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Method Article

Cancer-Associated Fibroblasts from Mouse Mammary Tumors as Tools for Molecular and Computational Studies

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DOI:

10.3791/68364

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July 3rd, 2025

 ,  ,  ,  ,  , 
1Department of Molecular Biotechnology and Health Science, University of Torino, 2Human Technopole, 3Department of Science and Technological Innovation, University of Piemonte Orientale

* These authors contributed equally

In This Article

Summary

This protocol outlines methods to culture, manipulate, and analyze murine CAFs, including in vitro/in vivo functional assays, transcriptomics, and computational analysis. Moreover, it describes a web-based platform developed to analyze the publicly available transcriptomes of laser capture micro-dissected stromal and epithelial components from a collection of human breast cancer tumors.

Abstract

A compelling body of research has confirmed the critical role of the tumor microenvironment (TME) in the growth and aggressiveness of breast cancer (BC). Cancer-associated fibroblasts (CAF), the predominant cell component of the TME, significantly influences its behavior through complex crosstalk with cancer and other stromal cells in response to tumor-derived activating signals. Murine models are extensively used to investigate the mechanisms underlying CAF's role in BC progression, as they allow for the evaluation of the endogenous TME and the assessment of immune system involvement using syngeneic xenograft studies. Here, we present a set of methods for culturing BC-derived CAFs, manipulating gene expression, and evaluating their pro-tumorigenic functions using both in vitro and in vivo approaches. We also describe protocols for CAF gene expression profiling and transcriptomic analysis, alongside a computational procedure for inferring transcriptional signatures in primary tumors using publicly available bulk RNA-seq data. Additionally, we introduce a user-friendly, web-based tool for analyzing the transcriptomes of stromal and epithelial components from laser capture micro-dissected BC tumors. Ultimately, these methods can provide valuable insights into the molecular mechanisms driving CAF-tumor interactions and hold potential for identifying novel therapeutic targets.

Introduction

Breast cancer (BC) is the most common malignancy affecting women and is characterized by significant molecular heterogeneity, with distinct molecular profiles guiding diagnosis, treatment decisions, and prognosis. The tumor microenvironment (TME) is also highly heterogeneous and plays a critical role in tumor progression, immunity, and treatment outcomes.

Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the TME and key regulators of tumor development. They contribute to extracellular matrix deposition and remodeling, angiogenesis, immune evasion, and drug resistance1,2,3,4,5,6. CAFs influence tumor cells through direct cell-cell contact, the release of extracellular vesicles7, and the secretion of various soluble factors2,5 to promote tumor growth, invasion, and therapy resistance8,9,10. Indeed, stroma-related gene signatures have predictive value for BC progression and therapy response and are associated with resistance to chemotherapy11,12,13,14. However, despite substantial evidence suggesting CAFs as an attractive therapeutic target, clinical trials targeting these cells have largely failed15. One major challenge is the limited understanding of the molecular mechanisms by which CAFs modulate tumor behavior. Recent advances in single-cell and spatial transcriptomic analyses have provided deeper insights into the complexity of CAFs in BC16,17,18,19,20,21, enabling the identification of distinct CAF subtypes with specific functional programs16,22 and further highlighting their complexity and plasticity23.

Genetically engineered mouse models (GEMMs), in which oncogenes or tumor suppressor genes are over-expressed or inactivated, are well-established models for studying human cancer24,25,26,27. Compared to syngeneic models, which involve grafting tumor cells in wild-type mice23,28, GEMMs more accurately mimic the multistep progression of BC and the interactions between cancer cells and the TME. Manipulating CAFs' activities in GEMMs has, however, been challenging due to their heterogeneity, which makes it difficult to target all CAF subtypes without off-target effects on other stromal or epithelial components5. Combining GEMMs with complementary approaches, such as in vitro CAF manipulation, can help generate cellular models for loss-of-function experiments in both in vitro and in vivo settings. However, developing stable cellular models to explore the functional roles of specific genes remains challenging due to the rapid onset of senescence.

Here, we describe a robust platform for stable loss-of-function experiments in CAFs, followed by functional in vitro and in vivo analysis and transcriptional profiling of the engineered cells; this approach offers a comprehensive strategy for dissecting the functional relevance of CAF-expressed genes in breast cancer, with advantages in specificity, reproducibility, and adaptability over existing methods. This approach is also complemented by a novel web-based tool for exploring transcriptomes of laser-capture microdissected stromal and epithelial compartments from human breast tumors, enabling cross-species comparisons and facilitating hypothesis generation from human data.

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Protocol

This study complies with all institutional guidelines regarding mouse handling and tissue sampling. All procedures were performed in accordance with national and international laws and policies. Mice were housed in a specific-pathogen-free transgenic unit under a 12 h light/dark cycle at 22 °C with 50% humidity and provided food and water ad libitum. Surgical procedures must follow institutional veterinary instructions to ensure appropriate anesthesia and analgesia, both during the procedure and in the postoperative period. The Table of Materials provides details on materials, reagents, and instruments used in this protocol.

