Patient-Derived Procoagulant Breast Fibroblasts Expressing Tissue Factor Promote Breast Cancer Cell Migration
Hadiyat A. Ogunlayi, John Castle, Emma L. Blower, Anne Armstrong, Robert B. Clarke, Cliona C. Kirwan
TL;DR
This study shows that fibroblasts in breast tissue, even those far from tumors, can take on a procoagulant state that helps cancer cells move and spread.
Contribution
The study reveals that distant fibroblasts can adopt a cancer-associated, procoagulant phenotype and that tissue factor inhibition reduces breast cancer cell migration.
Findings
Fibroblasts from distant breast tissue show CAF-like and procoagulant traits similar to tumor-associated fibroblasts.
Inhibiting tissue factor from fibroblasts significantly reduces MCF-7 breast cancer cell migration.
Combined inhibition of tissue factor and TGFβ1 further suppresses cancer cell migration.
Abstract
Cancer-associated fibroblasts (CAFs) play a key role in breast cancer progression and exhibit a procoagulant phenotype within the tumour microenvironment (TME). We hypothesised that this procoagulant phenotype correlates with a CAF-like phenotype and that fibroblasts distant from the immediate TME are less procoagulant. We also proposed that the procoagulant phenotype contributes functionally to breast cancer progression. Primary fibroblasts were cultured from human breast tumour tissue and matched normal breast tissue from regions distant to the tumour. Conditioned media (CM) from these cells were collected for analysis. We conducted immunocytochemistry, western blotting, transforming growth factor beta 1 (TGFβ1) ELISA, tissue factor (TF) activity and procoagulant activity assays. A positive correlation was found between the expression of TF and alpha-smooth muscle actin (α-SMA), a…
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Figure 6- —Cancer Research UK
- —Prevent Breast Cancer
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Taxonomy
TopicsAngiogenesis and VEGF in Cancer · Cell Adhesion Molecules Research · TGF-β signaling in diseases
Introduction
Breast cancer is the second most common cancer worldwide and the leading cause of cancer death among women [1]. The tumour microenvironment (TME) plays a key role in breast cancer progression making it an important potential target for breast cancer treatment [2]. Cancer associated fibroblasts (CAFs) are activated fibroblasts and they are the main cellular component of the TME [3]. CAFs contribute to cancer progression through the secretion of various cytokines such as transforming growth factor beta (TGFβ) [4]. TGFβ is a pluripotent cytokine and a multifunctional growth factor involved in various pro-tumorigenic mechanisms including wound healing, immunosuppression, invasion, migration, epithelial mesenchymal transition and apoptosis [5, 6]. Furthermore, TGFβ plays an important role in the activation and transformation of cellular precursors such as normal fibroblasts, into CAFs [7, 8]. CAFs also secrete more TGFβ compared to normal fibroblasts reflecting the function of TGFβ as a paracrine driver of fibroblast activation [4, 9]. The canonical signalling pathway of TGFβ leads to the expression of the TGFβ target protein, alpha smooth muscle actin (α-SMA) [6, 10], with α-SMA being an important CAF marker [11].
CAFs are phenotypically similar to myofibroblasts which are activated fibroblasts that are essential for wound healing [12]. Myofibroblasts also secrete more TGFβ and have an increased expression of α-SMA compared to normal fibroblasts [13–15]. This is significant given that tumours have been described as “wounds that do not heal” [16], with mechanisms involved in the wound healing response being dysregulated in cancer [17].
The first phase of wound healing is coagulation. Tissue factor (TF), the clotting factor that initiates the coagulation cascade, is overexpressed in various cancers including breast cancer, with TF overexpression in breast cancer cells correlating with a poor prognosis [18]. In vitro, TF in complex with the coagulation factor, FVII activates signalling pathways such as the MAPK and PI3K pathways via protease activated receptor 2 (PAR2), promoting breast cancer cell proliferation, migration and survival [19–21]. We have previously shown in human tissue that fibroblasts in the breast TME express more TF [22]. However, the functional significance of this was not investigated. We hypothesize that fibroblasts situated away from the tumour microenvironment (TME) are less procoagulant than those residing within it. Furthermore, we propose that the procoagulant activity of CAF-like fibroblasts contributes to the promotion of breast cancer progression.
Materials and Methods
Sample Collection
Invasive breast cancer and matched macroscopically normal breast tissue from the same breast, located ≥ 2 cm from the tumour (distant), were obtained from mastectomy specimens of 50 patients for ex vivo fibroblast culture. Freshly resected mastectomy specimens were macroscopically assessed by a pathologist. The oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status of breast tumour samples were determined using immunohistochemistry (IHC) according to standard protocols. Mammographic mapping was used to aid the localisation of the invasive breast cancer and the area of normal tissue distant from the tumour. The area of normal breast tissue sampled was at least 2 cm away from the breast tumour site to avoid the immediate tumour microenvironment. Breast tissue samples were transported in RPMI-1640 medium containing penicillin-streptomycin, to the research laboratory.
Additionally, 10 primary fibroblast samples (passage 3), cultured from breast tissue obtained during reduction mammoplasty surgeries of non-cancer patients, were provided by the “Breast Cancer Now” cell culture bank. The biobank performed identification and validation of these cells as fibroblasts according to their established standard procedures.
