Method Article

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

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DOI:

10.3791/68367

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August 1st, 2025

 ,  ,  ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of Molecular Biotechnology and Health Science, University of Torino, 2Department of Science and Technological Innovation, University of Piemonte Orientale, 3Division of Urology, Department of Surgical Sciences, University of Turin, 4Department of Surgical Sciences and Urology, Città della Salute e della Scienza di Torino, University of Turin, 5Department of Pathology, University of Turin, and AOU Città della Salute e della Scienza

Corresponding Authors: Valeria Poli <valeria.poli@unito.it>

In This Article

Summary

This protocol provides a comprehensive procedure to isolate, expand, and immortalize fibroblasts from radical prostatectomies. Moreover, it describes assays developed to assess the functional effects of fibroblast-tumor cell crosstalk, taking advantage of both treatment with conditioned medium and co-culturing.

Abstract

Tumor stroma can be actively shaped by tumor cells to adopt pro-tumorigenic properties, making it a focal point of cancer research. Among the stromal components, cancer-associated fibroblasts (CAFs) -- often the most abundant cell type in the tumor microenvironment (TME) -- play a pivotal role in promoting cancer progression through their interactions with tumor cells and other stromal components. Elucidating the mechanisms behind these interactions requires robust methods for isolating and characterizing CAFs, which can also lay the foundation for developing novel CAF-targeted cancer therapies. This study presents a method for isolating fibroblasts from both the tumor and the adjacent normal regions of radical prostatectomy specimens obtained from prostate cancer (PCa) patients. This approach enables the concurrent isolation of CAFs and their normal counterparts (NFs) from the same individual, providing a paired experimental system. Detailed protocols are provided for the maintenance of primary CAFs and NFs for downstream in vitro analyses, as well as for their immortalization to facilitate long-term studies. We also describe functional assays designed to compare the effects of CAFs and NFs on cancer cell behaviors, including proliferation, migration, and anchorage-independent growth. Two complementary approaches are detailed: treatment of cancer cells with fibroblast-derived conditioned media (CM), and direct co-culture of fibroblasts with tumor cells. Together, these methodologies represent a comprehensive toolkit for investigating the dynamic interplay between CAFs and tumor cells, not only in PCa but potentially across a range of human cancers. Moreover, molecular characterization of these interactions may reveal key mediators of CAF-driven oncogenesis, offering promising targets for therapeutic intervention.

Introduction

Prostate cancer (PCa) is the most frequently diagnosed cancer in men across nearly two-thirds of countries worldwide. In 2022, approximately 1.5 million new cases were reported, resulting in 3,97,000 deaths globally. These figures make PCa the second most common cancer and the fifth leading cause of cancer-related mortality among men1.

PCa is a multifocal and biologically complex disease, marked by substantial intertumoral heterogeneity2,3,4, diverse molecular subtypes, and variable clinical outcomes5,6, all of which significantly influence prognosis and therapeutic response7.

While the critical role of the tumor microenvironment (TME) in prostate tumor initiation and progression is well established8,9, the molecular interactions between tumor cells and the surrounding stromal compartment remain incompletely understood. While it is well established that the prostate tumor microenvironment (TME) plays a critical role in tumor initiation and progression, the molecular interactions between tumor cells and the surrounding stroma remain insufficiently defined. The TME comprises a variety of stromal cells, including those of mesenchymal and immune origin, which become activated in response to tumor-derived signals and subsequently acquire tumor-promoting functions10,11. Among these, cancer-associated fibroblasts (CAFs) often represent the most abundant stromal population. CAFs contribute to extracellular matrix (ECM) remodeling, promote tumor growth, and facilitate cancer cell invasion and migration4,11,12,13. Through secreted factors and direct cell-cell interactions, CAFs also influence proliferation, invasion, and therapy resistance. Notably, CAFs are a heterogeneous population. While generally associated with pro-tumorigenic functions, some subtypes may exert tumor-suppressive roles14,15. Importantly, the absence of unique markers for CAFs poses challenges for their precise identification and the functional dissection of their diverse roles12,16.

A robust approach to investigate the functional crosstalk between CAFs and tumor cells involves isolating primary CAFs and matched normal fibroblasts (NFs), then assessing their respective influence on tumor cell behaviors such as proliferation, migration, colony formation, and anchorage-independent growth17,18,19. This article describes a refined, highly reproducible protocol for isolating CAFs and NFs from radical prostatectomy specimens of high-risk PCa patients. A critical component of this protocol is the accurate identification and dissection of tumor and tumor-free regions by an expert uropathologist, allowing the derivation of CAFs and NFs from the same patient. This paired-sample approach helps control for intertumoral heterogeneity and individual patient-specific variables that can influence fibroblast phenotypes20,21,22. To ensure sufficient tumor tissue is available, we focus on specimens from patients with a Gleason score ≥7.

Reliable phenotypic characterization of the isolated fibroblasts requires the use of multiple markers to distinguish them from other cell types, particularly epithelial cells, which may share overlapping marker expression16. As primary PCa fibroblasts typically undergo senescence after 10-15 passages, complicating long-term analyses, we also present a protocol for cell immortalization via stable expression of the human telomerase reverse transcriptase (hTERT)23.

To explore the functional effects of CAFs and NFs on tumor cells, we tested and optimized several experimental strategies. Given that CAFs exert their influence through both secreted factors and direct cell-cell contact24, two complementary assay systems are developed: (1) treatment of cancer cells with fibroblast-conditioned medium (CM), and (2) direct co-culture of fibroblasts and tumor cells. These assays evaluate key cancer cell properties, including proliferation, anchorage-independent growth, and migration. To standardize the influence of secreted factors, conditioned media from CAFs and NFs were concentrated and quantified (conCM) based on total protein content.

