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Histopathology of OCI xenografts recapitulate the original human tumour. (a–c) Haematoxylin and eosin (H&E)-stained sections of primary human tumours used to establish OCI-P8p (papillary serous), OCI-E1p (endometrioid) and OCI-C3x (clear cell) cell lines. (d–f) H&E-stained sections of xenografts tumours derived by injecting SOC cells (ES2, SKOV3 and TOV-112D) subcutaneously into immunocompromised mice. The typical features of human adenocarcinomas such as glands, papillae, stromal cores and desmoplastic stroma are absent. (g–o) H&E-stained sections of xenograft tumours derived by injecting OCI cell lines (P5x, P7a, P9a, C5x, C3x, CSp and E1p) subcutaneously into immunocopromised mice. In papillary serous specimens, note the presence of stromal cores and papillary architecture (g–i). In the endometrioid specimen note the presence of glands (m) which were positive for oestrogen receptor (ER) and mucin (brown), respectively, consistent with the endometrioid phenotype (n,o). Scale bar, 100 μM. See Supplementary 4 for additional images.
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Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the...
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Citations
... In contrast with the consistent and reproducible behavior of several primary OC samples in the organotypic co-cultures, only the TYK-nu cells exhibited a strong TME-induced upregulation of FOXM1 among the cell lines tested (Supplementary Fig. 4C). While this somehow confirmed the limited potential of various classical OC lines to recapitulate certain aspects of the human disease [20,21], it pointed to TYK-nu cells as a suitable system for a loss-of-function approach. Lentiviral-mediated shRNA transduction was applied to assess the impact of ablating FOXM1 ( Supplementary Fig. 4D) on stem-related traits both in vitro and in vivo. ...
In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.
... cell carcinoma cell lines are reported in the literature [3,6]; however, most have not been extensively characterised, and less than half have been deposited in cell banks [3]. Furthermore, most of these cell lines do not necessarily retain phenotypes exhibited by the patient's original tumour tissue [7]. It is therefore important to establish additional clear cell carcinoma cell lines that retain characteristics of a patient's tumour. ...
... Intriguingly, but troublingly, cancer cells that proliferate autonomously (or uncontrollably) in the human body are often paradoxically difficult to culture in vitro [7]. Indeed, Verschraegen et al. report that of 90 ovarian tumour samples, only 11 could be established as cell lines [8]. ...
... In other words, whether or not a cancer cell line can be established from a patient's tumour tissue is based on chance, a concern not limited to ovarian carcinoma. It is reported that the success rate of cell line establishment can be increased by use of special media [7], but such media are often expensive and difficult to prepare. Thus, it is important to establish cell lines that can be cultured in standard media. ...
A human ovarian clear cell carcinoma cell line was established from a 46-year-old Japanese woman. That line, designated MTC-22, has proliferated continuously for over 6 months in conventional RPMI 1640 medium supplemented with 10% foetal bovine serum and has been passaged over 50 times. MTC-22 doubling-time is ~ 18 h, which is much shorter than most ovarian clear cell carcinoma lines reported to date. Morphologically, MTC-22 cells exhibit polygonal shapes and proliferate to form a monolayer in a jigsaw puzzle-like arrangement without contact inhibition. Ultrastructurally, cells exhibit numerous intracytoplasmic glycogen granules and well-developed mitochondria. G-band karyotype analysis indicated that cells have a complex karyotype close to tetraploid. We observed that the expression pattern of a series of ovarian carcinoma-related molecules in MTC-22 cells was identical to that seen in the patient’s tumour tissue. Notably, MTC-22 cells, and the patient’s carcinoma tissue, expressed low-sulphated keratan sulphate recognised by R-10G and 294-1B1 monoclonal antibodies, a hallmark of non-mucinous ovarian carcinoma, and particularly of clear cell ovarian carcinoma. Moreover, characteristic point mutations—one in ARID1A , which encodes the AT-rich interaction domain containing protein 1A, and the other in PIK3CB, which encodes the catalytic subunit of phosphoinositide 3-kinase—were seen in the patient’s tumour tissue and retained in MTC-22 cells. Collectively, these findings indicate that MTC-22 cells could serve as a valuable tool for investigating the pathophysiology of ovarian clear cell carcinoma, particularly that harbouring PIK3CB mutations, and for developing and validating new diagnostic and therapeutic approaches to this life-threatening malignancy.