Primary Breast Fibroblast Isolation and Culture
In-House Isolation of Breast Tumour-Derived and Matched Distant Fibroblasts
Breast tumour and their matched distant breast tissue samples were mechanically dissected in separate petri-dishes to which fibroblast cell culture medium (high glucose DMEM, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 500 mg/ml primocin) was added. The petri dishes were incubated in a humidified atmosphere at 37 °C and 5% CO_2_ to enable explant outgrowth. At approximately 50–60% confluency, the fibroblasts were passaged to passage 1 in a 75 cm^2^ flask with the addition of the fibroblast culture medium. Subsequent passages were carried out when the fibroblasts had reached approximately 90% confluency in the 75 cm^2^ flasks. Final experiments were carried out at passage 3.
Reduction Mammoplasty-Derived Normal Breast Fibroblasts (Breast Cancer Now Cell Culture Bank)
Primary fibroblasts from reduction mammoplasty (normal) breast tissue samples were cultured by the “Breast Cancer Now (BCN)” cell culture bank as follows: tissue samples were dissected into small pieces and enzymatically digested in RPMI-1640 medium supplemented with (25 mM HEPES, 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µg/ml amphotericin-B, 1 mg/ml collagenase 1 A, hyaluronidase) at 37 °C for 12–16 h. Following centrifugation, cell pellets were resuspended and cultured in (DMEM: F12, 10% FBS, 0.5 mg/ml hydrocortisone, 10 mg/ml apo-transferrin, 5 mg/ml insulin, 10 ng/ml EGF, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin-B) for the first two passages, then maintained in BCN fibroblast medium (DMEM: F12, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml amphotericin-B) from passage 3. Passage 3 fibroblasts were validated and confirmed to be fibroblasts by the BCN team using their standard protocols and cryopreserved in BCN fibroblast medium containing 40% FBS and 6% DMSO.
Upon receipt, cryovials were thawed at 37 °C, centrifuged, and resuspended in our primary fibroblast medium (high glucose DMEM, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 500 mg/ml primocin) before seeding into appropriate vessels for downstream experiments.
Cell Lines and Cell Culture
MCF-7 breast cancer cell line was purchased from the American Type Culture Collection (ATCC) and verified as mycoplasma free. 544R CAF cell line was derived by mixing human mammary fibroblasts with MCF-7-ras human breast carcinoma cells and injecting the mixture subcutaneously into immunodeficient nude mice [23]. TF overexpressing HMF-U19 cell line (TF-OE HMF-U19) is an immortalised human mammary fibroblast cell line that was transfected to stably overexpress TF. These cell lines were incubated in a humidified atmosphere at 37 °C and 5% CO_2_. MCF-7 cells were cultured in complete DMEM medium (DMEM, 10% FBS, 2 mM L-glutamine), the 544R CAF cell line in complete DMEM medium (DMEM, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) and the HMF-U19 cell line was cultured in RPMI medium (RMPI & 5% FBS).
Primary Fibroblast Conditioned Media Preparation
Primary fibroblasts conditioned media (CM) were prepared when the fibroblasts had reached ~ 90% confluency at passage 3. The media in the 75 cm^2^ cell flasks were replaced with 2% FBS fibroblast cell culture medium for 48 h. At 48 h, the media were collected and centrifuged at 1000 g for 10 min at 4 °C to remove cellular debris. The supernatant was aliquoted as CM.
Procoagulant Activity Assay
After CM collection, fibroblasts were trypsinised using trypsin-EDTA solution and the resulting cell solution was centrifuged at 600 g for 2 min. The cell pellet was resuspended in 300 µl of PBS. A cell count was performed using a haemocytometer and a cell solution was prepared at 1 × 10^6^ cells/ml in PBS.
To determine the procoagulant activity of the cells and CM, a human normal plasma solution was prepared using ddH_2_O and a lyophilised human normal plasma control vial (Routine control N, Helena Biosciences). A small steel ball, 100 µl of the 1 × 10^6^ cells/ml suspension or 100 µl of prepared CM and 100 µl of human normal plasma control was added into a cuvette that was placed in the Ceveron one coagulometer to be warmed for 4 min at 37 °C. Coagulation/clotting was initiated by adding 100 µl of 25 mmol/l CaCl_2_ and the time until clot formation was measured by the coagulometer.
Tissue Factor (TF) Activity Assay and Transforming Growth Factor Beta 1 (TGFβ1) ELISA
TF concentration of the primary fibroblast CM was determined using the TF activity assay (Abcam, ab108906). TGFβ1 concentration of the fibroblast CM was determined using the Human TGF-beta 1 DuoSet ELISA kit (DY240, R&D systems).
Western Blotting
Protein content of the fibroblast cell lysates was measured using Pierce BCA protein assay kit (ThermoFisher, 23227). The protein was separated by SDS-PAGE using the 10% Mini-PROTEAN TGX precast protein gels. The separated proteins were then transferred from the gel to a nitrocellulose membrane using the Mini Trans-Blot Cell (Bio-Rad). Primary antibodies were TF (Immbiomed, ADG4508) 1:500, α-SMA(Dako, M0851) 1:1000 and vinculin (Cell signalling, 13901) 1:1000. HRP secondary reaction was catalysed using the Luminata Classico Western HRP substrate (MerckMillipore, WBLUC0100). Western blot bands were quantified by volume densitometry analysis using the Image Lab Software Version 6.1.0 build 34 Standard Edition (2020, Bio-Rad Laboratories, Inc.).