Demonstrating significant functional effects through these assays can support the identification of critical mediators of CAF-driven tumorigenesis, offering potential targets for therapeutic intervention.

Protocol

This study was conducted in full compliance with Institutional guidelines for the use of clinical samples, as approved by the Bioethical Committee of the Città della Salute Molinette Hospital in Turin, Italy. All samples were obtained following the signing of informed consent by participating patients. The following section provides a detailed, step-by-step protocol for the isolation of cancer-associated fibroblasts (CAFs) and their matched normal fibroblast (NF) counterparts. The protocol includes dissection of the tumor and adjacent non-tumorous areas, tissue processing, cell culture, and both phenotypic and functional characterization of the isolated cells. All procedures must be performed under sterile conditions within a certified biosafety cabinet. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 using complete Dulbecco's Modified Eagle Medium (cDMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin, unless stated otherwise. All centrifugation steps are carried out with the brake engaged. Refer to the Table of Materials for a comprehensive list of reagents, materials, and equipment used throughout the protocol. This methodology has been optimized for high-risk prostate cancer specimens, with a Gleason score of 4+3 or higher.

1. Surgical sample dissection

NOTE: This procedure was performed on surgical specimens collected from radical prostatectomy procedures25 performed on patients with high-grade prostate cancer (PCa), and managed following standardized protocols, based on internal institutional guidelines and established international literature and best practices26,27,28.

  1. Place the prostate gland under a biosafety hood on a sterile drape, and orient it according to anatomical landmarks.
    NOTE: This procedure must be performed by an experienced uropathologist.
  2. Using a sterile blade, excise tissue slices from areas identified in the preoperative biopsy report as being neoplastic or free of tumor cells.
  3. Conduct a rapid cryostatic analysis29 on both tissue samples, by preparing 2.5-µm-thick tissue sections and staining them with hematoxylin and eosin (H&E) to verify the presence or absence of tumor cells in the selected regions, in accordance with the diagnostic criteria for prostatic adenocarcinoma of the latest edition of the WHO classification30 (see Figure 1A).
    NOTE: If discrepancies arise, repeat the sampling process until histological confirmation of both neoplastic and non-neoplastic areas is obtained.
  4. Excise approximately 10 mm2 samples from the neoplastic and non-neoplastic regions, place them in 15 mL conical tubes containing 7 mL of ice-cold Tissue Storage buffer, and keep them on ice for subsequent procedures.
    NOTE: Samples can be stored at 4 °C for up to 48 h in this buffer.

2. Fibroblasts isolation

NOTE: All following processing procedures must be performed on ice at 4 °C unless otherwise mentioned. See Figure 1 A for a scheme.

  1. Day 1: Weigh the tissue samples.
    NOTE: 70-100 mg are obtained on average.
  2. Move to 6 cm dishes and perform four sequential washes, twice in 4 mL of ice-cold PBS and twice in 4 mL of ice-cold cDMEM, both supplemented with 200 U/mL Penicillin, 200 µg/mL Streptomycin (2x), using sterile forceps to transfer the tissue from one dish to the next.
  3. Mechanically disrupt the tissue by placing it in a 6 cm plate on ice, with 1 mL of DMEM supplemented with 100 U/mL penicillin-G and 100 µg/mL streptomycin, then mince it into small fragments (<1 mm2) using scissors or blades.
  4. Transfer to a 15 mL conical tube containing 5 mL of ice-cold cDMEM. Centrifuge at 754 x g for 5 min at 4 °C.
  5. Use a 10 mL pipette to carefully remove the supernatant and resuspend the pellet in 5 mL of ice-cold cDMEM. Centrifuge again at 754 x g for 5 min at 4 °C.
  6. Remove supernatant as above and resuspend the pellet in Collagenase II (1mg/mL in DMEM), using 1 mL/100 mg of tissue. Move to a 1.5 mL microtube.
    NOTE: Due to the variability of Collagenase batches, test different batches and ensure that they have enough to perform all predicted isolations.
  7. Cover the tubes with paraffin film and digest samples O/N (8-12 h) at 37 °C with continuous rocking.
    NOTE: Perform the following steps on Day 2.
  8. Transfer the samples into 15 mL conical tubes and add 5 mL of ice-cold cDMEM to the cell suspension to inactivate collagenase, then centrifuge at 754 x g for 5 min at 4 °C.
    NOTE: Collagenase is disposed of by aspirating it into a detergent-containing container.
  9. Aspirate the supernatant using a 10 mL pipette and resuspend the pellet in 1 mL of 0.05% Trypsin/EDTA. Incubate for 5 min at 37 °C, shaking occasionally.
  10. Add 1 mL of DNase I to the samples and mix well. Use a freshly prepared 25 mg/mL solution in PBS.
    NOTE: DNase I treatment at this stage helps obtain the single-cell solution.
  11. Centrifuge at 754 x g for 5 min at 4 °C.
  12. Use a 10 mL pipette to aspirate the supernatant, and resuspend the pellet in 5 mL of cold cDMEM. Centrifuge at 754 x g for 5 min 4 °C.
  13. Resuspend cells in 20% FBS cDMEM. Plate and incubate at 37 °C, 5% CO2, and 95% humidity for at least 3 days. Take care not to move/tilt the dish. For 70-100 mg of tissue, distribute the cell suspension in 2 x 3.5 cm dishes, in 2 mL of medium. Downscale accordingly if necessary.
    NOTE: Less than 50 mg of tissue will result in inefficient cell derivation. The splitting and freezing scheme is shown in Figure 1B.
  14. After 3 days, check cells for morphology/viability and replace 2/3 of the medium to maintain cell-produced growth factors. It will take about 7-10 days for the cells to reach confluency, replacing the medium every other day.
  15. Once confluent, passage the cells into one 10 cm dish in 20% FBS cDMEM.
  16. Replace the medium every other day, using 15% FBS medium twice, then once, 12.5%, then 10%. For FACS analyses, RNA and protein extraction and freezing stocks follow the scheme in Figure 1B.
  17. Split cells when about 100% confluent (about 10 days). Wash 2-3 times with abundant PBS for 5 min each. Add 1% Trypsin/EDTA (2x) and place in the incubator, checking for cell detachment every few minutes.

