Epithelial ovarian cancer (EOC) is a lethal disease typically diagnosed at a late stage. There is an urgent need for treatment modalities that eliminate microscopic metastatic deposits missed by standard therapies while simultaneously engaging antitumor immunity. Photodynamic therapy (PDT) has demonstrated immune-enhancing effects, including photodynamic priming (PDP), wherein sublethal photodynamic stress remodels the tumor microenvironment to facilitate immune activation and infiltration. Here, we investigate cancer-targeted photoimmunotherapy (PIT), a molecularly targeted form of PDT, as a strategy to build upon and potentially enhance PDP by selectively depleting cancer cells while preserving immune effectors critical to antitumor responses. Using a 3D Matrigel dome model incorporating human ovarian cancer spheroids and allogeneic immune cells, we establish a broadly accessible imaging and analysis pipeline based on fluorescent labeling and 3D confocal microscopy to quantify cancer and immune cell viability. In this system, the presence of T cells or peripheral blood mononuclear cells enhances cancer depletion following PIT, consistent with stimulation of an antitumor immune response. Importantly, PIT spares significantly more T cells and NK cells compared to untargeted PDT and cetuximab at equivalent concentrations. PIT reduces spheroid size while preserving effector immune populations within the tumor microenvironment. Together, these findings suggest that targeted PIT may extend the immune-modulatory foundations established for PDT and PDP, offering a strategy to simultaneously eradicate residual tumor deposits and promote antitumor immune priming in EOC.

Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants of global concern that have been associated with a variety of adverse health outcomes, including diminished chemotherapy response. Previous studies in moderately chemosensitive ovarian cancer cells (OVCAR-3) have shown that the induction of chemoresistance from PFAS exposure is duration-dependent, with longer, more human-relevant exposure durations leading to worse outcomes. Mitochondrial content was also altered following chronic PFAS exposure, suggesting mitochondria as contributors to PFAS-induced chemoresistance. Here, chemotherapy response following chronic PFAS exposure in a chemoresistant human ovarian cancer cell line, OVCAR-8, was evaluated. Compared to OVCAR-3 cells, chemotherapy response was unaffected by chronic PFAS exposure in OVCAR-8 cells. As individuals gain awareness of sources of PFAS exposure, and associated harmful effects, actions can be taken to limit exposure using water filtration systems and/or safer alternatives to PFAS-containing consumer goods. Thus, we also explored the ability of PFAS-sensitive OVCAR-3 cells to recover from chronic exposure. Following 6 passages of chronic PFAS exposure, cells were “outgrown” in the absence of PFAS for 7 additional passages and proliferation, chemotherapy response, and mitochondria-related alterations were assessed. Compared to chronically-exposed cells, outgrown cells displayed heightened sensitivity to chemotherapy along with decreased superoxide production and mitochondrial content. Proliferation remained significantly elevated compared to controls, suggesting that not all PFAS-induced effects are abrogated by a recovery period. Together, these findings suggest that ovarian cancer cells differ in their PFAS sensitivities and that mitochondria-related alterations resulting from chronic PFAS exposure can be reversed following a “recovery period”, potentially resensitizing cancer cells to chemotherapy.

Resistance to chemotherapy is a major barrier to the effective treatment of ovarian cancer; however, the role of environmental exposures in the onset of chemoresistance remains elusive. Our previous work in ovarian cancer cells suggests that short-term perfluoroalkyl substances (PFAS) exposures induce chemoresistance, potentially by influencing mitochondrial parameters, but little is known about the effects of longer-term exposures, which are more human-relevant. Since mitochondria play critical roles in determining ovarian cancer chemotherapy response, it is also important to understand the role of environmental exposures in modulating mitochondrial function. This study explored how varying durations of PFAS exposure (2-35 days) affect mitochondrial parameters known to drive chemoresistance in human ovarian cancer cell lines. An ovarian cancer cell line (OVCAR-3) that was chronically exposed to PFAS (26-35 days) was generated. Compared to short-term PFAS exposure, chronic PFAS exposures significantly increased resistance to both carboplatin and doxorubicin. Chemotherapy resistance was accompanied by increased mitochondrial superoxide production, alterations in bioenergetics, and elevated mitochondrial content. These findings suggest that PFAS exposure induces chemotherapy resistance in ovarian cancer cells in a duration-dependent manner, worsened by human-relevant chronic exposures, and that mechanisms driving these effects are influenced by the modulation of mitochondrial parameters. Future studies should focus on targeting mechanisms underlying PFAS-induced chemotherapy resistance to improve survival outcomes.

