Name: a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay
Uploaded: 2018-04-12T14:15:00
Description: Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

This work was supported by grants from the Shanghai Committee of Science and Technology (15411951800, 15410723200). The authors wish to thank Prof. YuMei Wen and Prof. Chao Zhao of the Pathogenic Microorganism Department of Fudan University for their technical assistance.

This method was performed in accordance with the approved guidelines and regulations of the Research Ethics Committee of Huashan Hospital, Fudan University. Corresponding standard safety measures were followed in each step. For a schematic presentation, please refer to Figure 1.
1. Cell Culture and Plasmid Construct
<ol>
	<li>Routinely maintain the human umbilical vein endothelial cell (HUVEC) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal b...

<ol>
	<li>Lonser, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Hippel-Lindau+disease.">von Hippel-Lindau disease.</a> Lancet. 361 (9374), 2059-2067 (2003).</li><li>Hussein, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+capillary+haemangioblastoma:+the+pathologist's+viewpoint.">Central nervous system capillary haemangioblastoma: the pathologist's viewpoint.</a> Int J Exp Pathol. 88 (5), 311-324 (2007).</li><li>Ma, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemangioblastomas+might+derive+from+neoplastic+transformation+of+neural+stem+cells/progenitors+in+the+specific+niche.">Hemangioblastomas might derive from neoplastic transformation of neural stem cells/progenitors in the specific niche.</a> Carcinogenesis. 32 (1), 102-109 (2011).</li><li>Zhuang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+derived+vasculogenesis+in+von+Hippel-Lindau+disease-associated+tumors.">Tumor derived vasculogenesis in von Hippel-Lindau disease-associated tumors.</a> Sci Rep. 4, 4102 (2014).</li><li>Glasker, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VHL-deficient+vasculogenesis+in+hemangioblastoma.">VHL-deficient vasculogenesis in hemangioblastoma.</a> Exp Mol Pathol. 96 (2), 162-167 (2014).</li><li>Wizigmann-Voos, S., Breier, G., Risau, W., Plate, K. 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H., Kamijyo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+hemangioblastoma:+histogenesis+of+stroma+cells.">Cerebellar hemangioblastoma: histogenesis of stroma cells.</a> Cancer. 31 (6), 1528-1540 (1973).</li><li>Jurco, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemangioblastomas:+histogenesis+of+the+stromal+cell+studied+by+immunocytochemistry.">Hemangioblastomas: histogenesis of the stromal cell studied by immunocytochemistry.</a> Hum Pathol. 13 (1), 13-18 (1982).</li><li>Ma, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+tumorigenic+cells+and+implication+of+their+aberrant+differentiation+in+human+hemangioblastomas.">Identification of tumorigenic cells and implication of their aberrant differentiation in human hemangioblastomas.</a> Cancer Biol Ther. 12 (8), 727-736 (2011).</li><li>Ma, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD41+and+CD45+expression+marks+the+angioformative+initiation+of+neovascularisation+in+human+haemangioblastoma.">CD41 and CD45 expression marks the angioformative initiation of neovascularisation in human haemangioblastoma.</a> Tumour Biol. 37 (3), 3765-3774 (2016).</li><li>Sharifpanah, F., Sauer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Spheroid-Based+Sprout+Assay+in+Three-Dimensional+Fibrin+Scaffold:+A+Novel+In+Vitro+Model+for+the+Study+of+Angiogenesis.">Stem Cell Spheroid-Based Sprout Assay in Three-Dimensional Fibrin Scaffold: A Novel In Vitro Model for the Study of Angiogenesis.</a> Methods Mol Biol. 1430, 179-189 (2016).</li><li>Cai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+taurine+upregulated+1+enhances+tumor-induced+angiogenesis+through+inhibiting+microRNA-299+in+human+glioblastoma.">Long non-coding RNA taurine upregulated 1 enhances tumor-induced angiogenesis through inhibiting microRNA-299 in human glioblastoma.</a> Oncogene. 36 (3), 318-331 (2017).</li><li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+Conveniently+Screening+pLKO.1-TRC+Vector+Tagged+with+TurboGFP.">Construction of Conveniently Screening pLKO.1-TRC Vector Tagged with TurboGFP.</a> Appl Biochem Biotechnol. 181 (2), 699-709 (2017).</li><li>Laib, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid-based+human+endothelial+cell+microvessel+formation+in+vivo.">Spheroid-based human endothelial cell microvessel formation in vivo.</a> Nat Protoc. 4 (8), 1202-1215 (2009).</li><li>D'Alessio, A., Moccia, F., Li, J. H., Micera, A., Kyriakides, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+Vasculogenesis+in+Health+and+Disease.">Angiogenesis and Vasculogenesis in Health and Disease.</a> Biomed Res Int. 2015, 126582 (2015).</li><li>Finkenzeller, G., Graner, S., Kirkpatrick, C. J., Fuchs, S., Stark, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+in+vivo+vasculogenic+potential+of+endothelial+progenitor+cells+in+comparison+to+human+umbilical+vein+endothelial+cells+in+a+spheroid-based+implantation+model.">Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model.</a> Cell Prolif. 42 (4), 498-505 (2009).</li><li>Morin, K. T., Tranquillo, R. T. <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+models+of+angiogenesis+and+vasculogenesis+in+fibrin+gel.">In vitro models of angiogenesis and vasculogenesis in fibrin gel.</a> Exp Cell Res. 319 (16), 2409-2417 (2013).</li><li>Blacher, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+invasion+in+the+spheroid+sprouting+assay:+a+spatial+organisation+analysis+adaptable+to+cell+behaviour.">Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour.</a> PLoS One. 9 (5), 97019 (2014).</li><li>Straume, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+heat+shock+protein+27+induces+long-term+dormancy+in+human+breast+cancer.">Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer.</a> Proc Natl Acad Sci U S A. 109 (22), 8699-8704 (2012).</li><li>Naumov, G. N., Akslen, L. A., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+angiogenesis+in+human+tumor+dormancy:+animal+models+of+the+angiogenic+switch.">Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch.</a> Cell Cycle. 5 (16), 1779-1787 (2006).</li><li>Naumov, G. N., Folkman, J., Straume, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+dormancy+due+to+failure+of+angiogenesis:+role+of+the+microenvironment.">Tumor dormancy due to failure of angiogenesis: role of the microenvironment.</a> Clin Exp Metastasis. 26 (1), 51-60 (2009).</li><li>Wang, Y., Yang, J., Du, G., Ma, D., Zhou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effects+respond+to+cerebral+ischemia+without+susceptibility+to+HB-tumorigenesis+in+VHL+heterozygous+knockout+mice.">Neuroprotective effects respond to cerebral ischemia without susceptibility to HB-tumorigenesis in VHL heterozygous knockout mice.</a> Mol Carcinog. 56 (10), 2342-2351 (2017).</li><li>Stratmann, R., Krieg, M., Haas, R., Plate, K. 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Recently, multiple fields of vascular biology research were stimulated by the study of the angiogenic endothelium15. In this article, we developed an endothelial spheroid sprouting technique as an experimental model to study the vessel formation that originates from the VHL gene&#39;s loss of function in manipulated endothelial cells to identify novel candidate molecules of the angiogenic cascade. To the best of our knowledge, this is the first report to examine the angiogenic effects of the endog...