3. Culturing condition

  1. Passage the cells only when 100% confluent, no more than 1:3.
  2. Freeze aliquots corresponding to 50% of a confluent 10 cm dish in 0.5 mL of ice-cold freezing medium (10% DMSO in FBS). Thaw each aliquot in a 10 cm dish.
    NOTE: The average doubling time is 80-90 h.

4. FACS analysis

NOTE: This protocol ensures reliable characterization of the fibroblast population and identifies potential contamination from epithelial, endothelial, or immune cells. FACS analysis is performed in passage 2, as illustrated in Figure 2A.

  1. Harvest cells and resuspend them in FACS buffer (PBS plus 2% FBS) at a concentration of 106 cells/mL.
  2. Label flow cytometry tubes and add 1 x 105 cells in a 100 µL volume in each of them. For staining with 4 antibodies, prepare 11 tubes to account for both staining and gating, as follows (Table 1)
    NOTE: 1 tube, no antibodies (unstained control), to assess cell autofluorescence, 1 tube with antibody isotype control, to assess aspecific binding, 4 tubes, each with a single antibody, for compensation; compensation beads can replace cells, 4 Fluorescence Minus One (FMO) tubes for gating, each leaving out one of the antibodies, 1 tube with all 4 antibodies (actual sample). The following fluorescently labeled antibodies are used (see Table of Materials): (1) Anti-EpCAM-PE (Epithelial cell marker), (2) Anti-CD31-VioBlue (Endothelial cell marker), (3) Anti-CD45-FITC (Immune cell marker), (4) Anti-CD90-APC (Fibroblast cell marker). Antibodies conjugated to other fluorophores may be used, according to available flow cytometer filters. Perform all steps on ice to preserve cell viability.
  3. Incubate cells with Fragment Crystallizable receptors (FcR) blockers, such as purified anti-CD16/CD32 antibodies or blocking buffer (PBS 1%-2% BSA). Use 1-5 µL FcR Blocking Reagent per 100 µL of cell suspension, and incubate at 4 °C for 10-15 min.
  4. Proceed directly with the staining without washing. Add appropriate antibody dilutions or the corresponding isotype controls to the tubes, as specified in Table 1.
  5. Gently mix and incubate at 4 °C in the dark for 30 min.
  6. Wash with 2 mL of FACS buffer.
  7. Centrifuge for 5 min at 300 x g, at 4 °C.
  8. Discard the supernatant and resuspend cells in 1 mL of fresh FACS buffer.
  9. Add viability dye (propidium iodide) according to manufacturer's instructions (see Table of Materials).
  10. Acquire the samples on a flow cytometer.
  11. Use single-stained compensation controls to set up the compensation matrix. For data analysis, apply the following gating strategy on FACS density plots:
  12. Forward and side scatter gating: plot FSC-A vs. SSC-A and design a region around the main cell population to exclude cellular debris.
  13. For doublet exclusion: plot FSC-A vs. FSC-W (or FSC-H vs. FSC-A) and design a region to exclude clumps of cells, which will show increased width relative to the area (or increased area relative to the height). Select the single cells and perform the same procedure, plotting SSC-A vs. SSC-W (or SSC-H vs. SSC-A) to increase the purity of the single cell's population.
  14. Plot the viability dye (i.e., propidium iodide) vs. SSC-A and select negative live cells.
  15. On live cells, plot CD326 (x-axis) vs. CD31 (y-axis); use the corresponding FMO samples to accurately set gates. FMO allows one to account for the spread of fluorescence into a specific channel caused by the other fluorophores.
    1. Looking at the CD326 FMO sample, draw a quadrant gate that includes all cells in the left quadrants (i.e., negative for CD326). Repeat the same procedure using the CD31 FMO sample to adjust the gate in order to have all the FMO cells in the lower quadrants (i.e., negative for CD31). Use this quadrant to analyze the samples, and select cells in the lower left quadrant (CD31- CD326-).
  16. Repeat the same procedure plotting CD326 (x-axis) vs. CD45 (y-axis), using the CD326 FMO and the CD45 FMO to set the quadrant gate. Use this quadrant to analyze all the samples and select cells in the lower left quadrant (CD45- CD326-).
  17. Plot CD90 vs. SSC-A and use the CD90 FMO to set a region that excludes negative cells and includes only CD90+. Use this region to analyze all the samples, measuring the percentage of CD90+ cells. Representative results are shown in Figure 2A.

5. Fibroblasts immortalization

NOTE: Immortalization of CAF and NF populations is achieved by stable transduction with an hTERT-expressing retroviral vector. Below are the protocols to prepare infective pseudoviral particles and transduce cells.

