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. 2026 Jun 28;15(7):e72059. doi: 10.1002/cam4.72059

Microenvironmental Cues That Regulate Cancer Cell Dormancy in Estrogen‐Receptor Positive and Triple‐Negative Breast Cancer Cell Lines

Jinesh Maniar 1,2,3, Pulkit Datt 1,2, Prerana P Dange 1,2, Vaishali Kailaje 4, Mithlesh Lakhera 4, Mansi Samarth 4, Tanuja Durve 4, Anushree Kadam 1,2, Khushboo A Gandhi 1,2, Sudeep Gupta 1,2,3,✉
PMCID: PMC13310792  PMID: 42366462

ABSTRACT

The processes regulating cellular dormancy in tumor cells remain inadequately characterized. Further, there are very few models available that can recapitulate a dormancy‐like phenotype in vitro. In this study, we investigated the role of microenvironmental cues in inducing a dormancy‐like phenotype in estrogen receptor‐positive (MCF‐7) and triple‐negative (MDA‐MB‐231) breast cancer cell lines. We characterized an in vitro model in distinct conditions defined hierarchically by the following: (a) normoxia or true hypoxia, (b) dishes coated with fibronectin or laminin or none, and (c) presence or absence of 10 ng/mL basic fibroblast growth factor (FGF‐2) in the culture medium, added on day 0 of culture. Cells were cultured at clonogenic densities for 9 days (day −1, day 0, up to day +7). Cells were characterized for dormancy‐ like behavior using increased p‐p38 and decreased p‐ERK expression (dormant, p‐p38High and p‐ERKLow expression), reduced Ki67 expression, elevated p21 and p27 levels by immunofluorescence, absence of senescence using β‐galactosidase staining and resistance to doxorubicin. Fibronectin or laminin were sufficient for the induction of a dormancy‐like phenotype in MCF‐7 cells under hypoxic conditions, whereas the addition of FGF‐2 to fibronectin (but not laminin) could induce a dormancy‐like phenotype under normoxic conditions. FGF‐2 was required with laminin to induce dormancy in MDA‐MB‐231 cells under hypoxic conditions (FGF‐2 plus fibronectin were unable to induce dormancy), whereas laminin or fibronectin alone were sufficient under normoxic conditions. Overall, we established culture conditions that recapitulate an in vitro breast cancer dormancy‐like phenotype and reveal subtype‐specific differences in dormancy regulation.

1. Background

Metastatic disease is the major cause of death among breast cancer patients [1]. Distant site metastases can occur several years after eliminating the primary tumor. Late recurrence is a consequence of induction of a prolonged quiescent stage in disseminated tumor cells (DTCs). The DTCs likely enter a prolonged non‐proliferative state of reversible growth arrest (quiescence) at the site of the metastatic niche, often called cellular dormancy [2, 3, 4]. Another type of dormancy is tumor mass dormancy, where there is a lack of net tumor growth because of an equilibrium between the rates of cell proliferation and cell death [5, 6, 7, 8]. The cellular‐level dormant phenotype is clinically significant, as it enables DTCs to evade therapeutic elimination and persist over extended periods, ultimately contributing to disease relapse. For example, therapy‐induced dormancy in estrogen receptor‐positive breast cancer has been observed, where cells remain viable but growth‐arrested following anti‐estrogen treatment, implicating trefoil factor 3 (TFF3) as a key mediator of this process [9]. TFF3 has been shown to promote metastatic seeding and survival of breast cancer cells at distant sites, and is also associated with reduced sensitivity to chemotherapeutic agents such as doxorubicin, whereas its inhibition enhances chemosensitivity and can overcome acquired resistance [10, 11].

Cellular dormancy has been proposed to be regulated by the crosstalk between DTCs and their microenvironment but the regulatory processes involved in cellular dormancy remain insufficiently investigated. The fate of DTCs is regulated by immunomodulatory and microenvironmental stresses encountered at secondary organ sites, where they can persist in a dormant state, particularly within the bone marrow, and contribute to late disease recurrence [12, 13, 14, 15]. Subsequently, the extracellular matrix (ECM) plays a pivotal role in deciding the fate of DTCs during metastasis. ECM proteins promote integrin‐mediated adhesions influencing the cell's fate [16]. Therefore, it has been previously hypothesized that DTCs should land in a favorable site to grow. Extensive studies in many cancers have reported that dormant DTCs are present in the bone marrow, suggesting that this niche could provide a favorable microenvironment for DTCs to enter dormancy [17]. Prior studies have shown the presence of the perivascular and endosteal niche and the existence of hypoxic zones at short distances away from the blood vessels in the bone marrow [18, 19, 20], potentially providing a permissive microenvironment for induction and maintenance of dormancy in DTCs. However, the role of hypoxia signaling in the induction and maintenance of dormancy in DTCs is not well studied.

Hence, we planned this study to evaluate whether hypoxic stress induces cellular dormancy in DTCs in a microenvironment that is akin to the bone marrow. We characterized an in vitro model using the components abundantly found in bone marrow. The main elements of our model system include basic fibroblast growth factor (FGF‐2), fibronectin (F), and laminin‐111 (L). FGF‐2 plays an essential role in the morphogenic differentiation of mammary ducts and can halt the growth of breast cancer cells [21]. We used the ECM proteins fibronectin and laminin in this study because of their known relevance in integrin‐mediated adhesions [22]. Fibronectin and laminin are two structural proteins constituting a significant portion of the bone marrow cellular matrix [23]. In this study, we investigated how the FGF‐2, fibronectin, or laminin microenvironment affects the growth of estrogen receptor‐positive (MCF‐7) and triple‐negative (MDA‐MB‐231) breast cancer cells under normoxic conditions followed by hypoxic conditions of 1% oxygen concentration. Experimental analysis was performed in cells obtained on day +7 after exposure to various microenvironmental conditions to determine the possible scenarios involved in the regulation of cellular dormancy in these breast cancer cells (Figure 1a).

FIGURE 1.

FIGURE 1

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Demonstrates the (a) study design and (b) parameters used in the study.

2. Methods

2.1. Cell Lines

The human breast cancer cell lines MDA‐MB‐231 (RRID:CVCL_0062) and MCF‐7 (RRID:CVCL_0031) were procured from the American Type Culture Collection (ATCC). Cell line authentication was performed by short tandem repeat (STR) profiling using the PowerPlex 18D System (Promega) and profiles were confirmed to match reference databases. All cell lines were routinely tested for mycoplasma contamination and were confirmed to be mycoplasma‐free prior to use in all experiments.

