An in vitro hyaluronic acid hydrogel based platform to model dormancy in brain metastatic breast cancer cells.

Breast cancer cells (BCCs) can remain dormant at the metastatic site, which when revoked leads to formation of metastasis several years after the treatment of primary tumor. Particularly, awakening of dormant BCCs in the brain results in breast cancer brain metastasis (BCBrM) which marks the most advanced stage of the disease with a median survival period of ~4-16 months. However, our understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. To address this need, we developed an in vitro hyaluronic acid (HA) hydrogel platform to model dormancy in brain metastatic BCCs via exploiting the bio-physical cues provided by HA hydrogels while bracketing the normal brain and metastatic brain malignancy relevant stiffness range. In this system, we observed that MDA-MB-231Br and BT474Br3 brain metastatic BCCs exhibited a dormant phenotype when cultured on soft (0.4 kPa) HA hydrogel compared to stiff (4.5 kPa) HA hydrogel as characterized by significantly lower EdU and Ki67 positivity. Further, we demonstrated the nuclear localization of p21 and p27 (markers associated with dormancy) in dormant MDA-MB-231Br cells contrary to their cytoplasmic localization in the proliferative population. We also demonstrated that the stiffness-based dormancy in MDA-MB-231Br cells was reversible and was, in part, mediated by focal adhesion kinases and the initial cell seeding density. Finally, RNA sequencing confirmed the dormant phenotype in MDA-MB-231Br cells. This platform could further our understanding of dormancy in BCBrM and could be adapted for anti-metastatic drug screening. STATEMENT OF SIGNIFICANCE: Our understanding of dormancy associated with BCBrM remains obscure, in part, due to the lack of relevant in vitro platforms to model dormancy associated with BCBrM. Herein, we present a HA hydrogel-based platform to model dormancy in brain metastatic BCCs while recapitulating key aspects of brain microenvironment. We demonstrated that the biophysical cues provided the HA hydrogel mediates dormancy in brain metastatic BCCs by assessing both proliferation and cell cycle arrest markers. We also established the role of focal adhesion kinases and initial cell seeding density in the stiffness-mediated dormancy in brain metastatic BCCs. Further, RNA-seq. confirmed the dormant phenotype in brain metastatic BCCs. This platform could be utilized to further our understanding of microenvironmental regulation of dormancy in BCBrM.

Role of Surface Chemistry in Mediating the Uptake of Ultrasmall Iron Oxide Nanoparticles by Cancer Cells.

Ultrasmall iron oxide nanoparticles (USIONPs) (<4 nm) have recently attracted significant attention because of their potential as positive T1 magnetic resonance imaging (MRI) contrast agent contrary to larger superparamagnetic iron oxide nanoparticles (>6 nm) which act as negative T2 MRI contrast agents. However, studies on the cellular uptake behavior of these nanoparticles are very limited compared to their counterpart, larger-sized superparamagnetic iron oxide nanoparticles. In particular, the effects of specific nanoparticle parameters on the cellular uptake behavior of USIONPs by various cancer cells are not available. Here, we specifically investigated the role of USIONPs' surface functionalities [tannic acid (TA) and quinic acid (QA)] in mediating cellular uptake behavior of cancer cells pertaining to primary (U87 cells) and metastatic (MDA-MB-231Br cells) brain malignancies. Here, we chose TA and QA as representative capping molecules, wherein TA coating provides a general negatively charged nontargeting surface while QA provides a tumor-targeting surface as QA and its derivatives are known to interact with selectin receptors expressed on tumor cells and tumor endothelium. We observed differential cellular uptake in the case of TA- and QA-coated USIONPs by cancer cells. Both the cell types showed significantly higher cellular uptake of QA-coated USIONPs compared to TA-coated USIONPs at 4, 24, and 72 h. Blocking studies indicated that P-selectin cell surface receptors, in part, mediated the cellular uptake of QA-coated USIONPs. Given that P-selectin is overexpressed in cancer cells, tumor microenvironment, and at the metastatic niche, QA-coated USIONPs hold potential to be utilized as a platform for tumor-targeted drug delivery and in imaging and detection of primary and metastatic tumors.

