Zehua Wang

Extracellular vesicles from ovarian cancer cells induce senescent lipid-laden macrophages to facilitate omental metastasis

Ovarian cancer exhibits striking metastatic tropism for the omentum, where lipid-laden macrophages are key mediators that fuel disease progression. However, the mechanisms governing their formation and pro-metastatic functions remain poorly understood. As extracellular vesicles (EVs) have as critical regulators of tumor-stroma crosstalk in metastatic niches, we sought to define how ovarian cancer-derived EVs orchestrate macrophages and adipocytes, and their impact on omental metastasis, aiming to explore potential therapeutic interventions. Single-cell transcriptomics of ovarian cancer revealed a distinct lipid-laden macrophage population in omentum, whose abundance correlated with metastatic burden and poor survival. Proteomics revealed that EVs from highly metastatic ovarian cancer cells were enriched in lipid metabolism regulators. In vivo experiments demonstrated that these tumor-derived vesicles mediated macrophage reprogramming, driving the acquisition of a pro-metastatic phenotype. Quantitative lipidomic profiling and lipid staining approaches confirmed the progressive lipid-laden in EV-treated macrophages. Using a patient-derived omentum-macrophage co-culture system, we demonstrated that tumor-derived EVs stimulate lipid release from omental adipocytes, which macrophages subsequently internalize through CD36-dependent uptake to drive lipid accumulation. This metabolic reprogramming culminated in cellular senescence, as evidenced by classical biomarkers including SA-β-galactosidase activity, elevated p16-INK4A and p53 levels, and the development of a matrix metalloproteinase-enriched senescence-associated secretory phenotype. Immunohistochemistry of clinical specimens demonstrated overexpression of CD36 correlated with omental metastasis and poor survival in ovarian cancer. In vivo experiments demonstrated that CD36 inhibition and senolytic therapy attenuated omental metastasis. This study unveils an EV-driven mechanism of adipose tropism in ovarian cancer metastasis, where EVs promote the formation of senescent lipid-laden macrophages via CD36-mediated lipid uptake, remodeling the metastatic niche. Targeting CD36 and senescent cells offers a promising therapeutic strategy against omental metastasis.

A protocol for selection in mice of highly metastatic ovarian cancer cell with omental tropism

Preclinical models mimicking spontaneous omental metastasis from ovarian cancer (OC) can benefit the study of anti-metastatic therapies for OC patients. Here, we present a protocol to establish a highly metastatic (HM) mouse model with omental tropism by in vivo selection. We describe the processes of implanting OC cells in the ovaries of mice and obtaining HM sublines from their omental metastases. HM cells can metastasize from the ovary to the omentum within 2 weeks. For complete details on the use and execution of this protocol, please refer to Ying et al.

Omental cancer‐associated fibroblast‐derived exosomes with low microRNA‐29c‐3p promote ovarian cancer peritoneal metastasis

AbstractOvarian cancer (OC) is characterized by frequent widespread peritoneal metastasis. Cancer‐associated fibroblasts (CAFs) represent a critical stromal component of metastatic niche and promote omentum metastasis in OC patients. However, the role of exosomes derived from omental CAFs in metastasis remains unclear. We isolated exosomes from primary omental normal fibroblasts (NFs) and CAFs from OC patients (NF‐Exo and CAF‐Exo, respectively) and assessed their effect on metastasis. In mice bearing orthotopic OC xenografts, CAF‐Exo treatment led to more rapid intraperitoneal tumor dissemination and shorter animal survival. Similar results were observed in mice undergoing intraperitoneal injection of tumor cells. Among the miRNAs downregulated in CAF‐Exo, miR‐29c‐3p in OC tissues was associated with metastasis and survival in patients. Moreover, increasing miR‐29c‐3p in CAF‐Exo significantly weakened the metastasis‐promoting effect of CAF‐Exo. Based on RNA sequencing, expression assays, and luciferase assays, matrix metalloproteinase 2 (MMP2) was identified as a direct target of miR‐29c‐3p. These results verify the significant contribution of exosomes from omental CAFs to OC peritoneal metastasis, which could be partially due to the relief of MMP2 expression inhibition mediated by low exosomal miR‐29c‐3p.

