Questa ricerca è stata effettuata presso la Keck School of Medicine, University of California, Stati Uniti d&#39;America, e la University College di Londra, Regno Unito. KL è finanziato dal National Institute of Health sovvenzione 5 U19 CA148112-02. BG è stato finanziato da una borsa di studio del progetto dalla carità oncologia ginecologica Eve Appeal (Regno Unito). Parte di questo lavoro svolto in UCLH / UCL è stato in parte il finanziamento da parte del Dipartimento del regime di Salute di ricerca biomedica NIHR finanziamento Centre.

<ol>
	<li>Jelovac, D., Armstrong, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+diagnosis+and+treatment+of+ovarian+cancer.">Recent progress in the diagnosis and treatment of ovarian cancer.</a> CA Cancer J. Clin. , (2011).</li><li>Office for National Statistics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cancer Statistics registrations: registrations of cancer diagnosed in 2008. , (2011).</li><li>K&#246;bel, M., Kalloger, S. E., Santos, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+type+and+substage+predict+survival+in+stage+I+and+II+ovarian+carcinoma:+insights+and+implications.">Tumor type and substage predict survival in stage I and II ovarian carcinoma: insights and implications.</a> Gynecol. Oncol. 116, 50-56 (2010).</li><li>K&#246;bel, M., Kalloger, S. E., Boyd, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+carcinoma+subtypes+are+different+diseases:+implications+for+biomarker+studies.">Ovarian carcinoma subtypes are different diseases: implications for biomarker studies.</a> PLoS Med. 5, e232 (2008).</li><li>Smith, L. H., Morris, C. R., Yasmeen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer:+can+we+make+the+clinical+diagnosis+earlier.">Ovarian cancer: can we make the clinical diagnosis earlier.</a> Cancer. 104, 1398-1407 (2005).</li><li>Aviv, A., Hunt, S. C., Lin, J., Cao, X., Kimura, M., Blackburn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impartial+comparative+analysis+of+measurement+of+leukocyte+telomere+length/DNA+content+by+Southern+blots+and+qPCR.">Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR.</a> Nucleic Acids Res. 39, e134 (2011).</li><li>Kim, N. W., Wu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+quantification+and+characterization+of+telomerase+activity+by+the+telomeric+repeat+amplification+protocol+(TRAP.">Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP.</a> Nucleic Acids Res. 25, 2595-2597 (1997).</li><li>Lawrenson, K., Grun, B., Benjamin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescent+fibroblasts+promote+neoplastic+transformation+of+partially+transformed+ovarian+epithelial+cells+in+a+three-dimensional+model+of+early+stage+ovarian+cancer.">Senescent fibroblasts promote neoplastic transformation of partially transformed ovarian epithelial cells in a three-dimensional model of early stage ovarian cancer.</a> Neoplasia. 12, 317-325 (2010).</li><li>Lawrenson, K., Benjamin, E., Turmaine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+three-dimensional+modelling+of+human+ovarian+surface+epithelial+cells.">In vitro three-dimensional modelling of human ovarian surface epithelial cells.</a> Cell Prolif. 42, 385-393 (2009).</li><li>Lawrenson, K., Sproul, D., Grun, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+genetic+and+clinical+heterogeneity+in+epithelial+ovarian+cancers.">Modelling genetic and clinical heterogeneity in epithelial ovarian cancers.</a> Carcinogenesis. 32, 1540-1549 (2011).</li><li>Grun, B., Benjamin, E., Sinclair, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+cell+biology+models+of+ovarian+and+endometrial+cancer.">Three-dimensional in vitro cell biology models of ovarian and endometrial cancer.</a> Cell Prolif. 42, 219-228 (2009).</li><li>Zietarska, M., Maugard, C. M., Filali-Mouhim, A., Alam-Fahmy, M., Tonin, P. N., Provencher, D. M., Mes-Masson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+description+of+a+3D+in+vitro+model+for+the+study+of+epithelial+ovarian+cancer+(EOC.">Molecular description of a 3D in vitro model for the study of epithelial ovarian cancer (EOC.</a> Mol. Carcinog. 46, 872-885 (2007).</li><li>Shield, K., Ackland, M. L., Ahmed, N., Rice, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+spheroids+in+ovarian+cancer+metastases:+Biology+and+pathology.">Multicellular spheroids in ovarian cancer metastases: Biology and pathology.</a> Gynecol Oncol. 113, 143-148 (2009).</li><li>Dafou, D., Grun, B., Sinclair, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcell-mediated+chromosome+transfer+identifies+EPB41L3+as+a+functional+suppressor+of+epithelial+ovarian+cancers.">Microcell-mediated chromosome transfer identifies EPB41L3 as a functional suppressor of epithelial ovarian cancers.</a> Neoplasia. 12, 579-589 (2010).</li><li>Levanon, K., Crum, C., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+pathogenesis+of+serous+ovarian+cancer+and+its+clinical+impact.">New insights into the pathogenesis of serous ovarian cancer and its clinical impact.</a> J. Clin. Oncol. 26, 5284-5293 (2008).</li><li>Kurman, R. J., Shih, I. e. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+and+extraovarian+origin+of+epithelial+ovarian+cancer--shifting+the+paradigm.">Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer--shifting the paradigm.</a> Hum. Pathol. 42, 918-931 (2011).</li></ol>

