Novel therapies are urgently needed for breast cancer. While our previous work investigating the use of oncolytic viruses against the disease demonstrated its promise, heterogeneous responses were observed. Interestingly, breast cancer is classified into subtypes based on the expression of hormone receptors. We noted that triple-negative breast cancers (TNBCs), which lack the expression of the estrogen (E2) receptor (ER) were resistant to the oncolytic vesicular stomatitis virus (oVSV), whereas ER+ cancers were particularly sensitive. We found that E2 stimulation enhanced virus infection, while E2-antagonizing endocrine therapies counteract the virus. Further mechanistic studies revealed a defect of nuclear factor (NF)-κB activation with E2 stimulation. With the goal of enhancing oncolytic virotherapy independently of E2, we designed a treatment strategy that combines oVSV with the NF-κB inhibitors IKK16 and sulfasalazine and found that both drugs improved virus replication in TNBC cell lines and patient samples. Our data uncover a novel impact of E2 on oncolytic virotherapy and demonstrate that combining virus treatment with NF-κB inhibitors recapitulates the E2-mediated virus enhancement. Importantly, our treatment combinations could be beneficial to all breast cancers, including TNBCs, as well as other breast cancers that do not respond to E2.

Genes upregulated in each mouse macrophage cluster

The bone marrow microenvironment is highly saturated with bone marrow adipocytes (BMA), which differentiate from their precursor, mesenchymal stem cells (MSC). Evidence from patient trials suggests that bone marrow adiposity is increased in patients following some forms of chemotherapy. Moreover, it has been suggested that BMA can confer chemotherapeutic resistance to tumour cells, thereby ascribing a tumour-supportive role to BMA. We investigated the effect of chemotherapy on adipogenesis of human MSC in vitro, as well as potential underlying mechanisms leading to altered adipogenesis, and the effects in turn on tumour cell proliferation. Doxorubicin or carboplatin treatment of adipogenic differentiating MSC led to an increased percentage of mature BMA confirmed by increased gene expression of the adipocyte marker, PPARG. RNA-seq analysis identified significant increases in fibroblast growth factor (FGF) pathway genes in doxorubicin treated adipogenic differentiated MSC, which were validated at the mRNA and protein level. Notably, endogenous and secreted FGF2 was significantly increased with doxorubicin treatment. Furthermore, siRNA-mediated targeting of FGF2 impeded the doxorubicin-enhanced formation of lipid-containing mature BMA returning it to levels similar to vehicle control treated BMA. As FGF2 is a secreted protein we tested and confirmed that transfer of conditioned media from doxorubicin-treated BMA enhanced proliferation of tumour cells in vitro, a phenotype that was partially abrogated when FGF2 was depleted from adipogenic differentiating MSC. Our findings suggest that chemotherapy actively promotes adipogenesis, in part by alteration of FGF2 in the context of doxorubicin treatment, which directly enhances adipogenesis and in turn leads to enhanced tumour cell growth as a result.

Description of supplementary methods

Immunotherapies such as checkpoint inhibitors (i.e. anti-PD-1) and peptide-based therapies (DPX-Survivac) have strong potential for treatment of epithelial ovarian cancer, the most lethal gynecological malignancy. Magnetic resonance imaging (MRI) can be used to track tumor growth and iron-labeled immune cells longitudinally at the individual level. We studied MRI immune cell tracking in a murine model of ovarian cancer using a clinically relevant treatment combination of DPX-Survivac, anti-PD-1, and an intermittent low dose of Cyclophosphamide (CPA). HHD-DR1 mice were orthotopically implanted with mouse ovarian surface epithelial (MOSE) cancer cells. Myeloid and CD8+ cells were isolated from matched donor mice, labeled with superparamagnetic iron oxide (SPIO) and were scanned using MRI on days 42, 49 and 56. Tumor volumes in the treatment group as measured by MRI were significantly lower than in the control group (p < 0.01). The density of SPIO-labeled myeloid and CD8+ T cells in tumors was higher in the treatment group than in the control group. This study provides insights into how MRI can be used in concert with biological assays to study how immunotherapy and chemotherapy combinations exert their antitumor effects.