Loss of CBX2 causes genomic instability and Wnt activation in high grade serous ovarian carcinoma cells

AbstractHigh grade serous ovarian carcinoma (HGSOC) is lethal with insidious onset, rapid progression, poor prognosis, and limited treatment options. Polycomb repressor complexes (PRC) 1 and 2 are intimately involved in progression of many types of cancer including HGSOC. Unlike the consistent constitution of PRC2, PRC1 consists of diverse components whose clinical significance in HGSOC are not entirely clear. Here, prognosis‐associated PRC1 components were identified through data‐mining. CBX2 promoted proliferation and reduced apoptosis of HGSOC cell lines OVCAR4, OVCAR3, and CAOV3. Complete loss of CBX2 by CRISPR‐cas9 editing (CBX2KO) destabilized genome stability with increased spontaneous chromosomal breaks and tendency to polyploidy accompanied by disrupted cell cycle especially stalled G2/M transition and caused severe cell death. Wnt/β‐catenin/LEF1/TCF7L1 was activated in surviving OVCAR4‐CBX2KO clones to bypass the crisis caused by loss of CBX2. The relieve of TCF7L1 core‐promoter region occupied by CBX2 might be one of the possible explanations to TCF7L1 increase in OVCAR4‐CBX2KO clones. Subcutaneous tumor model further validated that depletion of CBX2 repressed HGSOC cell line derived tumor growth. High immunohistochemistry score of CBX2 in primary ovarian cancer tissue associated with advanced clinical stage (p = 0.033), poor overall survival (HR = 3.056, 95% CI: 1.024−9.123), and progression free survival (HR = 4.455, 95% CI: 1.513−13.118) in HGSOC. Overall, our results suggested that CBX2 was a promising prognostic factor and therapeutic target in HGSOC.

Rosiglitazone ameliorates senescence and promotes apoptosis in ovarian cancer induced by olaparib

Senescence mechanisms are vital to resistance to long-term olaparib maintenance treatment. Recently, peroxisome proliferator-activated receptor-γ agonists (e.g., rosiglitazone) have been reported to ameliorate the senescence-like phenotype by modulating inflammatory mediator production. This study examined synergistic effects on the anti-tumor activity of rosiglitazone combined with olaparib in ovarian cancer treatment. A2780 and SKOV3 mouse subcutaneous xenograft models were established for observing anti-tumor effects in living organisms and were randomly split into combination (both olaparib and rosiglitazone), rosiglitazone (10 mg/kg), olaparib (10 mg/kg), control (solvent) groups that received treatment once every 2 or 3 days (n = 6 per group). Cell counting kit-8 (CCK-8) assays were used to test the influences of rosiglitazone and olaparib on cell proliferation. PI and Annexin-V-FITC staining was used with flow cytometry to assess the cell cycle distribution and cell apoptosis. Senescence-associated β-galactosidase (SA-β-Gal) staining was used to observe cellular senescence. We performed quantitative real-time polymerase chain reaction assays to study the senescence-related secretory phenotype (SASP). Olaparib and rosiglitazone were observed to synergistically retard subcutaneous ovarian cancer growth in vivo, and synergistically suppress ovarian cancer cell proliferation in vitro. Compared with olaparib alone, the percentage of positive cells expressed SA-β-gal and SASP were significantly decreased in the treatment of combination of olaparib and rosiglitazone. Furthermore, olaparib plus rosiglitazone increased the percentage of apoptosis in ovarian cancer cell compared with olaparib alone. In A2780 cells, it showed lower expression of P53, phospho-p53 (Ser15), P21, and P18 protein in combination treatment compared with olaparib alone. While, in SKOV3 cells, it showed lower expression of phosphor-retinoblastoma protein (Rb) (Ser807/811), and higher expression of cyclin D1, P21, and P16 protein in combination treatment compared with olaparib alone. Rosiglitazone combined with olaparib can help manage ovarian cancer by ameliorating olaparib-induced senescence and improving anti-tumor effects.