1. Cell culture

  1. Culture cells at 37 °C in a 5% CO2 atmosphere with 95% relative humidity, in DMEM (HEK293T, CAFs, and immortalized CAFs (iCAFs)) or RPMI medium (4T1), supplemented with 100 U/mL Penicillin, 100 µg/mL Streptomycin (1% Pen/Strepto).
    NOTE: Complete media (cDMEM, cRPMI) composition: 10% heat-inactivated FBS, starvation media (sDMEM, sRPMI) composition: no FBS. Average doubling times for cells: HEK293T and iCAFs are 24 h; 4T1 cells are 12h.
  2. Passage cells when reaching 80% confluency, at no more than a 1:5 ratio, never more than 10 times after thawing to avoid phenotypic drifting, as described below.
    1. Aspirate the medium and gently add 7 mL of PBS to the side of a 100 mm dish. Aspirate the PBS, add 1 mL of trypsin-EDTA. Incubate for 2-3 min at 37 °C.
      CAUTION: EDTA is listed as a category 2 hazard compound for eye irritation. Handle in a biological/fume hood wearing proper PPE.
    2. Inactivate trypsin-EDTA with 5 mL of cDMEM, detach cells by pipetting up and down, and collect into a conical 15 mL tube. Centrifuge at 290 x g for 5 min.
    3. Carefully remove supernatant and resuspend the cell pellet in 5 mL of cDMEM. For a 1:5 splitting, seed 1 mL of resuspended cells in a 100 mm dish containing 9 mL of cDMEM.
  3. Cell counting: Load 10 µL of the cell suspension from step 1.2.3 on the side of a Neubauer counting chamber, count the cells in the big squares, and multiply the number obtained by 1 x 104 to calculate the number of cells contained in 1 mL of cell suspension.
  4. Cryopreservation: Freeze 50% of a 100 mm dish (around 3 x 106 cells) in 0.5 mL of ice-cold freezing medium (10% DMSO in heat-inactivated FBS) as described below.
    1. From a 100 mm dish, trypsinize the cells as described in steps 1.2.1 and 1.2.2, except that cells are resuspended in 1 mL of ice-cold freezing medium.
    2. Dispense 0.5 mL in each cryovial, and place at -80 °C in a container ensuring a cooling rate of about 1 °C/min. Each aliquot can be thawed in a 100 mm dish.
  5. Mycoplasma contamination test: Collect 1 mL of growing medium of confluent cells cultured for at least 3 days. Boil at 95 °C for 20 min. Spin at 16200 x g for 5 min at room temperature (RT). Add 5 µL of supernatant to 45 µL of PCR reaction mix (0.15 mM dNTPs, 1.5 mM MgCl2, 1x Reaction Buffer, 1 U Taq polymerase, 0.5 µM primers A and B, see Table 1). Perform PCR, including positive and negative controls, and separate PCR products on a 1% Agarose gel along with a DNA ladder. Mycoplasma-contaminated samples will show a 600 bp band.

2. CAFs' isolation from mammary tumors

NOTE: This step is optimized for advanced-stage tumors of 5-10 mm diameter (see reference29 ).

  1. Sacrifice the mouse by cervical dislocation. Isolate mammary tumors of at least 125 mm3. Excise the tumor carefully, removing the surrounding skin, mammary tissue, and visible necrotic areas. Using tweezers, collect in a 1.5 mL microtube placed on ice.
  2. Weigh the tumor with a precision balance, excise a cubic piece of 0.1-0.3 g with a scalpel, transfer to a new 1.5 mL microtube filled with ice-cold PBS, and keep on ice. Ensure that the tissue is submerged to avoid dehydration.
  3. Transfer to a 60 mm diameter dish, on ice. Finely chop in 1 mL of PBS with a scalpel until obtaining pieces of approximately 1-2 mm.
  4. Transfer to a 15 mL conical tube containing 5 mL per 0.1 g of tumor in cDMEM plus 10 µg/mL collagenase. Rinse the dish with 1 mL of the same medium, using a P1000 pipette with a cut tip to avoid cell damage. Secure the cap with a sealing film to avoid leakage.
  5. Incubate horizontally for 1 h at 37 °C on an orbital shaker. Place a 70 µm cell strainer on top of a 50 mL conical tube and transfer the homogenate with a 10 mL serological pipette. Use the back of a 5 mL syringe plunger to grind and push the sample through the filter.
  6. Use cDMEM (not exceeding 30 mL final volume) to recover everything from the strainer. Spin cells at 129 x g at 4 °C for 10 min and discard supernatant.
  7. Add 3 mL of Ammonium-Chloride-Potassium (ACK) lysis buffer to remove red blood cells (RBC). Close the tubes tightly and mix by inversion. Incubate for 5 min at RT on an orbital shaker.
  8. Stop RBC lysis by adding PBS to a final volume of 30 mL. Spin cells at 129 x g at 4 °C for 10 min and discard supernatant.
  9. Resuspend the cell pellet in 1 mL of cDMEM and count viable cells as in step 1.3 using 10 µL of methylene blue: living cells reduce the dye to a colorless product and become unstained, whereas dead cells are stained blue.
  10. Seed 5 x 105 cells/60 mm dish in 5 mL of cDMEM, incubate 20 min at 37 °C in a 5% CO2 atmosphere, collect nonadherent cells, and re-plate them overnight.
  11. After 48 h or 72 h, perform differential trypsinization. Since fibroblasts detach faster than epithelial and myoepithelial cells30, proceed as described in step 1.2. but closely monitor cell detachment under a microscope. Fibroblast-shaped cells will become round and detach while epithelial cells remain attached.
  12. Add cDMEM to inactivate trypsin and collect detached CAFs, transferring them to a new 60 mm dish in 5 mL of cDMEM. Passage primary CAFs 1:3 (see step 1.2) for three passages, then prepare early passage frozen aliquots as described in step 1.4.

