Abstract
Background/Aim: Previous studies have demonstrated that breast cancer cells secrete exosomes into the tumor microenvironment, promoting tumor progression. However, the paracrine influence of noncancerous breast epithelial cells on the growth of triple-negative breast cancer (TNBC) cells has largely been overlooked. We hypothesize that exosomes from noncancerous breast epithelial cells are secreted into the tumor microenvironment, stimulating TNBC growth. Materials and Methods: Exosome-containing media were prepared using exosomes isolated from triple-negative patient-derived xenografts (PDX) or noncancerous MCF-10A breast epithelial cells and used to treat MCF-7 or TNBC cells. Exosome-containing media from MCF-10A cells were characterized using ELISA. Subsequently, MDA-MB-231 and MDA-MB-468 cells treated with the MCF-10A exosome-containing media, and their impact on proliferation, migration, protein expression and gene expression were analyzed using Alamar blue assays, wound healing assays, western blotting, immunofluorescence, and gene expression arrays, respectively. Results: Exosomes extracted from the PDX and MCF-10A cells stimulated the growth of all examined cell lines. The MCF-10A exosome-containing media expressed CD9 and CD63; however, the MDA-MB-231 and MDA-MB-468 cells treated with these media exhibited differential expression of these proteins. Exposure of MDA-MB-231 and MDA-MB-468 cells to the MCF-10A exosome-containing media stimulated their growth and migration. Exposure of MDA-MB-231 cells to MCF-10A exosome-containing media caused down-regulation of genes involved in cell-cell adhesion, DNA damage response, epithelial-mesenchymal transition drivers, tumor suppression, and up-regulation of the MYC oncogene. Conclusion: Secreted factors from noncancerous cells, identified as exosomes, induce cancer cell proliferation and prime the tumor microenvironment by enhancing disease progression-associated pathways.
The tumor microenvironment is a complex region characterized by a diverse ecosystem comprised of various cell types, such as immune cells, fibroblasts, resident host cells, and cancer cells (1-5). Recognition of the tumor microenvironment impact on cancer progression has garnered interest, which prompted researchers to develop targeted therapeutics in contrast to broad-spectrum chemotherapeutic agents. Within this niche, cancerous and noncancerous cell interaction is mediated by direct cellular contact, chemokines, growth factors, and extracellular vesicles (6). Exosomes are extracellular vesicles ranging in size from 30-300 nm and are known to be secreted by most cell types across a wide range of species (7). Exosomes carry a wide range of cargo, such as miRNA, functional mRNAs, cytokines, chemokines, and other regulatory molecules that can alter the recipient cell phenotype (7). Exosomes are characteristically enriched in a group of membrane tetraspanin molecules, including CD63, CD81, CD9, and HSP70, which are molecules associated with metastasis and motility, as well as signal transduction (8-10). Exosomes play an essential role in cell-cell communication and the formation of a prometastatic phenotype that promotes epithelial-mesenchymal transition (11).
Several studies have focused on the pathological influence of cancer cells on other cancer cells alike and the tumor microenvironment as a whole (12-14). Cancer cells have the proclivity to coopt normal cells in a manner that causes them to display malignant properties. In the context of triple negative breast cancer, it has been demonstrated that exosomes derived from these cells promote proliferation, angiogenesis, invasion, metastasis, and drug resistance (1, 15). Higher levels of several factors specific to TNBC have been shown to be released via exosomes, including but not limited to TSP1 (16), ITGB4 (17), and MALAT1 (18). These factors are categorically associated with enhanced migration, phagocytosis induction, macrophage polarization, and metastasis in both an autocrine and paracrine fashion. Although TNBC cells have been reported to secrete exosomes to self-promote tumorigenesis (1, 15), and promote proliferation and drug resistance in non-tumorigenic breast cells (19), the role of exosomes secreted by the noncancerous microenvironment has not been well studied. Because the role of adjacent noncancerous cells in the tumor microenvironment is poorly understood, we decided to study how exosomes secreted by noncancerous breast epithelial cells impact the biology of triple negative breast cancer cells.
