MACL-1 Breast Cancer Secretome Reprograms Fibroblasts to Enhance Inflammation and Tumor Progression

Abstract

Tumor-secreted factors reprogram the stromal microenvironment to facilitate cancer progression. This study investigates the effects of the MACL-1 breast cancer cell secretome on CCD19-Lu fibroblasts. Fibroblasts treated with the MACL-1 secretome exhibited increased proliferation, altered morphology, and upregulated expression of vimentin, NFκB, and fibroblast-activating protein, indicative of an inflammatory, activated phenotype. Additionally, elevated mitochondrial superoxide levels and an enrichment of proteases were detected in the secretome, but not in corresponding cell lysates. These findings suggest that the MACL-1 secretome promotes fibroblast activation and inflammation, underscoring its potential role in tumor-stroma interactions and breast cancer progression.

Key words
Breast cancer; MACL-1 cells; CCD19-Lu fibroblasts; Vimentin; Oxidative stress

INTRODUCTION

The initiation and progression of cancer result from complex alterations in signaling pathways involving oncogenes and tumor suppressors. Among these changes, the tumor microenvironment plays a pivotal role in increasing the phenotypic plasticity of cells, including fibroblasts. These modified fibroblasts, known as cancer-associated fibroblasts (CAFs), acquire unique characteristics, including expression of molecules such as fibroblast activation protein (FAP), vimentin, desmin, tenascin C (TNC), and periostin (Augsten 2014, Polyak & Weinberg 2009).

In addition to fibroblasts, other cells within the tumor microenvironment secrete a variety of substances. Collectively, these secreted factors are termed the “secretome,” which encompasses soluble biomolecules (Zhao et al. 2023, Smart et al. 2013 ). Tumor cells rely on a sophisticated arsenal, and the tumor secretome plays a critical role in shaping the cancerous landscape.

Breast cancer cell lines have been instrumental in studying the secretome and its contribution to malignancy (Vargo-Gogola & Rosen 2007, Davis et al. 2014). However, many studies have focused on advanced-stage tumor cells (Yao et al. 2012, Swartz et al. 2012). The MACL-1 cell line, derived from primary human breast tumors, offers a unique model for studying tumor progression from its initial stages, particularly in terms of how tumor cells determine the transformation of nearby fibroblasts into CAFs (Hendrayani et al. 2014, Erez et al. 2010).

Normal fibroblasts within the breast stroma can be activated into CAFs by various substances, including cytokines secreted by cancer cells, further contributing to this transformation (Polyak & Weinberg 2009; Kalluri & Zeisberg 2006). In this study, we explored the ability of MACL-1 breast cancer cells to modify fibroblasts via their secretome and identified the molecular factors responsible for these alterations.

MATERIALS AND METHODS

Cell lines and cell culture maintenance

The MACL-1, MCF-10A, MDA-MB-231, and CCD-19 Lu cell lines were cultured following the guidelines provided by the American Type Culture Collection (ATCC). Cells were passaged when they reached approximately 80-90% confluence, and the culture medium was replaced every 2–3 days.

Transwell® Co-culture

MACL-1, MCF10A, MDA-MB-231, and CCD19-Lu cells were collected using trypsin (Gibco), centrifuged at 800 rpm for 5 min, and the pellets were resuspended in 1 mL DMEM (Sigma-Aldrich). After cell counting in a Neubauer chamber, cells were plated in triplicate on Transwell® plates (Corning) with 0.4 µm polyester membranes at 1.0 x 10⁴ cells/well. The cultures were maintained in a humid incubator with 5% CO₂ at 37°C for 5 days. Cells were observed and imaged daily with an inverted microscope (Zeiss Axiovert 200M, Carl Zeiss).

Cell Labeling for Fluorescence and Immunocytochemistry

After discarding the culture medium, cells were washed twice with 1x PBS and fixed with 4% paraformaldehyde for 1 h at room temperature. Cells were labeled with Alexa Fluor 488-conjugated phalloidin (Invitrogen), DAPI (Sigma-Aldrich), and a FAP labeling kit (Dako) following the manufacturer’s protocols. Fluorescence and conventional microscopic analysis were performed using a Carl Zeiss AxioVision Rel. 4.8 software.