3. Immortalization by viral transduction

NOTE: Some of the following steps must be performed in an environment suitable for handling retroviral vectors (e.g., BSL2+); check your institutional rules.

  1. Preparation of the recombinant virus
    1. Culture HEK293T cells and check for mycoplasma contamination (see step 1).
    2. Coat a 100 mm diameter dish by pipetting 6 mL of a 0.1 mg/mL solution of poly-L-lysine in ddH2O for 5 min at RT, aspirate, and allow to dry under the hood (around 10 min).
    3. Seed 3 x 106 HEK293T cells (low passage, mycoplasma negative) in 10 mL of cDMEM in the poly-L-lysine coated dish from step 3.1.2 (see step 1.3).
    4. The day after, prepare two tubes containing 0.75 mL of antibiotic-free transfection-optimized medium. To one of them, add 30 µL of cation lipid-mediated transfection reagent (T1). To the second, add 2 µg of pCL-Ampho (retroviral packaging vector) and 2 µg of retroviral pBABE-neo largeT cDNA vector (T2). Add T2 to T1, mix gently, and incubate for 10 min.
    5. Evenly distribute the medium over the cells after replacing it with antibiotic-free transfection-optimized medium + 10% heat-inactivated FBS. Move the plate to a Biosafety level 2+ (BSL-2+) tissue culture facility. Replace the medium with cDMEM after 6-12 h and incubate the cells for 24-48 h.
    6. Filter the medium containing the pseudoviral particles through a 0.45 µm syringe filter and store at 4 °C for a few days or -80 °C for long-term storage.
  2. CAF's viral transduction
    1. Seed 7.5 x 105 CAFs in each of two 60 mm dishes in 4 mL of cDMEM (see step 1.3) and culture them as in step 1.1.
    2. The day after, replace the medium of one dish with 2 mL of the pseudovirus-containing medium from step 3.1.6 (transduced CAFs), add 2 mL of cDMEM, and incubate for 24 h. Leave the other dish untouched (untransduced CAFs).
    3. The day after, repeat step 3.2.2 on the transduced CAFs. After 24 h, replace the medium of both dishes with 4 mL of cDMEM.
    4. After another 24 h, start selection by replacing the medium of both dishes with cDMEM supplemented with 400 µg/mL G418. Replace the G418-containing medium every 2-3 days until no live cells are left in the control dish of untransduced CAFs.
      NOTE: Untransduced cells are sensitive to G418 and will die within 7-10 days, indicating that only G418-resistant transduced cells will have survived in the transduced CAFs condition. Optimal antibiotic concentration must be determined for each cell type, performing kill curves (test a range of 50 to 1000 µg/mL G418 concentration for 7-10 days and select the dose killing all cells within 10 days).
    5. Passage the selected transduced cells 2x at a 1:3 ratio (see step 1.2) before exiting the BSL facility. Prepare cryopreserved aliquots of early passages as in step 1.4.
    6. Verify that cells continue to proliferate without showing a senescent phenotype for several passages and proceed to phenotypic and functional characterization (see steps 4 and 6-9).

4. Phenotypic characterization

NOTE: CAFs markers such as alpha smooth muscle actin (α-SMA), caveolin, fibroblast activating protein (FAP), N-cadherin, platelet derived growth factor beta (PDGFR-β), S100A4 and vimentin29 can be assessed by RT-qPCR and Western Blot.

  1. RT-qPCR: Extract total RNA with a monophasic solution of phenol and guanidine isothiocyanate as described in the reference15. Reverse transcribe 2 µg of total RNA in a volume of 20 µL, following the manufacturer's instructions for reaction conditions. Dilute cDNA 1:20 and use 3 µL to perform the RT-qPCR reactions, following the manufacturer's instructions (see Table 1 for primers).
    NOTE: We recommend preparing the RNA from a 12-well dish.
  2. Western blot
    1. Wash 2x with PBS as described in step 1.2.1, add 0.2 mL of ice-cold lysis buffer, detach cells with a scraper, and collect them in a 1.5 mL microtube as described in29. Leave for 20 min on ice.
    2. Spin the samples at 16,000 x g for 10 min at 4 °C and collect the supernatant. Quantify proteins by colorimetric assay according to the manufacturer's instructions. Use at least 20 µg of protein for the Western blot (see Table 2 for antibodies).
      NOTE: We recommend preparing proteins from no less than 6 wells in a dish.
  3. Analysis of secreted proteins
    1. Seed 2 x 106 iCAFs (see step 1.3) in a 100 mm diameter dish in 10 mL of cDMEM and culture them as described in step 1.1.
    2. After 24 h, remove the medium, wash 2x with 10 mL of PBS, and incubate for 48 h with 10 mL of sDMEM (see step 1.1) to generate conditioned medium (CM).
    3. After another 48 h, filter the CM through a 0.2 µm syringe filter into a 15 mL conical tube. Hold both the syringe and the tube tightly and be prepared for some resistance. CM can be stored at -80 °C for up to 6 months.
    4. Concentrate CM approximately 10x using protein concentrators according to the manufacturer's instructions, to generate concentrated CM (conCM). Quantify the conCM, boil, and perform Western Blot (see Table 2 for antibodies).