Tumour-derived exosomal piR-25783 promotes omental metastasis of ovarian carcinoma by inducing the fibroblast to myofibroblast transition

Ovarian carcinoma inherently possesses a distinct metastatic organotropism for the adipose-rich omentum, contributing to disease progression. Although the premetastatic microenvironment (PMM) has been known to often play a prometastatic role during the process, incomplete mechanistic insight into PMM formation has prevented its therapeutic targeting. Omental fibroblasts can be activated by tumour cells to differentiate into myofibroblasts, termed the fibroblast-to-myofibroblast transition (FMT), which, in turn, enhances cancer aggressiveness. Here, we report crosstalk between cancer cells and omental fibroblasts through exosomal piR-25783, which fuels tumour metastasis. Tumour cell-secreted exosomal piR-25783 activates the TGF-β/SMAD2/SMAD3 pathway in fibroblasts and promotes the FMT in the omentum along with the secretion of various cytokines and elevation of proliferative, migratory, and invasive properties, contributing to the formation of PMMs. Furthermore, piR-25783-induced myofibroblasts promote tumour implantation and growth in the omentum. In addition, the overexpression of piR-25783 in ovarian carcinoma is associated with unfavourable clinicopathological characteristics and shorter survival. In this study, we provide molecular, functional, and translational evidence suggesting that exosomal piR-25783 plays an important role in the formation of PMMs and the development of metastatic diseases in vitro and in vivo and may serve as a potential therapeutic target for ovarian carcinoma with metastasis.

Exosomes in ovarian cancer ascites promote epithelial–mesenchymal transition of ovarian cancer cells by delivery of miR-6780b-5p

AbstractThe poor prognosis of ovarian cancer is mainly due to metastasis, and the specific mechanism underlying ovarian cancer metastasis is not clear. Ascites-derived exosomes (ADEs) play an important role in the progression of ovarian cancer, but the mechanism is unknown. Here, we found that ADEs promoted ovarian cancer metastasis not only in vitro but also in vivo. This promotive function was based on epithelial–mesenchymal transition (EMT) of ovarian cancer cells. Bioinformatics analysis of RNA sequencing microarray data indicated that miR-6780b-5p may be the key microRNA (miRNA) in ADEs that facilitates cancer metastasis. Moreover, the expression of exosomal miR-6780b-5p correlated with tumor metastasis in ovarian cancer patients. miR-6780b-5p overexpression promoted and miR-6780b-5p downregulation suppressed EMT of ovarian cancer cells. These results suggest that ADEs transfer miR-6780b-5p to ovarian cancer cells, promoting EMT and finally facilitating ovarian cancer metastasis.

Identification of miR‐30c‐5p as a tumor suppressor by targeting the m6A reader HNRNPA2B1 in ovarian cancer

AbstractBackgroundmicroRNAs (miRNAs) and N6‐methyladenosine (m6A) play important roles in ovarian cancer (OvCa). However, the mechanisms by which miRNAs regulate m6A in OvCa have not been elucidated so far.MethodsTo screen m6A‐related miRNAs, Pearson's correlation analysis of miRNAs and m6A regulators was implemented using The Cancer Genome Atlas database (TCGA). To determine the level of m6A, RNA m6A quantitative assays were used. Then, colony formation assays, EdU assays, wound healing assays, and Transwell assays were performed. The dual‐luciferase reporter assay was used to confirm the miRNA target genes. Protein–protein interaction (PPI) analysis of the target genes was performed, and hub genes were discovered using the cytoHubba/Cytoscape software. The underlying molecular mechanisms were explored by bioinformatics and RNA stability assays.ResultsA total of 126 miRNAs were identified as m6A‐related miRNAs by Pearson's correlation analysis. Among them, the high level of miR‐30c‐5p was associated with good prognosis in OvCa patients. In vitro, the miR‐30c‐5p agomir lowered the m6A level and inhibited OvCa cell proliferation, migration, and invasion. The hub target genes of miR‐30c‐5p were identified as (i) XPO1, (ii) AGO1, (iii) HNRNPA2B1, of which m6A reader HNRNPA2B1 was highly expressed in OvCa tissues and related with poor prognosis. In vitro, knockdown of HNRNPA2B1 significantly reduced m6A level and hampered the proliferation and migration of OvCa cells. The inhibition of m6A reader HNRNPA2B1 attenuated the suppression of proliferation and migration and the low m6A level induced by the miR‐30c‐5p downregulation. Mechanistically, m6A reader HNRNPA2B1 might regulate CDK19 mRNA stability to alter m6A level.ConclusionsmiR‐30c‐5p inhibits OvCa progression and reduces the m6A level by inhibiting m6A reader HNRNPA2B1, thus providing new insights into the m6A regulatory mechanism in OvCa.

