Breast cancer is the most commonly diagnosed cancer worldwide surpassing lung cancer, with ∼2.3 million new cases reported in 2020, which accounted for 11.7% of all cancer diagnoses. 1,2 With an estimated 42,780 women dying from the disease, there are expected to be approximately 313,510 new cases of invasive breast cancer in the United States by 2024. 3 The aggressive nature of breast cancer, particularly its ability to metastasize to distant organs, is responsible for roughly 90% of breast cancer-related deaths. 4 Among the metastatic sites, the brain poses a particularly formidable challenge, with breast cancer being the second most common solid tumor to metastasize to the brain, following lung cancer. The metastasis process involves tumor cell detachment from the primary site, followed by tumor cells entering the bloodstream, surviving circulation, evading the immune system, adhering to capillaries, and colonizing distant organs. [5][6][7] Moreover, these cells can enter a state of temporary cell cycle arrest, remaining dormant for extended periods of time. This state of dormancy has significant implications for cancer recurrence and metastasis, whether in the primary tumor site or in secondary organs. [8][9][10] Breast cancer cells can enter a dormant state via a balance between proliferation and apoptosis or by the inability to proliferate in a new environment. 8,11,12 Additionally, dormancy can be induced and maintained, in part, by the tumor microenvironment. 13,14 As the noncellular element of the tumor microenvironment, the extracellular matrix (ECM), has been shown to control cancer cell dormancy via ECM stiffness, 15 and composition. 14 For example, the presence of particular ECM proteins including fibronectin, 16 laminin, 17 and collagen 18 can significantly influence cellular behavior, thereby promoting either active or dormant states by regulating cell adhesion, migration, and survival pathways. Changes in the microenvironment of the target organ can also awaken dormant disseminated tumor cells, thereby promoting their proliferation and subsequently disease recurrence. 10,19 Development of therapeutic approaches to target dormant cells and curb metastatic disease progression depends on an understanding of these interactions between breast cancer cells and their microenvironment.
To understand microenvironmental regulation of dormancy, hydrogels have emerged as an important biomaterial tool as they bridge the gap between traditional in vitro models and in vivo models. 20 The inherent flexibility of hydrogel design provides the opportunity to manipulate various aspects of the tumor microenvironment. [21][22][23] Both natural [24][25][26][27] and synthetic [28][29][30] hydrogels have been extensively utilized as in vitro models that mimic key features of the tumor microenvironment to study the mechanisms underlying metastatic breast cancer dormancy and the role of matrix properties in that process. In our previous studies on breast cancer brain metastasis (BCBM), we developed a biomimetic hyaluronic acid (HA) hydrogel-based in vitro platform to investigate how biophysical and biochemical cues regulate the dormant state of brain metastatic breast cancer cells. 31,32 Our findings demonstrated that cells cultured on soft (∼0.4 kPa) HA hydrogels, as opposed to stiff (∼4.5 kPa) ones, which were functionalized with the same levels of fibronectin-derived RGD peptide, exhibited dormancy. Additionally, we explored how varying concentrations of RGD peptide influenced dormancy vs proliferation in the soft HA hydrogel platform. However, the impact of laminin and laminin-derived peptides in modulating dormancy vs proliferation in this hydrogel culture platform was not studied.
To address this gap, in this work, we utilized ∼0.4 kPa HA hydrogels and functionalized them with varying concentrations of laminin I protein or laminin-derived peptides IKVAV/ YIGSR. We verified varying levels of surface functionalization of laminin I using the bicinchoninic acid (BCA) protein assay and to confirm varying levels of surface functionalized lamininderived peptides, fluorescently labeled peptides were employed. After functionalization, we seeded brain metastatic breast cancer cells onto HA hydrogels to determine the impact of these environments on cell morphology, spreading, and dormancy versus proliferation. Additionally, we examined if the dormant phenotype was reversible as well as the role of integrin αVβ3 in cells seeded on HA hydrogels functionalized with laminin through blocking studies.
Materials. Hyaluronic acid (66-90 kDa) was purchased from Lifecore Biomedical. Dulbecco's Modified Eagle's Medium (DMEM), Methacrylic anhydride, and Dithiothreitol (DTT) were purchased from Sigma-Aldrich. Penicillin-streptomycin (PS) was purchased from Gibco. Fetal bovine serum (FBS) was purchased from VWR. Paraformaldehyde and Triton X were purchased from Alfa Aesar. 4,6-diamidino-2-phenylindole (DAPI) was purchased from Invitrogen. Click-iT EdU Cell Proliferation Kit was purchased from Thermo Fisher Scientific. p-ERK [C#4370S], p-p38 [C#9216S] primary antibodies were purchased from Cell Signaling Technology. Secondary goat antirabbit antibody [A11034] and goat antimouse antibody [A11001] were purchased from Thermo Fisher Scientific. Cultrex mouse laminin I was purchased from Bio-Techne. Lamininderived peptides IKVAV with GCGYGIKVAVADR sequence and YIGSR with GCGYGYIGSR sequence were purchased from Gen-Script. Integrin αVβ3 antibody was purchased from Santa Cruz Biotech.