5. Gene knockdown analysis

NOTE: To stably interfere with gene expression, we transduce iCAFs with lentiviral particles expressing specifically designed shRNAs. These can be obtained by cloning the targeting sequences in a suitable lentiviral vector such as pLKO.1-Puro, following the protocols available at https://www.addgene.org/protocols/plko/#C. The specific targeting sequences can be selected using publicly available shRNA designing tools (i.e., GPP-Genetic Perturbation Portal by Broad Institute, https://portals.broadinstitute.org/gpp/public/). Alternatively, sets of pre-made targeting vectors are commercially available (https://en.vectorbuilder.com/products-services/service/shrna-knockdown-solutions.html, https://www.sigmaaldrich.com/IT/it/technical-documents/technical-article/genomics/gene-expression-and-silencing/mission-shrna-getting-started, https://www.origene.com/products/rnai/shrna-lentiviral-particles). We recommend designing 4-5 different shRNAs against each gene and selecting the best 2 for experiments. Generate also non-targeting vectors as negative controls. Some of the following steps must be taken in an environment suitable for handling HIV-derivative viruses (e.g., BSL2+). Check your institutional rules.

  1. Generation of lentiviral particles
    1. Seed 3 x 106 HEK293T cells as in steps 3.1.1-3.1.3. The following day, prepare the cation lipid-mediated transfection reagent-DNA complexes as in step 3.1.4, using 9 µg and 3 µg, respectively, of the packaging vectors pCMV-R8.74 and pCMV-VSVG, and 7 µg of the shRNA-containing pLKO.1 construct in 0.75 mL of antibiotic-free transfection-optimized medium, and proceed as described in steps 3.1.5 and 3.1.6.
  2. CAFs transduction
    1. Infect cells as described in steps 3.2.1, 3.2.2 and 3.2.4. After 24 h, remove the infection medium and start selection with 2 µg/mL puromycin in cDMEM. See note in step 3.2.5 for determining optimal concentration.
    2. Replace puromycin/cDMEM after 48 h. Selection is usually complete in 3 days. Expand and freeze cells as in step 3.2.6. Assess knockdown efficiency by both RT-PCR and Western blot as described in steps 4.1. and 4.2.

6. Super-activation of iCAFs with CM from tumor cells

NOTE: The following steps illustrate methods for the in vitro characterization of iCAFs pro-tumoral activities. Pro-tumoral activities of CAFs can be enhanced by incubating them with CM from mouse BC tumor cells (super-activation, s.a.), as described below.

  1. Count 4T1 murine BC cells as described in step 1.3 and seed 2 x 106 cells in a 100 mm diameter dish in 10 mL of cRPMI (see step 1.1).
  2. After 24 h, remove medium, wash 2x with 10 mL of PBS, and incubate for 48 h with 10 mL of sRPMI (see step 1.1) to generate CM.
  3. The day after, seed 2 x 106 iCAFs on a 100 mm diameter dish, in 10 mL of cDMEM (see step 1.3) and culture them as described in step 1.1. Filter the CM through a 0.2 µm syringe filter into a 15 ml conical tube as in step 4.3.3.
  4. Super-activation (s.a.): remove the medium from the iCAFs dish, wash 2x with 10 mL of PBS, and add the filtered 4T1 CM (8-10 mL). Incubate for 48 h to obtain s.a. iCAFs.