... 8,13 Tumor cell cultures were established from ascites tumor spheroids as described. 37,38 Tumor-associated lymphocytes (TALs) from ascites were separated from other cell types, including tumorassociated macrophages (TAMs) and tumor cells according to the published protocol. 8 Briefly, mononuclear cells were separated by Lymphocyte Separation Medium 1077 (Pro-moCell, Heidelberg, Germany) and density gradient centrifugation. ...
Background
IL‐17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation.
Methods
Flow‐cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL‐17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses.
Results
We identified Th17 cells as the main population of IL‐17A‐ and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL‐17A and its receptor subunit IL‐17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL‐17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro‐inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL‐17A and TNF synergistically induced the Th17‐promoting cytokines IL‐6 and IL‐1β as well as the Th17‐attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor‐promoting role of Th17 cells in OC.
Conclusions
Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL‐17A/TNF‐mediated induction of mesothelial‐mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.
... The ascites was centrifuged at 560 ×g for 5 min. The cell precipitation was resuscitated with DMEM/F12 medium (BasalMedia, Shanghai, China) containing 5% FBS (supplemented with insulin, hydrocortisone, epidermal growth factor, fibroblast growth factor; Sigma, St. Louis, Missouri, USA), and then cultured in a humidified atmosphere of 5% CO 2 at 37 • C. The cells were purified through a combination of differential adhesion method and low concentration trypsin digestion methods [23]. Briefly, cells with good growth status were digested with low concentration pancreatic enzyme. ...
Background
Although the constitutively activated Wnt/β‐catenin signaling pathway plays vital roles in gastric cancer (GC) progression, few Wnt inhibitors are approved for clinical use. Additionally, the clinical significance of long non‐coding RNAs (lncRNAs) in GC intraperitoneal dissemination (IPD) remains elusive. Here, we investigated the function and therapeutic potential of Wnt‐transactivated lncRNA, colon cancer‐associated transcript 5 (CCAT5), in GC metastasis.
Methods
LncRNA‐sequencing assay was performed to document abundance changes of lncRNAs induced by Wnt family member 3A (Wnt3a) and degradation‐resistant β‐catenin (S33Y mutated) in ascites‐derived GC cells with low Wnt activity. Luciferase reporter, Chromatin immunoprecipitation (ChIP)‐re‐ChIP assays were performed to determine how CCAT5 was transcribed. The clinical significance of CCAT5 was examined in 2 cohorts of GC patients. The biological function of CCAT5 was investigated through gain‐ and loss‐of‐function studies. The molecular mechanism was explored through RNA‐sequencing, mass spectrometry, and CRISPR/Cas9‐knocknout system. The therapeutic potential of CCAT5 was examined through RNAi‐based cell xenograft model and patient‐derived xenograft (PDX) model of IPD.
Results
We identified a novel Wnt‐regulated lncRNA, CCAT5, which was transactivated by the β‐catenin/transcription factor 3 (TCF3) complex. CCAT5 was significantly upregulated in GC and predicted poor prognosis. Functional studies confirmed the promotive role of CCAT5 in GC growth and metastasis. Mechanistically, CCAT5 bound to the C‐end domain of signal transducer and activator of transcription 3 (STAT3) and blocks Src homology 2 domain‐containing protein tyrosine phosphatase 1 (SHP‐1)‐mediated STAT3 Y705 dephosphorylation, leading to STAT3 nuclear entry and transactivation, thus accelerating GC progression. Furthermore, we demonstrated that both Wnt3a and β‐catenin acted as activator of STAT3 signaling pathway, and the interplay between CCAT5 and STAT3 was functionally essential for Wnt‐drived STAT3 signaling and tumor evolution. Finally, we revealed in vivo si‐CCAT5 selectively attenuated growth and metastasis of Wnt high GC, but not Wnt low GC. The combination of si‐CCAT5 and oxaliplatin displayed obvious synergistic therapeutic effects on Wnt high PDX mice.
