Patient samples were obtained from the tissue bank of the Unidad de Investigación en Virología y Cáncer, Hospital Infantil de México Federico Gómez, for which patients signed an informed consent, and the protocol was approved by the Scientific, Ethics, and Biosafety Institutional Review Boards. Tumor samples were taken from patients diagnosed with ductal carcinoma and with no previous neoadjuvant therapy before tissue resection.

The tumor tissue was rinsed with PBS and mechanically disaggregated with a scalpel in 1–2 mm fragments, which were subsequently digested for 2 h at room temperature (RT) with a mixture of 1 mg/mL collagenase type I and 100 U/mL hyaluronidase (both from Sigma; Saint Louis, MO, USA) in DMEM/F12 containing 100 U/mL penicillin and 100 μg/mL streptomycin, in constant stirring. The resulting suspension was filtered through a wide pore membrane and subsequently in a 100 μm pore membrane. The cells were pelleted and washed twice with sterile PBS and finally placed in their culture media. Cell morphology of primary isolates was analyzed and compared to the morphology of the commercial BrC cell lines in 2D (monolayer) and 3D culture conditions. Images were taken with an Olympus BX-41 microscope (Olympus Corporation, Tokyo, Japan). Primary cultures (PC) were given consecutive numbers; of 16 tumor tissues plated, eight PC were established for at least seven passages. These cultures were named UIVC-IDC-1, -4, -5, -6, -9, -10, -11, and UIVC-NIDC-1.

All commercial cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and culture media and supplements from Gibco Invitrogen Cell Culture (Carlsbad, CA, USA) unless specified differently. The human monocytic cell lines THP-1 (No. TIB-202), U937 (No. CRL-1593.2), and the BrC line T47D (HTB-133) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin, at 37°C in 5% CO₂. BrC cells MCF-7 (No. HTB-22) and MDA-MB-231 (No. HTB-26) were cultured in DMEM/F12 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37°C in 5% CO₂. BrC cells HS578T (No. HTB-126) were cultured in high glucose (4.5 g/L) DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37°C in 5% CO₂. MCF-10A cells (No. CRL-10317) were cultured in DMEM/F-12 medium supplemented with 20 ng/mL of epidermal growth factor (EGF; PeproTech, Rocky Hill, NJ, USA), 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin (all from Sigma Chemical Co., St. Louis, MO, USA), 5% horse serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Primary tumor cell isolates were cultured in DMEM/F12 supplemented with 5% horse serum, 100 U/mL penicillin and 100 μg/mL streptomycin, 100 ng/mL cholera toxin, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, and 20 ng/mL of EGF at 37°C in 5% CO₂ atmosphere.