Cell Pellet Formalin Fixed Paraffin Embedded (FFPE) Block Creation and Immunocytochemistry (ICC)
Primary fibroblasts were trypsinised and centrifuged to form cell pellets. Cell pellets were fixed in 4% formalin solution (Paraformaldehyde, 4% in PBS, Alfa Aesar, J61899.AP) at 4 °C overnight. The samples were centrifuged, and the fixed cell pellet was resuspended in PBS and embedded in paraffin. For ICC, the Leica BondMax and i6000 automated IHC platform were used. Separate sections were stained for: haematoxylin and eosin (H&E), TF (Immbiomed, ADG4508) 1:200, α-SMA (Dako, M0851) 1:500, fibroblast activation protein (FAP) (Cell Signalling, 66562 S) 1:200, vimentin (Dako, M0725) 1:100, cytokeratin 5 (Abcam, ab52635) 1:200 and pan-cytokeratin (Abcam, ab9377) 1:500, and quantified using the HALO (Indica Labs, New Mexico) software-based automated technique.
Scratch Wound Migration Assay
MCF-7 cells were seeded into each well of a 96-well plate. 24 h later, 2% mitomycin treatment in serum free medium was added to the cell monolayer in each well and incubated at 37 °C and 5% CO_2_ for 2 h. An incucyte wound maker was then used to scratch/wound the 100% confluent cell monolayers followed by the addition of the chosen fibroblast CM samples to their appropriate wells. Serum free medium and TF-OE HMF-U19 CM were used as the negative and positive controls, respectively. Time lapse imaging of all wells for 72 h was done using the Incucyte Live Cell Imaging System. Images of the wound sizes were analysed using a macro that was added to the Image J software.
Treatment of MCF-7 with Fibroblast Conditioned Media, 10H10 and SB431542 for Scratch Wound Migration Assay
The scratch wound migration assay was performed as previously described. For each selected fibroblast CM sample, MCF-7 cells were treated after scratching 100% confluent monolayers with one of the following: CM alone (negative control); CM with 1 µg/ml of the TF inhibitor, 10H10 (CD142 Monoclonal Antibody [TF9-10H10], ThermoFisher, MAI-83495); CM with 100 nM of the TGFβ1 receptor inhibitor, SB431542 (Tocris, 1614); or CM with both 1 µg/ml of 10H10 and 100 nM of SB431542.
To evaluate the effects of the inhibitors in the absence of fibroblast CM, serum free medium alone; or in combination with the same inhibitor treatments were applied to separate wells.
Software and Statistical Analysis
Statistical analysis was performed using GraphPad Prism Version 10. Shapiro-Wilk normality test was used for all the statistical analysis. Means were expressed ± standard deviation. Comparisons between groups were made using either the student’s t-test or Wilcoxon test, as appropriate. Correlation analyses were performed using Pearson’s correlation coefficient; non-normally distributed data were log_10_-transformed prior to correlation analysis. Differences were considered statistically significant at p < 0.05.
Results
Fresh breast tissue samples were obtained from 50 patients with invasive breast cancer for the ex vivo culture of primary fibroblasts. Fibroblasts were successfully cultured to passage 3 from 79% of samples, corresponding to 43/50 tumour samples and 36/50 matched distant breast tissue samples. Paired fibroblast cultures (tumour and distant) were successfully established from 32/50 patients, and experimental analyses were performed on fibroblasts from these paired samples (Table 1). In addition, passage 3 primary fibroblasts from non-cancer patients undergoing cosmetic reduction mammoplasty were obtained from the BCN Biobank to serve as controls (n = 10). The median age of this control group was 32 years (range: 19–46 years).
