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[Tianjiao Zeng](https://orcid.org/0000-0002-1286-0337), Huajian Chen, [Toru Yoshitomi](https://orcid.org/0000-0003-3847-1812), [Naoki Kawazoe](https://orcid.org/0000-0003-3916-0709), Yingnan Yang, [Guoping Chen](https://orcid.org/0000-0001-6753-3678)

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[Effect of Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells in 3D Culture](https://mdr.nims.go.jp/datasets/62490f30-afe4-4f99-965a-ae1433c7efc9)

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Effect of Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells in 3D CultureCitation: Zeng, T.; Chen, H.; Yoshitomi,T.; Kawazoe, N.; Yang, Y.; Chen, G.Effect of Hydrogel Stiffness onChemoresistance of Breast CancerCells in 3D Culture. Gels 2024, 10, 202.https://doi.org/10.3390/gels10030202Academic Editor: Aline F. MillerReceived: 5 March 2024Accepted: 12 March 2024Published: 17 March 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). gelsArticleEffect of Hydrogel Stiffness on Chemoresistance of Breast CancerCells in 3D CultureTianjiao Zeng 1,2 , Huajian Chen 1, Toru Yoshitomi 1 , Naoki Kawazoe 1 , Yingnan Yang 3and Guoping Chen 1,2,*1 Research Center for Macromolecules and Biomaterials, National Institute for Materials Science,Tsukuba 305-0044, Japan; zeng.tianjiao@nims.go.jp (T.Z.); chen.huajian@nims.go.jp (H.C.);yoshitomi.toru@nims.go.jp (T.Y.); kawazoe.naoki@nims.go.jp (N.K.)2 Graduate School of Science and Technology, University of Tsukuba, Tsukuba 305-8577, Japan3 Graduate School of Life and Environmental Science, University of Tsukuba, Tsukuba 305-8572, Japan;yo.innan.fu@u.tsukuba.ac.jp* Correspondence: guoping.chen@nims.go.jp; Tel.: +81-29-860-4496Abstract: Chemotherapy is one of the most common strategies for cancer treatment, whereas drugresistance reduces the efficiency of chemotherapy and leads to treatment failure. The mechanism ofemerging chemoresistance is complex and the effect of extracellular matrix (ECM) surrounding cellsmay contribute to drug resistance. Although it is well known that ECM plays an important role inorchestrating cell functions, it remains exclusive how ECM stiffness affects drug resistance. In thisstudy, we prepared agarose hydrogels of different stiffnesses to investigate the effect of hydrogelstiffness on the chemoresistance of breast cancer cells to doxorubicin (DOX). Agarose hydrogelswith a stiffness range of 1.5 kPa to 112.3 kPa were prepared and used to encapsulate breast cancercells for a three-dimensional culture with different concentrations of DOX. The viability of the cellscultured in the hydrogels was dependent on both DOX concentration and hydrogel stiffness. Cellviability decreased with DOX concentration when the cells were cultured in the same stiffnesshydrogels. When DOX concentration was the same, breast cancer cells showed higher viability inhigh-stiffness hydrogels than they did in low-stiffness hydrogels. Furthermore, the expression ofP-glycoprotein mRNA in high-stiffness hydrogels was higher than that in low-stiffness hydrogels.The results suggested that hydrogel stiffness could affect the resistance of breast cancer cells to DOXby regulating the expression of chemoresistance-related genes.Keywords: agarose hydrogel; stiffness; chemoresistance; breast cancer cells; 3D culture1. IntroductionBreast lumps are common in the diagnosis of breast diseases. Around 80% of breastcancer patients seek medical consultations for palpable breast lumps, as the breast cancertissue is more rigid than their surrounding normal tissue [1,2]. This pathological symptomindicates that an increase in matrix stiffness plays an important role during breast tumori-genesis and the progression of the disease. At the microscopic scale, the stiffness of thematrix, especially the stiffness of the extracellular matrix (ECM), has been found to have aclose relationship with cell behavior [3,4].Matrix stiffness is primarily induced by the rearrangement, cross-linking and depo-sition along with the degradation of specific ECM proteins. The ECM components incancers are mainly secreted by cancer-associated fibroblasts (CAFs) [5]. The expressionof lysyl oxidase (LOX) by CAFs that initiates the cross-linking of collagen can increaseECM stiffness and further affect cell functions [6,7]. For example, the stiffened ECM drivesthe activation and stabilization of vinculin and enhances Akt signaling to promote cancerprogression [8]. Moreover, ECM stiffness regulates transforming growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT), fostering cancer cell intravasationGels 2024, 10, 202. https://doi.org/10.3390/gels10030202 https://www.mdpi.com/journal/gelshttps://doi.org/10.3390/gels10030202https://creativecommons.org/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/gelshttps://www.mdpi.comhttps://orcid.org/0000-0002-1286-0337https://orcid.org/0000-0003-3847-1812https://orcid.org/0000-0003-3916-0709https://orcid.org/0000-0001-6753-3678https://doi.org/10.3390/gels10030202https://www.mdpi.com/journal/gelshttps://www.mdpi.com/article/10.3390/gels10030202?type=check_update&version=1Gels 2024, 10, 202 2 of 13and metastasis [9,10]. In light of the crucial role of ECM stiffness in regulating cancercell functions, extensive research has focused on developing innovative culture platformswith pathologically relevant stiffness in order to enable the in-depth exploration of matrixstiffness-mediated tumorigenesis and cancer progression [11–13].The ECM stiffness in normal breast tissue is less than 1 kPa, whereas it increasesto a range of 4 to 100 kPa for breast cancers [14–17]. Additionally, benign breast massesexhibit much lower stiffness (approximately 39.4 kPa) compared to malignant breast masses(around 100 kPa) [18–20]. In a word, during the development of breast cancers, there is awide-range change in ECM stiffness. The increase in stiffness has been used not only asa diagnostic factor of breast cancers, but also to assess the malignant level [19–22]. Thelarge variations in stiffness within different pathological grading reveal that cell functionsare highly related with ECM stiffness. However, many studies have reported the effectsof stiffness on breast cancer cell functions within a narrow range of stiffness that is either0.2 to 2 kPa [23] or 10 to 57 kPa [24]. There are no comparable results regarding the effectof stiffness across the entire spectrum of breast cancer stiffness in