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. Author manuscript; available in PMC: 2025 Aug 10.
Published in final edited form as: Cancer Lett. 2025 Apr 29;625:217738. doi: 10.1016/j.canlet.2025.217738

A necrosis inducer promotes an immunogenic response and destroys ovarian cancers in mouse xenografts and patient ascites organoids

Darjan Duraki a,1, Musarrat Jabeen a,1, Chengjian Mao a, Lawrence Wang a, Santanu Ghosh a, Xinyi Dai a, Junyao Zhu a, Matthew W Boudreau c,e, Erik R Nelson b,d,e,f, Paul J Hergenrother c,d,e, Georgina Cheng d,g,h, David J Shapiro a,d,*
PMCID: PMC12203437  NIHMSID: NIHMS2091908  PMID: 40311911

Abstract

Most ovarian cancer patients present with advanced disease and there are few targeted therapies; consequently, five-year survival for ovarian cancer remains below 50%. We described the anticipatory unfolded protein response (a-UPR) hyperactivator, ErSO, which induced profound and often complete regression of breast cancer in mouse models. Here we explore the effectiveness of ErSO against ovarian cancer. ErSO induced death of human PEO4 and Caov-3 ovarian cancer cells in vitro. In mouse xenografts, injected ErSO induced rapid complete, or near complete, regression of orthotopic metastatic PEO4 tumors and of Caov-3 ovarian tumors. Ovarian cancer patients often develop malignant ascites containing ovarian cancer organoids that drive metastasis. ErSO showed activity against 7/7 fresh patient derived ascites organoids (PDAOs). Low nanomolar ErSO destroyed 2/7 PDAOs. ErSO-mediated cell death in PDAOs occurred through the same a-UPR activation mechanism seen in cell culture. Moreover, ErSO family compound-induced a-UPR activation in ovarian cancer cells triggers necrotic cell death and release of damage associated molecular patterns (DAMPs), which strongly activated human macrophage and induced monocyte migration. These studies suggest ErSO has unusual potential for treatment of advanced ovarian cancer.

Keywords: Ovarian cancer, Patient ascites, Unfolded protein response, Immunogenic cell death, Organoid

1. Introduction

Epithelial ovarian cancer (EOC) usually presents at an advanced stage [1–5]. The metastases of these tumors are driven by clumps, or organoids, of ovarian cancer cells that are released into the peritoneal cavity, circulate in this “malignant ascites” and ultimately attach to other sites [6–9]. For patients who present with Stage III and Stage IV disease and malignant ascites, surgical debulking is often unable to remove all the tumor cells and the tumors recur. Although initially responsive to combination therapy using platinum agents and a taxane, after several treatment cycles, the tumors usually recur as resistant ovarian cancer with poor therapeutic options [1,10,11]. Despite recent improvements in treatment, including development of targeted therapy based on PARP inhibitors, most patients with recurrent disease die within 5 years [3,12,13].

In part because there are few common driver mutations, it has been challenging to develop targeted therapies for ovarian cancer. Most targeted therapies work by inhibiting a protein or pathway that contributes to tumor growth [5]. An alternative approach is to employ a turn-on strategy that works by inducing lethal overactivation of a pathway common in ovarian cancers. One such pathway is the anticipatory unfolded protein response (a-UPR) (simplified model: Fig. S1). We identified small molecule necrosis inducers, first-generation BHPI and second-generation ErSO, that induce robust regression of breast cancers in preclinical breast cancer cell-line derived xenografts and PDX models [14–23]. Whether ErSO would be effective as a single agent in ovarian cancer was unknown. We demonstrate that ErSO robustly activates the a-UPR in ovarian cancer cells, inducing ATP depletion and necrotic cell death and release of immune cell activating damage associated molecular patterns (DAMPs). Using sensitive bioluminescent imaging of large orthotopic ovarian tumors in immunocompromised mice, we show that ErSO, as a single agent, induces complete, or near-complete, regression of primary human ovarian tumors and metastases. Moreover, tumors treated with oral ErSO, that undergo incomplete regression and regrow, remain completely sensitive to re-treatment with injected ErSO.

We then explored a patient-derived ovarian cancer ascites model. Ovarian cancer ascites organoids exhibit stem cell like properties and have been challenging to target therapeutically [6–9,24]. We show that freshly isolated patient derived ascites organoids (PDAOs) from patients with metastatic Stage III and Stage IV disease are sensitive to killing by ErSO. These studies open the door to development of this turn-on therapy for metastatic ovarian cancer, and illustrate the potential of using an ovarian cancer patient’s ascites for a rapid personalized assessment of the patient’s suitability for a therapy.

2. Materials and methods

2.1. Cell lines and culture conditions

All cell lines were authenticated by genotyping (Univ. Arizona Genetics Core, USA). Cells were cultured at 37 °C with 5 % CO2. Caov-3 and Caov3-Luciferase cells: DMEM plus 10 % FBS and 1 % P/S. PEO4 and PEO4-Luciferase cells: DMEM plus 10 % FBS supplemented with insulin (10 μg/ml), 1:250 diluted Dulbecco’s Non-Essential Amino Acids, 1 mM glutamine, and 1 % P/S. PEO4-Luc and Caov3-Luc cells stably expressing luciferase were produced as we described for breast cancer cells [19, 23].

2.2. Cell proliferation assays

Cell proliferation assays and cell number determinations were performed as we described [19,23].

2.3. Western blots

Western blots of phospho-proteins and unphosphorylated proteins were performed as we recently described [23]. Imaging: iBright Imaging System (ThermoFisher) or Azure biosystems 400.

2.4. Antibodies

Phospho-PERK: CST 3172; total PERK: CST-3179, Phospho-eIF2α (Ser51): CST-3398, eIF2α: CST-5324, β-actin-HRP conjugate: CST-5125, α-Tubulin (DM1A) HRP conjugate: CST-12351, anti-HMGB1: CST-3935, anti-rabbit IgG HRP-linked: CST-7074 all from Cell Signaling Technology, USA, anti-mouse IgG HRP-linked.

2.5. Trypan Blue exclusion assay for cell death

Automated Trypan Blue exclusion assays for cell viability were as we described [17,19,23]. Briefly, 300,000 cells/well were plated (6-well plate). The next day, vehicle or indicated concentrations of ErSO or other compounds were added. After 24 h, cells were harvested, briefly spun down and resuspended in 100 μl of medium. 10 μl of resuspended cells was mixed with 10 μl of 0.4 % Trypan Blue dye, and stained (non-viable) and total cells quantitated (Countess II cell counter; Thermo Fisher USA).