Materials and Methods
Cell culture. Both MDA-MB-231 and MDA-MB-468 (TNBC epithelial cell lines) and MCF-10A (noncancerous epithelial cells) were purchased from American Type Culture Collection (ATCC) (Rockville, MD, USA). MDA-MB-231 and MDA-MB-468 cells were maintained in either DMEM (Thermo Fisher Scientific, Waltham, MA, USA) or RPMI-1640 media (VWR, Radnor, PA, USA) supplemented with 10% fetal bovine serum (FBS), 250 mg/ml Amphotericin B and 10,000 I.U./ml Penicillin/Streptomycin (Gibco, Thermo Fisher Scientific). MCF-10A cells were maintained in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F-12) (Gibco) and supplemented with 5-10% serum, 250 mg/ml amphotericin B, 10,000 I.U./ml penicillin/streptomycin (Gibco), 0.5 mg/ml hydrocortisone, 20 ng/ml EGF, and 10 μg/ml insulin. The cells were maintained in a tissue culture incubator in a humidified atmosphere of 5% CO2 and 95% air at 37°C. The cells were routinely sub-cultured every 2-3 days.
Isolation of exosomes from MCF-10A conditioned media using differential centrifugation. MCF-10A cells were cultured in standard growth media until they reached 70-80% confluency. After which, the media was decanted and replaced with conditioned media for 24-48 h. The conditioned media was prepared by replacing the standard growth media with DMEM/F-12 media supplemented with 0.5% exosome-depleted charcoal-stripped FBS (System Biosciences, Palo Alto, CA, USA) for 24-48 h. To isolate the exosomes, the media was harvested and subjected to differential centrifugation at 300-2,000 × g for 10 min followed by 60,000-100,000 × g from 2 h to overnight at 4°C. The supernatant was discarded, and the pellet (i.e., which contains the exosome fraction) was collected, washed, and resuspended in standard growth media supplemented with 0.5-2.5% exosome depleted FBS and used for further treatments. Hereafter this media will be referred to as exosome containing media. The exosome containing media was harvested and sterile filtered using 0.2 μm cellulose acetate filters. The exosome samples were analyzed using a ZetaView Nanoparticle tracking analyzer (Particle Metrix, Meerbusch, Germany) and ZetaView 8.06.01 software (Particle Metrix).
Exosome isolation from TU-BcX-4IC PDX model. As previously described, the TU-BcX-4IC primary cell line was established from the a 4IC patient derived xenograft (PDX) and maintained in DMEM supplemented with 10% FBS, insulin, non-essential amino acids (NEAA), minimal essential amino acids, antibiotic–antimycotic, and sodium pyruvate (20). The media from the 4IC PDX was generously donated by Dr. Matthew E. Burow. The Total Exosome Isolation Kit (Thermo Fisher Scientific) was used to extract exosomes from the media of either the TU-BcX-4IC PDX or MCF-10A cells according to the manufacturer’s instructions. The media was harvested and differentially centrifuged at 300 × g for 10 min and then 2,000 × g for 10-30 min to remove the cells and debris. The supernatant containing the cell-free culture media was transferred to a new tube, mixed with the Total Exosome Isolation reagent, and then thoroughly mixed until it was a homogenous mixture. The samples were incubated overnight at 4°C, and on the following day were centrifuged at 10,000 × g for 2 h at 4°C. The supernatant and pellet containing the exosomes was retained and then resuspended in PBS. The PDX-derived exosomal nanoparticles were detected and measured using the NanoSight (Malvern PanAnalytical, Malvern, UK).
ELISA. Exosome pellets were isolated from the MCF-10A conditioned media as described above. ExoELISA kits (System Biosciences) were used to detect tetraspanins reported to be enriched in exosomes: CD9, CD63, and CD81. Analyses were performed on exosome pellets and supernatant. The exosome count was extrapolated using a standard curve made from the provided ExoELISA protein standard. The ExoELISA protein standard was calibrated using NanoSight technology to enable the precise quantitation of exosomes. The absorbance of samples was measured at 450 nm. Data is reported as the mean±SEM of triplicate determinations.
