RANK in cancer-associated fibroblasts: A valuable prognostic determinant for metastasis in early-stage breast cancer patients

2.1.1Selection and characterization of breast cancer patients

A retrospective study was conducted, including 155 consecutive patients who underwent surgical treatment for breast cancer at the Hospital Italiano in Buenos Aires, Argentina. These patients had a confirmed histological diagnosis of early-stage invasive ductal carcinoma (stage I/II) based on the TNM classification system of the International Union Against Cancer [35]. A minimum follow-up period of 10 years after surgery was ensured. Patients who received neoadjuvant therapies, those with insufficient tissue samples, and those with previous primary tumors were excluded. After surgery, all patients received appropriate treatment, including hormonal therapy, radiotherapy, and/or chemotherapy. The treatment plan was determined based on the clinical and histopathological characteristics of each patient and the guidelines recommended by the European Society for Medical Oncology [36, 37]. This study was approved by the Ethics Committees of the Institute of Biology and Experimental Medicine (IBYME) and the Hospital Italiano. Informed consent was obtained from the patients or their family members (IBYME approval: CE 050 and Hospital Italiano approval: No. 5009). The research was conducted in accordance with the principles set out in the Declaration of Helsinki. To protect the privacy of the patients, medical records were anonymized using a numerical code.

The clinical characteristics of the patients, considered classical prognostic markers, were categorized according to the cutoff values specified in the Hospital Italiano protocols [38]. These characteristics included: a) Age (< 50 or ⩾ 50 years), b) Tumor size (⩽ 2 or > 2 cm), c) Histological grade according to the Scarff-Bloom-Richardson grading system [39], categorized as well-differentiated (G1), moderately differentiated (G2), or poorly differentiated (G3), d) Expression of estrogen receptors (ER), progesterone receptors (PR), and HER2/neu status, classified as negative or positive according to Wernicke M et al. [27], and e) Presence of regional lymph node metastasis, recorded as negative (no involvement in axillary dissection or sentinel lymph node) or positive (including micro-metastasis). Outcome data included local relapse, the occurrence of metastasis, the occurrence of bone metastasis, the occurrence of visceral metastasis, the occurrence of mixed metastasis (bone and visceral), local relapse-free survival (RFS), MFS, bone metastasis-free survival (BMFS), visceral metastasis-free survival (VMFS), mix metastasis-free survival (MMFS), and OS. MFS, BMFS, VMFS, and mix-MFS were defined as the time interval from the date of surgery to the first observation of tumor appearance (metastatic event and/or local relapse) or the last follow-up. Patients included in the mixed metastasis group were those who, at the time of follow-up, had both bone and visceral metastases, without differentiation of which event occurred first. OS was defined as the interval from the date of surgery to death or the last follow-up [40]. Supplementary Table 1 provides detailed information on the specific clinical characteristics of the patients and their corresponding outcome data. The site of breast cancer metastasis is described in Supplementary Table 2. Additionally, data on the presence of single or multiple foci of metastasis within the same organ were documented.

Figure 1.

Expression of RANK in CAFs from the primary tumor of BCPs. Left panel: representative example of RANK immunostaining (brown chromogen) in stromal cells assessed in the primary tumor tissue of a BCP. Right panel: isotype control. Nuclei were stained with hematoxylin (purple color). Original magnification: × 400. Scale bars represent 25 μm. B. Association of RANK expression with local relapse-free survival (RFS), metastasis-free survival (MFS), bone metastasis-free survival (BMFS), visceral metastasis-free survival (VMFS), mix metastasis-free survival (MMFS), and overall survival (OS) in early invasive ductal BCPs. Kaplan-Meier curves (univariate analysis) marked in green represent data from samples with high RANK expression, while blue curves represent samples with negative/low RANK expression. The Log Rank (Mantel-Cox) test was used to assess the Kaplan-Meier curves. ^*p-value < 0.050. C. Details of RFS, MFS, BMFS, VMFS, MMFS and OS for the negative/low and high RANK expression groups.

