΔΨ_m is lowered in PFAS-exposed OVCAR-3 and Caov-3 cells treated with PDP + carboplatin compared to PFAS-exposed + carboplatin treated cells (no PDP). More instances of lowered ΔΨ_m were present in BPD-PDP + carboplatin-treated cells compared to ALA-PpIX-PDP + carboplatin treated cells, further demonstrating increased efficacy of BPD compared to ALA-PpIX. These findings suggests that the decrease in ΔΨ_m after PDP shifts OVCAR-3 and Caov-3 cells towards death (potentially apoptosis), enabling the sensitization of ovarian cancer cells previously exposed to PFAS, to subsequent treatment with carboplatin.

Supplementary Material

SUPINFO

Figure S1. Absorbance spectra for the PFAS agents evaluated in the present study. Absorbance spectra for (a) PFOA, (b) PFHpA, and (c) PFPA demonstrated no overlapping absorbance at the wavelengths relevant to BPD-PDP (690 nm) or ALA-PpIX-PDP (630 nm).

Figure S2. Effect of 1% methanol on survival fraction post-BPD-PDT (green box) or ALA-PpIX-PDT (black box) in OVCAR-3 and Caov-3 cells. Survival fraction significantly decreased in OVCAR-3 cells after exposure to 1% methanol and treatment with (a) BPD-PDT (n=3 independent experiments in duplicate) in the 0.25 J/cm², 0.5 J/cm², and 1.0 J/cm² groups while survival fraction increased after exposure to 1% methanol and treatment with (c) ALA-PpIX-PDT at all light doses examined (n= at least 3 independent experiments in quadruplicate). In Caov-3 cells exposed to 1% methanol then treated with (b) BPD-PDT, increased survival fraction was noted in the light only group (hν: 1.0 J/cm²), while decreased survival fractions were noted in cells receiving 0.25 J/cm² and 0.5 J/cm² light (n=3 independent experiments in duplicate). Increased survival fractions were also noted in methanol-exposed Caov-3 cells receiving (d) ALA-PpIX-PDT at 5.0 J/cm², 10.0 J/cm², 15.0 J/cm², and 25.0 J/cm² (n= at least 3 independent experiments in duplicate). Data are expressed as mean ± standard deviation (SD) and are represented as a percentage of the no treatment (NT) group for each respective medium condition. NT = no photosensitizer or light, BPD or ALA only = cells were treated with 0.25 μM BPD or 1 mM ALA only (no irradiation), light only = cells received no photosensitizer but were irradiated at the highest light dose per group. Significant differences between exposure groups at each light dose are denoted by * (p < 0.05).

Figure S3. Effect of PFOA on BPD-PDT (green box) and ALA-PpIX-PDT (black box) dose-response curves in OVCAR-3 and Caov-3 cells. Relative to the vehicle control, a significant increase in survival fraction post-BPD-PDT was observed in (a) 500 nM PFOA-exposed OVCAR-3 cells after receiving 0.5 J/cm² light. Significant decreases in survival fraction were observed in (c) 500 nM PFOA-exposed Caov-3 cells in the light only (hν: 1.0 J/cm²) and 0.03 J/cm² treatment groups while survival fraction increased in the 0.25 J/cm² treatment group. Survival fraction also decreased in 2 μM PFOA-exposed Caov-3 cells after BPD-PDT at 0.03 J/cm², 0.05 J/cm², and 0.07 J/cm², but increased after 0.25 J/cm². No significant changes in survival fraction post-ALA-PpIX-PDT were observed in (b) OVCAR-3 or (d) Caov-3 cells. Data are expressed as mean ± SD and are represented as a percentage of the NT group for each respective medium condition (n= at least 3 independent experiments in duplicate). Significant differences between exposure groups at each light dose are denoted by * (p < 0.05).