Western blot analysis. MDA-MB-231 and MDA-MB-468 cell lines were cultured in either standard growth media supplemented with 0.5% exosome-depleted FBS (control) or exosome treated media supplemented with 0.5% exosome-depleted FBS (exosome containing media) for 48 h. Proteins were extracted using RIPA buffer and Halt protease inhibitor cocktail (Thermo Fisher Scientific). The resulting protein lysates were estimated using Pierce™ BCA protein assay kit (Thermo Fisher Scientific). Samples were denatured at 95°C for 5 min and 100 μg of total protein lysates were subjected to polyacrylamide gel electrophoresis and transferred to PVDF membrane at 70 V for 90 min. The membranes were blocked with 5% BSA for 2 h and incubated with the following primary antibodies: anti-CD9 (Rabbit IgG, D8O1A) (1:1,000) (Cell Signaling Technologies, Danvers, MA, USA), anti-CD63 (Rabbit IgG, E1W3T) (1:1,000) (Cell Signaling Technologies), anti-CD81 (Mouse IgG1, M38) (1:1,000) (Invitrogen, Rockford, IL, USA) overnight at 4°C. Afterwards, they were washed with 1X PBS Tween 20 and incubated in anti-rabbit or anti-mouse secondary antibodies at room temperature for 2 h. The Clarity Max Western ECL substrate was used to detect, and the Chemi Doc XRS imaging equipment (Bio-Rad, Hercules, CA, USA) was used to visualize the bands. The band density was analyzed using Image Lab software (Bio-Rad) and compared to vinculin which was used as a loading control.
AlamarBlue proliferation assay. The alamarBlue assay was performed as previously described (21). Briefly, the MDA-MB-231 or MDA-MB-468 cells were harvested and seeded at a density of 5,000 cells per well in a 96-well plate. The cells were allowed to attach for 24 h. On the following day, the cells were cultured in either standard growth media supplemented with 2.5% exosome-depleted FBS (control) or exosome treated media supplemented with 2.5% exosome-depleted FBS (exosome treated media) for 24, 48, and 72 h. The alamarBlue dye (Life Technologies, Grand Island, NY, USA) was added and incubated with the cells, after which the absorbance and background wavelengths were measured at 550 nm and 630 nm. The proliferative activity was measured using the following equation given as a percent value: Proliferative activity= [Absorbance of viable cells (cells cultured in standard RPMI media) − Absorbance of viable cells (cells cultured in conditioned media)]/Absorbance of viable cells (cells cultured in standard RPMI media) ×100%. Data are the mean ± the standard error of the mean (SEM) of triplicate determinations.
Immunofluorescent analysis. Approximately 30,000-40,000 MDA-MB-231 or MDA-MB-468 cells were seeded onto a coverslip in a 6-well plate and were left to attach overnight at 37°C and 5% CO2. The following day they were treated with either standard growth media supplemented with 2.5% exosome-depleted FBS (control) or exosome treated media supplemented with 2.5% exosome-depleted FBS (exosome treated media). Afterwards, they were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X 100 for 8 min at room temperature, and washed with PBS. Non-specific binding was blocked with 5% BSA in PBS-Tween 20 for 1 h at room temperature. Cells were then incubated overnight at 4°C with rabbit anti-Ki-67 primary antibody (1:250) (Thermo Fisher Scientific). The next day, they were washed three times with 1% BSA for 5 min each and subsequently incubated at room temperature for 2 h with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (2 mg/ml in 5% BSA; Invitrogen, Life Technologies, Carlsbad, CA, USA) in the dark. Afterwards the cells were washed with 1% BSA 3 times and stained with DAPI (Invitrogen, Life Technologies) for 15 min in the dark. Coverslips were mounted onto slides using Fluoromount-G (Invitrogen, Life Technologies) and the images were acquired using an Olympus BX41 microscope equipped with a DP72 CCD camera and DP2 software (Olympus, Center Valley, PA, USA). To ensure consistency, identical exposure settings were used for all fluorescence channels during imaging. No background fluorescence was observed in control cells processed without the primary antibody. The experiment was performed in triplicate using cells from at least three independent passages.
Gene expression arrays. MDA-MB-231 cells were cultured in either standard growth media supplemented with 2.5% exosome-depleted FBS (control) or exosome containing media supplemented with 2.5% exosome-depleted FBS (exosome treated media) for 48 h. Total RNA was extracted from cells using the RNeasy kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. Each array profiles the expression of a panel of 84 genes including seven internal controls and five housekeeping gene controls. For each array, 2 μg of RNA was reverse-transcribed into cDNA in the presence of gene-specific oligonucleotide primers using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) as described in the manufacturer’s protocol. The cDNA template was mixed with the appropriate ready-to-use PCR master mix (Bio-Rad). RT-qPCR was performed using the manufacturer’s protocols for the Tumor Metastasis (PAHS-028A) RT2 Profiler PCR Array (Qiagen). Relative gene expression was calculated using the 2−ΔΔCq method, in which Ct indicates the fractional cycle number where the fluorescent signal reaches the detection threshold. The “delta-delta” method uses the normalized ΔCt value of each sample, calculated using a total of five housekeeping gene control genes (18S rRNA, HPRT1, RPL13A, GAPDH, and ACTB) (22). Fold change values are presented as average fold change=2−(average ΔΔCt) for genes in treated samples, relative to control samples. Differences in gene expression between groups were calculated using Student’s t-test, in which ± 2-fold changes where p≤0.05 were considered significant. All experiments were performed with a minimum of three biological replicates.
