I have screened several tumor models (Panc02, 4T1, E0771 and EMT6) for CAF markers (such as FSP-1 and PDGF receptor) through flow cytometric analysis - the population of live CAFs in each model are about 1-3% CAFs.
Is there any way I could increase the content of CAFs in tumors? Could I simply co-inoculate mice with murine fibroblasts/cancer cell suspensions to reshape the stromal architecture of syngeneic tumor models?
Hi, James!!
Fibroblasts co-inoculation will increase CAFs to certain extent. That's because fibroblasts will partially differentiated into CAFs through interacting with cancer cells, as you know.
But I guess MSCs or bone marrow derived fibroblast-like cells may be better than normal fibroblasts from the point of view of differentiation ability into CAFs.
We previously performed the co-inoculation of MSCs (or bone-marrow derived fibroblast-like cells) with cancer cells to create the tumor having abundant stroma and CAFs. We confirmed the PDGFRβ expression and α-SMA expression as CAFs marker by immunohistochemistry and Western blotting in xenograft tissue.
Of course, FSP-1 and FAP are good marker as well, though we did not evaluate them.
Article Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Tran...
Article Multikinase inhibitor regorafenib inhibits the growth and me...
Article Stromal reaction inhibitor and immune-checkpoint inhibitor c...
MSCs are known to migrate into tumor cite and differentiate into CAFs by interacting with cancer cell.
Article Mesenchymal stem cells enhance growth and metastasis of colon cancer
I want to recommend MSCs use for your experiment, if available.
If you use normal fibroblast for your experiment, co-culture of cancer cells and fibroblast for certain period before co-inoculation may increase CAFs.
(You can also check the differentiation into CAFs in in vitro condition with immunostaining for α-SMA and FSP-1 with chamber slides.)
Best regards, Hidehiko.
"If you use normal fibroblast for your experiment, co-culture of cancer cells and fibroblast for certain period before co-inoculation may increase CAFs:" awesome, this was definitely my plan.
I guess it is good plan, and acceptable personally.
However, I am not sure whether the fibroblast cell lines co-cultured with cancer cells could be recognized as CAFs formally.
There are many reports showing that MSCs co-cultured with cancer cells differentiate into CAFs in in vitro and in vivo experiment .
However there are not so many research that fibroblast cell line (e.g. WI-38) co-cultured with cancer cells differentiate into CAFs in in vitro condition.
I want to show you one example.
Article Co-culture of 3D tumor spheroids with fibroblasts as a model...
This research shows that fibrblast cell line (CCD-18co and WI-38) co-cultured with cancer cell showed CAF-like activation.
If you use fibroblast in that experiment, you should confirm the CAF-like activation of fibroblast after co-culturing with cancer cells, by evaluating α-SMA, FAP, or FSP-1 expression.
In conclusion:
# The best is using MSCs.
# Using fibroblast cell line is acceptable personally. But I am not confident that it is definitely appropriate.
I am sorry I can not help you...
Best.
Hi James,
Did you end up co-culturing cancer cells with fibroblasts? Could you see an increase in the CAF markers you were looking for? What type of fibroblasts are you using? Did you use flow or western blot to detect the markers? Would you mind sharing your co-culture conditions with me?
I have been trying to transform my fibroblasts into CAFs in the past few weeks. I use established fibroblasts that we got from ATCC and I plate them in regular plastic cell culture dishes. Then I co-culture them with conditioned media from cancer cells. However, after doing a western blot, I see the same expression level of the markers in the treatment and control group. Not quite sure what the problem is. Any ideas?
Thanks!
Hello, Saeideh Maleki . We are actually going to inoculate those mice in the next few weeks, so I can get back to you regarding how successful co-inoculations are for generating CAFs. We are specifically looking for FAP+ CAFs, and we have seen that co-culture of murine fibroblasts with cancer cells does induce an activated state. We are hoping the activated phenotype can be recapitulated in vivo.
That's great, I hope that works in vivo as well. Any reasons you are specifically looking for FAP+ ones? I am looking at FAP, alpha-SMA, PDGFR-beta, CD29 and PDPN and I'm hoping to do Flow as well as western blot.
By the way, I found this very useful paper regarding CAF subsets that might be interesting to you too.
Article Fibroblast Heterogeneity and Immunosuppressive Environment i...
Good luck!
Saeideh Maleki We're targeting activated fibroblasts that express surface markers (such as FAP-alpha) for antibody based therapies. Alpha-SMA is less specific AND it's intracellular, but we do use it to characterize activated fibroblasts. We're specifically looking at FAP+ CAFs due to their immunosuppressive functions in the tumor microenvironment.
Article Isolation of Fibroblast-Activation Protein-Specific Cancer-A...
James D Barnett
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line