HA Hydrogel Fabrication. Hyaluronic acid methacrylate (HAMA) was prepared as described previously. 15,31 For HA hydrogel fabrication, a gel precursor solution with 5 wt % HAMA in serum-free DMEM was prepared. The cross-linker DTT (10 mM) was then added to the precursor solution. Subsequently, 75 μL of this solution was dispensed into each well of a 96-well plate followed by overnight incubation at 37 °C. This formulation results in HA hydrogels with a stiffness of ∼0.4 kPa as described previously. 15 To create HA hydrogels functionalized with various concentrations of laminin or laminin-derived peptides, the hydrogel surfaces were treated with different concentrations of laminin or laminin-derived IKVAV or YIGSR peptide. Laminin solutions were prepared by diluting the stock solution with ultrapure water to obtain concentrations of 0, 0.5, and 1 mg/mL. Similarly, IKVAV solutions at concentrations of 0, 5, and 10 mg/mL in serum-free DMEM and YIGSR solutions at concentrations of 0, 10, and 20 mg/mL in ultrapure water were prepared.
Next, 25 μL of the laminin, IKVAV, or YIGSR solution was applied to the surface of the HA hydrogels and the hydrogels were incubated overnight at 4 °C. Laminin attachment occurred likely through both physical and covalent bonding. Thiol groups on the free cysteine residues of the laminin molecule which are not involved in the Cterminal regions of the α, β, and γ chains that form the three-strand αhelical coiled coil can form covalent bonds with the methacrylated functional groups of HAMA via Michael-type addition. 33 Likewise, the peptides were covalently attached to the hydrogel surface through a Michael-type addition reaction, occurring between the methacrylated groups of HAMA and the thiol group of the cysteine amino acid within the peptide sequence (Figure S1). Following overnight incubation, the hydrogels were extensively rinsed with serum-free DMEM to eliminate any unbound laminin or laminin-derived peptides.
Quantification of Attached Laminin and Laminin-Derived Peptides on HA Hydrogels. Quantification of Laminin Attached to HA Hydrogels. To quantify the attachment of laminin to HA hydrogels, the Pierce BCA protein assay kit (Thermo Scientific) was employed to determine the concentration of unattached laminin. Following the fabrication of HA hydrogels, 25 μL of laminin solution at concentrations of 1 or 0.5 mg/mL were applied to the hydrogel surface. These functionalized hydrogels were incubated overnight at 4 °C. After incubation, the hydrogels were washed with 100 μL of phosphate-buffered saline (PBS) to remove any unattached laminin. The PBS wash was collected, and 50 μL of this solution, containing the unbound laminin, was analyzed using the BCA protein assay. Absorbance was measured using a FilterMax F5 multimode microplate reader at 550 nm. To determine the concentration of unattached laminin, standard curve was generated using known concentrations of laminin. The total concentration of laminin attached to the HA hydrogels was then calculated by subtracting the concentration of unbound laminin from the initial applied concentration.
Quantification of IKVAV and YIGSR Attached to HA Hydrogels. To quantify the attachment of IKVAV or YIGSR peptides to the hydrogel, we employed a previously established protocol. 32 Briefly, HA hydrogels were prepared using PBS as the solvent instead of DMEM. Next, 25 μL of a peptide solution, containing 5% fluorescently labeled IKVAV or YIGSR peptides at the previously mentioned total concentrations, were applied to the hydrogel surface. These functionalized hydrogels were incubated overnight at 4 °C to ensure proper peptide binding. Post incubation, the hydrogels underwent three washes with PBS to remove any unreacted fluorescently labeled peptides. The fluorescence intensity was subsequently quantified utilizing a FilterMax F5 multimode microplate reader. To quantify the peptide concentrations attached to the HA hydrogels, standard curves were generated for each peptide using known fluorescently labeled peptide concentrations. The overall concentration of functionalized peptides was determined by incorporating 5% fluorescently labeled IKVAV or YIGSR peptides into the solution.
Cell Culture. In this study, we utilized two brain metastatic breast cancer cell lines, MDA-MB-231Br and MDA-MB-361 cells. MDA-MB-231Br cells were kindly provided by Dr. Lonnie Shea (University of Michigan). MDA-MB-361 cells were obtained from ATCC. MDA-MB-361 and MDA-MB-231Br cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin and maintained in a 37 °C environment with 5% CO 2 . The cells were passaged at ∼80% confluency and subsequently seeded onto the HA hydrogel surface.
Optical Imaging, Single Cell Area, and Circularity Measurements. To assess the effect of varying laminin concentrations on cell spreading, 10 K cancer cells were plated onto HA hydrogels and visualized using an Olympus IX83 microscope equipped with a spinning confocal disc attachment. Images were acquired 30 min (day 0) and on day 5 after seeding. Cell area and perimeter were measured using ImageJ software, with cell boundaries manually outlined as described previously. 15 The circularity of cells was calculated by using the formula below:
A minimum of 10 images per condition were analyzed. The impact of varying concentrations of IKVAV or YIGSR on cell spreading was analyzed similarly.