Extraction of the Secretome from MACL-1 Cells

Upon reaching 80-90% confluence, MACL-1 cells were washed five times with 1x PBS, and high-glucose DMEM (Sigma-Aldrich) without supplements was added. The cells were then incubated in 5% CO₂ at 37°C for 24 h. The medium was collected, filtered (0.22 µm, Millipore), concentrated using a SpeedVac™ concentrator (Thermo Scientific), and desalted using a PD-10 Desalting Column (GE Healthcare). The protein concentration was measured using a 2D Quant Kit (Amersham Biosciences).

Extraction of Intracellular Proteins from MACL-1 and CCD19-Lu Cells

MACL-1 and CCD19-Lu cells at 80-90% confluence were washed thrice with ice-cold PBS, detached, and centrifuged at 830 rpm for 5 min. The pellets were resuspended in lysis buffer (Urea 8 M, Thiourea 2 M, CHAPS 4%, DTT 65 mM, Tris 40 mM, Bio-Lyte 0.2%, AEBSF 2 mM, EDTA 1 mM, Bestatin 130 µM, E-64 14 µM, Leupeptin 1 µM, and Aprotinin 0.3 µM) in a 1:3 (v/v) ratio and agitated for 2 h at room temperature. The solution was sonicated and centrifuged at 20,000 × g for 30 min, and the supernatant was collected. The protein concentration was determined using a 2D Quant Kit (Amersham Biosciences).

Cytokine Quantification by Cytometric Bead Array (CBA)

Cytokines (IL-2, IL-4, IL-6, IL-10, TGF-β, and TNF-α) in the supernatant of CCD19-Lu fibroblasts exposed or not exposed to the tumor secretome were quantified using the Cytometric Bead Array™ kit (BD Biosciences) according to the manufacturer’s instructions. Samples were analyzed using a Fortessa BD™ cytometer (BD Biosciences), and the results were expressed as the mean fluorescence intensity.

SDS-PAGE and Western Blotting

Lysates from CCD19-Lu fibroblasts, both exposed and not exposed to the tumor secretome, were analyzed by SDS-PAGE using 10% gels under non-reducing conditions. Proteins (35 µg per sample) were transferred to nitrocellulose membranes and blocked with 5% non-fat milk in PBS-Tween (0.3%) for 1 h. Membranes were then incubated with primary antibodies against desmin (1:500), vimentin (1:1000), and NF-κB (1:500) for 12 h. After washing, membranes were incubated with secondary antibodies and subjected to chemiluminescent detection (Amersham Biosciences).

MACL-1 Secretome Cytotoxicity Assay in CCD19-Lu Cells

CCD19-Lu cells were plated at 2.0 × 10⁵ cells/well in 96-well plates and exposed to different concentrations of MACL-1 secretome. After 24 and 48 h of incubation at 37°C with 5% CO₂, the cells were washed, labeled with the Live/Dead® kit (Molecular Probes), and analyzed by fluorescence microscopy (Zeiss Axiovert 200M, Carl Zeiss). Positive control groups were treated with hydrogen peroxide (2 mM).

Quantification of Mitochondrial Superoxide Production in CCD19-Lu Fibroblasts

CCD19-Lu cells were plated at 2.0 × 10⁵ cells/well in 96-well plates and incubated with MitoSOX® Red (5 µM, Molecular Probes) in HBSS for 30 min at 37°C. After washing with PBS, the cells were exposed to the MACL-1 secretome. Mitochondrial superoxide levels were quantified by flow cytometry (FACScan, Becton Dickinson).

SDS-PAGE with Copolymerized Gelatin Substrate

Electrophoresis with a gelatin-copolymerized polyacrylamide gel was performed as described previously. Samples included CCD19-Lu fibroblast lysate, lysate exposed to the MACL-1 secretome, and lysate exposed to the MACL-1 secretome plus 10 mM EDTA.

Statistical Analysis

Results are expressed as mean ± standard deviation. Statistical analyses included paired t-tests, Two-way ANOVA with Bonferroni post-test, Mann-Whitney, and unpaired t-tests with Welch’s correction, using Prism version 5.0.3 (GraphPad Prism Software). A p-value <0.05 was considered statistically significant.

RESULTS

MACL-1 secretome enhances CCD19-Lu cell proliferation and causes morphological changes.

