This video describes a procedure to generate carcinoma associated fibroblasts or calfs from primary cultured human memory. Fibroblasts carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the stromer. The tumor associated S stroma consists of extracellular matrix and a variety of stromal cells, including fibroblasts, myofibroblasts, endothelial cells, parasites and leukocytes carcinoma associated fibroblasts rich in myofibroblasts, a hallmark of activated fibroblasts present within the tumor.
Stromer play a major role in driving tumor progression. These can be generated in vitro by first isolating primary human mammary fibroblasts from healthy breast tissue obtained by reduction mammoplasty. The obtained tissue is dissociated into a single cell suspension, then a mortalized by viral transduction and engineered to express GFP and a puram mycin resistance gene.
The cells are then co injected with human breast carcinoma. MCF seven RAs cells subcutaneously into an immunodeficient mouse. The injected human memory fibroblasts will evolve into tumor promoting calf myofibroblasts.
Then the parental humor mammary fibroblasts are extracted from tumor xenografts and cultured in the presence of pur mycin. The resulting pur mycin resistant cells called experimentally generated calfs or exp calfs immunohistochemical analysis reveals that initially present normal human memory fibroblasts evolve into calf myofibroblasts within the tumor xenograft that have acquired the ability to promote tumorgenesis. The main advantage of this technique by existing method like extraction with primary cals from breast cancer patient is that we are eligible to experimentally generate calves from human breast fibroblast using co implantation tumor.
General model demonstrating procedure will be Kar a postdoctoral fellow in my laboratory and Ahmed Kar, a PhD student in the lab. Begin with one gram of healthy human breast tissue dissected from a reduction mammoplasty. Wash the tissue several times in PBS, then place the tissue into a 15 centimeter petro dish and use a sterile razor blade to mince it into small fragments less than 1.5 millimeter cubed in size.
Next, use the razor blade to transfer the tissue fragments into a 15 milliliter conical tube containing 10 milliliters of cell dissociation buffer for every half gram of tissue. Then vortex for one minute at maximum speed. Place the tube on a rocker in a 37 degree Celsius incubator and allow the tissue fragments to digest for 12 to 18 hours.
The next day there will still be many pieces of undigested tissue fragments remaining in the tube vortex. First at maximum speed, place the tube on the benchtop and incubate for five minutes at room temperature without agitation. Following the incubation, use a five milliliter physiological pipette to transfer the resulting stromal cell enriched SUP natant into a new conical tube.
Then centrifuge the stromal fraction for five minutes at 250 times gravity. After spinning, re suspend the cell pelus in PBS, then centrifuge your gain for five minutes at 250 times gravity. Then resuspend the pellet in DMEM supplemented with 10%FCS and transfer them to a 15 centimeter petri dish.
Incubate the cells at 37 degrees Celsius and 5%carbon dioxide. After eight to 10 days, the cell should be confluent. At this point, the cells are harvested and can be frozen for long-term storage.
To immortalize the primary human normal memory fibroblasts isolated, introduce a retroviral P MIG vector expressing both H turt and GFP culture, the cells for four to five days and then sort the GFP positive cells using flow cytometry. Introduce a retroviral P babe puro construct encoding a pure mycin resistance gene into the GFP labeled immortalized fibroblasts culture. The cells for five to seven days in the presence of pure mycin at a final concentration of one microgram per milliliter.
To isolate the GFP labeled PUR mycin resistant immortalized human memory fibroblasts. Next to examine whether the fibroblasts produce active viruses, which can lead to the horizontal transfer of genes encoding GFP and pur mycin resistance. Grow 2.5 times 10 to the five pur mycin resistant immortalized human memory fibroblasts in a six centimeter Petri dish for two days.
Collect and filter the fibroblasts conditioned medium with a pore size 0.45 micron syringe filter. Then add on to 10 t and a half mouse fibroblast cells and culture for 12 hours in the presence of five micrograms per milliliter protein sulfate, which increases the efficiency of the virus infection after 12 hours have passed. Change the medium to DMEM supplemented with 10%FCS and culture for an additional two days.
Examine the cells by fluorescence microscopy. 10 T and a half cells infected by GFP virus will be GFP positive. Now change the medium to treat the cells with one microgram per milliliter.
Pur mycin for five to seven days. Again, observe the cells by fluorescence microscopy to determine whether any pur mycin resistant tent and a half cells have formed colonies. Note that the horizontal gene transfer by active viruses produced by the parental pur mycin resistant GFP labeled human mammary fibroblasts might render the surrounding pur mycin sensitive murine cells and carcinoma cells.
Pur mycin resistant and GFP positive within the tumor xenograft. This may hinder the process of isolating the injected GFP labeled pure mycin resistant human mammary fibroblasts from the tumor xenograft to experimentally generate carcinoma associated fibroblasts for injection into an immunodeficient mouse. Begin by trypsin and counting the human mammary cell line just prepared along with the human breast carcinoma cell line MCF seven RAs in new micro refuse tubes.