Conclusions
We identified a novel Wnt‐transactivated lncRNA, CCAT5. Our study revealed a mechanism of STAT3 signaling regulation via canonical Wnt signaling and the functional significance of CCAT5 as critical mediator. We provided conceptual advance that lncRNAs serve as therapeutic targets reversing GC progression.
... Emanating from ascites tumor spheroids, permanent primary tumor cell cultures (OCMI tumor cells) were established from patients OC_37, OC_58 and OC_91, following the description of Ince et al. 70 with slight modifications as delineated previously. 69 The benefit of this culture method is to retain the original tumor characteristics and propagate the tumor cells long-term without the induction of cell culture crisis and genetic alterations. ...
A crucial requirement for metastasis formation in ovarian high-grade serous carcinoma (HGSC) is the disruption of the protective peritoneal mesothelium. Using co-culture systems of primary human cells, we discovered that tumor-associated NK cells induce TRAIL-dependent apoptosis in mesothelial cells via death receptors DR4 and DR5 upon encounter with activated T cells. Upregulation of TRAIL expression in NK cells concomitant with enhanced cytotoxicity toward mesothelial cells was driven predominantly by T-cell-derived TNFα, as shown by affinity proteomics-based analysis of the T cell secretome in conjunction with functional studies. Consistent with these findings, we detected apoptotic mesothelial cells in the peritoneal fluid of HGSC patients. In contrast to mesothelial cells, HGSC cells express negligible levels of both DR4 and DR5 and are TRAIL resistant, indicating cell-type-selective killing by NK cells. Our data point to a cooperative action of T and NK in breaching the mesothelial barrier in HGSC patients.
... SKOV3, A2780, TOV112D, and OVCAR3 cells are some representative cell lines of the many ovarian cancer cell lines that are commonly used in in vitro studies, of which the first two are cited the most. 59,60 To check if the 3D in vitro spheroid platform is versatile and can be exploited with all these cell lines, we also analyzed different starting cell densities. Figure 5a shows that homospheroids of SKOV3 resulted in the smallest spheroids (100−200 μm), while A2780-and TOV112D-based spheroids displayed larger sizes of 300−800 μm. ...
Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 ± 11 nm, while siRNA-based polyplexes displayed a size of 25 ± 2 nm. The polyplexes had a slightly positive zeta potential of +7–8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in ∼50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration..
... However, while cell lines are experimentally tractable, they have several weaknesses. Often, cell lines were established decades ago in sub-optimal culture conditions that may have selected specific phenotypes (Domcke et al. 2013;Ince et al. 2015;Nelson et al. 2020). Further propagation in vitro likely selects out the fitter subclones best adapted to cell culture conditions, possibly eliminating lesser fit clones that might only survive in vivo. ...
... The development of better experimental models to study ovarian cancer is a major research focus (Bowtell et al. 2015). Indeed, as reviewed recently (Tomas and Shepherd 2023), extensive effort has been applied to develop patient-derived 2D cell cultures, more complex spheroid, organoid or co-culture models, as well as xenografts (Bertozzi et al. 2006;Shepherd et al. 2006;Latifi et al. 2012;Sueblinvong et al. 2012;Thériault et al. 2013;Davidowitz et al. 2014;Lengyel et al. 2014;Ince et al. 2015;Liu et al. 2017;Thu et al. 2017;Hill et al. 2018;Kopper et al. 2019;Maru et al. 2019;Phan et al. 2019;Fritz et al. 2020;Hoffmann et al. 2020;Maenhoudt et al. 2020;Brodeur et al. 2021;Ito et al. 2023;Vias et al. 2023). Such efforts have been required because establishing primary cell cultures from tumours using traditional cell culture techniques has historically been challenging, with very low success rates due to tumour cell 'senescence' and with the emerging cell lines reflecting rare subclones (Ince et al. 2015). ...