Wound healing assays. Approximately 20,000-40,000 cells (MDA-MB-231 or MDA-MB-468) were seeded in cell culture inserts (Cat No: 80209; Ibidi, Fitchburg, WI, USA) within a 6-well plate and allowed to attach overnight at 37°C in a humidified 5% CO2 atmosphere. The next day, the inserts were removed to create a uniform wound, and cells were gently washed with PBS. The media was replaced with either standard growth media supplemented with 2.5% exosome-depleted FBS (control) or exosome containing media supplemented with 2.5% exosome-depleted FBS (exosome containing media). Wound closure was monitored by capturing images at 0, 24, and 48 h using an Olympus BX41 microscope equipped with a DP72 CCD camera and DP2 software (Olympus). Images of the wound were taken at three different locations along the scratch using a 10× objective to ensure reproducibility. The wound width was measured at multiple points, and the rate of closure was calculated as the percentage reduction in scratch width relative to the initial measurement at 0 h. All experiments were conducted in triplicate (n≥3), and the data were analyzed using GraphPad Prism Version 10.10 (Boston, MA, USA).
Statistical analysis. All in vitro results were confirmed in at least three independent experiments. The statistical significance of the results was analyzed using GraphPad Prism Version 10.10. Differences between groups were calculated using the student’s t-test and two-way ANOVA.
Results
Isolation of PDX-derived exosomes. Previous reports demonstrated that exosomes derived from breast tumors enhance the invasive and metastatic potential of adjacent breast cancer cells (19). Given that patient-derived xenografts (PDX) models more closely resemble the patient setting, we initially sought to isolate exosomes from the TU-BcX-4IC PDX media. This was chosen as it is a fully characterized PDX model for metaplastic breast cancer that is a clinically aggressive tumor exhibiting rapid growth in vivo, showing spontaneous metastases and chemotherapy resistance (20). Table I depicts the characteristics of the PDX tumor from which the exosomes were isolated from the media. The presence of exosomes was validated by measuring both the nanoparticle size and concentration using NanoSight, a nanoparticle tracking analysis instrument. The average nanoparticle size was 128.2±22.6 nm (Figure 1A), and the average concentration was 1.14×108/ml of media (Figure 1B), thus confirming the presence of exosomes.
Patient derived xenograft profile. This table presents the profile of the PDX model indicating the patient’s age, ethnicity, as well the clinicopathological features of the tumor including subtype, tumor grade, type, site of resection, lymph node (LN) involvement and treatment status.
Exosomal analysis of 4IC PDX media. (A) Indicates the concentration of nanoparticles derived from the TU-BcX-4IC PDX sample. (B) Analysis showing the intensity of the nanoparticles derived from the TU-BcX-4IC PDX sample using the NanoSight instrument as described in the methodology section.
PDX-derived exosomes drive the proliferation of breast cancer cells. After isolating and characterizing the exosomes from the conditioned media used to culture the TU-BcX-4IC PDX, we examined whether the exosomes could influence the proliferation of breast cancer cells. To measure proliferation, we exposed both hormone-dependent MCF-7 cells and hormone-independent triple negative MDA-MB-231 breast cancer cells to standard growth media, or exosome treated media from MCF-10A cells or the TU-BcX-4IC PDX model for 24-48 h and conducted a cell proliferation assay. As expected, the TU-BcX-4IC PDX-derived exosomes exhibited growth-promoting properties in both cell lines (Figure 2). Interestingly, compared to the MCF-7 cells, the MDA-MB-231 cells were more sensitive to the stimulatory effects of the TU-BcX-4IC PDX-derived exosomes. Given this finding, we investigated whether exosomes derived from MCF-10A noncancerous breast epithelial cells influenced either the MCF-7 or the MDA-MB-231 cells. While unexpected, the exosome containing media derived from the MCF-10A cells had a modest growth stimulatory effect on the MDA-MB-231 cells compared to standard growth media without causing a significant change in the MCF-7 cells.
