Molecular, Metabolic, and Subcellular Mapping of the Tumor Immune Microenvironment via 3D Targeted and Non-Targeted Multiplex Multi-Omics Analyses.

Most platforms used for the molecular reconstruction of the tumor-immune microenvironment (TIME) of a solid tumor fail to explore the spatial context of the three-dimensional (3D) space of the tumor at a single-cell resolution, and thus lack information about cell-cell or cell-extracellular matrix (ECM) interactions. To address this issue, a pipeline which integrated multiplex spatially resolved multi-omics platforms was developed to identify crosstalk signaling networks among various cell types and the ECM in the 3D TIME of two FFPE (formalin-fixed paraffin embedded) gynecologic tumor samples. These platforms include non-targeted mass spectrometry imaging (glycans, metabolites, and peptides) and Stereo-seq (spatial transcriptomics) and targeted seqIF (IHC proteomics). The spatially resolved imaging data in a two- and three-dimensional space demonstrated various cellular neighborhoods in both samples. The collection of spatially resolved analytes in a voxel (3D pixel) across serial sections of the tissue was also demonstrated. Data collected from this analytical pipeline were used to construct spatial 3D maps with single-cell resolution, which revealed cell identity, activation, and energized status. These maps will provide not only insights into the molecular basis of spatial cell heterogeneity in the TIME, but also novel predictive biomarkers and therapeutic targets, which can improve patient survival rates.

Proteogenomic analysis of chemo-refractory high-grade serous ovarian cancer.

To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.

Microfibril-associated protein 5 and the regulation of skin scar formation.

Many factors regulate scar formation, which yields a modified extracellular matrix (ECM). Among ECM components, microfibril-associated proteins have been minimally explored in the context of skin wound repair. Microfibril-associated protein 5 (MFAP5), a small 25 kD serine and threonine rich microfibril-associated protein, influences microfibril function and modulates major extracellular signaling pathways. Though known to be associated with fibrosis and angiogenesis in certain pathologies, MFAP5's role in wound healing is unknown. Using a murine model of skin wound repair, we found that MFAP5 is significantly expressed during the proliferative and remodeling phases of healing. Analysis of existing single-cell RNA-sequencing data from mouse skin wounds identified two fibroblast subpopulations as the main expressors of MFAP5 during wound healing. Furthermore, neutralization of MFAP5 in healing mouse wounds decreased collagen deposition and refined angiogenesis without altering wound closure. In vitro, recombinant MFAP5 significantly enhanced dermal fibroblast migration, collagen contractility, and expression of pro-fibrotic genes. Additionally, TGF-ß1 increased MFAP5 expression and production in dermal fibroblasts. Our findings suggest that MFAP5 regulates fibroblast function and influences scar formation in healing wounds. Our work demonstrates a previously undescribed role for MFAP5 and suggests that microfibril-associated proteins may be significant modulators of wound healing outcomes and scarring.

Comparative Tumor Microenvironment Analysis of Primary and Recurrent Ovarian Granulosa Cell Tumors.

Adult-type granulosa cell tumors (aGCT) are rare ovarian sex cord tumors with few effective treatments for recurrent disease. The objective of this study was to characterize the tumor microenvironment (TME) of primary and recurrent aGCTs and to identify correlates of disease recurrence. Total RNA sequencing (RNA-seq) was performed on 24 pathologically confirmed, cryopreserved aGCT samples, including 8 primary and 16 recurrent tumors. After read alignment and quality-control filtering, DESeq2 was used to identify differentially expressed genes (DEG) between primary and recurrent tumors. Functional enrichment pathway analysis and gene set enrichment analysis was performed using "clusterProfiler" and "GSVA" R packages. TME composition was investigated through the analysis and integration of multiple published RNA-seq deconvolution algorithms. TME analysis results were externally validated using data from independent previously published RNA-seq datasets. A total of 31 DEGs were identified between primary and recurrent aGCTs. These included genes with known function in hormone signaling such as LHCGR and INSL3 (more abundant in primary tumors) and CYP19A1 (more abundant in recurrent tumors). Gene set enrichment analysis revealed that primarily immune-related and hormone-regulated gene sets expression was increased in recurrent tumors. Integrative TME analysis demonstrated statistically significant depletion of cancer-associated fibroblasts in recurrent tumors. This finding was confirmed in multiple independent datasets. IMPLICATIONS: Recurrent aGCTs exhibit alterations in hormone pathway gene expression as well as decreased infiltration of cancer-associated fibroblasts, suggesting dual roles for hormonal signaling and TME remodeling underpinning disease relapse.