2.6. qRT-PCR analysis of mRNA levels

qRT-PCR of cytokine mRNAs in THP-1 cells and of spliced XBP1 mRNA in ovarian cancer cells was carried out as previously described [19,23]. The fold change was determined using the ΔΔCT method with 36B4 as an internal control.

2.7. ATP depletion and release assays

ATP depletion in cells and ATP release from the cells was determined as we recently described [23] using the ATPlite Luminescence Assay Kit (Perkin Elmer, USA).

2.8. Protein synthesis inhibition assay

Protein synthesis rates were determined by measuring incorporation of 35S-methionine into protein as we described [14–18,20,23].

2.9. Ovary weights and images

Intact ovaries were excised, placed on petri dishes and imaged. Ovaries were then cut from the uterine horns, briefly patted dry to remove any moisture, and weighed.

2.10. Orthotopic mouse ovarian xenografts

All mouse xenograft studies were approved by the Univ. Illinois IACUC (Protocol Number 20032). PEO4-Luc and Caov3-Luc xenografts: 1 million PEO4-Luc or Caov3-Luc cells suspended in Matrigel were engrafted into one ovary of NSG mice (Jackson Labs USA). After 10–12 weeks of tumor outgrowth, mice were randomized into groups of 4 and received daily treatment by intraperitoneal injection (IP), or by oral gavage, of vehicle or 40 mg/kg ErSO. Tumors were imaged by bioluminescent imaging (BLI) using luciferase (IVIS) on days 0, 3, 7 and 14 after treatment. To facilitate viewer comparison of images over the course of treatment, the scale of all images for each set of tumors is held constant. Therefore, some tumors that are not visible in the representative photos are still detected by the IVIS imager. Mice were weighed a minimum of once per week. After cessation of treatment, PEO4 and Caov-3 oral ErSO mice were allowed to grow for 14D, imaged and then re-treated with IP ErSO (40 mg/kg daily for 14 days) with imaging at indicated times. At the end of initial treatment for the vehicle mice, or after re-treatment, mice were humanly euthanized, ovaries were excised, gently blotted and weighed.

2.11. HMGB1 release

300,000 cells were plated in each well of a 6-well plate, in complete medium, and incubated overnight. The following day, the medium was replaced with 500 μl of complete medium containing 100 nM ErSO or vehicle and cells were incubated for 12, 24 and 48 h. Subsequently, the treated medium was collected, centrifuged at 1500 rpm for 5 min, and supernatant was collected, stored at −20 °C for no more than 2 weeks and HMGB1 levels visualized by Western blot.

2.12. Isolation of ovarian cancer organoids from patient samples and ascites viability assays

Ovarian cancer patient ascites samples were obtained after paracentesis or upfront surgery from normally discarded peritoneal fluid (with informed consent (IRB 20CCC3162), Carle Health, Urbana Illinois, USA). 200 ml ascites samples were transferred to 50 ml conical tubes and centrifuged (10×g for 1 min). The pellets were resuspended gently, pooled in serum-free DMEM/F-12 and then strained through a pre-washed 70 μm cell strainer. The organoids were then collected from the strainer in serum-free DMEM/F-12, supplemented with 0.2 % methylcellulose, 10 μg/ml insulin, 10 ng/ml Fibroblast Growth Factor (FGF), 20 ng/ml Epidermal Growth Factor (EGF), 0.1 % bovine serum albumin (BSA), and 1 % penicillin/streptomycin and transferred to ultra-low attachment (ULA) flat-bottom plates (Corning) and used for experiments without freezing. The ascites organoids were treated with indicated concentrations of ErSO, ErSO-DFP, ErSO-TFPy [21] or Vehicle (DMSO) in 96 well ULA flat-bottom plates for 72 h. After 72 h, AlamarBlue™ HS Cell Viability Reagent (Thermo Fisher, A50101) was added to each well and samples incubated at 37 °C for 2 h. Fluorescence was read using the 560/590 nm module (BMG PheraStar, Germany).

2.13. THP-1 migration assay

Cells were plated in a 6-well plate at 360,000 cells/well in complete medium. The following day, medium was replaced with serum-free medium containing vehicle, 100 nM ErSO-TFPy or 1 μM apoptosis inducer Raptinal [25]. THP-1 migration assays used a modification of the cell migration assay we described [26]. After 6 h, cells were washed once with ErSO-TFPy-free medium and fresh medium containing no ErSO-TFPy was added. After 18 h, the medium was collected. THP-1 human monocytes were seeded in 5 μm transwell inserts at 200,000 cells/well, in a 24-well plate, in no serum RPMI. 500 μl of vehicle medium, 10 % FBS medium, ErSO-TFPy-treated medium, or Raptinal-treated medium was pipetted into the bottom chamber as rapidly as possible. Migration was allowed to proceed for 4 h. The bottom chamber medium was transferred to a 96-well plate and an Alamar Blue assay was performed to measure the number of live cells that passed through the membrane.

2.14. Statistical analysis

Information on statistical analysis is in the legends to individual figures. Briefly, unpaired Student’s t-test was used to compare one variable between groups. All graphs are the mean plus or minus the standard deviation unless stated otherwise. Statistical significance: n.s.: not significant by Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

2.15. Data and materials availability

The data supporting the findings and conclusions of this study are available within the paper and its supplementary information. Resources described here and raw data are available from the corresponding author upon reasonable request. Correspondence and requests for materials should be addressed to: David J. Shapiro: djshapir@illinois.edu.

3. Results

3.1. ErSO kills ovarian cancer cells

Although the PARP inhibitor Olaparib has dramatically impacted therapy for a sub-set of ovarian cancer patients, the development of resistance is common. Moreover, several established ovarian cancer cell lines are not very sensitive to Olaparib [13,27]. We initially tested the effectiveness of Olaparib in PEO4 and Caov-3 human ovarian cancer cells [14,19]. Across a range of concentrations, Olaparib had negligible effects on proliferation of PEO4 and Caov-3 cells (Fig. 1A). In contrast, ErSO induced near quantitative cell death. In 4-day dose response studies, 100 nM ErSO nearly blocked proliferation and 250 nM ErSO killed most of the PEO4 and Caov-3 cells (Fig. 1B and C). We next used our automated Trypan Blue dye exclusion assay for cell death to evaluate ErSO’s ability to rapidly kill PEO4 and Caov-3 cells [17,20,23]. In 24 h, 500 nM ErSO killed about half the PEO4 cells and about 40 % of the Caov-3 cells (Fig. 1D and E).