  1. Retroviral particles production
    1. Day 1, poly-L-lysine coating and cells plating: Coat a 10 cm dish with 6 mL of poly-L-lysine solution (0.1 mg/mL in H2O), incubate 5 min under the TC hood, aspirate, and allow the opened dish to dry under the hood before plating 3 x 106 HEK293T cells.
    2. Day 2: Add 30 µL of cation lipid-mediated transfection reagent to 0.75 mL antibiotic-free lipofection-optimized medium.
    3. In parallel, add 2 µg of pCL-Ampho Retrovirus Packaging Vector and 2 µg of pBABE-puro-hTERT plasmids to another 0.75 mL of antibiotic-free lipofection-optimized medium.
    4. Incubate for 5 min at RT.
    5. Mix the two dilutions in a 15 mL conical tube and incubate for 20 min at RT.
    6. Meanwhile, replace the HEK293T medium with 6 mL of lipofection-optimized medium supplemented with 10% FBS.
    7. Gently distribute the DNA complexes on the dish surface and incubate for 6 h to O/N in a humidified CO2 incubator.
    8. Day 3: Replace the medium with 7 mL of cDMEM and incubate for 48 h.
      NOTE: Starting from this step, work must be performed in a Biosafety level 3 (BSL3) tissue culture facility, following institutional rules.
    9. On day 5, collect the virus-containing medium and filter through a 0.22 µm syringe filter.
    10. Aliquot and store at -80 °C.
  2. Viral transduction
    1. Day 1: Plate low passage fibroblasts at 60% confluence in 2 wells of a 6-well plate.
    2. Day 2: Replace the medium with 1 mL of the previously produced pBABE-puro-hTERT retroviral preparation containing 8 µg/mL of polybrene and incubate for 12-16 h.
    3. Day 3: Replace the virus-containing medium with 2 mL of cDMEM.
    4. Day 4: After 24 h, start two 48 h rounds of puromycin selection (2 µg/mL).
      NOTE: The concentration should be empirically determined for each batch of antibiotic and cell type, and the lowest concentration should be selected to kill all cells within 4 days of selection.
    5. Day 8: Check that the uninfected control cells have all died. Replace the medium with cDMEM and let infected cells grow until confluent before passaging them 1:3. Let cells reach confluency and split again 1:3. At this stage, the cells can exit the BLS3 facility.
      NOTE: Prepare frozen aliquots as soon as possible. To ensure immortalization has occurred, passage the cells 5-6 times and score their morphology and duplication rates, which should not change with passages. Functionally compare immortalized cells to their primary counterparts, using the methods described below.

6. Fibroblasts characterization

  1. Phenotypically analyze the isolated cells for the expression of CAF/NF markers, and functionally characterize for their potential pro-oncogenic activities on tumor cells.
    NOTE: The qPCR oligonucleotides and antibodies optimized to assess marker expression levels at both RNA and protein levels are reported in Table 2 and Table of Materials, respectively. Beware that antibodies against fibroblast activation protein (FAP) are often non-specific. Make sure to include appropriate positive and negative controls31,32. Different methods can be used to assess the crosstalk between fibroblasts and tumor cells. Here, two alternative approaches are described, consisting of either treating tumor cells with conditioned medium (CM) derived from CAFs or NFs, or co-culturing fibroblasts and tumor cells. The efficacy of either approach to enhance the proliferation of tumor cells is described.

7. Preparation and concentration of conditioned medium

NOTE: Conditioned medium is concentrated and accurately dosed after collection to facilitate the comparison between different cells.

  1. Plate the cells at 70% confluence in a 15 cm dish in cDMEM.
  2. The following day, switch to 15 mL of starvation medium (DMEM with no FBS) and incubate for 48 h to produce the CM.
  3. Collect the CM, centrifuge at 300 x g for 5 min at RT, and filter through a 0.22 µm filter to eliminate cell debris and sterilize.
  4. Transfer to concentrating tubes (MWCO 10,000 Da), and centrifuge 20 min at 2000 x g and 4 °C, or according to the manufacturer's instructions, to obtain about 1 mL of the concentrated CM (conCM), i.e. 15 x concentration.
  5. Aliquot the conCM and store at -80 °C.
  6. Quantify the protein content of the collected conCM using a colorimetric protein assay.
    NOTE: The average concentration obtained is 0.5 and 2 µg/µL. Lower concentrations can still be used.

8. Proliferation assay

NOTE: Cell proliferation (using DU145 or LNCaP human PCa cell lines) was assessed using a live cell analysis instrument under two different settings, either treating the tumor cells with conCM, or co-cultivating them with CAFs or NFs. In this case, fluorescent tumor cells must be used. Representative results are shown in Figure 3.

  1. Proliferation assay upon treatment with conCM
    NOTE: This protocol is optimized for DU145 cells; different cells may require adjustments.
    1. Plate tumor cells in cDMEM in a 96-well plate (750 cells/well), considering at least 3 replicates per condition, and incubate O/N.
    2. Replace cDMEM with starvation medium, supplemented or not, with 50 µg/mL of conCM.
    3. Transfer the plate to the live cell analysis instrument chamber within the incubator.
    4. Let warm at 37 °C for 30 min, then start the first scanning.
    5. Use the phase contrast channel, choosing an appropriate objective and number of desired fields/well (10x objective and 5 fields/well are recommended), and standard scan type. Scan every 6 h for 4-5 days (duration must be empirically determined for conditions and cell types).
    6. Perform the analysis according to manufacturer's instructions (see Table of Materials).
    7. Calculate for each well (technical replicates for each condition) the fold change relative to its value at time zero. Generate the corresponding graphs and assess statistical significance using appropriate software.
  2. Proliferation assay upon co-culturing
    1. Co-plate 750 RFP-expressing DU145 cells and CAFs or NFs at a 1:3 ratio in cDMEM in a 96-well plate.
    2. The following day, switch to 2% FBS DMEM. Transfer the plate to the live cell analysis instrument chamber in the incubator and start standard scanning every 6 h for 4 days, using both the phase contrast and orange channel.
    3. Perform proliferation analysis according to manufacturer's instructions (see Table of Materials).
    4. Calculate for each well (technical replicates for each condition) the fold change relative to its value at time zero. Generate the corresponding graphs and assess statistical significance using appropriate software.

9. Wound healing migration assay

NOTE: Here, an automated version of the classical scratch assay33 is presented to measure cell migration, using a live cell analysis instrument.