three-dimensional (3D)culture [25]. Given the wide spectrum of ECM stiffness, it is challenging to accurately andcomprehensively recapitulate the effect of ECM stiffness on breast cancer development andeven the occurrence of chemoresistance of breast cancer cells.On the other hand, chemotherapy is one of the most common strategies for cancertreatment, whereas drug resistance reduces the chemotherapy efficiency seriously and evencauses the failure of chemotherapy [26,27]. The mechanism of chemoresistance emerging iscomplex, which includes, while not limited to DNA repair, apoptosis and/or autophagyinhibition, expression change in miRNA and overexpression of ATP-binding cassette (ABC)drug transporter proteins [28–31]. Among them, the enhanced drug efflux out of thecells through the ABC drug transporter protein family, especially overexpression of P-glycoprotein (P-gP, also known as ABCB1), is regarded as the major factor that contributesto drug resistance [32]. Doxorubicin (DOX) is a commonly used first-line drug for breastcancer chemotherapy, while it is also one of the substrates of P-gP [33]. P-gP can triggerDOX efflux out of the cells and therefore induce DOX-resistance in breast cancers [34].In an early study, chemoresistance was more considered to be the cellular mechanismbut with little relationship to the surrounding environment [35]. However, some recentstudies have revealed the important role of ECM in the generation of drug resistance [36,37].For instance, interaction between acute myelogenous leukemia and fibronectin in bonemarrow reduces chemosensitivity of the cells [38] and the chemoresistance mediated byreceptor–ligand interaction of the cells with ECM is termed as cell-adhesion-mediated drugresistance (CAM-DR) [39]. Although numerous studies have reported the effect of ECM onchemoresistance, the relationship between ECM stiffness and chemoresistance occurrenceis unknown. Investigating the effect of ECM stiffness on the chemoresistance of breastcancer cells can help to deepen our understanding of the mechanism of chemoresistanceand to improve the treatment of drug-resistant cancers.Hydrogels are ideal materials to create ECM conditions because they can be fabricatedfrom various artificial or natural polymers and can span the range of stiffness seen intissues and organs. Agarose hydrogel is one of the most common hydrogels that has beenused in biomedical research. Agarose is a linear polysaccharide extracted from seaweedand has good biocompatibility for cell culture and tissue engineering applications [40–42].Unlike the gelation process of other hydrogels, agarose hydrogels can be easily formedthrough temperature controlling [43,44]. At high temperatures, agarose exists in the formof random coils in aqueous solutions [45]. As the temperature decreases to the gelationpoint, the agarose helices are associated with long fiber-like aggregates that eventually forma percolating network during the sol–gel transition [46,47]. After gelation, the hydrogelsremain stable at 37 ◦C and there is no need to introduce other cross-linking agents. Moreimportantly, the mechanical properties of agarose hydrogels, such as stiffness, are highlydependent on the concentration and molecular weight [45,46,48]. Thus, the properties ofagarose hydrogel can be easily tuned to recapitulate the mechanical characteristics of theGels 2024, 10, 202 3 of 13ECM in breast cancers. Agarose hydrogels are considered as a suitable platform to studythe effect of stiffness on breast cancer cell function in 3D culture.Therefore, in this study, agarose hydrogels with a wide range of stiffness (1.5 kPa~112.3 kPa)were prepared to cover the whole stiffness range of breast cancers and used to encapsulatebreast cancer cells for 3D culture. The effect of agarose hydrogel stiffness on the resistanceof breast cancer cells to DOX was investigated. The possible mechanism was elucidatedthrough analyzing the expression of P-gP mRNA (Figure 1).Gels 2024, 10, x FOR PEER REVIEW  3  of  13   molecular weight [45,46,48]. Thus, the properties of agarose hydrogel can be easily tuned to  recapitulate  the mechanical  characteristics  of  the  ECM  in  breast  cancers. Agarose hydrogels are considered as a suitable platform to study the effect of stiffness on breast cancer cell function in 3D culture. Therefore,  in  this  study,  agarose  hydrogels  with  a  wide  range  of  stiffness  (1.5 kPa~112.3 kPa) were prepared  to cover  the whole stiffness range of breast cancers and used  to  encapsulate breast  cancer  cells  for  3D  culture. The  effect of  agarose hydrogel stiffness on  the  resistance of breast cancer cells  to DOX was  investigated. The possible mechanism was elucidated through analyzing the expression of P-gP mRNA (Figure 1).  Figure 1. Usage of agarose hydrogels to mimic the ECM stiffness of breast cancers to investigate the effect of ECM stiffness on chemoresistance of breast cancer cells. 2. Results and Discussion 2.1. Preparation and Characterization of Agarose Hydrogels The agarose hydrogels were prepared from 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose aqueous  solutions. Their gross appearance  is shown  in Figure 2a. The hydrogels were transparent with increasing turbidity as the concentration of agarose increased. The SEM observation  of  the  lyophilized  agarose  hydrogels  showed  that  all  the  hydrogels  had similar  porous  structures  (Figure  2b,c).  The  porous  structures  were  formed  after lyophilization.  The  SEM  images  only  showed  the  microporous  structures  of  the lyophilized hydrogels, not the mesh size of the hydrated hydrogels.  