Fear of recurrence, emotional well-being and quality of life among long-term advanced ovarian cancer survivors.

OBJECTIVE: Although advanced stage epithelial ovarian cancer is widely considered life-threatening, 17% of women with advanced disease will survive long-term. Little is known about the health-related quality of life (QOL) of long-term ovarian cancer survivors, or how fear of recurrence might affect QOL. METHODS: 58 long-term survivors with advanced disease participated in the study. Participants completed standardized questionnaires to capture cancer history, QOL, and fear of recurrent disease (FOR). Statistical analyses included multivariable linear models. RESULTS: Participants averaged 52.8 years at diagnosis and had survived >8 years (mean:13.5); 64% had recurrent disease. Mean FACT-G, FACT-O, and FACT-O-TOI (TOI) scores were 90.7 (SD:11.6), 128.6 (SD:14.8), and 85.9 (SD:10.2) respectively. Compared to the U.S. population using T-scores, QOL for participants exceeded that of healthy adults (T-score (FACT-G) = 55.9). Overall QOL was lower in women with recurrent vs. non-recurrent disease though differences did not reach statistical significance (FACT-O = 126.1 vs. 133.3, p = 0.082). Despite good QOL, high FOR was reported in 27%. FOR was inversely associated with emotional well-being (EWB) (p < 0.001), but not associated with other QOL subdomains. In multivariable analysis, FOR was a significant predictor of EWB after adjusting for QOL (TOI). A significant interaction was observed between recurrence and FOR (p = 0.034), supporting a larger impact of FOR in recurrent disease. CONCLUSION: QOL in long-term ovarian cancer survivors was better than the average for healthy U.S. women. Despite good QOL, high FOR contributed significantly to increased emotional distress, most notably for those with recurrence. Attention to FOR may be warranted in this survivor population.

Spatial Transcriptomics Depict Ligand-Receptor Cross-talk Heterogeneity at the Tumor-Stroma Interface in Long-Term Ovarian Cancer Survivors.

Advanced high-grade serous ovarian cancer (HGSC) is an aggressive disease that accounts for 70% of all ovarian cancer deaths. Nevertheless, 15% of patients diagnosed with advanced HGSC survive more than 10 years. The elucidation of predictive markers of these long-term survivors (LTS) could help identify therapeutic targets for the disease, and thus improve patient survival rates. To investigate the stromal heterogeneity of the tumor microenvironment (TME) in ovarian cancer, we used spatial transcriptomics to generate spatially resolved transcript profiles in treatment-naïve advanced HGSC from LTS and short-term survivors (STS) and determined the association between cancer-associated fibroblasts (CAF) heterogeneity and survival in patients with advanced HGSC. Spatial transcriptomics and single-cell RNA-sequencing data were integrated to distinguish tumor and stroma regions, and a computational method was developed to investigate spatially resolved ligand-receptor interactions between various tumor and CAF subtypes in the TME. A specific subtype of CAFs and its spatial location relative to a particular ovarian cancer cell subtype in the TME correlated with long-term survival in patients with advanced HGSC. Also, increased APOE-LRP5 cross-talk occurred at the stroma-tumor interface in tumor tissues from STS compared with LTS. These findings were validated using multiplex IHC. Overall, this spatial transcriptomics analysis revealed spatially resolved CAF-tumor cross-talk signaling networks in the ovarian TME that are associated with long-term survival of patients with HGSC. Further studies to confirm whether such cross-talk plays a role in modulating the malignant phenotype of HGSC and could serve as a predictive biomarker of patient survival are warranted. SIGNIFICANCE: Generation of spatially resolved gene expression patterns in tumors from patients with ovarian cancer surviving more than 10 years allows the identification of novel predictive biomarkers and therapeutic targets for better patient management. See related commentary by Kelliher and Lengyel, p. 1383.

Ryanodine receptor 1-mediated Ca2+ signaling and mitochondrial reprogramming modulate uterine serous cancer malignant phenotypes.