Nessun conflitto di interessi dichiarati.

La biologia di stadio precoce del cancro ovarico epiteliale (EOC) è poco conosciuta. Forse uno dei principali ostacoli in questo campo per molti anni è stata la mancanza di comprensione delle origini tessuto specifiche che la malattia, e del significato del ruolo del microambiente nello sviluppo EOC. Nel corso degli ultimi anni, è diventato chiaro che EOC è una malattia eterogenea con più sottotipi distinti histophathological, probabilmente con differenti origini cellulari per diversi sottotipi. Ad esempio, i dati ...

<table border="1"><tbody><tr><td> Reagente </td><td> Fornitore </td><td> Numero di catalogo </td></tr><tr><td> PolyHEMA, adatto per coltura cellulare </td><td> Sigma Aldrich </td><td> P3932 </td></tr><tr><td> Biologia molecolare etanolo grado </td><td> Sigma Aldrich </td><td> E7023 </td></tr><tr><td> Acqua sterile per colture cellulari </td><td> VWR </td><td> 12001-356 </td></tr><tr><td> MCDB105 </td><td> Sigma Aldrich </td><td> M6395 </td></tr><tr><td> Medium 199 </td><td> Sigma Aldrich </td><td> M2154 </td></tr><tr><td> Hyclone siero fetale bovino </td><td> Thermo Scientific </td><td> SH30088.03 </td></tr><tr> &lt;td&gt; Gentamicina </td><td> Sigma Aldrich </td><td> G1397 </td></tr><tr><td> Amfotericina B </td><td> Sigma Aldrich </td><td> A2942 </td></tr><tr><td> pBABE-igro-hTERT </td><td> Addgene </td><td> 1773 </td></tr><tr><td> PBS </td><td> VWR </td><td> 12001-766 </td></tr><tr><td> 0,25% tripsina-EDTA </td><td> Invitrogen </td><td> 25200-072 </td></tr><tr><td> Cella filtro (40 o 70 micron) </td><td> VWR </td><td> 21008-949 21008-952 </td></tr><tr><td> Anti-fibroblasti proteina anticorpi di superficie, clone 1B10 </td><td> Sigma </td><td> F4771 </td></tr><tr><td> Anti-pan-citocheratina anticorpo (C11) </td><td> Santa Cruz </td><td> sc-8018 </td></tr><tr><td> Polibrene (hexadimethrine bromuro) </td><td> Sigma </td><td> 107689 </td></tr><tr><td> TeloTAGGG telomerasi PCR ELISA PLUS </td><td> Roche </td><td> 12013789001 </td></tr><tr><td> TeloTAGGG Assay Lunghezza dei telomeri </td><td> Roche </td><td> 12209136001 </td></tr></tbody></table> Reagenti Tabella 1. E apparecchiature di cui al presente studio.

heterotypic three-dimensional in vitro modeling of stromal-epithelial interactions during ovarian cancer initiation and progression