Fig. 1. ErSO kills human ovarian cancer cells in vitro.

Fig. 1.

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(A) Dose-response study of the effect of Olaparib on proliferation of PEO4 (red bars) and Caov-3 (blue bars) human ovarian cancer cells. 5000 cells/well were plated in 96 well plates and allowed to attach overnight and then maintained in medium containing DMSO (Veh.) or the indicated concentrations of Olaparib. Medium was replaced after 2 days. After 4 days absorbance was measured using MTS assay. Vehicle (Veh.) was set to 100 %. (B,C) Dose response study of the effect of ErSO on proliferation of PEO4 (B) and Caov-3 (C) cells. 2000 cells/well were plated in 96 well plates, allowed to attach overnight and then treated with vehicle (Veh.) or the indicated concentrations of ErSO. Medium was changed after 2 days; after 4 days, cell proliferation was determined using AlamarBlue. Cell number was from a standard curve of fluorescence versus cell number for each cell line. Dashed line indicates day 0 cell number (2000 cells/well); values below the dashed line indicate cell death. (D–E) ErSO rapidly kills ovarian cancer cells. 300,000 cells/well (6 well plate) were treated with the indicated concentrations of ErSO; after 24 h cell death was evaluated by our automated Trypan Blue exclusion assay. (D) PEO4, (E) Caov-3 (A–E) All data are mean ± s.d. Statistical comparisons are to the vehicle control. (A) n = 6, (B,C) n = 8; (D,E) n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns: not significant.

3.2. ErSO induces profound regression in orthotopic ovarian cancer mouse xenografts

Since ErSO effectively killed PEO4 and Caov-3 cells in culture, we next explored its ability to induce regression in orthotopic mouse ovarian cancer xenograft models. PEO4 and Caov-3 cells were stably transfected to express luciferase, enabling us to use bioluminescent imaging (BLI) to follow growth and regression of ovarian tumors in live mice. Immune compromised NSG mice were grafted into one ovary, with the other ovary used as a control. After tumor outgrowth (no estrogen supplementation [28]), mice were treated with vehicle or ErSO (40 mg/kg daily) for 14 days either by IP injection, or oral gavage (p.o.)

Vehicle-treated PEO4 tumors exhibited continued growth (Fig. 2A–C,D, Fig. S2B). Notably, all 4 mice treated with daily IP ErSO regressed by more than 98.5 % in just 3 days and these tumors had regressed to undetectable, or nearly undetectable (>99 %) at day 14 (Fig. 2B and C(red bars),D, Fig. S2B). Importantly, ErSO was effective against both the primary ovarian tumor and against likely peritoneal spread (Fig. 2B, mice 3 and 4). The mice with PEO4 tumors treated with oral ErSO exhibited complete cessation of tumor growth and robust, but incomplete regression (Fig. 2C(blue bars),D, Fig. S1A and B), likely because the oral bioavailability (%F) for ErSO is 47 % [20].

Fig. 2. ErSO destroys primary and metastatic PEO4 ovarian tumors in a mouse xenograft model.

Fig. 2.

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(A–D) PEO4 human ovarian cancer cells were engrafted into one ovary of NOD scid gamma (NSG) mice and tumor outgrowth was quantitated using flux values from luciferase-based bioluminescent imaging (BLI). After 10–12 weeks of tumor outgrowth, randomized groups of 4 mice received injected IP vehicle (A,C,D) or 40 mg/kg ErSO daily (B–D), or by oral gavage (C,D, see also Fig. S2A). Tumors were imaged on days 0, 3, 7 and 14 after initiating treatment. (A,B) Representative BLI images at Day 0 and Day 14 of vehicle-treated (A) and IP ErSO-treated tumors (B). Vehicle-treated tumors showed continued outgrowth and increased metastases (mice 2 and 4). (B) In the ErSO-treated mice, both primary ovarian tumors (mice 1–4) and metastases (mice 3 and 4) are no longer visible. (C) Waterfall plot showing % change in tumor flux after 14 days. For each mouse, % change is relative to the tumor in that mouse at day 0 (grey bars: vehicle-treated mice; blue bars: oral ErSO; red bars: injected ErSO; see also flux values in (Fig. S2B). (D) Percent change in tumor flux over the course of treatment. Flux at day 0 (start of treatment) set to 100 %. No vehicle or ErSO-treated mice were dropped from the study.

In some reports, Caov-3 cells display partial resistance to platinum and/or taxanes [29]. We therefore evaluated the ability of ErSO to block tumor growth and induce regression in orthotopic Caov-3 ovarian tumors. Mice were treated for 14 days with daily IP or oral ErSO. While oral ErSO blocked tumor growth and induced strong regression in 2/4 mice (Fig. S3A and B), injected ErSO was far more effective. Remarkably, in just 3 days IP ErSO induced complete regression in 4/4 mice and light output remained undetectable in all 4 mice at days 7 and 14 (Fig. 3A–D, Fig. S3B). There were no significant differences in weight changes in vehicle and ErSO-treated mice bearing PEO4 and Caov-3 tumors (Figs. S2C and S3C).

Fig. 3. Injected ErSO eradicates Caov-3 ovarian tumors.

Fig. 3.

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Caov-3 cells were engrafted into one ovary, allowed to grow out and imaged using BLI (A–D) (see Fig. 2 legend). (A,B) Representative BLI images of vehicle-treated mice (A) and IP ErSO-treated tumors (B) in mice at day 0 and day 14. Tumors in vehicle-treated mice (A) experienced robust growth, while tumors in all 4 ErSO-treated mice (40 mg/kg daily IP for 14D) (B) regressed to undetectable. (C) Waterfall plot showing % change in tumor flux at the end of treatment (14 days). For each mouse, % change is relative to the tumor in that mouse at day 0 (grey bars: vehicle-treated; blue: oral ErSO; red: injected ErSO; flux values are in Fig. S3B). (D) Percent change in tumor flux during treatment. Day 0 flux set to 100 %. One mouse became ill and had to be euthanized early in the study, reducing vehicle numbers to 3. No mice were dropped from the study.

Since oral ErSO did not eradicate the ovarian tumors, we assessed whether these tumors had acquired resistance to ErSO. After cessation of oral 14-day ErSO treatment, the tumors were allowed to regrow for 14 days with no treatment and then treated for 14 days with ErSO injected daily (40 mg/kg IP). All of the re-treated PEO4 tumors regressed by >85 % (Fig. S4A and C) and several of the re-treated Caov-3 tumors regressed to undetectable (Fig. S4B and D). These data indicate that tumors treated with oral ErSO that exhibited incomplete regression over the initial 14-day treatment did not acquire resistance.

