Chemotherapy remains the primary treatment for ovarian cancer (OvCa), and chemoresistance drives patient mortality. Cellular quiescence, reversible exit from the cell cycle, increases chemotherapy resistance as chemotherapies primarily target rapidly proliferating cells. Here, we report that CHD4 and MBD3, components of the nucleosome remodeling and deacetylase (NuRD) complex, are downregulated in quiescent OvCa cells (qOvCa). We find that either CHD4 or MBD3 knockdown or histone deacetylase inhibitors (HDACi), induce quiescence in OvCa cells. RNA-Seq and ATAC-seq analysis of HDACi-treated cells confirmed expression changes consistent with induction of quiescence. Additionally, HDACi-treated cells revealed downregulation of the RHO/RAC pathways. Suggesting downregulation of the RAC pathway could play a role in HDACi-mediated quiescence, RAC inhibitors (RACi) similarly induced quiescence. Both HDACi and RACi resulted in nuclear-to-cytoplasmic shifting of the pro-proliferative transcription factor MRTFA. which has been linked to quiescence. Further analysis of HDACi qOvCa indicated multiple alterations in proteostasis, including increased proteasome activity and autophagy. We find qOvCa cells are dependent on these pathways for survival, such that there is profound synergistic OvCa cell death with HDACi and proteasome- or autophagy-inhibitor combination therapy. Combined, this work supports HDACi as pharmacologic means to induce a quiescent state in OvCa cells and sensitize them to proteostasis-targeting drugs.

Distribution of immune cells and tumor cells across different histologies in the malignant mesothelioma (MM) cohort.

Comparison of cell–cell contact score differences between malignant pleural mesothelioma (MPM) and malignant peritoneal mesothelioma (MPeM).

Comparison of differences in cell–cell contact scores between malignant pleural mesothelioma (MPM) and malignant peritoneal mesothelioma (MPeM) tumors.

Cytotoxic chemotherapy remains the primary treatment for ovarian cancer (OvCa). Development of chemoresistance typically results in patient death within two years. As such, understanding chemoresistance is critical. One underexplored mechanism of chemotherapy resistance is quiescence. Quiescent cells, which have reversibly exited the cell cycle, are refractory to most chemotherapies which primarily target rapidly proliferating cells. Here, we report that CHD4 and MBD3, components of the nucleosome remodeling and deacetylase (NuRD) complex, are downregulated in quiescent OvCa cells (qOvCa). Indicating a direct role for NuRD complex downregulation in the induction of quiescence, either CHD4 or MBD3 knockdown or histone deacetylase inhibitors (HDACi), such as vorinostat, induce quiescence in OvCa cells. RNA-Seq analysis of HDACi-treated cells confirmed expression changes consistent with induction of quiescence. We also find that both primary qOvCa and vorinostat-induced qOvCa demonstrate altered proteostasis, including increased proteasome activity and autophagy, and combination therapy of HDACi and proteasome inhibitors or autophagy inhibitors demonstrated profound synergistic death of OvCa cells. Finally, we overlapped RNA-Seq signatures from quiescent ovarian cancer cells with genes essential for quiescence in yeast to identify a "quiescent cell core signature." This core quiescent cell signature appeared to be conserved across multiple cancer types, suggesting new therapeutic targets.

MM intra-tumor heterogeneity and survival analysis. (A) Comparison of TMA core cell-type proportions within patients and across patients. Each value in the boxplot represents the Pearson correlation coefficients between cell-type proportions across cores from different patients and cores within individual patients. (B) Kaplan-Meier survival curves of MM patients that categorized into two groups based on intra-tumor heterogeneity: those with high intra-tumor heterogeneity (Shannon entropy > 0.6 quantile) shown in blue, and those with low intra-tumor heterogeneity (Shannon entropy < 0.4 quantile) depicted in yellow.

Comparison of cell type distributions between malignant pleural mesothelioma (MPM) and malignant peritoneal mesothelioma (MPeM) within the same histologic subtypes.

Example of 7-plex healthy donor lymphoid tissue as a control and for validation. All channels combined as well as Individual marker channels (pCK: pan-cytokeratin).

The association of immune cells and cellular neighborhood proportion with overall survival in all malignant mesothelioma (MM) as well as for malignant pleural mesothelioma (MPM) and malignant peritoneal mesothelioma (MPeM) separately.

Clinical features, cell count, tumor percentage, and density associated with each core, including marker percentage, density and intensity for each core.

Comparison of clinical parameters between malignant pleural mesothelioma (MPM) and malignant peritoneal mesothelioma (MPeM) for various cell types.

