In my lab we are interested in fighting glioblastoma pretty hard. Now we are using two pan-tyrosine kinase inhibitors, namely Sunitinib and Axitinib. Because one of the major in vivo tumor impacts is the reduction of angionesis in the tumors, we would like to check first in vitro if your strategy of miRNA modulation+drug also reduces the tumor cells pro-angiogenic features.
Is it enough to look for VEGF in the medium or the levels of VEGFR/Epherins in the cell's membrane? The co-culture with endothelial cells can give us some good results?
Or do you think the CAM assay is more aproppriate?
Thank you
Just looking at VEGF in media is not enough because VEGF is in FBS. Presence of VEGFR/Ephrins doesn't mean they are activated. Needs a functional assay.
CAM assay is good but sometimes people argue that it is on Chick CAM and is far in the evolutionary tree from humans. Sometime people do this assay in mouse ears to overcome that question.
In vitro tube formation assay is one good technique (among in vitro) and Xenograft CD31 microvessel density is more appropriate in vivo technique (among in vivo).
Just looking at VEGF in media is not enough because VEGF is in FBS. Presence of VEGFR/Ephrins doesn't mean they are activated. Needs a functional assay.
CAM assay is good but sometimes people argue that it is on Chick CAM and is far in the evolutionary tree from humans. Sometime people do this assay in mouse ears to overcome that question.
In vitro tube formation assay is one good technique (among in vitro) and Xenograft CD31 microvessel density is more appropriate in vivo technique (among in vivo).
As Dr.Goodwin G. Jinesh have mentioned above, angigenesis needs several growth factors including VEGF, basic fibroblast growth factor(bFGF),angiopoietins, hepatocyte growth factor(HGF), epidermal growth factor(EGF), placental―derived growth factor(PlGF). However, it is difficult to distinguish whether or not these factors are really derived from tumor cells, cancer-associated fibroblasts (CAF) or tumor-associated macrophages (TAM). Tumor endothelial markers are critical to tumor angiogenesis. If you perform co-culture system with tumor endothelial cells (TECs), you should evaluate the expression of TEM8 and CD13, both of which are specific to TECs.
Hi Pedro, the main in vitro angiogenesis assay is the endothelial tube forming assay that Goodwin mentioned.
http://link.springer.com/article/10.1007/s10456-009-9146-4
To do CAM assays (which is in vivo angiogenesis), you need specific incubators and skilled personnel to perform the experiments.
First of all thank you very much for the replies.
I think I will try first to implement in the lab the co-culture technique. But I have some questions that came to my mind:
1
a) I didn't said before but I want to evaluate the pro-angiogenic features of CSCs isolated from 2 GBM cell lines. Those cells grow in suspension and the medium is supplemente among other with bFGF. Would you expect any problems given these conditions?
( I can perhaps grow the CSCs in adherent conditions by coating the wells/flasks with laminin and possibly use always the exact same amount of bFGF)
2
a) Regarding the TECs that Dr. Go J Yoshida kindly mensioned, do they need to be from Glioblastoma?
b) There are some existing human cell line models that we can acquire/ask from? TEM8 and CD13 would be decrease in those TECs when co-cultured with cancer cells correct?
c) There are some specific released factor that can be analysed by ELISA from the medium?
The most standard angiogenic assay used is the sprouting assay of tubulogenesis assay. This could be performed over a Matrigel Matrix, which is an artificial matrix that mimicks the host environment and the cells that normally behave as non-angiogenic they do form sprouts when cultured over this kind of matrix. If you seed the normal cells and then the isolated CSCs then you can determine the pro-angiogenic potential. The usage of bFGF it's not a problema because I have used it in the media to grow this kind of cells under these conditions.
I am not sure about the usage of TECs but there should be some cell lines obtained form angiomas which are cancer types that most of the tumour cells are endothelial as well.
Thank you! That's wonderfull news. And then I should slice the matrigel, stain for those endothelial cells and quantify the ramification level with a software such as ImageJ?
Pedro, the Matrigel it's not an stain, it's a matrix coating that you have to prepare into the dish or the plate that you think you're gonna culture your cells and then seed them on top of them. Then you can use ImageJ to analyze the pictures that you take under a normal microscope. Here I attach a pic that I took last week from my melanoma tumour cell line growth on top of matrigel.
Dear Pedro
I'm not completely sure whether you plan to quantify the ability of your glioblastoma cells to form blood vessels themselves, or to induce endothelial cells to grow as vessels into the tumor.
In the first case, the proposal to plate your cells onto matrigel and quantify their ability to form tubes (nicely shown in the image posted by Francisco above) seems good.
In the second case (which seemed to me to be the original question at the beginning of the discussion), however, you actually want to assess the ability of the factors secreted by your cells to stimulate normal endothelial cells to grow into blood vessels and a completely different experimental design would be necessary. In fact, if you do not know, or do not want to make assumptions, about what factors are responsible for your effect, you would use conditioned media from your cells (conatining all the secreted factors) and use them to treat normal endothelial cells plated on Matrigel: the amount of tube formation is then a measure of the total pro-angiogenic activity of the media. Since bFGF is a potent pro-angiogenic factor in vitro and could easily make all your conditions equally positive, thereby masking your experimental effect, conditioned media are typically made culturing your cells for a few hours with a base assay medium that would not be angiogenic per se (low serum, no extra factors) and then the classic experimental design would be:
base medium alone (negative control)
base medium + VEGF or bFGF (positive control)
base medium conditioned by test cells (experimental conditions)
Hope it helps!
Best regards,
Andrea
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