Objective Endometrioid ovarian carcinoma (ENOC) is increasingly recognized as a distinct disease entity, yet treatment still largely parallels high-grade serous disease management. We aimed to assemble a large, well-annotated ENOC cohort allowing us to study real-world treatment patterns with special interest on lymph node (LN) metastasis in presumed early-stage disease. Methods ENOC cases from 22 centers across five countries underwent IHC-supported central pathology review. Standardized chart review captured patient characteristics, detailed surgical and adjuvant treatment data. Results A total of 721 centrally confirmed ENOC cases diagnosed between 1984 and 2020 was assembled. Median age was 55.6 years; 86.0% presented with pelvic-confined disease, and 45.1% had grade 1 tumors. Complete resection was achieved in 96.5% of pT1/2 and 62.6% of pT3 cases. LN surgery (LNS) was performed in 58.3% of presumed early-stage cases, revealing nodal metastases in 2.6%, occurring in 4/255 (1.6%) after sampling and 5/95 (5.3%) after systematic LNS. No nodal metastases were observed in grade 1 pelvic-confined tumors (0/171). Adjuvant chemotherapy was administered in 68.0% of FIGOI/II cases and 95.5% of advanced-stage disease. Multivariable analyses revealed grade (p = 0.0006), stage (p = 0.0030) and chemotherapy (p = 0.0219) as independent prognosticators. Conclusions This multinational initiative enabled detailed analyses in a large cohort of validated ENOC.According to our results, LNS may be safely omitted in patients with pelvic-confined G1 tumors, however, if LNS is deemed necessary, a systematic approach seems to result in higher detection rates. The findings presented herein may help to shape type-specific treatment, ultimately aiming to reduce not only ovarian carcinoma mortality but also treatment-associated morbidity.

Table of antibodies used in flow cytometry and immunohistochemistry.

Up- and down-regulated differentially expressed genes in HMOX1-hi human macrophages

Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer, and those affected are in urgent need of new therapeutic strategies. Standard treatment is surgery followed by taxane- and platinum-based chemotherapy. However, the rate of relapse is high, and the 5-year survival is only 45%. Oncolytic viruses (OV) are a promising approach to EOC therapy through remodeling the immune composition of the tumor microenvironment. Treatment response in EOC tumors can differ based on the presence of key tumorigenic mutations. This study evaluated the impact of specific tumor mutations on the response to the current standard-of-care carboplatin, two promising OV candidates VSVΔM51 and MG1, an infected cell vaccine (ICV-MG1) regimen, and the antiangiogenic drug Fc3TSR. Mice with tumors harboring constitutive K-Ras activation showed an enhanced response to carboplatin and VSVΔM51 treatment. Additionally, VSVΔM51 treatment prolonged survival of syngeneic mice bearing tumors with mutations in Pten and Kras, Pten and Trp53, or Trp53 and Brca2 with increased activation of CD4+ and CD8+ T lymphocytes in the peritoneal tumor microenvironment. To enhance OV potency, an MG1-based infected cell vaccine inducing the expression of IL21 or IL15 + IL21 was developed and found to enable strong and long-lasting antitumoral immunity in two carboplatin-refractory syngeneic models, ID8-Trp53-/- and STOSE. VSVΔM51 combined with the antiangiogenic Fc3TSR enhanced efficacy in the ID8 model. In summary, OV-based immunotherapy has shown promise in diverse murine models of EOC-bearing clinically relevant mutations, thus laying the foundation for developing new OV-based strategies to target a large spectrum of EOC genotypes.