Table 1. Clinicopathological details for studied invasive breast cancer patientsAge (years) median (range)60.5 (25–92)Clinicopathological characteristicsn = 32 (%)Invasive cancer pathology Invasive ductal carcinoma (IDC)25 (78) Invasive lobular carcinoma (ILC)7 (22)Grade 10 (0) 215 (47) 317 (53)ER status Positive28 (88) Negative4 (12)PR status^a^ Positive23 (72) Negative8 (25)HER2 receptor status Positive3 (9) Negative29 (91)^a^Data unavailable for one patient
Characterisation of Breast Tissue – Derived Tumour- and Distant-Associated Fibroblasts
Primary cells isolated from breast tumour and their matched distant breast tissue exhibited the spindle-shaped morphology typical of mesenchymal cells such as fibroblasts when cultured ex vivo (Fig. 1A). Immunocytochemical staining was performed prior to functional experiments to validate fibroblast identity and exclude epithelial contamination. Both tumour and distant breast– derived cells demonstrated strong vimentin positivity across passages 1–3, consistent with a mesenchymal cell phenotype (Fig. 1B – C). Additional fibroblast markers, α-SMA and FAP, were assessed at passage 3 for both tumour and distant breast – derived cells and were also positive (Fig. 1D). In contrast, cytokeratin 5 (CK5) and pan-cytokeratin (Pan-CK) staining were negative across passages 1–3 (Fig. 1B – C) in both cell populations, indicating an absence of myoepithelial or epithelial cell contamination in the cultured fibroblast populations. Fig. 1. Characterisation of breast tissue – derived tumour- and distant-associated fibroblasts. A Ex vivo primary fibroblast culture from mechanically dissected breast tissue (Scale bar: 100 μm). Yellow arrows indicate breast tissue, and the purple box highlights the spindle shaped mesenchymal cells migrating out from the tissue. B Representative images of immunocytochemical staining of breast tissue – derived distant cells across passages 1–3 (Scale bar: 100 μm). Vimentin staining shows strong positivity across all passages, whereas cytokeratin 5 (CK5) and pan-cytokeratin (Pan-CK) staining are negative at all passages. Brown chromogen indicates positive staining. C Representative images of immunocytochemical staining of breast tissue – derived tumour cells across passages 2–3 (Scale bar: 100 μm). Vimentin staining is strongly positive, while CK5 and Pan-CK are negative at both passages. Brown chromogen indicates positive staining. D Representative images of immunocytochemical staining of breast tissue – derived tumour and their matched distant cells showing strong alpha – smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) positivity at passage 3 (Scale bar: 100 μm). Brown chromogen indicates positive staining
Fibroblast Procoagulant Activity is Driven by Fibroblast Tissue Factor Expression
Using the trypsinised fibroblasts and their conditioned media, the time to clot formation (shorter clotting time reflecting increased procoagulant activity) was assessed with the procoagulant activity assay. Procoagulant activity correlated with the TF expression of cultured primary breast fibroblasts (n = 31, Pearson r = -0.32, p = 0.04) (Fig. 2A). Procoagulant activity and TF concentration of fibroblast conditioned media also correlated (n = 33, Pearson r = -0.44, p = 0.006) (Fig. 2B). This highlights that fibroblast TF expression and secretion is functionally procoagulant.
Fig. 2. Tissue factor drives procoagulant activity in primary breast fibroblasts.** A** Correlation of fibroblast TF expression (immunocytochemistry) and procoagulant activity (clotting times) (log_10_ – transformed). B Correlation of fibroblast conditioned media TF concentration (TF activity assay) and procoagulant activity (clotting times) (log_10_ – transformed). TF (tissue factor), CM (conditioned medium)
CAF-Like Fibroblasts Exhibit Increased Procoagulant Activity
We hypothesized that fibroblasts acquiring a CAF-like phenotype exhibit enhanced procoagulant activity. Having established that fibroblast TF expression is functionally procoagulant, we investigated the relationship between the fibroblast expression of α-SMA, a CAF and fibroblast activation marker, and TF expression. α-SMA expression positively correlated with TF levels (n = 19, Pearson r = 0.67, p = 0.002) (Fig. 3A). Similarly, secretion of the fibroblast activator, TGFβ1 inversely correlated with clotting times of the fibroblast CM (n = 46, Pearson r = -0.45, p = 0.0008), indicating that a higher TGFβ1 level is associated with increased procoagulant activity (shorter clotting times) (Fig. 3B). Together these findings support the hypothesis that fibroblast activation toward a CAF-like state is associated with elevated tissue factor expression and procoagulant activity.
Fig. 3. Procoagulant and activation phenotypes correlate in primary breast fibroblasts. A Correlation of fibroblast TF and α-SMA expression (western blotting). B Correlation of TGFβ1 concentration (ELISA) and clotting times (procoagulant activity assay) in fibroblast conditioned media (CM) (log10 – transformed). TF (tissue factor), TGFβ1 (transforming growth factor beta 1)
Fibroblasts Distant from the Breast Tumour are Procoagulant and CAF-Like
Having evidenced that as fibroblasts become more CAF-like, they exhibit increased procoagulant activity, we investigated whether this was an immediate TME effect or more consistent with a field change. We compared the procoagulant and activation phenotypes of fibroblasts cultured from invasive breast cancer tissue (tumour) with those cultured from macroscopically normal tissue ≥ 2 cm from the tumour within the same breast (distant), as well as with fibroblasts from normal breast tissue of non-cancer patients (normal).
When comparing tumour fibroblasts to matched distant fibroblasts, there was no difference in the procoagulant activity of the fibroblasts (median [tumour = 39.9 s, distant = 40.9 s, ]; p = 0.55) (Fig. 4D) or their CM (median [tumour = 482.1 s, distant = 454.5 s, ]; p = 0.93) (Fig. 4E). Conversely, normal breast fibroblasts and CM from non-cancer patients exhibited significantly lower procoagulant activity compared to both tumour and distant fibroblasts ([median: normal 53.2 s vs. tumour 39.9 s, p = 0.004; vs. distant 40.9 s, p = 0.007]; [CM, median: normal 835.9 s vs. tumour 482.1 s, p = 0.002; vs. distant 454.5 s, p = 0.01]) (Fig. 4D and E).
