Bioimpedance Spectra Confirm Breast Cancer Cell Secretome Induces Early Changes in the Cytoskeleton and Migration of Mesenchymal Stem Cells

Abstract

Mesenchymal stem cell (MSC) treatments take advantage of the ability of these cells to migrate to target sites, although they have been shown to move in response to tumor influence. Currently, tools are being developed to detect these opportune changes in cellular behavior patterns. No reports of such changes in the morphological patterns or migration of MSCs in the presence of a tumor environment, which would provide information of high diagnostic value, have been made. We determined the changes in the cytoskeleton and migration of MSCs exposed to the secretome of breast tumor cells via bioimpedance records. MSCs were cultured and incubated in the presence of 24 and 48 h secretomes of the MCF-7 tumor cell line. The proliferation, migration, morphology, cytoskeleton, and electrical bioimpedance were evaluated at 48 h for cells treated with 24 and 48 h secretomes. Secretomes induced early morphological changes related to the migration of MSCs, directly confirmed via bioimpedance, but no changes in cell proliferation were found. These changes cannot be related to a transformation or malignancy phenotype. The modification of the bioimpedance patterns recorded from the first hours suggests that this method can be applied in an innovative way to detect early changes in a cellular population in the clinical diagnostic setting.

1. Introduction

Mesenchymal stem cell (MSC) are undifferentiated cells that possess significant self-renewal, proliferation, and differentiation potential [1,2] characteristics and are currently considered suitable candidates for applications in regenerative medicine [3,4,5]. MSCs can be isolated from different sources [6] such as the umbilical cord, which has been proposed as a potential therapeutic source of MSCs [7].

MSCs are currently used in treatments for a large number of diseases a nd conditions, such as diabetic retinopathy [8], neurodegenerative diseases [9] and chronic wounds with healing problems [10]. Among the properties of MSCs that facilitate their regenerative and immunomodulation efficacy is their ability to efficiently migrate to the target site [11], including to tumoral tissues [12]. This has been well-described for breast cancer, where MSCs have the ability to migrate and counteract the damage [13]. Although some in vitro works have suggested the transformation of MSCs to sarcomas [14], they still have not elucidated whether migration inducing a malignant phenotype over a long period of time is an unusual event [15]. Therefore, we must focus our attention on the subtle changes that MSCs undergo during regenerative medicine treatments in tumor environments.

Breast cancer (BC) is a heterogeneous disease characterized by the uncontrolled proliferation of breast cells [16,17]. BC represents one of the most prominent causes of death in women [18,19,20] which is in part a consequence of unfortunate diagnoses in its late stages [21,22,23]. This is particularly important as BC is one of the most prevalent types of cancer in Mexico [24]. For this reason, the secretome of the MCF-7 breast cancer cell line was used in this work to provide a tumoral environment to explore the stability and capacity of MSCs.

Cell morphology is an emergent property of the cell phenotype and an indicator of physiological state [25,26] determined by the components of the cytoskeleton, which includes actin [27,28,29]. MSCs represent a homogeneous cell population that can present different morphologies [30,31,32] depending on the state undergoing adhesion and migration [29].

In response to pathological stimuli, MSCs can develop adaptations or morphological alterations [33] in relation to their architecture, surface area, volume, nucleus–cytoplasm ratio, shape, density, and homogeneity [34]. These changes can affect their physiology and indicate degeneration, cell death, and even the development of cell dysplasia [35]. Recently, it has been proposed that bioelectric impedance can describe the timely changes in physiological states [36,37] and could be useful for diagnosis in tumoral progression. For this reason, bioimpedance has emerged as a useful method for diagnosis.

The aim of this study was to evaluate the early changes in the morphological and bioimpedance patterns of MSCs induced by secretomes from the MCF-7 cell line in the presence of a tumoral environment. An understanding of these characteristics represents a great contribution to the clinical field, using bioengineering tools to discern the specific influence of BC tumoral stimuli on MSCs.

2. Materials and Methods

2.1. Obtaining the Cell Culture of MSCs

MSCs were isolated from the Wharton’s jelly of expanded umbilical cords, which were remnants of clinical and surgical interventions. The MSCs were characterized according to the criteria of the International Society for Cell Therapy (ISCT) [38,39]. Cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C and 5% CO₂.