2.2. Coating and Cell Culture

Thermo‐ScientificTM Nunc 35 mm petri‐dishes (C#150318) and 35 mm glass bottom dish (C# D35‐14‐1.5‐N) were used for ECM coating with Corning Fibronectin, Human, 5 mg (C#356008)/Sigma‐Aldrich Laminin (#L2020). The stock coating solution was diluted further in PBS at a concentration of 10 μg/mL for coating the 35 mm petri dishes. One milliliter of coating solution was added to a single 35 mm petri dish. After adding the coating solution, the petri dishes were kept at 4°C overnight. On the next day, the excess solution was removed and petri dishes were allowed to air dry for at least 45 min at room temperature inside the biosafety cabinet. These petri dishes were further used for performing the cell culture. Both the cell lines, MCF‐7 and MDA‐MB‐231, were maintained in GibcoTM Dulbecco's Modified Eagle's Media (C#11995065) supplemented with 10% (v/v) Gibco Fetal Bovine Serum (C#16140071) and 1% (v/v) Gibco Antibiotic‐Antimycotic (100×) (C#15240062). In this study, we cultured MCF‐7 and MDA‐MB‐231 cells, respectively, for 9 days (day −1, day 0, up to day +7) at a clonogenic density to ensure that their primary interaction was with the substratum and not with each other. On day −1, 2000 cells were seeded on 35 mm petri dishes coated with fibronectin, laminin, and none, respectively. At day 0, these cells were kept under normoxic (~20% O2, 5% CO2). Subsequently, 10,000 cells were seeded under hypoxic conditions (1% O2, 5% CO2) using a hypoxia workstation (INVIVO2 400, UK). Basic fibroblast growth factor (FGF‐2; 10 ng/mL, C#354060, Corning) was added once at day 0 where indicated and was not replenished during the culture period. Cells were maintained under these conditions until day +7, at which point they were harvested and analyzed for all experiments, except for calcein‐AM staining assays.

2.3. Crystal Violet Staining

Sigma‐Aldrich Crystal Violet (C#6158) was used to stain these breast cancer cells to determine their viability and adhesion. After 7 days of culture, cells were stained with a freshly made 0.1% crystal violet in 2% ethanol/10 mM sodium borate (pH 9.0) solution. Media was aspirated, and 1 mL of crystal violet solution was added for 10 min. Plates were rinsed with water and kept for drying overnight. Colonies were counted optimally at 40× magnification in an inverted phase contrast microscope. Colonies of > 30 cells as growing and colonies of 12 or less cells were considered dormant.

2.4. Immunofluorescence Assays

A phospho‐p38 (p‐p38) mitogen‐activated kinase (MAPK) (Thr180/Tyr182) mouse antibody (C#9216), Ki67 mouse antibody (C#9449) and a phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) rabbit antibody (C#4370) and p21 (12D1, C#2947) were purchased from Cell Signaling Technology, and p27 (PA5‐27188) was purchased from Invitrogen. After 7 days in culture, cells were fixed using 4% paraformaldehyde for 10 min and blocked with blocking buffer overnight at 4°C. Blocking buffer was made with 5% (w/v) goat serum (C#642921), 0.3% (v/v) Triton X‐100 in PBS. These cells were then incubated overnight with the p‐p38 primary antibody (1:100 in dilution buffer, 1% (w/v) BSA, 0.3% (v/v) Triton X‐00 in PBS) at 4°C, rinsed with PBS three times, and incubated with a secondary antibody (goat anti‐mouse IgG‐Alexa Fluor 568: 1:1000 in dilution buffer, 1% (w/v) BSA, 0.3% (v/v) Triton X‐100 in PBS) for 2 h at room temperature in the dark, rinsed with PBS three times, and then incubated with the p‐ERK antibody (1:400 in dilution buffer, 1% (w/v) BSA, 0.3% (v/v) Triton X‐100 in PBS) overnight at 4°C, rinsed with PBS three times, and incubated with a secondary antibody (goat‐anti rabbit IgG‐Alexa Fluor 488: 1:1000 in dilution buffer, 1% (w/v) BSA, 0.3% (v/v) Triton X‐100 in PBS) for 1 h and then rinsed with PBS three times. Incubated for 1 min with DAPI (1 μg/mL) in dilution buffer. Similarly, for the Ki67 mouse antibody (C#9449S), p21 (C#2947) and p27 (PA5‐27188) (1:400 in dilution buffer, 1% (w/v) BSA, 0.3% (v/v) Triton X‐100 in PBS) was used for staining a new set of experiments, where cells were stained in a similar manner. Labeled cells were stored in PBS. Images were acquired using a Zeiss LSM 780 confocal microscope (RRID:SCR_020922) with ZEN software. All images within a given experiment were captured under identical acquisition settings (laser power, gain, and exposure) across all conditions to ensure comparability. Any auto fluorescence was negated by using secondary antibody control. FIJI: ImageJ version 1.53t (http://imagej.nih.gov/ij) (RRID:SCR_003070) software was used to quantify the mean fluorescence intensity of each z‐stack to quantify p‐p38, p‐ERK, and the p‐p38 to p‐ERK ratio. For each image stack, the mean fluorescence intensity of a maximum intensity z‐projection was measured for p‐p38 and for p‐ERK using FIJI: ImageJ version 1.53t (http://imagej.nih.gov/ij) software. To account for background noise, intensity measurements of five background regions (regions in each corner and near the center of the image) that did not contain cells were quantified and averaged for each image. Background measurements were subtracted, and normalized intensity values from 10 individual z‐stacks were averaged to obtain the p‐ERK and p‐p38 intensity values. A minimum of 10 z‐stacks from every culture scenario were imaged and quantified.

2.5. Senescence Staining Assay

Senescence‐associated β‐galactosidase activity was measured for all scenarios on day +7 using the Senescence β‐Galactosidase Staining Kit (Cell Signaling Technology). We used a similar protocol provided by the manufacturer. Briefly, cells were washed with PBS and fixed with 1× fixative solution for 15 min. Then, the β‐galactosidase staining solution with a final pH between 5.9 and 6.1 was prepared and added to fixed cells. Samples were sealed with parafilm to prevent evaporation and placed in a dry 37°C incubator overnight. Imaging was performed using a Nikon Eclipse Ti Microscope. For the positive control of senescence, MCF‐7 and MDA‐MB‐231 cells were treated with 12.5 μM etoposide (Cell Signaling Technology) for 4 days and allowed to recover for 2 days in normal growth media.