For both PEO4 and Caov-3 tumors, analysis of ovarian weights confirmed the imaging data. Compared to the control ovary, the injected tumor bearing vehicle ovary exhibited a dramatic increase in weight (Fig. S5A–C). Ovaries from mice treated with oral ErSO, allowed to regrow, and then treated with injected ErSO exhibited no significant difference in weight between the control and injected ovaries (Fig. S5D–F).

3.3. ErSO is effective in fresh ovarian cancer patient-derived ascites organoids

Since ErSO was effective against large primary ovarian tumors and metastases in orthotopic mouse xenografts, we next evaluated ErSO in a physiologically relevant patient-centric context. Patients who present with Stage III and Stage IV ovarian cancer often have malignant ascites that is removed by paracentesis [6–9]. With informed consent, we obtained fluid from 7 patients who underwent drainage of their ascites and isolated their ascites organoids (PDAOs) (Fig. 4A), maintained the PDAOs in serum-free medium containing growth factors and immediately evaluated their response to ErSO. In several cases, the AlamarBlue assays required using all the fresh isolated organoids. Therefore, additional biochemical characterization (see below) is limited to the samples from which a larger pool of PDAOs was isolated. By histologic evaluation, 6/7 samples were from patients with high grade serous carcinoma (Fig. 4B–G) and one was a relatively rare mixed Mϋllerian adenocarcinoma (Fig. 4H). This type of tumor contains more than one cell type – the proportions of the two cancer cell types in this PDAO and their respective sensitivity to ErSO are unknown. While the control samples remained healthy, these fresh samples displayed little or no growth during the 3 days of our experiments. Therefore, a decline in AlamarBlue fluorescence suggests a loss of PDAOs and death of the cancer cells. As expected for heterogenous patient samples, the response to ErSO varied (Fig. 4B–H). Several of the PDAOs exhibited a dose-dependent decline in fluorescence with a decline of 40–60 % at 250 nM ErSO (Fig. 4B,C,F). Notably, two of the PDAOs from patients with relatively advanced disease exhibited a near-complete response at 50 nM ErSO (Fig. 4E and G, Stage IIIC and IV, respectively). These PDAOs did not exhibit a further decline in fluorescence at 250 and 1000 nM ErSO. Since these are native ovarian cancer organoids isolated intact from patient ascites, we speculate that they likely contain a few cells that are not ovarian cancer cells [24] and are not sensitive to killing by ErSO. These data demonstrate robust effectiveness of ErSO in a sub-set of fresh ovarian cancer samples from patients with metastatic disease.

Fig. 4. ErSO is effective in fresh ovarian cancer patient derived-ascites organoids (PDAOs).

Fig. 4.

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(A) Representative image showing the heterogeneity of isolated organoids from the fresh ascites sample in panel (B). (B–H) Following paracentesis, with informed consent, ascites patient organoids were rapidly isolated and fresh (never frozen) PDAOs were maintained in serum-free medium and treated with vehicle or 50, 250 or 1000 nM ErSO. After 3 days, cell viability was evaluated by AlamarBlue assay. n = 8 wells of PDAOs from that patient. Tumor stage is shown below each sample and was from histologic evaluation by a pathologist and a surgical oncologist. All patient samples from which we were able to isolate PDAOs are shown. (I–L) ErSO activates the UPR in PDAOs. (I) ErSO inhibits protein synthesis in a PDAO. Incorporation of 35S-methionine into protein in a PDAO (n = 5). (J) Western blot showing rapid ErSO mediated phosphorylation of PERK (p-PERK) and eIF2α (p-eIF2α). (K,L) Progressive decline in ATP levels in ErSO-treated PDAOs (n = 3). (B-I, K,L) Data are mean ± s.d. Statistical comparisons are to the vehicle control. ***p < 0.001, ****p < 0.0001.

Using both PEO4 cells and two of the patient ascites (samples G and H in Fig. 4), we also evaluated the effect of two recently described close derivatives of ErSO, ErSO-DFP and ErSO-TFPy with potentially improved potency or specificity [21]. In Caov-3 cells the dose-response data for all three compounds is similar at high concentrations, with modestly improved potency of ErSO-TFPy at low concentrations (Fig. S6A). In the two PDAOs, ErSO-TFPy was about as effective as ErSO, with near complete response in one PDAO at 50 nM and no further decline at higher concentrations, and ErSO-DFP was slightly less effective (Fig. S6B). Neither ErSO-TFPy nor ErSO-DFP exhibited increased potency in the mixed Mϋllerian adenocarcinoma (Fig. S6C).

3.4. In cells and PDAOs, ErSO-induced necrosis hyperactivates the a-UPR

The necrosis inducers BHPI and ErSO work by hyperactivating the a-UPR, inducing strong and sustained cell death in breast cancer cells (Model Fig. S1) [15,17,20,23]. Important for cell death are activation of the PERK-eIF2α arm of the UPR (Fig. S1), resulting in robust inhibition of protein synthesis and depletion of intracellular ATP [15,16,19,20,23]. ErSO induced near quantitative inhibition of protein synthesis in Caov-3 and PEO4 cells and robust inhibition of protein synthesis in a patient ascites sample (Fig. 4I and 5A,C). Consistent with the inhibition of protein synthesis resulting from activation of the PERK arm of the UPR, ErSO induced rapid phosphorylation of PERK and eIF2α in Caov-3 cells, PEO4 cells and a patient ascites sample (Fig. 4J and 5B,D). ErSO-mediated activation of the a-UPR results in release of calcium stored in the endoplasmic reticulum into the cell body. To restore calcium homeostasis, the cell activates ATP-dependent pumping of calcium into the lumen of the endoplasmic reticulum. Because ErSO causes strong and sustained UPR activation, the calcium leaks out causing a futile cycle of ATP-dependent SERCA calcium pumping and release that depletes intracellular ATP (Model Fig. S1, SERCA) [15,16,19,20,23]. ErSO depleted intracellular ATP in PEO4 cells, Caov-3 cells and PDAOs (Fig. 4K and L 5G,H). Supporting activation of the IRE1α arm of the UPR, ErSO rapidly induced the widely used marker for IRE1α activation, spliced XBP1 mRNA (Fig. 5E and F). These data indicate that ErSO kills ovarian cancer cells in culture and in ovarian cancer patient ascites organoids by hyperactivating the a-UPR.

Fig. 5. ErSO activates the a-UPR in ovarian cancer cells.

Fig. 5.