2.2. Culture of Breast Cancer Cells and Obtaining Its Secretome

The breast cancer cell line MCF-7 was donated by the National Institute of Cancerology (Mexico City) and was cultured in DMEM/F12 medium supplemented with 5% FBS at 37 °C and 5% CO₂. The cell lines were characterized and confirmed in the laboratory through the expression profiles of specific molecules using RT-PCR and flow cytometry, as previously demonstrated by our working group [40]. A vial containing 1 × 10⁶ cells/mL was thawed, and 1 mL was added to 4 mL of DMEM/F12 medium with 5% FBS. The cells were cultured to 85% confluence, and this procedure was maintained through the third passage. Once the desired confluence was achieved, the monolayers were washed six times with HEPES buffer (8.8 g/L, pH 7.4) to remove serum components and incubated at 37 °C and 5% CO₂ in serum-free DMEM/F12 for 24 and 48 h. From the conditioned media or secretomes, 5 mL was collected and referred to as the 24- and 48-h secretomes, respectively (Figure 1). A control medium (not exposed to MCF-7 cells) was also prepared in serum-free DMEM/F12.

2.3. Migration of MSCs and Cell Morphology

For the migration assay, MSCs were first seeded at a density of 2000 cells/cm² in Lab-Tek chambers (Nunc, Waltham, MA, USA, catalog no. 177380) and allowed to adhere for 24 h. After this period, three experimental conditions were evaluated: (Figure 2a) negative control, cells incubated with DMEM/F12 without phenol red supplemented with 10% FBS; (Figure 2b) secretome collected after 24 h of conditioned media incubation (24 h secretome); and secretome collected after 48 h of conditioned media incubation (48 h secretome) (Figure 2c).

A wound-healing assay was performed to assess cell migration under these conditions, during which a scratch was made with a fine tip to create a gap in the monolayer of cells (Figure 2e). Following an additional 48-h incubation at 37 °C and 5% CO₂, the cells were fixed and stained with crystal violet to analyze their morphology, which was observed under light microscopy (Figure 2f,g). The experiment was performed in triplicate to ensure reproducibility.

2.4. Cytoskeleton Structure Analysis

To visualize the cytoskeleton organization, MSCs were seeded at a density of 2000/cm² in Lab-Tek chambers and subjected to three experimental conditions: (a) a negative control, where cells were cultured without a secretome; (b) stimulation with a 24-h secretome; and (c) stimulation with a 48-h secretome (Figure 3d) and incubated additionally for 48 h at 37 °C and 5% CO₂. All samples were performed in triplicate for each condition. After that, the slides were stained with Phalloidin Green 488 (Biolegend, San Diego, CA, USA, catalog no. 424201). To visualize the nuclei, VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA) was used as the mounting medium. Images were acquired using a fluorescence confocal microscope (LSM 5 Exciter, Zeiss, Jena, Germany) in triplicate, and the best representative image for each condition was selected for presentation.

2.5. Assay of Bioimpedance for the Early Detection of Changes Induced by the Secretome Treatment on MSCs

To verify the early changes in cells treated with tumoral secretomes, an alternative technique based on bioimpedance spectroscopy was used. This technique registers the resistance to the flow of electrical currents with values directly related to intracellular and membrane conformations when the equivalent impedance of a single cell comprises the capacitance of the cell membrane and the resistance of the cytoplasm. The bioimpedance of cells can reflect subtle stages before presenting physiological, molecular, and morphological changes [36,41].

For the bioimpedance measurements, MSCs were seeded in Lab-tek chambers and incubated at 37 °C and 5% CO₂ for 48 h under different conditions: control medium and 24 and 48 h secretomes (Figure 4a–c). After incubation, MSCs were washed with PBS and mechanically harvested using a scraper (Figure 4e) and centrifuged. A total of 35,000 cells were placed in electroporation cuvettes (BTX, Holliston, MA, USA, gap: 2 mm) containing 100 μL of PBS (Figure 4f). To obtain the registers, the cuvettes were adapted to Sciospec^TM (Bennewitz, Germany) multifrequency impedance equipment, which injected a potential difference of 100 mV while measuring spectra of bioimpedance over a frequency range of 100 Hz to 1 MHz in 126 logarithmically spaced steps. A PC laptop was used to program the Sciospec™ instrument and store the data (Figure 4g).