Photodynamic therapy (PDT) is a photochemistry‐based treatment modality that synergizes with traditional agents and can overcome chemoresistance. Eighty percent of ovarian cancer patients develop chemoresistant disease, highlighting the need to identify sources of treatment failure and develop rational combinations. Studies have shown that perfluoroalkyl substances (PFAS) induce chemoresistance in a duration‐dependent manner in OVCAR‐3 cells. PFAS are widespread drinking water contaminants present in the blood of nearly all Americans. The present study evaluated the ability of photodynamic priming (PDP), a sub‐cytotoxic variant of PDT, in combination with chemotherapy to overcome chemoresistance in two OVCAR‐3 cell cohorts: PFAS chronically‐exposed and outgrown (allowed to “recover” from chronic PFAS exposure). Effectiveness of benzoporphyrin derivative‐ (BPD‐) or aminolevulinic acid‐induced protoporphyrin IX‐PDP (ALA‐PpIX‐PDP) was assessed in combination with carboplatin and doxorubicin. In PFAS chronically‐exposed cells, BPD‐PDP + carboplatin reduced survival fraction compared to carboplatin alone. Mitochondrial membrane potential also decreased significantly in both cohorts following ALA‐PpIX‐PDP‐based combinations. PDP + doxorubicin also successfully overcame chemoresistance arising from chronic PFAS exposure but was less effective than PDP + carboplatin. Together, these findings demonstrate the efficacy of PDP‐based combinations in overcoming chronic PFAS exposure‐induced chemoresistance and should be explored in pre‐clinical models of ovarian cancer.

Significance:Intralipid, a soybean oil-based lipid emulsion, is widely used in photomedicine to enhance light distribution due to its strong scattering properties. Although the optical characteristics of Intralipid are well documented, interactions with the reactive molecular species (RMS) generated during photodynamic therapy (PDT) and the impact of such interactions on therapeutic outcomes remain poorly understood. We reveal that Intralipid actively influences PDT response in vitro, beyond its role as a scattering agent. Aim:We examined how Intralipid affects the optical and photodynamic behavior of benzoporphyrin derivative (BPD), a clinical photosensitizer, in solution and across four ovarian cancer cell lines. Approach:The photodynamic properties of BPD, with and without Intralipid, were analyzed using fluorescence spectrometry and RMS probes, and PDT-induced oxidation of Intralipid components was characterized using LC-MS. The effects of Intralipid on BPD-PDT were evaluated under various conditions. Results:Intralipid reduced BPD photobleaching and RMS generation, suggesting RMS quenching. Extensive oxidation of Intralipid components was observed following PDT. Finally, Intralipid significantly modified BPD-PDT efficacy across all four cell lines, depending on photosensitizer-light interval, dose, and incubation time. Conclusions:Intralipid acts as a bioactive modulator of PDT response, highlighting the need for further investigations both in vitro and in vivo.