7. Tumor cell proliferation

  1. Pre-treat 4T1 cells for 48 h with iCAFs CM, prepared as in steps 4.3.1, 4.3.2, 4.3.3.
  2. Trypsinize and count them, then seed 4 x 103 cells/well of a 96-well plate in 100 µL of cRPMI as described in step 1.3 and culture as in step 1.1. Plate technical triplicate.
    1. Prepare one 96-well plate per day of the time course. The number of cells suggested here is optimized for a 4-day protocol; scale it down for longer times.
  3. After 4 h, or as soon as cells are attached, remove medium, wash with PBS by gently adding 100 µL, taking care not to detach cells by resting the tip against the edge of the well, with a multichannel pipette and using a sterile reservoir. Remove PBS, then similarly add 100 µL of sDMEM (control) or iCAF CM, both supplemented with 2% heat-inactivated FBS (see steps 4.3.1, 4.3.2, 4.3.3).
  4. After 4 h, stop the day 0 plate as described from point 7.5. For the other time points, replace the medium every other day until the end of the experiment.
  5. To assess cell density, remove the 96-well plate from the incubator and wash the wells 2x with 100 µL of PBS, as described in step 7.3.
  6. Under a fume hood, add 100 µL of 4% Paraformaldehyde (PFA) to each well and fix the cells for 10 min at RT.
    CAUTION: PFA can cause serious eye damage and skin irritation; it is harmful if swallowed and inhaled and is suspected of causing genetic defects and cancer. Handle in a fume hood wearing proper Personal Protective Equipment (PPE).
  7. Wash 3x with 100 µL of PBS as in step 7.3 and let dry under the fume hood (about 10 min).
  8. Incubate with 50 µL of 0.1% crystal violet in 20% methanol for 10 min at RT.
    CAUTION: Crystal violet can cause serious eye damage and is reported as a category 4 hazard for oral acute toxicity and a category 2 hazard as a carcinogen. Handle it in a fume hood wearing proper PPE.
  9. Wash 3x with 100 µL of ddH2O, empty the wells and tap the plate on absorbent pad or paper towel to completely remove liquid.
  10. Dissolve crystal violet in 100 µL of 10% acetic acid for 15 min at RT and measure optical density (O.D.) at 600 nm wavelength in a microplate reader, using 100 µL of 10% acetic acid as a blank. Normalize the O.D. for that day to 0 per condition.

8. Migration and invasion

  1. Pipette 700 µL of DMEM supplemented with 1% heat-inactivated FBS, 1% Pen/Strepto, into the wells of a 24-well plate. Place an 8 µm pore transwell insert, coated (invasion) or not (migration) with biologically active matrix, in each well. Leave the plate in the incubator to equilibrate at 37 °C while collecting and counting 4T1 cells as in step 1.3.
  2. Detach 4T1 cells pre-treated for 48 h with iCAFs CM from a 100 mm diameter dish as in steps 4.3.1, 4.3.2, and 4.3.3, using 1 mL of 5 mM EDTA instead of trypsin-EDTA.
  3. Add 5 mL of cDMEM, harvest the cells in a 15 mL conical tube, and pellet by centrifuging as in step 1.2. Resuspend cells in sDMEM, count as in step 1.3, and dilute to 1 x 105 cells/100 µL.
  4. Add 100 µL of the cell suspension to the transwell's upper chamber. As a plating control, seed 100 µL of cells in a 24-well plate, in 500 µL of cDMEM. Incubate for 16 h (migration) or 24 h (invasion), and stop the experiment as described below.
    NOTE: The incubation times must be empirically determined if using different cells.
  5. Label the inserts on their upper side with a thin marker and submerge them in a 50 mL conical tube filled with PBS for washing using tweezers. Carefully clean the internal part of the transwell with a cotton swab.
  6. Under a fume hood, incubate samples in 700 µL of 4% PFA for 10 min at RT (see step 7.6).
  7. Wash 3x with 700 µL of PBS and let dry under the hood (about 10 min). Incubate inserts with 700 µL of 0.1% crystal violet in 20% methanol for 10 min at RT (see step 6.10).
  8. Wash inserts 3x submerging them in a 50 mL conical tube filled with ddH2O using tweezers. Carefully clean the internal part of the transwell with a cotton swab. Store samples at RT and take pictures up to 3 days later.
  9. Using a phase-contrast microscope, take one picture with a 10x objective imaging most of the well surface, then acquire pictures from 5 independent areas with a 20x objective.
  10. Dissolve crystal violet in 10% acetic acid as follows: for wells, add 100 µL of acetic acid and mix on an orbital shaker for 10 min at RT; for transwells, lay them, lower surface up, on a bench, deposit 50 µL of acetic acid on the surface and mix by pipetting until crystal violet is completely dissolved.
  11. Read the absorbance at 600 nm in a 96-well plate using 50 µL of the dissolved material from the above step. Use 10% acetic acid as a blank.
  12. Normalize the optical density (O.D.) of the plating control from step 8.5 and express migration or invasion as relative to the control condition.

9. Primary tumor growth and lung metastases formation

NOTE: The following step illustrates methods for the in vivo characterization of CAFs' pro-tumoral activities. This step is optimized for evaluating CAFs' in vivo pro-tumorigenic features by co-injection in syngeneic mouse models. As both the CAFs and 4T1 cells are from the BALB/c mouse background, BALB/c mice are used.