CCD19-Lu cells co-cultured with MCF-10A or MDA-MB-231 cells showed similar proliferation rates to the control (Figure 1a and 1b). In contrast, CCD19-Lu cells co-cultured with MACL-1 cells exhibited a significant increase in cell numbers compared to the control (Figure 1b). Additionally, fibroblasts exposed to the MACL-1 secretome displayed morphological changes (Figure 2).

Figure 1
CCD19-Lu cells exposed to the secretome of MCF-10A, MACL-1, and MDA-MB-231 cells. (a) Representative microscopy images from the proliferation assay over time under the same conditions. DMEM represents the control condition. (b) Proliferation of CCD19-Lu cells cultured in a transwell system with secretomes from MCF-10A, MACL-1, and MDA-MB-231 cells on polyester (PET) membranes over 96 hours. Cell counts related to the control were performed using a Neubauer chamber, with data representing the mean ± SD from at least three independent experiments conducted in triplicate. ** p < 0.001; *** p<0.0001 and **** p<0.00001.

Figure 2
Morphological changes in CCD19-Lu fibroblasts exposed to the MACL-1 secretome. (a) Representative images of CCD19-Lu fibroblasts exposed to the secretome of MACL-1 breast cancer cells (CCD19-Lu+Sec) compared to normal fibroblasts (CCD19-Lu). (a,b,c and d) - Images were captured using lower magnification (20X) and higher magnification (40X). White arrows (Panel d) point to nucleoli. Scale bar: 20 µm.

MACL-1 secretome induces Vimentin, NF-κB, and FAP expression in CCD19-Lu cells

We found an increase in vimentin expression in the CCD19-Lu cells exposed to MACL-1 secretome compared to the control (Figure 3a and 3d). In contrast, desmin expression did not change (Figure 3a and 3c). Additionally, NF-κB expression was significantly higher in CCD19-Lu fibroblasts exposed to the MACL-1 secretome compared to those that were unexposed (Figure 3b and 3e). Fibroblasts exposed to the MACL-1 secretome also showed fibroblast-activating protein (FAP) expression, unlike the controls (Figure 4a and 4b).

Figure 3
Figure 3. Protein expression of vimentin, desmin, and NFkB in CCD19-Lu fibroblasts exposed to the MACL-1 secretome. (a-b) Immunoblot analysis depicting the expression levels of vimentin, desmin, and NFkB in CCD19-Lu fibroblasts exposed to the MACL-1 secretome (CCD19-Lu+Sec) compared to unexposed fibroblasts (CCD19-Lu). (c-e) Quantitative comparison of protein expression levels, represented as a bar graph. Data are expressed as mean ± SD. * p < 0.05.

Figure 4
Expression of fibroblast-activating protein (FAP) in CCD19-Lu cells exposed to the MACL-1 secretome. (a) Representative microscopy images from Transwell® assays showing CCD19-Lu fibroblasts not exposed to the MACL-1 secretome and stained with anti-FAP antibody. No detectable FAP expression was observed under these conditions (ai and aii). (b) Representative microscopy images from Transwell® assays illustrating CCD19-Lu fibroblasts exposed to the MACL-1 secretome and stained with anti-FAP antibodies. All cells exhibited positive FAP staining, indicating a consistent expression across the population (bi and bii).

MACL-1 secretome increases IL-6 expression and mitochondrial superoxide anion (O₂⁻•) in CCD19-Lu fibroblasts

Our results show that CCD19-Lu cells exposed to the MACL-1 secretome exhibited a significant increase in IL-6 expression (Figure 5), while levels of other cytokines, including IL-2, IL-4, IL-10, TGF-β, and TNF-α, remained unchanged (Data not shown).

Figure 5
Expression of IL-6 in cell culture supernatants. The graph represents IL-6 expression quantified by ELISA in the supernatants of cell cultures, including CCD19-Lu fibroblasts (Sec CCD19-Lu), MACL-1 cells (Sec MACL-1), CCD19-Lu fibroblasts exposed to the secretome of non-tumorigenic breast cells (Sec CCD19-Lu+MCF-10A), CCD19-Lu fibroblasts exposed to the secretome of breast tumor cells (Sec CCD19-Lu+MACL-1), and culture medium. Data are presented as mean ± SD. * p < 0.05.

To assess cytotoxic effects, we tested different concentrations of the MACL-1 secretome. At a concentration of 50 µg/mL for 24 h, cell viability remained high (Figure 6). However, concentrations ranging from 100 to 200 µg/mL caused a significant increase in cell death (Figure 6).