Figure S4. Effect of PFAS mixtures on BPD-PDT (green box) and ALA-PpIX-PDT (black box) dose-response curves in OVCAR-3 and Caov-3 cells. Relative to the vehicle control, decreased survival fractions post-BPD-PDT were observed in OVCAR-3 cells exposed to (a) 1 μM PFOA + 1 μM PFHpA and (b) 1 μM PFOA + 1 μM PFPA at 0.10 J/cm² and 0.10 J/cm² and 0.25 J/cm², respectively. Decreased survival fraction in Caov-3 cells post-BPD-PDT was noted for (c) 1 μM PFOA + 1 μM PFHpA and (d) 1 μM PFOA + 1 μM PFPA at 0.10 – 1.0 J/cm². Changes in survival fraction were not observed in (e, f) mixture-exposed OVCAR-3 cells after ALA-PpIX-PDT. Caov-3 survival fraction post-ALA-PpIX-PDT increased after exposure to (g) 1 μM PFOA + 1 μM PFHpA and (h) 1 μM PFOA + 1 μM PFPA at light doses ranging from 5.0 – 25.0 J/cm² and 2.5 – 25 J/cm², respectively. Data are expressed as mean ± SD and are represented as a percentage of the NT group for each respective medium condition (n= at least 3 independent experiments in duplicate). Significant differences between exposure groups at each light dose are denoted by * (p < 0.05).

Figure S5. Survival fraction decreased in PFAS-exposed OVCAR-3 and Caov-3 cells post-BPD-PDP and ALA-PpIX-PDP + carboplatin. Relative to the vehicle control (dashed line), survival fraction significantly decreased in OVCAR-3 cells exposed to (a) 500 nM PFHpA and (b) 2 μM PFHpA after treatment with BPD-PDP (hν: 0.03 J/cm²) and 50 – 100 μM or 50 – 200 μM carboplatin, respectively. Survival fraction also decreased in OVCAR-3 cells exposed to (c) 500 nM PFPA after BPD-PDP (hν: 0.03 J/cm²) + 100 μM carboplatin and in (d) 2 μM PFPA-exposed cells after BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 – 200 μM carboplatin. After (e,g) 500 nM PFOA exposure, survival fraction decreased post-BPD-PDP (hν: 0.03 J/cm²) + 50 – 100 μM carboplatin and ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 μM carboplatin in OVCAR-3 cells and BPD-PDP (hν: 0.03 J/cm²) + 100 – 400 μM carboplatin in Caov-3 cells. Survival fraction was unchanged in (f) 2μM PFOA-exposed OVCAR-3 cells but decreased significantly in (h) 2 μM PFOA-exposed Caov-3 cells after BPD-PDP (hν: 0.03 J/cm²) + 100 – 200 μM carboplatin. Grey bars represent previously published PFAS + carboplatin only (no PDP) data that has been re-analyzed (all treatment groups normalized to the vehicle control (no carboplatin) instead of vehicle control at each respective carboplatin dose). Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n= at least 3 independent experiments in duplicate). BPD-PDP was performed using 690 nm light at an energy density of 0.03 J/cm²; ALA-PpIX-PDP was performed using 630 nm light at an energy density of 0.1 J/cm² for OVCAR-3 cells and 1.0 J/cm² for Caov-3 cells. Significant differences between PDP groups and the PFAS + carboplatin only group (grey bar) at each carboplatin dose are denoted by * (p < 0.05).