BACKGROUND: Uterine serous cancer (USC) is the most common non-endometrioid subtype of uterine cancer, and is also the most aggressive. Most patients will die of progressively chemotherapy-resistant disease, and the development of new therapies that can target USC remains a major unmet clinical need. This study sought to determine the molecular mechanism by which a novel unfavorable prognostic biomarker ryanodine receptor 1 (RYR1) identified in advanced USC confers their malignant phenotypes, and demonstrated the efficacy of targeting RYR1 by repositioned FDA-approved compounds in USC treatment. METHODS: TCGA USC dataset was analyzed to identify top genes that are associated with patient survival or disease stage, and can be targeted by FDA-approved compounds. The top gene RYR1 was selected and the functional role of RYR1 in USC progression was determined by silencing and over-expressing RYR1 in USC cells in vitro and in vivo. The molecular mechanism and signaling networks associated with the functional role of RYR1 in USC progression were determined by reverse phase protein arrays (RPPA), Western blot, and transcriptomic profiling analyses. The efficacy of the repositioned compound dantrolene on USC progression was determined using both in vitro and in vivo models. RESULTS: High expression level of RYR1 in the tumors is associated with advanced stage of the disease. Inhibition of RYR1 suppressed proliferation, migration and enhanced apoptosis through Ca2+-dependent activation of AKT/CREB/PGC-1α and AKT/HK1/2 signaling pathways, which modulate mitochondrial bioenergetics properties, including oxidative phosphorylation, ATP production, mitochondrial membrane potential, ROS production and TCA metabolites, and glycolytic activities in USC cells. Repositioned compound dantrolene suppressed USC progression and survival in mouse models. CONCLUSIONS: These findings provided insight into the mechanism by which RYR1 modulates the malignant phenotypes of USC and could aid in the development of dantrolene as a repurposed therapeutic agent for the treatment of USC to improve patient survival.

Dissection of PIK3CA Aberration for Cervical Adenocarcinoma Outcomes.

Personalized treatment of genetically stratified subgroups has the potential to improve outcomes in many malignant tumors. This study distills clinically meaningful prognostic/predictive genomic marker for cervical adenocarcinoma using signature genomic aberrations and single-point nonsynonymous mutation-specific droplet digital PCR (ddPCR). Mutations in PIK3CA E542K, E545K, or H1047R were detected in 41.7% of tumors. PIK3CA mutation detected in the patient's circulating DNA collected before treatment or during follow-up was significantly associated with decreased progression-free survival or overall survival. PIK3CA mutation in the circulating DNA during follow-up after treatment predicted recurrence with 100% sensitivity and 64.29% specificity. It is the first indication of the predictive power of PIK3CA mutations in cervical adenocarcinoma. The work contributes to the development of liquid biopsies for follow up surveillance and a possibility of tailoring management of this particular women's cancer.

SIO: A Spatioimageomics Pipeline to Identify Prognostic Biomarkers Associated with the Ovarian Tumor Microenvironment.

Stromal and immune cells in the tumor microenvironment (TME) have been shown to directly affect high-grade serous ovarian cancer (HGSC) malignant phenotypes, however, how these cells interact to influence HGSC patients' survival remains largely unknown. To investigate the cell-cell communication in such a complex TME, we developed a SpatioImageOmics (SIO) pipeline that combines imaging mass cytometry (IMC), location-specific transcriptomics, and deep learning to identify the distribution of various stromal, tumor and immune cells as well as their spatial relationship in TME. The SIO pipeline automatically and accurately segments cells and extracts salient cellular features to identify biomarkers, and multiple nearest-neighbor interactions among tumor, immune, and stromal cells that coordinate to influence overall survival rates in HGSC patients. In addition, SIO integrates IMC data with microdissected tumor and stromal transcriptomes from the same patients to identify novel signaling networks, which would lead to the discovery of novel survival rate-modulating mechanisms in HGSC patients.

Quality of Life and Adverse Events: Prognostic Relationships in Long-Term Ovarian Cancer Survival.