2.1.2Analysis of RANK expression

Breast tissue samples were processed following the methodology outlined by Labovsky et al. [10]. In order to assess the levels of RANK expression in spindle-shaped stromal cells not associated with vasculature, we employed an immunohistochemical protocol as described in previous studies [10]. The immunohistochemical signal was evaluated using the Allred scoring system [41]. Cells with membranous staining, nuclear counterstaining, and displaying characteristic fibroblastic morphology (spindle shape), not associated with the vasculature, were counted within the intratumoral stroma. Positive cell percentages were assigned scores according to the following categories: 0 (< 10%), 1 (10–30%), 2 (31–60%), 3 (61–90%), and 4 (> 90%). Staining intensity was scored on a scale of 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong), based on the relative intensity of low molecular weight cytokeratin AE1-AE3 expression [10]. The final staining score, ranging from 0 to 7, was calculated by combining the percentage of positive cells and the staining intensity score. Enumeration was performed in five representative optical fields per tissue section at a magnification of 400x. The evaluation was independently conducted by two pathologists, with an 88.4% agreement in the immunohistochemical assessment between observers (Kappa value = 0.867) (Fig. 1).

2.1.3Intra-tumoral stromal characteristics

The histological characteristics of the tumor stroma, such as the percentage (%) of intratumoral stroma, the quantity of fibroblasts, collagen deposition, lymphocytic infiltration, myxoid changes, and blood and lymphatic vascularization, were assessed through hematoxylin and eosin staining. Intratumoral stroma was assessed as a percentage and categorized as low (< 50%) or high (⩾ 50%) in quantity. Pathologists scored the presence of fibroblasts, collagen deposition, lymphocytic infiltration, myxoid changes, blood and lymphatic vascularization using a scale of absent (0%, score 0), scanty (< 30%, score 1), moderate (30–50%, score 2), or abundant (⩾ 50%, score 3). The degree of desmoplasia was also documented based on information obtained from the patients’ medical records, categorized as low/moderate or severe.

2.1.4Statistical analysis

The statistical analysis of RANK expression and its correlation with clinical-pathological characteristics was conducted following the methodology described by Labovsky et al. [10]. To determine the optimal cutoff value for receptor expression, we used the values of the first quartile (Q1), median, and third quartile (Q3) for sample classification. Associations between categorized RANK expression and patient OS were evaluated through univariable analysis. The cutoff value that provided the lowest p-value was selected. The optimal cutoff values for receptor expression were as follows: RANK = 1 (Median), quantity of fibroblasts = 2 (Q1), collagen deposition = 1 (Q1), lymphocytic infiltration = 1 (Q3), myxoid changes = 0 (Q1), blood and lymphatic vascularization = 0 (Q1). The Fisher exact test was used to assess the association between RANK expression and classical prognostic markers, as well as the occurrence of metastasis, bone metastasis, visceral metastasis, mixed metastasis, and local occurrence. Survival analyses, including RFS, MFS, BMFS, VMFS, mix-MFS, and OS, were performed using the Kaplan-Meier method, and differences were assessed with the log-rank (Mantel-Cox) test [37]. Multivariate survival analysis was conducted using the Cox proportional hazards model with backward stepwise selection (likelihood ratio). Only significant variables identified in the univariable analysis were considered. A significance level of less than 0.0500 was established for all analyses. The statistical analysis was carried out by an experienced statistician using SPSS software (version 18.00, Chicago, Illinois).

2.2.1Patient selection

A prospective study was conducted using tumor-associated fibroblasts obtained from tumor tissue during surgery from 9 patients with early invasive ductal breast carcinoma (stages I/II, luminal type). Patients who had received neoadjuvant therapy, had a history of previous tumors, or had insufficient sample size (< 1 cm) were excluded. The samples were provided by the Breast Pathology Service of the Hospital Italiano, CABA. This study was approved by the Ethics Committees of the Institute of Biology and Experimental Medicine (IBYME) and the Hospital Italiano. Informed consent was obtained from the patients or their family members (IBYME approval: CE 050 and Hospital Italiano approval: No. 5009). The research was conducted in accordance with the principles outlined in the Declaration of Helsinki. To protect the privacy of the patients, medical records were anonymized using a numerical code.