Exosomes influence the proliferation of breast cancer cells. Alamar blue proliferation studies were conducted according to the methodology and either MCF-7 or MDA-MB-231 cells were exposed to standard growth media, or exosome-containing media from MCF-10A cells or TU-BcX-4IC PDXs.
Confirmation of the presence of putative exosomal markers in the conditioned media. Given the modest growth promoting effect that the exosomes derived from the MCF-10A cells had on the MDA-MB-231 cells, we characterized and quantified the composition of putative exosomal surrogate markers (i.e., CD9, CD63, and CD81) present in the conditioned media. As such, we performed an ELISA, where we analyzed the type and quantity of the exosomes in the conditioned media pellet and supernatant (Figure 3A). Of the total CD9+ exosomes, 63.5% (or 1.43×109 exosomes) were found in the pellet, and 36.5% (or 8.2×108 exosomes) were present in the supernatant. Regarding the CD63+ exosomes, 77.7% (or 1.63×1010 exosomes) were found in the pellet, and 22.3% (or 4.7×109 exosomes) were present in the supernatant. Interestingly, there were no CD81+ exosomes in either the pellet or the supernatant of the conditioned media, indicating that the exosomal profile of the MCF-10A cells was CD9+/CD63+/CD81−. Given these findings, we examined whether the exosome containing media derived from the MCF-10A cells could alter the exosome protein profile of the triple negative breast cancer cells. Thus, immunoblots were conducted where the cells were cultured in either standard growth media supplemented with 2.5% exosome-depleted FBS (control) or exosome-containing media supplemented with 2.5% exosome-depleted FBS (exosome treated media). In both cell lines, the control treated cells expressed CD9, CD63, and CD81; however, the exosome-containing media, caused a significant increase in the expression of CD9, CD63 and CD81 in the MDA-MB-468 cells while only CD9 was increased in the MDA-MB-231 cells (Figure 3B).
Exosome profile in supernatant, pellet, and triple negative breast cancer cells exposed to exosome-containing media. (A) Exosomes were extracted from noncancerous MCF-10A cells as described and the supernatant and the exosome pellet were retained for the quantification of CD9 and CD63 in both fractions using an ELISA. (B) MDA-MB-231 and MDA-MB-468 cells were treated with exosome-containing media for 48 h and assayed using immunoblot with antibodies directed against CD9, CD63, and CD81. Vinculin was used as an internal loading control. Representative immunoblots are shown. The graphs below depict the quantitative analysis showing the fold changes in CD9, CD63, and CD81 after normalizing to the loading control. Two-way ANOVA analysis was performed, and treatments were compared to control. Results are expressed as the mean unit ±SEM (****p<0.0001, **p<0.01), and data are representative from one of at least two independent experiments.
Exosomes from normal breast epithelial cells stimulate the proliferation of TNBC cells. While it is evident that triple negative breast cancer cells secrete exosomes which provide a survival advantage for breast cancer cells, the impact of exosomes secreted from the noncancerous tumor microenvironment is unclear. Based on our result in Figure 2, we studied the influence of exosomes secreted from noncancerous breast epithelial cells on the proliferation of triple-negative breast cancer cells. Therefore, we isolated exosomes from the noncancerous MCF-10A breast epithelial cells and resuspended them in standard growth media (i.e., exosome containing media). Afterwards, we exposed the MDA-MB-231 and MDA-MB-468 triple-negative breast cancer cell lines to the RPMI standard growth media or exosome containing media for 24, 48, or 72 h and measured cell proliferation. The exosome containing media significantly increased the growth of both cell lines (Figure 4A and B). As Ki67 is a surrogate marker for the proliferative index, immunofluorescent staining was conducted where both TNBC cell lines were exposed to exosome containing media and results indicated that Ki67 staining was more prevalent and intense when cells were treated with exosome containing media (Figure 4C). To our knowledge, this is the first documented report demonstrating that exosomes secreted from noncancerous breast epithelial cells adversely impact the growth of triple-negative breast cancer cells from diverse cellular backgrounds.
