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The identification of tumor-initiating cells (TICs) has traditionally relied on surface markers including CD133, CD44, CD117, and the aldehyde dehydrogenase (ALDH) enzyme, which have diverse expression across samples. A more reliable indication of TICs may include the expression of embryonic transcription factors that support long-term self-renewal, multipotency, and quiescence. We hypothesize that SOX2, OCT4, and NANOG will be enriched in ovarian TICs and may indicate TICs with high relapse potential. We evaluated a panel of eight ovarian cancer cell lines grown in standard 2-D culture or in spheroid-enriching 3-D culture, and correlated expression with growth characteristics, TIC marker expression, and chemotherapy resistance. RNA-sequencing showed that cell cycle regulation pathways involving SOX2 were elevated in 3-D conditions. HGSOC lines had longer doubling-times, greater chemoresistance, and significantly increased expression of SOX2, OCT4, and NANOG in 3-D conditions. CD117+ or ALDH+/CD133+ cells had increased SOX2, OCT4, and NANOG expression. Limiting dilution in in vivo experiments implicated SOX2, but not OCT4 or NANOG, with early tumor-initiation. An analysis of patient data suggested a stronger role for SOX2, relative to OCT4 or NANOG, for tumor relapse potential. Overall, our findings suggest that SOX2 may be a more consistent indicator of ovarian TICs that contribute to tumor repopulation following chemotherapy. Future studies evaluating SOX2 in TIC biology will increase our understanding of the mechanisms that drive ovarian cancer relapse.

Background Epithelial ovarian cancer (EOC) is a global health burden, with the poorest five-year survival rate of the gynecological malignancies due to diagnosis at advanced stage and high recurrence rate. Recurrence in EOC is driven by the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that are supported by a complex extracellular matrix and immunosuppressive microenvironment. To target TICs to prevent recurrence, we identified genes critical for TIC viability from a whole genome siRNA screen. A top hit was the cancer-associated, proteoglycan subunit synthesis enzyme UDP-glucose dehydrogenase (UGDH). Methods Immunohistochemistry was used to characterize UGDH expression in histological and molecular subtypes of EOC. EOC cell lines were subtyped according to the molecular subtypes and the functional effects of modulating UGDH expression in vitro and in vivo in C1/Mesenchymal and C4/Differentiated subtype cell lines was examined. Results High UGDH expression was observed in high-grade serous ovarian cancers and a distinctive survival prognostic for UGDH expression was revealed when serous cancers were stratified by molecular subtype. High UGDH was associated with a poor prognosis in the C1/Mesenchymal subtype and low UGDH was associated with poor prognosis in the C4/Differentiated subtype. Knockdown of UGDH in the C1/mesenchymal molecular subtype reduced spheroid formation and viability and reduced the CD133 + /ALDH high TIC population. Conversely, overexpression of UGDH in the C4/Differentiated subtype reduced the TIC population. In co-culture models, UGDH expression in spheroids affected the gene expression of mesothelial cells causing changes to matrix remodeling proteins, and fibroblast collagen production. Inflammatory cytokine expression of spheroids was altered by UGDH expression. The effect of UGDH knockdown or overexpression in the C1/ Mesenchymal and C4/Differentiated subtypes respectively was tested on mouse intrabursal xenografts and showed dynamic changes to the tumor stroma. Knockdown of UGDH improved survival and reduced tumor burden in C1/Mesenchymal compared to controls. Conclusions These data show that modulation of UGDH expression in ovarian cancer reveals distinct roles for UGDH in the C1/Mesenchymal and C4/Differentiated molecular subtypes of EOC, influencing the tumor microenvironmental composition. UGDH is a strong potential therapeutic target in TICs, for the treatment of EOC, particularly in patients with the mesenchymal molecular subtype.