Robust whole slide image analysis for cervical cancer screening using deep learning

Abstract Computer-assisted diagnosis is key for scaling up cervical cancer screening. However, current recognition algorithms perform poorly on whole slide image (WSI) analysis, fail to generalize for diverse staining and imaging, and show sub-optimal clinical-level verification. Here, we develop a progressive lesion cell recognition method combining low- and high-resolution WSIs to recommend lesion cells and a recurrent neural network-based WSI classification model to evaluate the lesion degree of WSIs. We train and validate our WSI analysis system on 3,545 patient-wise WSIs with 79,911 annotations from multiple hospitals and several imaging instruments. On multi-center independent test sets of 1,170 patient-wise WSIs, we achieve 93.5% Specificity and 95.1% Sensitivity for classifying slides, comparing favourably to the average performance of three independent cytopathologists, and obtain 88.5% true positive rate for highlighting the top 10 lesion cells on 447 positive slides. After deployment, our system recognizes a one giga-pixel WSI in about 1.5 min.

Survival after laparoscopic radical surgery for stage IA–IIB cervical cancer: 1316 consecutive cases from a national laparoscopic training center in China

To investigate the survival of cervical cancer patients undergoing laparoscopic radical hysterectomy (LRH) in a minimally invasive gynecology center. A consecutive series of patients undergoing first LRH for cervical cancer from May 2008 to December 2017 at a national laparoscopic training center was retrospectively analyzed. The overall survival (OS) and progression-free survival (PFS) were compared between groups. In total, 1316 women with FIGO (2009) stage IA-IIB cervical cancer received LRH. Among them, 1114 (84.7%) were followed up for 3 months or longer; the median follow-up period was 48 months (range 3-144 months). In patients with stage IA, IB1 (≤ 2 cm), IB1 (> 2 cm), IB2, IIA1 and IIA2-IIB tumors, the 4-year PFS rates were 98.6, 94.5, 87.4, 65.6, 80.0 and 67.4%, respectively, and the 4-year OS rates were 98.6, 96.8, 91.1, 77.4, 85.6 and 76.2%, respectively. The 4-year PFS and OS were as high as 96.2 and 97.5%, respectively, in patients with squamous cell carcinoma of 2 cm or smaller in diameter. A stable high 4-year OS and PFS was achieved after completing 100 LRHs. In patients operated on by the same surgeon, an improvement in survival was observed after 40 LRHs. Favorable oncologic outcomes can be achieved in patients with IA-IB1 cervical cancer after LRH in a center with a high surgery volume.

Neoadjuvant chemotherapy followed by radical surgery reduces radiation therapy in patients with stage IB2 to IIA2 cervical cancer