Figure S6. Survival fraction remained largely unchanged in PFAS mixture-exposed OVCAR-3 and Caov-3 cells post-BPD-PDP or ALA-PpIX-PDP. Relative to the vehicle control (dashed line), survival fraction was unchanged in OVCAR-3 and Caov-3 cells exposed to (a,c) 1 μM PFOA + 1 μM PFHpA after treatment with BPD-PDP (hν: 0.03 J/cm²) or ALA-PpIX-PDP (hν: 0.10 J/cm² for OVCAR-3 and 1.0 J/cm² for Caov-3) + carboplatin. After 1 μM PFOA + 1 μM PFPA exposure, (b) OVCAR-3 cell survival fraction decreased after BPD-PDP (hν: 0.03 J/cm²) + 200 μM carboplatin while (d) Caov-3 cell survival fraction decreased post-BPD-PDP (hν: 0.03 J/cm²) + 100 – 200 μM carboplatin. Grey bars represent previously published PFAS + carboplatin only (no PDP) data that has been re-analyzed (all treatment groups normalized to the vehicle control (no carboplatin) instead of vehicle control at each respective carboplatin dose). Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n= at least 4 independent experiments in duplicate). BPD-PDP was performed using 690 nm light at an energy density of 0.03 J/cm²; ALA-PpIX-PDP was performed using 630 nm light at an energy density of 0.1 J/cm² for OVCAR-3 cells and 1.0 J/cm² for Caov-3 cells. Significant differences between PDP groups and the PFAS + carboplatin only group (grey bar) at each carboplatin dose are denoted by * (p < 0.05).

Figure S7. In OVCAR-3 cell exposure groups where platinum resistance was observed, photosensitizer efficacy for PDP in combination with 100 μM carboplatin did not differ between BPD and ALA-PpIX. The interaction between BPD-PDP (hν: 0.03 J/cm²) + 100 μM carboplatin and ALA-PpIX-PDP (hν: 0.10 J/cm²) + 100 μM carboplatin was not significant in (a) 500 nM PFOA- [p = 0.064], (b) 500 nM PFHpA- [p = 0.275], (c) 2 μM PFHpA- [p = 0.652], (d) 500 nM PFPA- [p = 0.969], (e) 2 μM PFPA- [p = 0.270], (f) 1 μM PFOA + 1 μM PFHpA- [p = 0.133], (g) 1 μM PFOA + 1 μM PFPA- [p = 0.557], (h) 1 μM PFHpA + 1 μM PFPA- [p = 0.529], and (i) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed OVCAR-3 cells [p = 0.087].

Figure S8. In Caov-3 cell exposure groups where platinum resistance was observed, photosensitizer efficacy for PDP in combination with 100 μM carboplatin did not differ between BPD and ALA-PpIX. The interaction between BPD-PDP (hν: 0.03 J/cm²) + 100 μM carboplatin and ALA-PpIX-PDP (hν: 1.0 J/cm²) + 100 μM carboplatin was not significant in (a) 2 μM PFOA- [p = 0.943], (b) 500 nM PFHpA- [p = 0.364], (c) 500 nM PFPA- [p = 0.445], (d) 2 μM PFPA- [p = 0.152], (e) 1 μM PFHpA + 1 μM PFPA- [p = 0.080], and (f) 750 nM PFOA + 750 nM PFHpA + 750 nM PFPA-exposed Caov-3 cells [p = 0.104].

Figure S9. Survival fraction in OVCAR-3 cells was largely unaffected by 630 nm or 690 nm light after PFAS exposure. Relative to the vehicle control (dashed line), survival fraction was unchanged (a) post-PFOA and (b) PFHpA exposure and irradiation by 630 nm light (hν: 0.10 J/cm²). Survival fraction increased after exposure to (c) 2 μM PFPA and irradiation by 630 nm light (hν: 0.10 J/cm²). After irradiation with 690 nm light (hν: 0.03 J/cm²), survival fraction decreased in (d) 2 μM PFOA-exposed cells + 200 μM carboplatin treatment, increased in (e) 500 nM PFHpA-exposed cells + 400 μM carboplatin, and was unchanged in (f) PFPA-exposed cells. Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n=3 independent experiments in duplicate). Significant differences between exposure groups at each light dose are denoted by * (p < 0.05).

Figure S10. Survival fraction in Caov-3 cells was not affected by 630 nm or 690 nm light after PFAS exposure. Relative to the vehicle control (dashed line), survival fraction was unchanged post-PFAS exposure and irradiation by (a-c) 630 nm (hν: 1.0 J/cm²) or (d-f) 690 nm (hν: 0.03 J/cm²) light. Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n=3 independent experiments in duplicate).