2.2.2Isolation and expansion of CAFs from primary beast cancer tissue

Immediately after mastectomy, the tissues were placed in DMEM-F12 (#12500-062, Gibco). They were subsequently washed with the same medium supplemented with an antibiotic-antimycotic solution (ATB/ATM) (Gibco, Cat.15240), which contained a final concentration of 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 25 μg/ml of amphotericin B, and 2 mM of L-glutamine (hereinafter referred to as supplemented medium). The tissue was dissected with a scalpel onto a tissue culture dish and treated with 0.1% type III collagenase/hyaluronidase (Sigma-Aldrich, St. Louis, MO) overnight at 37^∘C with gentle agitation. After this period, the sample was centrifuged at 40g for 2 minutes. The resulting pellet, rich in organelles, as well as the undispersed tissue, was discarded. The supernatant obtained was transferred and centrifuged again at 100g for 2 minutes. The pellet obtained at this step is enriched in epithelial cells, while the supernatant is enriched in fibroblasts, the cell fraction of interest. A final centrifugation of the supernatant was performed at 200g for 5 minutes. The resulting pellet, rich in fibroblasts, was resuspended in supplemented α medium. Cell counting was performed using a 3% acetic acid solution in water, and fibroblast viability was determined using the trypan blue exclusion test with 0.04% trypan blue in PBS. For primary cultures, 375,000 cells were incubated per 25 cm² culture flask in 10 ml of supplemented α medium with 20% fetal bovine serum (FBS) (Natocor). After 24 hours, the medium was changed to remove non-adherent cells. Incubations were carried out at 37^∘C, 5% CO2, and humidity. The medium was renewed every 7 days, and when the culture reached 70–80% confluence, adherent cells were treated with a trypsin-EDTA solution (0.05%–0.02% in PBS; Gibco, Cat. 15400) to detach them at 37^∘C, 5% CO2 humidified environment for 10 minutes. The cells obtained in this first subculture are mesenchymal cells mostly composed of differentiated stromal cells, particularly CAFs. For simplicity, they will be referred to as CAFs from now on. To reduce cell density, CAFs from the first subculture were split into two 25 cm² culture flasks and incubated in supplemented α medium with 20% FBS. The culture medium was renewed every 7 days until they reached 70–80% confluence again. At that point, CAFs from the second subculture were trypsinized, and the CD34(-) fibroblast population was separated using a magnetic separation column (Anti-PE Multisort Kit: #130-091-271, MACS Miltenyibiotec, Ab anti CD34-PE: #130-098-140, MACS Miltenyibiotec). These step is important to remove the endothelial progenitors and endothelial cells. Subsequently, the CD34(-) CAFs fraction was incubated at a concentration of 240 viable cells/cm². Low cell density promotes the self-renewal of MSCs and, therefore, fibroblasts derived from them. The third subcultures were incubated with supplemented α medium with 20% FBS and the culture medium was renewed every 7 days until they reached 70–80% confluence again. Then the cells were trypsinized and to increase cell yield, CD34(-) fibroblasts were seeded at 3,000 viable cells/cm2 and maintained by changing the medium every 7 days until they reached 70–80% confluence (fourth subculture). This cell fraction was used for the remaining assays.

2.2.3Phenotypic characterization of CD34(-) fibroblast populations

A total of 3 × 10⁵ cells from 4^th subculture was centrifuged at 1,100 rpm for 5 minutes and re-suspended in 50 μl of PBS with 1% bovine serum albumin (BSA; Santa Cruz, Cat. sc-2323). Subsequently, the cells were incubated with specific monoclonal antibodies (Abs) conjugated with different fluorochromes targeting the following human antigens for 30 minutes at room temperature: RANK, CD105, CD34, CD90, CD73, FAP, CD19, and CD14. To analyze the co-expression of CD105, RANK, and CD34, a triple labeling was performed. The details of each Ab, along with their respective isotype controls, are summarized in Supplementary Table 3. The concentrations recommended by the manufacturers for each Ab were used in the flow cytometry analysis. Controls were simultaneously evaluated using the same protein concentration as the corresponding primary Abs. After incubation, the cells in each tube were washed twice with 1% PBS-BSA and centrifuged at 1,100 rpm for 4 minutes in each wash. Finally, the cells were re-suspended in 100 μl of PBS, and 10,000 events were analyzed in each case using flow cytometry (FACScalibur, BD Biosciences). FlowJo software was used to analyze the data, with isotype controls used to properly position the analysis quadrants and obtain relative fluorescence indices (RFI: specific surface molecule fluorescence index/specific isotype control fluorescence index). Each sample was performed in duplicate.