Tumori ovarici epiteliali (EOCS) sono la principale causa di morte per neoplasia ginecologica nelle società occidentali. Nonostante i progressi nei trattamenti chirurgici e il miglioramento delle chemioterapie a base di platino, si è registrato pochi miglioramenti nei tassi di sopravvivenza EOC per più di quattro decenni 1,2. Mentre stadio I tumori sono a 5 anni tasso di sopravvivenza&gt; 85%, i tassi di sopravvivenza per la fase III / IV della malattia sono &lt;40%. Pertanto, gli alti tassi di mortalità per tumore ovarico epiteliale potrebbe essere significativamente ridotta se i tumori sono stati rilevati in precedenza, più trattabili, fasi 3-5. Allo stato attuale, la base molecolare genetica e biologica della malattia in stadio precoce sviluppo è poco conosciuta. Più in particolare, si sa poco circa il ruolo del microambiente tumorale durante l&#39;inizio, ma i fattori di rischio noti per EOCS (ad esempio età e parità) suggeriscono che il microambiente svolge un ruolo chiave nella genesi precoce di EOCS. Abbiamo quindi sviluppato modelli eterotipiche tridimensionalisia dell&#39;ovaio normale e di tumori ovarici primi stadi. Per l&#39;ovaio normale, abbiamo co-coltura superficie epiteliale ovarico normale (Iose) e normale fibroblasti stromale (INOF) cellule, immortalata da trasduzione retrovrial della subunità catalitica della telomerasi holoenzyme umana (hTERT) per estendere la durata di vita di queste cellule in coltura. Per modellare le prime fasi di trasformazione cellulare ovarico epiteliale, sovraespressione del CMYC oncogene nelle cellule Iose, di nuovo co-coltura con cellule INOF. Questi modelli eterotipiche sono stati utilizzati per studiare gli effetti di invecchiamento e senescenza sulla trasformazione e l&#39;invasione delle cellule epiteliali. Qui descriviamo le fasi metodologiche in sviluppo di questi modello tridimensionale; queste metodologie non sono specifici per lo sviluppo di ovaio normale e tessuti di cancro ovarico, e potrebbe essere usato per studiare tipi di tessuto in cui le interazioni cellule stromali ed epiteliali sono un fondamentale aspetto del tessuto e di manutenzioneSease sviluppo.

Watch this Scientific Journal Video about Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression at JoVE.com

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Descriviamo metodologie per la creazione di<em&gt; In vitro</em&gt; Eterotipiche modelli tridimensionali comprendenti fibroblasti ovariche e di superficie ovarica normale o cellule di cancro ovarico epiteliale. Discutiamo l&#39;uso di questi modelli per studiare stroma-epiteliali interazioni che si verificano durante lo sviluppo del cancro ovarico.

Eterotipica tridimensionale<em&gt; In Vitro</emModellazione&gt; di stromale-epiteliali interazioni Durante iniziazione e la progressione del cancro ovarico

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

heterotypic-three-dimensional-vitro-modeling-stromal-epithelial

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

15.0K Views.  University of Southern California. Descriviamo metodologie per la creazione di In vitro Eterotipiche modelli tridimensionali comprendenti fibroblasti ovariche e di superficie ovarica normale o cellule di cancro ovarico epiteliale. Discutiamo l'uso di questi modelli per studiare stroma-epiteliali interazioni che si verificano durante lo sviluppo del cancro ovarico.

Video: Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro test Clearance mesoteliali che modella i primi passi di metastasi del cancro ovarico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Ricerca

Medicina

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Un nuovo modello di spheroid del tumore strutturato in 3D modulato come fibromastro per studiare l'interazione tumora-stroma e la scoperta di farmaci

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Ricerca sul cancro

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

Preparazione e analisi di<em&gt; In Vitro</em&gt; Tridimensionale carcinoma mammario mondo dei replicanti

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

Bioingegneria

Stabilire un modello 3D ex vivo per l'interazione cancro ovarico-omento

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Autore in primo piano: Modello avanzato ex vivo per lo studio dell'interazione cancro-microambiente adiposo 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Un modello ex vivo di metastasi peritoneali del carcinoma ovarico con omento umano

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Modellazione Comportamento Ovarian Cancer multicellulare Spheroid in un Microdevice dinamica 3D peritoneale

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensionale Co-cultura Modello di tumore stromale-Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

Cellule stromali eteromulticellulari in colture 3D prive di scaffold di cellule tumorali epiteliali per guidare l'invasione

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

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Cellule stromali eteromulticellulari in colture 3D prive di scaffold di cellule tumorali epiteliali per guidare l'invasione