First-line treatment for advanced-stage or recurrent endometrial cancer consists of platinum- and taxane-based chemotherapy, to which many patients will develop resistance. Determining the factors that contribute to platinum resistance and developing alternate treatment options for patients with advanced-stage gynecologic malignancies is critical to improving survival outcomes. Recently, we published the first study evaluating the contribution of perfluoroalkyl substances (PFAS) exposure to platinum resistance in endometrial cancer cell lines and found that select PFAS induce carboplatin resistance, potentially by dysregulating mitochondrial function. The present study expands upon those findings by examining the efficacy of photodynamic priming (PDP) in combination with carboplatin to overcome PFAS-induced platinum resistance. Due to the suspected role of mitochondrial dysfunction in platinum resistance, two clinically approved photosensitizers that, in part, localize to mitochondrial membranes or are synthesized in mitochondria were evaluated: benzoporphyrin derivative (BPD) and aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX), respectively. Combination of ALA-PpIX-mediated PDP + carboplatin resulted in a greater reduction in survival fraction than the same combination with BPD. While PDP with both photosensitizers reduced mitochondrial membrane potential, the reduction was greater with BPD-PDP than ALA-PpIX-PDP. These findings demonstrate that BPD-PDP and ALA-PpIX-PDP in combination with carboplatin can be used to overcome PFAS-induced platinum resistance in endometrial cancer cells.

Resistance to apoptosis-inducing chemotherapy is a major factor contributing to treatment failure and poor survival outcomes in high-grade serous ovarian cancer (HGSOC). Ferroptosis, a regulated form of cell death driven by lipid peroxidation, has emerged as a promising effector mechanism because it remains available in HGSOC cells with impaired apoptosis signaling. While most research has focused on pharmacological ferroptosis inducers, there is growing interest in strategies that could trigger lipid autoxidation through externally delivered energy, such as photons. Photodynamic therapy (PDT), which utilizes light and light-activatable photosensitizers to generate reactive molecular species, offers a means of initiating lipid peroxidation with a high degree of precision and minimal systemic toxicities. However, the precise lipid targets of PDT, the influence of varying tumor lipidomic landscapes, and the role of ferroptosis sensitivity on PDT-lipid interactions have yet to be elucidated. In this study, we systematically compare PDT to ferroptosis induced by the inhibition of glutathione peroxidase 4, focusing on lipid redox states and composition in HGSOC cell lines. While PDT was similarly effective in both ferroptosis-sensitive and -resistant cells, its effects on cellular lipidomes differed markedly. PDT robustly induced lipid radical formation in both cell types; however, a dose-dependent accumulation of lipid hydroxides and hydroperoxides was only observed in ferroptosis-sensitive cells rich in unsaturated phospholipids. Further analysis revealed a significant overlap in lipid oxidation targets between PDT and ferroptosis. Notably, in both cell types, and in vivo, PDT upregulated ceramides, a lipid class strongly associated with mitochondrial apoptosis. In summary, PDT exhibited comparable efficacy in both ferroptosis-sensitive and -resistant cells by triggering a combination of lipid peroxidation and ceramide upregulation, suggesting the activation of both ferroptosis and apoptosis pathways. Further studies are needed to explore the role of PDT-induced lipidomic changes in the initiation of various cell death pathways and in overcoming chemoresistance in HGSOC.

Over 75% percent of ovarian cancer patients are diagnosed with advanced-stage disease characterized by unresectable intraperitoneal dissemination and the presence of ascites, or excessive fluid build-up within the abdomen. Conventional treatments include cytoreductive surgery followed by multi-line platinum and taxane chemotherapy regimens. Despite an initial response to treatment, over 75% of patients with advanced-stage ovarian cancer will relapse and succumb to platinum-resistant disease. Recent evidence suggests that fluid shear stress (FSS), which results from the movement of fluid such as ascites, induces epithelial-to-mesenchymal transition and confers resistance to carboplatin in ovarian cancer cells. This study demonstrates, for the first time, that FSS-induced platinum resistance correlates with increased cellular protoporphyrin IX (PpIX), the penultimate downstream product of heme biosynthesis, the production of which can be enhanced using the clinically approved pro-drug aminolevulinic acid (ALA). These data suggest that, with further investigation, PpIX could serve as a fluorescence-based biomarker of FSS-induced platinum resistance. Additionally, this study investigates the efficacy of PpIX-enabled photodynamic therapy (PDT) and the secretion of extracellular vesicles under static and FSS conditions in Caov-3 and NIH:OVCAR-3 cells, two representative cell lines for high-grade serous ovarian carcinoma (HGSOC), the most lethal form of the disease. FSS induces resistance to ALA-PpIX-mediated PDT, along with a significant increase in the number of EVs. Finally, the ability of PpIX-mediated photodynamic priming (PDP) to enhance carboplatin efficacy under FSS conditions is quantified. These preliminary findings in monolayer cultures necessitate additional studies to determine the feasibility of PpIX as a fluorescence-based indicator, and mediator of PDP, to target chemoresistance in the context of FSS.