Hemangioblastomas (HBs) are benign vascular tumors that are found exclusively within the human central nervous system (CNS). They develop in patients with either von Hippel-Lindau (VHL) disease or sporadic lesions. VHL-HBs are difficult to cure through surgical treatment due to the frequent recurrence and multiple lesions that result from this genetic disorder1. Although the inactivation of the VHL tumor suppressor gene has been considered the root cause of the tumorigenesis of VHL-HBs, the cytological origin (including neovascularization) and evolutionary process of HBs remain largely controversial2. Therefore, a better understanding of HB-neovascular biological mechanisms may provide useful insights into the most promising anti-vascular strategies for VHL-HBs.
Recent research has suggested that HB-neovascularization is similar to the embryologic vasculogenesis3,4,5. Classic vascular endothelial growth factor (VEGF)-mediated angiogenesis that originated from the vascular endothelium and that is driven by VHL loss of function resulted in proliferation and neovascular formation, which has been challenged6. In 1965, Cancilla and Zimmerman found, using electron microscopy, that HBs originated from the endothelium7. Later it was found that stromal cells are derived from vasoformative element8. In 1982, Jurco et al. found that stromal cells are of endothelial origin9. Therefore, we hypothesized that human vascular endothelial cells are the original cells of HB-neovascularization10. Although it is better to use the primary cultures from HB cells derived from VHL patient surgeries, our previous research indicated that primary cultures from HB are not stable, and cell lines could not be established3. Moreover, the primary cultures in the 3D environment could not identify the cytological origin of HB-neovascularization because they include the progenitors of HB-vascular ingredients10,11. Therefore, as a primitive and classic model of endothelial cells, human vascular endothelial cells (HUVEC) could serve as an alternative cellular model for HBs.
The spheroid sprouting assay is a new model in tissue engineering12,13. In this paper, a 3D collagen-based coculture system in vitro using the spheroid sprouting assay was developed, with an overall goal to evaluate whether classic tumor angiogenesis exists in HBs, as well as its role in HBs.

