Bo R. Rueda

Flower-Win/Lose signaling underlies a tumor–host fitness conflict in ovarian cancer, in which tumor-derived exosomal Tu-stroma reduces the fitness of surrounding host cells, favoring tumor expansion within host tissues. High-grade serous carcinoma (HGSC), the most prevalent histotype of epithelial ovarian cancer and leading cause of ovarian-cancer-related mortality, arises from serous tubal intraepithelial carcinoma (STIC), a noninvasive precursor typically identifiable only by histopathologic examination of excised fallopian tube tissue. Here we evaluated Tu-stroma, a tumor-associated long noncoding RNA enriched in serum extracellular vesicles (EVs), as a circulating biomarker in STIC and early-stage HGSC, representing the earliest histopathologically defined phases of disease. Tu-stroma was quantified in serum EVs from 506 individuals with histologically normal adnexa, benign-adnexal lesions, isolated STIC and HGSC across International Federation of Gynecology and Obstetrics (FIGO) stages I–IV. Normalized EV Tu-stroma ΔC_T values were elevated in STIC (n = 43) and HGSC (n = 232) versus controls (n = 143) and benign-adnexal lesions (n = 88), including premenopausal women (controls = 57, benign = 34, STIC = 32, HGSC = 88). In paired receiver operating characteristic analyses, Tu-stroma showed higher discrimination than cancer antigen 125 (CA125) for STIC versus controls (area under the curve (AUC) = 0.99 versus 0.59, P < 0.001) and benign-adnexal masses (0.99 versus 0.31, P < 0.001), and for HGSC versus controls (AUC = 0.99 versus 0.96, P = 0.001) and benign-adnexal masses (0.98 versus 0.84, P < 0.001). Tu-stroma showed no systematic elevation across inflammatory or physiologic comparators (n = 116) or nonovarian malignancies (n = 160), each assessed against healthy controls (n = 100), supporting disease-context specificity. Modeled risk-enriched contexts yielded predictive values compatible with intended-use populations. These findings identify Tu-stroma as a candidate EV-RNA marker associated with pre-invasive and early HGSC biology. Prospective evaluation in consecutively enrolled real-world cohorts is required to validate diagnostic performance, predictive value and clinical utility.

Detecting low-abundance proteins is essential in liquid biopsy applications, particularly for extracellular vesicle (EV)-based diagnostics. Here, we introduce RapidEx (Rotationally amplified protein intensity detection of Extracellular vesicles), a disc-fluidic system engineered for highly sensitive protein assays. RapidEx performs two key functions within a single disc cartridge: i) enzymatic chemical reactions for target labeling and ii) efficient washing to enhance signal-to-background contrast.

Ovarian cancer stem cells (CSCs) can seed recurrent drug-resistant disease. Likewise, non-CSCs can acquire CSC phenotypic properties. How this process is orchestrated is of interest to inform how it might be prevented. We tested the hypothesis that ovarian CSC and/or drug-resistant tumor cells confer stem-like properties via extracellular vesicles (EVs). We focused our investigation on how EVs might mediate EZH2 signaling to promote a phenotypic change in drug-sensitive, non-CSCs. To accomplish this, we utilized paired PARP inhibitor-sensitive and - resistant ovarian cancer (OvCa) cell lines, EZH2 knockdown lines, and patient-derived organoids (PDOs) originating from recurrent high-grade serous OvCa. Small EVs isolated from drug-sensitive, CSC and/or drug-resistant enriched cultures, PARP inhibitor (olaparib) resistant lines, or drug-treated (olaparib or carboplatin) lines were cultured with treatment naïve or sensitive lines for defined time points. The impact of small EV exposure was determined by assessing cell number, metabolic activity, viability, sphere and colony-forming capacity, ALDH activity, DNA damage, and changes in associated signaling pathways. We found that EVs from CSC or drug-resistant enriched cell fractions communicate CSC-like phenotypes to the more sensitive tumor cells via EZH2 canonical and non-canonical signaling pathways, promoting stemness. We conclude that EV-mediated activation of EZH2 signaling represents a targetable mechanism contributing to stemness-associated drug resistance in OvCa. Graphical

Ovarian cancer is one of the deadliest gynecological malignancies, where most patients become clinically symptomatic at advanced stages of disease due to the lack of effective diagnostic screening. Despite recent advances in surgical resection and chemotherapy, recurrent ovarian cancer remains largely refractory to treatment, resulting in poor prognosis. The ovarian cancer tumor microenvironment (TME) is highly abnormal and presents a significant barrier to successful therapy. A combination of abnormal vasculature, desmoplastic extracellular matrix, and aberrantly activated hypoxic and immune-suppressive pathways culminates in promoting tumor growth, dissemination, chemoresistance, and immunosuppression. Whilst immune checkpoint inhibitors have shown success in other cancers, their application in ovarian cancer, particularly at advanced stages, remains limited. In this review, we discussed the application of tumor extracellular matrix normalizing therapies in preclinical models of advanced ovarian cancer, and their synergistic benefit to chemotherapy and immunotherapy. Collectively, these insights underscore TME normalization as a promising therapeutic strategy with the potential to improve ovarian cancer management.