Background: Advanced epithelial ovarian cancer (EOC) has high recurrence rates due to disseminated initial disease presentation. Cytotoxic phototherapies, such as photodynamic therapy (PDT) and photoimmunotherapy (PIT, cell-targeted PDT), have the potential to treat disseminated malignancies due to safe intraperitoneal delivery. Methods: We use in vitro measurements of EOC tumour cell and T cell responses to chemotherapy, PDT, and epidermal growth factor receptor targeted PIT as inputs to a mathematical model of non-linear tumour and immune effector cell interaction. The model outputs were used to calculate how photoimmunotherapy could be utilised for tumour control. Results: In vitro measurements of PIT dose responses revealed that although low light doses (<10 J/cm2) lead to limited tumour cell killing they also increased proliferation of anti-tumour immune effector cells. Model simulations demonstrated that breaking up a larger light dose into multiple lower dose fractions (vis-& agrave;-vis fractionated radiotherapy) could be utilised to effect tumour control via stimulation of an anti-tumour immune response. Conclusions :There is promise for applying fractionated PIT in the setting of EOC. However, recommending specific fractionated PIT dosimetry and timing will require appropriate model calibration on tumour-immune interaction data in human patients and subsequent validation of model predictions in prospective clinical trials.

Resistance to platinum-based chemotherapies remains a significant challenge in advanced-stage high-grade serous ovarian carcinoma, and patients with malignant ascites face the poorest outcomes. It is, therefore, important to understand the effects of ascites, including the associated fluid shear stress (FSS), on phenotypic changes and therapy response, specifically FSS-induced chemotherapy resistance and the underlying mechanisms in ovarian cancer. This study investigated the effects of FSS on response to cisplatin, a platinum-based chemotherapy, and doxorubicin, an anthracycline, both of which are commonly used to manage advanced-stage ovarian cancer. Consistent with prior research, OVCAR-3 and Caov-3 cells cultivated under FSS demonstrated significant resistance to cisplatin. Examination of the role of mitochondria revealed an increase in mitochondrial DNA copy number and intracellular ATP content in cultures grown under FSS, suggesting that changes in mitochondria number and metabolic activity may contribute to platinum resistance. Interestingly, no resistance to doxorubicin was observed under FSS, the first such observation of a lack of resistance under these conditions. Finally, this study demonstrated the potential of photodynamic priming using benzoporphyrin derivative, a clinically approved photosensitizer that localizes in part to mitochondria and endoplasmic reticula, to enhance the efficacy of cisplatin, but not doxorubicin, thereby overcoming FSS-induced platinum resistance. Resistance to platinum-based chemotherapies poses a major challenge in high-grade serous ovarian carcinoma, especially in patients with malignant ascites. This study examined the effects of fluid shear stress (FSS) on cisplatin, doxorubicin, and benzoporphyrin derivative (BPD)-enabled photodynamic therapy (PDT) in an in vitro perfusion model. Significant resistance to cisplatin and BPD-PDT was observed in OVCAR-3 and Caov-3 cells grown under FSS, while, importantly, no resistance to doxorubicin was noted. Photodynamic priming with BPD enhanced cisplatin efficacy, abrogating FSS-induced platinum resistance. These results elucidate approaches to target FSS-induced platinum resistance in ovarian cancer, particularly roles for photodynamic priming and doxorubicin.image