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			<td height="40" style="height:40px;width:265px;">human umbilical vein endothelial cell</td>
			<td style="width:147px;">Fudan IBS Cell Center</td>
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			<td height="40" style="height:40px;">dulbecco&#8217;s modified eagle&#8217;s medium&#160;</td>
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			<td>Addgene</td>
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			<td height="40" style="height:40px;">packing plasmid psPAX2&#160;</td>
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			<td>Addgene</td>
			<td>#12259</td>
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			<td height="40" style="height:40px;">3D round-bottom 96-well plates</td>
			<td>S-Bio</td>
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			<td>BD Biosciences</td>
			<td>354234</td>
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			<td height="40" style="height:40px;">Opti-MEM medium</td>
			<td>Gibco</td>
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			<td style="width:761px;">reduced serum medium&#160;</td>
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			<td height="40" style="height:40px;">15-well plate</td>
			<td>Ibidi</td>
			<td>81501</td>
			<td>Air bubbles in the gel can be reduced by equilibrating the &#956;&#8211;Slide angiogenesis before usage inside the incubator overnight</td>
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			<td height="40" style="height:40px;">endothelial cell growth supplements</td>
			<td>Sciencell</td>
			<td>#1052</td>
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			<td height="40" style="height:40px;">10-cm culture dish</td>
			<td>Corning</td>
			<td>Scipu000813</td>
			<td></td>
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			<td height="40" style="height:40px;">Puromycin</td>
			<td>Gibco</td>
			<td>A1113802</td>
			<td></td>
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			<td height="40" style="height:40px;">typsin-EDTA</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
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			<td height="40" style="height:40px;">Automated Cell Counter System &#160;</td>
			<td>BioTech</td>
			<td></td>
			<td></td>
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			<td height="40" style="height:40px;">Image Analysis software&#160;</td>
			<td>Winmasis</td>
			<td style="width:343px;"></td>
			<td>http://mywim.wimasis.com&#160;</td>
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a comprehensive procedure to evaluate the in vitro performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

Original images are taken by inverted light microscope. The typical images of the control group and the VHL group are shown in Figure 2-A1 and Figure 2-A2. The sprouting length of the control group is shorter than that of the VHL group.
After uploading the Images, the online platform provides analysis results directly. The output consis...

Watch this Scientific Journal Video about A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay at JoVE.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