Figure S11. Survival fraction in OVCAR-3 cells was not affected by 630 nm or 690 nm light after exposure to PFAS mixtures. Relative to the vehicle control (dashed line), survival fraction was unchanged post-PFAS exposure and irradiation by (a-d) 630 nm (hν: 0.10 J/cm²) or (e-h) 690 nm (hν: 0.03 J/cm²) light. Data are expressed as mean ± SD and are represented as a percentage of the vehicle control for each respective medium condition (n=3 independent experiments in duplicate).

Figure S12. Survival fraction in Caov-3 cells was not affected by 630 nm or 690 nm light after exposure to PFAS mixtures. Relative to the vehicle control (dashed line), survival fraction was unchanged post-PFAS exposure and irradiation by (a-d) 630 nm (hν: 1.0 J/cm²) or (e-h) 690 nm (hν: 0.03 J/cm²) light. Data are expressed as mean ± SD and are represented as a percentage of the vehicle control) for each respective medium condition (n=3 independent experiments in duplicate).

Figure S13. ΔΨ_m decreased in PFOA-exposed OVCAR-3 and Caov-3 cells after BPD-PDP (green box) or ALA-PpIX-PDP (black box) and carboplatin treatment. Relative to the PFAS + 400 μM carboplatin group (no PDP), ΔΨ_m decreased (a) post-BPD-PDP (hν: 0.03 J/cm²) and carboplatin treatment at all doses tested in 2 μM PFOA-exposed OVCAR-3 cells and (b) post-ALA-PpIX-PDP (hν: 0.10 J/cm²) in 2 μM PFOA-exposed (100 – 200 μM carboplatin) OVCAR-3 cells. Decreased ΔΨ_m was also observed in Caov-3 cells exposed to (c) 500 nM PFOA after BPD-PDP (hν: 0.03 J/cm²) and carboplatin treatment, while no significant differences were observed in (d) 500 nM or 2 μM-exposed Caov-3 cells post-ALA-PpIX-PDP (hν: 1.0 J/cm²) and carboplatin treatment. Data are expressed as mean ± SD and are represented as either a percentage of the vehicle control (PFAS + 400 μM carboplatin, no PDP group) or as a percentage of the PFAS + 400 μM carboplatin (no PDP) group for each respective medium condition (n=2 in duplicate for grey bars, n=3 independent experiments in duplicate for colored bars). BPD-PDP was performed using 690 nm light at an energy density of 0.03 J/cm²; ALA-PpIX-PDP was performed using 630 nm light at an energy density of 0.1 J/cm² for OVCAR-3 cells and 1.0 J/cm² for Caov-3 cells. Significant differences between the vehicle control and PFOA-exposed cells are denoted by * (p < 0.05), while significant differences between PDP-exposed groups and the PFOA + 400 μM carboplatin (no PDP) group are denoted by # (p < 0.05).

Figure S14. ΔΨ_m decreased in PFAS mixture-exposed OVCAR-3 and Caov-3 cells after BPD-PDP (green box) or ALA-PpIX-PDP (black box) and carboplatin treatment. Relative to the PFAS + 400 μM carboplatin group (no PDP), ΔΨ_m decreased post-BPD-PDP (hν: 0.03 J/cm²) and carboplatin treatment in (b) 1 μM PFOA + 1 μM PFPA-exposed OVCAR-3 cells and (d) 1 μM PFOA + 1 μM PFPA-exposed Caov-3 cells (100 – 200 μM carboplatin). Caov-3 cells exposed to (h) 1 μM PFOA + 1 μM PFPA and treated with ALA-PpIX-PDP (hν: 1.0 J/cm²) + 100 – 200 μM carboplatin demonstrated decreased ΔΨ_m compared to PFAS + carboplatin only controls. Data are expressed as mean ± SD and are represented as either a percentage of the vehicle control (PFAS + 400 μM carboplatin, no PDP group) or as a percentage of the PFAS + 400 μM carboplatin (no PDP) group for each respective medium condition (n=2 in duplicate for grey bars, n=3 independent experiments in duplicate for colored bars). BPD-PDP was performed using 690 nm light at an energy density of 0.03 J/cm²; ALA-PpIX-PDP was performed using 630 nm light at an energy density of 0.1 J/cm² for OVCAR-3 cells and 1.0 J/cm² for Caov-3 cells. Significant differences between the vehicle control and PFAS mixture-exposed cells are denoted by * (p < 0.05), while significant differences between PDP-exposed groups and the PFAS mixture + 400 μM carboplatin (no PDP) group are denoted by # (p < 0.05).