  1. At 2 days before injection, number 6 weeks old female BALB/c mice with metal ID ear tags and shave the 4th mammary gland region with a shaver.
  2. Collect and count 4T1 cells and s.a. iCAFs, prepared as described in step 6. In a 15 mL conical tube, mix 1 x 105 4T1 cells and 3 x 105 s.a. iCAFs/mouse, considering an excess of at least 20%. Use 4T1 only as a control.
  3. Centrifuge at 290 x g for 5 min, resuspend in a volume of PBS calculated to obtain the above number of cells/mice in 50 µL, transfer to a 1.5 mL microtube, and keep on ice.
    NOTE: For example, to inject 10 mice, mix 1.2 x 106 4T1 cells with 3.6 x 106 s.a. iCAFs, pellet cells, and resuspend in 600 µL of PBS. Inject 50 µL of cell suspension per mouse.
  4. Prepare the working area by cleaning and disinfecting the surface of a laminar flow hood. Cover a foam surgery board with a bench protector sheet.
  5. Anesthetize the mice one at a time, according to institutional procedures (we use intraperitoneal injection of tiletamine base/zolazepam base + xylazine 70 mg/kg + 5 mg/kg). Apply veterinary ointment on the eyes to prevent dryness. Using a cotton tip, disinfect the skin with a povidone-iodine solution.
  6. Use the toe pinch test to ensure that the mice are under proper anesthesia. With a hypodermic needle, inject 50 µL of the cell suspension into the 4th mammary fat pad. Use the nipple as a reference and inject about 3 mm toward the ipsilateral leg.
  7. Inspect the mice for a few hours after the injection and in the following days. The tumors become palpable after 4 days.
  8. Measure tumor size every 48 h with a caliper, determining the maximum and minimum diameters. Calculate the tumor volume using the formula: (length x width2)/2.
  9. After 10 days of injection, surgically remove the primary tumors as described below.
    1. Prepare the surgical area as in step 9.5. Sterilize tools by autoclaving and place them on a sterile surface while working. Sterilize tools between animals using a hot bead sterilizer.
    2. Anesthetize the mice as in steps 9.6 and 9.7. Using a scalpel blade, make a 5-8 mm incision close to the tumor and gently pull the skin away, creating a small pocket to access the tumor. Cauterize any visible blood vessel with a low-temperature cauterizing pen to avoid hemorrhages when dissecting.
    3. Use the scalpel to remove the tumor, trying not to remove any skin. Place the skin flaps back in their original position, suture them together using 5-0 surgical thread with a 17 mm 1/2c needle, and add surgical glue to completely seal the wound.
    4. Do not leave the mice unattended until they have regained sufficient consciousness to maintain sternal recumbency. Animals that underwent surgery are not returned to the company of other animals until they are fully recovered.
  10. Closely monitor the animals after surgery and in the following days, administer analgesic treatment if necessary, according to Institutional protocols. We use subcutaneous injections of Carprofen (3-5 mg/kg).
  11. Weigh the tumor using a precision balance as in step 2.2. Section the tumor in half using the scalpel. Place one half in a 1.5 mL microtube, snap-freeze in liquid nitrogen, and store at -80 °C for molecular analyses. Fix the other half in 5 mL of 4% PFA in a conical 15 mL tube for 24 h at 4 °C for histological analysis.
  12. At day 21 post-injection, sacrifice the mice by CO2 exposure and collect the lungs upon intratracheal infusion with 4% PFA as described below. Work under a fume hood.
    1. Open the chest cavity using surgical scissors. Remove skin from the submandibular area and the salivary glands to clearly expose the trachea.
    2. Prepare a 22G needle syringe containing 4% PFA solution. Insert the needle into the mouse trachea, directing it toward the lungs while clamping the trachea toward the head. Inject the solution until the lungs swell (around 2 mL).
    3. Dissect the lungs and place them into a 15 mL tube containing a 4% PFA solution. Gently invert the tube a few times, and allow it to fix for 24 h at 4 °C.
  13. Perform semi-serial sections of the lungs, and stain with Hematoxylin and Eosin (H&E), one slice every 50/100 µm until the specimen is exhausted, as described in reference31.
  14. Score metastases upon software-guided analysis of microphotographs of the H&E-stained sections.

10. Gene expression signatures

  1. Define a gene signature as described below.
    1. Depending on the experimental setting and the specific biological question investigated, obtain a gene signature in several ways. Most commonly, assess the differential expression between two conditions (e.g., CAFs vs normal counterparts, or treated in several ways (e.g., super-activated or silenced) vs untreated.
    2. Alternatively, identify the most variably expressed or informative genes across the samples analyzed (e.g., genes with high variance or with high loadings on the first components of a principal component analysis). Here, we will use a STAT3-regulated signature as an example. This signature was defined as the genes downregulated upon STAT3 silencing in BC CAFs29, as described below. For a thorough description of RNA sequencing, see references32,33.
  2. Extract total RNA in technical triplicate from each sample as in step 4.1.1 and assess RNA quality to check RNA integrity (RIN ≥ 9).
  3. Prepare libraries from 2 µg of poly(A) selected RNA and sequence them. We used the NextSeq 500 platform.
  4. Map reads to the Mus musculus mm9 reference assembly using TopHat v2.0. 1034.
  5. Compute gene counts using the htseq-count tool from the HTSeq package (http://www.huber.embl.de/HTSeq/doc/overview.html). 
  6. Perform differential expression analysis using R and the DESeq2 package (http://www.bioconductor.org/packages/devel/bioc/html/DESeq2.html). Filter down-regulated genes with FDR < 0.05 to define the STAT3 signature.