Figure 2. Gross appearance (a) and SEM image of agarose hydrogels (b,c) prepared from 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose aqueous solutions. Scale bar: 1 mm (a), 500 µm (b) and 100 µm (c). Figure 1. Usage of agarose hydrogels to mimic the ECM stiffness of breast cancers to investigate theeffect of ECM stiffness on chemoresistance of breast cancer cells.2. Results and Discussion2.1. Preparation and Characterization of Agarose HydrogelsThe agarose hydrogels were prepared from 0.35%, 0.5%, 1%, 2%, 3% and 4% agaroseaqueous solutions. Their gross appearance is shown in Figure 2a. The hydrogels weretransparent with increasing turbidity as the concentration of agarose increased. The SEMobservation of the lyophilized agarose hydrogels showed that all the hydrogels had similarporous structures (Figure 2b,c). The porous structures were formed after lyophilization.The SEM images only showed the microporous structures of the lyophilized hydrogels, notthe mesh size of the hydrated hydrogels.Gels 2024, 10, x FOR PEER REVIEW  3  of  13   molecular weight [45,46,48]. Thus, the properties of agarose hydrogel can be easily tuned to  recapitulate  the mechanical  characteristics  of  the  ECM  in  breast  cancers. Agarose hydrogels are considered as a suitable platform to study the effect of stiffness on breast cancer cell function in 3D culture. Therefore,  in  this  study,  agarose  hydrogels  with  a  wide  range  of  stiffness  (1.5 kPa~112.3 kPa) were prepared  to cover  the whole stiffness range of breast cancers and used  to  encapsulate breast  cancer  cells  for  3D  culture. The  effect of  agarose hydrogel stiffness on  the  resistance of breast cancer cells  to DOX was  investigated. The possible mechanism was elucidated through analyzing the expression of P-gP mRNA (Figure 1).  Figure 1. Usage of agarose hydrogels to mimic the ECM stiffness of breast cancers to investigate the effect of ECM stiffness on chemoresistance of breast cancer cells. 2. Results and Discussion 2.1. Preparation and Characterization of Agarose Hydrogels The agarose hydrogels were prepared from 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose aqueous  solutions. Their gross appearance  is shown  in Figure 2a. The hydrogels were transparent with increasing turbidity as the concentration of agarose increased. The SEM observation  of  the  lyophilized  agarose  hydrogels  showed  that  all  the  hydrogels  had similar  porous  structures  (Figure  2b,c).  The  porous  structures  were  formed  after lyophilization.  The  SEM  images  only  showed  the  microporous  structures  of  the lyophilized hydrogels, not the mesh size of the hydrated hydrogels.  Figure 2. Gross appearance (a) and SEM image of agarose hydrogels (b,c) prepared from 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose aqueous solutions. Scale bar: 1 mm (a), 500 µm (b) and 100 µm (c). Figure 2. Gross appearance (a) and SEM image of agarose hydrogels (b,c) prepared from 0.35%, 0.5%,1%, 2%, 3% and 4% agarose aqueous solutions. Scale bar: 1 mm (a), 500 µm (b) and 100 µm (c).The stiffness of agarose hydrogels was measured using a compressive strength analysis.As presented in Figure 3, the stiffness increased with the increase in agarose concentration.The 0.35% agarose hydrogel had the lowest stiffness (1.5 ± 0.2 kPa) and 4% had the higheststiffness (112.3 ± 3.6 kPa). The increase in stiffness should be due to the dense agaroseGels 2024, 10, 202 4 of 13networks in the hydrogels of high concentrations. The stiffness of these agarose hydrogelscould cover the ECM stiffness range of breast cancers (4 kPa~100 kPa). The hydrogels wereused to encapsulate breast cancer cells for 3D culture to investigate the effect of hydrogelstiffness on their resistance to DOX.Gels 2024, 10, x FOR PEER REVIEW  4  of  13   The  stiffness  of  agarose  hydrogels was measured  using  a  compressive  strength analysis. As presented  in Figure 3,  the  stiffness  increased with  the  increase  in agarose concentration. The 0.35% agarose hydrogel had the lowest stiffness (1.5 ± 0.2 kPa) and 4% had the highest stiffness (112.3 ± 3.6 kPa). The increase in stiffness should be due to the dense agarose networks  in  the hydrogels of high concentrations. The stiffness of  these agarose hydrogels could cover the ECM stiffness range of breast cancers (4 kPa~100 kPa). The hydrogels were used to encapsulate breast cancer cells for 3D culture to investigate the effect of hydrogel stiffness on their resistance to DOX.  Figure 3. Young’s modulus of agarose hydrogels prepared from different concentrations of agarose. Data are the means ± S.D. (n = 5). 2.2. Effect of Agarose Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells to DOX MDA-MB-231  cells were  encapsulated  in  the  agarose  hydrogels with  or without DOX. The agarose hydrogels with DOX were prepared by mixing DOX  in  the agarose solutions  before  gelation.  The  cells  encapsulated  in  the  hydrogels  were  cultured  in medium  supplemented with  or without DOX  for  24  h. Afterward,  cell  viability was investigated using live/dead staining (Figure 4). Almost all  the breast cancer cells cultured  in  the agarose hydrogels without DOX were  alive  (green  fluorescence)  (Figure  4a).  The  results  indicated  that  the  agarose hydrogels without DOX were nontoxic to cells. When DOX was added, some dead cells (red fluorescence) were observed. The number of dead cells was dependent on both DOX concentration and agarose hydrogel stiffness  (Figure 4). When DOX concentration was low (5 mg L−1), a small number of dead cells were observed in the 0.35% and 0.5% agarose hydrogels, whereas few dead cells were observed in the agarose hydrogels prepared with concentrations of 1%, 2%, 3% and 4% (Figure 4b). When DOX concentration increased to 10 mg L−1, more dead cells were observed in the 0.35% and 0.5% agarose hydrogels and a small number of dead cells were observed in the 1% agarose hydrogel, while few dead cells were observed in the 2%, 3% and 4% agarose hydrogels (Figure 4c). With the further increase in DOX concentration to 20 mg L−1 and 50 mg L−1, most cells were dead in the 0.35% agarose hydrogels. A large number of dead cells were observed in the 0.5% and 1% agarose hydrogels. Some dead cells were also observed  in  the 2%, 3% and 4% agarose hydrogels (Figure 4d,e). The live/dead staining results indicated that the number of live cells decreased with DOX concentration, while they increased with the hydrogel stiffness, as summarized in Figure 4f. Figure 3. Young’s modulus of agarose hydrogels prepared from different concentrations of agarose.Data are the means ± S.D. (n = 5).2.2. Effect of Agarose Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells to DOXMDA-MB-231 cells were encapsulated in the agarose hydrogels with or withoutDOX. The agarose hydrogels with DOX were prepared by mixing DOX in the agarosesolutions before gelation. The cells encapsulated in the hydrogels were cultured in mediumsupplemented with or without DOX for 24 h. Afterward, cell viability was investigatedusing live/dead staining (Figure 4).Gels 2024, 10, x FOR PEER REVIEW  5  of  13    Figure 4. Live/dead staining of breast cancer cells after 24 h culture in 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose hydrogels without (a) or with different concentrations of DOX (b–e). A brief summary illustration of  live/dead staining results  (f). Scale bar: 200 µm. Green fluorescence:  live cells; red fluorescence: dead cells. Cell viability was furthered quantified with an AlamarBlue assay (Figure 5). At the same concentration of DOX, cell viability increased with the increase in hydrogel stiffness. When DOX  concentration was fixed at  5 mg L−1, 70% of  cells were  alive  in  the  0.35% agarose  hydrogel,  which  was  significantly  lower  than  that  observed  in  other  high concentrations of agarose hydrogels  (Figure 5a). The  cell viability  in  the 0.5% agarose hydrogel was around 80%, also significantly lower than that in 1%, 2%, 3% and 4% agarose hydrogels.  