Consistent with these findings, there was no difference between the levels of TF secreted by the tumour and distant fibroblasts into their CM (median [tumour = 2.24 pM, distant = 2.64 pM]; p = 0.65) (Fig. 4F). In contrast, normal breast fibroblasts from non-cancer patients secreted significantly lower levels of TF into their CM compared to both tumour and distant fibroblasts (median: normal 0.796 pM vs. tumour 2.24 pM, p = 0.002; vs. distant 2.64 pM, p = 0.006) (Fig. 4F).
When tumour fibroblast expression of TF was compared to paired distant fibroblasts, surprisingly, there was a significantly lower TF expression in the tumour fibroblasts (n = 12, median H-score [tumour = 60.6, distant = 68.4], p = 0.01 (Fig. 4C).
In addition, consistent with the demonstrated correlation between the fibroblast procoagulant and activation phenotypes, there was no significant difference between the expression levels of the CAF marker, α-SMA in the distant fibroblasts and their paired tumour fibroblasts (n = 12, median H-score [tumour = 119.4, distant = 111.6], p = 0.38) (Fig. 4A). There was also no significant difference between the expression levels of the CAF marker, FAP in distant fibroblasts and their paired tumour fibroblasts (n = 17, median H-score [tumour = 175.3, distant = 181.4], p = 0.80) (Online resource 1, Fig. 1). In line with these results, there was no significant difference between the concentration of the fibroblast activator, TGFβ1, in the CM of the distant fibroblasts and their paired tumour fibroblasts (n = 15, median [tumour = 71.9 pg/ml, distant = 85.3 pg/ml; ], p = 0.44) (Fig. 4B). In contrast, normal breast fibroblasts from non-cancer patients secreted significantly lower levels of TGFβ1 into their CM compared to both tumour and distant fibroblasts (median: normal 37 pg/ml vs. tumour 71.9 pg/ml, p < 0.0001; vs. distant 85.3 pg/ml, p < 0.0001) (Fig. 4B).
The similar CAF-like and procoagulant phenotype between the fibroblasts from the immediate TME and fibroblasts from elsewhere in the same breast implies that there may be a field change effect.
Fig. 4. Procoagulant and activation phenotype comparison in invasive breast cancer fibroblasts vs. matched distant breast tissue-derived fibroblasts vs. normal breast fibroblasts from non-cancer patients.** A** Immunocytochemical analysis comparing α-SMA expression in distant fibroblasts and breast tumour fibroblasts (n = 12). B Quantification of TGFβ1 levels in CM from normal fibroblasts (non-cancer patients) (n = 10), distant fibroblasts (n = 15) and breast tumour fibroblasts (n = 15) using a TGFβ1 ELISA. C Immunocytochemical analysis comparing TF expression in distant fibroblasts and breast tumour fibroblasts (n = 12). D Procoagulant activity assay comparing clotting times of normal breast fibroblasts from non-cancer patients (n = 10), distant fibroblasts (n = 32) and breast tumour fibroblasts (n = 32). E Procoagulant activity assay comparing clotting times of CM of normal breast fibroblasts from non-cancer patients (n = 10), distant fibroblasts (n = 15) and breast tumour fibroblasts (n = 15). F Quantification of TF concentration in CM of normal breast fibroblasts from non-cancer patients (n = 10), distant fibroblasts (n = 12) and breast tumour fibroblasts (n = 11) using a TF activity assay. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ICC (immunocytochemistry), TF (tissue factor), CM (conditioned medium), TGFβ1 (transforming growth factor beta 1)
Procoagulant Fibroblasts in the Breast TME Promote Breast Cancer Cell Migration
CAFs promote breast cancer cell migration, a fundamental requirement for breast cancer progression. Given the demonstrated correlation between the CAF-phenotype and procoagulant activity, we hypothesized that this procoagulant activity is functional in breast cancer cell migration, and therefore progression. Using the scratch wound assay, the effects of the fibroblast CM on the rate of migration of the breast cancer cell line, MCF-7, was assessed. Immunocytochemical analysis confirmed the absence of detectable TF expression in MCF-7 cells, whereas the 544R CAF cell line, included as a positive control for TF expression, demonstrated strong TF staining (Fig. 5A). Analysis of CM from primary fibroblasts showed that TF concentration correlated positively with MCF-7 breast cancer cell migration (n = 16, Pearson r = 0.53, p = 0.02) (Fig. 5B).
Given that both TF and TGFβ1 promote cell migration [24, 25] and that fibroblast procoagulant activity correlates with TGFβ1 expression, we hypothesized that simultaneous inhibition of both factors may produce an additive effect on cell migration. We therefore investigated the effects of inhibiting TF (using the inhibitor, 10H10 [TFi]) and inhibiting TGFβ1 (using the TGFβ type 1 receptor inhibitor, SB431542 [TGFβR1i]), both individually and in combination, on MCF-7 migration in the presence of fibroblast CM. To determine whether these inhibitors also influence MCF-7 migration independently of fibroblast-derived factors, we additionally assessed their effects in the absence of fibroblast CM under serum free conditions.
We confirmed that these inhibitors do not influence MCF-7 migration in the absence of fibroblast CM. There was no significant difference in MCF-7 migration rates when TFi, TGFβR1i or both inhibitors were added to serum free medium (SFM) compared with SFM alone (SFM vs. + TFi, p = 0.35; SFM vs. + TGFβR1i, p = 0.45; SFM vs. + both inhibitors, p = 0.50) (Fig. 5C and D).