2.2.4Evaluation of RANK and CD105 co-expression in CAFs isolated from fresh breast tissue and paraffin samples by immunofluorescence

CAFs isolated from breast tumor tissue were seeded onto slides at a density of 350,000 cells/ml (from 4^th subculture). They were then fixed with methanol for 15 minutes and subsequently hydrated with 0.1% TBS-TW20 for 10 minutes. To perform double-label immunofluorescence, firstly, they were incubated over-night with the primary anti-RANK Ab (#MAB683, RyD Systems). On the second day, extensive washes were carried out with 1X PBS, followed by incubation with a secondary anti-mouse Ab labeled with Alexa 488 (715545150, Jackson-immunoresearch). Then, overnight incubation was performed with the primary anti-CD105 Ab (#AF1097, RyD Systems). On the third day, two washes with 1X PBS with 0.1% Tween 20 were done, followed by incubation with a secondary anti-goat Ab labeled with Alexa-647 (705605147, Jackson Immunoresearch). Subsequently, counterstaining was done using DAPI, followed by a wash with distilled water, and finally, the samples were mounted using Vectashield. CAFs expressing RANK (+) and CD105 (+) were visualized using a confocal microscope (40X). To perform immunofluorescence on breast tumor tissue embedded in paraffin, the same procedure was followed, with the difference that the initial processing of the sample was carried out as previously mentioned in the retrospective study [10]. Each sample was performed in duplicate.

2.2.5Quantitative RT-PCR

CD34(-) stromal cells (CAF-like) for the 4^th subculture was again seeded at 3,000 cells/cm² to obtain enough cells density for the RNA isolation. The culture conditions were as follow: i) 10 ml of supplemented α-medium with 5% FBS, and ii) 10 ml of supplemented α-medium with 5% FBS and 25 ng/ml hrRANKL. The culture was maintained until 70–80% of confluence at 37^∘C and 5% CO2 humidified environment. Subsequently, total RNA was extracted from 3 × 10⁵ cells using EasyPure RNA kit (#ER101-01, Transgen biotech, Beijing, Chinese). A total of 1 μg of RNA was reverse-transcribed into cDNA using random primers (#4368814, High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Foster City, CA, USA). Samples were analyzed using FS UNIVERSAL SYBR GREEN MASTER ROX master mix (cat. 04913850001, ROCHE, Mannheim, Germany) on a CFX96TM TOUCH REAL-TIME PCR system (Bio-Rad, Hercules, CA, USA) following standard cycling conditions and a subsequent melting curve analysis. The threshold cycle (Ct) values were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the data are presented as the fold change in gene expression of OCT4, SOX-2 and DKK-1. Primer sequences are provided in Supplementary Table 4. Each sample was assayed in duplicate.

2.2.6Fibroblastic colony-forming unit (CFU-F) assay

Isolated CD34(-) stromal cells (CAFs-like) were plated at a density of 100 cells of 4^th subculture/cm² in 25 cm² culture flasks containing: i) 10 ml of supplemented α-medium with 5% FBS and ii) 10 ml of supplemented α-medium with 5% FBS and 25 ng/ml hrRANKL and iii) 10 ml of supplemented α-medium with 20% FBS (standard positive control). Cells were incubated in a humidified environment at 37^∘C with 5% CO2 for 7 days. After this period, the culture medium was refreshed with/without hrRANKL. After another 7 days, stromal cells were washed twice with PBS, fixed with 100% methanol (Merck, Darmstadt, Germany) for 15 minutes, and finally stained with pure Giemsa (cat. 48900 Sigma, Biopure, St. Louis, MA, USA) for 5 minutes at room temperature. Colonies containing more than 50 spindle-shaped cells were identified under a microscope and recorded as CFU-F. The frequency of CFU-F was represented as the colony-forming efficiency, calculated as the number of CFU-F obtained for every 2,500 seeded CAFs. Each sample was performed in duplicate. The number of CAFs per CFU-F field (referred to as stromal cell density) was calculated by capturing ten images of different CFUF culture fields and processing them with FIJI software [42]. Morphological changes in CFU-F cultures were also evaluated. Analysis of area, ellipse longitudinal, and horizontal axis values were carried out using three pictures obtained from three typical regions (three optical fields, 200X) of each CFU-F culture, evaluating 10 cells per photo and analyzed by FIJI Software [43].