The Hippo signaling pathway, through the core kinases LATS1 and LATS2, plays a critical role in regulating tissue homeostasis, proliferation, and organ size by phosphorylating and inactivating the transcriptional coactivator YAP (Yes-associated protein). While LATS1/2 are often described as tumor suppressors in various solid cancers, their mechanistic contribution to ovarian cancer remains incompletely characterized. The prevailing model posits that low LATS1/2 expression leads to YAP activation and transcription of oncogenic genes, thereby promoting tumor progression. However, our study identifies an unexpected dual role of LATS1/2 in ovarian cancer, revealing context-dependent effects on cell survival and YAP function. This study aims to define the mechanisms underlying that dichotomy. To investigate the functional consequences of LATS1/2 expression in ovarian cancer, we used two high-grade serous ovarian cancer cell lines with low endogenous LATS1/2 expression—TOV3041G and OV1946—to assess the impact of LATS1 or LATS2 overexpression, introduced via lentiviral transduction. Functional assays demonstrated significantly reduced proliferation, migration, and invasion in both cell lines upon LATS1 or LATS2 overexpression, supporting their canonical tumor-suppressive roles. Surprisingly, the knockdown of LATS1/2 via siRNA in OVCAR8 cells resulted in decreased proliferation—opposite to expectations based on known Hippo-YAP pathway functions. To explore this paradox, we performed RNA-sequencing combined with TCGA, which revealed enrichment of apoptotic signatures in LATS1/2 knockdown cells. Subsequent validation confirmed increased apoptotic activity, including elevated caspase-3/7 activity (measured by Caspase-Glo 3/7 assay), increased Annexin V binding, and 7-AAD uptake, consistent with early and late apoptotic events. To investigate whether the altered phenotype was associated with dysregulated YAP signaling, we assessed YAP subcellular localization using immunofluorescence and nuclear-cytoplasmic fractionation. These analyses revealed a marked failure of YAP to translocate into the nucleus in LATS1/2 knockdown cells, despite unchanged total YAP protein levels. In addition, qPCR analysis showed reduced expression of canonical YAP target genes, CTGF and CYR61, indicating suppressed YAP-driven transcription in the knockdown cells. These findings suggest that in certain ovarian cancers, loss of LATS1/2 expression does not activate YAP but instead disrupts its nuclear localization and function, leading to apoptotic cell death. This highlights a previously unrecognized context-specific vulnerability where ovarian cancer cells may rely on minimal LATS1/2 activity to sustain YAP function and survival. Our results challenge the simplistic view of LATS1/2 as uniformly tumor-suppressive and suggest that their role in ovarian cancer may be more nuanced and cell-type dependent. Understanding this dualistic behavior could uncover therapeutic opportunities for selectively targeting YAP signaling in ovarian tumors with divergent LATS1/2 dependencies. Yalun Zhu, Barbara Vanderhyden. Dual roles of LATS1/2 in ovarian cancer reveal critical context dependence of hippo pathway signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr B018.

High-grade serous ovarian cancer (HGSOC) is the deadliest and most common subtype of ovarian cancer. Unfortunately, most patients develop recurrence and, ultimately, resistance to standard platinum chemotherapy. Large tumor suppressors LATS1 and LATS2, the core Hippo signaling kinases, have been implicated in various cancer types, including ovarian cancer. The mechanism by which LATS1/2 suppresses ovarian cancer progression is currently elusive, but the expression of LATS1/2 is frequently reduced or lost in these cancers. In this study, we demonstrate that the inactivation of LATS1/2 is sufficient to transform normal mouse ovarian epithelium into tumorigenic cells associated with increased cell proliferation, invasion, and stemness and epithelial-mesenchymal transition (EMT) characteristics. The knockout of Lats1/2 in the epithelial cells also leads to higher expression levels of the immune checkpoint molecule PD-L1, suggesting a regulatory role of LATS1/2 in modulating immune responses and immune evasion. In addition to the loss of LATS1/2 activating the downstream transcriptional coactivators YAP and TAZ, PI3K-AKT activity was also increased, likely contributing to enhanced tumor proliferation and survival. The stimulatory effect of Lats1/2 knockout on cell proliferation can be partially reversed by treatment with the AKT inhibitor MK2206. Treatment with verteporfin, a potent inhibitor of YAP/TAZ, decreases ovarian tumor progression and reduces the activated AKT in the tumors. In summary, this study uncovers several biological mechanisms for the initiation of HGSOC and identifies LATS1/2 as potential prognostic indicators and therapeutic targets.