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(A–J) Cells and PDAOs were treated with vehicle or 1000 nM ErSO for the indicated times (Blue: Caov-3; Red: PEO4). (A–D) ErSO robustly inhibits protein synthesis and activates the PERK-eIF2α arm of the UPR in ovarian cancer cells (A,C) Incorporation of 35S-methionine into protein in (A) Caov-3 (C) PEO4 (B,D) Western blot showing a rapid increase in p-PERK and p-eIF2α (model in Fig. S1) in Caov-3 (B) and PEO4 (D) cells after ErSO treatment. (E,F) ErSO rapidly induces the UPR marker spliced XBP1 mRNA. qRT-PCR showing the fold increase in sp-XBP1 mRNA in Caov-3 (E) and PEO4 (F) cells after ErSO treatment. (G,H) Progressive decline in ATP levels in ErSO-treated (G) Caov-3 (H) PEO4 cells (A,C) n = 5; (E–H) n = 3. Data are mean ± s. d. (A,C, G-H) Statistical comparisons are to vehicle which is set to 100 %. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3.5. ErSO induces necrosis of ovarian cancer cells and release of immune-cell-activating HMGB1

A feature of BHPI and ErSO family compounds is that lethal hyperactivation of the a-UPR kills breast cancer cells not by inducing apoptosis or autophagy, but rather by inducing necrosis that is dependent on the calcium-activated, ATP-inhibited plasma membrane TRPM4 ion channel [17,20,23]. We therefore explored the effect of inhibitors of apoptosis and necroptosis on ErSO-induced death of PEO4 and Caov-3 ovarian cancer cells. The broad-spectrum caspase inhibitor, Q-VD-OPh, which blocks apoptosis, had no effect on ErSO-induced death of ovarian cancer cells (Fig. 6A). Since aspects of RIP-kinase-mediated necroptosis, including cell swelling and caspase-independent cell death resemble necrosis, we explored the effect of the RIP kinase inhibitors Nec1 and GSK872 on ErSO-induced inhibition of PEO4 cell proliferation and cell death [30–32]. Nec1 and GSK872 did not block ErSO’s inhibition of PEO4 and Caov-3 proliferation and cell death (Fig. 6B). When cells die by necrosis they release cell contents, termed Damage Associated Molecular Patterns (DAMPs) [33,34]. A widely used marker is release of the prototypical DAMP, HMGB1, into the medium. PEO4 and Caov-3 cells treated with ErSO released HMGB1 (Fig. 6C and D).

Fig. 6. Medium from ErSO-treated ovarian cancer cells activates human immune cells.

Fig. 6.

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(A) ErSO does not kill ovarian cancer cells by inducing apoptosis. Caov-3 and PEO4 cells were pre-treated for 1 h with vehicle, the pancaspase inhibitor Q-VD-OPh (20 μM) and then treated for 24 h with 1 μM ErSO and cell viability evaluated using our automated Trypan Blue exclusion assay. (B) Necroptosis inhibitors do not block ErSO-induced cell death. Caov-3 and PEO4 cells were pre-treated for 1 h with Vehicle (Con) or 30 μM Nec1 or 5 μM GSK872, individually or in combination, and control and necroptosis inhibitor-treated cells were maintained with or without ErSO for 24 h and cell viability assayed using Trypan Blue exclusion. (C,D) Western blot showing progressive release of HMGB1 into the medium by Caov-3 and PEO4 cells treated with 1 μM ErSO for the indicated times. (E) ErSO-free medium from ErSO-treated Caov-3 cells activates THP-1 macrophage. qRT-PCR analysis of RNA from PMA-differentiated human THP-1 macrophage showing relative expression levels of IL-1β, IL-6, IL-8, TNFα, CD80, CXCL10 and HLA-DR mRNAs after exposing the THP-1 cells to medium from Caov-3 cells treated with vehicle (set to 1) ErSO, or the apoptosis inducer Raptinal [23]. To exclude potential effects of ErSO or Raptinal on the THP-1 macrophage, Caov-3 cells were treated with 100 nM ErSO or 1 μM Raptinal, after 4 h the medium was removed and replaced with treatment-free medium for another 20 h. Medium was collected and the THP-1 macrophage were incubated in medium and RNA was isolated and analyzed by qRT-PCR. (F) ErSO-TFPy-free medium increases migration of THP-1 monocytes. Migration of undifferentiated THP-1 monocytes across an uncoated 5 μm membrane filter. Caov-3 cells were maintained in: No Serum, 10 % FBS medium (their standard medium) or ErSO-TFPy or Raptinal medium, as in (E). The medium was placed in the bottom chamber as attractant and the THP-1 cells were placed in the top chamber (above the membrane). After 4 h, to measure the number of cells that have migrated across the membrane, the medium from the bottom chambers was transferred to a 96 well plate and assayed using AlamarBlue. Relative fluorescence changes are shown with no Serum set to 1. (A,B,E,F) Shown is the mean ± s.d.; n = 3. (A) ns: no significant difference between ErSO-treated and ErSO + OPh (Q-VD-OPh: apoptosis inhibitor) cells. (E) ErSO-treated and Raptinal-treated cell medium compared to Vehicle (set to 1). (F) Medium from no serum cells set to 1. ErSO-TFPy medium compared to no serum and to Raptinal-treated medium. *p < 0.05, **p < 0.01, ****p < 0.0001; ns: not significant.

Since ErSO-treated ovarian cancer cells released the DAMP HMGB1 into the medium, we tested the effect of medium from Caov-3 cells treated with ErSO family compounds on immune cell activation and migration. Caov-3 cells were treated with an ErSO compound, that was then washed out; the cells were incubated in medium lacking ErSO family compound, this medium was used to treat PMA-differentiated human THP-1 macrophages. Medium from Caov-3 cells treated with the ErSO family compound, but not from cells treated with the apoptosis inducer Raptinal [24], induced a rapid increase in levels of several mRNAs encoding pro-inflammatory cytokines (Fig. 6E).

Many tumors fail to respond to immunotherapy in part because they do not contain certain immune cells. We therefore used a modified version of our Invasion-Dissociation-Rebinding assay [26] to explore the ability of medium from Caov-3 cells treated with ErSO family compounds to promote migration of undifferentiated THP-1 monocytes across a filter. Notably, medium from the ErSO-family-treated Caov-3 cells induced a highly significant ~3-fold increase in migration while medium from Caov-3 cells treated with the powerful apoptosis inducer, Raptinal, was no more effective than serum in inducing THP-1 migration (Fig. 6F). These data highlight the immunotherapeutic potential of our necrosis inducers in potentially addressing obstacles to immunotherapy in ovarian cancer.