2.2.7Proliferation assay

Viable CD34(-) fibroblasts were seeded at a density of 5 × 10³ cells per well and cultured in 96-well plates (cat. 4430100, Orange Scientific, Belgium) with 200 μl of supplemented α-MEM with 20% FBS for 24 hours. Subsequently, the cultures were washed with PBS and incubated for 48 hours in α-MEM supplemented without phenol red and FBS (α-MEM without RF, cat. 41061029, Gibco, Grand Island, NY, USA). Finally, the cells were washed with PBS and incubated for an additional 48 hours at 37^∘C, 5% CO2, and humidity in: i) α-MEM without RF supplemented with 5% FBS, ii) α-MEM without RF supplemented with 10% FBS (i and ii positive control), iii) α-MEM without RF supplemented with 25 ng/ml hrRANKL and iv) α-MEM without RF and FBS (negative control). Cell proliferation was assessed using the Non-Radioactive CellTiter 96 AQueous Cell Proliferation Assay (cat. G5421, Promega, Madison, WI, USA) following the manufacturer’s instructions. Optical density (OD) was measured at 490 nm using a microplate reader. The value for each sample was calculated by subtracting the OD of the negative control from its respective value. The data was analyzed and plotted as percentage increase relative to baseline (negative control) All experiments were performed in triplicate for each sample.

2.2.8Migration assay

Experiments were conducted using transwell membranes with an 8 μm pore size (PI8P01250, Millipore) in 24-well plates. Each well was filled with i) supplemented α-medium with 10% FBS (positive control), ii) supplemented α-medium without FBS (basal control), iii) supplemented α-medium with 50 ng/ml hrRANKL (# GF091, RyD Systems), iv) supplemented α-medium with 50 ng/ml hrRANKL and 3.3 μg/ml anti-RANKL antibody (#MAB626, RyD Systems) or v) supplemented α-medium with 50 ng/ml hrRANKL and 5 μg/ml anti-RANK antibody (#MAB683, RyD Systems). In this particular assay, we use 50 ng/ml instead 25 ng/ml because we did not observe effect with this last dose. Subsequently, transwells were seeded with 4 × 10⁴ CD34(-) fibroblastic cells. The assay was stopped at 14 hours by fixing the porous membranes in pure methanol for 10 minutes at room temperature. Then, the membranes were washed with water, and the remaining non-migrated cells on the membrane surface were removed with a wet cotton swab. The membranes were allowed to dry, stained with 0.05% crystal violet for 10 minutes, and subsequently observed under an inverted fluorescence microscope, where photos of 5 defined fields were taken at a magnification of 200X. The migrated cells were counted using Image J software. Each sample was performed in duplicate.

2.2.9Statistical analysis

Results were presented as the mean ± standard error (SE). Data normality was assessed using the Shapiro-Wilk test. Parametric data were analyzed using an unpaired t-test with Welch correction to determine differences between groups. All statistical tests were two-tailed. Statistical analysis was performed using GraphPad 8 Prism software (GraphPad Prism version 8.01, GraphPad Software, La Jolla, CA, USA). Statistical significance was defined as p< 0.0500.

3.1.1Expression of RANK in spindle-shaped stromal cells, not associated with vasculature, and its association with the clinical-pathological characteristics of breast cancer patients

Out of a total of 155 BCPs diagnosed with invasive ductal breast cancer (stage I/II), 43 (27.75%) samples were found to have high RANK expression while 112 (72.25%) samples were found to have low RANK expression. Within the high expression group of RANK, the average percentage of fibroblasts expressing RANK was 46.90 ± 5.12% with an intensity of 1.89 ± 0.12. Whereas in the low expression group, the average percentage of RANK expression was 1.95 ± 0.28% with an average intensity of 0.38 ± 0.05.

There were no significant differences found regarding the association between RANK expression in spindle-shaped stromal cells, not associated with vasculature, “fibroblasts-like”, and clinical parameters such as ER, PR, Her2/neu status, tumor size, age, histological grade, and regional lymph node status (Table 1). However, it was found that RANK expression in these stromal cells was related to the occurrence of metastasis in BCPs at early stages (I/II). High RANK expression was significantly associated with a higher risk of developing metastasis (p= 0.006, Table 1). Although no significant association was found between RANK expression and the occurrence of metastasis at specific sites, there was a tendency of association with bone metastasis events. RANK expression was also associated with the number of metastatic foci per organ (p= 0.005) (Table 2). Patients with high RANK expression had multiple foci within the same organ (Table 2). RANK expression was significantly related to MFS, BMFS, MMFS, and OS (p= 0.033, p= 0.003, p= 0.042, and p= 0.025, respectively) (Fig. 1). Patients with high RANK expression had shorter times for MFS (169.59 ± 9.55 months), BMFS (186.65 ± 10.53 months), MMFS (176.95 ± 2.13 months), and OS (178.03 ± 8.99 months) compared to the group of patients with low RANK expression (205.05 ± 5.39, 261.28 ± 1.5, 219.07 ± 9.88, and 226.67 ± 6.58 months, respectively) (Fig. 1).

