This work was supported by a Grant-in-Aid for Scienti&#64257;c Research C (grant no. 19K09834 to K.N.) from the Ministry of Education, Science, and Culture, Japan.

All procedures were performed under the protocol approved by the Ethics Committee for human welfare. All patients provided written informed consent for the research use of their samples, and the collection and use of tissues for this study were approved by the Human Genome, Gene Analysis Research Ethics Committee at Teikyo University.
1. Isolation and culture of ovarian cancer stem cells (OCSCs) from Patients with ovarian cancer and ascites in a level 2 biosafety cabinet
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The presented protocol describes how to mimic the tumor microenvironment of OCSCs in an in vitro setting. The primary component of the method constitutes the highly reproducible coculture model obtained using the NICO-1 system, an indirect Transwell co-culture system. Many of the currently available coculture models examine the effects of direct cell-cell contact on cocultured cell populations12,13,14,<sup class="xref...

Ovarian cancer is the eighth most common malignancy in women worldwide, with approximately 300,000 new diagnoses and an estimated 180,000 deaths annually1. At initial diagnosis, ovarian cancer often presents with severe symptoms, with about 75% of patients already at stage III-IV. Accordingly, the 5-year survival rate is &#60;30% and the mortality rate is the highest among gynecological cancers2, with the efficiency of treatment for ovarian cancer being highly dependent on clinical factors such as the successful accomplishment of debulking surgery, resistance to chemotherapy, and recurrence after the initial therapy.
Ovarian cancer tissues are hierarchically organized, with not all tumor components being equally capable of generating descendants. The only cells able to self-renew and produce a heterogeneous tumor cell population are considered to represent cancer stem cells (CSCs)3. CSC self-renewal and tumor initiation are accompanied by the promotion of angiogenesis to remodel their tumor microenvironment for the purpose of maintaining a supportive niche. However, previous models could not be utilized for in vitro analyses because of the limited reproducibility of cultivating CSCs derived from clinical samples owing to the disruption of spheroids after multiple passaging. More recently, experimental methods to cultivate CSCs from patients have been developed for several applications4,5,6,7. In particular, by exploiting the characteristic of CSCs to grow by forming spheroids in ultra-low attachment plates with serum-free medium, the cultivated CSCs are induced to express a stem-cell surface marker that is not expressed in normal tumor cells with multilineage differentiation potential8,9.
Recent data have shown that the persistence of dormant ovarian (O)CSCs visualized as dissemination at the peritoneum is associated with their regeneration as recurrent tumors10. Understanding the molecular and biological features of OCSCs may thus allow for effective targeting and eradication of these cells, resulting in potential tumor remission. In particular, little is known regarding the cellular and molecular mechanistic features of CSCs roles in angiogenesis11. Therefore, in the present protocol we used patient-derived OCSCs in an in vitro setting to investigate the angiogenic property of endothelial cells using the co-culture model, which may mimic the tumor microenvironment of CSCs and endothelial cells at the metastatic site in the clinical setting. Ultimately, as neovascularization constitutes a critical process necessary to support tumor growth and metastasis, a better understanding of its mechanism will allow the development of a novel targeting therapy for OCSCs at the metastatic site.
Here, we present a protocol to visualize the neovascularization step surrounding the CSCs in a time course manner. The advantage of the protocol includes allowing fully reproducible investigations using the 3D co-culture system, NICO-1, thereby permitting observation of the effects on patients of the OCSC-derived tumor-initiation capability during endothelial cell angiogenesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025% Trypsin&#160;</td>
			<td>Thermo</td>
			<td>R001100</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pipet</td>
			<td>Thermo</td>
			<td>170356N</td>
			<td></td>
		</tr>
		<tr>
			<td>1250 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T112XLRS-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Nunc</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Pipet tip</td>
			<td>QSP</td>
			<td>T110RS-NEW</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Thermo (Gibco)</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pipet</td>
			<td>Thermo</td>
			<td>170366N</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuMAX</td>
			<td>Innovative Cell Technologies</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoatTM Collagen I 60mm Dish</td>
			<td>Corning</td>
			<td>356401</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>KUBOTA</td>
			<td>2800</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2&#160;</td>
			<td>PromoCell</td>
			<td>C-22011&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>WAKO</td>
			<td>057-00456</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-Basic</td>
			<td>Thermo (Gibco)</td>
			<td>PHG0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz</td>
			<td>SIGMA</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>HUEhT-1 cell</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB1458</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCP Filter 0.6 &#181;m</td>
			<td>Ginrei Lab.</td>
			<td>2525-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, human</td>
			<td>SIGMA (Roche)</td>
			<td>11376497001</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>PerkinElmer</td>
			<td>ARVO MX-flad</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>Corning</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Yokogawa</td>
			<td>CQ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NICO-1</td>
			<td>Ginrei Lab.</td>
			<td>2501-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPlate-96</td>
			<td>PerkinElmer</td>
			<td>6005290</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 Pipet</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 Pipet</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo (Gibco)</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro hESC SFM</td>
			<td>Thermo (Gibco)</td>
			<td>A1000701</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipet</td>
			<td>FALCON</td>
			<td>357575</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>WAKO</td>
			<td>253-00513</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

We collected ascites fluids obtained from patients with advanced ovarian cancer during surgery or paracentesis for the purpose of performing a long-term stable culture for spheroids. Here, we present cases of a long-term spheroid culture of ovarian CSCs termed CSC1 and CSC2. Both cell lines carry the same diagnosis and histological profiles. The mechanistic roles of OCSCs underlying the interaction with endothelial cells necessary to induce the neovascularization of endothelial cells surr...