The overall goal of this experiment is to evaluate the performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay in vitro. The method can help answer the key questions in the angiogenic field, such as tumor angiogenesis. The main advantage of this technique is that the product of a comprehensive procedure to evaluate in vitro where the classic tumor angiogenesis exists in hemangioblastoma and it grows in hemangioblastoma.The implication of this technique extends toward therapy of hemangioblastumor and vasculaturement because the result highlight the complexity of hemangioblastumor neovascularization, and that suggests that this common form of angiogenesis is only a complementary mechanism. So this method can provide insight into the study of hemangioblastumor neovascularization. It can also be applied to tumors, angiogenesis, and vasculogenesis related application in some solid tumor, such as tumor vasculogenic mimicry in glass tumor.Real demonstration of this method is critical as the generation of these manipulated endothelial cell spheroid step are difficult to learn. Because the suitable condition are important. First, culture the HUVEC endothelial cells in DMEM culture medium supplemented with ten percent fetal bovine serum, penicillin, and streptomycin.Then, maintain the culture in the incubator at 37 degrees Celsius with five percent carbon dioxide. Next, to synthesize the the shRNA fragment, digest the plasmid with ApaI and EcoRI restriction enzymes. Then to the digested plasmid, add both the forward and reverse oligos, 10x NEB buffer, and double distilled water to a final volume of 50 microliters.After adding all the reagents, heat the mixture at 98 degrees Celsius for four minutes. Then, gradually cool down to room temperature in a span of few hours. Use T4 ligase to ligate the annealed oligos and the plasmid.Incubate the tube at four degrees Celsius for overnight. The following day, add five microliters of ligation mix to 25 microliters of DH5alpha competent cells. In 500 microliters of DMEM medium, add the lentiviral vector or the scrambled vector, along with other packaging plasmids.Then, incubate the lentiviral mixture for 25 minutes at 37 degrees Celsius. Post incubation, add 7 milliliters of the DMEM medium to the scrambled or lentiviral mixed solution. Culture the 293FT cells in DMEM medium without fetal bovine serum in a 10 centimeter culture dish.Add one milliliter of the lentiviral or scrambled solution to the 293FT cells in the culture dish. After six hours, aspirate the old medium from the culture dish. Then, replace the old medium with fresh DMEM medium containing 10 percent fetal bovine serum.After 48 hours, collect the medium. Transfer the HUVEC endothelial cells in the lentiviral medium and maintain for 72 hours. Next, add two micrograms per milliliter of puromycin to the HUVEC cell culture medium.Finally, incubate the culture for another 24 hours. The next day, add one milliliter of trypsin EDTA to trypsinize HUVEC cells. Then resuspend the trypsinized cell suspension in DMEM medium with 10 percent fetal bovine serum.Count the number of cells using a cell counter. After counting, seed the cells in a 3D round bottom 96-well plate. After seeding, incubate the cells at 37 degrees Celsius with five percent carbon dioxide for 72 hours continuously.After 36 hours, replace half of the culture medium with fresh medium. First, thaw the gel solution at four degrees Celsius, and then dilute it at a ratio of one to five with the reduced serum medium. Suck the spheroids from the DMEM medium using a micropipette.Next, wash the spheroids with five milliliters of reduced serum medium. Carefully transfer the suspended spheroids and the diluted gel. In a 15 well plate, embed 300 microlitters of the mixed liquid of spheroids and diluted gel.Incubate the plate at 37 degrees Celsius and five percent carbon dioxide for one hour. Next, add 400 microliters of reduced serum medium to a single well. To prevent evaporation, fill the surrounding of the well with sterile water.Then, culture the cells at 37 degrees Celsius, five percent carbon dioxide, and 100 percent humidity for an hour. After the incubation, aspirate the old medium and add 600 microliters of reduced serum medium with one percent endothelial cell growth supplement to the well and incubate for a day. Capture images using an inverted light microscope.Here, spheroid sprouting of the cells is captured using inverted light microscope to study the effect of VHL gene silencing on the angiogenic potential of the endothelial cells. A spheroid is seen to sprout 12 hours after lentiviral treatment in both the control and VHL silenced endothelial cells. Next, statistical analysis is performed to quantify the length of the sprouts generated by the VHL gene silenced spheroids.The mean sprout length seem to be about 125 micrometers in the VHL silenced groups while only being about 65 micrometers in the control group. The VHL silenced group's average cumulative sprout length is approximately 1250 micrometers while the control group's is 680 micrometers. These results indicate a two-fold sprout length increase in the VHL silenced groups.Once mastered, this technique can be done in 48 hours if it's performed properly. Following this procedure and method like capillary formation assay can be performed in order to answer additional questions. Like exploring the angiogenic ability of vascular endothelial cells.After this development, this technique paved the way for researchers in the field of angiogenesis to explore tumor inner vasculorization. After watching this video, you should have a good understanding of which genes loose a function in manipulated endothelial cells used for exploding assay.

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Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...