Peripheral blood mononuclear cells (PBMCs) were isolated according to the following protocol: 40 mL of blood of healthy volunteers was extracted, diluted in a 1:3 proportion with sterile endotoxin-free PBS (Gibco Invitrogen Cell Culture), and subjected to density gradient centrifugation with Histopaque^®-1077 (Sigma-Aldrich Inc., St. Louis, MO, USA) per 30 min at 2,000 rpm. PBMCs were then carefully retrieved from the gradient and washed three times with PBS, each time followed by slower centrifugation (1,500, 1,250, and 1,000 rpm). To obtain the monocyte-enriched fraction, PBMCs were subjected to negative selection with the Monocyte Isolation Kit II Human (Miltenyi Biotec Inc., Auburn, CA, USA) following the manufacturer’s recommendations as we briefly describe next. PBMCs were washed once with diluted 1:20 MACS BSA Stock Solution (Miltenyi Biotec Inc., Auburn, CA, USA), counted, and adjusted to a density of 10⁷ cells per 30 μL of buffered solution; 10 μL of FcR-blocking reagent and 10 μL of monocyte biotin-antibody cocktail were then added for every 10⁷ cells to be labeled; cells were mixed and incubated for 15 min at 4°C. An additional 30 μL of buffered solution was added plus 20 μL of anti-biotin microbeads for every 10⁷ cells to be labeled; cells were mixed and incubated for 20 min at 4°C. Cells were then washed once with buffered solution, centrifuged at 1,500 rpm for 5 min, and resuspended in 1.5 mL of buffered solution for magnetic separation. Suspension of cells was passed through a pre-rinsed LS column (Miltenyi Biotec Inc., Auburn, CA, USA) and 7 mL of buffered solution was added. The monocyte-enriched cells were collected in a conical 15 mL tube, counted, and if not cultured immediately, monocytes were frozen at a density of 2 × 10⁶ in 1 mL of DMEM/F12 medium supplemented with 50% FBS and 10% DMSO at −80°C. PM used for experimentation were never frozen for more than 2 months after isolation. Cultures were maintained in DMEM/F12 medium supplemented with 6% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37°C in 5% CO₂ atmosphere. Each set of experiments was performed utilizing PM of at least two different donors (each donor isolates independently because pooling resulted in monocyte activation). The phenotype of purified monocytes was very homogeneous between different isolates: CD34^neg CD11b^pos CD14^pos CD64^pos CD68^neg CD16^neg, and also with both monocytic cell lines, which corresponds to the phenotype of a non-activated immature monocyte (15).

A total of 20,000 cells of the primary isolates were plated on 8-well chamber slides (Nunc^® Lab-Tek^® Chamber Slide System, Sigma), cultured for 48 h in their correspondent media, rinsed with PBS, and fixed with paraformaldehyde at RT for 10 min. Cells were then hydrated with PBS and permeabilized with 0.05% triton X-100 for 10 min at 4°C. Endogenous peroxidase activity was blocked by incubating the slides in peroxidase blocking solution (Dako, Inc., Carpinteria, CA, USA). Non-specific antibody binding was blocked by incubation with 8% albumin in PBS for 20 min. The slides were incubated with the following primary antibodies at an optimized concentration: mouse monoclonal anti-human anti-PanCytokeratin (Clone AE1/AE3; Biocare Medical; Concord, CA, USA), mouse monoclonal anti-human anti-mucin 1 (MUC-1) [epithelial membrane antigen (EMA; Clone Mc-5; Biocare Medical)], and mouse monoclonal anti-human anti-epithelial cell adhesion molecule (EpCAM, clone AUA1; Biocare Medical). Cells were incubated overnight in a moist chamber at 4°C. The EnVision Detection Kit (Dako; Carpinteria, CA, USA) was employed as the detection system. Cells were counterstained with hematoxilin for 5 min followed by 5 min 0.9% ammonium hydroxide at RT, rinsed with distilled water, and left to dry at RT and permanently cover slipped. Slides were analyzed and photographed with an Olympus BX-41 microscope (Olympus Corporation, Tokyo, Japan).

4 × 10⁵ cells from primary isolates were seeded in 4 mL of low glucose (1 g/L) DMEM medium (Gibco Invitrogen), with sodium pyruvate 110 mg/L, without HEPES and supplemented with 15% FBS, 1% l-glutamine, and 100 U/mL penicillin and 100 μg/mL streptomycin and incubated at 37°C in 5% CO₂. Cells were maintained for 5–7 days in the described conditions and their growth evaluated. Results were scored as none when no growth was observed, one cross when cells attached and formed ≈40% of a confluent layer, two crosses 80–90% of confluence, and three crosses when cells were passaged and proliferate in a new culture flask with mesenchymal cell medium. Bone marrow mesenchymal cells were used as positive control and MCF-10A cells as negative control.