Supplementary Data consisting of table 1-4 and supplementary figure 1-4

Antibody–peptide epitope conjugates (APEC) are a new class of modified antibody–drug conjugates that redirect T-cell viral immunity against tumor cells. APECs contain a tumor-specific protease cleavage site linked to a patient-specific viral epitope, resulting in presentation of viral epitopes on cancer cells and subsequent recruitment and killing by CD8+ T cells. Here we developed an experimental pipeline to create patient-specific APECs and identified new preclinical therapies for ovarian carcinoma. Using functional assessment of viral peptide antigen responses to common viruses like cytomegalovirus (CMV) in patients with ovarian cancer, a library of 192 APECs with distinct protease cleavage sequences was created using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Each APEC was tested for in vitro cancer cell killing, and top candidates were screened for killing xenograft tumors grown in zebrafish and mice. These preclinical modeling studies identified EpCAM-MMP7-CMV APEC (EpCAM-MC) as a potential new immunotherapy for ovarian carcinoma. Importantly, EpCAM-MC also demonstrated robust T-cell responses in primary ovarian carcinoma patient ascites samples. This work highlights a robust, customizable platform to rapidly develop patient-specific APECs.Significance:This study develops a high-throughput preclinical platform to identify patient-specific antibody–peptide epitope conjugates that target cancer cells and demonstrates the potential of this immunotherapy approach for treating ovarian carcinoma.

Pulmonary lymphangioleiomyomatosis (LAM) is a metastatic sarcoma, but the mechanisms of LAM metastasis are unknown. Extracellular vesicles (EVs) regulate cancer metastasis; however, their roles in LAM have not yet been thoroughly investigated. Here, we report the discovery of distinct LAM-EV subtypes derived from primary tumor or metastasizing LAM cells that promote LAM metastasis through ITGα6/β1-c-Src-FAK signaling, triggered by the shuttling of ATP synthesis to cell pseudopodia or the activation of integrin adhesion complex, respectively. This signaling leads to increased LAM cell migration, invasiveness, and stemness and regulates metastable (hybrid) phenotypes that are all pivotal for metastasis. Mouse models corroborate in vitro data by demonstrating a significant increase in lung metastatic burden upon exposure to EVs through distinct mechanisms involving either lung resident fibroblasts or metalloproteinases' activation that are EV subtype dependent. The clinical relevance of these findings is underscored by increased EV biogenesis in LAM patients and the enrichment of these EV cargo with lung-tropic integrins and metalloproteinases. These findings establish EVs as a novel therapeutic target in LAM, warranting future clinical studies.

Background: Tumor heterogeneity and antigen escape are mechanisms of resistance to chimeric antigen receptor (CAR)-T cell therapy, especially in solid tumors. Targeting multiple antigens with a unique CAR construct could be a strategy for a better tumor control than monospecific CAR-T cells on heterogeneous models. To overcome tumor heterogeneity, we targeted mesothelin (meso) and Mucin 16 (MUC16), two antigens commonly expressed in solid tumors, using a tandem CAR design. Methods: We designed a series of tandem CAR constructs based on various anti-meso (SS1) and anti-MUC16 ectodomain (MUC16ecto) (4H11) single-chain variable fragment (scFv) arrangements and G4S linker lengths. Then we determined the best tandem CAR design based on binding of soluble antigens, steric hindrance, avidity and functionality against cell lines expressing one or both antigens in vitro. Finally, we compared the tandem CAR to monospecific CAR-T cells in mixed tumor models in vitro (two-dimensional and three-dimensional models) and in vivo. Results: We show that the scFv arrangement and linker length impacted antigen binding and CAR expression in T cells. Tandem CAR configuration (TanCAR1) (with SS1 scFv located distally and one G4S repeat as the linker between scFvs) had the best binding and activation profile in vitro and outperformed SS1 and 4H11 monospecific CAR-T cells in mixed tumor models in vitro and in vivo, showing an antigen-driven killing of tumor cells based on antigen density. Moreover, acoustic force microscopy, using tumor cells with different levels of antigen expression, revealed that TanCAR1-T cells likely bind to one antigen at a time rather than simultaneously. Conclusions: This is the first time using a tandem CAR design targeting meso and MUC16, and demonstrating a benefit on tumor control over monospecific CAR-T cells. Tandem CAR-T cells targeting meso and MUC16ecto could be employed as a strategy to overcome tumor cell heterogeneity in ovarian and pancreatic tumors, and may help to design therapeutic approaches relying on its one-antigen-at-a-time binding properties and on its antigen-driven killing of tumor cells based on antigen density.

Pulmonary lymphangioleiomyomatosis (LAM) is metastatic sarcoma but mechanisms of LAM metastasis are unknown. Extracellular vesicles (EV) regulate cancer metastasis but their roles in LAM have not yet been thoroughly investigated. Here, we report the discovery of distinct LAM-EV subtypes derived from primary tumor or metastasizing LAM cells that promote LAM metastasis through ITGα6/β1-c-Src-FAK signaling, triggered by shuttling ATP synthesis to cell pseudopodia or the activation of integrin adhesion complex, respectively. This signaling leads to increased LAM cell migration, invasiveness, and stemness and regulates metastable (hybrid) phenotypes that are all pivotal for metastasis. Mouse models corroborate in vitro data by demonstrating a significant increase in metastatic burden upon the exposure to EV through distinct mechanisms involving either lung resident fibroblasts or metalloproteinases’ activation that are EV subtype dependent. The clinical relevance of these findings is underscored by increased EV biogenies in LAM patients and the enrichment of these EV cargo with lung tropic integrins and metalloproteinases. These findings establish EV as novel therapeutic target in LAM, warranting the future clinical studies.