In  agarose  hydrogels with  concentrations  of  1%,  2%,  3%  and  4%,  the  cell viability was over  90%. As  the  concentration  of DOX  increased  to  10 mg L−1,  the  cell viability  significantly  decreased  in  all  samples  (Figure  5b)  and  exhibited  a  gradual decrease  corresponding  to  the  decreasing  concentration  of  agarose  hydrogel. A  low concentration of agarose hydrogel was associated with  low cell viability, while a high concentration  of  agarose  hydrogel was  associated with  high  cell  viability. When  the concentration of DOX increased to 20 mg L−1, the decrease in cell viability became more evident  (Figure  5c). More  than  50%  of  cells  were  dead  in  0.35%  and  0.5%  agarose hydrogels,  while  around  43%  of  cells  were  dead  in  1%  agarose  hydrogel.  As  the concentration of DOX  increased  to 50 mg L−1,  the cell viability  in all agarose hydrogels decreased continuously (Figure 5d). All these results suggested that cells cultured in the high-stiffness agarose hydrogels were less sensitive to DOX. The increase in stiffness led to a decrease in cell sensitivity to DOX, emphasizing the effect of hydrogel stiffness on the cellular response to the anti-cancer drug. Figure 4. Live/dead staining of breast cancer cells after 24 h culture in 0.35%, 0.5%, 1%, 2%, 3% and4% agarose hydrogels without (a) or with different concentrations of DOX (b–e). A brief summaryillustration of live/dead staining results (f). Scale bar: 200 µm. Green fluorescence: live cells; redfluorescence: dead cells.Gels 2024, 10, 202 5 of 13Almost all the breast cancer cells cultured in the agarose hydrogels without DOX werealive (green fluorescence) (Figure 4a). The results indicated that the agarose hydrogels with-out DOX were nontoxic to cells. When DOX was added, some dead cells (red fluorescence)were observed. The number of dead cells was dependent on both DOX concentration andagarose hydrogel stiffness (Figure 4). When DOX concentration was low (5 mg L−1), a smallnumber of dead cells were observed in the 0.35% and 0.5% agarose hydrogels, whereas fewdead cells were observed in the agarose hydrogels prepared with concentrations of 1%, 2%,3% and 4% (Figure 4b). When DOX concentration increased to 10 mg L−1, more dead cellswere observed in the 0.35% and 0.5% agarose hydrogels and a small number of dead cellswere observed in the 1% agarose hydrogel, while few dead cells were observed in the 2%,3% and 4% agarose hydrogels (Figure 4c). With the further increase in DOX concentrationto 20 mg L−1 and 50 mg L−1, most cells were dead in the 0.35% agarose hydrogels. A largenumber of dead cells were observed in the 0.5% and 1% agarose hydrogels. Some dead cellswere also observed in the 2%, 3% and 4% agarose hydrogels (Figure 4d,e). The live/deadstaining results indicated that the number of live cells decreased with DOX concentration,while they increased with the hydrogel stiffness, as summarized in Figure 4f.Cell viability was furthered quantified with an AlamarBlue assay (Figure 5). At thesame concentration of DOX, cell viability increased with the increase in hydrogel stiffness.When DOX concentration was fixed at 5 mg L−1, 70% of cells were alive in the 0.35% agarosehydrogel, which was significantly lower than that observed in other high concentrations ofagarose hydrogels (Figure 5a). The cell viability in the 0.5% agarose hydrogel was around80%, also significantly lower than that in 1%, 2%, 3% and 4% agarose hydrogels. In agarosehydrogels with concentrations of 1%, 2%, 3% and 4%, the cell viability was over 90%. Asthe concentration of DOX increased to 10 mg L−1, the cell viability significantly decreasedin all samples (Figure 5b) and exhibited a gradual decrease corresponding to the decreasingconcentration of agarose hydrogel. A low concentration of agarose hydrogel was associatedwith low cell viability, while a high concentration of agarose hydrogel was associated withhigh cell viability. When the concentration of DOX increased to 20 mg L−1, the decreasein cell viability became more evident (Figure 5c). More than 50% of cells were dead in0.35% and 0.5% agarose hydrogels, while around 43% of cells were dead in 1% agarosehydrogel. As the concentration of DOX increased to 50 mg L−1, the cell viability in allagarose hydrogels decreased continuously (Figure 5d). All these results suggested that cellscultured in the high-stiffness agarose hydrogels were less sensitive to DOX. The increasein stiffness led to a decrease in cell sensitivity to DOX, emphasizing the effect of hydrogelstiffness on the cellular response to the anti-cancer drug.2.3. Effect of Agarose Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells after DifferentCulture TimeThe chemoresistance of breast cancer cells cultured in the agarose hydrogels for differ-ent times (12~60 h) was further investigated by fixing the DOX concentration at 10 mg L−1(Figure 6). A concentration of 10 mg L−1 DOX was used because this concentration ofDOX showed a moderate cytotoxic effect to breast cancer cells cultured in the agarosehydrogels (Figures 4 and 5). In addition to the effect of the stiffness on chemoresistanceto different concentrations of DOX, the effect of the stiffness on cellular chemoresistancebecame more pronounced with prolonged incubation time. After 12 h of culturing at aDOX concentration of 10 mg L−1, no significant difference was observed and cell viabilityin all agarose hydrogels were higher than 90% (Figure 6a). This should be because that thedoubling time of MDA-MB-231 cells is around 24 to 25 h [49] and the cytotoxic effect ofDOX could not be observed within the 12 h culture. However, after 24 h of culturing, cellviability