High-grade serous cancer (HGSC) remains the most lethal subtype of ovarian cancer, due in part to its complex tumor microenvironment (TME) and resistance to current therapies. The complexity of this problem necessitates the identification of new therapeutic targets. One target of interest is PAX8, a lineage-specific transcription factor expressed in the majority of HGSCs as well as in normal fallopian tube tissue. Studies have shown that loss of PAX8 in non-tumorigenic cells does not significantly compromise viability, making it a compelling therapeutic target. In this study, we investigated how PAX8 drives tumor progression by orchestrating a signaling axis involving TGF-β, SOX17, and KRAS. We hypothesized that PAX8 interacts with distinct protein partners in tumor versus normal cells, reprogramming the TME to favor cancer growth. Using two murine cell models (MOE PTENshRNA KRASG12V and STOSE), we silenced PAX8 expression via shRNA and observed a reduction in TGF-β secretion, as measured by ELISA. Subsequent western blot analyses revealed that PAX8 loss destabilizes mutant KRAS, but not wild-type KRAS; such stabilization was reversed by recombinant TGF-β treatment, suggesting PAX8 – TGF-β collaborate to stabilize oncogenic KRAS. Further investigation identified SOX17, a transcription factor critical for angiogenesis regulation, as a downstream effector of PAX8 – TGF-β signaling. Antibody array profiling demonstrated that PAX8 knockdown downregulates SOX17, shifting the angiogenic balance toward inhibition (e.g., increased Serpin F1 and Pentraxin-3, decreased Coagulation factor III and Proliferin). Strikingly, the PAX8–TGF-β–SOX17 axis also regulates interferon gamma receptor 1 expression, leading to reduced MHC class I levels. Since MHC class I is essential for CD8+ T cell recognition, this pathway enables tumor immune evasion—a finding corroborated by enhanced immune infiltration in PAX8-deficient tumors. In immune-competent FVB/N mice, PAX8-deficient tumors exhibited significantly prolonged survival and slower growth compared to controls across both models. Further analysis of tumors from the MOE PTENshRNA KRASG12V group revealed increased infiltration of CD45+ immune cells and reduced stromal density relative to controls, indicating that PAX8 influences both cellular and structural aspects of the TME. Our findings establish PAX8 as a central driver of HGSC progression through three interconnected mechanisms: (1) TGF-β–dependent stabilization of oncogenic KRAS, (2) SOX17-mediated angiogenic reprogramming, and (3) MHC class I–dependent immune evasion. These results position PAX8 as a promising therapeutic target whose inhibition could simultaneously disrupt mutant KRAS signaling, normalize angiogenesis, and restore anti-tumor immunity, addressing key clinical challenges in HGSC treatment. Joohyun Im, Amrita Salvi, Yi-Chien Wu, Dan Lantvit, Steve Seung-Young Lee, Barbara Vanderhyden, Joanna Burdette. PAX8-dependent signaling networks as drivers of progression and immune evasion in high grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A011.

Rationale: The chronic lung disease bronchopulmonary dysplasia (BPD) remains the most common complication of extreme prematurity (<28 wk of gestation). Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) represent an opportunity for autologous cell therapy, as UC-MSCs have been shown to improve lung function and structure in experimental BPD. However, characterization and repair capacity of UC-MSCs derived from donors with pregnancy- related complications associated with prematurity remain unexplored. Objectives: To characterize UC-MSCs' transcriptome and determine if pregnancy-related complications (preeclampsia and chorioamnionitis) alter their therapeutic potential. Methods: Single-cell RNA sequencing was used to compare the transcriptome of UC-MSCs derived from 5 term donors, 16 preterm donors, and human neonatal dermal fibroblasts (control cells of mesenchymal origin) and correlated with their therapeutic potential in experimental BPD. Using publicly available neonatal lung single-nucleus RNA sequencing data, we also determined putative communication networks between UC-MSCs and resident lung cell populations. Measurements and Main Results: Most UC-MSCs displayed a similar transcriptome despite their pregnancy-related conditions and mitigated hyperoxia-induced lung injury in newborn rats. Conversely, human neonatal dermal fibroblasts and one term and two preterm with preeclampsia UC-MSC donors exhibited a distinct transcriptome enriched in genes related to fibroblast function and senescence and were devoid of therapeutic benefit in hyperoxia-induced BPD. Conversely, therapeutic UC-MSCs displayed a unique transcriptome active in cell proliferation and distinct cell-cell interactions with neonatal lung cell populations, including NEGR (neuronal growth regulator 1) and NRNX (neurexin) pathways. Conclusions: Term and preterm UC-MSCs are lung protective in experimental BPD. Single-cell RNA sequencing allows us to identify donors with a distinct UC-MSC transcriptome characteristic of reduced therapeutic potential.

Group 2 innate lymphoid cells (ILC2s) are emerging players in tumor immunity. ILC2 heterogeneity confers tissue-specific outcomes, with sometimes opposing effects on the prognosis and progression of various cancers. The role of ILC2s in breast and ovarian cancer is an active area of investigation, with research aimed at leveraging these cells to enhance immunotherapy.

