Epithelial ovarian cancer (EOC) exhibits a unique mode of metastasis, involving spheroid formation in the peritoneum. Our research on EOC spheroid cell biology has provided valuable insights into the signaling plasticity associated with metastasis. We speculate that EOC cells modify their biology between tumour and spheroid states during cancer dormancy, although the specific mechanisms underlying this transition remain unknown. Here, we present novel findings from direct comparisons between cultured EOC spheroids and organoids. Our results indicated that AMP-activated protein kinase (AMPK) activity was significantly upregulated and protein kinase B (Akt) was downregulated in EOC spheroids compared to organoids, suggesting a clear differential phenotype. Through RNA sequencing analysis, we further supported these phenotypic differences and highlighted the significance of cell cycle regulation in organoids. By inhibiting the G2/M checkpoint via kinase inhibitors, we confirmed that this pathway is essential for organoids. Interestingly, our results suggest that specifically targeting aurora kinase A (AURKA) may represent a promising therapeutic strategy since our cells were equally sensitive to Alisertib treatment as both spheroids and organoids. Our findings emphasize the importance of studying cellular adaptations of EOC cells, as there may be different therapeutic targets depending on the step of EOC disease progression.

Late-stage epithelial ovarian cancer (EOC) involves the widespread dissemination of malignant disease throughout the peritoneal cavity, often accompanied by ascites. EOC metastasis relies on the formation of multicellular aggregates, called spheroids. Given that Liver Kinase B1 (LKB1) is required for EOC spheroid viability and LKB1 loss in EOC cells decreases tumor burden in mice, we investigated whether the LKB1 complex controls the invasive properties of human EOC spheroids. LKB1 signalling was antagonized through the CRISPR/Cas9 genetic knockout of LKB1 and/or the RNAi-dependent targeting of STE20-related kinase adaptor protein (STRAD, an LKB1 activator). EOC spheroids expressing nuclear GFP (green) or mKate2 (red) constructs were embedded in Matrigel for real-time live-cell invasion monitoring. Migration and invasion were also assessed in spheroid culture using Transwell chambers, spheroid reattachment, and mesothelial clearance assays. The loss of LKB1 and STRAD signalling decreased cell invasion through Matrigel and Transwell membranes, as well as mesothelial cell clearance. In the absence of LKB1, zymographic assays identified a loss of matrix metalloproteinase (MMP) activity, whereas spheroid reattachment assays found that coating plates with fibronectin restored their invasive potential. A three-dimensional EOC organoid model demonstrated that organoid area was greatly reduced by LKB1 loss. Overall, our data indicated that LKB1 and STRAD facilitated EOC metastasis by promoting MMP activity and fibronectin expression. Given that LKB1 and STRAD are crucial for EOC metastasis, targeting LKB1 and/or STRAD could disrupt the dissemination of EOC, making inhibitors of the LKB1 pathway an alternative therapeutic strategy for EOC patients.

Epithelial ovarian cancer (EOC) is a leading cause of gynecological cancer mortality, driven largely by late diagnosis and chemoresistance. While autophagy is critical for EOC spheroid survival during metastasis, the role of ULK1, a key regulator of autophagy, in EOC progression remains unclear. To investigate this, we utilized CRISPR/Cas9 technology to delete ULK1 in EOC cell lines OVCAR8, HEYA8, ES2 and the fallopian tube epithelial cell line FT190. ULK1 loss and autophagy disruption were confirmed in EOC spheroids, with reduced Beclin-1 phosphorylation, impaired LC3 processing, and p62 accumulation. ULK1 knockout decreased EOC spheroid cell viability via increased apoptosis, and impaired matrix-bound organoid growth, offering new insights into ULK1 activity in affecting EOC tumor growth and spread. These findings were supported by in vivo xenograft models, in which ULK1 loss significantly reduced tumor burden and metastatic potential. ULK1 requirement during metastasis was supported by diminished invasive capacity of ULK1 knockout spheroid cells in mesothelial clearance assays. To investigate ULK1 mechanisms contributing to EOC tumor progression and metastasis, we conducted proteomic analyses of OVCAR8 spheroids, which revealed ULK1 loss disrupted critical pathways, including MEK-MAPK, PI3K-AKT-mTOR, and apoptosis regulation. Although ULK1 knockout failed to synergize with standard-of-care chemotherapeutics, it significantly enhanced sensitivity to MEK and mTOR inhibition. Analysis of ovarian cancer datasets demonstrates that high ULK1 mRNA correlates with a poorer 10-year overall and progression-free survival; in fact, its expression is further elevated in metastases as compared with primary tumors and normal tissue. Treatment of metastatic patient-derived organoids with the clinical ULK1 inhibitor DCC-3116, MEK inhibitor trametinib, or mTORC1/2 inhibitor AZD-8055 reduced viability in a subset of these samples, reflecting inter-patient heterogeneity and need for biomarker-guided selection. Overall, this study highlights ULK1 as a critical regulator of multiple steps of EOC disease progression, underscoring its potential as a therapeutic target in advanced ovarian cancer.