in all samples decreased and the cell viability had a more pronounced decrease inthe low-stiffness hydrogel (Figure 6b). After 36 h of culturing, the cell viability in the 0.35%agarose hydrogel was lower than 50%. The cell viability in the 0.5% agarose hydrogel wassignificantly higher than that in the 0.35% agarose hydrogel, but significantly lower thanthat in the other high concentration agarose hydrogels. The cell viability in the 1% agaroseGels 2024, 10, 202 6 of 13hydrogel was around 64%, significantly lower than that in the 2%, 3% and 4% agarosehydrogels. The cell viability in the 2% agarose hydrogel was around 70% and that in the3% and 4% agarose hydrogels was over 80% (Figure 6c). After 48 h of culturing, the cellviability was lower than 50% in both 0.35% and 0.5% agarose hydrogels, and significantlylower than that in the 1%, 2%, 3% and 4% agarose hydrogels. (Figure 6d). When the culturetime was prolonged to 60 h, the cell viability in most hydrogels decreased rapidly, except inthe 3% and 4% agarose hydrogels, where approximately 50% of the cells remained alive(Figure 6e).Gels 2024, 10, x FOR PEER REVIEW  6  of  13    Figure 5. Quantified viability of breast cancer cells after 24 h culture in 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose hydrogels with different DOX concentrations of 5 mg L−1 (a), 10 mg L−1 (b), 20 mg L−1 (c) and 50 mg L−1 (d). The data were normalized to the cell viability in the respective agarose hydrogels without DOX. Data are the means ± S.D. (n = 3). Significant differences: * p < 0.1; ** p < 0.01; *** p < 0.001. 2.3. Effect of Agarose Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells after Different Culture Time The  chemoresistance  of  breast  cancer  cells  cultured  in  the  agarose  hydrogels  for different times (12~60 h) was further investigated by fixing the DOX concentration at 10 mg L−1 (Figure 6). A concentration of 10 mg L−1 DOX was used because this concentration of DOX showed a moderate cytotoxic effect to breast cancer cells cultured in the agarose hydrogels (Figures 4 and 5). In addition to the effect of the stiffness on chemoresistance to different  concentrations of DOX,  the  effect of  the  stiffness on  cellular  chemoresistance became more pronounced with prolonged  incubation  time. After 12 h of culturing at a DOX concentration of 10 mg L−1, no significant difference was observed and cell viability in all agarose hydrogels were higher than 90% (Figure 6a). This should be because that the doubling time of MDA-MB-231 cells is around 24 to 25 h [49] and the cytotoxic effect of DOX could not be observed within the 12 h culture. However, after 24 h of culturing, cell viability in all samples decreased and the cell viability had a more pronounced decrease in the low-stiffness hydrogel (Figure 6b). After 36 h of culturing, the cell viability in the 0.35%  agarose  hydrogel was  lower  than  50%.  The  cell  viability  in  the  0.5%  agarose hydrogel  was  significantly  higher  than  that  in  the  0.35%  agarose  hydrogel,  but significantly lower than that in the other high concentration agarose hydrogels. The cell viability in the 1% agarose hydrogel was around 64%, significantly lower than that in the 2%,  3%  and  4%  agarose hydrogels. The  cell viability  in  the  2%  agarose hydrogel was around 70% and that in the 3% and 4% agarose hydrogels was over 80% (Figure 6c). After Figure 5. Quantified viability of breast cancer cells after 24 h culture in 0.35%, 0.5%, 1%, 2%, 3% and4% agarose hydrogels with different DOX concentrations of 5 mg L−1 (a), 10 mg L−1 (b), 20 mg L−1 (c)and 50 mg L−1 (d). The data were normalized to the cell viability in the respective agarose hydrogelswithout DOX. Data are the means ± S.D. (n = 3). Significant differences: * p < 0.1; ** p < 0.01;*** p < 0.001.The results indicated that the cell viability decreased both dose-dependently andincubation time-dependently. Furthermore, the sensitivity of breast cancer cells to DOXwas dependent on the stiffness of agarose hydrogels. There were more dead cells in thelow-stiffness agarose hydrogels than in the high-stiffness agarose hydrogel. High-stiffnesshydrogels could decrease the sensitivity to DOX, while low-stiffness hydrogels couldincrease the sensitivity to DOX for all dosages of DOX and all culture times. Therefore,it is reasonable to hypothesize that breast cancer cells in stiff ECM could have a higherpossibility for emerging chemoresistance ability than the cells in soft ECM.Gels 2024, 10, 202 7 of 13Gels 2024, 10, x FOR PEER REVIEW  7  of  13   48 h of culturing, the cell viability was lower than 50% in both 0.35% and 0.5% agarose hydrogels, and significantly lower than that in the 1%, 2%, 3% and 4% agarose hydrogels. (Figure  6d). When  the  culture  time was  prolonged  to  60  h,  the  cell  viability  in most hydrogels  decreased  rapidly,  except  in  the  3%  and  4%  agarose  hydrogels,  where approximately 50% of the cells remained alive (Figure 6e). The  results  indicated  that  the  cell viability decreased both dose-dependently and incubation time-dependently. Furthermore, the sensitivity of breast cancer cells to DOX was dependent on the stiffness of agarose hydrogels. There were more dead cells in the low-stiffness agarose hydrogels than in the high-stiffness agarose hydrogel. High-stiffness hydrogels  could decrease  the  sensitivity  to DOX, while  low-stiffness  hydrogels  could increase the sensitivity to DOX for all dosages of DOX and all culture times. Therefore, it is  reasonable  to hypothesize  that breast  cancer  cells  in  stiff ECM  could have a higher possibility for emerging chemoresistance ability than the cells in soft ECM.  Figure 6. Quantified viability of breast cancer cells after 12  (a), 24  (b), 36  (c), 48  (d) and 60 h  (e) culture in 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose hydrogels containing 10 mg L−1 DOX. The data were normalized to the cell viability in the respective agarose hydrogels without DOX. Data are the means ± S.D. (n = 3). Significant differences: * p < 0.1; ** p < 0.01; *** p < 0.001. n.s. = no significant difference. 2.4. Effect of Agarose Hydrogel Stiffness on Gene Expression of P‐Glycoprotein To investigate the possible mechanism of the effect of stiffness on chemoresistance, the expression of P-gP mRNA was analyzed using RT-qPCR. After the breast cancer cells were cultured in hydrogels without or with DOX for 36 h, the expression level of P-gP mRNA was  analyzed.  