Figure S15. ΔΨ_m was unchanged in OVCAR-3 (blue) and Caov-3 (red) cells pre- and post-BPD-PDP or ALA-PpIX-PDP, photosensitizer incubation, and irradiation. Relative to the vehicle (NT) control, ΔΨ_m was unchanged in OVCAR-3 cells (a) pre- and post-ALA-PpIX-PDP (hν: 0.10 J/cm²) and Caov-3 cells (b) pre- and post-BPD-PDP (hν: 0.03 J/cm²) or (c) pre- and post-ALA-PpIX-PDP (hν: 1.0 J/cm²). ΔΨ_m was not significantly different from the NT control in OVCAR-3 and Caov-3 cells treated with (d,e) BPD ± 690 nm light (hν: 0.03 J/cm²) or (f,g) ALA ± 630 nm light (hν: 0.10 J/cm² for OVCAR-3 cells and 1 J/cm² for Caov-3 cells). Data are expressed as mean ± SD and as a percentage of the NT control (n=1 independent experiment with at least 24 replicates for graphs a-c, n=3 independent experiments in sextuplicate for graphs d-g).

Table S1. Summary of results. Data illustrating platinum resistance, PDP overcoming PFAS-induced platinum resistance, BPD-PDP (hν: 0.03 J/cm²) potency with or without carboplatin, and ΔΨ_m post-BPD-PDP (hν: 0.03 J/cm²) and ALA-PpIX-PDP (hν: 0.10 J/cm² for OVCAR-3 cells and 1 J/cm² for Caov-3 cells) are shown in Figure 6, Figures 3, 4, and 6, Figures 5, S7, and S8, and Figures 7, 8, S13, and S14, respectively.

NIHMS1838711-supplement-SUPINFO.pdf^{(5.3MB, pdf)}

ACKNOWLEDGEMENTS:

This research was supported by the National Institutes of Health (NIH), National Institute of Environmental Health Sciences (NIEHS) (Z01-ES102785 to SEF), a pre-doctoral traineeship from (National Research Service Award T32 ES007126 to BPR) from NIEHS, a NIH T32 award to the Certificate in Translational Medicine Program at UNC-Chapel Hill: grant number GM122741 (to BPR), as well as funding from the NC Translational and Clinical Sciences Institute (NC TraCS) at UNC-Chapel Hill supported by the National Center for Advancing Translational Sciences (NCATS), NIH through Grant Award Number UL1TR002489 (to IR), the Center for Environmental Health and Susceptibility (CEHS) at UNC-Chapel Hill supported by the NIEHS through Grant Award Number P30ES010126 (to IR), and UNC-NC State Joint Department of Biomedical Engineering Startup Funds (to IR). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article:

^†

This article is part of a Special Issue celebrating the 50^th Anniversary of the American Society for Photobiology.

