An in vitro hyaluronic acid hydrogel based platform to model dormancy in brain metastatic breast cancer cells. 2020

Akshay A Narkhede, and James H Crenshaw, and David K Crossman, and Lalita A Shevde, and Shreyas S Rao
Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, AL 35487-0203, USA.

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.

UI MeSH Term Description Entries
D010641 Phenotype The outward appearance of the individual. It is the product of interactions between genes, and between the GENOTYPE and the environment. Phenotypes
D001932 Brain Neoplasms Neoplasms of the intracranial components of the central nervous system, including the cerebral hemispheres, basal ganglia, hypothalamus, thalamus, brain stem, and cerebellum. Brain neoplasms are subdivided into primary (originating from brain tissue) and secondary (i.e., metastatic) forms. Primary neoplasms are subdivided into benign and malignant forms. In general, brain tumors may also be classified by age of onset, histologic type, or presenting location in the brain. Brain Cancer,Brain Metastases,Brain Tumors,Cancer of Brain,Malignant Primary Brain Tumors,Neoplasms, Intracranial,Benign Neoplasms, Brain,Brain Neoplasm, Primary,Brain Neoplasms, Benign,Brain Neoplasms, Malignant,Brain Neoplasms, Malignant, Primary,Brain Neoplasms, Primary Malignant,Brain Tumor, Primary,Brain Tumor, Recurrent,Cancer of the Brain,Intracranial Neoplasms,Malignant Neoplasms, Brain,Malignant Primary Brain Neoplasms,Neoplasms, Brain,Neoplasms, Brain, Benign,Neoplasms, Brain, Malignant,Neoplasms, Brain, Primary,Primary Brain Neoplasms,Primary Malignant Brain Neoplasms,Primary Malignant Brain Tumors,Benign Brain Neoplasm,Benign Brain Neoplasms,Benign Neoplasm, Brain,Brain Benign Neoplasm,Brain Benign Neoplasms,Brain Cancers,Brain Malignant Neoplasm,Brain Malignant Neoplasms,Brain Metastase,Brain Neoplasm,Brain Neoplasm, Benign,Brain Neoplasm, Malignant,Brain Neoplasms, Primary,Brain Tumor,Brain Tumors, Recurrent,Cancer, Brain,Intracranial Neoplasm,Malignant Brain Neoplasm,Malignant Brain Neoplasms,Malignant Neoplasm, Brain,Neoplasm, Brain,Neoplasm, Intracranial,Primary Brain Neoplasm,Primary Brain Tumor,Primary Brain Tumors,Recurrent Brain Tumor,Recurrent Brain Tumors,Tumor, Brain
D001943 Breast Neoplasms Tumors or cancer of the human BREAST. Breast Cancer,Breast Tumors,Cancer of Breast,Breast Carcinoma,Cancer of the Breast,Human Mammary Carcinoma,Malignant Neoplasm of Breast,Malignant Tumor of Breast,Mammary Cancer,Mammary Carcinoma, Human,Mammary Neoplasm, Human,Mammary Neoplasms, Human,Neoplasms, Breast,Tumors, Breast,Breast Carcinomas,Breast Malignant Neoplasm,Breast Malignant Neoplasms,Breast Malignant Tumor,Breast Malignant Tumors,Breast Neoplasm,Breast Tumor,Cancer, Breast,Cancer, Mammary,Cancers, Mammary,Carcinoma, Breast,Carcinoma, Human Mammary,Carcinomas, Breast,Carcinomas, Human Mammary,Human Mammary Carcinomas,Human Mammary Neoplasm,Human Mammary Neoplasms,Mammary Cancers,Mammary Carcinomas, Human,Neoplasm, Breast,Neoplasm, Human Mammary,Neoplasms, Human Mammary,Tumor, Breast
D002467 Cell Nucleus Within a eukaryotic cell, a membrane-limited body which contains chromosomes and one or more nucleoli (CELL NUCLEOLUS). The nuclear membrane consists of a double unit-type membrane which is perforated by a number of pores; the outermost membrane is continuous with the ENDOPLASMIC RETICULUM. A cell may contain more than one nucleus. (From Singleton & Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d ed) Cell Nuclei,Nuclei, Cell,Nucleus, Cell
D003593 Cytoplasm The part of a cell that contains the CYTOSOL and small structures excluding the CELL NUCLEUS; MITOCHONDRIA; and large VACUOLES. (Glick, Glossary of Biochemistry and Molecular Biology, 1990) Protoplasm,Cytoplasms,Protoplasms
D003857 Deoxyuridine 2'-Deoxyuridine. An antimetabolite that is converted to deoxyuridine triphosphate during DNA synthesis. Laboratory suppression of deoxyuridine is used to diagnose megaloblastic anemias due to vitamin B12 and folate deficiencies. (beta 1-(2-Deoxyribopyranosyl))thymidine
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006820 Hyaluronic Acid A natural high-viscosity mucopolysaccharide with alternating beta (1-3) glucuronide and beta (1-4) glucosaminidic bonds. It is found in the UMBILICAL CORD, in VITREOUS BODY and in SYNOVIAL FLUID. A high urinary level is found in PROGERIA. Amo Vitrax,Amvisc,Biolon,Etamucine,Healon,Hyaluronan,Hyaluronate Sodium,Hyvisc,Luronit,Sodium Hyaluronate,Acid, Hyaluronic,Hyaluronate, Sodium,Vitrax, Amo
D015415 Biomarkers Measurable and quantifiable biological parameters (e.g., specific enzyme concentration, specific hormone concentration, specific gene phenotype distribution in a population, presence of biological substances) which serve as indices for health- and physiology-related assessments, such as disease risk, psychiatric disorders, ENVIRONMENTAL EXPOSURE and its effects, disease diagnosis; METABOLIC PROCESSES; SUBSTANCE ABUSE; PREGNANCY; cell line development; EPIDEMIOLOGIC STUDIES; etc. Biochemical Markers,Biological Markers,Biomarker,Clinical Markers,Immunologic Markers,Laboratory Markers,Markers, Biochemical,Markers, Biological,Markers, Clinical,Markers, Immunologic,Markers, Laboratory,Markers, Serum,Markers, Surrogate,Markers, Viral,Serum Markers,Surrogate Markers,Viral Markers,Biochemical Marker,Biologic Marker,Biologic Markers,Clinical Marker,Immune Marker,Immune Markers,Immunologic Marker,Laboratory Marker,Marker, Biochemical,Marker, Biological,Marker, Clinical,Marker, Immunologic,Marker, Laboratory,Marker, Serum,Marker, Surrogate,Serum Marker,Surrogate End Point,Surrogate End Points,Surrogate Endpoint,Surrogate Endpoints,Surrogate Marker,Viral Marker,Biological Marker,End Point, Surrogate,End Points, Surrogate,Endpoint, Surrogate,Endpoints, Surrogate,Marker, Biologic,Marker, Immune,Marker, Viral,Markers, Biologic,Markers, Immune

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