Having established that these inhibitors do not affect MCF-7 migration under serum free conditions, we next assessed the effects of fibroblast CM – with and without inhibitor treatment – on MCF-7 migration rates. There was a slower rate of MCF-7 migration when TF was inhibited in the tumour fibroblast CM (tumour fibroblast CM, n = 3; TFi vs. negative control, p = 0.008) (Fig. 5C). This was also the case when TF was inhibited in the distant fibroblast CM (distant fibroblast CM, n = 3; TFi vs. negative control, p = 0.02) (Fig. 5D). There was no difference in the rate of MCF-7 migration when TGFβ1 effect was inhibited in the tumour fibroblast CM (tumour fibroblast CM, n = 3; TGFβR1i vs. negative control, p = 0.20) (Fig. 5C). There was also no difference when it was inhibited in the distant fibroblast CM (distant fibroblast CM, n = 3; TGFβR1i vs. negative control, p = 0.27) (Fig. 5D).
The rate of MCF-7 cell migration was significantly decreased when TF and TGFβ1 were inhibited in combination in the tumour fibroblast CM compared to the negative control (tumour fibroblast CM, n = 3; negative control vs. TFi + TGFβR1i, p = 0.04), but not to the inhibitors used individually (TFi vs. TFi + TGFβR1i, p = 0.50; TGFβR1i vs. TFi + TGFβR1i, p = 0.50) (Fig. 5C). The rate of MCF-7 migration was also significantly decreased when TF and TGFβ1 were inhibited in combination in the distant fibroblast CM (distant fibroblast CM, n = 3; control vs. TFi + TGFβR1i, p = 0.002), with the decrease in migration being significantly greater than when the inhibitors were used individually (TFi vs. TFi + TGFβR1i, p = 0.049; TGFβR1i vs. TFi + TGFβR1i, p = 0.01) (Fig. 5D).
These findings support the hypothesis that procoagulant fibroblasts facilitate breast cancer cell migration through the secretion of TF into the breast TME. Our results also show that any additive effect of fibroblast-derived TF and TGFβ1 on breast cancer cell migration is not consistently observed.
Fig. 5. Inhibiting fibroblast-secreted tissue factor reduces breast cancer cell migration.** A** Immunocytochemical staining of TF expression in MCF-7 and 544R CAF cell lines (Scale bars: 100 μm, top panels; 50 μm, bottom panels). Brown chromogen indicates positive staining. B Correlation between TF concentration in primary breast fibroblast CM and rate of MCF-7 migration (wound closure per hour) (log_10_ – transformed). C Bar graphs showing MCF-7 migration measured by scratch wound assays. Left: effects of tissue factor inhibitor (TFi; 1 µg/mL 10H10) and TGFβR1 inhibitor (100 nM SB431542), under serum free conditions. Right: effects of tumour fibroblast CM, alone and in combination with TFi and TGFβR1 inhibitor, on MCF-7 cell migration. D Bar graphs showing MCF-7 migration measured by scratch wound assays. Left: effects of tissue factor inhibitor (TFi; 1 µg/mL 10H10) and TGFβR1 inhibitor (100 nM SB431542), under serum free conditions. Right: effects of distant fibroblast CM, alone and in combination with TFi and TGFβR1 inhibitor, on MCF-7 cell migration. Data represent mean results ± standard deviation of three independent experiments, each with four technical replicates per condition. Where normally distributed, the paired t-test was used for comparisons and where the distribution was non-parametric, the Wilcoxon test was used for comparisons (*P < 0.05, **P < 0.01). CM (conditioned medium), SFM (serum free medium), TFi (tissue factor inhibitor), TGFβR1i (transforming growth factor beta type 1 receptor inhibitor)
Discussion
In this study, we investigated the relationship between the procoagulant and CAF phenotypes of fibroblasts in the breast TME, and the potential contribution of procoagulant CAFs to breast cancer progression. Primary fibroblasts were cultured from breast tumour tissue and their matched distant breast tissue from 50 invasive breast cancer patients, up to passage 3. Fibroblasts were successfully established from both tumour and matched distant tissue in 32 patients, while the remaining samples yielded insufficient viable cells, likely reflecting differences in tissue viability, sample sizes or intrinsic variation in fibroblast proliferation.
The culture conditions employed, high-glucose DMEM supplemented with 10% FBS favour mesenchymal/ stromal cell growth over epithelial and myoepithelial cells [26–28]. Serial passaging to passage 3 likely eliminates residual epithelial and myoepithelial cells, and immunocytochemistry using cytokeratin 5 and pan-cytokeratin antibodies indicated that these populations are largely absent as early as passage 1 [29]. Morphologically, the cultures were dominated by spindle-shaped cells without epithelial-like clusters, consistent with a stromal identity [30, 31], and immunocytochemical analysis confirmed strong expression of the fibroblastic markers, vimentin, α-SMA, and fibroblast activation protein, confirming the stromal nature of our cultured cells [32–34].