The current tools for validating dose delivery and optimizing new radiotherapy technologies in radiation therapy do not account for important dose modifying factors (DMFs), such as variations in cellular repair capability, tumor oxygenation, ultra-high dose rates and the type of ionizing radiation used. These factors play a crucial role in tumor control and normal tissue complications. To address this need, we explored the feasibility of developing a transportable cell culture platform (TCCP) to assess the relative biological effectiveness (RBE) of ionizing radiation. We measured cell recovery, clonogenic viability and metabolic viability of MDA-MB-231 cells over several days at room temperature in a range of concentrations of fetal bovine serum (FBS) in medium-supplemented gelatin, under both normoxic and hypoxic oxygen environments. Additionally, we measured the clonogenic viability of the cells to characterize how the duration of the TCCP at room temperature affected their radiosensitivity at doses up to 16 Gy. We found that (78±2)% of MDA-MB-231 cells were successfully recovered after being kept at room temperature for three days in 50% FBS in medium-supplemented gelatin at hypoxia (0.4±0.1)% pO2, while metabolic and clonogenic viabilities as measured by ATP luminescence and colony formation were found to be (58±5)% and (57±4)%, respectively. Additionally, irradiating a TCCP under normoxic and hypoxic conditions yielded a clonogenic oxygen enhancement ratio (OER) of 1.4±0.6 and a metabolic OER of 1.9±0.4. Our results demonstrate that the TCCP can be used to assess the RBE of a DMF and provides a feasible platform for assessing DMFs in radiation therapy applications.

Spatially fractionated radiotherapy (SFRT) offers a promising approach for debulking large tumors by delivering high-dose radiation to a fraction of the tumor volume. However, the complex tumor microenvironment necessitates models beyond traditional 2D cultures and resource-intensive animal studies for SFRT investigations. Three-dimensional (3D) scaffold-based models with an adequate cross-sectional area have emerged as uniquely suited platforms to bridge this gap, by providing a more realistic platform for GRID-based SFRT research. In this study, we employed a 3D co-culture scaffold model to dissect the contributions of the radiation-induced bystander effect, abscopal effect, and immune system response on clonogenic survival following GRID irradiation. MDA-MB-231 breast cancer cells were seeded on commercial 3D scaffolds and irradiated at a 20 Gy peak dose using lead grids with three- and six-hole patterns, exposing ~12.8% and 25.7% of the scaffold area, respectively. An assessment of reproductive cell survival revealed a significant bystander effect, as the survival was notably lower than predicted based solely on the directly irradiated fraction. Evidence of an abscopal effect was observed by culturing non-irradiated cells in media exposed to GRID irradiation. Furthermore, a co-culture with allogeneic peripheral blood mononuclear cells (PBMCs) modulated clonogenic survival, with an additive effect observed when combined with SFRT. These findings underscore the presence of a bystander effect in GRID radiotherapy and indicate an abscopal immune component, particularly with the three-hole GRID configuration. This study established the utility of in vitro 3D co-culture scaffolds as an effective model system for elucidating complex SFRT-mediated biological responses.

Background Metastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies. Treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC, and the complexity of this disease implies multi-faceted pathway interactions. Given that IKKε behaves as an oncogene in breast cancer, we hypothesized that IKKε regulates NF-κB signaling to control diverse oncogenic functions in TNBC. Methods Vector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε, in conjunction with p52 RNA interference or MEK inhibition. Results This study found that non-canonical NF-κB p52 levels are inversely proportional to ΙΚΚε, and growth of TNBC cells in anchorage supportive, high-attachment conditions requires IKKε and activated MEK. Growth of these cells in anchorage resistant conditions requires IKKε and activated MEK or p52. In this model, IKKε and MEK cooperate to support overall viability whereas the p52 transcription factor is only required for viability in low attachment conditions, underscoring the contrasting roles of these proteins. Conclusions This study illustrates the diverse functions of IKKε in TNBC and highlights the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC.