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PERMALINK

Photodynamic Priming Overcomes Per- and Polyfluoroalkyl Substance (PFAS)-Induced Platinum Resistance in Ovarian Cancer†

Brittany P Rickard

Xianming Tan

Suzanne E Fenton

Imran Rizvi

Abstract

Graphical Abstract

INTRODUCTION

MATERIALS AND METHODS

Cell culture:

Preparation of PFAS stocks:

Preparation of photosensitizer stocks:

Evaluation of PDT efficacy at baseline:

Evaluation of PDT efficacy post-PFAS exposure:

Evaluation of PDP in combination with carboplatin treatment:

Evaluation of ΔΨm post-PFAS exposure, PDP, and/or carboplatin treatment:

Statistical analysis:

RESULTS

PFAS exposure altered the efficacy of BPD-PDT or ALA-PpIX-PDT at select light doses in OVCAR-3 or Caov-3 cells

BPD-PDT:

Figure 1. Effect of PFHpA and PFPA on light-dose-dependent responses to BPD-PDT (green box) or ALA-PpIX-PDT (black box) in OVCAR-3 and Caov-3 cells.

ALA-PpIX-PDT:

Exposure to mixtures of PFAS similarly affected the efficacy of BPD-PDT or ALA-PpIX-PDT at select light doses in OVCAR-3 and Caov-3 cells

Figure 2. Effect of PFAS mixtures on light-dose-dependent responses to BPD-PDT (green box) or ALA-PpIX-PDT (black box) in OVCAR-3 and Caov-3 cells.

BPD-PDT:

ALA-PpIX-PDT:

BPD-PDP was more effective than ALA-PpIX-PDP at reducing survival fraction in combination with carboplatin in PFAS-exposed ovarian cancer cell lines

Figure 3. Survival fraction decreased in PFAS-exposed OVCAR-3 and Caov-3 cells post-BPD-PDP or ALA-PpIX-PDP + carboplatin.

BPD-PDP:

Figure 4. In OVCAR-3 and Caov-3 cells exposed to PFAS mixtures, BPD-PDP was more effective than ALA-PpIX-PDP at reducing survival fraction in combination with carboplatin.

ALA-PpIX-PDP:

Figure 5. In OVCAR-3 and Caov-3 cell exposure groups where platinum resistance was not observed, photosensitizer efficacy for PDP differed between BPD and ALA-PpIX.

Table 1.

Combination of BPD-PDP or ALA-PpIX-PDP with carboplatin significantly decreased survival fraction and overcame PFAS-induced platinum resistance in OVCAR-3 and Caov-3 cells

Figure 6. BPD-PDP and ALA-PpIX-PDP overcame resistance to carboplatin induced by PFAS and PFAS mixtures in OVCAR-3 (blue) and Caov-3 (red) cells.

BPD-PDP:

ALA-PpIX-PDP:

Mitochondrial membrane potential (ΔΨm) decreased post-PDP + carboplatin in PFAS-exposed OVCAR-3 and Caov-3 cells

Figure 7. ΔΨm decreased in PFAS-exposed OVCAR-3 and Caov-3 cells after BPD-PDP (green box) or ALA-PpIX-PDP (black box) in combination with carboplatin.

BPD-PDP:

Figure 8. ΔΨm decreased after BPD-PDP (green box) or ALA-PpIX-PDP (black box) in combination with carboplatin in OVCAR-3 and Caov-3 cells exposed to PFAS mixtures.

ALA-PpIX-PDP:

DISCUSSION

Supplementary Material

ACKNOWLEDGEMENTS:

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Photodynamic Priming Overcomes Per- and Polyfluoroalkyl Substance (PFAS)-Induced Platinum Resistance in Ovarian Cancer^†

Evaluation of ΔΨ_m post-PFAS exposure, PDP, and/or carboplatin treatment:

Mitochondrial membrane potential (ΔΨ_m) decreased post-PDP + carboplatin in PFAS-exposed OVCAR-3 and Caov-3 cells

Figure 7. ΔΨ_m decreased in PFAS-exposed OVCAR-3 and Caov-3 cells after BPD-PDP (green box) or ALA-PpIX-PDP (black box) in combination with carboplatin.

Figure 8. ΔΨ_m decreased after BPD-PDP (green box) or ALA-PpIX-PDP (black box) in combination with carboplatin in OVCAR-3 and Caov-3 cells exposed to PFAS mixtures.