2.6. Calcein‐AM Staining

Cells were initially cultured under the indicated microenvironmental conditions for 7 days to establish two distinct states: (i) proliferative conditions, where cells continued to expand, and (ii) dormancy‐inducing conditions, where cells remained growth‐arrested (Ki‐67−, p‐p38high/p‐ERKlow) but viable. Following this 7‐day induction phase, doxorubicin hydrochloride (2 μM) was added directly to the existing cultures without re‐plating, and cells were maintained under the same conditions for an additional 7 days. Invitrogen Calcein‐AM (C#C1430) was used to stain cells on day +14. Cells from all the culture scenarios were stained with 2 mM Calcein AM by removing the existing medium and putting in new culture medium without FBS for 30 min containing Calcein AM and then immediately evaluated by confocal microscopy (LSM780, Zeiss). This assay was used to assess persistence of viable cells under sustained cytotoxic stress rather than direct measurement of apoptosis or clonogenic survival. In this system, survival following doxorubicin exposure was evaluated based on the presence of Calcein‐positive cells at the endpoint, reflecting the ability of cells to persist under prolonged drug treatment.

2.7. Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 9 (http://www.graphpad.com) (RRID:SCR_002798) software. For in vitro experiments, a minimum of three replicates repeated at least three times were used to achieve reproducibility. For the dormant breast cancer cells detection experiments, the p‐values were estimated using an Ordinary one‐way ANOVA non‐parametric test with Dunnett's multiple comparison p‐values ≤ 0.05 considered significant (95% confidence). Represented with: p‐value: ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3. Results

3.1. Tumor Microenvironment Regulates Induction of Cellular Dormancy‐Like Phenotype in Breast Cancer Cells

We investigated 24 culture scenarios to identify microenvironmental cues that could induce a cellular dormancy‐like phenotype in breast cancer cells. These 24 culture scenarios were determined according to the following parameters (Figure 1b). First, there were four culture conditions as follows: (i) MCF‐7 cells cultured in normoxia; (ii) MCF‐7 cells cultured in hypoxia; (iii) MDA‐MB‐231 cells cultured in normoxia; and (iv) MDA‐MB‐231 cells cultured in hypoxia. Each of these four culture conditions was further subjected to the following six culture scenarios for 7 days for a total of 24 culture conditions. The following example depicts six culture conditions for MCF‐7 cells grown under normoxic conditions for 7 days: (i) MCF‐7 cells grown on fibronectin with FGF‐2 (MCF‐7, FGF + F); (ii) MCF‐7 cells grown on fibronectin without FGF‐2 (MCF‐7, F); (iii) MCF‐7 cells grown on laminin with FGF‐2 (MCF‐7, FGF + L); (iv) MCF‐7 cells grown on laminin without FGF‐2 (MCF‐7, L); (v) MCF‐7 cells grown on uncoated dishes with FGF‐2 (MCF‐7, FGF); (vi) MCF‐7 cells grown on non‐coated dishes without FGF‐2 (MCF‐7, control). Likewise, similar culture scenarios were reproduced for MCF‐7 cells in hypoxic conditions, MDA‐MB‐231 in normoxic conditions, and MDA‐MB‐231 in hypoxic conditions, for a total of 24 culture conditions. This framework helped us to identify specific combinations of the microenvironmental cues involved in the induction of a cellular dormancy‐like phenotype in breast cancer cells.

We performed crystal violet staining to study how different microenvironmental factors affect the growth pattern of MCF‐7 and MDA‐MB‐231 cells, as shown in Figures 2 and 3, respectively. Under the normoxic condition on day +7, we observed that fibronectin and laminin prevented formation of big colonies in both cell lines. The cell count per colony for MDA‐MB‐231 was seven on fibronectin and eight on laminin coated dishes while for MCF‐7, it was 66 on fibronectin and only 16 on laminin coated dishes, which is indicative of the absence of big colonies (Figures 2 and 3). When these cells were grown only with FGF‐2 on non‐coated dishes under normoxia, both the cell lines formed big colonies with a high population density. The cell count per colony for MDA‐MB‐231 was 136 whereas for MCF‐7 it was > 500 cells. Further, crystal violet staining on day +7 led us to primarily identify the formation of possible dormant colonies with ≤ 12 cells as reported by Tivari et al. [24]. To confirm whether these cells were dormant or not, we performed immunofluorescence staining on all of the scenarios demonstrated later in this study.

FIGURE 2.

FIGURE 2

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The appearance of growing and dormant MCF‐7 cell colonies stained with crystal violet in different scenarios. Growing colonies contain > 30 cells, and dormant colonies have 12 or fewer cells. (a) MCF‐7 cells grown on fibronectin with FGF‐2 in normoxia; (b) MCF‐7 cells grown on fibronectin without FGF‐2 in normoxia; (c) MCF‐7 cells grown on uncoated dishes with FGF‐2 in normoxia; (d) MCF‐7 cells grown on laminin with FGF‐2 in normoxia; (e) MCF‐7 cells grown on laminin without FGF‐2 in normoxia; (f) MCF‐7 cells grown on uncoated dishes without FGF‐2 in normoxia; (g) MCF‐7 cells grown on fibronectin with FGF‐2 in hypoxia; (h) MCF‐7 cells grown on fibronectin without FGF‐2 in hypoxia; (i) MCF‐7 cells grown on uncoated dishes with FGF‐2 in hypoxia; (j) MCF‐7 cells grown on laminin with FGF‐2 in hypoxia; (k) MCF‐7 cells grown on laminin without FGF‐2 in hypoxia; (l) MCF‐7 cells grown on uncoated dishes without FGF‐2 in hypoxia.

FIGURE 3.