Cell Viability. Cell viability was qualitatively evaluated by staining cells with Calcein AM, as previously described. 34 Briefly, hydrogels with seeded cells were incubated in 100 μL fresh medium containing 4 × 10 -6 M Calcein AM at 37 °C with 5% CO 2 for 1 h on day 5. After incubation, the samples were subjected to a PBS wash. Fluorescence images were then captured using an Olympus IX83 microscope equipped with a spinning disk confocal attachment.
EdU Cell Proliferation Assay. Cell proliferation on HA hydrogels was quantified using the Click-iT EdU Cell Proliferation Kit, in accordance with previously established methods. 35,36 In brief, ∼ MDA-MB-231Br or MDA-MB-361 cells were seeded onto HA hydrogels functionalized with different laminin concentrations. After 4 days, 10 μM EdU was added to the media and incubated overnight. On day 5, the media was removed, and cells were detached via 5 min trypsinization, transferred to a 96-well plate, and fixed with 4% paraformaldehyde for 20 min at room temperature (RT). Permeabilization was performed using 0.25% Triton-X for 15 min at RT, followed by blocking with 5% BSA at 4 °C for 30 min. Cells were then incubated with the reaction cocktail (30 min) at RT in the dark, and nuclei were stained with DAPI. Between steps, cells were washed with PBS, and the well plate was centrifuged at 1000g for 1 min to settle cells before removing liquids. An Olympus IX83 microscope with a spinning disk confocal attachment was used for imaging, and EdU-positive cells were counted manually using ImageJ. The percentage of EdU-positive cancer cells on hydrogels functionalized with varying concentrations of IKVAV or YIGSR were determined similarly.
Immunofluorescence Staining. The expression of p-ERK and p-p38 was detected via immunofluorescence staining. Cells were seeded onto HA hydrogels as described above. On day 5, the cells were trypsinized to detach them from the HA hydrogels. Following previously described techniques, 30,37 the detached cells were then transferred to a 96-well plate for fixation, followed by permeabilization, and blocking. Next, the cells were incubated with primary antibody solutions (p-ERK [1:200]; p-p38 [1:200]) overnight at 4 °C in independent experimental set-ups. The following day, the cells were fluorescently labeled by incubating with secondary antibodies (goat antirabbit antibody [1:1000] for p-ERK, and goat antimouse antibody [1:1000] for p-p38) for 1 h at 4 °C. The cells were counterstained with DAPI. Imaging was carried out using an Olympus IX83 microscope equipped with a spinning disk confocal attachment. The percentage of positive cells for each marker were determined using ImageJ software.
Integrin αVβ3 Staining and Blocking Studies. For integrin αVβ3 staining, the culture medium was removed from cell-seeded hydrogels and the cells on the hydrogel were washed with PBS. The cells were then fixed with 4% paraformaldehyde for 15 min at RT, followed by washing with PBS. The cells were then permeabilized with 0.2% Triton X-100 in PBS for 10 min at RT and subsequently washed with PBS. The cells were then blocked via incubation with 5% BSA for 1 h at RT. The primary antibody Integrin αV/β3 (sc-7312) was diluted 1:200 in PBS, added to the cells, and incubated overnight at 4 °C. On the next day, the cells were washed with PBS, following which, Alexa Fluor 488 goat antimouse IgG secondary antibody diluted in PBS [1:1000] was added to the cells, followed by incubation for 1 h at 4 °C in the dark. The cells were then washed with PBS followed by DAPI staining to label the nuclei.
For integrin αVβ3 blocking experiments, 10 K MDA-MB-231Br cells were seeded onto HA hydrogels functionalized with 1 mg/mL laminin. After 4 days of culture, integrin αVβ3 antibody was added to the culture media at a concentration of 5 μg/mL, and cells were incubated for 24 h with the antibody. Cell spreading area was quantified through imaging, while proliferation was quantified using the Click-iT EdU Cell Proliferation Kit, as described above.
Statistical Analysis. Each experiment was repeated at least 2 times with a minimum of two replicates in each experiment. All data are reported as the mean ± standard deviation unless otherwise indicated. Statistical analyses were conducted using the PRISM software package. Student's t-test or ordinary one-way ANOVA was utilized for sample comparisons, and Tukey-HSD test was conducted for post-ANOVA comparisons.
We used the BCA protein assay to measure the density of laminin on the surface of HA hydrogels. To determine the attached protein density, a standard curve was first created using known laminin concentrations in PBS (Figure S2). This standard curve enabled us to quantify the concentration of unattached laminin in the PBS wash solution, which was used to remove any remaining unbound laminin from the hydrogel surfaces. We calculated the difference to determine the laminin density on the hydrogels. The results indicated that the laminin concentration in the applied solution affected laminin attachment to the HA hydrogel surface, with higher solution concentrations leading to increased laminin density (Table 1).