Background/Objectives: Epithelial ovarian cancer (EOC) is often diagnosed at advanced stages, with metastasis driven by spheroid dissemination within the peritoneal cavity. We previously demonstrated that autophagy supports spheroid cell survival and suggest that it contributes to chemoresistance. Unc-51-like autophagy activating kinase 1 (ULK1), a key regulator of autophagy, has emerged as a promising therapeutic target. Here, we evaluated the effects of ULK1 inhibition via MRT68921, alone and in combination with afatinib—a tyrosine kinase inhibitor (TKI) known to induce pro-survival autophagy—in EOC. Methods: High-grade serous (HGSOC) and ovarian clear cell carcinoma (OCCC) cell lines were cultured under adherent and spheroid conditions. Immunoblotting confirmed on-target effects and modulation of autophagy. Autophagic flux was assessed using mCherry-eGFP-LC3 reporter assays. We assessed 96 dose combinations of MRT68921 and afatinib using drug combination matrices, with synergy evaluated via Synergy Finder. Promising combinations were evaluated across multiple EOC spheroid models and patient ascites-derived organoids. Results: MRT68921 inhibited ULK1 activity and reduced autophagic flux in a context-dependent manner while afatinib alone induced autophagy. Their combination produced synergistic effects at select concentrations, impairing spheroid reattachment and viability. However, MRT68921 alone significantly reduced viability across multiple EOC models, including patient ascites-derived organoids. Conclusions: This study is the first to evaluate the combined effects of MRT68921 and afatinib in epithelial ovarian cancer. Our findings demonstrate that ULK1 inhibition via MRT68921 consistently reduces cell viability across multiple ovarian cancer models, supporting ULK1 as a promising therapeutic target. In contrast, combination with afatinib produced limited and context-dependent effects, indicating that further investigation is needed to identify optimal combination strategies for ULK1-targeted therapies.

PARP inhibitors (PARPis) are new targeted agents that exploit homologous recombination DNA repair deficiencies (HRDs), which are present in 50% of high-grade serous carcinoma (HGSC) cases. Currently, olaparib is approved as maintenance therapy for BRCA1/2-mutated HGSC, and niraparib is approved for platinum-sensitive recurrent disease. However, research is currently expanding their potential as front-line agents or in combination with carboplatin, a standard HGSC chemotherapeutic. Immortalized ovarian cancer (iOvCa) cell lines, developed from HGSC patient ascites, were treated with carboplatin, olaparib and niraparib to determine their sensitivity. Immunofluorescence analysis of RAD51 was conducted for HRD testing of all the cell lines. The cell lines were cultured as three-dimensional organoids and spheroids to mimic tumor growth and metastasis, respectively, and then treated to assess the effects of different drug combinations. The half-maximal inhibitory concentrations of olaparib and niraparib varied across our iOvCa cell lines, with iOvCa195 BRCA1-mutant line exhibiting the expected high sensitivity to both PARPis. Direct combination of carboplatin with olaparib or niraparib enhanced cell killing, yet achieved cell viability levels to those of carboplatin alone. In sequential experiments, carboplatin followed by either PARPi or vice versa showed no significant difference in cell viability to carboplatin alone, except in iOvCa195 organoids when treated with a PARPi first. Overall, first-line carboplatin treatment remains ideal, yet there may be select utility for PARPi prior to chemotherapy. Using patient-derived tumor models such as spheroids and organoids may provide insights for on-going and future clinical trials to enhance therapeutic outcomes for HGSC patients.

The goal of this study was to (a) determine the minimum selection concentrations of tetracycline family antibiotics necessary to maintain plasmids carrying tetracycline-resistant genes and (b) correlate these results to environmental hotspot concentrations reported in previous studies. This study used two plasmids (pT295A and pT413A) originating from dairy manure in a surrogate Escherichia coli host CV601. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined as follows: 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, and 0.13–0.25 mg/L for tetracycline. Mixing oxytetracycline and chlortetracycline had minimum selection concentration values increased 2-fold compared to those in single antibiotic tests. Minimum selection concentrations found in this study were lower than reported environmental hotspot concentrations, suggesting that tetracycline family antibiotics were likely to be the driver for the selection and maintenance of these plasmids. Relatively high plasmid loss rates (>90%) were observed when culturing a strain carrying a tetracycline-resistant plasmid in antibiotic-free nutrient-rich and nutrient-defined media. Overall, results suggested that these plasmids can be maintained at concentrations environmentally relevant in wastewater treatment plants, sewage, manure, and manured soil; however, they are unstable and easily lost in the absence of antibiotics.