FIGURE 3

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The appearance of growing and dormant MDA‐MB‐231 cell colonies stained with crystal violet in different scenarios. Growing colonies contain > 30 cells and dormant colonies contain 12 or fewer cells. (a) MDA‐MB‐231 cells grown on fibronectin with FGF‐2 in normoxia; (b) MDA‐MB‐231 cells grown on fibronectin without FGF‐2 in normoxia; (c) MDA‐MB‐231 cells grown on uncoated dishes with FGF‐2 in normoxia; (d) MDA‐MB‐231 cells grown on laminin with FGF‐2 in normoxia; (e) MDA‐MB‐231 cells grown on laminin without FGF‐2 in normoxia; (f) MDA‐MB‐231 cells grown on uncoated dishes without FGF‐2 in normoxia; (g) MDA‐MB‐231 cells grown on fibronectin with FGF‐2 in hypoxia; (h) MDA‐MB‐231 cells grown on fibronectin without FGF‐2 in hypoxia; (i) MDA‐MB‐231 cells grown on laminin with FGF‐2 in hypoxia; (j) MDA‐MB‐231 cells grown on laminin without FGF‐2 in hypoxia.

The observations for each of the six culture scenarios of MCF‐7 and MDA‐MB‐231 cells in normoxic and hypoxic conditions are shown in Figures 2 and 3, respectively. We observed growth arrest of MCF‐7 cells under normoxia only when they were grown on fibronectin along with FGF‐2 with a cell count of nine per colony. In contrast, MDA‐MB‐231 cells demonstrated growth‐arrest when grown on fibronectin coated dishes without FGF‐2 with seven cells per colony and on laminin coated dishes without FGF‐2 with eight cells per colony. Under hypoxic conditions, we observed complete cell death for both cell lines with a lower cell seeding density of 2000 cells in 3 mL medium in a 35 mm dish. Hence, to support cell survival and maintenance of clonal growth under hypoxic stress for 7 days we increased the seeding density to 10,000 cells in 3 mL medium in a 35 mm plate for both cell lines. Although most cells survived for 7 days, we observed some cell death due to hypoxic stress. In hypoxic conditions, MCF‐7 cells survived in all six culture scenarios as indicated in Table 1 and Figure 2. However, MDA‐MB‐231 cells only survived for 7 days when grown on ECM‐coated dishes but not when grown without fibronectin or laminin (two culture conditions) (Table 2 and Figure 3).

TABLE 1.

Cell count per colony corresponding to Figures 2 and 3.

Cell lines MCF‐7 MDA‐MB‐231
Hypoxia Normoxia Hypoxia Normoxia
Control 26 299 — 107
Laminin coated (L) 13 16 9 8
Fibronectin coated (F) 14 66 13 7
FGF‐2 35 > 500 — 136
L + FGF‐2 15 40 1 17
F + FGF‐2 18 9 19 120
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TABLE 2.

Ratios of p‐p38 to p‐ERK for each condition.

Breast cancer cell type Microenvironmental cues p‐p38/p‐ERK signaling ratio Phenotypic states
MDA‐MB‐231 Normoxia, none (Control) 0.39 Proliferative
MDA‐MB‐231 Normoxia, Fibronectin 1.36 Dormant
MDA‐MB‐231 Normoxia, Laminin 1.69 Dormant
MDA‐MB‐231 Normoxia, FGF‐2, Fibronectin 0.21 Proliferative
MDA‐MB‐231 Normoxia, FGF‐2, Laminin 0.72 Proliferative
MDA‐MB‐231 Hypoxia, FGF‐2, Laminin 1.05 Dormant
MDA‐MB‐231 Hypoxia, FGF‐2, Fibronectin 0.26 Proliferative
MDA‐MB‐231 Hypoxia, Laminin 0.27 Proliferative
MDA‐MB‐231 Hypoxia, Fibronectin 0.33 Proliferative
MCF‐7 Normoxia, none (Control) 1.00 Proliferative
MCF‐7 Normoxia, FGF‐2, Fibronectin 1.44 Dormant
MCF‐7 Normoxia, FGF‐2, Laminin 1.00 Proliferative
MCF‐7 Normoxia, Fibronectin 0.86 Proliferative
MCF‐7 Hypoxia, none (Control) 0.93 Proliferative
MCF‐7 Hypoxia, Fibronectin 2.45 Dormant
MCF‐7 Hypoxia, Laminin 1.3 Dormant
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Figure 2 shows that MCF‐7 cells demonstrated growth arrest when grown on fibronectin coated dishes with 14 cells per colony or laminin coated dishes with 13 cells per colony under hypoxic conditions. On the other hand, growth arrest was observed in MDA‐MB‐231 cells when grown with FGF‐2 on laminin coated dishes with one cell per colony (Figure 3). After observing these results, we proceeded with 22 culture‐competent scenarios under which cells survived till day 7 for further investigation via immunostaining to evaluate whether cell lines were growth‐arrested and whether the phenomenon observed was cellular dormancy.

3.2. Dormancy‐Like Phenotype Induction Marked by Phosphorylated p38High /ERKLow Expression Ratio and Expression of Cell Cycle Inhibitor Proteins

Immunofluorescence staining of phospho‐p38 (p‐p38) and phospho‐ERK (p‐ERK) was performed to determine the protein expression levels for confirmation of cellular dormancy. In vivo, studies performed by Aguirre‐Ghiso et al. showed that increased p‐p38 and decreased p‐ERK expression are associated with transition of cancer cells from a state of tumorigenicity to dormancy [12, 25]. There are several lines of evidence that demonstrate the function of p‐p38 lies in the induction of a G0‐G1 cell cycle arrest phase in the cancer cells, whereas p‐ERK acts as a promoter of proliferation. Therefore, a high p‐p38 and low p‐ERK expression has been recognized as a potential marker for the dormant state of cancer cells [12, 25, 26, 27]. Further, it has also been confirmed that this mechanism supports the overexpression of several genes, leading to the suppression of p‐ERK protein for promoting dormancy [26, 27, 28, 29, 30]. Furthermore, we also analyzed the expression of Ki67, a proliferation marker, for all culture scenarios. The expression of Ki67 has been widely used for pathological investigations and to determine whether the cancer cells are proliferating [31]. Lastly, we confirmed high expression of the cyclin‐dependent kinase inhibitors p21 (CDKN1A) and p27 (CDKN1B) which operate at the G1 checkpoint, to further confirm growth arrest (if any) of the cells [32].