... Indeed, as reviewed recently (Tomas and Shepherd 2023), extensive effort has been applied to develop patient-derived 2D cell cultures, more complex spheroid, organoid or co-culture models, as well as xenografts (Bertozzi et al. 2006;Shepherd et al. 2006;Latifi et al. 2012;Sueblinvong et al. 2012;Thériault et al. 2013;Davidowitz et al. 2014;Lengyel et al. 2014;Ince et al. 2015;Liu et al. 2017;Thu et al. 2017;Hill et al. 2018;Kopper et al. 2019;Maru et al. 2019;Phan et al. 2019;Fritz et al. 2020;Hoffmann et al. 2020;Maenhoudt et al. 2020;Brodeur et al. 2021;Ito et al. 2023;Vias et al. 2023). Such efforts have been required because establishing primary cell cultures from tumours using traditional cell culture techniques has historically been challenging, with very low success rates due to tumour cell 'senescence' and with the emerging cell lines reflecting rare subclones (Ince et al. 2015). A major breakthrough was the development of highly specialised cell culture conditions capable of propagating isolated CRC cells as organoids (Sato et al. 2011), an approach then adapted to breast (Sachs et al. 2018) and epithelial ovarian cancers (Kopper et al. 2019;de Witte et al. 2020). ...
Chromosome instability (CIN) is a cancer hallmark that drives tumour heterogeneity, phenotypic adaptation, drug resistance and poor prognosis. High-grade serous ovarian cancer (HGSOC), one of the most chromosomally unstable tumour types, has a 5-year survival rate of only ~30% — largely due to late diagnosis and rapid development of drug resistance, e.g., via CIN-driven ABCB1 translocations. However, CIN is also a cell cycle vulnerability that can be exploited to specifically target tumour cells, illustrated by the success of PARP inhibitors to target homologous recombination deficiency (HRD). However, a lack of appropriate models with ongoing CIN has been a barrier to fully exploiting disease-specific CIN mechanisms. This barrier is now being overcome with the development of patient-derived cell cultures and organoids. In this review, we describe our progress building a Living Biobank of over 120 patient-derived ovarian cancer models (OCMs), predominantly from HGSOC. OCMs are highly purified tumour fractions with extensive proliferative potential that can be analysed at early passage. OCMs have diverse karyotypes, display intra- and inter-patient heterogeneity and mitotic abnormality rates far higher than established cell lines. OCMs encompass a broad-spectrum of HGSOC hallmarks, including a range of p53 alterations and BRCA1/2 mutations, and display drug resistance mechanisms seen in the clinic, e.g., ABCB1 translocations and BRCA2 reversion. OCMs are amenable to functional analysis, drug-sensitivity profiling, and multi-omics, including single-cell next-generation sequencing, and thus represent a platform for delineating HGSOC-specific CIN mechanisms. In turn, our vision is that this understanding will inform the design of new therapeutic strategies.
... FN-silk networks offer a fibrous structure that could support the novel Wood and PB cells, which phenocopy the original primary tumors thanks to a defined and optimized medium. 35 The protocol for FN-silk network formation was proved to be flexible and capable of sustaining cell viability even in the case of minimal initial material with slowly proliferating cells, such as the one obtained by superficial scraping of breast cancers. 36 This is valuable since low cell number and slow proliferation are a challenge for spheroids formation. ...
... We confirmed that the decrease of TJP1 can be detected already after 24 h and is maintained over time (Figure 4e). Afterward, we performed staining for the protein coded by TJP1, zonula occludens-1 (ZO-1), revealing that MCF-7 cultured in 2D formed monolayer clusters in which ZO-1 was always detectable, whereas in FN-silk networks some clusters did not express ZO-Figure last part of the study, we investigated if it is possible to use the FN-silk networks for the culture of novel breast cancer cell lines as well as primary cells obtained from fresh human breast tumors.First, we tested if FN-silk networks could support the growth of the Wood and PB cells, two lines with phenotypic properties which faithfully reproduce the original cancer tissues.35 These cells complement canonical immortalized cells by better representing the variability observed among cancer patients. ...