4. Discussion

In part due to the lack of clear symptoms in early-stage epithelial ovarian cancer (EOC), most ovarian cancers are diagnosed at stages III and IV. Standard of care therapy, surgical debulking and chemotherapy with a platinum agent and a taxane often do not achieve long-term remission of these stage III and IV tumors [3,4,11,35]. Recently, the poly ADP Ribose Polymerase (PARP) inhibitor Olaparib has demonstrated effectiveness in the sub-set of ovarian cancer patients with BRCA1/2 mutations [5,13,27]. Immunotherapies, including bevacizumab, which inhibits angiogenesis and immune checkpoint inhibitors have also shown promise [36,37]. Recently, an antibody drug-conjugate (MIRV) has been shown to extend survival of platinum-resistant ovarian cancer patients [38]. In general, these treatments are only effective in a sub-set of patients and resistance often develops over time [35]. The limitations of current therapies and poor long-term survival of patients with EOC indicates that new approaches are needed. Here we demonstrate that a turn-on approach using the a-UPR activator, ErSO, induces complete or near complete regression in diverse ovarian cancer models. ErSO was effective in killing both proliferating PEO4 and Caov-3 cells and in fresh patient ascites samples, which show very little proliferation in the chemically defined medium used in our 3-day experiments. Although ErSO induces sp-XBP1 mRNA, a marker for activation of the protective chaperone inducing IRE1α arm of the UPR, we showed previously that the near quantitative inhibition of protein synthesis we observe with ErSO compounds blocks production of the UPR-induced protective oncogenic chaperone protein, BiP [15], and of the UPR-regulated apoptosis inducer, CHOP [17]. Thus, PERK activation, which triggers robust inhibition of protein synthesis, ATP depletion and TRPM4 mediated cell swelling [23] lead to plasma membrane rupture and release of immunogenic DAMPs in ErSO-treated ovarian cancer cells.

Notably, we did not observe ErSO-resistant tumors in our studies. This is likely due to several factors. Many current therapies block proliferation, but do not rapidly kill nearly all the cancer cells. In contrast, in just 3 days ErSO kills nearly all the cancer cells, so there is not a large pool of surviving ovarian cancer cells in which resistance can be selected for. Importantly, for conventional inhibitor therapies a single mutation that renders the inhibitor unable to bind is usually sufficient to restore tumor growth [16,20,39,40]. In contrast, for this activation-based therapy a mutation in one of the two copies of a gene in the initiating complex, or in the a-UPR pathway (Fig. S1), will likely only moderately reduce the activity of ErSO. Therefore, if development of resistance to ErSO occurs at the genome level, it will likely be a rare event requiring mutation or inactivation of both copies of an essential gene in the pathway. The plasma membrane sodium channel TRPM4 is not essential for cell viability and tumor formation and knockout of TRPM4 blocks the influx of sodium that triggers and sustains necrotic cell death [23]. Notably, in these ovarian tumors and in our earlier work with breast tumors we did not observe resistant tumors suggestive of complete silencing of TRPM4 expression.

Ovarian cancer patient ascites containing clumps or organoids of metastatic ovarian cancer cells are removed from patients as a standard of care procedure called paracentesis. While it has been recognized that these ascites ovarian cancer patient organoids are an important tool for research and for application of personalized medicine, the PDAOs have stem cell-like properties and are challenging to target therapeutically [6–9,24]. Unlike PDOs grown in culture in Matrigel, which are pure populations of cancer cells, the PDAOs we isolate and evaluate have not been dissociated in the laboratory, are used fresh, and exhibit very little proliferation during the 3-day culture period in chemically defined medium. Therefore, the PDAOs contain some associated cells that are not ovarian cancer cells [24], and will likely not be killed by ErSO. We therefore hypothesize that in the two patient PDAOs in which 50 nM ErSO reduced the signal by ~90 % and there was no further reduction at higher concentrations of ErSO, we may have achieved complete, or near-complete destruction of the ovarian cancer cells in the organoids.

Importantly, while reduction in the other organoids was generally in the range of 40–60 %, this may be sufficient for a robust response in patients. Unlike most chemotherapy agents that induce apoptosis, which often does not activate immunogenic cell death, ErSO kills cells by inducing necrosis. We show that ErSO induces release of the DAMP, HMGB1, and that medium from ovarian cancer cells treated with ErSO family compounds robustly activates undifferentiated human monocytes and induces their migration. Notably, induction of UPR stress translocates calreticulin to the plasma membrane, where it is recognized by the NK cell receptor NKp46, facilitating immunogenic cell death [41]. Therefore, it may only be necessary for ErSO to induce necrosis in a fraction of the ovarian cancer cells, with activated immune cells inducing immunogenic cell death of surviving ovarian cancer cells.

Single cell sequencing demonstrates that spheroids from ascites recapitulate features of the original ovarian cancer metastases [42]. Since drainage of ascites is widely performed on ovarian cancer patients as a standard of care treatment, the approach we describe of rapid evaluation of sensitivity of ovarian cancer organoids to ErSO provides a way to identify patients most likely to benefit from therapy using ErSO and can be extended to rapid evaluation of the effectiveness of other ovarian cancer therapies.

We describe an approach to personalized medicine using rapid drug testing in fresh ovarian cancer patient organoids, show that a single agent, the necrosis inducing a-UPR hyperactivator, ErSO, induces complete, or near complete, regression in primary and metastatic ovarian cancer xenografts and destroys a sub-set of ascites ovarian cancer patient organoids. With its unusual ability to both directly target cancer cells and to activate immunogenic cell death, a-UPR hyperactivation is a promising strategy for targeting ovarian cancer.

Supplementary Material

Supplementary Material

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.canlet.2025.217738.

Acknowledgements

This study was supported by National Institutes of Health grants RO1DKO71909 (DJS) RO1CA265333 (DJS); RO1CA258746 (PJH), R35CA283859 (PJH), an unrestricted donation from Systems Oncology (DJS, PJH), University of Illinois (PJH), RO1 CA234025 (ERN); Carle Stephens Family Clinical Research Institute (GC). predoctoral fellowships and traineeships: DD and SG, Dept of Biochemistry, Univ. Illinois, MWB: Chemistry-Biology Interface Training Program (T32-GM1336629), ACS Medicinal Chemistry, NCI F99 (F99-CA253731). We are grateful to Dr. M. Livezey for preliminary studies using ovarian cancer cell lines, to the University of Illinois Cell/Media facility for preparation of cell culture medium and to the ovarian cancer patients who consented to use of samples for this study.

