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1. Matrix stiffness and cluster size collectively regulate dormancy versus proliferation in brain metastatic breast cancer cell clusters

   https://doi.org/10.1039/d0bm00969e  

   Kondapaneni, Raghu Vamsi ; Rao, Shreyas S. ( November 2020 , Biomaterials Science)

   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. 

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2. A Biomimetic Hyaluronic Acid Hydrogel Models Mass Dormancy in Brain Metastatic Breast Cancer Spheroids

   https://doi.org/10.1002/adbi.202200114  

   Kondapaneni, Raghu Vamsi ; Shevde, Lalita A. ; Rao, Shreyas S. ( November 2022 , Advanced Biology)

   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.

    

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3. Cell Communication Network Factor 4 (CCN4/WISP1) Shifts Melanoma Cells from a Fragile Proliferative State to a Resilient Metastatic State

   https://doi.org/10.1007/s12195-019-00602-2  

   Deng, Wentao ; Fernandez, Audry ; McLaughlin, Sarah L. ; Klinke, David J. ( April 2020 , Cellular and Molecular Bioengineering)

   Introduction—Cellular communication network factor 4 (CCN4/WISP1) is a secreted matricellular protein that stimulates metastasis in multiple malignancies but has an unclear impact on phenotypic changes in melanoma. Recent data using cells edited via a double-nickase CRISPR/Cas9 approach suggest that CCN4/WISP1 stimulates invasion and metastasis of melanoma cells. While these data also suggest that loss of CCN4/WISP1 increases cell proliferative, the CRISPR approach used may be an alternative explanation rather than the loss of gene function. Methods—To test whether CCN4/WISP1 also influences the proliferative phenotype of melanoma cells, we used mouse melanoma models and knocked out Ccn4 using a homologydirected repair CRISPR/Cas9 system to generate pools of Ccn4-knockout cells. The resulting edited cell pools were compared to parental cell lines using an ensemble of in vitro and in vivo assays. Results—In vitro assays using knockout pools supported previous findings that CCN4/WISP1 promoted an epithelial– mesenchymal-like transition in melanoma cells and stimulated invasion and metastasis. While Ccn4 knockout also enhanced cell growth in optimal 2D culture conditions, the knockout suppressed certain cell survival signaling pathways and rendered cells less resistant to stress conditions. Tumor cell growth assays at sub-optimal conditions in vitro, quantitative analysis of tumor growth assays in vivo, and transcriptomics analysis of human melanoma cell lines were also used to quantify changes in phenotype and generalize the findings. Conclusions—In addition to stimulating invasion and metastasis of melanoma cells, the results suggested that CCN4/WISP1 repressed cell growth and simultaneously enhanced cell survival. 

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4. Breast cancer dormancy: need for clinically relevant models to address current gaps in knowledge

   https://doi.org/10.1038/s41523-021-00269-x  

   Bushnell, Grace G. ; Deshmukh, Abhijeet P. ; den Hollander, Petra ; Luo, Ming ; Soundararajan, Rama ; Jia, Dongya ; Levine, Herbert ; Mani, Sendurai A. ; Wicha, Max S. ( December 2021 , npj Breast Cancer)

   null (Ed.)

   Abstract Breast cancer is the most commonly diagnosed cancer in the USA. Although advances in treatment over the past several decades have significantly improved the outlook for this disease, most women who are diagnosed with estrogen receptor positive disease remain at risk of metastatic relapse for the remainder of their life. The cellular source of late relapse in these patients is thought to be disseminated tumor cells that reactivate after a long period of dormancy. The biology of these dormant cells and their natural history over a patient’s lifetime is largely unclear. We posit that research on tumor dormancy has been significantly limited by the lack of clinically relevant models. This review will discuss existing dormancy models, gaps in biological understanding, and propose criteria for future models to enhance their clinical relevance. 

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5. Varying the RGD concentration on a hyaluronic acid hydrogel influences dormancy versus proliferation in brain metastatic breast cancer cells

   https://doi.org/10.1002/jbm.a.37651  

   Goodarzi, Kasra ; Lane, Rachel ; Rao, Shreyas S. ( November 2023 , Journal of Biomedical Materials Research Part A)

   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.

    

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