Traditional cancer models rely on 2D cell cultures or 3D spheroids, which fail to recapitulate cell‐extracellular matrix (ECM) interactions, a key element of tumor development. Existing hydrogel‐based 3D alternatives lack mechanical support for cell growth and often suffer from low reproducibility. Here we report a novel strategy to make 3D models of breast cancer using a tissue‐like, well‐defined network environment based on recombinant spider silk, functionalized with a cell adhesion motif from fibronectin (FN‐silk). With this approach, the canonical cancer cells SK‐BR‐3, MCF‐7, and MDA‐MB‐231, maintain their characteristic expression of markers (i.e., ERα, HER2, and PGR) while developing distinct morphology. Transcriptomic analyses demonstrate how culture in the FN‐silk networks modulates the biological processes of cell adhesion and migration while affecting physiological events involved in malignancy, such as inflammation, remodeling of the ECM, and resistance to anticancer drugs. Finally, we show that integration in FN‐silk networks promotes the viability of cells obtained from the superficial scraping of patients' breast tumors.
... The monolayers are artificial because they reflect the 222 response of cells to substrates with stiffness far beyond tissues where OC develops 223 and grows after dissemination. However, the establishment of patient-derived OC 224 cell lines frequently involves 2D cultures, and recently, a combination of 2D 225 cultures and defined cell culture media led to the derivation of 25 OC cell lines, 226 which phenocopied genetic landscapes of type I or type II tumors [84]. These cell 227 lines have been used in multiple studies [85-88] and represent well-validated and 228 important cellular models of OC. . ...
Determination of the frequency of somatic mutations, copy number variations, and the composition of single-nucleus transcriptomes in epithelial ovarian cancer (OC) biopsies have provided crucial information about the heterogeneity of cancer cell assemblies and their various tumor microenvironments (TMs). Translating this information into tumor biology for the discovery of new treatments could be accomplished by the engineering tissue culture models of OC within its TM.KeywordsBiomaterialDisseminationEndometriumFallopian tubesMesotheliumMicrofluidicOrganoidOrganotypic culture
... The PI3K-Akt-mTOR pathway is one of the most widely mutated and activated signaling pathways in human cancers, since it has the capacity to promote cell proliferation, protein translation, cell motility and migration, and enhance epithelial-mesenchymal transition, to name just a few [25]. Mutations are observed in this pathway in human EOC, with activating missense mutations in PIK3CA, loss of function deletions in PTEN, and amplifications in AKT1, AKT2, and AKT3, among the different histotypes of this disease [26,27]. Our results however showed reduced phosphorylation of Akt at serine 473 in HGSC cell lines and concomitant downstream signaling effects of lower phospho-p70S6K and phospho-4EBP1 [25,28]. ...
Epithelial ovarian cancer (EOC) research has become more complex as researchers try to fully understand the metastatic process. Especially as we delve into the concept of tumour dormancy, where cells transition between proliferative and dormant states to survive during disease progression. Thus, the in vitro models used to conduct this research need to reflect this vast biological complexity. The innovation behind the many three-dimensional (3D) spheroid models has been refined to easily generate reproducible spheroids so that we may understand the various molecular signaling changes of cells during metastasis and determine therapeutic efficacy of treatments. This ingenuity was then used to develop the 3D ex vivo patient-derived organoid model, as well as multiple co-culture model systems for EOC research. Although, researchers need to continue to push the boundaries of these current models for in vitro and even in vivo work in the future. In this review, we describe the 3D models already in use, where these models can be developed further and how we can use these models to gain the most knowledge on EOC pathogenesis and discover new targeted therapies.