For 3D individual cultures, a 40 μL base of Matrigel was added per well of an 8-well chamber slide system (8-well plates, Lab-Tek Chamber Slide System, Nalgene Nunc International, Rochester, NY, USA), incubated for 30 min at 37°C and 800 cells were added in 400 μl of the correspondent culture medium supplemented with 4 ng/mL of EGF and 2% Matrigel. For 3D co-cultures, 4 × 10⁵ monocytes contained in 1 mL of their correspondent medium supplemented with 2% Matrigel and 2% FBS for U937 and THP-1 monocytes, or 2% Matrigel and 6% FBS for PM, were plated per well of a 24-well flat-bottom culture plate. A transwell cell culture insert with pore size of 0.4 μm (Thermo Fisher Scientific™ Nunc^TM; Waltham, MA, USA) was placed in each well containing 1 mL of a 4 × 10⁵ BrC cells in their correspondent medium supplemented with 2% Matrigel and 2% FBS (for commercial cell lines) or 5% horse serum for PC. Controls of individual cell cultures with the same media were included. After 5 days of culture, the supernatants from bottoms and tops of the 3D co-cultures were recovered, mixed, aliquoted, and kept at −20°C until use.

The following analytes were determined in the supernatants of the cultures: granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage-colony-stimulating factor (GM-CSF), interleukin (IL)-1 beta (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17, interferon-alpha 2 (INF-α2), monocyte chemoattractant protein-1 (MCP-1) also known as chemokine CCL2, regulated on activation normal T cell expressed and secreted (RANTES) also known as chemokine CCL5, EGF, vascular endothelial growth factor (VEGF), and a panel of the MMP-1, -2, -7, -9, and -10. The determinations were done with the multiplexing assay platform from MILLIPLEX (EMD Millipore Corporation, Billerica, MA, USA) following the manufacturer’s recommended procedure. Briefly, in each well of a 96-well flat-bottom culture plate 25 μL of assay buffer was mixed with 25 μL of supernatants or controls and 25 μL of the detection microbeads cocktail. The mixture was incubated at 4°C overnight with orbital agitation. Wells were then washed twice with washing buffer, 25 μL of the detection antibodies mix was added to each well, and the plate was incubated at RT with orbital agitation for 1 h. After incubation, 25 μL of streptavidin-phycoerythrin was added to each well followed by 30 more minutes of incubation at RT with orbital agitation. The wells were then washed twice with washing buffer, 150 μL of PBS was added to each well to proceed with the analysis in Luminex MAGPIX multiplexing instrument, and the analysis of data was performed in the xPONENT^® Software. Three biological replicates were analyzed.

Migration assays with U937, THP-1, and fresh PM were performed in 24-well plates using polycarbonate membrane transwell inserts with 8-μm pores (Corning Costar, USA) filled with Matrigel. 1.5 × 10⁵ monocytes were resuspended in 200 μL of RPMI without serum and placed in the upper chamber. Then, transwells were placed in a 24-well culture dish containing 800 μL of RPMI without serum but supplemented with either 100 ng/mL of GM-CSF, MCP-1, or RANTES (all from PeproTech, Rocky Hill, NJ, USA) that was used as chemoattractant. Cell migration was allowed to progress for 24 h at 37°C in a humidified 5% CO₂ environment. The migrating cells were counted at 2, 4, 6, and 24 h using a microscope Motic AE31, and images were acquired with a digital camera (Moticam 5.0 MP). The mean cell count from three random fields (at 100× magnification) was used for the analysis. For the BrC cell lines invasion assays, a total of 6 × 10⁵ cells were resuspended in 200 μL of their respective media without sera and placed in the upper chamber of the Transwell filled with Matrigel (as for the monocytes). The Transwells were placed in a 24-well culture dish containing 800 μL of their respective media with sera or without sera but supplemented with 100 ng/mL of IL-8 as chemoattractant (PeproTech, Rocky Hill, NJ, USA). The experiment proceeded as for monocytes and after 48 h, invading cells were stained with crystal violet and observed under the microscope. The mean cell count from three random fields (at 100× magnification) was used for the analysis of Figure 6C and the integrated optical density (IOD) for the analysis of Figure S1 in Supplementary Material. Three biological replicates were performed.