Although other mesenchymal cell types, including mesenchymal stem cells (MSCs) and pericytes, contribute to the breast tissue stroma and express similar markers to fibroblasts [35–39], several lines of evidence suggest that fibroblasts constitute the predominant cell type in our cultures. First, fibroblasts are known to proliferate more rapidly and tolerate standard in vitro culture conditions better than other mesenchymal cells such as MSCs or pericytes [40, 41]. In contrast, MSCs and pericytes typically require low-glucose, serum-free, or otherwise specialised media to maintain their phenotypic identity, whereas the high-glucose medium supplemented with 10% FBS used here preferentially supports fibroblast growth [42, 43]. Taken together, these considerations strongly suggest that fibroblasts are the main constituent of our cell population.
For comparison, passage 3 primary fibroblasts from non-cancer patients undergoing reduction mammoplasty were obtained through the BCN biobank and cultured under similar conditions (DMEM/F12 (high glucose), 10% FBS). These normal fibroblasts displayed spindle-shaped morphology and a stromal marker profile characterised by expression of vimentin, fibronectin, and caveolin-1, and absence of cytokeratins 8 and 14. Additionally, they were negative for α-SMA, as expected of normal fibroblasts, confirming their suitability as a control group for our experiments.
Having confirmed the fibroblast identity of our primary cultures, we explored the fibroblast expression of the procoagulant marker tissue factor (TF), which has been shown to increase progressively from normal breast tissue through DCIS to invasive breast cancer [22]. Here, we demonstrate that TF expression by fibroblasts exerts a functional procoagulant effect, with TF expression and secretion by primary breast fibroblasts correlating with increased procoagulant activity. We also found correlations between fibroblast expression of TF and the CAF marker, α-SMA and between fibroblast procoagulant activity and secretion of the CAF-inducer, TGFβ1. These associations support the hypothesis that the transition of fibroblasts to a more CAF-like phenotype is accompanied by the acquisition of a more procoagulant phenotype. Our findings align with a previous study that demonstrated that TGFβ1 upregulates TF expression in human lung fibroblasts [44]. Notably, TGFβ1 plays a key role in the differentiation of normal fibroblasts into CAFs. Therefore, our findings add to the accumulating evidence implicating TGFβ1 signalling in both CAF differentiation and the induction of a procoagulant phenotype, suggesting a mechanistic link between fibroblast transition to a CAF-like state and the emergence of a procoagulant phenotype.
Given that normal fibroblasts are not activated and thus do not express the CAF marker, α-SMA and produce lower levels of TGFβ1 than CAFs [4, 13, 45], we hypothesised that fibroblasts that are distant from the tumour by ≥ 2 cm and presumed normal will be less activated and less procoagulant than breast cancer fibroblasts. Interestingly, we found that distant fibroblasts expressed similar levels of α-SMA and TGFβ1 and had a similar procoagulant phenotype compared to breast tumour fibroblasts or CAFs. One potential explanation for this observation is our cell culture method: culturing fibroblasts on stiff plastic surfaces in high glucose and high serum medium (10% FBS) has been shown to promote a transition from quiescent fibroblast to a myofibroblast-like phenotype particularly the increased expression of α-SMA [46, 47]. However, both distant and tumour-derived fibroblasts demonstrated significantly higher procoagulant activity, with increased secretion of TF, as well as significantly elevated secretion of the CAF inducer, TGFβ1, compared to fibroblasts from normal breast tissue of non-cancer patients which were also cultured on plastic surfaces in high glucose and high serum medium (10% FBS). This suggests that culture conditions alone cannot fully account for the observed activation of the distant fibroblasts.
Another possible explanation for our findings is the concept of field cancerization, whereby histologically normal tissue distant from the immediate TME exhibits molecular and functional alterations consistent with a pre-cancerous state [48, 49]. For instance, pro-tumorigenic genes are upregulated in histologically normal breast tissue as far as 4 cm from the breast tumour site [50]. These changes have been seen across several cancer types with significant involvement of genes related to the TGFβ signalling pathway [50]. Furthermore, in breast cancer, fibroblasts in histologically normal breast tissue located up to 6 cm from the breast tumour have an increased expression of TGFβ [51]. Overall, our findings suggest that the breast tumour may promote a procoagulant phenotype in fibroblasts extending beyond the immediate TME.
To assess the clinical relevance of our findings, we investigated whether the procoagulant phenotype of CAFs was simply a bystander effect or has a functional role in breast cancer progression. We observed that procoagulant breast CAFs secrete TF into the TME, which correlates with an increased rate of breast cancer cell migration, a critical step in the processes of invasion and metastasis. We further demonstrated that the inhibition of the secreted TF using 10H10 monoclonal antibody resulted in a reduced rate of breast cancer cell migration. Although, an isotype control was not included in this assay, the specificity and functional activity of 10H10 have been well established in multiple independent studies [52, 53], supporting the conclusion that the observed effect is likely due to targeted TF inhibition. Furthermore, the lack of TF expression in MCF-7 cells indicates that the observed effect is mediated specifically via TF present in the fibroblast conditioned media. Mechanistically, the TF-FVIIa-FXa complex can activate PAR2, which activates the pro-migratory MAPK signalling pathway, upregulating genes involved in epithelial-mesenchymal transition [54–57]. This provides a potential explanation for how procoagulant CAFs enhance breast cancer cell migration.