Exosome containing media from normal breast epithelial cells promote the growth of triple negative breast cancer cells. Both (A) MDA-MB-231 and (B) MDA-MB-468 cells were cultured in exosome-containing media as described in the methodology and Alamar blue proliferation assays were conducted. The cells cultured in standard growth media supplemented with 2.5% exosome-depleted FBS (control) served as a control for cells cultured in exosome-containing media supplemented with 2.5% exosome-depleted FBS (exosome treated media). Significance was determined by comparing the two groups where, ****p<0.0001. (C) Immunofluorescence staining of Ki67 in MDA-MB-231 and MDA-MB-468 cells. Both cell lines were treated with either control or exosomes containing media for 48 h. The left panels show DAPI (blue) nuclear staining; the middle panels display anti-Ki67 (green) FITC staining; and the right panels present the merged images.
Exosome treated media promotes a novel gene signature associated with enhanced proliferation. Since noncancerous derived exosomes enhanced triple-negative breast cancer cell proliferation, we performed a focused gene array to investigate potential genes that may be involved or associated with this phenomenon. Given that triple-negative breast cancer cells are highly invasive, we chose to measure the expression of genes involved in metastasis by culturing the MDA-MB-231 cells in exosome containing media compared to cells cultured in standard growth media. Among the panel of 84 genes, 19 genes were significantly altered; 16 genes were significantly down-regulated (p<0.01) and three genes were significantly up-regulated (p<0.001). Table II depicts eight genes that were changed ± 2-fold. Most notable were that many genes associated with tumorigenesis were down regulated [i.e., genes involved in cell-cell adhesion (CDH1, CDH11), DNA damage response (CDH4), cell adhesion and tumor suppression (FAT1) (23), metastasis suppression (KISS1) (24), tumor necrosis factor (TNFSF10), and angiogenesis (VEGF)] while the MYC oncogene was up-regulated. This indicated that the exosomes derived from noncancerous cells further prime cells which are already aggressive for enhanced dysregulated growth.
Selected up- and down-regulated genes significantly altered by MDA-MB-231 cells exposed to MCF-10A exosome-containing media. MDA-MB-231 cells were cultured in standard growth media or exosome treated media from MCF-10A cells and a focused gene expression array was conducted as described in the methodology. Select genes with ± 2-fold are shown.
Noncancerous exosomes enhance the motility of triple negative breast cancer cells. Given the impact of the exosome containing media on the genetic signature of the MDA-MB-231 cells, we conducted a proof of principle experiment to determine whether the exosome treated media enhanced the migratory potential of the cells. To this end, both MDA-MB-231 and MDA-MB-468 cells were cultured in either standard growth media supplemented with 2.5% exosome-depleted FBS (control) or exosome containing media supplemented with 2.5% exosome-depleted FBS (exosome treated media) for 0, 24, or 48 h and results demonstrated that the exosome treated media significantly enhanced the migratory properties of both cell lines in a comparable manner (Figure 5), which may suggest that exosomes from noncancerous cells may play a more complex role in the tumor microenvironment than previously considered.
Exosome containing media from normal breast epithelial cells promote the migration of triple negative breast cancer cells. Both (A) MDA-MB-231 and (B) MDA-MB-468 cells were cultured in exosome-containing media as described in the methodology and wound healing assays were conducted. (C) The quantitated results of the wound healing assays are depicted. Significance was determined by comparing the cells cultured in standard growth media to those cultured in exosome-containing media for each cell line where, ++++p<0.0001, +p<0.05 where MDA-MB-231 exosome treated cells are compared to MDA-MB-231 controls. ***p<0.001 and *p<0.05 where MDA-MB-468 exosome treated cells are compared to MDA-MB-468 controls.
Discussion
The tumor microenvironment is a complex interconnected network of immune cells, cancer associated fibroblasts, pericytes, endothelial cells as well as many other cell types that cooperatively influence tumorigenesis. While for many years the tumor microenvironment was not considered to play a role in the pathogenesis of cancer exosomes in particular, play an essential role. Li et al. found that breast and gastric cancer cells enhance their own proliferation via the secretion of exosomes (25). Also, Greening et al., reported that MCF-7 cell-secreted exosomes promote tumor growth, pro-metastatic ECM degradation, and migration via a Rab27b dependent pathway (26). Thus, cancer-derived exosomes possess the ability to accelerate cancer progression. After isolating and characterizing the exosomes, we initially sought to validate this finding by culturing both MCF-7 cells and MDA-MB-231 cells in the conditioned media from an aggressive TNBC PDX which represented a stage 3 metaplastic carcinoma. As expected, the exosome containing media dramatically induced the proliferation of both cell lines compared to standard media (Figure 2). This supported the findings of others and suggested that exosomes carry cargo that influence the aggressive phenotype of TNBC (19, 27).