3 × 10⁴ cells of each of the cell lines were seeded on coverslips for 24 h in their respective media. Then, cells were fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 in PBS for 20 min. Cells were blocked with blocking buffer (10% goat serum, 1% BSA, 0.2% triton X-100, and PBS 1X) for 1 h and then stained with the primary antibodies: mouse monoclonal anti-E-Cadherin antibody (Clone: 36/E-cadherin; BD Biosciences, San José, CA, USA), rabbit monoclonal anti-Vimentin antibody-Alexa Fluor-594 (Clone: EPR3776; Abcam, Cambridge, MA, USA), overnight at 4°C. After that, cells were incubated with the secondary antibody goat anti-mouse-IgG-FITC antibody (Sigma-Aldrich Co., St. Louis, MO, USA) for 30 min. Nuclei were stained with DAPI for 25 min. Cells were observed using a fluorescence microscope Olympus BX51, and images were acquired with a digital camera (Camedia C4040, Olympus).

Statistical comparison of values from the different conditions tested was performed with the GraphPad Prism 5 Software, using Kruskal–Wallis test and Mann–Whitney test to compare all data columns. Statistical significance (p) ≤0.05 was indicated with *, ≤0.01 was indicated with **, and ≤0.001 with ***.

We have previously generated evidence that aggressive BrC cells modulate the expression of monocytes to favor conditions for tumor invasion (14). We define the aggressive potential of BrC cells as follows: to present a cancer stem cell-like phenotype CD44⁺ CD24^−/low; to present the epithelial-to-mesenchymal transition (EMT), which is characterized by positivity to vimentin (mesenchymal marker) and negativity to E-cadherin (epithelial marker), and to be invasive in transwell assays. According to these characteristics, MCF-7 and T47D cells were classified as non-aggressive and MDA-MB-231 and HS578T cells as aggressive. This characterization correlates with the ability of MDA-MB-231 and HS578T cells, but no of MCF-7 and T47D cells, to form metastasis in immunodeficient mice (16–19). To extend our initial observations, we used three different types of monocytes: commercial U937 and THP1 monocytes, which were obtained from patients with diffuse histiocytic lymphomas and have been widely used as models of macrophage differentiation (20–23), and PM isolated from healthy donors. All the different monocytes presented an immature non-activated phenotype (15).

The co-cultures with the commercial BrC cell lines and monocytes were done in a Matrigel 3D matrix, after which the content of several cytokines, chemokines, and growth factors (G-CSF, GM-CSF, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17, INF-α2, MCP-1, RANTES, EGF, and VEGF) were measured in the supernatants. Consistent readings were obtained for the following analytes: RANTES, MCP-1, G-CSF, GM-CSF, IL-8, IL-1β, INFα2, and IL12p70, all related to inflammatory processes, although only the first six have been previously associated with pro-tumor effects. Figure 1A shows three independent assays of the three types of monocytes in co-culture with the commercial BrC cells. It can be observed that IL-1β and IL-8 levels were increased upon co-culture, while the opposite happened with INF-α2 and IL-12p70, which presented the highest levels in some of the monocytes cultured individually. The most relevant result observed was that of IL-1β and IL-8 (Figure 1B). In the case of IL-1β, we found statistical differences between co-cultures and individual cultures. Interestingly, the concentration of IL-1β also allowed distinguishing between co-cultures of the most aggressive BrC cells (average of 183.7 pg/mL) and the non-aggressive cells (94.0 pg/mL). Similarly, IL-8 basal levels (3D) perfectly segregate non-aggressive (average of 30.83 pg/mL) from aggressive tumor cells (average of 9,654 pg/mL). Also, the levels of IL-8 increased significantly when non-aggressive tumor cells were co-cultured with monocytes (average of 6,737 pg/mL), although we did not observe a significant difference between co-cultures of the BrC aggressive cells and co-cultures of the non-aggressive cells.