TGFβ has also been reported in different studies to have a pro-migratory effect in several cancer types including breast cancer [58–60]. Therefore, we investigated whether TGFβ1 secreted by CAFs, promotes breast cancer cell migration. Contrary to a previous study [61], we found that breast CAF-derived TGFβ1 did not promote breast cancer cell migration. This discrepancy may be attributed to methodological differences, particularly the higher concentration of TGFβ type 1 receptor inhibitor used in that study. Furthermore, data from the Broad Institute Cancer Cell line Encyclopaedia indicate that MCF-7 cells express TGFβ1, suggesting that autocrine TGFβ1 production may reduce the efficacy of SB431542 (TGFβ type 1 receptor inhibitor) at the concentration used in our experiments.
When assessing the potential additive effect of CAF-secreted TF and TGFβ1, our results were mixed. Conditioned media (CM) from distant fibroblasts showed a modest reduction in pro-migratory response when both TF and TGFβ1 were inhibited, compared with inhibition of either factor alone. A similar trend was observed with CM from breast tumour fibroblasts; however, combined inhibition did not produce a greater effect than individual inhibitors, suggesting the absence of a true additive effect. This aligns with a previous study [44], in human lung fibroblasts, which reported that TF depletion did not alter TGFβ1- induced fibroblast migration, further questioning a functional interaction between these factors in promoting migration. Nonetheless, our mixed results may also reflect insufficient inhibition of TGFβ signalling at the SB431542 concentration used, leaving open the possibility that CAF-derived TGFβ1 could contribute to breast cancer cell migration under conditions of more complete receptor blockade.
One limitation of this study is that while we compared the procoagulant and CAF phenotypes of breast tumour fibroblasts with those from macroscopically normal breast tissue located distant from the tumour, we lacked data on the exact distance of each normal tissue sample from the tumour. This prevented the analysis of how tumour proximity affects fibroblast procoagulant phenotype. Additionally, the distant breast tissue samples not being consistently collected from the same relative distance across cases, may have introduced bias into the results. Another limitation of our study is that the normal fibroblast controls from non-cancer donors were obtained from a separate biobank and cultured under different conditions than the tumour and distant fibroblasts. The control fibroblasts were isolated from enzymatically digested tissue and cultured in medium supplemented with hydrocortisone, apo-transferrin, insulin, EGF, and amphotericin B for the first two passages, whereas the tumour and distant fibroblasts were isolated from mechanically dissected tissue and cultured in medium lacking these supplements. Importantly, at passage 3, when experiments were performed, both the normal fibroblast controls and the tumour and distant fibroblasts were cultured in medium lacking these supplements. Nevertheless, these methodological differences may influence fibroblast phenotype and should be considered when interpreting comparisons with the normal controls. However, all fibroblasts in our study were cultured on plastic in high-glucose DMEM with 10% FBS, conditions reported to induce fibroblast activation, indicating that the culture conditions exerted comparable effects across all samples [46, 47].
It should also be noted that only a single concentration of the TF inhibitor and the TGFβ type 1 receptor inhibitor was used in the scratch-wound assay. The concentration of 1 µg/ml 10H10, a TF inhibitor, was chosen based on previous unpublished data from our laboratory, which showed its effectiveness at inhibiting the pro-migratory effects of TF present in the CM derived from a TF-over expressing human mammary fibroblast cell line. The concentration of 100 nM SB431542, a TGFβ type 1 receptor inhibitor, was selected based on previous studies showing that it was not toxic to cancer cell lines [62–64]. Additionally, this concentration is approximately 7000-fold higher than the highest concentration of TGFβ1 measured in the fibroblast CM samples, making it a reasonable and effective choice for competitive inhibition of TGFβ signalling. Nevertheless, dose-response experiments would have been useful for determining the optimal concentrations of each inhibitor for effective reduction in cell migration.
In conclusion, our study highlights the link between a CAF phenotype and a procoagulant phenotype. We also found that fibroblasts from macroscopically normal breast tissue distant from the tumour exhibited a similar CAF and procoagulant phenotype to CAFs, suggesting the influence of field cancerization, in which histologically normal tissue beyond the immediate TME displays tumour-associated alterations. Furthermore, we showed that fibroblast-secreted TF promotes breast cancer cell migration, whereas the role of fibroblast-secreted TGFβ1 in cell migration remains inconclusive. These findings suggest that the procoagulant phenotype of CAFs contributes to breast cancer progression and highlight TF as a potential therapeutic target in breast cancer. Notably, TF has been explored in targeted chemotherapy delivery through anti-TF-antibody drug conjugates [65, 66], and the inhibition of the TF-FVIIa-(FXa)-PAR2 signalling pathway has shown anti-tumour effects in preclinical models [53, 67–69]. This signalling pathway is currently being investigated in a phase II clinical trial that aims to determine whether the inhibition of FXa in early breast cancer downregulates pro-tumorigenic mechanisms [70]. While this is promising, additional clinical trials are warranted considering the encouraging pre-clinical and in vitro evidence including findings from this study, that support the potential of anticoagulant treatment targeting this pathway in breast cancer.
Supplementary Information
Supplementary Material 1.
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