Figure 6
Impact of MACL-1 Secretome on CCD19-Lu Cell Viability. Representative fluorescence microscopy images showing CCD19-Lu fibroblasts stained with calcein-AM (green, live cells) and propidium iodide (red, dead cells) after exposure to different concentrations of the MACL-1 secretome (50 µg/mL, 100 µg/mL, 200 µg/mL) for 24 hours. (a) Positive control with 2 mM H₂O₂ showing complete cell death. (b) Negative control with DMEM showing primarily viable cells. (c-e) Progressive increases in red fluorescence (arrows) corresponding to dead cells were observed with rising secretome concentrations, with significant cell death at 200 µg/mL. (f) Quantification of live and dead CCD19-Lu cells exposed to H₂O₂, DMEM, and 50 µg/mL, 100 µg/mL, and 200 µg/mL of the MACL-1 secretome. Data are presented as mean ± SD (n ≥ 3). * p<0.05.

Reactive oxygen species (ROS) play a dual role in cancer progression, either promoting or inhibiting it. Before exposure, CCD19-Lu fibroblasts had low basal superoxide anion levels, as shown by fluorescence microscopy (Figure 7a). After exposure to the secretome, superoxide levels increased progressively, with red fluorescence intensifying over time (Figure 7b and 7c). This was further confirmed by flow cytometry, which revealed rapid superoxide generation in fibroblasts exposed to 50 µg/mL MACL-1 secretome within 1500 s (Figure 7d).

Figure 7
ROS generation in CCD19-Lu fibroblasts exposed to the MACL-1 secretome over time. (a-c) Representative fluorescence images showing reactive oxygen species (ROS) levels in CCD19-Lu fibroblasts exposed to 50 g/mL MACL-1 secretome at 0 s (a), 900 s (b), and 1500 s (c). ROS levels were detected using a fluorescent probe (MitoSox Red), with increased fluorescence intensity correlating with elevated ROS. (d) Quantitative analysis of ROS levels over time, expressed as a percentage. CCD19-Lu fibroblasts in the presence of 50 g/mL MACL-1 secretome exhibited a significant increase in ROS levels at 1500 seconds compared to earlier time points. Data represents the mean ± SD from at least three independent experiments. * Indicates statistical significance at p < 0.05.

Protease levels are significantly higher in the MACL-1 secretome compared to the cell lysate.

To examine the protein profile of the MACL-1 secretome and cell lysates, we performed polyacrylamide gel electrophoresis. As expected, no protein bands were observed in DMEM control (M). In contrast, distinct protein bands appeared in all other samples (Figure 8a). The MACL-1 secretome (SM) exhibited a range of proteins with varying molecular weights, many of which were not fully reflected in the cell lysates (IM). A comparison between CCD19-Lu fibroblast lysates exposed to the MACL-1 secretome (FS) and unexposed CCD19-Lu fibroblast lysates (FN) revealed clear differences in band number and density, highlighting the secretome’s impact on CCD19-Lu fibroblasts protein expression (Figure 8a).

Figure 8
Identification of proteases in CCD19-Lu fibroblast lysates by zymography. (a) SDS-PAGE electrophoresis of the secretome and cell lysates. (P) Protein ladder with molecular markers ranging from 220 to 10 kDa. (M) non-supplemented DMEM culture medium; (SM) MACL-1 cell secretome (30 µg/mL), (IM) MACL-1 cell lysate (30 µg/mL), (FN) lysate of CCD19-Lu fibroblasts not exposed to the MACL-1 secretome (30 µg/mL), and (FS) lysate of CCD19-Lu fibroblasts exposed to the MACL-1 secretome (30 µg/mL). Electrophoresis was performed for approximately 2 h at 100 V, followed by staining with Coomassie Blue. (b) (FN) Zymography of CCD19-Lu fibroblast lysates and (FSE) lysates from CCD19-Lu cells exposed to the MACL-1 secretome in the presence of 10 mM EDTA, and (FS) CCD19-Lu cells exposed to the MACL-1 secretome. The gel was prepared with 12.5% polyacrylamide-SDS in Tris-HCl pH 8.8 and 0.1% gelatin (protease substrate) incorporated into the gel. The electrophoresis was run at 100 V for approximately 2 h, followed by SDS removal using Triton X-100 (2.5%), incubation in 0.2 M phosphate buffer pH 8.0 at 37°C for 4 h, and staining with Coomassie Blue. The presence of protease activity is indicated by clear bands of proteolysis within the gel (Black arrows), corresponding to the degradation of the gelatin substrate.