To extend these observations, the experiments were repeated using primary tumor cells isolated from BrC Mexican patients. The tumor cells were derived from eight female patients with an average age of 56.8 years (range 42–75). All tumors were classified as ductal, with seven out of eight already showing tissue infiltration and three patients already presenting lymph node involvement. Seven tumors were classified as histological grade II and one as grade III, and seven were clinical stages IIA or IIB and one stage I (Table 1).

For the duration of this study, PC were maintained for up to seven passages in mammary epithelial cell growth medium. We determined their identity as epithelial cells after positive staining to three well known epithelial markers: a panel of cytokeratins, mucin 1, and EpCAM (Figure 2A; Table 2). The primary tumor cells expanded in epithelial cell growth medium were also plated in medium specialized to grow mesenchymal cells, we thought that this was important to exclude the possibility of culture contamination with other resident cells of the tumor stroma that could have pro-tumoral activities. We found that cultures UIVC-IDC-1, -4, -6 and UIVC-NIDC-1 proliferated to different extents in mesenchymal medium (Figure 2B; Table 2). However, cells recovered after expansion in this medium still were positive for epithelial markers (Figure 2C). We concluded that cells expanding in this medium were not mesenchymal cells contaminating the primary isolates, but rather that these tumor epithelial cells acquired mesenchymal characteristics that allowed them to adapt to mesenchymal culture conditions. MCF-10A non-transformed cells were unable to expand in mesenchymal medium (not shown).

We assessed the morphology of cells in monolayer (2D) according to the capacity to form cobblestone or spindle-like shapes (Figure 3A; Table 2). Only culture UIVC-IDC-5, -10 and UIVC-NIDC-1 formed an organized monolayer with polygonal-shaped cells and well-delimited cell-to-cell interactions. Cells from the rest of the cultures had an elongated shape, formed less organized monolayers and in some regions cells stacked randomly on top of each other, a characteristic of cells that have lost contact inhibition. We also compared the 3D colonies of the primary isolates with the ones formed by MCF-10A, and the non-aggressive and aggressive cell lines. MCF-10A non-transformed cells form well-organized spherical structures in which cells are polarized with the basal side facing the extracellular matrix (ECM) and the basolateral side facing the hollow lumen of the sphere. Only cultures UIVC-IDC-5 and -11 formed medium size structures resembling spheres, cultures UIVC-IDC-6, -9, and -10 formed smaller structures and cultures UIVC-IDC-1, UIVC-IDC-4, and UIVC-NIDC-1 did not organized in any kind of recognizable sphere-like shape (Figure 3B; Table 2). Based on the 2D and 3D morphology, we conclude that the primary isolates more closely resemble the highly aggressive BrC cells, which is in contrast with most of the patients’ clinical profiles.

We co-cultured the primary BrC isolates with the PM. The results showed that MCP-1, G-CSF, RANTES, GM-CSF, INF-α2, and IL12-p70 analytes gave no differences between the 3D individual cultures and the 3D co-cultures (data not shown). However, IL-1β and IL-8, which in commercial lines allowed to distinguish between aggressive and non-aggressive BrC cells, showed again a significant increased concentration in co-culture (Figure 4). IL-1β increased from 3.3 pg/mL (individual 3D culture) to 16.5 pg/mL (co-culture), while IL-8 increased from 8,886.3 pg/mL (3D) to 12,027.4 pg/mL (CC).