Our study demonstrates that different microenvironmental conditions significantly influence p‐p38 and p‐ERK expression patterns in MCF‐7 and MDA‐MB‐231 cells. Figure 4 shows the expression patterns of p‐p38 and p‐ERK in the six culture scenarios for MCF‐7 cells cultured in normoxic conditions. It demonstrates that the expression of p‐p38 was significantly higher than p‐ERK (mean intensity: 23.5 vs. 16.3; p < 0.0001) when the MCF‐7 cells were grown on fibronectin‐coated dishes with FGF‐2 for 7 days, which was accompanied by negative expression of Ki67. Simultaneously, the ratio of p‐p38 to p‐ERK was also calculated. If the observed ratio was above one, it was considered a positive induction of dormancy. The observed ratio of p‐p38 to p‐ERK for MCF‐7 cells grown on fibronectin‐coated dishes with FGF‐2 under normoxia was 1.44, which indicated that this microenvironment scenario is associated with the induction of dormancy in MCF‐7 cells. Further, as can be seen in Figure 4, none of the other five culture scenarios for MCF‐7 cells grown in normoxic conditions induced dormancy.

FIGURE 4.

FIGURE 4

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Protein expression in MCF‐7 cells cultured under distinct culture scenarios in normoxia condition for 7 days. (a) Phosphorylated p38 and ERK expression (Scale bar 200 μm). (b) Ki67 expression (Scale bar 20 μm). (c) p‐p38 and p‐ERK mean fluorescence intensity representation graph. p‐value summary: p‐p38 MCF‐7, Normoxia Day 7: (1) Control versus FGF‐2 + Fibronectin (****p < 0.0001), (2) Control versus FGF‐2 + Laminin (***p = 0.0005), (3) Control versus Laminin (****p < 0.0001). p‐ERK MCF‐7 Normoxia Day 7: (1) Control versus FGF‐2 + Fibronectin (****p < 0.0001), (2) Control versus FGF‐2 + Laminin (**p = 0.0035).

Figure 5 shows the expression of p‐p38 and p‐ERK in the six culture scenarios for MCF‐7 cells grown under hypoxic conditions. MCF‐7 cells grown on fibronectin coated dishes without FGF‐2 resulted in a growth‐arrest state, with high expression of p‐p38 and relatively similar expression of p‐ERK when compared to normal culture conditions (mean intensity of p‐p38: 36.7 vs. 10.9; p < 0.0001), (mean intensity of p‐ERK: 13.1 vs. 11.5, NS) with p‐p38 to p‐ERK ratio of 2.45, along with no expression for Ki67, which all together were suggestive of dormancy induction. Similarly, MCF‐7 cells cultured on laminin‐coated dishes without FGF‐2 also showed a growth‐arrest state, with high p‐p38 expression and p‐ERK expression compared to normal culture conditions (mean intensity of p‐p38: 34.4 vs. 10.9; p < 0.0001; mean intensity of p‐ERK: 26.6 vs. 11.5, p < 0.0001). The ratio of p‐p38 to p‐ERK was 1.3, alongside the absence of Ki67 expression, collectively indicating the induction of dormancy. None of the other four culture conditions of MCF‐7 cells grown under hypoxia resulted in an induction of dormancy.

FIGURE 5.

FIGURE 5

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Protein expression in MCF‐7 cells cultured under distinct culture scenarios in hypoxia condition for 7 days. (a) Phosphorylated p38 and ERK ratio expression (Scale bar 200 μm). (b) Ki67 expression (Scale bar 20 μm). (c) p‐p38 and p‐ERK mean fluorescence intensity representation graph. p‐value summary: p‐p38 MCF‐7, Hypoxia Day 7: (1) Control versus Fibronectin (****p < 0.0001), (2) Control versus FGF‐2 (**p = 0.0014), (3) Control versus FGF‐2 + Laminin (****p < 0.0001), (4) Control versus Laminin (****p < 0.0001). p‐ERK MCF‐7 Hypoxia Day 7: (1) Control versus FGF‐2 (***p = 0.0005), (2) Control versus FGF‐2 + Laminin (****p < 0.0001), (3) Control versus Laminin (****p < 0.0001).

Figure 6 shows the expression of p‐p38 and p‐ERK in the six culture scenarios for MDA‐MB‐231 cells grown under normoxic conditions. MDA‐MB‐231 cells, when grown on either fibronectin or laminin‐coated dishes showed a high p‐p38 and low p‐ERK expression as compared to normal culture conditions (mean intensity of p‐p38: 39.4 vs. 24.7; p < 0.0001; mean intensity of p‐ERK: 29.1 vs. 63.8, p < 0.0001 for fibronectin coated dishes and mean intensity of p‐p38: 41.5 vs. 24.7; p < 0.0001; mean intensity of p‐ERK: 15.8 vs. 63.8, p < 0.0001 for laminin‐coated dishes) with p‐p38 to p‐ERK ratio of 1.36 and 1.69 respectively, along with no expression of Ki67. These results indicate that microenvironment containing fibronectin or laminin can induce dormancy in MDA‐MB‐231 cell under normoxic condition. Surprisingly, FGF‐2 was seen to increase the p‐ERK expression in MDA‐MB‐231 cells as compared to normal culture conditions (mean intensity: 83.6 vs. 63.8; p = 0.0052) suggesting that it prevented the cells from dormancy induction in normoxic culture conditions.

FIGURE 6.

FIGURE 6

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Protein expression in MDA‐MB‐231 cells cultured under distinct culture scenarios in normoxia condition for 7 days. (a) Phosphorylated p38 and ERK ratio expression (Scale bar 200 μm). (b) Ki67 expression (Scale bar 20 μm). (c) p‐p38 and p‐ERK mean fluorescence intensity representation graph. p‐value summary: p‐p38 MDA‐MB‐231, Normoxia Day 7: (1) Control versus FGF‐2 + Fibronectin (****p < 0.0001), (2) Control versus Fibronectin (****p < 0.0001), (3) Control versus FGF‐2 + Laminin (**p = 0.0054), (4) Control versus Laminin (****p < 0.0001). p‐ERK MDA‐MB‐231, Normoxia Day 7: (1) Control versus Fibronectin (****p < 0.0001), (2) Control versus FGF‐2 (**p = 0.0052), (3) Control versus FGF‐2 + Laminin (*p = 0.0219), (4) Control versus Laminin (****p < 0.0001).

Further, Figure 7 shows the expression of p‐p38 and p‐ERK in MDA‐MB‐231 cells cultured in four hypoxic conditions, since these cells couldn't survive the hypoxic stress without an ECM protein (i.e., either fibronectin or laminin, two culture scenarios). For MDA‐MB‐231 cells grown under hypoxia condition only one microenvironmental condition induced dormancy in the cells where MDA‐MB‐231 cells were grown on laminin‐coated dishes along with FGF‐2, which induced growth arrest and also showed a high p‐p38 and p‐ERK expression as compared to normal culture conditions under normoxia (mean intensity of p‐p38: 106.3 vs. 24.7; p < 0.0001; mean intensity of p‐ERK: 101.3 vs. 63.8, p = 0.0003) with p‐p38 to p‐ERK ratio of 1.05, along with no expression of Ki67.