  1. Plate tumor cells in a 96-well plate suitable for the live cell analysis instrument to obtain a confluent monolayer.
  2. The following day, treat the cells with 10 ug/mL of mitomycin C for 2 h.
    NOTE: This step is essential to avoid cell proliferation during the assay, which would confound the results.
  3. Apply the scratch on the wells following manufacturer's instructions (see Table of Materials).
  4. Wash with 100 µL of PBS to remove detached cells.
  5. Add starvation medium, with 50 mg/mL or without (control).
  6. Place the plate in the live cell analysis chamber in the incubator.
  7. After 30 min, program the recommended wound healing scanning protocol, every 4 h for 1 day or longer, depending on cells and conditions.
  8. Analyze the results according to manufacturer's instructions (see Table of Materials).

10. Anchorage-independent growth by soft agar assay

  1. Plate CAFs or NFs at 70% confluence in 12-well plates, leaving three empty wells as a control.
  2. The following day, prepare a 4x soft agar stock solution (3.6%) by adding 0.45 g of low-melting agar to 12.5 mL PBS in a 50 mL conical tube under sterile conditions.
  3. Dissolve in a microwave oven. Beware of excessive boiling that would cause the solution to concentrate.
  4. Cool down at 37 °C, then prepare a 1x working solution by diluting 1:4 with prewarmed cDMEM.
  5. Aspirate the medium from the 12-well plate and add 500 µL of 1x agar solution to each well.
  6. Let it solidify for 20 min at 4 °C, while storing the rest of the agar solution at 37 °C.
  7. Meanwhile, trypsinize the tumor cells and prepare a cell suspension of 2 x 104 cell/mL.
  8. Mix equal volumes of the cell suspension and the agar 1x solution by pipetting multiple times, and dispense 500 µL (i.e., 5000 cells) on top of the first agar layer in each well. Make sure to prepare at least a 10% excess of cell suspension.
  9. Let the agar solidify by incubating for 20 min at 4 °C.
  10. Add 1 mL of cDMEM to each well and renew it every other day by gently aspirating the medium from the edge of the well and adding the fresh medium to the center, almost dropwise to avoid displacing the soft agar layer.
  11. Incubate until easily visible colonies appear. DU145 cells usually take about 12 days, with slight variations.
  12. Discard the medium and stain colonies by adding 200 µL of nitroblue tetrazolium chloride solution (1 mg/mL in PBS). Incubate overnight at 37 °C in a humidified incubator.
  13. Once the colonies are stained, discard the dye solution and acquire images using a stereomicroscope. Place the plate on the microscope stage and set the magnification to its lowest value (0.8x) to ensure the entire well is visible. Use the coarse focus knob to bring the colonies into sharp focus, then adjust the lighting and mode settings before capturing the images.
  14. Use appropriate software34 to quantify the colony number and occupied area by following the steps provided in Supplementary File 1.
  15. Score the number and size distribution of the colonies (see Figure 4).

Results

CAF and NF pairs from 7 high-grade PCa patients were successfully isolated with this protocol. Patients' age at surgery and Gleason scores are summarized in Table 3. To evaluate the purity of the derived fibroblast populations, passage two primary CAFs and NFs were detached and stained with the indicated fluorescent antibodies or with propidium iodide to assess apoptosis. Representative results from the subsequent FACS analysis are shown in Figure 2A, highlighting the gating strategy and the percentages of cells positive for each marker. The analysis included markers for epithelial cells (EpCAM), endothelial cells (CD31), immune cells (CD45), and activated fibroblasts (CD90). The results confirm the successful isolation of pure fibroblasts, as cells were mostly negative for epithelial, endothelial, or immune cell markers, and showed variable positivity for CD90, which is considered a marker of CAF activation35,36. In many cases, particularly at early passages, CAFs displayed a more spindle-like morphology compared to NFs, as shown in Figure 2B. However, these morphological differences tended to diminish with subsequent passages.

To evaluate the effects of CAFs and NFs on DU145 cell proliferation, proliferation assays were performed using two alternative approaches: either treating PCa cells with conCM from CAF/NF pairs (Figure 3A,B), or co-plating PCa cells and fibroblasts at a 1:3 ratio (Figure 3C,D). The use of fluorescent RFP-DU145 cells, which can specifically be detected by the live cell analysis instrument orange filter, allowed to distinguish between the two cell types. The experiments shown were performed using CAFs and NFs derived from the same patient. Both approaches could demonstrate fibroblasts pro-proliferative activity. However, the differences between CAFs and NFs were evident mostly with the conCM approach, as clearly shown in the graphs (Figure 3B,D). The variable effects of conCM from different CAF/NF pairs are illustrated in Figure 3E.

Anchorage-independent growth is a feature often considered as a proxy for metastatic ability. Among several methods to assess CAFs/NFs effects on anchorage-independent growth of PCa cells, the best results were obtained by co-culturing fibroblasts and tumor cells without direct contact. Fibroblasts were seeded in 12-well plates and covered with a layer of 0.9% soft agar in cDMEM, on top of which tumor cells in 0.45% soft agar cDMEM were layered. Colony formation was monitored over 12 days. Representative images of the DU145 colonies obtained are shown in Figure 4A, along with corresponding graphs analyzing colony numbers of different sizes. Of note, both CAFs and NFs were similarly able to stimulate the colony-forming ability of DU145 cells as compared to the control cells plated without a fibroblast feeder layer. Colonies also tended to grow larger, suggesting increased proliferation. One problem we have occasionally encountered with this assay is the early detachment of the fibroblast layer, possibly due to incorrect soft agar density or temperature when plating. In these cases, the experiment was discontinued (Figure 4B).