Quantification of Peptide Density on the Surface of HA Hydrogels. We used fluorescently labeled IKVAV and YIGSR peptides to determine the density of these peptides on HA hydrogel surfaces. To quantify the peptide density on the hydrogel surfaces, we first created a standard curve using known concentrations of the labeled peptides in PBS (Figures S3 andS4). This standard curve enabled us to determine the concentrations of IKVAV and YIGSR attached to the HA hydrogels. As expected, higher concentrations of IKVAV
Table 1. Quantification of Laminin Density on the Surface of HA Hydrogels solutions resulted in increased IKVAV density on the surface of the HA hydrogel (Table 2).
In a similar vein, increasing concentrations of YIGSR solution led to a higher YIGSR density on the HA hydrogel surface (Table 3).
Laminin-Derived Peptide Concentration. We initially evaluated the morphology and spreading of MDA-MB-231Br cancer cells in response to varying concentrations of laminin. After 5 days of culture, in the absence of laminin, the cells displayed a rounded morphology. In contrast, when laminin was present, the cells adopted a spindle-shaped morphology.
We found that cell spreading increased with an increase in applied laminin concentration, showing a positive correlation between the amount of functionalized laminin and cell spreading area (Figure 1A,B). Specifically, in hydrogels without laminin, the cell spreading area (average) was 527 ± 132 μm 2 . When laminin was applied at concentrations of 0.5 and 1 mg/ mL, the cell spreading area increased to 1251 ± 453, and 1412 ± 529 μm 2 , respectively. Cell circularity assessments also revealed that the morphology of the cells changed significantly in the presence of laminin as compared to laminin-deficient condition (Figure S5). These findings align with previous experimental evidence, 38,39 suggesting that a laminin-rich microenvironment influences the morphology of breast cancer cells.
Additionally, we investigated the proliferation of MDA-MB-231Br cancer cells in response to varying laminin concentrations. To assess cancer cell proliferation, we employed EdU staining, which is commonly used to assess cancer cell proliferation. 40 To evaluate the impact of the HA hydrogel platform on inducing a relative dormancy phenotype in MDA-MB-231Br cells, we cultured cells on Tissue Culture Plastic (TCP) as a positive control for 5 days and performed EdU staining. The results demonstrated that the percentage of EdUpositive cells on TCP was significantly higher than that of EdU-positive cells on HA hydrogels in the absence of proteins/ peptides. This finding illustrates the dormancy-inducing effect of the HA hydrogel platform. Specifically, the percentage of EdU-positive cells on day 5 cultured on TCP was 71.6 ± 10.6%, which decreased dramatically to 9.6 ± 1.4% when the cells were cultured on protein/peptide-deficient HA hydrogel surfaces (Figure S6). These results indicate a shift from a proliferative state to a dormant state when transitioning from TCP to the protein/peptide-deficient HA hydrogel platform.
In addition, on day 5, EdU staining revealed a significantly lower percentage of EdU-positive cells in hydrogels without laminin compared to those with laminin concentrations of 0.5 and 1 mg/mL. Specifically, the percentage of EdU-positive cells was 9.6 ± 1.4% in the absence of laminin, 18.3 ± 0.5% in the presence of 0.5 mg/mL laminin, and 24.6 ± 6.7% with 1 mg/mL applied laminin concentration (Figure 1C,D). These findings suggest that cells cultured on hydrogels with higher laminin concentrations exhibited a proliferative phenotype, whereas cells in laminin-free condition demonstrated a nonproliferative or dormant phenotype. The increase noted in the percentage of EdU-positive cells also aligned with the cell area measurements (Figure 1A,B). We also evaluated the morphology, spreading, and proliferation of a human epidermal growth factor 2 (HER2) positive cell line, MDA-MB-361. 41 We found that the morphology and proliferation of these cells were not substantially influenced in the presence of laminin. The MDA-MB-361 cells displayed similar morphology and comparable levels of proliferation after being cultured on HA hydrogels functionalized with 0, 0.5, or 1 mg/mL applied laminin concentration for 5 days (Figure S7).
To explore the mechanisms underlying the effects of laminin on cell morphology and proliferation, we utilized HA hydrogels functionalized with varying concentrations of laminin-derived peptides, IKVAV and YIGSR. Also, we selected MDA-MB-231Br cells for further studies because of their responsiveness to laminin. We first assessed the morphology of MDA-MB-231Br cells by seeding ∼10 K cells on HA hydrogels functionalized with varying concentrations of IKVAV, a peptide derived from the α1 chain of laminin. 42 After 5 days of culture, cells on IKVAV-functionalized hydrogels displayed significantly greater spreading compared to those on IKVAVdeficient hydrogels, where the cells exhibited a rounded morphology. Cell area measurements revealed that the cell spreading area increased with an increase in the applied concentration of the IKVAV peptide. Specifically, the average cell spreading area was 1861 ± 971, and 1187 ± 722 μm 2 for cells cultured on hydrogels with 10 and 5 mg/mL IKVAV, respectively. These values were significantly higher than the average cell area of 527 ± 132 μm 2 observed in cells cultured on hydrogels without IKVAV (Figure 2A,B). The changes in cell morphology were also confirmed via circularity measurements (Figure S5).