Our initial findings also support that aggressive MDA-MB-231 cells promote secretion of MMPs in co-culture with U937 monocytes (14). To extend those results, we analyzed the levels of MMP-1, MMP-2, MMP-7, MMP-9, and MMP-10 present in the primary isolates individually cultured or co-cultured with PM. We did not find significant differences in the secretion levels of MMP-7 and MMP-9 (data not shown). Supernatants obtained from the primary isolates UIVC-IDC-1, UIVC-IDC-4, UIVC-IDC-6, and UIVC-NIDC-1 did not show detectable levels of MMP-7 and MMP-9. Primary culture UIVC-IDC-11 secreted the highest basal level of MMP-7 with 297,006 and 339,296 pg/mL in its correspondent co-culture. UIVC-IDC-11 was also the only primary isolate secreting detectable levels of MMP-9. More interestingly, MMP-1, MMP-2, and MMP-10 showed significant differences between co-cultures and individual 3D cultures (Figure 5). In the case of MMP-1 and -2, the basal levels in individual cultures were average of 19,264 pg/mL (MMP-1) and of 12,474 pg/mL (MMP-2), which in co-culture with monocytes was further increased 3- and 1.9-fold, respectively. For MMP-10, the average level found in supernatants from individual cultures was of 219.3 pg/mL and in the co-cultures with monocytes increased 2.6-fold.

In an attempt to understand the cytokine profile of tumor cells that promote an immunological shift or immunoediting in monocytes (24), we analyzed the basal expression levels of the commercial and primary BrC cells (Figure 6A). We found an interesting inflammatory profile promoted by the PC, which consisted of high concentrations of MCP-1/CCL2 (average of 9,277.09 pg/mL), G-CSF/SDF3 (average of 3,999.37 pg/mL) and RANTES (average of 953.19 pg/mL) and, lower levels of INF-α2 (average of 27.07 pg/mL) and IL-12p70 (average of 6.415 pg/mL). For GM-CSF, two cultures were polarized from the rest, UIVC-IDC-1 had 5,022.8 pg/mL and UIVC-IDC-9 had 573.6 pg/mL, the rest of the primary isolates ranged from 4.5 to 51.2 pg/mL. Interestingly, this profile is in good correlation with the profile found in the commercial BrC cell lines, with aggressive cells exhibiting higher levels of MCP-1 and G-CSF, and GM-CSF appreciably separating aggressive from non-aggressive cells. On the contrary, primary and commercial BrC cells showed low levels of INF-α2 and IL-12p70 (Figure 6A). Other cytokines were either or both, not significantly different between aggressive and non-aggressive cells or they had no detectable levels, such as IL-2 and IL-4 (data not shown).

An important observation taken from this experiment was that the most enriched cytokines are critical monocyte chemoattractants. Because of the increased secretion of MMPs, migration assays were performed with commercial and PM in response to GM-CSF, MCP-1, and RANTES in Matrigel filled Transwell inserts. We observed that U937 and THP-1 cells were capable to migrate in response to GM-CSF (average numbers of migrating cells/field were 115 for U937 and 11.5 for THP-1) and to MCP-1 (average numbers of migrating cells/field were 99.7 for U937 and 12.8 for THP-1) and low to null response to RANTES. PM showed high basal migratory properties, giving potent responses to MCP-1 (average number of migrating cells/field of 54,666.7). Although, we observed migration in response to GM-CSF and RANTES the number of migrating cells was not different than the control without chemoattractant (Figure 6B). U937 cells also exhibited migratory properties in the absence of chemoattractant (average number of migrating cells/field 10.7), although lesser than PM (18,666.7 cells). On the contrary, THP-1 cells were not intrinsically migratory in our experimental conditions even without Matrigel.

Interleukin-8 is another potent chemokine. Since IL-8 and MMPs are enriched upon co-culturing of the monocytes and the BrC cell lines, we decided to test whether IL-8 could facilitate invasion of the tumor cells. Figure 6C shows that the aggressive BrC cell lines responded to IL-8 (average number of invading cells/field were 11.5 for HS578T and 20.5 for MDA-MB-231 cells). We did not observe invasive properties in the absence of IL-8 or with the non-aggressive cell lines (Figure 6C). These data argue that the pro-inflammatory profile of the most aggressive BrC cells serve them to attract monocytes, and the BrC-monocyte association further enhances the tumor cell invasive properties.