IHC staining for WT1 (purple) and SMA (brown in top panels; teal in bottom panels) in representative crocidolite-induced and chrysotile-induced MMs from Bap1+/- mice. WT1 stains MM cells, whereas SMA stains CAFs, the latter typically more abundant in chrysotile-induced than crocidolite-induced MMs, as in the examples shown here.

Primary antibodies used for IHC, IF, and immunoblot analyses

CAMILLA is a basket trial (NCT03539822) evaluating cabozantinib plus the ICI durvalumab in chemorefractory gastrointestinal cancer. Herein, are the phase II colorectal cohort results. 29 patients were evaluable. 100% had confirmed pMMR/MSS tumors. Primary endpoint was met with ORR of 27.6% (95% CI 12.7-47.2%). Secondary endpoints of 4-month PFS rate was 44.83% (95% CI 26.5-64.3%); and median OS was 9.1 months (95% CI 5.8-20.2). Grade≥3 TRAE occurred in 39%. In post-hoc analysis of patients with RAS wild type tumors, ORR was 50% and median PFS and OS were 6.3 and 21.5 months respectively. Exploratory spatial transcriptomic profiling of pretreatment tumors showed upregulation of VEGF and MET signaling, increased extracellular matrix activity and preexisting anti-tumor immune responses coexisting with immune suppressive features like T cell migration barriers in responders versus non-responders. Cabozantinib plus durvalumab demonstrated anti-tumor activity, manageable toxicity, and have led to the activation of the phase III STELLAR-303 trial.

High-grade serous ovarian carcinoma (HGSOC) is a heterogeneous disease, and a highstromal/desmoplastic tumor microenvironment (TME) is associated with a poor outcome. Stromal cell subtypes, including fibroblasts, myofibroblasts, and cancer-associated mesenchymal stem cells, establish a complex network of paracrine signaling pathways with tumor-infiltrating immune cells that drive effector cell tumor immune exclusion and inhibit the antitumor immune response. In this work, we integrate single-cell transcriptomics of the HGSOC TME from public and in-house datasets ( n = 20) and stratify tumors based upon high vs. low stromal cell content. Although our cohort size is small, our analyses suggest a distinct transcriptomic landscape for immune and non-immune cells in high-stromal vs. low-stromal tumors. High-stromal tumors have a lower fraction of certain T cells, natural killer (NK) cells, and macrophages, and increased expression of CXCL12 in epithelial cancer cells and cancer-associated mesenchymal stem cells (CA-MSCs). Analysis of cell-cell communication indicate that epithelial cancer cells and CA-MSCs secrete CXCL12 that interacte with the CXCR4 receptor, which is overexpressed on NK and CD8+ T cells. Dual IHC staining show that tumor infiltrating CD8 T cells localize in proximity of CXCL12+ tumor area. Moreover, CXCL12 and/or CXCR4 antibodies confirm the immunosuppressive role of CXCL12-CXCR4 in high-stromal tumors.

Immunotherapies have shown modest clinical benefit thus far for malignant mesothelioma (MM). A deeper understanding of immune cell spatial distribution within the tumor immune microenvironment (TIME) is needed to identify interactions between tumor and different immune cell types that might impact the effectiveness of potential immunotherapies. We performed multiplex immunofluorescence (mIF) using tissue microarrays (TMAs, n=3) of samples from patients with malignant peritoneal (n=25) and pleural (n=88) mesothelioma (MPeM and MPM, respectively) to elucidate the spatial distributions of major immune cell populations and their association with LAG3, BAP1, NF2, and MTAP expression, the latter as a proxy for CDKN2A/B. We also analyzed the relationship between the spatial distribution of major immune cell types with MM patient prognosis and clinical features. The distribution of immune cells within the TIME is similar between MPM and MPeM. However, there is a higher level of interaction between immune cells and tumor cells in MPM than MPeM. Within MPM tumors, there is increased amount of interaction between tumor cells and CD8+ T cells in BAP1-low than in BAP1-high expressing tumors. The cell-cell interactions identified in this investigation have potential implications for the immune response against MM tumors and could be a factor in the different behaviors of MPM and MPeM. Our findings provide a valuable resource for the MM cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. Our mesothelioma spatial atlas mIF dataset is available at https://mesotheliomaspatialatlas.streamlit.app/.