To investigate the impact of the laminin-derived IKVAV peptide on cell proliferation, we employed EdU staining. EdU staining results revealed that cells cultured on IKVAVfunctionalized hydrogels exhibited higher proliferation compared to those on IKVAV-deficient hydrogels. Moreover, increasing the concentration of IKVAV on the hydrogels significantly enhanced the proliferation of MDA-MB-231Br cells. Specifically, the percentage of EdU-positive cells on hydrogels functionalized with 10 mg/mL IKVAV was 31.0 ± 5.8%, while the percentage of EdU-positive cells on hydrogels functionalized with 5 mg/mL IKVAV was 23.1 ± 3.2% (Figure 2C,D).
Next, we investigated the effects of YIGSR, a peptide derived from the β1 chain of laminin, 42 on the behavior of MDA-MB-231Br cells. On day 5, we evaluated cell morphology and found that the MDA-MB-231Br cells exhibited a rounded morphology across all hydrogels, regardless of functionalization with YIGSR. Cell area measurements revealed significantly smaller spreading areas for cells on YIGSR-functionalized hydrogels, with averages of 324 ± 82, and 301 ± 71 μm 2 for hydrogels treated with 10 and 20 mg/mL YIGSR, respectively. These values were considerably lower than the cell area observed in the YIGSR-deficient condition, which was 527 ± 132 μm 2 (Figure 3A,B). The results of cell circularity measurements indicated no significant difference between YIGSR-deficient HA hydrogels and HA hydrogels functionalized with varying concentrations of YIGSR (Figure S5). Further, the assessment of cell proliferation revealed that the average percentage of EdU-positive cells was 3.9 ± 5.4% and 5.1 ± 5.5% for hydrogels functionalized with 10 and 20 mg/mL YIGSR, respectively. These values indicate that the presence of YIGSR, even at higher concentrations, did not significantly enhance the proliferation of MDA-MB-231Br cells compared to the YIGSRdeficient hydrogels (Figure 3C,D). Nonetheless, in all conditions tested, MDA-MB-231Br cells were viable as tested via the Calcein-AM assay (Figure S8).
Cell Phenotype. To elucidate the underlying mechanism regulating cell phenotype via cell-matrix interactions in the presence of laminin, we examined the role of integrin αVβ3 based on its dominant role in cell adhesion and interactions with the ECM-derived laminin protein. 42 In particular, we blocked integrin αVβ3 using an antibody in MDA-MB-231Br cells seeded on HA hydrogels that were modified with a 1 mg/ mL solution of laminin that showed significantly higher levels of cell spreading and cell proliferation compared to the laminin-deficient condition (Figure 1). We found that the average cell spreading area without blocking integrin αVβ3 was 1525 ± 878 μm 2 which significantly decreased to 740 ± 235 μm 2 after incubation with integrin αVβ3 antibody. Similarly, we observed a significant reduction in percentage of EdU positive cells from 24.1 ± 5.2% without blocking integrin αVβ3 to 17.1 ± 5.0% after blocking integrin αVβ3 (Figure 4A,B). Furthermore, αvβ3 immunostaining qualitatively confirmed the expression of this integrin in cells on hydrogels functionalized with 1 mg/mL laminin. Cells cultured on HA hydrogels without laminin (0 mg/mL functionalization) did not express αvβ3. Also, hydrogels functionalized with 1 mg/ mL laminin and treated with αvβ3 integrin-blocking antibody for 24 h qualitatively exhibited reduced expression relative to no antibody controls (Figure S9). These findings demonstrated that integrin αVβ3, at least, in part, played a role in promoting cell spreading, adhesion and proliferation in the HA hydrogel platform modified with laminin.
To determine dormancy vs proliferation, we probed the percentage of p-ERK and p-p38 positive cell populations as a function of varying laminin and laminin-derived peptide concentration. The balance between these pathways is critical in determining whether cancer cells remain dormant or become proliferative. 43,44 The ERK pathway is well-known for promoting cell proliferation and driving cell cycle progression, which aids in tumor growth. 45 The p38 pathway, on the other hand, has been linked to cellcycle arrest, which inhibits proliferation while increasing cell survival. 46 Also, a lower ratio of %p-ERK positive cells to %p-p38 positive cells has also been reported as an indicator of dormancy. 30,47 We quantified the percentage of MDA-MB-231Br cells positive for phosphorylated ERK and p38 on day 5 using immunostaining. Our analysis revealed that the presence of laminin in the HA hydrogels increased the percentage of p-ERK-positive cells. Specifically, in HA hydrogels functionalized with 1 and 0.5 mg/mL laminin, the percentage of p-ERK positive cells were 23.1 ± 5.3% and 21.5 ± 6.4%, respectively.
In contrast, the percentage of p-ERK positive cells decreased to 12.8 ± 7.3% in the absence of laminin (Figure 5A,B).