BACKGROUND: There is a critical need to identify patient characteristics associated with long-term ovarian cancer survival. METHODS: Quality of life (QOL), measured by the Functional Assessment of Cancer Therapy-Ovarian-Trial Outcome Index (FACT-O-TOI), including physical, functional, and ovarian-specific subscales, was compared between long-term survivors (LTS) (8+ years) and short-term survivors (STS) (<5 years) of GOG 218 at baseline; before cycles 4, 7, 13, 21; and 6 months post-treatment using linear and longitudinal mixed models adjusted for covariates. Adverse events (AEs) were compared between survivor groups at each assessment using generalized linear models. All P values are 2-sided. RESULTS: QOL differed statistically significantly between STS (N = 1115) and LTS (N = 260) (P < .001). Baseline FACT-O-TOI and FACT-O-TOI change were independently associated with long-term survival (odds ratio = 1.05, 95% confidence interval = 1.03 to 1.06 and odds ratio = 1.06, 95% confidence interval = 1.05 to 1.07, respectively). A 7-point increase in baseline QOL was associated with a 38.0% increase in probability of LTS, and a 9-point increase in QOL change was associated with a 67.0% increase in odds for LTS. QOL decreased statistically significantly with increasing AE quartiles (cycle 4 quartiles: 0-5 vs 6-8 vs 9-11 vs ≥12 AEs, P = .01; cycle 21 quartiles: 0-2 vs 3 vs 4-5 vs ≥6 AEs, P = .001). Further, LTS reported statistically significantly better QOL compared with STS (P = .03 and P = .01, cycles 4 and 21, respectively), with similar findings across higher AE grades. CONCLUSIONS: Baseline and longitudinal QOL change scores distinguished LTS vs STS and are robust prognosticators for long-term survival. Results have trial design and supportive care implications, providing meaningful prognostic value in this understudied population.

Correction: Hu, W., et al. Targeting Dopamine Receptor D2 by Imipridone Suppresses Uterine Serous Cancer Malignant Phenotype. Cancers 2020, 12, 2436.

In the original article, there was a mistake in Figure 2B as published [...].

Targeting Dopamine Receptor D2 by Imipridone Suppresses Uterine Serous Cancer Malignant Phenotype.

Uterine serous cancer (USC) is an aggressive subtype of endometrial cancer, with poor survival and high recurrence rates. The development of novel and effective therapies specific to USC would aid in its management. However, few studies have focused solely on this rare subtype. The current study demonstrated that the orally bioavailable, investigational new drug and novel imipridone ONC206 suppressed USC cell proliferation and induced apoptosis both in vitro and in vivo. Disruption of the DRD2-mediated p38MAPK/ERK/PGC-1α network by ONC206 led to metabolic reprogramming and suppression of both glycolysis and oxidative phosphorylation. ONC206 also synergized with paclitaxel in reducing USC cell viability. In addition, DRD2 overexpression correlated with poor overall survival in patients. This study provides the first evidence that ONC206 induced metabolic reprogramming in USC cells and is a promising therapeutic agent for USC treatment. These findings support further development of ONC206 as a promising therapeutic agent and improves survival rates in patients with USC.

Disabled-2: a positive regulator of the early differentiation of myoblasts.

Dab2 is an adaptor protein and a tumor suppressor. Our previous study has found that Dab2 was expressed in early differentiating skeletal muscles in mouse embryos. In this study, we determined the role of Dab2 in the skeletal muscle differentiation using C2C12 myoblasts in vitro and Xenopus laevis embryos in vivo. The expression of Dab2 was increased in C2C12 myoblasts during the formation of myotubes in vitro. Knockdown of Dab2 expression in C2C12 myoblasts resulted in a reduction of myotube formation, whereas the myotube formation was enhanced upon overexpression of Dab2. Re-expression of Dab2 in C2C12 myoblasts with downregulated expression of Dab2 restored their capacity to form myotubes. Microarray profiling and subsequent network analyses on the 155 differentially expressed genes after Dab2 knockdown showed that Mef2c was an important myogenic transcription factor regulated by Dab2 through the p38 MAPK pathway. It was also involved in other pathways that are associated with muscular development and functions. In Xenopus embryos developed in vivo, XDab2 was expressed in the myotome of somites where various myogenic markers were also expressed. Knockdown of XDab2 expression with antisense morpholinos downregulated the expression of myogenic markers in somites. In conclusion, this study is the first to provide solid evidence to show that Dab2 is a positive regulator of the early myoblast differentiation.

ITLN1 modulates invasive potential and metabolic reprogramming of ovarian cancer cells in omental microenvironment.

Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin's effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression.

Pathogenesis and Clinical Management of Uterine Serous Carcinoma.

Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer that has not been well characterized. It accounts for less than 10% of all endometrial cancers and 80% of endometrial cancer-related deaths. Currently, staging surgery together with chemotherapy or radiotherapy, especially vaginal cuff brachytherapy, is the main treatment strategy for USC. Whole-exome sequencing combined with preclinical and clinical studies are verifying a series of effective and clinically accessible inhibitors targeting frequently altered genes, such as HER2 and PI3K3CA, in varying USC patient populations. Some progress has also been made in the immunotherapy field. The PD-1/PD-L1 pathway has been found to be activated in many USC patients, and clinical trials of PD-1 inhibitors in USC are underway. This review updates the progress of research regarding the molecular pathogenesis and putative clinical management of USC.

Expression of long noncoding RNAs in cancer-associated fibroblasts linked to patient survival in ovarian cancer.

Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment and are responsible for producing the desmoplastic reaction that is a poor prognostic factor in ovarian cancer. Long non-coding RNAs (lncRNAs) have been shown to play important roles in cancer. However, very little is known about the role of lncRNAs in the tumor microenvironment. We aimed to identify lncRNAs expressed in ovarian CAFs that were associated with patient survival and used computational approaches to predict their function. Increased expression of 9 lncRNAs and decreased expression of 1 lncRNA in ovarian CAFs were found to be associated with poorer overall survival. A "guilt-by-association" approach was used to predict the function of these lncRNAs. In particular, MIR155HG was predicted to play a role in immune response. Further investigation revealed high MIR155HG expression to be associated with higher infiltrates of immune cell subsets. In conclusion, these data indicate expression on several lncRNAs in CAFs are associated with patient survival and are likely to play an important role in regulating CAF function.

Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) Promotes Uterine Serous Cancer Cell Proliferation and Cell Cycle Progression.

Uterine serous carcinoma (USC) is the most aggressive form of endometrial cancer, with poor survival rates and high recurrence risk. Therefore, the purpose of this study was to identify therapeutic targets that could aid in the management of USC. By analyzing endometrial cancer samples from The Cancer Genome Atlas (TCGA), we found Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) to be highly expressed in USC and to correlate with poorer overall survival. UCHL1 silencing reduced cell proliferation in vitro and in vivo, cyclin B1 protein levels and cell cycle progression. Further studies showed that UCHL1 interacts with cyclin B1 and increases cyclin B1 protein stability by deubiquitination. Treatment of USC-bearing mice with the UCHL1-specific inhibitor reduced tumor growth and improved overall survival. Our findings suggest that cyclin B1 is a novel target of UCHL1 and targeting UCHL1 is a potential therapeutic strategy for USC.

The Impact of Stroma Admixture on Molecular Subtypes and Prognostic Gene Signatures in Serous Ovarian Cancer.

BACKGROUND: Recent efforts to improve outcomes for high-grade serous ovarian cancer, a leading cause of cancer death in women, have focused on identifying molecular subtypes and prognostic gene signatures, but existing subtypes have poor cross-study robustness. We tested the contribution of cell admixture in published ovarian cancer molecular subtypes and prognostic gene signatures. METHODS: Gene signatures of tumor and stroma were developed using paired microdissected tissue from two independent studies. Stromal genes were investigated in two molecular subtype classifications and 61 published gene signatures. Prognostic performance of gene signatures of stromal admixture was evaluated in 2,527 ovarian tumors (16 studies). Computational simulations of increasing stromal cell proportion were performed by mixing gene-expression profiles of paired microdissected ovarian tumor and stroma. RESULTS: Recently described ovarian cancer molecular subtypes are strongly associated with the cell admixture. Tumors were classified as different molecular subtypes in simulations where the percentage of stromal cells increased. Stromal gene expression in bulk tumors was associated with overall survival (hazard ratio, 1.17; 95% confidence interval, 1.11-1.23), and in one data set, increased stroma was associated with anatomic sampling location. Five published prognostic gene signatures were no longer prognostic in a multivariate model that adjusted for stromal content. CONCLUSIONS: Cell admixture affects the interpretation and reproduction of ovarian cancer molecular subtypes and gene signatures derived from bulk tissue. Elucidating the role of stroma in the tumor microenvironment and in prognosis is important. IMPACT: Single-cell analyses may be required to refine the molecular subtypes of high-grade serous ovarian cancer.