3.1.2Expression of RANK in spindle-shaped stromal cells, not associated with vasculature, and its association with intratumoral stromal characteristics

The expression of RANK was associated with the percentage of intratumoral stroma, as well as with blood and lymphatic vascularization (p= 0.029, p= 0.013, and p= 0.006, respectively, Table 3). Among patients with high RANK expression, 58.14% had a high percentage of intratumoral stroma (Table 3). Regarding vascularization, high RANK expression was associated with a greater amount of blood and lymphatic vascularization (Table 3).

3.1.3Association between classical prognostic markers and tumor progression

The ER status, PR status, tumor size, and histological grade were significantly associated with a worse prognosis in BCPs. Patients with ER (-) had lower MFS, BMFS, VMFS, and OS (p= 0.0001, p= 0.001, p= 0.001, p= 0.0001, respectively). Additionally, patients with PR (-) had lower MFS, BMFS, and OS (p= 0.003, p= 0.013, and p= 0.001, respectively). Those patients with a tumor size > 2 cm had lower MFS, BMFS, VMFS, and OS (p= 0.0001, p= 0.003, p= 0.001, and p= 0.0001, respectively). Furthermore, BCPs with a high histological differentiation grade (G3) had lower MFS and OS (p= 0.002 and p= 0.006, respectively) (Data not shown).

Figure 2.

Forest plot showed odds ratios for the multivariate association between classical prognosis factors and RANK, and metastasis-free survival (A), bone metastasis-free survival (B), visceral metastasis-free survival (C), and overall survival (D) in early invasive ductal BCPs.

3.1.4Multivariate analysis

RANK expression was an independent prognostic factor for MFS and OS in our BCPs (p= 0.007 and 0.004, respectively) (Fig. 2).

3.2.1Phenotypic characterization of CD34(-) spindle-shaped stromal cells isolated from BCPs

Figure 3.

Phenotypic characterization of CD34(-) CAFs. A. Representative flow cytometry surface antigen histograms of CAFs from a representative BCP. Isotype control (). B. Co-expression of RANK and CD105 in CAFs of breast tumors. A representative dot plot of RANK-CD105 co-expression, CD34-CD105, and CD34-RANK in fibroblasts isolated from the primary tumors of BCPs (I/II). C. Dual staining of RANK (green) and CD105 (red) by immunofluorescence in CD34(-) fibroblasts isolated from the primary tumors of BCPs. Counterstained with DAPI. Magnification 400X. The scale corresponds to 50 μm. D. Dual staining of RANK (green) and CD105 (red) by immunofluorescence in paraffin-embedded breast tissue from BCPs. Magnification 200X. The scale corresponds to 200 μm.

Phenotypic analysis conducted through flow cytometry revealed that the studied CD34(-) spindle-shaped stromal cells exhibited the expression of markers associated with both CAFs and MSCs. We observed that our cell population had an expression of 97.19 ± 1.13% for CD90, 95.85 ± 0.95% for CD73, 20.42 ± 3.18% for CD105, and 66.91 ± 6.89% for FAP (Fig. 3). Regarding the RANK marker, we found that it was expressed in 37.14 ± 8.36% (Fig. 3). As for the markers CD34, CD14, and CD19, we found a low percentage of expression, considered as negative (1.51 ± 0.26%, 2.25 ± 0.62%, and 2.68 ± 0.44%, respectively) (Fig. 3). Furthermore, it was identified that 1516 ± 3.29% of the stromal cells isolated from the primary breast tumor co-expressed both RANK and CD105 markers (Fig. 3, panels B and C). This same co-expression was evident in breast cancer tissue embedded in paraffin (Fig. 3, panel D).

Figure 4.