As tumor cells grow adjacent to noncancerous breast epithelial cells, we were interested in testing whether exosomes secreted from noncancerous breast epithelial cells present in the tumor microenvironment had any influence on cell-cell communication of TNBC cells. Upon characterization of the exosome profile of the MCF-10A cells, they were found to be CD9+/CD63+/CD81− which was similar to that found by us and others (28). The exosome-containing media derived from the MCF-10A cells was used to treat the triple negative breast cancer cells and the CD9/CD63/CD81 protein profile was examined. Interestingly, the conditioned media increased the expression of CD9, CD63, and CD81 in the MDA-MB-468 cells, but only CD9 was increased in the MDA-MB-231 cell. Positivity for the exosome surrogate markers confirmed that cells and the exosome containing media were enriched in exosomes that were within the expected exosomal size. While it remains unclear why not all surrogate markers are present in the pellet or in the MDA-MB-231 cells, previous reports indicate that overexpression of CD9 (i.e., which was present in all samples) is associated with invasiveness, tumorigenicity, and chemoresistance (29, 30).
The conditioned media from the MCF-10A breast epithelial cells were used to culture the MCF-7 and MDA-MB-231 cells and while the growth of both cell lines was increased, the MDA-MB-231 cells were more responsive to the proliferative influence of the noncancerous conditioned media (Figure 3). Although the exosomes secreted from the MCF-10A cells did not cause as much proliferation as the exosomes secreted from the PDX model, to our knowledge this is the first demonstration that exosomes secreted from noncancerous breast cells could drive the proliferation of ER+ and TNBC cells. Given this result, we examined whether this effect would extend to additional TNBC cells and if the response was temporary. As such, both MDA-MB-231 and MDA-MB-468 cells were cultured in the conditioned media from noncancerous breast epithelial cells which had a robust impact on proliferation in both cell lines compared to standard media (Figure 3). A focused gene array was conducted to provide mechanistic insights into the effect of the conditioned media from the noncancerous cells, and results indicated that the gene signature was associated with down-regulation of cell adhesion, DNA damage response, and tumor suppression, as well as with an increase in the MYC oncogene. Collectively these factors suggest the secreted factors from noncancerous cells carry cargo that initiates a cellular program that decreases cellular adhesion, prevents DNA repair and increases oncogenic activity contributing to an aggressive phenotype.
Conclusion
Although the role of exosomes from the noncancerous microenvironment is still poorly understood, our data suggest that these exosomes adversely impact the progression of this disease. Future studies will be required to examine the precise factors within the noncancerous exosomes that are responsible for altering the tumor microenvironment. Taken together, our findings provide insight into the possible mechanism by which secreted factors from noncancerous cells can influence tumor cells and may not function solely as innocent bystanders.
Acknowledgements
Research reported in this publication was supported by the National Institute of Health grant numbers U54MD008149, sub-award 13-14-MB-G007RN0A-XU-KPL, G12MD007595, SC1GM125617 and U54MD007582. Technical support was provided by use of the Cellular and Molecular Biology Core facilities, supported by funding from the Louisiana Cancer Research Consortium (LCRC). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the LCRC or the NIH. This project was also supported, in part, by funds from the Bureau of Health Professions (BHPr), Health Resources and Services Administration (HRSA), and the Department of Health and Human Services (DHHS) under grant number D34HP00006. This research was partly supported with funding from the U.S. Department of Education, Title III Part B, Strengthening Historically Black Graduate Institutions Programs (HBGI), awarded to Florida A&M University. The Authors declare they have no competing interests.
Footnotes
Authors’ Contributions
Conceptualization: K.P.L.; methodology: J.O., L.A.Y., M.K., A.O., B.B., and M.P.; formal analysis: L.A.Y., M.K., A.O., B.B., J.P., and S.L.T.; investigation: L.A.Y., P.R., J.J., K.P.; resources:, J.O., K.P.L. and S.L.T.; data curation: L.A.Y., M.K., A.O., B.B., M.P.; writing – original draft preparation: L.A.Y., M.K., A.O., B.B., S.L.T., and K.P.L.; writing – review and editing: A.O., S.L.T.; visualization: M.K.; supervision: K.P.L. and S.L.T.; project administration: K.P.L.; funding acquisition: K.P.L. and S.L.T. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received December 21, 2024.
- Revision received January 7, 2025.
- Accepted January 9, 2025.
- Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).