Abbreviations:

a-UPR

Anticipatory Unfolded Protein Response

PARP

Poly ADP Ribose Polymerase

PDAO

Patient Derived Ascites Organoid

DAMPs

Damage Associated Molecular Patterns

eIF2α

eukaryotic Initiation Factor 2α

BLI

bioluminescent imaging

HMGB1

High Mobility Group Protein 1

EOC

Epithelial Ovarian Cancer

P/S

Penicillin-Streptomycin

Footnotes

CRediT authorship contribution statement

Darjan Duraki: Writing – review & editing, Investigation, Formal analysis, Data curation. Musarrat Jabeen: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Chengjian Mao: Writing – review & editing, Investigation. Lawrence Wang: Writing – review & editing, Investigation. Santanu Ghosh: Writing – review & editing, Investigation. Xinyi Dai: Writing – review & editing, Investigation. Junyao Zhu: Writing – review & editing, Investigation. Matthew W. Boudreau: Writing – review & editing, Investigation. Erik R. Nelson: Investigation, Funding acquisition. Paul J. Hergenrother: Writing – review & editing, Resources, Funding acquisition. Georgina Cheng: Writing – review & editing, Project administration. David J. Shapiro: Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

Ethics approval and consent to participate

Written informed consent for use of normally discarded ovarian cancer ascites samples was obtained from all patients that provided samples in accordance with Carle Health IRB approved protocol (20CCC3162) (Carle Health, Urbana Illinois, USA). Mouse xenograft studies were performed under protocol 20032 (approved by the Institutional Animal Use and Care Committee (IACUC) of the University of Illinois at Urbana-Champaign.

Consent for publication

All authors approved the final manuscript and submission to this journal.

Declaration of competing interest

The Univ. of Illinois has filed patent applications on some compounds described here on which DJS, MWB and PJH are co-inventors (US Patent No 12,180,159; Title: Activators of the Unfolded Protein Response). Some compounds described herein have been licensed to Systems Oncology and Cureteq AG. PJH is a consultant for Systems Oncology and is on the Systems Oncology Scientific Advisory Board. DD is currently employed by Pfizer.

Data and materials availability

The data supporting the findings and conclusions of this study are available within the paper and its supplementary information. Resources described here and raw data are available from the corresponding authors upon reasonable request. Correspondence and requests for materials should be addressed to: David J. Shapiro: dj shapir@illinois.edu.