Epithelial ovarian cancer (EOC) remains the fifth leading cause of cancer-related death in women worldwide, partly due to the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that promote disease relapse. We previously described a role for the NF-κB pathway in promoting TIC chemoresistance and survival through NF-κB transcription factors (TFs) RelA and RelB, which regulate genes important for the inflammatory response and those associated with cancer, including microRNAs (miRNAs). We hypothesized that NF-κB signaling differentially regulates miRNA expression through RelA and RelB to support TIC persistence. Inducible shRNA was stably expressed in OV90 cells to knockdown RELA or RELB; miR-seq analyses identified differentially expressed miRNAs hsa-miR-452-5p and hsa-miR-335-5p in cells grown in TIC versus adherent conditions. We validated the miR-seq findings via qPCR in TIC or adherent conditions with RELA or RELB knocked-down. We confirmed decreased expression of hsa-miR-452-5p when either RELA or RELB were depleted and increased expression of hsa-miR-335-5p when RELA was depleted. Either inhibiting miR-452-5p or mimicking miR-335-5p functionally decreased the stem-like potential of the TICs. These results highlight a novel role of NF-κB TFs in modulating miRNA expression in EOC cells, thus opening a better understanding toward preventing recurrence of EOC.

Background/Objectives: Ovarian cancer (OC) remains the most lethal gynecologic malignancy, largely due to late-stage diagnosis and high rates of recurrence following platinum-based chemotherapy. Growing evidence implicates cancer stem-like cells (CSCs) in OC relapse, as these cells exhibit enhanced chemoresistance, stemness, epithelial–mesenchymal transition (EMT), and the capacity to remodel the tumor microenvironment. Inhibitors of DNA-binding (ID) 1-4 proteins are transcription factors with known redundancy; however, their collective role in OC chemotherapy response remains poorly defined. Here, we examined how ID family signaling responds to chemotherapy and contributes to CSC-associated features and microenvironment remodeling. Methods: Publicly available patient data, OC cell lines, and a subcutaneous xenograft mouse model were used to correlate changes in ID1-4 expression with CSCs, EMT, and the tumor microenvironment (TME). OC cell lines were used for in vitro assays to evaluate CSC features and IL-6 production in the presence of carboplatin and/or a small molecule inhibitor of ID proteins, AGX51. Results: Analysis of clinical datasets, cell lines, and in vivo models revealed enrichment of ID1-4 following chemotherapy, with additive increases across treatment cycles. In vivo ID2 and ID4 expression was associated with IL-6 secretion and loss of anti-tumoral macrophages. Pan-ID inhibition demonstrated that cumulative ID activity minimally supports CSC maintenance during chemotherapy, while more strongly regulating IL-6 secretion. Conclusions: IL-6 production from cancer cells was at least partially dependent on ID proteins, linking collective ID signaling to microenvironment remodeling and relapse potential in ovarian cancer.

Functional expression of voltage-gated Na + channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis -acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

Disease recurrence is the major cause of morbidity and mortality of ovarian cancer (OC). In terms of maintenance therapies after platinum-based chemotherapy, PARP inhibitors significantly improve the overall survival of patients with BRCA mutations but is of little benefit to patients without homologous recombination deficiency (HRD). The stem-like tumor-initiating cell (TIC) population within OC tumors are thought to contribute to disease recurrence and chemoresistance. Therefore, there is a need to identify drugs that target TICs to prevent relapse in OC without HRD. RNA sequencing analysis of OC cells grown in TIC conditions revealed a strong enrichment of genes involved in drug metabolism, oxidative phosphorylation and reactive oxygen species (ROS) pathways. Concurrently, a high-throughput drug screen identified drugs that showed efficacy against OC cells grown as TICs compared to adherent cells. Four drugs were chosen that affected drug metabolism and ROS response: disulfiram, bardoxolone methyl, elesclomol and salinomycin. The drugs were tested in vitro for effects on viability, sphere formation and markers of stemness CD133 and ALDH in TICs compared to adherent cells. The compounds promoted ROS accumulation and oxidative stress and disulfiram, elesclomol and salinomycin increased cell death following carboplatin treatment compared to carboplatin alone. Disulfiram and salinomycin were effective in a post-surgery, post-chemotherapy OC relapse model in vivo, demonstrating that enhancing oxidative stress in TICs can prevent OC recurrence.