FIGURE 7.

FIGURE 7

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Protein expression in MDA‐MB‐231 cells cultured under distinct culture scenarios in hypoxia condition for 7 days. (a) Phosphorylated p38 and ERK ratio expression. (Scale bar 200 μm). (b) Ki67 expression. (Scale bar 20 μm). (c) p‐p38 and p‐ERK mean fluorescence intensity representation graph. p‐value summary: p‐p38 MDA‐MB‐231, Hypoxia: (1) Normoxia Control versus FGF‐2 + Laminin (****p < 0.0001). p‐ERK MDA‐MB‐231, Hypoxia: (1) Normoxia Control versus FGF‐2 + Laminin (***p = 0.0003), (2) Normoxia Control versus Laminin (****p < 0.0001).

In summary, we observed that six out of a total of 22 studied culture scenarios showed a dormancy‐like pattern for cell growth with high p‐p38 expression, low p‐ERK expression, and no Ki67 expression. In the remaining 16 culture scenarios, the cells were proliferative.

Further we evaluated expression of p21 and p27 in both the cell lines under previously described conditions. Figure 8 shows the expression of p21 and p27 in MCF‐7 cells under normoxic and hypoxic conditions. Under normoxic conditions, MCF‐7 cells cultured on fibronectin in the presence of FGF‐2 showed a significant increase in p21 expression compared to both control and fibronectin‐alone conditions (mean intensity: 145.0 vs. 99.03 and 103, p < 0.0001). Similarly, p27 expression was significantly increased in MCF‐7 cells cultured on fibronectin compared to control (mean intensity: 168.5 vs. 105.9, p < 0.0001), and was further increased in the presence of FGF‐2 (mean intensity: 206.3 vs. 168.5, p < 0.0001). Under hypoxic conditions, p21 expression was significantly increased in MCF‐7 cells cultured on fibronectin‐coated dishes compared to control (mean intensity: 243.6 vs. 171.7, p < 0.0001). However, the addition of FGF‐2 to fibronectin resulted in a reduction of p21 expression compared to fibronectin alone (mean intensity: 174.5 vs. 243.6, p < 0.0001), bringing levels comparable to control conditions. A similar trend was observed for p27 expression, where fibronectin significantly increased p27 levels compared to control (mean intensity: 218.2 vs. 155.3, p < 0.0001), while the addition of FGF‐2 resulted in a significant reduction in p27 expression as compared to fibronectin alone (mean intensity: 184.9 vs. 218.2, p < 0.0001).

FIGURE 8.

FIGURE 8

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Protein expression in MCF‐7 cells cultured under distinct culture scenarios under normoxia or hypoxia condition for 7 days. (a) p21 expression under normoxia (Scale bar 100 μm). (b) p27 expression under normoxia (Scale bar 100 μm). (c) p21 expression under hypoxia (Scale bar 100 μm). (d) p27 expression under hypoxia (Scale bar 100 μm). (e) p21 and p27 mean fluorescence intensity representation graph. p‐value summary: ****p < 0.0001, ns, non‐significant.

Figure 9 shows the expression of p21 and p27 in MDA‐MB‐231 cells under normoxic and hypoxic conditions. Under normoxic conditions, p21 expression showed a slight increase in cells cultured on fibronectin‐coated dishes compared to control conditions (mean intensity: 183.9 vs. 119.9), although this difference was not statistically significant. When FGF‐2 was added to fibronectin, a small reduction in p21 expression was observed compared to fibronectin alone (mean intensity: 163 vs. 183.9); however, this change was also not statistically significant, and overall levels remained similar to control conditions.

FIGURE 9.

FIGURE 9

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Protein expression in MDA‐MB‐231 cells cultured undert distinct culture scenarios under normoxia or hypoxia condition for 7 days. (a) p21 expression under normoxia (Scale bar 100 μm). (b) p27 expression under normoxia (Scale bar 100 μm). (c) p21 expression under hypoxia (Scale bar 100 μm) (d) p27 expression under hypoxia (Scale bar 100 μm). (e) p21 and p27 mean fluorescence intensity representation graph. p‐value summary: ***p = 0.0001, ****p < 0.0001, ns, non‐significant.

However, p27 expression was significantly increased in cells cultured on fibronectin compared to control (mean intensity: 187 vs. 139.7, p < 0.0001), while the presence of FGF‐2 showed a significant decrease compared to fibronectin alone (mean intensity: 146.1 vs. 187, p < 0.0001). Under hypoxic conditions, p21 expression was increased in MDA‐MB‐231 cells cultured on fibronectin‐coated dishes compared to control (mean intensity: 173.6 vs. 152.7, p < 0.05). The addition of FGF‐2 slightly reduced (mean intensity: 152.6 vs. 173.6, p < 0.05) p21 expression, indicating a limited modulatory effect. A similar trend was observed for p27 expression, where fibronectin significantly increased p27 levels compared to control (mean intensity: 186.7 vs. 146.6, p < 0.0001), and the presence of FGF‐2 maintained or slightly increased p27 expression relative to fibronectin alone.

3.3. Insights Into Senescence and Chemoresistance in Cancer Dormancy

To investigate whether these cells are going into permanent growth arrest (senescence) or dormancy. We performed senescence staining experiments and observed no senescence‐associated beta‐galactosidase activity in the six culture scenarios associated with dormancy induction in these breast cancer cells (Figure 10).

FIGURE 10.