Reus suspend one times 10 to the six mc seven seven RAs human breast carcinoma cells and three times 10 to the six GFP labeled pur mycin resistant immortalized. Human mammary fibroblasts in 400 microliters of culture media with 50%matrigel per injection. Place the tubes on ice as a control.
Also prepare the same human mammary fibroblasts without the MCF seven RAs cells. Next, using a 27 gauge needle, inject the cell mixture into a subcutaneous site of an immunodeficient nude mouse to examine the intratumoral variation of calfs phenotypes. Isolate calfs and control fibroblasts from a few different tumor xenografts.
Then implant each mixture into right and left lengths of each mouse. Allow the tumor or fibroblasts xenografts to grow for 42 days after injection. The incubation period of the fibroblasts within the tumor xenograft should be optimized based on the myofibroblast phenotype acquired in the parental fibroblasts in tumor.
To extract the calfs or control fibroblasts from the euthanized mouse place at least 0.5 grams of the xenograft tissue and at least 0.1 grams of fibroblasts xenograft tissue in separate dishes. Next, use a sterile razor blade to mince the tissue into small tissue fragments less than 1.5 millimeter cubed. Then use the razor blade to transfer the tissue fragments into a 15 milliliter conical tube containing the cell dissociation buffer and vortex for one minute At maximum speed, incubate the cell suspension for three hours at 37 degrees Celsius with continuous slow agitation centrifuge to the cell suspension.
Dissociated either from tumor or fiberglass sano graft for five minutes at 250 times gravity after the spin. Resuspend the cell pellet in PBS and repeat the centrifugation. Then resuspend the resulting cell palette in DMEM supplemented with 10%FCS to isolate pur mycin resistant parental human mammary fibroblasts culture.
The cells derived from the tumor or fibroblasts raft in a 15 centimeter Petri dish in the presence of one microgram per milliliter. Puram mycin a 37 degrees Celsius and 5%carbon dioxide. This will eliminate any contaminating carcinoma cells and or murine.
Stromal cells propagate the cells until cofluent store the cells at minus 80 degrees Celsius using freezing medium. Note that the resulting pur mycin resistant cells designated 42 day old XP calf one or control fibroblast one cells are immortal and positive for GFP Human memory fibroblasts increase their tumor promoting myofibroblastic phenotype within the tumor xenograft by interacting with carcinoma cells during tumor progression. Mix one times 10 to the six MCF seven RAs cells and three times 10 to the 6 42 day old P calf.
One cells in 400 microliters of culture medium with 50%matrigel power injection in a 1.5 milliliter eend orph tube. Also prepare three times 10 to the 6 42 day old control fibroblasts one cells without carcinoma cells in an einor tube to further boost tumor promoting myofibroblastic phenotypes of exp calf one cells inject a mixture of these cells with CF seven RAs cells subcutaneously into a nude mouse. Also inject control fibroblasts one cells alone without cancer cells to prepare the control cells if required.
Incubate exp calf one cells for additional periods of 200 days within the tumor xenograft to generate exp C two cells to enhance the tumor promoting myofibroblastic phenotype. Also generate control. Fibroblasts two cells as a control against exp calf two cells by injecting control fibroblasts one cells without carcinoma cells into a mouse, remove tumors and place them into tissue culture dishes.
Dissect and dissociate the tumor or fibroblast seno graft into a single cell suspension and culture. The cells in a 15 centimeter petro dish with 10%F-C-S-D-M-E-M in the presence of pur mycin propagate the cells until they're complement trypsin eyes and store the cells at minus 80 degrees Celsius using freezing medium. The resulting pur mycin resistant cells are named 242 day old exp calf two cells or control fibroblasts two cells GFP labeled exp calf two and control fibroblasts two cells extracted from breast tumor xenografts were analyzed immunohisto chemically as shown in this image, the cells stain strongly positive for mesenchymal markers including human specific fermenting prolo four hydroxylase collagen one A fibronectin S 100, A four, and fibroblasts surface protein.
These results indicate a human origin and mesenchymal nature of these cells. In contrast, cytokeratin a marker for epithelial cells was not stained in these GFP positive fibroblasts taken together. These findings suggest that EXP C two and control fibroblasts two cells are originated from the parental human mammary fibroblasts initially introduced to mouse xenografts.
Importantly, far increased numbers of XP calf two cells stained positive for alpha SMA and extracellular matrix glycoprotein to nasin C, both of which are markers of myofibroblasts compared to control fibroblasts two cells. Note that a higher proportion of alpha smma positive myofibroblasts is present in exp calf two cells compared to that observed in 42 day old exp calf one cells. These data demonstrate that resident human memory fibroblasts progressively involve into calf myofibroblasts within tumor xenografts during tumor progression After its development.
The complementation tumor xenograft model paved the way for researchers working the tumor microenvironment to study the processes of evolution of normal fibroblast into cancer. So say fibroblast.