For  the  cells  cultured  in  agarose  hydrogels without DOX,  the expression  level  of  P-gP  mRNA  had  no  significant  difference  among  the  agarose hydrogels of different concentrations (Figure 7a). However, when cells were cultured in agarose hydrogels with DOX, the expression level of P-gP mRNA increased compared to the cell cultured in agarose hydrogels without DOX. The expression of P-gP mRNA in 3% and  4%  agarose  hydrogels was  significantly  higher  than  that  in  0.35%,  0.5%  and  1% agarose hydrogels (Figure 7b). These results demonstrated that stiffness had no effect on the expression of P-gP mRNA without DOX, while stiffness had a positive effect on the expression level of P-gP mRNA at the presence of DOX. The expression of P-gP mRNA in Figure 6. Quantified viability of breast cancer cells after 12 (a), 24 (b), 36 (c), 48 (d) and 60 h (e)culture in 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose hydrogels containing 10 mg L−1 DOX. Thedata were normalized to the cell viability in the respective agarose hydrogels without DOX. Dataare the means ± S.D. (n = 3). Significant differences: * p < 0.1; ** p < 0.01; *** p < 0.001. n.s. = nosignificant difference.2.4. Effect of Agarose Hydrogel Stiffness on Gene Expression of P-GlycoproteinTo investigate the possible mechanism of the effect of stiffness on chemoresistance, theexpression of P-gP mRNA was analyzed using RT-qPCR. After the breast cancer cells werecultured in hydrogels without or with DOX for 36 h, the expression level of P-gP mRNAwas analyzed. For the cells cultured in agarose hydrogels without DOX, the expressionlevel of P-gP mRNA had no significant difference among the agarose hydrogels of differentconcentrations (Figure 7a). However, when cells were cultured in agarose hydrogels withDOX, the expression level of P-gP mRNA increased compared to the cell cultured inagarose hydrogels without DOX. The expression of P-gP mRNA in 3% and 4% agarosehydrogels was significantly higher than that in 0.35%, 0.5% and 1% agarose hydrogels(Figure 7b). These results demonstrated that stiffness had no effect on the expression ofP-gP mRNA without DOX, while stiffness had a positive effect on the expression levelof P-gP mRNA at the presence of DOX. The expression of P-gP mRNA in high-stiffnesshydrogel increased compared to that in low-stiffness hydrogel. It was worth noting that theexpression level of P-gP mRNA increased gradually but showed no significant differenceamong the 0.35%, 0.5%, 1% and 2% agarose hydrogels. This might be because the effectof stiffness on the expression level of P-gP mRNA had a cumulative effect following theincrease in stiffness. The low-stiffness hydrogel lacked a significant effect on the expressionof P-gP mRNA. When the stiffness of hydrogels increased, the expression level of P-gPmRNA could increase gradually and become significant.Gels 2024, 10, 202 8 of 13Gels 2024, 10, x FOR PEER REVIEW  8  of  13   high-stiffness hydrogel increased compared to that in low-stiffness hydrogel. It was worth noting  that  the  expression  level  of  P-gP mRNA  increased  gradually  but  showed  no significant difference among the 0.35%, 0.5%, 1% and 2% agarose hydrogels. This might be because the effect of stiffness on the expression level of P-gP mRNA had a cumulative effect following the increase in stiffness. The low-stiffness hydrogel lacked a significant effect on  the expression of P-gP mRNA. When  the stiffness of hydrogels  increased, the expression level of P-gP mRNA could increase gradually and become significant.  Figure 7. Quantified expression level of P-gP mRNA in breast cancer cells after 36 h culture in 0.35%, 0.5%, 1%, 2%, 3% and 4% agarose hydrogels without DOX (a) or with 10 mg L−1 DOX (b). The data relative to GAPDH were normalized to the expression level in 0.35% agarose hydrogel. Data are the means ± S.D. (n = 3). Significant differences: * p < 0.1; ** p < 0.01. n.s. = no significant difference. ECM components play an important role in orchestrating cell functions through cell-matrix cross-talks [50]. In this study, agarose hydrogels of different concentrations were successfully prepared to provide a large range of stiffness (1.5 kPa~112.3 kPa) for 3D cell culture, allowing recapitulation of the wide range of pathological ECM stiffness in breast cancers. Moreover, since the stiffened ECM can act as a physical barrier that limits drug penetration  [51], DOX was mixed with  the agarose solution before gelation. Therefore, DOX was impregnated in the hydrogels to avoid the diffusion problem. The breast cancer cells were encapsulated  in  the agarose hydrogels and  cultured  in  the DOX-containing medium in 3D to investigate the effect of stiffness on chemoresistance to anti-cancer drug. Increase in hydrogel stiffness changed the sensitivity of breast cancer cells to DOX. A stiffened  hydrogel  was  found  to  decrease  the  sensitivity  of  cells  to  drug,  which  is consistent with previous reports [23,52,53]. Furthermore, the expression of P-gP mRNA increased when cells were incubated in the agarose hydrogels with DOX, which should be  because  cancer  cells  exposing  to  DOX  can  stimulate  P-gP  expression  [34].  The interesting result is that the increased level of P-gP mRNA in different stiffness hydrogels was different. In the high-stiffness agarose hydrogel, the P-gP expression level increased more compared to the low-stiffness agarose hydrogel. This upregulation of P-gP mRNA might lead to a high expression of P-gP protein, thereby failing to make DOX accumulate and causing the chemoresistance of breast cancer cells in stiffer hydrogel [32]. 3. Conclusions In this study, agarose hydrogels of a wide range of stiffness (1.5 kPa~112.3 kPa) were prepared from different concentrations of agarose aqueous solutions. The hydrogels were used for the 3D culture of breast cancer cells to investigate the effect of hydrogel stiffness on the chemoresistance of breast cancer cells. Cell viability was dependent on hydrogel stiffness,  DOX  concentration  and  culture  time.  