FIGURE 10

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Cells stained for senescence‐associated β‐galactosidase (SA‐β‐Gal). In particular, there was no senescence activity observed in the dormant cancer cells. Although there were a few cases where MCF‐7 cells displayed a slightly higher percentage of senescent cells, but only when grown in the presence of FGF‐2. Subsequently, MDA‐MB‐231 cells demonstrated noticeable SA‐β‐Gal staining when the cells were cultured in the presence of FGF‐2. (a) MCF‐7 cells grown on fibronectin with FGF‐2 in normoxia. (b) MCF‐7 cells grown on fibronectin without FGF‐2 in normoxia. (c) MCF‐7 cells grown on uncoated dishes with FGF‐2 in normoxia. (d) MCF‐7 cells grown on laminin with FGF‐2 in normoxia. (e) MCF‐7 cells grown on laminin without FGF‐2 in normoxia. (f) MCF‐7 cells grown on uncoated dishes without FGF‐2 in normoxia. (g) MCF‐7 cells grown on fibronectin with FGF‐2 in hypoxia. (h) MCF‐7 cells grown on fibronectin without FGF‐2 in hypoxia. (i) MCF‐7 cells grown on uncoated dishes with FGF‐2 in hypoxia. (j) MCF‐7 cells grown on laminin with FGF‐2 in hypoxia. (k) MCF‐7 cells grown on laminin without FGF‐2 in hypoxia. (l) MCF‐7 cells grown on uncoated dishes without FGF‐2 in hypoxia. (m) MDA‐MB‐231 cells grown on fibronectin with FGF‐2 in normoxia. (n) MDA‐MB‐231 cells grown on fibronectin without FGF‐2 in normoxia. (o) MDA‐MB‐231 cells grown on uncoated dishes with FGF‐2 in normoxia. (p) MDA‐MB‐231 cells grown on laminin with FGF‐2 in normoxia. (q) MDA‐MB‐231 cells grown on laminin without FGF‐2 in normoxia. (r) MDA‐MB‐231 cells grown on uncoated dishes without FGF‐2 in normoxia. (s) MDA‐MB‐231 cells grown on fibronectin with FGF‐2 in hypoxia. (t) MDA‐MB‐231 cells grown on fibronectin without FGF‐2 in hypoxia. (u) MDA‐MB‐231 cells grown on laminin with FGF‐2 in hypoxia. (v) MDA‐MB‐231 cells grown on laminin without FGF‐2 in hypoxia. Further for senescence control, (w) Etoposide treated MCF‐7 cells, and (x) Etoposide treated MDA‐MB‐231 cells.

Further, we evaluated the chemoresistance properties and viability of the cells obtained from the culture‐competent 22 scenarios. The baseline viability of cells under normoxia conditions was confirmed using Calcein AM staining (Figure S1). Subsequently, the cells were treated with doxorubicin hydrochloride (2 μM concentration) for 7 days, and cellular viability was evaluated by staining them with calcein‐AM (Figure 11). We observed that only the dormant cancer cells survived and retained their cellular viability, demonstrating their chemoresistant properties. On the other hand, we observed complete cell death in the remaining 16 culture scenarios. This confirms that cells obtained from these culture scenarios were indeed proliferative in nature and were susceptible to doxorubicin hydrochloride, leading to their death.

FIGURE 11.

FIGURE 11

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Cell viability assay (Scale bar 400 μm). Breast cancer cells were stained with calcein‐AM after treatment with doxorubicin hydrochloride (2 μM) for a period of 7 days. (a) Represents MCF‐7 cells cultured at non‐dormant (proliferative) culture scenarios where cells well grown on uncoated petridish under normoxia (b) MCF‐7 cells cultured with fibronectin in hypoxia. (c) MCF‐7 cells cultured with FGF‐2 and fibronectin in normoxia. (d) MCF‐7 cells cultured with laminin in hypoxia. (e) Represents MDA‐MB‐231 cells cultured at non‐dormant culture scenarios where cells well grown on uncoated petridish under normoxia (f) MDA‐MB‐231 cells cultured with fibronectin in normoxia. (g) MDA‐MB‐231 cells cultured with FGF‐2 and laminin in hypoxia. (h) MDA‐MB‐231 cells cultured with laminin in normoxia.

4. Discussion

Several past studies have shown that tumor microenvironmental factors, particularly ECM remodeling, tumor hypoxia, as well as therapy‐induced stress, contribute to the induction of dormancy in breast cancer metastatic niche [33]. In the present study, we found that specific combinations of extracellular matrix proteins (like fibronectin and laminin), growth factor signaling (FGF‐2), and oxygen availability can induce a dormancy‐like phenotype in two breast cancer cell lines, MCF‐7 and MDA‐MB‐231, representing estrogen receptor positive and triple negative breast cancer, respectively. To recapitulate key features of the bone marrow microenvironment, we incorporated FGF‐2, a growth factor abundantly present in the bone marrow niche and known to regulate mammary cell behavior. Previous studies have shown that FGF‐2 exposure can induce a non‐proliferative state in breast cancer cells, accompanied by altered integrin expression and enhanced interactions with ECM [21, 34]. Similarly, strong adhesion to ECM proteins has been reported to support survival of growth‐arrested cells through activation of pro‐survival pathways such as PI3K/Akt signaling [35]. In addition to fibronectin, laminin is a major component of the bone marrow microenvironment, particularly enriched in periosteal and vascular niches. Laminin‐mediated signaling has been implicated in promoting growth arrest and cellular differentiation in breast cancer cells, whereas disruption of these interactions can facilitate tumor progression [36, 37, 38, 39, 40]. However, the molecular mechanisms underlying laminin‐driven regulation of tumor cell dormancy remain incompletely understood. Consistent with these observations, our findings suggest that laminin, in combination with other microenvironmental cues such as FGF‐2 and hypoxia, may play a critical role in regulating the induction and maintenance of a dormancy‐like phenotype in DTCs. Furthermore, our experimental conditions were designed to simulate key features of the hypoxic bone marrow niche [19]. While these experimental conditions do not fully capture the complexity of the bone marrow metastatic microenvironment, which includes stromal, endothelial, and immune components, they enable the identification of causal relationships between defined extracellular signals and dormancy induction.

Additionally, our study used true hypoxic conditions, unlike previous studies which used CoCl2‐induced hypoxia in in vitro models [41]. Current literature on cancer dormancy focuses predominantly on cellular and molecular factors like ECM [42, 43], while non‐cellular factors, such as oxidative stresses in the bone microenvironment, are under‐studied [34, 44]. To our knowledge, the combined role of true hypoxia and laminin‐111 in regulating dormancy in estrogen‐receptor positive and triple‐negative breast cancer cells has not been systematically examined in this way. Therefore, this study improves our understanding of how cellular dormancy is regulated in both normoxic and hypoxic conditions. Key molecular players driving hypoxia‐induced dormancy in breast cancer include HIF‐1α‐dependent pathways as well as HIF‐independent regulators such as NRF2, which enhances antioxidant defenses to counteract ROS‐mediated stress [45, 46]. In parallel, hypoxia modulates cell cycle control through downregulation of proliferative markers (e.g., Ki67, CDKs) and upregulation of CDK inhibitors (p16, p27), collectively enforcing a quiescent, survival‐adapted state [47, 48].