IntroductionOvarian cancer is the most lethal gynecological malignancy. Deepening our knowledge of the interactions within the tumor microenvironment (TME) is important for discovering new targeted treatment strategies. Transglutaminase 2 (TG2) is a protein implicated in many biological and pathophysiological processes, including promoting tumor progression in ovarian cancer. Its role in disease progression has been studied in ovarian cancer cells; however, its role in the ovarian TME is less understood.MethodsIn this study, for the first time, we assessed the therapeutic potential of novel covalent irreversible small molecule TG2 inhibitors in xenograft models of ovarian cancer. We further elucidated the role of TG2 in ovarian cancer cells and syngeneic tumors by immune phenotyping using flow cytometry, RNA sequencing, and immunohistochemistry to characterize the contribution of TG2 in the TME to the metastatic process of ovarian cancer.ResultsTo investigate the transamidation catalytic and GTP binding activities of TG2 in cancer cells, we used several TG2 inhibitors, some of which decreased invasiveness of human ovarian cancer cell lines in vitro and lengthened survival of the SKOV3 xenograft model. Using the ID8 Trp53-/- Brca1-/- and KPCA.B syngeneic mouse models of ovarian cancer, we defined the contribution of TG2 in the TME to the metastatic process. Lack of TG2 in the TME prolonged survival in the ID8 Trp53-/- Brca1-/- metastatic model, but it did not affect survival in the non-metastatic KPCA.B model. Through extensive analysis of the immune composition in both the primary tumor and metastatic ascites in the ID8 Trp53-/- Brca1-/- model, we discovered that the lack of host TG2 resulted in decreased frequency of immunosuppressive tumor-associated macrophages, and increased frequency of T cells, NK cells, and B cells. RNA sequencing of the primary tumors with or without TG2 present in the TME, revealed an enrichment of pathways related to B cell activation and regulation.DiscussionThese findings highlight the importance of TG2 in the TME for ovarian cancer metastasis, potentially by activation of humoral immunity and specifically highlight a crucial role for TG2 in modulating B cells to prolong survival in mouse models of ovarian cancer.

Background/Objectives: Type II ovarian cancer, including high-grade serous carcinoma (HGSC), is genetically unstable and exhibits frequent mutations in the tumor suppressor genes. Mutations of TP53 and BRCA1 genes have been associated with HGSC, which has been suggested as a subtype that arises from the fallopian tube lesion called serous tubal intraepithelial carcinoma (STIC). Although TP53 and BRCA1 genes are well-known tumor suppressor genes, the actual effects of TP53 and BRCA1 mutations in enhancing the development of ovarian cancer initiated from STIC are poorly understood. Methods: In this study, we knocked out Trp53 and Brca-1 in epithelial cell clones derived from mice fallopian tube tissues (known as oviducts) and investigated the potential involvement of these two mutations in inducing cancer stem-like cells as cancer-initiating cells. Results: We have shown that the knockout of Trp53 induced oviduct cells to undergo EMT and acquire stem cell characteristics. Conclusions:Trp53 mutation may induce the early stage of precursor lesions formation at the distal end of the oviducts.

Rationale: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. Objectives: To determine and correlate single-cell UC-MSC transcriptomic profiles with therapeutic potential. Methods: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs; control cells of mesenchymal origin) transcriptomes were investigated using single-cell RNA sequencing (scRNA-seq) analysis. The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested in vivo in hyperoxia-induced neonatal lung injury in rats. Measurements and Main Results: UC-MSCs showed limited transcriptomic heterogeneity but were different from HNDFs. Gene Ontology enrichment analysis revealed distinct (progenitor-like and fibroblast-like) UC-MSC subpopulations. Only treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of nontherapeutic cells and associated with decreased lung retention. Conclusions: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.

The tumour microenvironment is infiltrated by immunosuppressive cells, such as regulatory T cells (Tregs), which contribute to tumour escape and impede immunotherapy outcomes. Soluble fibrinogen-like protein 2 (sFGL2), a Treg effector protein, inhibits immune cell populations, via receptors FcγRIIB and FcγRIII, leading to downregulation of CD86 in antigen presenting cells and limiting T cell activation. Increased FGL2 expression is associated with tumour progression and poor survival in several different cancers, such as glioblastoma multiforme, lung, renal, liver, colorectal, and prostate cancer. Querying scRNA-seq human cancer data shows FGL2 is produced by cells in the tumour microenvironment (TME), particularly monocytes and macrophages as well as T cells and dendritic cells (DCs), while cancer cells have minimal expression of FGL2. We studied the role of FGL2 exclusively produced by cells in the TME, by leveraging Fgl2 knockout mice. We tested two murine models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer and melanoma. We show that absence of FGL2 leads to a more activated TME, including activated DCs (CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 leads to more activated natural killer cells (DNAM-1+, NKG2D+) in the TME. Furthermore, the absence of FGL2 leads to prolonged survival in the B16F10 melanoma model, while the absence of FGL2 synergizes with oncolytic virus to prolong survival in the ID8-p53−/−Brca2−/− ovarian cancer model. In conclusion, targeting FGL2 is a promising cancer treatment strategy alone and in combination immunotherapies.