Effect of RANKL-RANK system on the self-renewal, proliferation abilities, expression of pluripotency factors and migration of CD34(-) CAFs in BCPs. A. Gene expression of self–renewal and pluripotency factors in CAFs from BCPs treatment with and without RANKL. Expression of OCT-4, SOX-2 and DKK-1 by quantitative real-time polymerase chain reaction (RT-PCR). All the results were normalized against a set of reference genes. B. CFU-F Assay: The self-renewal capacity of CD34(-) CAFs from BCPs was assessed in αMEM basal medium supplemented with 5% fetal bovine serum (FBS) and in the presence of hrRANKL (25 ng/ml). The CFU-F assay was also conducted in the presence of 20% FBS as a standard positive control (#CFU-F/2500 CAFs =29.22 ± 5.11). C. Stromal cell density per optical microscope field in each CFU-F. D. Area of stromal cells in typical regions of each CFU-F culture. E. Length of stromal cells in typical regions of CFU-F cultures. F. Width of stromal cells in typical regions of CFU-F cultures. G. CFU-F size observed for a representative BCP in supplemented αMEM added with 5% FBS and in supplemented α MEM added with 5% SBF + 25 ng/ml hrRANKL. Giemsa staining (40X). H. Proliferation of CD34(-) CAFs in BCPs was evaluated in supplemented αMEM added with 5% and 10% SBF without hrRANKL, and in the presence of 25 ng/ml hrRANKL. Percentage increase relative to baseline (negative control) is plotted. All values are expressed as mean ± SE. Unpaired t-test with Welch’s correction was used for statistical analysis. Asterisks indicate a significant difference (^{*p <} 0.0500). I and K. Migration of CD34(-) CAFs in BCPs was assessed in supplemented basal medium (α MEM) with I) 10% FBS (positive control), II) 50 ng/ml of hrRANKL and III) 50 ng/ml of hrRANKL and 3.3 μg/ml of anti-RANKL Ab. Values are expressed as mean ± standard error (SE). Unpaired t-test with Welch’s correction was used for statistical analysis, (^**p= 0.0089). J and L. Migration of CD34(-) CAFs in BCP was assessed in supplemented basal medium (αMEM) with I) 10% FBS (positive control), II) 50 ng/ml of hrRANKL and III) 50 ng/ml of hrRANKL and 5 μg/ml of anti-RANK Ab. Values are expressed as mean ± standard error (SE). Unpaired t-test with Welch’s correction was used for statistical analysis, (^**p= 0.0049).

3.2.2Gene expression of self-renewal and multipotency

We observed that CD34 (-) fibroblasts treated with 25 ng/ml hrRANKL had increased expression of genes related to cell multipotentiality as well as self-renewal, such as OCT4, SOX-2 and DKK-1, compared to CD34(-) fibroblasts that did not receive treatment (Fig. 4, panel A).

3.2.3Capacity to generate fibroblastic colony-forming units

We did not find differences in the capacity to form CFU-F between CD34(-) spindle-shaped stromal cells treated with αMEM + 5% FBS + 25 ng/ml hrRANKL and those without RANKL treatment (αMEM + 5% FBS). (Fig. 4, panel B and G). Additionally, no differences were observed in the number of stromal cells per optical field of CFU-F (stromal cell density), cell area, or ellipse longitudinal, and horizontal axis (Fig. 4, panel C, D, E and F). Thus, in our experimental conditions in the presence of 5% of FBS, hrRANKL does not promote CD34(-) spindle-shaped stromal cells self-renewal and proliferation inside the CFU-F.

3.2.4Proliferation capacity

Regarding the proliferation analysis performed using the MTS assay, we observed an increase in CD34(-)spindle-shaped stromal cells proliferation when we cultured these stromal cells with 25ng/ml hrRANKL alone. However, the stimulating effect of hrRANKL was lower than the values obtained with FBS (5% and 10%) (Fig. 4, panel H).

3.2.5Migration capacity

The CD34(-) spindle-shape stromal cells had migratory capacity when stimulated with 50 ng/ml of hrRANKL. Furthermore, this migratory capacity was inhibited when adding an anti-RANKL Ab to the assay (Fig. 4, panel I and K). Similarly, when the study was conducted with the blocking of the RANKL receptor, RANK, using an anti-RANK Ab, a similar reduction in migration was observed (Fig. 4, panel J and L). Thus, in our experimental conditions, hrRANKL promoted CD34(-) spindle-shaped stromal cells migration.