3. Results

3.1. Morphological Changes and Modification in Motility (Migration) Induced by the Secretome Treatment on MSCs

The morphology of control cells predominantly exhibited traditional triangular and fusiform shapes (Figure 5a). Notably, exposure to secretomes induced pleomorphism in MSCs, characterized by changes in the cell morphology (Figure 5b,c). Cells treated with the 24-h secretome displayed a variety of shapes, including triangular, trapezoidal, stellate, and fusiform forms (Figure 5b). In contrast, cells treated with the 48-h secretome showed no significant morphological differences compared to the unstimulated control group, which maintained a more uniform morphology (Figure 5a–c). Proliferation was measured by a crystal violet assay and statistically evaluated via the Kruskal–Wallis method with p < 0.05 determining statistical significance (Figure 6a).

Secretomes did not affect the nucleus-to-cytoplasm ratio in MSCs. However, treated cells exhibited hyperchromatism and aberrant mitosis. Despite these observations, crystal violet staining confirmed that the cells did not acquire malignant morphologies or transformation characteristics. Additionally, the effect of secretomes on cell migration was evaluated using a wound-healing assay, which involved observing the closure of gaps created in a monolayer cell culture (Figure 5d–f).

Interestingly, the migration patterns changed significantly with secretome treatment (Figure 6b). Cells treated with the 24-h secretome showed enhanced migration compared to the control group, accompanied by increased morphological diversity (Figure 5b). In contrast, cells treated with the 48-h secretome demonstrated reduced migration, even lower than that of the control cells (Figure 5c). Cell migration in the wound-healing assay was quantified using ImageJ software (version 1.53e, National Institutes of Health, Bethesda, MD, USA).

Images of the wound area stained with crystal violet were processed by converting them to binary format using the “Adjust → Threshold” function to clearly delineate the gap. A region of interest (ROI) corresponding to the wound area was defined, and the reduction in the gap area over time was measured using the “Analyze → Measure” function. This method provided an objective and reproducible assessment of wound closure, allowing for the quantification of cell migration. The percentage of gap closure was calculated by comparing the initial and final wound areas, and statistical analysis was performed to determine significant differences (p < 0.05) between the experimental groups (Figure 6b).

3.2. Tumoral Secretome Modified Actin Fiber Organization

Secretomes induced changes in the disposition of cytoskeletons, as evidenced by the green fluorescence of phalloidin staining and the blue fluorescence of DAPI staining for nuclei staining (Figure 7b,c). The results showed changes in the arrangement of actin fibers in the presence of 24 and 48 h secretomes (Figure 7e,f) that differed from that of the negative control cells (Figure 7a,d), which presented the typical triangular shape without nuclear polarization. Figure 7d–f schematically illustrates the changes in the fibers of actin with a typical abundant comet tail in the membrane surface and the structures highlighted for obtaining a different morphological pattern.

The MSCs incubated with the 24 h secretome (Figure 7b) showed a long cytoplasmic prolongation and nuclear polarity. Figure 7e illustrates how dorsal actin fibers converged in a common point of focal adhesion that extends since the nucleus area is along almost the entire major cell axis. It also shows ventral fibers oriented near the edge of the cytoplasmic prolongation that contained several cytoplasmic extensions or filopodia (FPs).

Figure 7c shows the actin changes in MSCs incubated with the 48-h secretome, resulting in a condensed morphology due to the shorter disposition of stress fibers (SFs). This produced a rounded projection structure with an increased number of filopodia (FPs) and microspikes (MSs). Figure 7f illustrates the increase in short SFs arranged in a random disposition, contributing to the expanded morphology. Additionally, the number of MSs was higher.