Abstract Platinum resistance is a major barrier to the effective treatment of advanced-stage ovarian cancer; however, factors leading to the development of resistance are not well understood. In fact, the role of environmental endocrine disrupting chemicals, such as perfluoroalkyl substances (PFAS), have seldom been explored. This is important because select PFAS have been linked to adverse reproductive outcomes in females including endometriosis, oocyte apoptosis, infertility, and increased even ovarian cancer risk at very high exposure levels. The relationship between PFAS and adverse female reproductive outcomes, as well as reports of other endocrine disruptors leading to therapy resistance, led, in the current study, to the evaluation of the effect of PFAS exposure on response to carboplatin in ovarian cancer cells. We recently showed that select PFAS, at human-relevant concentrations, induced carboplatin resistance in OVCAR-3 and Caov-3 cells; however, the mechanism underlying the onset of resistance remains largely unknown. Since platinum-resistant ovarian cancer is characterized, in part, by increased mitochondrial networks and enhanced bioenergetic capacities, the central hypothesis of this study is that PFAS induce alterations to mitochondrial biology. Mitochondrial endpoints of interest included redox ratio and superoxide production. To measure redox ratio, nicotinamide dinucleotide (NADH) and flavin adenine dinucleotide (FAD), which are inherently fluorescent, were measured using a fluorescence plate reader. Since NADH and FAD are metabolites produced via oxidative phosphorylation and glycolysis, evaluating redox ratio can provide an understanding of which pathway is preferred in the presence of environmental stimuli like PFAS. In OVCAR-3 and Caov-3 cells, baseline NADH:FAD ratios were 3.3 and 3.5, respectively. After 48 hours of PFAS exposure, NADH:FAD ratios of OVCAR-3 and Caov-3 cells ranged from 8-10 or 6-10, respectively. This increase after PFAS exposure could suggest enhanced energy production capabilities that warrant further investigation. As an additional measure, superoxide production was measured using flow cytometry. OVCAR-3 and Caov-3 cells were exposed to PFAS for 6 days prior to isolating cell pellets and incubating them with MitoSOX (mitochondrial) and SYTOX Blue (nuclear) dyes. After 15–30-minute incubations, samples were run on the flow cytometer. Compared to controls, superoxide production in PFAS-exposed ovarian cancer cells increased by up to 200%. Often, cancer cells have elevated levels of reactive oxygen species due to enhanced energy production and signaling pathways, thus this finding further suggests that PFAS affect mitochondrial energy pathways. Altogether, these data show that PFAS exposure, which can induce carboplatin resistance in OVCAR-3 and Caov-3 cells, increases redox ratios and superoxide production in ovarian cancer cells. Therefore, targeting alterations in mitochondrial biology arising from PFAS exposure using precision medicine could be effective in overcoming platinum resistance in ovarian cancer. Citation Format: Brittany P. Rickard, Marta Overchuk, Vesna A. Chappell, Carl D. Bortner, Victoria L. Bae-Jump, Suzanne E. Fenton, Imran Rizvi. Perfluoroalkyl substances (PFAS) induce platinum resistance in ovarian cancer through altering mitochondrial function [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B100.