The evaluation of phosphorylated p38 expression revealed a significant reduction in expression levels in hydrogels functionalized with the highest concentration of laminin. In particular, the percentage of p-p38 positive cells was relatively consistent between cells seeded on HA hydrogels functionalized with 0.5 mg/mL laminin and those on laminin-free hydrogels, at 34.1 ± 1.5% and 36.2 ± 8.8%, respectively. However, increasing the laminin concentration to 1 mg/mL resulted in a significant decrease in p-p38 positivity, with the percentage dropping to 21.4 ± 6.8% (Figure 5C,D). We found that with an increase in laminin concentration, the ratio of %p-ERK positive cells to % p-p38 positive cells also increased (Table S1).
We then evaluated the impact of laminin-derived peptides, IKVAV and YIGSR, on the expression levels of the p-ERK and p-p38 in MDA-MB-231Br cancer cells seeded on HA hydrogels. We found that the percentage of p-ERK positive cells increased with an increase in the applied IKVAV concentration. Specifically, the average percentage of p-ERK positive cells was 12.8 ± 7.3% in the absence of IKVAV. This percentage increased significantly when cells were seeded on hydrogels functionalized with IKVAV, with a value of 22.3 ± 5.0% for hydrogels containing 5 mg/mL of IKVAV and 29.3 ± 5.5% for hydrogels containing 10 mg/mL of IKVAV (Figure 6A,B).
The evaluation of p-p38 expression levels revealed a decreasing trend in response to increasing concentrations of IKVAV on HA hydrogels. Although the percentage of p-p38 positive cells on hydrogels functionalized with 5 mg/mL IKVAV was lower, averaging 26.6 ± 4.7%, compared to the IKVAV-deficient hydrogels, which had an average of 36.2 ± 8.8%, this difference was not statistically significant. However, when the concentration of IKVAV was increased to 10 mg/ mL, the percentage of p-p38 positive cells further declined to 24.8 ± 6.3%, a reduction that was statistically significant compared to the IKVAV-deficient condition (Figure 6C,D). We found that with an increase in IKVAV concentration, the ratio of %p-ERK positive cells to %p-p38 positive cells also increased (Table S2).
The evaluation of MDA-MB-231Br cancer cells on HA hydrogels with varying concentrations of YIGSR revealed that the presence of this peptide did not significantly influence p-ERK expression levels, even at high concentration. Across all conditions tested, the percentage of p-ERK positive cells remained within a similar range. Specifically, the percentage of p-ERK positive cells was 12.8 ± 7.3% for YIGSR-deficient hydrogels, and 14.3 ± 5.9% and 13.6 ± 5.7% for hydrogels functionalized with 10 and 20 mg/mL YIGSR, respectively (Figure 7A,B). A similar trend was observed for p-p38 expression levels. The percentage of p-p38 positive cells on YIGSR-free HA hydrogels was 36.2 ± 8.8%, and this value showed no significant variation in the presence of YIGSR, with percentages of 31.1 ± 4.0% and 31.3 ± 7.1% for hydrogels functionalized with 10 and 20 mg/mL YIGSR, respectively (Figure 7C,D). The ratio of %p-ERK positive cells to %p-p38 positive cells in all hydrogels functionalized with different concentrations of YIGSR was similar, indicating that the presence of different concentrations of YIGSR did not have a significant effect (Table S3). These findings suggest that the laminin as well as IKVAV concentration modulates dormant vs proliferative phenotype, in contrast to YIGSR, wherein a proliferative phenotype is not observed.
Reversibility of the Dormant Tumor Cell Phenotype. To determine if the dormant phenotype observed in MDA-MB-231Br cells could be reversed, we seeded 10 K cells on HA hydrogels in the absence of IKVAV functionalization and cultured them for a period of 5 days. Following this, the cells were carefully retrieved and transferred to freshly prepared HA hydrogels that were either IKVAV-deficient or functionalized with 10 mg/mL IKVAV solution. The cells were then cultured for an additional 5 days.
MDA-MB-231Br cells maintained their rounded shape after being transferred to the IKVAV-deficient hydrogels, with an average cell spreading area of 443 ± 102 μm 2 . However, a shift from dormant state to a proliferative state was noted when the cells were transferred to IKVAV-enriched hydrogels, characterized by a spindle-shaped morphology and a notable increase in cell spreading. The average cell spreading area in this condition reached 991 ± 390 μm 2 , significantly higher than that observed in the IKVAV-deficient hydrogels by day 10 (Figure 8A,B).
These observations were further supported by EdU staining analysis. Cells cultured on IKVAV-enriched hydrogels for 10 days exhibited a significantly higher percentage of EdU-positive cells, at 16.6 ± 3.2%, compared to the 8.5 ± 3.6% observed in the IKVAV-deficient hydrogels (Figure 8C,D). These results indicate that the cells transitioned to a proliferative state upon exposure to IKVAV. Collectively, these findings demonstrate the reversibility of the dormant phenotype in brain metastatic breast cancer cells mediated via the IKVAV peptide.