3.3. Changes in Electrical Bioimpedance Induced by the 48 h Secretome but Not the 24 h Secretome

The bioimpedance spectrum of cell cultures was determined using a 1 MHz focused resonance device, with readings at 200 KHz identified as a useful bandwidth [41]. The treatments with the 24-h and 48-h tumor cell secretomes on MSCs (Figure 8 and Figure 9, respectively) altered the bioimpedance spectra, causing shifts in both the magnitude (Z-axis) and phase (degree axis), which were represented on logarithmic scales. The use of logarithmic scaling provided a clearer visualization of these modifications, which were more pronounced following treatment with the 48-h secretome, while the 24-h secretome induced only minor changes.

Within the explored bioimpedance frequency range, changes in the magnitude and phase signatures were indicative of modifications to the volumetric cell structure and cell membrane thickness, respectively. These logarithmic shifts appeared to correlate closely with the morphological and cytoskeletal changes described in Figure 5, as well as with the alterations in actin fiber organization that contributed to microstructural changes upon the membrane surface, as presented in Figure 7.

The cell disintegration index (Z_p) based on multifrequency bioimpedance measurements has been widely used to monitor cell structure changes and disruption [42]. Here, we proposed the use of Z_p to quantitively estimate bioimpedance variations with cell morphological changes. Z_p was calculated on the basis of the measurement of the absolute impedance value of the control (Z_c) and secretome-treated (Z_tr) groups (at 24 and 48 h) in the low (1 kHz) and high (150 kHz) frequency ranges, in accordance with [43,44], as follows:

$$Z_p={\displaystyle \frac{\left|Z_{c\left(1\mathrm{k}\mathrm{H}\mathrm{z}\right)}\right|-\left|Z_{tr\left(1\mathrm{k}\mathrm{H}\mathrm{z}\right)}\right|}{\left|Z_{c\left(1\mathrm{k}\mathrm{H}\mathrm{z}\right)}\right|-\left|Z_{tr\left(150\mathrm{k}\mathrm{H}\mathrm{z}\right)}\right|}}$$

(1)

where the value of Z_p varies between 0 for intact cells and 1 for fully permeabilized cells.

The results indicate the following index: Z_p(24h) = 0.14 y Z_p(48h) = 0.27. Thus, a greater cell structural change is evident after the 48 h secretome treatment. As a first intuitive approach, secretomes induced early morphological changes related to the migration of MSCs. The modification of the bioimpedance patterns recorded and the Z_p index increments as a function of secretome time suggest that this method can be applied in an innovative way to detect early changes in a cellular population in the clinical diagnostic setting.

3.4. Molecular Pattern of MSCs Under Tumor-Cell-Conditioned 48 h Media over 48 and 72 h

To investigate the effect of tumor-cell-conditioned 48 h media on the molecular pattern of MSCs after 48 and 72 h of treatment, RT-PCR was performed to detect CD73, CD90, and CD105 as mesenchymal cell markers (Figure 10). Additionally, IL-10 and TGF-β were evaluated as anti- and pro-inflammatory molecules, respectively. NANOG and OCT4 were used as markers of malignant transformation. The results revealed a decreased expression of CD90 at 24 h, which was evidently reduced at 72 h. ImageLab, version 6.1.0, build 7 (2020, Bio-Rad laboratories, Inc., Hercules, CA, USA) was used for the analysis of the obtained images.

4. Discussion

Currently, there are no reports about the changes that MSCs undergo when exposed to a tumor environment. In this work, we specifically studied the changes in the morphology, cytoskeleton, and migration of exposed MSCs into the secretome of cancer cells verified by impedance. In the 24 h secretome, the results showed a morphological diversity, greater migration (Figure 5 and Figure 6) development, and greater organization of the cytoskeleton (Figure 7) due to evident changes in the arrangement of actin fibers and the cell polarity of the exposed MSCs to the 24 h secretome. This was not observed in cultures that were exposed to the 48 h secretome since the cells had a homogeneous morphology, a marked inhibition of migration, and a different disposition of the cytoskeleton.