Abstract Background: Over 75% of patients with disseminated ovarian cancer develop resistance to platinum-based chemotherapies (carboplatin or cisplatin). Patients with a high volume of malignant ascites or excessive fluid buildup in the peritoneum are more likely to present with primary platinum-resistant disease. It is hypothesized that the physical forces generated by ascites may contribute to the dissemination and progression of ovarian cancer. Our research group has shown that fluid shear stress (FSS) induces carboplatin resistance and a motile and aggressive phenotype in monolayer cultures and 3D models for adherent ovarian cancer. In this study, the effects of FSS on treatment response to cisplatin and doxorubicin were examined. The latter is used to manage recurrent platinum-resistant disease. Changes in mitochondrial function under FSS as they relate to platinum resistance were also characterized. Finally, the use of photodynamic therapy (PDT), which utilizes 690 nm light and a clinical photosensitizer benzoporphyrin derivative (BPD) to locally generate cytotoxic reactive molecular species as a means of overcoming FSS-induced platinum resistance, was explored. Methods: Human epithelial ovarian adenocarcinoma cells OVCAR-3 and Caov-3 (ATCC) were cultured in tissue culture plates (static culture) or chamber slides (ibidi) connected to a perfusion pump for 48 h (flow rate: 0.11 mL/min, shear stress: 0.41 dyn/cm2). Cells in static and flow cultures were exposed to cisplatin, doxorubicin, BPD-PDT or combinations thereof and survival fractions were measured 72 h post-treatment. Mitochondrial membrane potential was measured using JC-1 fluorescence assay, and mitochondrial DNA (mtDNA) copy number was quantified using qPCR. ATP content per cell was measured with a CellTiterGlo luminescence assay. Results: OVCAR-3 and Caov-3 cells grown under FSS developed significant resistance to cisplatin. Specifically, 3.13 µM cisplatin induced a 50% decrease in the survival fraction in OVCAR-3 static cultures but failed to decrease the survival fraction under flow. In contrast to these findings with platins, no resistance to doxorubicin was observed under flow. Evaluation of the mitochondrial function revealed increased ATP content and mtDNA copy number in flow cultures, suggesting that increased mitochondrial activity may contribute to platinum resistance. Finally, low-dose BPD-PDT (IC10, 0.15 J/cm2) enhanced the efficacy of cisplatin in OVCAR-3 cells under flow, indicating that PDT is a viable strategy to sensitize tumors exposed to FSS to platinum-based chemotherapy. Conclusions: Our data suggest that ovarian cancer cells that acquired resistance to platinum due to FSS remain sensitive to doxorubicin. Changes in mitochondrial function, including increased ATP content per cell and mtDNA copy number, have been identified as potential contributing factors to platinum resistance under FSS. Finally, low-dose BPD-PDT can be used to re-sensitize cells to cisplatin and overcome platinum resistance. Citation Format: Marta Overchuk, Brittany P. Rickard, Justin Tulino, Frances S. Ligler, Imran Rizvi. Mechanism-informed photochemical strategies to overcome fluid shear stress-induced platinum resistance in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B111.

Ovarian cancer is the most lethal gynecologic cancer, largely due to the development of platinum-based chemotherapy resistance. Understanding sources of chemoresistance and the development of mechanism-based treatments are critical. In a recent study, we reported that perfluoroalkyl substances (PFAS), endocrine disruptors that pollute drinking water supplies worldwide, induce carboplatin resistance in ovarian cancer cells, potentially by enhancing mitochondrial function. The ability of photodynamic priming (PDP) to overcome PFAS-induced resistance using photosensitizers that, in part, localize to mitochondria, specifically benzoporphyrin derivative (BPD) or aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX)), will be presented. Ovarian cancer cells were exposed to PFAS, then treated with either BPD- or ALA-PpIX-PDP followed by carboplatin. Under PFAS exposure conditions that previously induced chemoresistance, both BPD- and ALA-PpIX-PDP sensitized ovarian cancer cells to carboplatin. A concomitant decrease in mitochondrial membrane potential was observed.

Platinum-resistant human epithelial ovarian cancer cell lines OVCAR-5 and OVCAR-4 expresses tyrosyl-DNA phosphodiesterase 1 (Tdp1). The Tdp1 protein expression levels in human epithelial ovarian carcinoma cell lines OVCAR-5 (derived from the ascitic fluid of EOC patient without prior treatment; tumor characterized to be platinum-resistant) and OVCAR-4 (derived from the ascitic fluid of platinum-refractory EOC patient) were evaluated by Western Blot. Tdp1 expression was found higher in the OVCAR-4 cells compared to that in the OVCAR-5 cells.