For protease detection, we utilized a gelatin zymography assay, revealing two prominent bands at approximately 60 kDa in the FS sample, indicating significant protease activity (Figure 8b). No protease activity was detected in the FN and FS samples, as no bands were clearly visible (Figure 8b).

DISCUSSION

This study provides novel insights into the role of the MACL-1 breast cancer cell secretome in modifying fibroblast behavior, emphasizing its ability to induce fibroblast activation, morphological changes, and a pro-inflammatory phenotype. The MACL-1 secretome significantly upregulated markers such as vimentin, NF-κB, and fibroblast-activating protein (FAP) in CCD19-Lu fibroblasts. These markers are widely recognized as key indicators of fibroblast activation and are commonly associated with cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) (Zhao et al. 2023, Martinez-Outschoorn et al. 2010). Activated fibroblasts play a crucial role in remodeling the extracellular matrix (ECM), facilitating tumor growth, invasion, and metastasis. Our results align with previous studies, which have demonstrated that cancer cell secretome can reprogram fibroblasts into an activated state that supports tumor progression. This suggests that the MACL-1 secretome has the potential to influence tumor-stromal interactions, enhancing our understanding of how breast cancer cells influence their surrounding stromal cells.

In many cancers, ROS act as biochemical signaling, promoting cell proliferation, survival, and metastasis, as well as inducing an inflammatory environment. In our study, the increase in superoxide anion production correlates with the observed fibroblast activation and suggests a mechanism by which the MACL-1 secretome may promote fibroblast-mediated ECM remodeling and contribute to tumor progression. Interestingly, while ROS are often implicated in tumor suppression through DNA damage and cell death, our findings point to a more complex role, where superoxide anion may be driving fibroblast activation and promoting tumor-stroma crosstalk rather than inhibiting it.

Previous studies have demonstrated that NF-κB activation in fibroblasts contributes to the production of pro-inflammatory cytokines, growth factors, and proteases that support tumor growth and metastasis. Our findings suggest that the MACL-1 secretome may stimulate fibroblasts to adopt a pro-inflammatory phenotype, which could enhance the inflammatory microenvironment in breast cancer. These results are consistent with the emerging concept that fibroblast-mediated inflammation is a key driver of cancer progression, with activated fibroblasts secreting cytokines and growth factors that support the survival and metastasis of cancer cells (Zhao et al. 2023, Martinez-Outschoorn et al. 2010).

Another important observation was the expression of fibroblast-activating protein (FAP) in almost all fibroblasts exposed to the MACL-1 secretome. FAP is a cell surface enzyme involved in ECM remodeling and is considered a marker of activated fibroblasts in the TME. Its expression is associated with a fibrotic response, and FAP-positive fibroblasts have been shown to promote cancer cell invasion and metastasis. The presence of FAP in our study suggests that the MACL-1 secretome not only activates fibroblasts but also primes them to participate in the remodeling of the ECM, potentially contributing to a more permissive microenvironment for tumor growth (Choi et al. 2014, Smart et al. 2013).

Our study also observed a marked increase in protease expression in fibroblasts exposed to the MACL-1 secretome, a finding that points to the potential role of these enzymes in ECM degradation and remodeling. Proteases, such as matrix metalloproteinases (MMPs), are critical mediators of ECM turnover and are often upregulated in cancer-associated fibroblasts (Bauvois 2012, Valencia et al. 2014, Pavlides et al. 2012). The secretome-induced protease expression observed in our study could be involved in facilitating tumor cell invasion and metastasis. Interestingly, the protease activity was observed in the secretome but not in the cell lysates, suggesting that the secretory factors released by MACL-1 cells specifically trigger this proteolytic response in fibroblasts.

The results from this study highlight the dynamic interplay between cancer cells and fibroblasts, driven by soluble factors secreted by tumor cells. This interaction has been increasingly recognized as a key factor in the progression of solid tumors, including breast cancer (Marx 2008). The MACL-1 secretome appears to play a crucial role in reprogramming fibroblasts, promoting inflammation, oxidative stress, and ECM remodeling, which could ultimately contribute to tumor progression.