The MSCs had no alterations in their nucleus/cytoplasm ratio, and neither cell hyperchromatism nor aberrant mitoses induced by the tumor secretome were observed. Since cell morphology is not a determining criterion to establish a neoplastic phenotype, it cannot be concluded with determination that MSCs undergo malignant transformation. Although no morphological changes related to cellular dysplasia were observed, the homogeneity of cellular morphology with a singular triangular predominance in the experimental conditions could be appreciated. This triangular morphology can be observed when MSCs are cultivated in vitro and is generally seen after a short time in cultures [45].

To study the arrangement of actin filaments (responsible for cell morphology) upon the secretome stimulation of MSCs, fluorescence microscopy was chosen. MSCs were stained with a protein related to actin fibers (phalloidin coupled to Green 488 fluorophore), which are closely related to cell morphology, as actin fibers orchestrate the control of cell shape dynamics as well as cell polarity [46,47].

In some cell systems, changes in MSCs, such as cell asymmetry, are a result of cell polarization, which is a fundamental step prior to cell migration [46]; therefore, the characteristics of fiber organization and cell polarity presented by MSCs exposed to a secretome are indicative that the secretome has components that could induce MSC migration, reflecting early changes in the morphological pattern. It has been reported that the secretome of MCF-7 contains molecules such as vascular endothelial growth factor (VEGF) and interleukin 8 [48,49] which could explain this effect. Vallenius [29] mentioned that migration processes are due to branched networks of actin arranged in filipodia and lamellipodia and that these processes are actin-dependent. According to our results, the MSCs exposed to secretomes presented a greater organization of the dorsal fibers in the lamellipodia compared to the control, which are indications of migration. It might be interesting to investigate whether molecules such as Rho A are involved in this migration process, as described in the stromal tissue of prostate and ovarian cancer [50].

Recently, some techniques from a bioengineering perspective have emerged to describe the cellular state; this is the case for electrical bioimpedance [51] which allows us to detect differences between benign cells and malignant cells due to the different lineages of physiological characteristics [40,45,46,47,48,49,50,51,52]. Moonen et al. (2021) reported that bioimpedance in a cell population is of a low frequency when electric currents do not penetrate cell membranes, resulting in measuring only water impedance [53]. They also proposed that the grade of hydration in the cells directly affects the polymerization of the cytoskeleton in living cells. In this context, changes in the actin fibers could be related to hydration [54]. Yizeng Li and Sean X. Sun (2018) [55] documented that cell migration is a complex process orchestrated by actin dynamics, focal adhesions, and water flux. This agrees with our results, where actin fibers were rearranged and translated as changes when MSCs were cultivated in the presence of a tumoral secretome.

Therefore, bioimpedance allows us to investigate the subtle changes that are not visible to the naked eye. We concluded that untreated cells respond differently to the external electric field. This is because they possess different electrical properties provided by membrane capacitance and cytosol resistance [36]. Since MSCs have not shown evidence of tumoral malignancy with the classic techniques used previously, bioimpedance spectroscopy only indicated changes related to the effect of the secretome; it probably changes the components in the membrane or cytoplasm, as well as the water contained as an adaptive response. Nevertheless, tumoral behavior could be reflected by specific bioimpedance spectra patterns (involving unique conditions of the capacitance and resistance of molecular expression) [40], although this was not found in our results.

While protein profiling of the secretome was not performed, mRNA expression was evaluated via agarose gel electrophoresis in an effort to understand the cellular alterations. Specific markers, such as NANOG, OCT4, IL-10, TGF-β, CD73, CD90, and CD105, were analyzed to identify the key biological responses.

CD90 is a marker of MSCs; however, the overexpression of CD90 can indicate the tumor transformation of stem cells [56]. In this study, it does not appear that the changes in characteristics were related to tumor transformation, as CD90 decreased. Previous studies have demonstrated that CD90 decreases while other markers increase due to differentiation processes [57]. In this case, the migration processes might be related to the reduced CD90. Recently, it was reported by Mancarella, S (2023) [58] that the downregulation of CD90 is associated with a decrease in the proliferation rate, which was also observed in this work.