BackgroundOncolytic virus (OV)-based therapies have demonstrated considerable promise for treating various solid tumor types. However, mounting evidence indicates OVs are negatively impacted by Type I interferon(s) (IFN) produced in solid tumors with functional cytosolic nucleic acid sensing mechanisms, including the cGAS/STING and/or RIG-I pathways.1 2 VAX014 is a novel, clinical stage oncolytic agent based on recombinant bacterial minicells (rBMCs) and is designed to target and deliver a pre-formed oncolytic protein toxin, perfringolysin O (PFO), directly to integrin-expressing tumor cells.3 4 Here we characterize the interaction and impact of tumor intrinsic STING and RIG-I on the antitumor activity of VAX014 in vitro and in vivo.MethodsTargeted deletion of STING or RIG-I in wild type MB49 murine urothelial carcinoma cells was performed via CRISPR-Cas9. Following extensive orthogonal in vitro functional characterization to confirm genotype/phenotype, MB49STING KO and MB49RIG-I KO tumor cell lines were utilized in in vivo preclinical pharmacology studies in wild type C57BL/6 mice to assess any influence of tumor intrinsic STING and/or RIG-I on the efficacy of VAX014 following intratumoral (i.t.) administration to intradermal (i.d.) tumors. Individual tumor growth rates and survival curves were plotted against respective saline and wild type controls.Results In vitro analysis demonstrated wild type MB49 cells express STING and RIG-I (but not cGAS) and upregulate PD-L1 and MHC-I in a Type I IFN and Tank-binding kinase-1 (TBK-1) dependent manner following treatment with VAX014. Targeted genetic ablation of either STING or RIG-I (MB49STING KO and MB49RIG-I KO) reduced this response. In MB49STING KO cells, pharmacologic inhibition of TBK-1 led to complete elimination of Type I IFN production and subsequent PD-L1/MHC-I upregulation, as did pharmacologic inhibition of STING in MB49RIG-I KO cells. In vivo, both the MB49STING KO and MB49RIG-I KO tumor cell lines had similar i.d. tumor growth rates to wild type MB49. Consistent with previous work, weekly i.t. administration of VAX014 to wild type MB49 tumors led to a 100% durable complete response (CR) rate. In contrast, the loss of tumor intrinsic STING or RIG-I reduced CR rates and lengthened the time to respond in MB49STING KO or MB49RIG-I KO tumors.ConclusionsVAX014 activates both the murine STING and RIG-I pathways and the presence of tumor-intrinsic STING and/or RIG-I leads to optimal antitumor activity of VAX014 following i.t. administration. This unique mechanism pairs STING and RIG-I agonism and subsequent Type I IFN production with oncolysis-mediated availability of tumor antigens, which together, may lead to better antitumor T cell priming.AcknowledgementsWe would like to acknowledge Steven N. Fiering, Ph.D and Trevor Hallam, Ph.D for useful discussions, review, and interpretation of study data.Ethics ApprovalThis study was approved by San Diego State University’s Institutional Animal Care and Use Committee under approved Animal Protocol number IACUC-22–064.ReferencesKaufman HL, Shalhout SZ, Iodice G. Talimogene Laherparepvec: Moving From First-In-Class to Best-In-Class. Front. Mol. Biosci. 2022;9:834841Li Q, Tan F, Wang Y, Liu X, Kong X, Meng J, Yang L, Cen S. The gamble between oncolytic virus therapy and IFN. Front. Immunol. 2022;13:971674Tsuji S, Reil KA, Nelson K, Proclivo VH, McGuire KL, Giacalone MJ. Intravesical VAX014 Synergizes with PD-L1 Blockade to Enhance Local and Systemic Control of Bladder Cancer. Cancer Imunol. Res. 2022;10:978–95.Reil KA, Tsuji S, Molina E, Nelson KL, McGuire KL, Giacalone MJ. Intralesional administration of VAX014 facilitates in situ immunization and potentiates immune checkpoint blockade in immunologically cold tumors. J. Immunother Cancer. 11:e006749