In our system, hypoxia‐dependent dormancy required careful optimization of initial cell seeding density. At low density, cells failed to maintain viability under hypoxia, whereas an empirically determined higher density enabled survival with minimal proliferation. Under these optimized conditions, cells remained viable, with no overt cell death, and displayed a Ki‐67–negative, p‐p38high/p‐ERKlow phenotype, consistent with a dormant state as previously defined by the Aguirre‐Ghiso group [12].

MCF‐7 cells entered dormancy in normoxic conditions in the presence of FGF‐2 and fibronectin together, we have used p‐p38 as a marker here, but studies have also highlighted that both FGF‐2 and fibronectin can regulate other dormancy markers such as p21 and p27, which promote growth arrest through Rb dephosphorylation and inhibition of cyclin‐dependent kinases that might be promoting the dormant phenotype [35, 49, 50]. Consistent with this, we observed increased expression of p21 and p27 under normoxic conditions, supporting their role in inducing a dormancy‐like phenotype. In hypoxic conditions, MCF‐7 cells entered into dormancy in the presence of either fibronectin or laminin and activated p‐p38 whilst lowering the levels of p‐ERK to induce dormancy. This was further reinforced by elevated p21 and p27 levels, particularly in fibronectin‐containing conditions under hypoxia. Studies have also shown that hypoxic stress can activate dormancy markers such as p21 via suppression of c‐Myc [51, 52]. Cellular interactions with fibronectin and laminin have also been shown to initiate the pro‐survival PI3K/Akt signaling pathway [35], which may explain how our dormant phenotype was resistant to doxorubicin.

In contrast, MDA‐MB‐231 cells exhibited distinct, subtype‐specific regulation of dormancy. Under normoxic conditions, either fibronectin or laminin alone was sufficient to induce dormancy, consistent with increased p‐p38 signaling. This was accompanied by elevated p21 and p27 expression, suggesting effective induction of cell cycle arrest. In hypoxic conditions, MDA‐MB‐231 cells in the presence of either fibronectin or laminin were highly proliferative in nature and demonstrated a significantly high expression of p‐ERK. However, when FGF‐2 and laminin were present together, MDA‐MB‐231 cells demonstrated dormant behavior. This suggests that hypoxic stress could be involved in regulating dormancy‐associated mechanisms at a molecular level. Furthermore, fibronectin consistently increased p27 levels under both normoxic and hypoxic conditions, but this alone did not lead to dormancy under hypoxia, suggesting that other signals are also required. However, in this study, we have not performed any experiments to define the regulatory mechanisms responsible for dormancy induction for which further investigations are required. This indicates that although p21 and p27 play a role in growth arrest, they may not be sufficient on their own to drive dormancy, particularly in more aggressive cells. The results from our study provide an opportunity for researchers to develop an experimental model targeting the integrin proteins present in these dormant cancer cells, which interact with ECM. This could potentially reverse dormancy and make them susceptible to chemotherapy.

We also demonstrate that cells in a dormancy‐like state exhibit resistance to doxorubicin, a commonly used chemotherapeutic agent. This is consistent with prior reports indicating that dormant or slow‐cycling breast cancer cells are less sensitive to cytotoxic therapies that target proliferating populations [53]. The persistence of viable cells following prolonged drug exposure in our system supports the concept that dormancy contributes to therapy resistance and disease recurrence.

An important observation from our study is that, at the same seeding density, cells grown under normoxic conditions did not enter dormancy but instead remained proliferative. This may be due to increased cell–cell interactions and contact‐mediated signaling, which support continued growth even in the presence of ECM components. Therefore, these conditions likely reflect a distinct proliferative state rather than the absence of dormancy.

Finally, while our study highlights the role of the microenvironment in dormancy induction, it has a few limitations. The lack of in vivo validation and the use of only two cell lines limit generalizability. Additionally, upstream signaling pathways and downstream survival mechanisms were not directly investigated. From a therapeutic perspective, targeting dormant cancer cells remains a significant challenge. Strategies either to maintain dormancy or selectively eliminate dormant cells by disrupting survival signaling pathways (e.g., integrin/FAK/PI3K axis) may hold promise in preventing metastatic relapse.

5. Conclusion

To conclude, we were able to create culture conditions where specific interactions of ECM components—fibronectin and laminin with FGF‐2 in normoxic and true hypoxic conditions can lead to a dormancy‐like phenotype in breast cancer cell line. The specific combinations leading to a dormancy‐like phenotype were different in the two cell types and differed between normoxia and hypoxia, suggesting that the induction of cellular dormancy is cell and microenvironment context dependent.

Author Contributions

Jinesh Maniar: investigation, methodology, validation, visualization, software, formal analysis, data curation, writing – review and editing. Pulkit Datt: methodology, investigation, software, data curation, formal analysis, validation, writing – original draft, writing – review and editing, visualization, conceptualization. Prerana P. Dange: methodology, data curation, writing – review and editing. Vaishali Kailaje: methodology, data curation, writing – review and editing. Mithlesh Lakhera: methodology, data curation, writing – review and editing. Mansi Samarth: methodology, data curation, writing – review and editing. Tanuja Durve: methodology, data curation, writing – review and editing. Anushree Kadam: methodology, data curation, writing – review and editing. Khushboo A. Gandhi: formal analysis, writing – review and editing, methodology, data curation. Sudeep Gupta: conceptualization, funding acquisition, writing – original draft, visualization, software, formal analysis, supervision, project administration, resources, writing – review and editing.

Funding

This study was funded by the Department of Atomic Energy, Government of India. Dr. Gupta acknowledges philanthropic donations from Mr. Akhil Gupta and Mr. Sunil Gupta to his Clinician Scientist Laboratory at ACTREC/TMC for procuring consumables, establishing laboratory infrastructure, and the salary of research staff.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Calcien AM staining showing control scenario for (a) MCF‐7 and (b) MDA MB‐231 before the doxoxrubicin treatment.

CAM4-15-e72059-s001.docx (468.5KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: Calcien AM staining showing control scenario for (a) MCF‐7 and (b) MDA MB‐231 before the doxoxrubicin treatment.

CAM4-15-e72059-s001.docx (468.5KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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