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.

BACKGROUND Capnography is one of the most important respiratory monitoring tools used in EMS because changes to end-tidal CO2 (ETCO2) generally precede blood oxygen desaturation, and waveform morphology can be used to assess bronchial patency. Many of capnography’s indications overlap with those of continuous positive airway pressure (CPAP) therapy. However, there are currently no convenient methods to administer CPAP while using capnography, and no peer-reviewed studies have examined combining the two technologies in the prehospital setting.METHODS & FINDINGS Two types of capnography sampler were used to investigate how ETCO2, capnogram shape, and respiration rate (RR) are affected by applying a CPAP mask both with and without oxygen flowing. A traditional nasal-oral cannula (NC) sampler, as well as a novel sampler that does not break the seal between the CPAP mask and the patient’s face (called the CPAP-Capnography Adapter, or CCA), were evaluated on a Human Patient Simulator (HPS). No significant differences between the NC and CCA were found for ETCO2 and RR percent error. Placement of a CPAP mask without turning on O2 increased ETCO2. During CPAP therapy, the two samplers maintained their RR measurement accuracy, but ETCO2 values were significantly reduced and the capnogram depicted an increased β-angle with severe blunting of the inspiratory downstroke.CONCLUSIONS The HPS had set-point ETCO2 and PaCO2 values, indicating that the local CO2 concentration at the nares was diluted during CPAP and no longer accurately reflected the simulated subject’s breathing. The compromise in waveform morphology could be overcome by further research into interpreting capnogram differences during CPAP between healthy patients and those with respiratory pathologies. However, at this time, our results show that ETCO2 values and capnogram shape should be interpreted with caution during CPAP.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis work was funded by the Abrams Scholarship from the UNC/NCSU Joint Department of Biomedical Engineering, as well as significant in-kind support from the UNC School of Nursing Simulation Center, UNC/NCSU Joint Department of Biomedical Engineering, and Durham Technical Community College EMS Education program.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors.* EMS : Emergency Medical Services CO2 : Carbon Dioxide CPAP : Continuous Positive Airway Pressure ETCO2 : End-Tidal Carbon Dioxide (mmHg) PaCO2 : Partial Pressure of Arterial Carbon Dioxide (mmHg) SpO2 : Blood Oxygen Saturation RR : Respiration Rate (breaths/min) NC : Nasal-Oral Cannula CCA : CPAP-Capnography Adapter MAP : Mean Arterial Pressure HPS : Human Patient Simulator PEEP : Positive End Expiratory Pressure

Preliminary analysis of costs related to the side effect management for irinotecan-based combination therapies.

Supplementary Methods Supplemental Figure 1: The MIA-PaCa-2 + PCAF model exhibits histologic features consistent with desmoplasia 90 days post implantation Supplemental Figure 2: 1 μM calcipitriol activates the vitamin D receptor (VDR) in cancer associated fibroblasts Supplemental Figure 3: VDAR activation + PDP enable a 75% dose reduction in nal-IRI Supplemental Figure 4: Inhibition of the CXCL12/CXCR7 by CAL+PDP is lost by day 90 post-implantation Supplemental Figure 5: Calcipotriol and photodynamic priming cytotoxicity curves Supplemental Figure 6: MRC-5 conditioned medium does not affect the baseline metabolic activity of PDAC cell lines Supplemental Figure 7: PDP increases intratumoral nal-IRI via increased vascular and parenchymal permeability Supplemental Figure 8: Treatment with CAL+PDP+5 mg/kg nal-IRI induces global transcriptomic shifts in pancreataic tumors that correlate with a less aggressive and more responsive treatment profile compared to treatment with PDP+20 mg/kg nal-IRI

Supplementary Figure 2 from Ki-67 as a Molecular Target for Therapy in an In vitro Three-Dimensional Model for Ovarian Cancer