We are supplementing RPMI meida with human plasma instead of serum for culture of human breast cancer cells.
When PRMI is supplemented with 10% human plasma, the media looks fine, however once cells are added into this media a gel forms.
This seems to be a reproducible phenomenon with plasma from different donors.
Do you think its concentration dependent? or are the coagulation factors somehow clumping our cells?
Human liver cancer has been occurred by the co-infection of HIV-1 and HCV (please see file; The Fascio effect). Therefore, defending mechanism against invading microbes seems to be an important issue in the cancer biology.
I have searched for Coagulation factor and Complement in my protein database (please see file; HepG2 Fucoidan).
Fetal hepatocyte Hc has Vitamin K-dependent protein C/Protein C at 5.0 μg/mg cell protein.
Hepatoma HepG2 (cultured without fucoidan) has Complement C4-B at 12.0 μg/mg cell protein. Healed hepatocyte HepG2 (cultured with fucoidan) does not have such proteins.
LC tissue (with leprosy) has Coagulation factor V/Activated protein C cofactor/Proaccelerin, labile factor at 5.5, and Complement component C8 alpha chain at 2.3 μg/mg tissue protein, respectively.
HCC tissue (with PBC) has Complement component C7/Complement C7 at 7.2 μg/mg tissue protein.
LC tissue (named as No.6) has Coagulation factor V/Activated protein C cofactor/Proaccelerin, labile at 3.5, and Complement C2 at 1.3 μg/mg tissue protein, respectively.
HCC tissue (No.6) has Coagulation factor V/Activated protein C cofactor/Proaccelerin, labile factor at 0.23, Complement C7 at 2.0, and Complement factor D/Adipsin at 0.21 μg/mg tissue protein, respectively.
Serum of GSD-1b patient (with biotin deficiency) has Complement factor H-related protein 3 at 4.2 μg/mg serum protein.
Healthy serum (with light haemolysis in his gut; 7mo1w, male) has Coagulation factor VIII at 4.9 μg/mg serum protein.
Healthy serum (with light haemolysis; 1y4mo, female) has Complement factor B at 8.0 μg/mg serum protein.
The serum of the above girl (excess biotin, anorexia; 1y11mo) has Complement decay-accelerating factor/CD55 at 2.4 μg/mg serum protein.
Serum of common cold (12mo, female) has Thrombospondin type-1 domain-containing protein 7B at 7.2 μg/mg serum protein.
Gene expression seems to be under the control of virus.
Therefore, expression of Coagulation factor and Complement gene (protein) and expression of viral protein are quantitatively searched and compared.
It is found that Coagulation factor and Complement genes seem to be up-regulated by HPV, Coronavirus/CoV, and Baculovirus, and down-regulated by Marburg virus/MARV and Vesicular stomatitis Indiana virus/VSIV.
By the way, we are now estimating that Breast cancer is induced by co-infection of HIV-1 and enveloped +ssRNA virus (Flavivirus of Dengue virus/DENV or Zika virus/ZIKV and/or Togavirus of Sindbis virus/SINV or Chikungunya virus/CHIKV). It is important that these virus are transfered by Mosquitoes.
By using similar estimation method, we are estimating that Pancreatic cancer is induced by co-infection of HIV-1 and HCoV (our unpublished results).
Salma,
In my opinion, Yes. The coagulation factors in the plasma may act as mediators that stimulate the breast cancer cells or macrophages or other cancer cells in culture together with RPMI media and may promote growth and invasion by expression of various cytokines. These cytokines may also cause expression of tissue factor and other thromboplastic substances that may tend to form a gel.
Iqbal
Some cell lines express or release tissue factors, which will activate coagulation and the gel formation you described
This most likely will happen at even very low concentration of plasma (0.1%)
I used tissue plasminogen activator on a PC 12 culture and observed that the cells promptly dissociate from the bottom of the culture flask and float. However they are still alive as monitored by trypan blue exclusion. I guess therefore that coagulation factors may play a role in the attachment of some cells to the matrix.
What type of plasma are you using ? Citrated, EDTA, or heparinized ? If you use calcium-chelators their action might be neutralized by calcium ions in RPMI + cells-medium, then the gel should be fibrin.
Interesting problem. Is the gelation uniform or in clumps? If it is in clumps, have you taken one out and had a look under microscope? If you have, what does it consist of? Cells or just gelatinous/fibrinous material? If the clumps have cells enmeshed, a likely explanation is that the cells are producing some kind of protease that is cleaving fibrinogen that is present in the plasma to generate fibrin. It would be interesting to stain them for fibrin and FDP. Another interesting thing to do would be to take the supernatant and add it to normal plasma and see whether that generates fibrin/FDP. My suspicion is that the cells are producing proteins with enzymatic activity. I will be interested to know what you find though. Good luck.
Siva
Thank you Dr Sivakumaran,
its a homogeneous gel and if we do not plate the cell suspension fast enough it becomes unpipettable.
Under the microscope we see a fibrous clump with a lot cells embedded through out.
Sure, coagulation factors solved in plasma (e.f. FX, FVII or asTF) may interact wirth/ activate cells leading to different reactions, such as disregulated signaling, protein generation etc...
Many different cancer cell types are known to induce coagulation pathways directly. Expression of tissue factor (TF) is one of the main mechanisms described. This paper actually depicts methodology and shows that this is both reproducible as you've mentioned, and also does dependent (more cancer cells = more coagulation).
https://www.frontiersin.org/articles/10.3389/fonc.2012.00110/full
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