Abstract Background To investigate whether carboplatin-liposomal paclitaxel neoadjuvant chemotherapy (NACT) benefits patients with locally advanced cervical cancer (LACC) through avoiding or delaying postoperative radiation. Methods A total of 414 patients with cervical cancer of International Federation of Gynecology and Obstetrics (FIGO 2009) stages IB2–IIA2 were included in the retrospective cohort study, who had received carboplatin-liposomal paclitaxel chemotherapy followed by radical surgery (NACT group) or primary radical surgery (PRS group) between 2007 and 2017 at our hospital. The baseline clinicopathological characteristics at diagnosis, postoperative pathological risk factors, and oncological outcomes after surgery, including postoperative radiation (as adjuvant treatment or treatment of recurrent diseases), progression-free survival (PFS), and overall survival (OS), were compared between the groups. Before treatment, the patients in the NACT group had significantly more advanced tumor stages and larger tumor sizes than those in the PRS group. Results The NACT reduced the tumor volumes remarkedly with a response rate of 62.4%, and the tumors in the NACT group were smaller than those in the PRS group when the patients were subjected to radical surgery. Furthermore, postoperative pathology examination revealed less frequent deep stromal invasion in the NACT group than in the PRS group. According to the presence of pathological risk factors for recurrence, 54.82% of women in the NACT group needed adjuvant radiotherapy, while 60.87% in the PRS group, and in fact, 33.00% of NACT patients and 40.09% of PRS patients received adjuvant radiation. In addition, 8.12% of NACT patients and 9.68% of PRS patients underwent radiotherapy after relapse. The cumulative postoperative radiation rate was significantly lower in the NACT group (P = 0.041), while the differences in 5-year OS and PFS were not statistically significant between the groups. Conclusions NACT reduces the pathological risk factors and the use of radiation without compromising survival in patients with LACC, which may protect younger patients from radiation-related side effects and subsequently improve the quality of life. Trial registration ISRCTN Registry, ISRCTN24104022

A meta-analysis of survival after minimally invasive radical hysterectomy versus abdominal radical hysterectomy in cervical cancer: center-associated factors matter

Abstract Purpose To explore the possible factors that contributed to the poor performance of minimally invasive surgery (MIS) versus abdominal surgery regarding progression-free survival (PFS) and overall survival (OS) in cervical cancer. Methods MEDLINE, EMBASE, Cochrane Library and Web of Science were searched (January 2000 to April 2021). Study selection was performed by two researchers to include studies reported oncological safety. Summary hazard ratios (HRs) and 95% confidence intervals (CIs) were combined using random-effect model. Subgroup analyses were stratified by characteristics of disease, publication, study design and treatment center. Results Sixty-one studies with 63,369 patients (MIS 26956 and ARH 36,049) were included. The overall-analysis revealed a higher risk of recurrence (HR 1.209; 95% CI 1.102–1.327) and death (HR 1.124; 95% CI 1.013–1.248) after MIS versus ARH expect in FIGO IB1 (FIGO 2009 staging) patients with tumor size less than 2 cm. However, subgroup analyses showed comparable PFS/DFS and OS in studies published before the Laparoscopic Approach to Cervical Cancer (LACC) trial, published in European journals, conducted in a single center, performed in centers in Europe and in centers with high sample volume or high MIS sample volume. Conclusion Our findings highlight possible factors that associated with inferior survival after MIS in cervical cancer including publication characteristics, center-geography and sample volume. Center associated factors were needed to be taken into consideration when evaluating complex surgical procedures like radical hysterectomy.

Tumor cell-derived exosomes deliver TIE2 protein to macrophages to promote angiogenesis in cervical cancer

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (TIE2)-expressing macrophages (TEMs) are an angiogenesis-promoting subset of tumor-associated macrophages that have been demonstrated to be increased in solid tumors and associated with the progression of cervical cancer. However, the induction mechanism of TEMs remains unclear. Here, based on multicolor immunofluorescence of 58 cervical cancer tissues and the GEPIA database, we found that TEMs were increased in TIE2-high cervical cancer and related to shorter survival. In vitro and in vivo experiments verified that exosomes derived from TIE2-high cervical cancer cells transferred TIE2 protein directly to macrophages, thereby inducing TEMs. Similar to primary TEMs, TEMs induced by tumor-derived exosomes promoted angiogenesis, could be induced by angiopoietin-2, and possessed an M2-like phenotype. In conclusion, exosomes derived from TIE2-high cervical cancer cells induce TEMs by directly transporting TIE2 to promote tumor angiogenesis.