Most  importantly,  the  high-stiffness Figure 7. Quantified expression level of P-gP mRNA in breast cancer cells after 36 h culture in 0.35%,0.5%, 1%, 2%, 3% and 4% agarose hydrogels without DOX (a) or with 10 mg L−1 DOX (b). The datarelative to GAPDH were normalized to the expression level in 0.35% agarose hydrogel. Data are themeans ± S.D. (n = 3). Significant differences: * p < 0.1; ** p < 0.01. n.s. = no significant difference.ECM components play an important role in orchestrating cell functions through cell-matrix cross-talks [50]. In this study, agarose hydrogels of different concentrations weresuccessfully prepared to provide a large range of stiffness (1.5 kPa~112.3 kPa) for 3D cellculture, allowing recapitulation of the wide range of pathological ECM stiffness in breastcancers. Moreover, since the stiffened ECM can act as a physical barrier that limits drugpenetration [51], DOX was mixed with the agarose solution before gelation. Therefore, DOXwas impregnated in the hydrogels to avoid the diffusion problem. The breast cancer cellswere encapsulated in the agarose hydrogels and cultured in the DOX-containing mediumin 3D to investigate the effect of stiffness on chemoresistance to anti-cancer drug.Increase in hydrogel stiffness changed the sensitivity of breast cancer cells to DOX. Astiffened hydrogel was found to decrease the sensitivity of cells to drug, which is consistentwith previous reports [23,52,53]. Furthermore, the expression of P-gP mRNA increasedwhen cells were incubated in the agarose hydrogels with DOX, which should be becausecancer cells exposing to DOX can stimulate P-gP expression [34]. The interesting result isthat the increased level of P-gP mRNA in different stiffness hydrogels was different. Inthe high-stiffness agarose hydrogel, the P-gP expression level increased more comparedto the low-stiffness agarose hydrogel. This upregulation of P-gP mRNA might lead to ahigh expression of P-gP protein, thereby failing to make DOX accumulate and causing thechemoresistance of breast cancer cells in stiffer hydrogel [32].3. ConclusionsIn this study, agarose hydrogels of a wide range of stiffness (1.5 kPa~112.3 kPa)were prepared from different concentrations of agarose aqueous solutions. The hydrogelswere used for the 3D culture of breast cancer cells to investigate the effect of hydrogelstiffness on the chemoresistance of breast cancer cells. Cell viability was dependent onhydrogel stiffness, DOX concentration and culture time. Most importantly, the high-stiffness hydrogel containing DOX increased cell viability and promoted expression of P-gPmRNA. The results suggested that ECM stiffness might contribute to the development ofchemoresistance of breast cancer cells. Stiffened ECM could induce chemoresistance inbreast cancer cells through upregulating the expression of P-gP mRNA.4. Materials and Methods4.1. Preparation and Characterization of Agarose HydrogelsDifferent concentrations of agarose solutions ((0.35%, wt/v), (0.50%, wt/v), (1.00%,wt/v), (2.00%, wt/v), (3.00%, wt/v) and (4.00%, wt/v)) were prepared through dissolvingGels 2024, 10, 202 9 of 13autoclave-sterilized agarose powder (A2576, Sigma, St. Louis, MO, USA) in sterilizedPBS (1X) under an oil bath at 110 ◦C for 15 min. Subsequently, the agarose solutionswere transferred to a 37 ◦C water bath to equilibrate the temperature overnight. Aftertemperature equilibration, the agarose solutions were pipetted into a silicon frame having acentral hole of φ6 mm × H5 mm. Then, the samples were transferred to a 4 ◦C refrigerator,enabling gelation for 30 min. After gelation, the silicon frame was removed and the grossappearances of the agarose hydrogels with different concentrations were observed usingan optical microscope (Olympus, Tokyo, Japan). Afterward, the agarose hydrogels weretransferred to a −80 ◦C refrigerator for freezing and lyophilized in a freeze-drying machine.The pore structures of the lyophilized agarose hydrogels were examined using scanningelectron microscopy (SEM; Hitachi S-4800, Tokyo, Japan).4.2. Stiffness Measurement of Agarose HydrogelsThe stiffness of agarose hydrogels was measured using a static compression test aspreviously described [54]. Briefly, round discs of agarose hydrogels (φ10 mm × H5 mm)were prepared for the measurement. The preparation of agarose solution was the sameas described above. After gelation, a 10 mm diameter biopsy punch was used to cut thehydrogel into discs with 5 mm height. Subsequently, the hydrogel discs were placed atroom temperature overnight for temperature equilibration before the mechanical testing.The stress–strain curves of the agarose hydrogel discs were obtained by using a TA.XT. plusTexture Analyzer (Hamilton, MA, USA, test speed = 0.1 mm/s). The Young’s modulus wasdetermined by fitting a line to the linear region of the stress–strain curve. Five sampleswere used for each measurement.4.3. Cell CultureThe Triple Negative Breast Cancer Cell line MDA-MB-231-Luc (JCRB, Osaka, Japan)were cultured in DMEM serum medium containing 10% fetal bovine serum (FBS, Gibco,Norristown, PA, USA), L-glutamine (Sigma) and antibiotics (100 U mL−1 penicillin and100 µg mL−1 streptomycin) in a humidified incubator (5% CO2, 37 ◦C).4.4. In Vitro Anticancer Effect of Doxorubicin in Agarose HydrogelsThe effect of hydrogel stiffness on the anticancer effect of DOX was investigatedthrough culturing the breast cancer cells in agarose hydrogels of different concentrations.All samples were prepared on a clean bench to avoid contamination. DOX at a concentrationof 1 g L−1 was prepared through dissolving DOX powder into sterilized water and dilutingto the required concentration when used. Different concentrations of agarose solutions((0.41%, wt/v), (0.58%, wt/v), (1.17%, wt/v), (2.33%, wt/v), (3.50%, wt/v), (4.67%, wt/v))were prepared as described above. After temperature equilibration, 90 µL of agarosesolution was mixed with a pre-warmed 10 µL DOX solution to prepare agarose hydrogelsat a final DOX concentration of 5, 10, 20 and 50 mg L−1. As for the control group withoutDOX, PBS (1×) with the same volume was used as a replacement for DOX.Silicon discs with a dimension of φ35 mm × H1 mm and silicon frames having acentral hole of φ6 mm × H2 mm were sterilized via autoclaving at 120 ◦C for 20 min. Nylonmesh (250 µ, Iwai Chemicals Company, Tokyo, Japan) was used to support the agarosehydrogels. The Nylon mesh was cut into round discs (φ6 mm), sterilized with 70% ethanoland washed with PBS (1×) three times. After removing the PBS from the Nylon mesh disc,it was placed on the silicon disc. And then, the silicon frame (φ6 mm × H2 mm) was placedon the Nylon mesh disc. The subcultured MDA-MB-231-Luc cells were detached from theculture plate using trypsin-EDTA solution and re-suspended in DMEM serum medium at