Approximately 90% of breast cancer related mortalities are due to metastasis to distant organs. At the metastatic sites, cancer cells are capable of evading death by exhibiting cellular or mass dormancy. However, the mechanisms involved in attaining dormancy at the metastatic site are not well understood. This is partly due to the lack of experimental models to study metastatic site‐specific interactions, particularly in the context of brain metastatic breast cancer (BMBC). Herein, an in vitro hyaluronic acid (HA) hydrogel‐based model is developed to study mass dormancy in BMBC. HA hydrogels with a stiffness of ≈0.4 kPa are utilized to mimic the brain extracellular matrix. MDA‐MB‐231Br or BT474Br3 BMBC spheroids are prepared and cultured on top of HA hydrogels or in suspension for 7 days. HA hydrogel induced a near mass dormant state in spheroids by achieving a balance between proliferating and dead cells. In contrast, these spheroids displayed growth in suspension cultures. The ratio of %p‐ERK to %p‐p38 positive cells is significantly lower in HA hydrogels compared to suspension cultures. Further, it is demonstrated that hydrogel induced mass dormant state is reversible. Overall, such models provide useful tools to study dormancy in BMBC and could be employed for drug screening.
more » « less- Award ID(s):
- 1749837
- PAR ID:
- 10379729
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Biology
- Volume:
- 7
- Issue:
- 1
- ISSN:
- 2701-0198
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
null (Ed.)Breast cancer cells can metastasize either as single cells or as clusters to distant organs from the primary tumor site. Cell clusters have been shown to possess higher metastatic potential compared to single cells. The organ microenvironment is critical in regulating the ultimate phenotype, specifically, the dormant versus proliferative phenotypes, of these clusters. In the context of breast cancer brain metastasis (BCBM), tumor cell cluster–organ microenvironment interactions are not well understood, in part, due to the lack of suitable biomimetic in vitro models. To address this need, herein, we report a biomaterial-based model, utilizing hyaluronic acid (HA) hydrogels with varying stiffnesses to mimic the brain microenvironment. Cell spheroids were used to mimic cell clusters. Using 100–10 000 MDA-MB-231Br BCBM cells, six different sizes of cell spheroids were prepared to study the impact of cluster size on dormancy. On soft HA hydrogels (∼0.4 kPa), irrespective of spheroid size, all cell spheroids attained a dormant phenotype, whereas on stiff HA hydrogels (∼4.5 kPa), size dependent switch between the dormant and proliferative phenotypes was noted ( i.e. , proliferative phenotype ≥5000 cell clusters < dormant phenotype), as tested via EdU and Ki67 staining. Furthermore, we demonstrated that the matrix stiffness driven dormancy was reversible. Such biomaterial systems provide useful tools to probe cell cluster–matrix interactions in BCBM.more » « less
-
Abstract A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA‐MB‐231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA‐MB‐231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle‐like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal‐regulated kinase 1/2 (p‐ERK) to phosphorylated p38 (p‐p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel‐induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.
-
Abstract The role of hydrogel properties in regulating the phenotype of triple negative metastatic breast cancer is investigated using four cell lines: the MDA‐MB‐231 parental line and three organotropic sublines BoM‐1833 (bone‐tropic), LM2‐4175 (lung‐tropic), and BrM2a‐831 (brain‐tropic). Each line is encapsulated and cultured for 15 days in three poly(ethylene glycol) (PEG)‐based hydrogel formulations composed of proteolytically degradable PEG, integrin‐ligating RGDS, and the non‐degradable crosslinker N‐vinyl pyrrolidone. Dormancy‐associated metrics including viable cell density, proliferation, metabolism, apoptosis, chemoresistance, phosphorylated‐ERK and ‐p38, and morphological characteristics are quantified. A multimetric classification approach is implemented to categorize each hydrogel‐induced phenotype as: 1) growth, 2) balanced tumor dormancy, 3) balanced cellular dormancy, or 4) restricted survival, cellular dormancy. Hydrogels with high adhesivity and degradability promote growth. Hydrogels with no adhesivity, but high degradability, induce restricted survival, cellular dormancy in the parental line and balanced cellular dormancy in the organotropic lines. Hydrogels with reduced adhesivity and degradability induce balanced cellular dormancy in the parental and lung‐tropic lines and balanced tumor mass dormancy in bone‐ and brain‐tropic lines. The ability to induce escape from dormancy via dynamic incorporation of RGDS is also presented. These results demonstrate that ECM properties and organ‐tropism synergistically regulate cancer cell phenotype and dormancy.
-
Abstract Dormant, disseminated tumor cells (DTCs) can persist for decades in secondary tissues before being reactivated to form tumors. The properties of the premetastatic niche can influence the DTC phenotype. To better understand how matrix properties of premetastatic niches influence DTC behavior, three hydrogel formulations are implemented to model a permissive niche and two nonpermissive niches. Poly(ethylene glycol) (PEG)‐based hydrogels with varying adhesivity ([RGDS]) and degradability ([N‐vinyl pyrrolidinone]) are implemented to mimic a permissive niche with high adhesivity and degradability and two nonpermissive niches, one with moderate adhesivity and degradability and one with no adhesivity and high degradability. The influence of matrix properties on estrogen receptor positive (ER+) breast cancer cells (MCF7s) is determined via a multimetric analysis. MCF7s cultured in the permissive niche adopted a growth state, while those in the nonpermissive niche with reduced adhesivity and degradability underwent tumor mass dormancy. Complete removal of adhesivity while maintaining high degradability induced single cell dormancy. The ability to mimic reactivation of dormant cells through a dynamic increase in [RGDS] is also demonstrated. This platform provides the capability of inducing growth, dormancy, and reactivation of ER+ breast cancer and can be useful in understanding how premetastatic niche properties influence cancer cell fate.
-
Abstract Breast cancer brain metastasis marks the most advanced stage of breast cancer no longer considered curable with a median survival period of ∼4–16 months. Apart from the genetic susceptibility (subtype) of breast tumors, brain metastasis is also dictated by the biophysical/chemical interactions of tumor cells with native brain microenvironment, which remain obscure, primarily due to the lack of tunable biomimetic
in vitro models. To address this need, we utilized a biomimetic hyaluronic acid (HA) hydrogel platform to elucidate the impact of matrix stiffness on the behavior of MDA‐MB‐231Br cells, a brain metastasizing variant of the triple negative breast cancer line MDA‐MB‐231. We prepared HA hydrogels of varying stiffness (0.2–4.5 kPa) bracketing the brain relevant stiffness range to recapitulate the biophysical cues provided by brain extracellular matrix. In this system, we observed that the MDA‐MB‐231Br cell adhesion, spreading, proliferation, and migration significantly increased with the hydrogel stiffness. We also demonstrated that the stiffness based responses of these cells were mediated, in part, through the focal adhesion kinase‐phosphoinositide‐3 kinase pathway. This biomimetic material system with tunable stiffness provides an ideal platform to further the understanding of mechanoregulation associated with brain metastatic breast cancer cells. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1832–1841, 2018.