11. Estimation of the signature's expression in primary tumors

NOTE: The following steps describe a procedure to computationally estimate the expression of the STAT3-regulated signature29 across publicly available transcriptomic data of BC primary tumors, testing its relationship with the estimated stromal proportion in the same set of samples.

  1. Install R (https://cran.r-project.org/) and RStudio (https://posit.co/products/open-source/rstudio/). Start it and install the required libraries: GSVA35, TCGAbiolinks36.
  2. Download primary tumor gene expression data, such as TCGA data from https://portal.gdc.cancer.gov/, METABRIC data from http://synapse.org/ (syn1757063), or other transcriptomic data from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) as pre-normalized expression matrices. Data must be formatted as a numeric matrix with rows as genes and columns as samples, and normalized depending on the platform and log-scaled using an offset of 1.
    1. Download the list of mouse-human gene orthologs from Biomart at https://www.ensembl.org/biomart/martview/, select the database: ensembl genes, choose the human genes dataset, and click on the attributes selection in the left tab.
    2. From the pre-selected Features, open the EXTERNAL tab and flag the HGNC symbol, select Homologues in the box at the top of the page, and flag Mouse orthologues / Mouse gene name.
    3. Click the Results button at the top left corner, select the CSV format, and click the GO button to download the file.
  3. Load the gene expression signature (a set of genes) in R. In this case, we use the list of genes downregulated upon STAT3 silencing in murine CAFs.
  4. Load the list of mouse-human gene orthologs in R and map the STAT3 signature to the corresponding human orthologs via the Biomart table.
  5. Load primary tumor gene expression data in R and call the GSVA function with parameters as an ssgseaParam object, including primary tumor data, and a list comprising the STAT3 signature.

12. Test the relationship between the gene signature's expression and clinical features

  1. Identify the variables of interest. Stromal percentage represents the proportion of cells in the sequenced tumor sample belonging to tumor stroma. Use the following clinical features often associated with omic data: tumor size, stage, patient's age at diagnosis, lymph node status, metastasis status, etc. Pre-computed stromal percentage estimates are available for TCGA.
    NOTE: Specifically, available stromal estimates are: EDec Immune/Stromal = Epigenomic Deconvolution of stromal/immune percentage37; Immune Score = ssGSEA based on gene expression profiles of 141 immune genes38; LUMP = Leukocytes Unmethylation for Purity, which averages 44 non-methylated immune-specific CpG sites39; Stromal score = ssGSEA based on gene expression profiles of 141 stromal genes38; IHC = estimated by image analysis of haematoxylin and eosin-stained slides produced by the Nationwide Children's Hospital Biospecimen Core Resource40; ABSOLUTE = based on somatic copy-number data41.
  2. Download EDEc data from http://genboree.org/theCommons/documents/569, Stromal and Immune scores from https://bioinformatics.mdanderson.org/estimate/disease.html (RNASeqV2), and Tumor purity data (LUMP, IHC, ABSOLUTE) from TCGAbiolinks. For other datasets, compute the percentage of stroma making use of the GSVA package, computing the ssGSEA for stroma and immune signatures obtained from Yoshihara et al.38.
  3. Compute the correlation between the numeric vector of the estimated stromal proportion and the numeric vector of the STAT3 signature expression across primary tumor samples. This can be achieved with the cor.test function in R.
  4. Download clinical data associated with the primary tumors' transcriptomic dataset as a table with samples in rows and clinical features in columns.
  5. Load the clinical data in R and check that samples in the transcriptomic matrix and in the metadata table are in the same order as the match function.
  6. For numeric variables (e.g., tumor size), compute the correlation between the numeric vector of the STAT3 signature expression across primary tumor samples and the vector of clinical features'values with the cor.test function.
  7. For categorical variables (e.g., metastasis status), test the difference in STAT3 signature expression between two groups of samples (e.g., metastatic vs non-metastatic tumors) with the non-parametric Wilcoxon rank-sum test (assuming that the two groups of samples are independent). This can be achieved with the wilcox.test function in R.

13. Testing the signature's regulation in tumor stroma

NOTE: The expression of a stromal signature in primary tumors analyzed through bulk RNA-sequencing has the advantage of relying on large patient cohorts with detailed clinical annotations. On the other hand, only separating stroma and epithelium allows for defining gene expression changes in either of the two cellular compartments in the tumor when compared to normal tissue42. MetaLCM is a ShinyApp that facilitates the analysis of transcriptomic datasets of laser-capture microdissected breast tumors. Through this tool, the user can search for robust differentially expressed genes between two conditions across transcriptomic datasets of laser-capture microdissected breast tumors and test the regulation of previously defined gene signatures in tumor stroma. The following steps describe how to use MetaLCM.

  1. Access the app at the link https://aurorasavino.shinyapps.io/metalcm/. To start the analysis, select the features of the compared conditions.
  2. Choose the direction for the comparison. By choosing up-regulated genes, the genes with higher expression in condition 1 are shown.
  3. Choose the p-value threshold for differential expression and press Run to run the analysis, and each time the settings are changed.
  4. To restrict the analysis to a pre-defined gene list, either type gene symbols separated by commas or import a CSV file containing gene symbols in one single column.
  5. Press Submit gene list to import gene symbols. Press Reset to restore the analysis using all genes. Save the list of differentially expressed genes: when a gene list is provided, multiple testing correction is performed on those genes only. The table comprises the average logFC, collapsed p-value, and individual logFC and p-values for differential expression in each available dataset.
  6. Plot a heatmap with the fold changes of differentially expressed genes. The heatmap shows only the differentially expressed genes with the completely coherent sign of differential expression across datasets.