References

  • [1].Friedrich M, et al. , Multimodal treatment of primary advanced ovarian cancer, Anticancer Res. 41 (2021) 3253–3260. [DOI] [PubMed] [Google Scholar]
  • [2].Kroeger PT Jr., Drapkin R, Pathogenesis and heterogeneity of ovarian cancer, Curr. Opin. Obstet. Gynecol 29 (2017) 26–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Narod S, Can advanced stage ovarian cancer be cured? Nat. Rev. Clin. Oncol 13 (2016) 255–261. [DOI] [PubMed] [Google Scholar]
  • [4].Penny SM, Ovarian cancer: an overview, Radiol. Technol 91 (2020) 561–575. [PubMed] [Google Scholar]
  • [5].McMullen M, Karakasis K, Rottapel R, et al. , Advances in ovarian cancer, from biology to treatment, Nat. Cancer 2 (2021) 6–8, 2021. [DOI] [PubMed] [Google Scholar]
  • [6].Tan DS, et al. , Mechanisms of transcoelomic metastasis in ovarian cancer, Lancet Oncol. (2006), 10.1016/1470-2045(06)70939-1. [DOI] [PubMed] [Google Scholar]
  • [7].Ahmed N, et al. , Getting to know ovarian cancer ascites: opportunities for targeted therapy-based translational research, Front. Oncol (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Ford CE, Werner B, Hacker NF, Warton K, The untapped potential of ascites in ovarian cancer research and treatment, Br. J. Cancer 123 (2020) 9–16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Kipps E, Tan DS, Kaye SB, Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research, Nat. Rev. Cancer 13 (2013) 273–282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Marchetti C, De Felice F, Romito A, Lacobelli V, Sassu CM, Corrado G, et al. , Chemotherapy resistance in epithelial ovarian cancer: mechanisms and emerging treatments, Semin. Cancer Biol 77 (2021) 144–166. [DOI] [PubMed] [Google Scholar]
  • [11].Christie EL, Bowtell DDL, Acquired chemotherapy resistance in ovarian cancer, Ann. Oncol 28 (2017) viii13–viii15, suppl 8. [DOI] [PubMed] [Google Scholar]
  • [12].Torre LA, Trabert B, Desantis CE, Miller KD, Samimi G, Runowicz CD, et al. , Ovarian cancer statistics, 2018, CA Cancer J. Clin 68 (4) (2018) 284–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].O’Malley DM, Krivak TC, Kabil N, Munley J, Moore KN, PARP inhibitors in ovarian cancer: a review, Target. Oncol (2023), 10.1007/s11523-023-00970-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Andruska N, Zheng X, Yang X, Helferich WG, Shapiro DJ, Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor α positive breast cancer, Oncogene 34 (29) (2015) 3760–3769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Andruska N, Zheng X, Yang X, Mao C, Cherian M, Mahapatra L, Helferich WG, Shapiro DJ, An estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis and induces tumor regression, Proc. Natl. Acad. Sci. USA 112 (15) (2015) 4737–4742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Mao C, Livezey M, Kim JE, Shapiro DJ, Antiestrogen resistant cell lines expressing estrogen receptor α mutations upregulate the unfolded protein response and are killed by BHPI, Sci. Rep (2016), 10.1038/srep34753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Livezey M, Huang R, Hergenrother PJ, Shapiro DJ, Strong and sustained activation of the anticipatory unfolded protein response induces necrotic cell death, Cell Death Differ. 25 (10) (2018) 1796–1807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Yu L, Wang L, Kim J-E, Shapiro DJ, Src couples estrogen receptor to the anticipatory unfolded protein response and regulates cancer cell fate under stress, Biochim Biophys Acta Mol Cell Res Oct 1867 (10) (2020) 118765, 10.1016/j.bbamcr.2020.118765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Zheng X, Andruska N, Lambrecht M, He S, Parissenti A, Hergenrother P, Nelson ER, Shapiro DJ, Targeting multidrug resistant ovarian cancer through estrogen receptor α dependent hyperactivation of the UPR, Oncotarget 9 (2016) 14741–14753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Boudreau MW, Duraki D, Wang L, Mao C, Kim JE, Henn MA, Tang B, Fanning SW, Kiefer J, Tarasow TM, Bruckheimer EM, Moreno R, Mousses S, Greene GL, Roy EJ, Park BH, Fan TM, Nelson ER, Hergenrother PJ, Shapiro DJ, A small molecule activator of the unfolded protein response eradicates human breast tumors in mice, Sci. Transl. Med (2021), 10.1126/scitranslmed.abf1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Boudreau MW, Mulligan MP, Shapiro DJ, Fan TM, Hergenrother PJ, Activators of the anticipatory unfolded protein response with enhanced selectivity for estrogen receptor positive breast cancer, J. Med. Chem 10 (5) (2022) 3894–3912, 65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Livezey M, Kim J-E, Shapiro DJ, A new role for estrogen receptor α in cell proliferation and cancer: activating the anticipatory unfolded protein response, Front. Endocrinol (2018), 10.3389/fendo.2018.00325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Ghosh S, Yang R, Duraki D, Zhu J, Kim JE, Jabeen M, Mao C, Dai X, Livezey MR, Boudreau MW, Park BH, Nelson ER, Hergenrother PJ, Shapiro DJ, Plasma membrane channel TRPM4 mediates immunogenic therapy-induced necrosis, Cancer Res. 83 (18) (2023) 3115–3130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Latifi A, et al. , Isolation and characterization of tumor cells from the ascites of ovarian cancer patients: molecular phenotype of chemoresistant tumors, PLoS One 7 (10) (2012) e46858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Palchaudhuri R, Lambrecht MJ, Botham RC, Partlow KC, van Ham TJ, Putt KS, et al. , A small molecule that induces intrinsic pathway apoptosis with unparalleled speed, Cell Rep. 13 (2015) 2027–2036, 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Yu L, Wang L, Mao C, Duraki D, Kim JE, Huang R, Helferich WG, Nelson ER, Park BH, Shapiro DJ, Estrogen-independent myc overexpression confers endocrine therapy resistance on breast cancer cells expressing ERαY537S and ERαD538G mutations, Cancer Lett. 442 (2019) 373–382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Hirschl N, Leveque W, Granitto J, Sammarco V, Fontillas M, Penson RT, PARP inhibitors: strategic use and optimal management in ovarian cancer, Cancers (Basel) 16 (5) (2024) 932, 10.3390/cancers16050932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Spillman MA, Manning NG, Dye WW, Sartoriius CA, Post MD, Harrell JC, Jacobsen BM, Horwitz KB, Tissue-specific pathways for estrogen regulation of ovarian cancer growth and metastases. 2010, Cancer Res. 70 (21) (2010), 8927–8036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Freeburg EM, Goyeneche AA, Seidel EE, Telleria CM, Resistance to cisplatin does not affect sensitivity of human ovarian cancer cells to mifepristone cytotoxicity, Cancer Cell Int. (2009), 10.1186/1475-2867-9-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Sprooten J, De Wijngaert P, Vanmeerbeerk I, Martin S, Vangheluwe P, Schlenner S, et al. , Necroptosis in immuno-oncology and cancer immunotherapy, Cells 9 (8) (2020) E1823, 10.3390/cells9081823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Shahsavari Z, Karami-Tehrani F, Salami S, Shikonin induced necroptosis via reactive oxygen species in the T-47D breast cancer cell line, Asian Pac. J. Cancer Prev. APJCP 16 (16) (2015) 7261–7266. [DOI] [PubMed] [Google Scholar]
  • [32].Pawlikowska M, Jędrzejewski T, Brożyna AA, Wrotek S, Protein-bound polysaccharides from coriolus versicolor induce RIPK1/RIPK3/MLKL-mediated necroptosis in ER-positive breast cancer and amelanotic melanoma cells, Cell. Physiol. Biochem 54 (4) (2020) 591–604, 2020 Jun 13. [DOI] [PubMed] [Google Scholar]
  • [33].Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P, Immunogenic cell death and DAMPs in cancer therapy, Nat. Rev. Cancer 12 (12) (2012) 860–875, 2012. [DOI] [PubMed] [Google Scholar]
  • [34].Koren E, Fuchs Y, Modes of regulated cell death in cancer, Cancer Discov. 11 (2) (2021) 245–265. [DOI] [PubMed] [Google Scholar]
  • [35].De Felice F, Romito A, Iacobelli V, Sassu CM, Corrado G, et al. , Chemotherapy resistance in epithelial ovarian cancer: mechanisms and emerging treatments, Semin. Cancer Biol 77 (2021) 144–166. [DOI] [PubMed] [Google Scholar]
  • [36].Le Saux O, Ray-Coquard I, Labidi-Galy SI, Challenges for immunotherapy for the treatment of platinum resistant ovarian cancer, Semin. Cancer Biol 77 (2021) 127–143. [DOI] [PubMed] [Google Scholar]
  • [37].Ang ZB, Zhang X, Fang C, Liu XT, Liao QJ, Wu N, Wang L, Immunotherapy and the ovarian cancer microenvironment: exploring potential strategies for enhanced treatment efficacy, Immunology (2024), 10.1111/mmm.13793. [DOI] [PubMed] [Google Scholar]
  • [38].Moore KN, Angelergues A, Konency GE, et al. , Van gorp, t and gynecology Oncology group partners and the European network of gynaecological oncological trial groups, N. Engl. J. Med 389 (23) (2023) 2162–2174.38055253 [Google Scholar]
  • [39].Jeselsohn R, Buchwalter G, De Angelis C, Brown M, Schiff R, ESR1 mutations – a mechanism for acquired endocrine resistance in breast cancer, Nat. Rev. Clin. Oncol 12 (10) (2015) 573–583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Perner F, et al. Cai SF, MEN1 mutations mediate clinical resistance to menin inhibition, Nature 615 (2023) 913–919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Sen Santara S, Lee D-J, Crespo A, Hu JJ, Walker C, et al. Liberman J, The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells, Nature 616 (2023) 348–356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Velletri T, Villa CE, Cilli D, Barzhagi B, Lo Riso P, et al. Testa G, Single-cell derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer, Cell Death Differ. 29 (3) (2022) 614–626. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material
NIHMS2091908-supplement-Supplementary_Material.pdf (808.9KB, pdf)

Data Availability Statement

The data supporting the findings and conclusions of this study are available within the paper and its supplementary information. Resources described here and raw data are available from the corresponding authors upon reasonable request. Correspondence and requests for materials should be addressed to: David J. Shapiro: dj shapir@illinois.edu.

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