aconcentration of 1 × 107 cells mL−1. Afterward, a 5 µL cell suspension solution was addedinto the agarose/DOX solution and mixed well. The cell/agarose/DOX mixture solutionwas pipetted into the silicon frame. The construct was then placed in a 4 ◦C refrigeratorrapidly, enabling gelation for 30 min. After that, the silicon frame was removed, and thecells/agarose/DOX construct together with the Nylon mesh disc were transferred to aGels 2024, 10, 202 10 of 1324-well cell culture plate and cultured for 24 h. For the control group, the procedures werethe same as the above mentioned, aside from changing DOX to an equal volume of PBS.4.5. Cell Viability AssaysThe cell viability in agarose hydrogel was analyzed by using live/dead stainingassay and AlamarBlue cell viability quantification. For live/dead staining, the cells werelabeled with a live/dead kit (Dojindo, Kumamoto, Japan). Following the staining of thesamples with calcein-AM and propidium iodide in serum-free DMEM medium for 10 minat 37 ◦C, the hydrogels were washed 3 times with PBS and immediately analyzed by usingfluorescence microscopy (Olympus, Japan).AlamarBlue (Thermo Fisher Scientific, Inc., Tokyo, Japan) was used to assess themetabolic activity of cells in 3D culture. The reduction in AlamarBlue resazurin to pinkcolored resorufin was determined through fluorescence measurement (λex: 530 nm, λem:590 nm). All measurements were conducted in a microplate reader (Spark MultimodeMicroplate Reader, Tecan) using a 96-well black-clear flat bottom plate (Nunclon Delta-Treated, Flat-Bottom Microplate, Thermo Fisher Scientific, Inc., Japan). For the details, afterthe cell culturing at different times, the cells/agarose and cells/agarose/DOX constructswere transferred to a new 24-well plate and 1 mL AlamarBlue working solution (100 µL ofAlamarBlue diluted with 900 µL fresh DMEM medium) was added to each well, followedby incubation at 37 ◦C for 4 h. Subsequently, 100 µL of the working solution was transferredto the 96-well black-clear flat bottom plate to measure the fluorescence intensity. The valueof the fluorescence intensity was used to calculate the cell viability. The cell viability of thecells/agarose/DOX hydrogels was normalized to the cell viability in the respective agarosehydrogel without DOX. Three samples were used for each measurement to calculate themeans and standard deviations.4.6. Real-Time PCR Analysis (RT-qPCR Analysis)The P-glycoprotein mRNA expression levels in the cells/agarose and cells/agarose/DOXconstructs were analyzed after cell culturing for 36 h using real-time PCR according to a pre-vious method [55,56]. The samples were immersed in 1 mL of a Sepasol solution (NacalaiTesque, Kyoto, Japan) for extraction of total RNA. A high-capacity cDNA reverse transcrip-tion kit (Applied Biosystems, Foster City, CA, USA) was used to reverse transcription ofthe complementary DNA (cDNA) from 1 µg of purified total RNA. The cDNA served as atemplate for RT-qPCR analysis and the amplification of glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) and P-glycoprotein (ABCB1) was conducted using a QuantStudios3 Real-Time PCR system (Thermo Fisher Scientific). The pre-designed primer and probesequences of ABCB1 (Hs00184500_m1) were used and GAPDH (Hs99999905_m1) was usedas the housekeeping gene (Thermo Fisher, Japan). The relative expression of each gene wascalculated using a 2−∆∆Ct method with an endogenous control (GAPDH). The expressionlevel of ABCB1 in each group was normalized using the respective gene expression of thecells cultured in a 0.35% concentration of agarose hydrogel as a reference. Three sampleswere used for each measurement to calculate the means and standard deviations.4.7. Statistical AnalysisAll quantitative experiments were repeated in triplicate or quintuplicate to calculatethe means and standard deviations (S.D.). Statistical analysis of the experimental data wasperformed using GraphPad Prism software. One-way ANOVA with Dunnett’s multiplecomparison test were used to compare the samples. The p value was used to determine thelevel of significance: * p < 0.05, ** p < 0.01, and *** p < 0.001.Gels 2024, 10, 202 11 of 13Author Contributions: Conceptualization, G.C. and N.K.; methodology, G.C., N.K., T.Y., Y.Y. and T.Z.;software, T.Z. and N.K.; validation, G.C., N.K., T.Z. and H.C.; formal analysis, T.Z. and H.C.; investi-gation, T.Z. and H.C.; resources, G.C., N.K. and T.Y.; data curation, T.Z. and H.C.; writing—originaldraft preparation, T.Z. and G.C.; writing—review and editing, T.Z., H.C., T.Y., N.K., Y.Y. and G.C.;visualization, T.Z. and H.C.; supervision, G.C.; project administration, G.C. and N.K. All authorshave read and agreed to the published version of the manuscript.Funding: This research was supported by JSPS KAKENHI Grant Number 19H04475, 21H03830and 22K19926.Institutional Review Board Statement: Not applicable.Informed Consent Statement: Not applicable.Data Availability Statement: All data and materials are available on request from the correspondingauthor. 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Biomaterials 2024, 307, 122511.[CrossRef] [PubMed]Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.https://doi.org/10.1021/acsmacrolett.9b00258https://doi.org/10.1021/bm005583jhttps://doi.org/10.1007/s00396-020-04698-1https://doi.org/10.1007/s10439-014-1183-5https://doi.org/10.1007/s10544-019-0450-5https://doi.org/10.3389/fonc.2020.00397https://www.ncbi.nlm.nih.gov/pubmed/32351878https://doi.org/10.1016/j.jconrel.2017.02.035https://doi.org/10.3390/polym8040112https://doi.org/10.1039/D3BM00598Dhttps://doi.org/10.1021/acsabm.3c00159https://www.ncbi.nlm.nih.gov/pubmed/37061939https://doi.org/10.1002/adhm.202202604https://doi.org/10.1016/j.biomaterials.2024.122511https://www.ncbi.nlm.nih.gov/pubmed/38401482 Introduction  Results and Discussion  Preparation and Characterization of Agarose Hydrogels  Effect of Agarose Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells to DOX  Effect of Agarose Hydrogel Stiffness on Chemoresistance of Breast Cancer Cells after Different Culture Time  Effect of Agarose Hydrogel Stiffness on Gene Expression of P-Glycoprotein  Conclusions  Materials and Methods  Preparation and Characterization of Agarose Hydrogels  Stiffness Measurement of Agarose Hydrogels  Cell Culture  In Vitro Anticancer Effect of Doxorubicin in Agarose Hydrogels  Cell Viability Assays  Real-Time PCR Analysis (RT-qPCR Analysis)  Statistical Analysis  References