TGF-β and its receptors can be strongly induced in the presence of carcinogens that pretend to disrupt cell homeostasis to produce an oncogenic transformation [59]. Our results showed that the tumoral environment induced the high expression of TGF-β mRNA but did not influence cell proliferation. TGF-β is involved in malignant hepatocellular progression through interaction with CD105 [60]. While protein profiling of the secretome was not performed, mRNA expression was evaluated via agarose gel electrophoresis to confirm the biological activity.

Our results indicate that CD105 expression did not change when cultures were exposed to a tumoral environment (Figure 10a). Fernández Penas et al. (2017) [61] reported that in monkeys, when CD105 was exacerbated, cell migration occurred in response to damage induced by previously produced osteoarthritis. Therefore, the high expression of TGF-β mRNA seems to be involved in an inflammatory process occasioned by the tumoral medium in cell cultures since TGF-β promotes wound healing by participating in the inflammation step during the stage of proliferation and remodeling [59]. Evidently, the cytoskeletal changes shown in this work are related to this process of maintaining homeostasis under a tumoral environment.

NANOG mRNA was not detected under any conditions. Various studies have proposed that downregulated NANOG is associated with decreased proliferation and migration. Furthermore, NANOG has been proposed as a predictor of poor prognosis in people with breast or kidney cancer [62]. In our study, NANOG was not affected, so it does not appear to be related to the effect produced by the tumor secretome (Figure 10a).

It has been reported that OCT4 expression increases in inflammatory processes that induce migration [63]. However, OCT4 remained constant in all conditions, indicating that OCT4 is not involved in the changes observed in MSCs in the presence of a tumor secretome.

IL-10 is a cytokine involved in modulating inflammation [61], although it has been related to tumoral growth [64]. In our work, secretomes did not affect proliferation, so the role that IL-10 probably had is related to the counteraction of inflammatory effects produced by the tumoral secretome. It has been observed that the downregulation of TGF-β inhibits the motility of cancer cells. However, in cells cultivated in the presence of a secretome, TGF-β increases, but motility is decreased, so the results in this study cannot be related to tumoral transformation [65].

All the results together suggest that the tumoral environment induces the inhibition of proliferation and significant changes in the disposition of actin fibers, which, in turn, alter the morphology of MSCs and directly affect bioimpedance values. Changes in the magnitude and phase signatures of bioimpedance indicate modifications in the volumetric cell structure and cell membrane thickness, respectively. These logarithmic shifts in the bioimpedance parameters appear to correlate with morphological and cytoskeletal changes, as well as with alterations in actin fiber organization, which promote the formation of microstructures on the membrane surface.

Interestingly, TGF-β expression does not appear to be related to tumor transformation but rather seems to represent a natural anti-inflammatory response counteracting the tumor stimuli (secretome). Future studies could explore other molecules associated with bioimpedance changes to better understand the different cellular responses involved.

These molecular analyses not only explained the observed morphological changes but also provided detailed information on the signaling pathways involved in the response to the tumor environment. The results obtained underline the importance of integrating both morphological and molecular characterization to obtain a more complete picture of cellular responses.

This work suggests that the secretome derived from breast cancer tumor cells (MCF-7 line) induces early morphological changes in MSCs. These changes are associated with the tumor environment but are not indicative of malignancy. This finding is highly relevant to regenerative medicine, as MSCs are widely used in therapies that often overlook the potential influence of tumor environments, which could alter their morphology and, consequently, their migratory capacity. However, the observed changes are more likely related to anti-inflammatory responses rather than cellular transformation.

On the other hand, it was possible to prove that bioimpedance spectroscopy is a sensitive technique that is helpful in detecting early subtle changes. This demonstrates that bioimpedance represents an emerging, fast, and inexpensive technique that does not require a large number of cells for analysis [66]. These results, using knowledge of biomedical engineering, are pioneering in describing the short-term morphological changes in MSCs produced by a tumor secretome. However, an area for improvement is the absence of a secretome from non-tumor cells as a control. Including this in future experiments will help determine the tumor-specific effects on MSC morphology and migration. Since the main criteria of this study focus on evaluating morphology, future studies will include proteomic analyses to further characterize the secretome and validate these findings, as well as additional molecular studies to determine their safety for use in regenerative medicine.