Watch this Scientific Journal Video about Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1 at JoVE.com

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Ovarian cancer has a high mortality rate among gynecological malignancies. Ovarian cancer stem cells and their vascular niche are able to remodel tumor microenvironment. However, previous ovarian cancer stem cell models have not permitted the in-detail analysis because of their limited reproducibility.The advantage of our protocol include the reproducibility of its 3D co-culture system, which facilitate the study of the effects of ovarian cancer stem cells-derived tumor initiation capabilities in patient during angiogenesis. Demonstrating the procedure will be Yuko Miyagawa, a research fellow from our laboratory. To isolate cancer stem cells from human ovarian cancer ascites obtained via paracentesis, Collect 100 to 250 milliliters of patient ascites and centrifuge the sample within 24 hours of acquisition.Resuspend the pellets in two milliliters of OCSC medium and add eight milliliters of 30%non-ionic density gradient medium in PBS to each sample. Transfer the cell suspensions to individual 15 milliliter tubes and carefully layer two milliliters of additional OCSC medium onto the cell solution in each tube. Separate the cells by density gradient centrifugation and carefully transfer the OCSC at the interphase into a new 15 milliliter tube.Wash the collected cells with a full volume of PBS and resuspend the pellets in fresh OCSC medium. Seed the cells onto ultra low attachment culture dishes and incubate the cultures at 37 degrees Celsius and 5%carbon dioxide. After five days of culture, collect the cells in one 15 milliliter tube per plate by centrifugation.After a second centrifugation in PBS, resuspend the cells in one milliliter of proteolytic and collagenolytic enzyme cell detachment solution for 10 minutes at 37 degrees Celsius. At the end of the incubation, mix well by pipetting and return the cells to 37 for another five minutes. At the end of the incubation, mix the cells again by pipetting and fill the tube with PBS for washing by centrifugation.Then, resuspend the pellet in OCSC medium and seed the cells on new ultra low attachment culture dishes. To set up a HUEhT-1 endothelial cell culture, wash the HUEhT-1 cells with PBS and treat the cells with 0.025%trypsin for three minutes at room temperature. When the cells have detached, stop the reaction with five milliliters of endothelial cell growth medium two, and collect the cells in a 15 milliliter tube for centrifugation.Then, resuspend the pellet in HUEhT-1 medium at a 1.5 by 10 to the fifth concentration and seed the cells on collagen coated culture dishes. To set up an interactive co-culture plate for a tube formation assay, assemble the NICO-1 device according to the manufacturer's instructions and place the system on ice. Wash the assembly with 300 microliters of cold PBS before adding 300 microliters of chilled extracellular matrix based hydrogel to the device for a 60 minute incubation at 37 degrees Celsius.At the end of the incubation, immerse a sterile 13 millimeter filter in 100%ethanol followed by a one minute submersion in PBS. At the end of the incubation, place an O-ring and the equilibrated filter into the appropriate chambers in the body of the assembly. When the device has been assembled, resuspend the HUEhT-1 cells at 1.25 by 10 to the fifth cells per milliliter of endothelial cell growth medium supplemented with 2%fetal calf serum concentration and add 1.2 milliliters of cells to each extracellular matrix based hydrogel coated well.Then, add 1.5 milliliters of five day cultured OCSC to one well and place the NICO-1 system in the cell culture incubator. Tube formation can be observed by light microscopy and the formation cell networks on the extracellular matrix based hydrogel can be measured by quantifying the number of branches that form within the co-culture over time. Here, a comparison of the tube formation activity induced by two different CSC can be observed.In this analysis, the number of formed vascular tubes dramatically increased over time during co-culture with the second CSC population. In these images, the angiogenic property of HUEhT-1 after vascular endothelial growth factor treatment without CSC2 co-culture can be observed as a positive control. In this OSCS endothelial cell co-culture model using the NICO-1 system, HUEhT-1 cells were co-cultured with CSC2 for 20 hours.As observed via time lapse video imaging, the HUEhT-1 cells formed vascular tubes over the course of the 20 hour CSC co-culture. Recent discovery have led to the clinical use of PARP inhibitors for ovarian cancer, defective in homologous recombination repair, as well as anti-angiogenic bevacizumab. Our protocol using 3D co-culture system can provide insight regarding the angiogenic property of ovarian cancer stem cells in tumor metastasis.

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2.4K visualizações.  Teikyo University School of Medicine. Ovarian cancer has a high mortality rate among gynecological malignancies. Ovarian cancer stem cells and their vascular niche are able to remodel tumor microenvironment. However, previous ovarian cancer stem cell models have not permitted the in-detail analysis because of their limited reproducibility.The advantage of our protocol include the reproducibility of its 3D co-culture system, which facilitate the study of the effects of ovarian cancer stem cells-derived tumor initiation capa...