Abstract Asbestos and BAP1 germline mutations are risk factors for malignant mesothelioma (MM). While it is well accepted that amphibole asbestos is carcinogenic, the role of serpentine (chrysotile) asbestos in MM has been debated. To address this controversy, we assessed whether minimal exposure to chrysotile could significantly increase the incidence and rate of MM onset in germline Bap1-mutant mice. With either crocidolite or chrysotile, and at each dose tested, MMs occurred at a significantly higher rate and earlier onset time in Bap1-mutant mice than in wild-type littermates. To explore the role of gene–environment interactions in MMs from Bap1-mutant mice, we investigated proinflammatory and protumorigenic factors and the tumor immune microenvironment (TIME). IHC and immunofluorescence staining showed an increased number of macrophages in granulomatous lesions and MMs. The relative number of CD163-positive (CD163+) M2 macrophages in chrysotile-induced MMs was consistently greater than in crocidolite-induced MMs, suggesting that chrysotile induces a more profound immunosuppressive response that creates favorable conditions for evading immune surveillance. MMs from Bap1-mutant mice showed upregulation of CD39/CD73-adenosine and C-C motif chemokine ligand 2 (Ccl2)/C-C motif chemokine receptor 2 (Ccr2) pathways, which together with upregulation of IL6 and IL10, promoted an immunosuppressive TIME, partly by attracting M2 macrophages. Interrogation of published human MM RNA sequencing (RNA-seq) data implicated these same immunosuppressive pathways and connections with CD163+ M2 macrophages. These findings indicate that increased M2 macrophages, along with upregulated CD39/CD73-adenosine and Ccl2/Ccr2 pathways, contribute to an immunosuppressive TIME in chrysotile-induced MMs of Bap1-mutant mice, suggesting that immunotherapeutic strategies targeting protumorigenic immune pathways could be beneficial in human BAP1 mutation carriers who develop MM. Significance: We show that germline Bap1-mutant mice have enhanced susceptibility to MM upon minimal exposure to chrysotile asbestos, not only amphibole fibers. Chrysotile induced a more profound immune tumor response than crocidolite in Bap1-mutant mice by upregulating CD39/CD73-adenosine and Ccl2/Ccr2 pathways and recruiting more M2 macrophages, which together contributed to an immunosuppressive tumor microenvironment. Interrogation of human MM RNA-seq data revealed interconnected immunosuppressive pathways consistent with our mouse findings.

Anatomical image of peritoneal MMs (white long arrows) induced by chronic intraperitoneal injections of chrysotile in a Bap1+/- mouse. MMs developing in Bap1+/- and Bap1+/+ (wild type) littermates injected with either chrysotile or crocidolite generally were diffuse peritoneal lesions sometimes accompanied by ascites. Note that the MMs shown in this animal are unusually large for illustrative purposes. Most tumors in this model were solitary, much smaller, and diffuse. Irregular liver surface (yellow rectangle) is likely indicative of fibrosis. L, liver; LI, large intestine; M, mesothelioma.

Transcription factors (TFs) drive significant cellular changes in response to environmental cues and intercellular signaling. Neighboring cells influence TF activity and, consequently, cellular fate and function. Spatial transcriptomics (ST) captures mRNA expression patterns across tissue samples, enabling characterization of the local microenvironment. However, these datasets have not been fully leveraged to systematically estimate TF activity governing cell identity. Here, we present STAN ( S patially informed T ranscription factor A ctivity N etwork), a linear mixed-effects computational method that predicts spot-specific, spatially informed TF activities by integrating curated TF-target gene priors, mRNA expression, spatial coordinates, and morphological features from corresponding imaging data. We tested STAN using lymph node, breast cancer, and glioblastoma ST datasets to demonstrate its applicability by identifying TFs associated with specific cell types, spatial domains, pathological regions, and ligand‒receptor pairs. STAN augments the utility of STs to reveal the intricate interplay between TFs and spatial organization across a spectrum of cellular contexts.

Immune cell phenotypes associated with clinical features. A, Prevalence of all cell types in MPM epithelioid (n = 45), biphasic (n = 16), and sarcomatoid (n = 7) subtypes (left) and MPeM epithelioid (n = 12) and biphasic (n = 3) subtypes (right) presented as the proportion of total cells. Statistical analysis was performed using one-sided Multiple Mann–Whitney nonparametric U tests. *, P < 0.05. B, Bubble plot illustrating significance levels, in which circle size corresponds to the level of significance, and circle color indicates which of the two comparisons on the y-axis shows higher levels of the cell type plotted on the x-axis. C, Box plots displaying cell-type proportion across different clinical subgroups in patients with MPM and MPeM. Statistical analysis was conducted using one-sided multiple Mann–Whitney U tests. P < 0.05 was considered significant. The center of the box plot represents the median, with the box boundaries indicating the 25th and 75th percentiles. Whiskers extend to the minimum and maximum values in the dataset.