In this study, we elucidated the role of laminin and laminin derived peptides IKVAV and YIGSR in regulating brain metastatic breast cancer cell dormancy vs proliferation by employing a biomimetic HA hydrogel culture platform. By varying laminin concentrations, we aimed to understand how varying levels of laminin influence tumor cell morphology, spreading, and dormancy versus proliferation. Additionally, to examine the specific active sites of laminin that regulate tumor cell phenotypes and their response to the microenvironment, we focused on the laminin-derived peptides IKVAV and YIGSR. This allowed us to determine which peptide plays a significant role in mediating cancer cell interactions with laminin particularly focusing on dormancy vs proliferation. Previous studies on the role of microenvironmental cues in modulating dormancy in brain metastatic breast cancer cells in vitro have been limited, with studies primarily focusing on the impact of biophysical cues. 15,25,30,31,48 To address this gap, our research group previously explored the role of varying levels of adhesivity, though this investigation was limited to the RGD peptide. 32 To further expand our understanding of other ECM proteins, herein we focused on examining the impact of laminin and laminin-derived IKVAV and YIGSR peptide on brain metastatic breast cancer cell dormancy versus proliferation.
The brain tumor microenvironment is a key determinant in the progression of breast cancer brain metastasis. The normal brain ECM is rich in glycosaminoglycans, such as HA, and contains minimal levels of fibrotic ECM proteins like collagen and laminin. 49 However, in primary and metastatic brain tumors, the ECM composition is altered, with shifts in the balance of ECM proteins that influence regulation of dormancy and tumor progression. 50,51 There is a limited understanding of how these biochemical cues influence metastatic tumor cell dormancy and reawakening. Our study advances this understanding by utilizing biomimetic HA hydrogels functionalized with laminin and laminin-derived peptides, which are found in higher concentrations in the remodeled brain tumor ECM, 49 to explore the role of these biochemical cues in regulating dormancy and the transition from a dormant to proliferative state.
Our results indicate a relationship between the concentration of laminin applied to the HA hydrogel surface and the level of cell spreading in MDA-MB-231Br cells. These observations are consistent with prior studies on the influence of laminin-rich environments on breast cancer cell adhesion. [52][53][54] Moreover, a notable increase in cell proliferation was observed on HA hydrogels functionalized with higher laminin concentrations. This is likely attributed to the interaction between laminin and integrins on the MDA-MB-231Br cell surface, which play a crucial role in activating signaling pathways that drive cellular proliferation. 54,55 To confirm this, we conducted integrin αVβ3 blocking studies. Blocking αVβ3 integrin for 24 h resulted in a significant reduction in cell spreading area and cell proliferation, indicating that αVβ3 integrin signaling, at least, in part, mediates cell phenotype. This finding is also likely cell specific, as we did not observe any laminin-dependent effects in MDA-MB-361 cells.
We investigated the effects of the laminin-derived peptide IKVAV, finding that increasing IKVAV concentrations significantly enhanced MDA-MB-231Br cell spreading and proliferation. In contrast, the laminin-derived peptide YIGSR did not influence cell proliferation. Cells cultured on YIGSRfunctionalized hydrogels showed reduced level of cell spreading and did not show a significant increase in proliferation, even at higher concentrations. These findings are consistent with previous research on other cancer types, which found a similar cell response to the presence of IKVAV and YIGSR peptides in their microenvironments. 42,[56][57][58][59][60] Our results suggest that IKVAV plays a more important functional role than YIGSR in mediating the interaction between laminin and brain metastatic breast cancer cells.
Previous research has shown that dormant cancer cells typically exhibit low levels of p-ERK, which is linked to cellular proliferation, and high levels of p-p38, which is associated with cellular dormancy. 9,47,61 Cancer cells that continuously proliferate often display constant activation of ERK, enabling cell division. 62,63 However, during this ERK-driven proliferation, elevated activity of p38 mitogen-activated protein kinase acts as a counter-regulatory mechanism. This increased p38 activity inhibits ERK, stopping cell proliferation and inducing dormancy. 64,65 In this study, we found that the presence of laminin in HA hydrogels led to a significant increase in the percentage of p-ERK-positive MDA-MB-231Br cells, indicating that cells in a laminin-enriched microenvironment adopted a proliferative state. Additionally, MDA-MB-231Br cancer cells cultured on hydrogels with the highest laminin concentration exhibited significantly lower percentages of p-p38 positive cells compared to those on laminin-deficient hydrogels, where cells adopted a dormant state in the absence of laminin. These findings demonstrate the role of laminin as a biochemical cue to modulate brain metastatic breast cancer cell phenotypes. Additionally, our investigation into the effects of lamininderived peptides, IKVAV and YIGSR, revealed that increasing concentrations of IKVAV resulted in a higher percentage of p-ERK positive cells, indicating a higher percentage of cells in a proliferative state. As the IKVAV concentration increased, the percentage of p-p38 positive cells decreased, also indicative of a proliferative state. On the other hand, YIGSR did not significantly impact the expression levels of either p-ERK or p-p38, suggesting that IKVAV plays a more prominent role in influencing these signaling pathways in brain metastatic breast cancer cells. These results highlight the crucial role of laminin and laminin-derived peptides within the tumor microenvironment in determining the dormant or proliferative state of MDA-MB-231Br cells by regulating specific biochemical interactions and modulating cellular responses to microenvironmental changes. A key component of disease recurrence at metastatic sites is the reactivation of dormant tumor cells. Eventually, dormant cancer cells could re-enter the cell cycle and adapt to new microenvironments, ultimately causing metastases. 9,20 This process is heavily influenced by specific cues within the tumor microenvironment. The ECM plays a key role in this process, not only by providing structural support but also by actively participating in cell signaling. Alterations in the ECM's composition, stiffness, or associated signaling molecules can trigger the reactivation of dormant cancer cells, leading to metastatic growth. 19,51,[66][67][68] In our study, cell spreading, and proliferation significantly increased following 5 days of culture when MDA-MB-231Br cells were transferred from IKVAVdeficient HA hydrogel to the hydrogel functionalized with the highest IKVAV concentration. Still, the level of cell spreading, and proliferation was less than that observed for cells grown straight on HA hydrogels with 10 mg/mL applied IKVAV concentration (Figures 8 vs 2). This could be due to the initial dormancy phase that the cells underwent when first cultured on IKVAV-deficient hydrogels. This initial dormancy period may have delayed the cells' ability to immediately transition to a fully proliferative state after being transferred to IKVAV-rich hydrogels.
