This protocol makes it possible to successfully isolate and culture primary cancer associated fibroblasts from murin breast cancer, to screen novel nanoparticles designed to target the tumor micro environment. Primary CAF represent the most reliable and physiological model for in vitro studies of tumor stroma. In this protocol, we describe how to achieve an optimal yield.
To begin, prepare a digestion mix for tumor dissociation, and soak the three to five millimeter tumor fragments in the solution after tightly closing the tube. Turn the tube upside down, checking that all the pieces of tumor are at the bottom of the tube toward the cap. Then attach the tube to a mechanical dissociater in the proper housing, and run a dissociation program specifically designed for tough tumors.
After the run, detach the tube, and place it upside down to incubate the sample at 37 degrees Celsius for 40 minutes with gentle shaking. Then reattach the tube to the dissociater in the proper housing, and run the dissociation program for tough tumors twice, ensuring no large tissue pieces remain at the end of the procedure. Filter the sample through a 40 micron cell strainer in a 50 milliliter tube.
Then wash the filter with 10 milliliters of RPMI 1640 medium and centrifuge. After centrifugation, resuspend the pellet in PBE buffer, composed of PBS, 0.5%BSA, and two millimolar EDTA, and count the cells with Trypan Blue. Repair the dead cell removal binding buffer by diluting the 20X binding buffer stock solution with sterile double distilled water.
Wash the cells with five milliliters of 1X binding buffer and centrifuge. After centrifugation, resuspend the cell pellet with 0.1 milliliter of dead cell removal microbeads for up to 10 to the seventh cells, and incubate at room temperature for 15 minutes. During the incubation, prepare a magnetic stand under the hood, and hang ferromagnetic separation columns to it, with the tips pointing down.
Equilibrate the columns with 0.5 milliliters of cold binding buffer, and wait until the solution has flowed through. After incubation, add 400 microliters of cold binding buffer to the bead and cell suspension. Load the entire volume onto the column and collect the effluent in a 15 milliliter tube.
Wash the column four times with 0.5 milliliters of cold binding buffer, and collect the total effluent in the same tube. For depletion of non-cancer associated fibroblasts, filter the cell suspension using a 70 micron cell strainer moistened with PBS, and centrifuge the cells. Remove the supernatant and resuspend the pellet in 80 microliters of cold PBE buffer.
Add 20 microliters of non tumor-associated fibroblast depletion cocktail. Mix well, and incubate the sample at 4 degrees Celsius for 15 minutes in the dark. During the incubation with beads, prepare ferromagnetic depletion columns on a magnetic stand.
Equilibrate the columns with two milliliters of cold PBE buffer and wait until the solution has flowed through. After incubation, add 400 microliters of buffer to the bead and cell suspension. Load the entire volume onto the column and collect the effluent in a 15 milliliter tube.
Wash the column twice with two milliliters of PBE. Collect the total effluent into the same tube, and centrifugate the effluent. For the positive selection of cancer-associated fibroblasts, resuspend the pellet in 80 microliters of cold PBE buffer.
Then at 20 microliters of tumor-associated fibroblasts microbeads, and incubate the samples at 4 degrees Celsius for 15 minutes in the dark. During incubation, prepare ferromagnetic separation columns on a magnetic stand and equilibrate them with 0.5 milliliters of cold PBE buffer. After incubation, add 400 microliters of PBE to the bead and cell suspension.
Load the entire volume onto the column, and let the unlabeled cells flow through into a 15 milliliter tube. Wash the column three times with 0.5 milliliters of PBE buffer, and collect the total effluent in the same tube. Remove the column from the magnet and place it in a 1.5 milliliter tube.
Add one milliliter of PBE onto the column, and immediately push the plunger into the column to flush the cells out. After centrifugation, resuspend the cell pellet in an appropriate volume of DMEM, and seed the cells in a tissue culture plate. Check the cell density under a microscope, and place the plate at 37 degrees Celsius, and 5%carbon dioxide to allow the cells to adhere and grow.
The injection of 4T1 luciferase cells into the mammary fat pad of female mice led to the growth of a detectable tumor mass five days after implantation. To find a sacrifice window that is adequate for CAF isolation, an optimal compromise was sought between higher tumor size and bioluminescence imaging on the one hand, and an emerging tumor ulceration and necrosis on the other hand. Day 20 was set as the time point to optimize cell recovery, after the isolation process.
Two more passages were needed to isolate the population of CAFs, from the panel of collected viable cells, the depletion of non tumor associated fibroblasts, and the enrichment of tumor associated fibroblasts. CAF cells revealed large spindle shaped morphology, typical of fibroblasts, and were different from 4T1 tumor cells. FAP expression followed over five passages confirm that primary CAF culture maintained its original characteristics.
When duration and temperature of the incubations with microbeads were not maintained, clones with different morphology were found among the isolated culture. These contaminant cells grew faster and prevailed over the primary CAF culture. CAFs binding with Human Ferritin Hfn-FAP functionalized nano drug increased at one-to-one, and one-to-five antibody fragment concentrations by threefold, compared to bare HFN.
On the contrary, for tumor 4T1 cells, bare HFN showed higher binding than functionalized HFN. To increase the yield impurity of isolated CAF, keep buffers cold, respect incubation time with beads, and pull up to four tumors per column before the final enrichment step. The isolated CAFs can be used to screen several candidate nano drugs in term of binding, uptake, and pharmacological efficacy before proceeding with preclinical in vivo experiments.
CAF isolation allowed us to test if nanoparticles can be used to target the tumor microenvironment. Thus paving the way to new targeted therapies working in synergy with chemotherapy.