CAFs/NFs-induced anchorage-independent growth was also assessed upon treatment with conCM. Briefly, DU145 cells plated in soft agar were layered with 1 mL of 5% FBS DMEM, plus or minus 50 µg/ml of conCM, replacing it every 4 days until completion of the assay. Under these conditions, however, DU145 cells were unable to form good-sized colonies, and no improvement was observed upon conCM treatment, as shown in Figure 4C.

Tissue processing and cell culture workflow diagram; dissection, washing, digestion, centrifugation steps.
Figure 1: Fibroblast isolation and culturing schemes. (A) Stepwise representation of the procedure for dissection and isolation of fibroblasts from PCa patients' samples. The images represent the H&E cryosections identifying the tumor and normal tissue regions to be dissected. (B) Schematic representation of the splitting and freezing scheme. Please click here to view a larger version of this figure.

Flow cytometry analysis diagram showing NF and CAF cell separation using propidium iodide and CD markers.
Figure 2: FACS analysis and representative pictures of isolated fibroblasts. (A) Fibroblasts from patient 11 were derived according to the procedure described in Figure 1A, and cytofluorimetric analysis was performed on passage 2 cells as described in Figure 1B. Representative FACS density plots showing the consecutive gating strategies (red arrows) used to evaluate the expression of the CD45, CD31, EpCAM, and CD90 in the cell population derived from tumor or non-tumor areas (CAFs and NFs, respectively). CD326 = EpCAM, Epithelial cell marker; CD31, Endothelial cell marker; CD45, immune cell marker; CD90, a marker of fibroblast activation. Cells were negative for the first three markers and variably positive for CD90. (B) Representative images of the isolated pair 11 NFs and CAFs during passage 2. Scale bars = 2 µm. Please click here to view a larger version of this figure.

Proliferation assay results, fold change graphs, and CAF/NF conCMs treatment analysis on DU145 cells.
Figure 3: Proliferation assays on DU145 PCa cells. (A,B) Treatment with conCM. 750 DU145 cells were plated in 96-well plates and, the following day, switched to starvation medium containing or not 50 µg/mL of pair 11 CAF/NF conCM as described in the text and placed in a live cell analysis instrument. (A) shows five scanned fields/wells at the indicated times. The relevant proliferation graph, calculated as fold change in cell density for the three conditions, each normalized to its time 0 value, is shown in (B). (C,D) 750 RFP-expressing DU145 cells were plated in 96-well plates in cDMEM, either alone or together with either CAFs or NFs from the same patient at a 1:3 ratio. The following day, cells were switched to 2% FBS DMEM and placed in a live cell analysis instrument. 5 scanned fields of the fluorescent cells/well at the indicated times are shown in (C), while (D) shows the relevant proliferation graph, calculated as fold change in the orange object area, representing the tumor cells, for the three conditions, each normalized to its time 0 value. Variable effects of conCM derived from 7 different patients CAF/NF is represented in (E). Data are shown as mean+/-SEM of triplicate wells. Asterisks indicate significant differences between the three conditions (Two-Way ANOVA on repeated measures with Tukey post-test). *p < 0.05; ***p < 0.001; ****p < 0.0001. Scale bars = 1µm. Please click here to view a larger version of this figure.

Cell colony formation analysis with bar graphs; A: Ctrl, CAF, NF samples; experiment results.
Figure 4: Soft agar assays. (A) Anchorage-independent growth of DU145 cells was assessed by seeding tumor cells in an agar matrix on top of a feeder layer of either CAFs or NFs from the same pair that had been plated the day before, or without feeder cells as control (Ctrl), refreshing the medium with 10% FBS DMEM every other day as described in the text. The graphs represent the mean +/- SEM of the number of colonies, either scoring all colonies ≥8 × 103 µm2, or ≥16 × 103 µm2 in size. (B) shows an experiment as in (A), which had to be discontinued due to the detachment of the feeder layer. See text for details. (C) DU145 cells were seeded in soft agar as described in the text, and treated/or not with conCM derived from CAFs or NFs in 5% FBS DMEM. Medium was not refreshed. Under these conditions, colonies grew significantly less than when using CAFs/NFs feeder cells. Please click here to view a larger version of this figure.

Table 1: Preparation of FACS samples. The table specifies how to prepare flow cytometry tubes for staining with four antibodies, setting up the gating, and designing the protocol for FACS analysis. Please click here to download this Table.

Table 2: Primer sequences for qRT-PCR. The table lists the forward and reverse primer sequences used to assess the expression of known CAF marker genes. Please click here to download this Table.

Table 3: Patients' information. The table reports the ages at radical prostatectomy (RP) and Gleason scores of all patients from whom CAF and NF pairs were derived. Please click here to download this Table.

Supplementary File 1: Image Calibration for soft-agar assay. Step-by-step image analysis procedure to score the colonies and measure the total occupied area. Please click here to download this File.

Discussion

The derivation and analysis of cells from PCa tissue provide a relevant experimental model, particularly considering that available animal models do not fully replicate human prostate anatomy and disease progression37. Chimpanzees' prostate glands are anatomically similar to humans but show a slow, stochastic disease progression, in addition to implying ethical limitations. Dogs differ significantly in prostate structure and pathobiology and develop sporadic, age-related prostate disease. Common laboratory models like rats and mice differ in prostate anatomy, epithelial-stromal ratios, and stem cell marker expression. Additionally, mice are resistant to prostate malignancy induction, while rats require hormonal carcinogens, making these models suboptimal for studying human prostate disease37. In vivo, human-to-mouse xenografts provide a valuable model, particularly for preclinical therapy testing. However, their principal limitation lies in the absence of functional human immune cells and other key components of the TME, such as CAFs, which play a pivotal role in tumor progression (reviewed in Sasaki et al.13).