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Results

In vitro assessment of CAFs' activities
The contribution of mammary tumor-derived cancer-associated fibroblasts (CAFs) to the aggressive features of breast cancer (BC) was studied using immortalized CAFs (iCAFs) generated according to step 3. iCAFs were super-activated as described in step 6, then starved in sDMEM to collect their CM, which was used to treat 4T1 cells for 48 h (see step 7.1). This treatment was followed by in vitro functional assays.

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Discussion

In this study, we present optimized protocols for the immortalization of murine CAFs and the characterization of their pro-tumorigenic activities both in vitro and in vivo. Rather than selectively isolating specific CAF subtypes based on marker expression, we chose to retain the entire cell population to preserve heterogeneity. The cells were functionally characterized as a whole, and their gene expression profiles were analyzed to capture their diversity. Immortalizing CAFs was crucial for this researc...

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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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1Kb plus DNA ladderThermoFisher Scientific10787018
24 well plateSarstedt833922
96 well plateSarstedt833924
Acetic acidSigma Aldirch695092
AgeI-HFNew England BiolabsR3552Store at -20 °C
BetadineMeda Pharma23907076
Bürker Counting chamber VWR40443703
Cautery Low Temp FineTip DispKeelerAX10339KL
CentrifugeEppendorf5810R
Conical tube 15 mLSarstedt62554502
Conical tube 50 mLSarstedt62547254
Cryopure 2 mL microtubesSarstedt72380992
Crystal violetSigma AldirchC0775Store at RT, use in a Fume hood 
Culture plate 100 mm diameter Sarstedt833902
Culture plate 60 mm diameter Sarstedt833920
ddH20HomemadeStore at RT, use in a Biological hood
Digital CaliperPreciva02-301-2
Digital MicroscopeLeicaDM6B
Dimethyl sulfoxideSigma AldirchD8418Use in a Biological hood
Dulbecco’s Modified Eagle’s Medium (DMEM)Gibco11965092Store at 4 °C, use in a Biological hood
EcoRI-HFNew England BiolabsR3101Store at -20 °C
Ethylenediaminetetraacetic acid (EDTA)Sigma AldirchE6758Store at RT, use in a Biological hood
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
Filtropur S Plus 0,45 µm Sarstedt831826
Fine scissors2Biol1406009
Forcep2Biol1102412
GeneticinGibco10131035Store at 4 °C, use in a Biological hood
Lipofectamine 2000 Transfection ReagentThermoFisher Scientific11668019Store at 4 °C, use in a Biological hood
Matrigel invasion chambers with 8 μm PET membrane Corning354480
Microplate reader (600 nm absorbance)PromegaGM3500
Microtubes 1.5 mL Sarstedt7269001
OptimemGibco31985047Store at 4 °C, use in a Biological hood
Paraformaldehyde (PFA)Sigma Aldirch158127Store at RT, use in a Fume hood
pBABE-neo largeTcDNA Addgene1780Store at -20 °C, use in a Biological hood
pCL-AmphoNovus Biological 18746853Store at -20 °C, use in a Biological hood
pCMV-R8.74Addgene22036Store at -20 °C, use in a Biological hood
pCMV-VSVG Addgene8454Store at -20 °C, use in a Biological hood
Penicillin-Streptomycin (Pen Strepto, 10,000 μg/mL) Gibco15140122Store at 4 °C, use in a Biological hood
Phase contrast microscope Olympus BX42
Phosphate Buffered Saline (PBS)HomemadeStore at RT, use in a Biological hood
pLKO.1-Puro Addgene10878Store at -20 °C, use in a Biological hood
Poly-L-lysine solutionSigma AldirchP8920Store at 4 °C, use in a Biological hood
Puromycin dihydrochloride from Streptomyces albonigerSigma AldirchP8833
rCutSmart Buffer New England BiolabsB6004Store at -20 °C
Rodent shaverAesculapGT416
Roswell Park Memorial Institute (RPMI)Gibco61870010Store at 4 °C, use in a Biological hood
Round-tip Thirsty-SwabsSigma AldirchZ744127
ScalpelGima27041
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
Stitches 5-0Ethicon1866G
Syringe 10 mLPentaferte00202250E14
Syringe 5 mLPentaferte00202250E12
Syringe Omnican Insulin 1 mLBraun9151125S
Tissue adesive3M Vetbond1469SB
Transwell inserts 8 µm pore Falcon353097
Transwell Matrigel inserts 8 µm pore Corning354480
Trypsin-EDTA (0.5%), no phenol redGibco15400054
Vivaspin 20 centrifugal concentrator MWCO 10,000 Da Sigma AldirchZ614602

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