Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts

We aim to understand the mechanism by which Cancer-Associated Fibroblast contribute to tumor progression in prostate cancer with the goal to neutralize this crosstalk as a therapeutic strategy. Distinct populations of CAFs with different properties can be assessed in 3D organoids obtained using tumor and stromal cells upon various manipulations. Our protocol allows to reliably generate CAFs for these purposes.

Main challenges are the reliable isolation of tumor and normal cells from the same patients'microenvironments, heterogeneity, absence of specific CAF markers, CAFs'plasticity, and the limited in vitro models recapitulating TME complexity. With similar methods, we characterized the fundamental pro-tumor role of the transcription factor STAT3 and its target genes in murine breast cancer CAFs, demonstrating their efficacy as therapeutic targets. An optimal isolation of fibroblasts from the tumor and the adjacent normal regions of radical prostatectomy specimens obtained from prostate cancer patients using small amount of the tissue.

To begin, weigh the tissue samples on the balance on day one for fibroblast isolation. Using sterile forceps, transfer the tissue into six centimeter dishes. Wash the tissue twice in four milliliters of ice cold PBS and twice in four milliliters of ice cold complete DMEM.

Now, transfer the tissue into a six centimeter plate on ice. Add one milliliter of antibiotic supplemented DMEM to the plate. Then use scissors or blades to mince the tissue into fragments smaller than one square millimeter.

Transfer the minced tissue to a 15 milliliter conical tube containing five milliliters of ice cold complete DMEM. Then centrifuge the suspension at 754 g for five minutes at four degrees Celsius. Using a 10 milliliter pipette, remove the supernatant.

Resuspend the pellet in five milliliters of ice cold complete DMEM and centrifuge again. After removing the supernatant, resuspend the pellet in collagenase II solution. Transfer the suspension into 1.5 milliliter microtubes.

Seal the microtubes with paraffin film. Then place them at 37 degrees Celsius for overnight digestion with continuous rocking for eight to 12 hours. The next day, transfer the digested samples to 15 milliliter conical tubes.

Pipette five milliliters of ice cold complete DMEM to inactivate collagenase. Then centrifuge the suspension at 754 g for five minutes at four degrees Celsius. Resuspend the pellet in one milliliter of 0.05%trypsin-EDTA after pipetting out the supernatant.

Incubate for five minutes at 37 degrees Celsius with occasional shaking. Next, pipette one milliliter of freshly prepared DNase I solution to the samples and mix well. After centrifuging and removing the supernatant, resuspend the pellet in five milliliters of cold complete DMEM and centrifuge again.

Resuspend the resulting cells in complete DMEM supplemented with 20%fetal bovine serum. Plate the cells and incubate at 37 degrees Celsius with 5%carbon dioxide and 95%humidity for at least three days. After three days, examine the cells for morphology and viability.

Once the cells reach confluency, passage them into a 10 centimeter dish containing complete DMEM with 20%fetal bovine serum. Plate cancer-associated fibroblasts or normal fibroblasts at 70%confluency in 12 well plates. On the following day, add 0.45 grams of low melting point agar to 12.5 milliliters of PBS in a 50 milliliter conical tube under sterile conditions.

Dissolve the mixture using a microwave oven. Cool the agar solution down to 37 degrees Celsius. Then dilute in a one to four ratio with pre-warmed complete DMEM to prepare a working solution.

Aspirate the medium from the 12 well plate. Pipette 500 microliters of the working agar solution into each well. Incubate for 20 minutes at four degrees Celsius to allow for agar solidification.

Meanwhile, keep the remaining agar solution at 37 degrees Celsius. Trypsinize the tumor cells and prepare a cell suspension of 20, 000 cells per milliliter. Mix equal volumes of the tumor cell suspension and the agar solution to obtain a final volume of seven milliliters, accounting for three technical replicates per each condition.

Pipette up and down multiple times to ensure even mixing. Then dispense 500 microliters of this mixture containing approximately 5, 000 cells on top of the solidified base layer in each well. After letting the agar solidify for 20 minutes at four degrees Celsius, add one milliliter of complete DMEM to each well.

Change the medium every other day by aspirating gently from the edge of the well and adding fresh medium to the center. Incubate until colonies become easily visible. Once colonies are visible, discard the culture medium.

Then stain the colonies with 200 microliters of Nitro blue tetrazolium chloride solution. Incubate overnight at 37 degrees Celsius in a humidified incubator. The next day, discard the staining solution.

Acquire images of stained colonies using a stereo microscope. Pure fibroblasts were successfully isolated. In many cases, particularly at early passages, cancer-associated fibroblasts displayed a more spindle-like morphology compared to normal fibroblasts.

Quantitative analysis confirmed that proliferation of DU145 cells treated with cancer-associated fibroblast conditioned media was significantly higher over 120 hours than those treated with normal fibroblast conditioned media or left untreated. DU145 cells co-cultured directly with cancer-associated fibroblasts and normal fibroblasts also showed increased proliferation over 96 hours relative to the controls. Corresponding growth curves demonstrated that co-culture with cancer-associated and normal fibroblasts resulted in a similar increase in DU145 proliferation over time.

The effect of conditioned media on DU145 proliferation varied across cancer-associated fibroblast and normal fibroblast pairs with early passage pairs showing significantly stronger effects than later ones. In soft agar colony formation assays, both cancer-associated and normal fibroblasts increased the number and size of DU145 colonies compared to controls, indicating enhanced anchorage-independent growth. Detached fibroblast layers formed due to technical issues prevented colony formation in some replicates.

Conditioned media alone did not promote anchorage-independent colony formation in DU145 cells.