Name: evaluating the angiogenetic properties of ovarian cancer stem-like cells using the three-dimensional co-culture system, nico-1
Uploaded: 2020-12-05T13:30:00
Description: 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

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.

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	<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 ...

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