However, there are several limitations to this study that should be considered. First, our experiments were conducted using a single fibroblast cell line (CCD19-Lu), which may not fully represent the diversity of fibroblast subtypes present in the breast cancer stroma. Future studies could investigate whether the responses observed in CCD19-Lu fibroblasts are consistent across different fibroblast populations or in more complex in vivo models. Additionally, while we have identified key markers of fibroblast activation and inflammation, the precise signaling pathways and factors involved in the MACL-1 secretome’s effects remain unclear. Further investigation into the molecular mechanisms underlying these changes is necessary to identify potential therapeutic targets.

Our findings suggest disrupting the signaling between cancer cells and fibroblasts could limit tumor progression by preventing fibroblast activation and ECM remodeling, a great promise for cancer treatment by therapeutic targeting of tumor-stromal interactions. Future research should explore the potential of targeting specific secreted factors or signaling pathways involved in these interactions. Moreover, strategies aimed at reducing oxidative stress or blocking the inflammatory response in the TME could offer new avenues for cancer therapy (Smart et al. 2013).

In breast cancer, the tumor microenvironment is crucial for the onset and progression of carcinoma (Vargo-Gogola & Rosen 2007, Davis et al. 2014). The interactions between cancer cells and other cell types within the tumor microenvironment significantly influence tumor malignancy and affect both tumor and non-tumor cells (Kuzet & Gaggioli, 2016, Swartz et al. 2012). Fibroblasts, key components of the stroma, play essential roles in inflammation and tissue repair. In tumors, they can be linked to non-healing wounds, with approximately 80% of stromal fibroblasts in breast cancer adopting an aggressive phenotype (Polyak & Weinberg 2009) and can “reprogram” the tumor microenvironment (Pavlides et al. 2012). No significant changes in proliferation of fibroblasts exposed to MDA-MB-231 or MCF-10A cells secretome indicate no effect, which aligns with Smart et al. (2013), who noted that some cells in co-cultures exhibit higher metabolic activity, consuming more nutrients. Exposure of CCD19-Lu fibroblasts to the MACL-1 secretome significantly enhanced their proliferation, indicating that the secretome actively stimulates fibroblast growth. This finding aligns with previous studies reporting similar effects in MCF-7 tumor cells, likely mediated by the release of growth factors (Yao et al. 2012).

Morphological analysis revealed that normal fibroblasts, characterized by their thin, wavy shape and minimal intracytoplasmic fibers, differ from cancer-associated fibroblasts (CAFs), which often appear larger, fusiform, with pronounced intracytoplasmic fibers and prominent nucleoli (Giannoni et al. 2012, Pavlides et al. 2012). The morphological changes of fibroblasts exposed to MACL-1 secretome indicate the potential alteration in their phenotype. While desmin levels remained unchanged, which might be due to its role in late-stage cellular changes (Pavlides et al. 2012), vimentin, a marker of mesenchymal cells, increased significantly, suggesting a responsibility of phenotypic shift in fibroblasts, corroborating findings from other studies (Kuzet & Gaggioli 2016, Erez et al. 2010).

Fibroblast-activating protein (FAP) also increased in CCD19-Lu fibroblasts exposed to MACL-1 secretome, further indicating a shift towards a malignant fibroblast phenotype (Giannoni et al. 2012). Despite these changes, we did not detect TGF-β expression, possibly due to low secretion levels or limitations of our detection methods. However, IL-6 levels increased in the supernatant of fibroblasts exposed to MACL-1 secretome, suggesting a response to phenotypic changes, as reported in other studies (Martinez-Outschoorn et al. 2010, Kalluri & Zeisberg 2006). IL-6, a cytokine produced by CAFs, can promote cell proliferation and inhibit apoptosis by inactivating c-Myc (Smart et al. 2013).

The increase in NF-κB expression in fibroblasts exposed to MACL-1 secretome indicates activation of this transcription factor, which is known to promote inflammation and support tumor progression (Valencia et al. 2014, Erez et al. 2010). Elevated NF-κB levels are associated with various cancer-related processes and may contribute to the observed phenotypic changes. Additionally, ROS production increased in fibroblasts exposed to MACL-1 secretome, consistent with findings by Karin & Greten (2005), which can induce oxidative stress, leading to further changes in fibroblast phenotype and supporting the differentiation into CAFs, as observed by Martinez-Outschoorn et al. (2010).