In sum, we have demonstrated that varying concentration of laminin protein on HA hydrogels significantly influences brain metastatic breast cancer cell morphology, spreading, and dormancy vs proliferation. This was mediated, in part, via the αVβ3 integrin. To determine which laminin motifs might be involved in mediating cancer cell phenotype, we examined laminin-derived peptides, IKVAV and YIGSR, finding that the presence of IKVAV captures the effects seen in the presence of laminin. However, we acknowledge the following limitations of our study: (1) While unlikely, it is possible that surface stiffness may be altered with peptide/protein conjugation, which was not tested in this work. (2) Future studies would examine how varying laminin and laminin-derived peptides regulate dormancy vs proliferation in cancer cells encapsulated in three-dimensional (3D) environments. (3) Future studies also would investigate the role of laminin and laminin-derived peptides in modulating cancer cell invasion and migration. (4) In the future, we will investigate the effect of other ECM derived proteins, such as Tenascin-C and Collagen-IV, both found in the brain tissue microenvironment 69,70 on dormancy versus proliferation. (5) In future studies, we will examine the mechanistic link between cell adhesion and dormancy phenotype through further analysis of adhesion molecules such as FAK, vinculin, talin, and mechanotransducers such as YAP/TAZ, which would provide deeper insights into dormancy regulation.
In this study, we examined the effect of laminin and lamininderived peptides on brain metastatic breast cancer cell morphology, spreading, and dormancy versus proliferation. Our results showed that the presence of laminin and the laminin-derived peptide IKVAV increased cell spreading with cells transitioning from a round to a spindle-like morphology. Further, in the presence of laminin and IKVAV peptide, cell proliferation was enhanced. In contrast, the presence of laminin-derived YIGSR peptide did not influence cell spreading and proliferation. We demonstrated that αVβ3 integrin was, in part, involved in mediating cell-matrix interactions, influencing cell morphology, spreading, and proliferation. The presence of laminin or IKVAV led to an increase in p-ERK positivity and a decrease in p-p38 positivity compared to cells cultured on hydrogels in the absence of laminin or IKVAV, while the presence of YIGSR had no impact. We also found that modulating the culture environment via laminin-derived peptide IKVAV reversed HA hydrogel-induced cellular dormancy. Overall, the HA hydrogel culture platform could be utilized to examine the effects of various biochemical cues, such as proteins or peptides, on the regulation of dormancy versus proliferation in brain metastatic breast cancer cells in vitro.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.4c01386. Schematic representation of the functionalization reaction; standard calibration curve of laminin, IKVAV, and YIGSR peptide concentration; evaluation of circularity in brain metastatic breast cancer cells; morphology, cell spreading, and proliferation of brain metastatic breast cancer cells on TCP and protein/ peptide-deficient HA hydrogels; morphology, cell spreading, and proliferation of MDA-MB-361 cells on HA hydrogels as a function of laminin concentration; representative fluorescent images of Calcein AM staining of MDA-MB-231Br cells; representative fluorescent images of integrin αvβ3 immunostaining of MDA-MB-231Br cells; %p-ERK/%p-p38 positive cells in hydrogels with varying laminin, IKVAV, or YIGSR concentration (PDF)
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AUTHOR INFORMATION Corresponding Author Shreyas S. Rao -Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama, United States; orcid.org/0000-0001-7649-0171; Phone: (205) 348-6564; Email: srao3@eng.ua.edu; Fax: (205) 348-7558
https://doi.org/10.1021/acsabm.4c01386 ACS Appl. Bio Mater. 2025, 8, 2824-2837