Bioengineered models to study tumor dormancy.

The onset of cancer metastasis is the defining event in cancer progression when the disease is considered lethal. The ability of metastatic cancer cells to stay dormant for extended time periods and reawaken at later stages leading to disease recurrence makes treatment of metastatic disease extremely challenging. The tumor microenvironment plays a critical role in deciding the ultimate fate of tumor cells, yet the mechanisms by which this occurs, including dormancy, is not well understood. This mini-review discusses bioengineered models inspired from tissue engineering strategies that mimic key aspects of the tumor microenvironment to study tumor dormancy. These models include biomaterial based three dimensional models, microfluidic based models, as well as bioreactor based models that incorporate relevant microenvironmental components such as extracellular matrix molecules, niche cells, or their combination to study microenvironmental regulation of tumor dormancy. Such biomimetic models provide suitable platforms to investigate the dormant niche, including cues that drive the dormant to proliferative transition in cancer cells. In addition, the potential of such model systems to advance research in the field of tumor dormancy is discussed.

A combined compression molding, heating, and leaching process for fabrication of micro-porous poly(ε-caprolactone) scaffolds.

Biomaterial scaffolds have been increasingly used for tissue engineering applications as well as three dimensional (3D) cell culture models. Herein, we report a simple procedure combining compression molding, heating, and leaching methods for the fabrication of 3D micro-porous poly(ε-caprolactone) (PCL) biomaterial scaffolds. In this procedure, PCL micro particles are mixed with NaCl of defined sizes and compression molded, followed by heating and subsequent leaching of NaCl particles. This technique eliminates the gas foaming method, which is commonly used in the fabrication of PCL scaffolds. Process and scaffold parameters (i.e., heating time, NaCl concentration, and NaCl particle size) were varied and analyzed to determine their impact on the overall scaffold structural and mechanical properties. An increase in NaCl particle size led to an increase in pore area but did not significantly impact the mechanical properties of the scaffolds. Additionally, NaCl concentration did not show a significant effect on pore area, but considerably impacted the mechanical properties, water absorption capacity and porosity of the scaffolds. Variations in the heating time did not have an effect in the pore area, porosity, water absorption capacity or mechanical properties of the scaffolds. We also demonstrated the ability of these scaffolds to support the proliferation of breast cancer cells. Overall, these results elucidated structure-property relationships in the fabricated micro-porous PCL scaffolds. Further, this procedure could be potentially scaled up for the fabrication of micro-porous PCL scaffolds.

The influence of matrix stiffness on the behavior of brain metastatic breast cancer cells in a biomimetic hyaluronic acid hydrogel platform.

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.

Biomimetic strategies to recapitulate organ specific microenvironments for studying breast cancer metastasis.

The progression of breast cancer from the primary tumor setting to the metastatic setting is the critical event defining Stage IV disease, no longer considered curable. The microenvironment at specific organ sites is known to play a key role in influencing the ultimate fate of metastatic cells; yet microenvironmental mediated-molecular mechanisms underlying organ specific metastasis in breast cancer are not well understood. This review discusses biomimetic strategies employed to recapitulate metastatic organ microenvironments, particularly, bone, liver, lung and brain to elucidate the mechanisms dictating metastatic breast cancer cell homing and colonization. These biomimetic strategies include in vitro techniques such as biomaterial-based co-culturing techniques, microfluidics, organ-mimetic chips, bioreactor technologies, and decellularized matrices as well as cutting edge in vivo techniques to better understand the interactions between metastatic breast cancer cells and the stroma at the metastatic site. The advantages and disadvantages of these systems are discussed. In addition, how creation of biomimetic models will impact breast cancer metastasis research and their broad utility is explored.