A useful model for studying functional interactions between PCa cells and CAFs, as well as the role of CAFs in supporting tumor growth and progression, is based on isolating CAFs and their normal counterparts (NFs) from patient tumors, followed by functional characterization of their pro-tumorigenic properties both in vitro and in vivo. Standardized and optimized isolation procedures are crucial for successfully generating patient-derived fibroblasts. The protocol described here ensures high-purity CAF populations suitable for downstream analyses and provides a robust foundation for studying their crosstalk with PCa cells. The multifocality of prostate cancer poses a challenge for collecting purely normal or cancerous tissue. Previous protocols38, which we also tested, employed needle biopsies to obtain prostatic tissue. However, this approach implies unpredictable tumor cell content and does not allow reliable selection of tumoral and normal tissue. Indeed, in our hands this method frequently failed to yield sufficient tissue to reliably isolate fibroblast cells.

The protocol described here, developed in close collaboration with urologists and pathologists, greatly improved cell yields and allowed reliable isolation of either CAF or NF cells from the same prostate sample. The downside of this approach is that it can only be applied to high-grade tumors, as sizable tumor dimensions are required. A significant improvement in cell preparation quality was the introduction of a commercially available tissue storage buffer (see Table of Materials) specifically formulated to prevent degradation or apoptosis by incubating the dissected samples before processing.

Various dissociation methods have been described for isolating fibroblasts from PCa tissue17,37,39,40,41, involving either mechanical disruption combined with enzymatic digestion using different enzymes (e.g., collagenase types I, II, IV17,39,40, alone42 or combined with liberase37 or hyaluronidase43), or purely mechanical dissociation followed by direct plating, sometimes including CD90+ cell sorting41. Although purely mechanical methods better preserve cell surface markers, they often yield low cell numbers, requiring more passages and accelerating senescence. Strand et al.37 demonstrated the superior performance of overnight collagenase II digestion in preserving cell surface markers while maintaining optimal cell viability37, while liberase digestion compromised surface markers critical for flow cytometry. Additionally, Collagenase II's slow digestion kinetics allows more flexibility in the length of digestion44,45. This protocol was based on these observations.

It was also observed that cell survival, adhesion, and proliferation after dissociation were improved when initially plating cells in a medium supplemented with 20% FBS. Retaining approximately one-third of the culture medium during medium changes further supported cell growth, likely due to fibroblast-secreted factors. A gradual reduction in FBS concentration subsequently helped maintain cell viability.

Both CAFs and NFs from PCa tumors proliferated extremely slowly (average doubling time of approximately 80-90 h), poorly tolerated low-density seeding, and easily became stressed and senescent. Cell viability upon thawing frozen aliquots was particularly critical. Using a commercially available fibroblast-specific medium greatly improved the viability and recovery time of primary CAFs and NFs. These specialized media typically contain fibroblast growth factor (FGF)46, along with a low percentage of FBS and undisclosed growth factors. To avoid potential interference with downstream analyses, we gradually transitioned cells to complete DMEM over 2-3 medium changes prior to assays.

Obtaining control NFs from the same patient is essential to account for inter-tumor variability and patient-specific features. However, several of our NF populations appeared to possess or acquire functional CAF features in culture, despite relatively homogeneous patient age and Gleason scores (Table 3). Indeed, NFs displayed variable but sizable positivity for the CD90 antigen, a marker of CAF activation35,36. Moreover, both CAFs and NFs were capable of stimulating proliferation or anchorage-independent growth of human PCa cells, as shown in Figure 3 and Figure 4, although in some pairs, CAFs were more potent (see proliferation assay with conCM, Figure 3B,E). This may be explained by fibroblasts becoming activated under standard culture conditions on tissue culture plastic47. Additionally, fibroblasts isolated from histologically normal areas may be influenced by nearby tumor cells.

The results clearly demonstrate that using conCM or co-culturing yields different outcomes depending on the assay. While both methods stimulated PCa cell proliferation, enhanced colony formation in soft agar was observed only when fibroblasts were used as a feeder laye, despite the lack of direct contact between fibroblasts and tumor cells. This underscores the importance of assessing multiple interaction models when studying CAF/NF-tumor crosstalk and suggests that secretome analysis could identify factors responsible for tumor cell activation. Whenever possible, the conCM approach is recommended, as it is easily standardized and allows direct analysis of secreted factors. In contrast, co-culturing poses the risk of inadvertently activating NFs, potentially masking differences between CAFs and NFs. This may explain the different proliferation results obtained with conCM as compared to co-culturing. Interestingly, Paland et al.18 did not observe enhanced proliferation of PC-3 cells in response to CM or co-culturing with NFs or CAFs. Differences may be attributed to the use of different cell lines (PC-3 vs. DU145) or experimental conditions such as time points or cell quality.

Given the high proliferation rates of PCa cells, we found it useful to perform proliferation assays under reduced FBS conditions. In this study, the best results were achieved using a serum-free medium for conCM assays and 2% FBS for co-culture assays. Optimal conditions should be empirically determined for each cell line. Moreover, concentrating the CM prior to the use of standardized protein input across experiments improves comparability between CAF and NF samples. Another important technical consideration was fibroblast seeding density in soft agar assays, as over-seeding led to early confluency and detachment. Seeding at approximately 70% confluence worked best, although optimal conditions may vary across populations.

Finally, CAFs and NFs immortalization helps overcome their limited proliferative capacity and early senescence. Both hTERT and SV40-mediated immortalization were tested, and hTERT provided the best results. Importantly, immortalized cells maintained similar functional properties compared to their primary counterparts, exhibited stably increased proliferative capacity (estimated doubling time 50-60 h), and avoided senescence, providing a more stable cellular model easily amenable to gene expression manipulation.

This article presents a limited number of functional in vitro assays to characterize CAFs' pro-tumorigenic functions. These cells can also be used for additional assays both in vitro and in vivo, including xenografts generated by co-injecting PCa cells with NFs or CAFs17. Genetic manipulation can further help identify the roles of specific genes in supporting tumor growth, enhancing the number and viability of circulating tumor cells48, promoting metastases, and inducing drug resistance13.