In line with Giannoni et al. (2012), who reported that ROS induce epithelial-mesenchymal transition (EMT) and resistance through COX-2/NFκB/HIF-1 signaling, our results show that increased ROS production and NFκB activation in fibroblasts are linked to potential phenotypic changes. ROS can also lead to the release of matrix metalloproteinases (MMPs), which are associated with EMT and tumor progression (Davis et al. 2014). Our study observed protease activity in fibroblasts exposed to MACL-1 secretome, which decreased upon EDTA treatment, suggesting that MMPs are involved in substrate degradation (Pavlides et al. 2012).

Supporting the findings of Choi et al. (2014), it is believed that CAFs, through elevated expression of MMPs, may play a crucial role in the metastatic process by aiding tumor cell survival and proliferation at secondary sites during metastasis (Martin et al. 2013). This aligns with the “seed and soil” hypothesis, which suggests that the tumor microenvironment is essential for metastasis (Kuzet & Gaggioli 2016). However, the precise role of CAFs in tumor formation and progression requires further investigation. While much research has traditionally focused on tumor cells themselves, there is growing recognition of the tumor microenvironment’s co-mediating role in cancer development (Zhao et al. 2023, Martinez-Outschoorn et al. 2010). To address this, various techniques were employed to better understand the mechanisms driving phenotypic changes in fibroblasts exposed to MACL-1 tumor cell secretions, building on previous research (Davis et al. 2014, Smart et al. 2013).

Although this study provides insights into the phenotypic alterations in fibroblasts, it is important to recognize that the transforming potential of the secretome on normal fibroblasts remains an ongoing challenge. CAFs are known to be a heterogeneous population, often characterized by the expression of multiple markers. This heterogeneity arises from their diverse origins, with most CAFs derived from the activation of resident fibroblasts (Martinez-Outschoorn et al. 2010, Pavlides et al. 2012). Our findings support the notion that factors secreted by tumors contribute to this heterogeneity, as observed in the present study.

Study Limitations

We recognize that, like any scientific study, this work has its limitations. However, we believe these limitations do not undermine the value of our findings but instead provide valuable insights and highlight directions for future research. Below, we outline the major limitations of our study.

A key limitation of our study is that it predominantly describes the secretome of MACL-1 cells and its effects on fibroblast activation, without providing a comprehensive mechanistic analysis of the underlying pathways. While we have identified cytokines and factors that likely play a role in stromal activation, we would like to point out that further studies are needed to investigate the detailed molecular mechanisms. This would undoubtedly strengthen our conclusions and is an important avenue for future work.

The use of the MACL-1 cell line (Correa, Bertollo & Goes 2009), which is less widely characterized compared to other commercial breast cancer cell lines, presents another challenge. While we believe this cell line offers important insights into tumor-stroma interactions, its novelty means that many aspects of its molecular and phenotypic behavior remain underexplored. This presents both a limitation and an opportunity for further investigation, and we hope our study will encourage additional research using this cell line. While the role of epithelial-to-mesenchymal transition (EMT) in stromal activation is acknowledged, this study did not focus on a detailed examination of EMT in MACL-1 cells.

The study is based on in vitro experiments, which, while informative, cannot fully replicate the complexities of the in vivo tumor microenvironment. As such, while our findings provide useful insights into the effects of the MACL-1 secretome on fibroblast activation, the behavior of these cells in an actual tumor context may differ. Future studies incorporating in vivo models will be necessary to validate and expand upon the current findings.

In conclusion, while these limitations exist, we believe our study represents an important first step in understanding the role of the MACL-1 secretome in stromal activation. We are confident that our findings provide a valuable foundation for further research in this area, and we look forward to future investigations that will address the questions raised by this work.

Acknowledgements

This work was supported by Conselho Nacional de Pesquisa e Desenvolvimento Cientifico e Tecnológico (CNPq - #312939/2021-3 and INCT-INOVATOX to JSC), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), and Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES – Financial Code 001). CD received funding from CNPq scholarship. The funding bodies were not involved in the design of the study, data collection, analysis, interpretation, or writing of the manuscript.

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Publication Dates

• Publication in this collection
  04 Aug 2025
• Date of issue
  2025

History

• Received
  4 Feb 2025
• Accepted
  24 Apr 2025