Disclosures

The authors declare no competing interests.

Acknowledgements

This work was supported by the Italian Cancer Research Association (AIRC), IG16930 and IG24851 to V.P; the Italian Ministry of University and Research (MIUR PRIN 2017 and 2022 to V.P.); the Truus and Gerrit van Riemsdijk Foundation, Liechtenstein, donation to V.P.; Piedmont Region (Deflect). A.S. was supported by an Italian Cancer Research Foundation (FIRC) post-doctoral fellowship, and L.A. was supported by Fondazione Umberto Veronesi. Figure 2 was created with BioRender.com.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
12 well plateSarstedt833921
24 well plateSarstedt833922
6 well plateSarstedt833920
96 well plateSarstedt833924
Anti-GAPDH Mouse mAb (6C5)Sigma AldrichCB1001Source: Mouse; Dilution for WB: 1:1000
aSMASigma AldrichA5228 Source: Mouse; Dilution for WB: 1:3000
CaveolinCell Signalling TechnologyCST3267Source: Rabbit; Dilution for WB: 1:1000
CD31-VioBlueMilteny Biotec130-119-980Flow Cytometry Ab, Endothelial cell marker
CD326-PE (EpCAM)Milteny Biotec130113-826Flow Cytometry Ab, Epithelial cell marker
CD45-FITCMilteny Biotec130-110769Flow Cytometry Ab, Pan leukocyte marker
CD90-APCMilteny Biotec130-114-903Flow Cytometry Ab, Fibroblast marker
Collagen VI (COL6A1)Abcamab182744Source: Rabbit; Dilution for WB: 1:1000
Conical tube 15 mLSarstedt62554502
Conical tube 50 mLSarstedt62547254
Cryopure 2 mL microtubesSarstedt72380992
Culture plate 100 mm diameter Corning353003
Culture plate 100 mm diameter Corning353025
Culture plate 60 mm diameter Corning353002
Dimethyl sulfoxide (DMSO)Sigma AldirchD8418Use in a Biological hood
Dulbecco’s Modified Eagle’s Medium (DMEM)Gibco11965092Store at 4 °C, use in a Biological hood
FAPAbcamab53066Source: Rabbit; Dilution for WB: 1:500
FcR Blocking Reagent, humanMilteny Biotec130-059-901Fc Blocker
Fetal Bovine Serum (FBS)Gibco16000044Store at 4 °C, use in a Biological hood
Filtered tips 10 µLSarstedt703010255
Filtered tips 100 µLSarstedt703030255
Filtered tips 1000 µLSarstedt703060255
Filtered tips 20 µLSarstedt703030265
Filtered tips 200 µLSarstedt703031255
Filtropur S Plus 0,20 µm Sarstedt831826102
Forcep2Biol1102412
FSP1/S100A4Cell Signalling TechnologyD9F9D (13018)Source: Rabbit; Dilution for WB: 1:2000
IL6Abclonal technologyA0286Source: Rabbit; Dilution for WB: 1:1000
ImageJNational Inst. of Healthversion 1.53cSoftware for image analysis
incucyte imagelock 96-well plateSartoriousBA-04857
Incucyte® SX5SartoriousSX5Live cell analysis instrument 
Lipofectamine 2000 Transfection ReagentThermoFisher Scientific11668019cation lipid-mediated transfection reagent
MACS® Tissue Storage SolutionMilteny Biotec130-100-008
Microplate reader (600 nm absorbance)PromegaGM3500
Microtubes 1.5 mL Sarstedt7269001
NcadAbcamab18203Source: Rabbit; Dilution for WB: 1:1000
Nitro Blue Tetrazolium Sigma Aldirchn5514
Parafilm Sigma AldirchHS234526B
pBABE-puro-hTERTAddgene1771Store at -20 °C, use in a Biological hood
pCL-AmphoNovus Biological NBP2-29541Store at -20 °C, use in a Biological hood
PDGFRBSanta Cruzsc-432Source: Rabbit; Dilution for WB: 1:1000
Penicillin-Streptomycin (PS, 10,000 μg/mL) Gibco15140122Store at 4 °C, use in a Biological hood
Phase contrast microscope Olympus BX42
Phosphate Buffered Saline (PBS)HomemadeNaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 10 mM;KH2PO4, 1.8 mM. Autoclave and Store at RT
Poly-L-lysine solutionSigma AldirchP8920Store at 4 °C, use in a Biological hood
Puromycin dihydrochlorideSigma AldirchP8833
Secura Semi Micro BalanceSartoriusSECURA125-1S
Serological pipette 10 mL Sarstedt861254001
Serological pipette 2 mL Sarstedt861252001
Serological pipette 25 mL Sarstedt861256001
Serological pipette 5 mL Sarstedt861253001
STAT3CST9139Source: Mouse; Dilution for WB: 1:1000
STAT3 Y705CST9131Source: Rabbit; Dilution for WB: 1:1000
stereomicroscopeLeicaMZ125
Syringe 10 mLPentaferte00202260D12
TermoMixerEppendorph22331
Trypsin-EDTA (0.5%), no phenol redGibco15400054
VimSanta Cruzsc-6260Source: Mouse; Dilution for WB: 1:2000
Vivaspin 20 centrifugal concentrator Sigma AldirchZ614602MWCO 10,000 Da 
Wagner scissors2Biol14070-12

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Prostate Cancer FibroblastsFibroblast IsolationTumor MicroenvironmentConditioned MediaAnchorage-Independent GrowthSoft Agar AssayFibroblast Co-CultureTumor StromaFibroblast Characterization
Isolation, Culture, and Characterization of Prostate Cancer-Associated Fibroblasts
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