(I-352) Probing the effects of ECM stiffness on hypersialylation and drug response in breast cancer

Introduction::

The cases of breast cancer diagnosed annually worldwide have approached 2 million, claiming approximately 500,000 lives each year as a result of metastasis¹. Finding a cure for metastasis thus requires an understanding of the dynamic cellular interactions on the extracellular matrix (ECM), that enhance the process. Previous studies have identified that there is a significant influence of ECM on the migration and proliferation of cancer cells that results in metastasis when cells are cultured on tissues with higher stiffness². However, these studies often focus on the effect of bulk mechanics or migration or metastatic transformation, which while highly relevant to cancer progression in response to a stiffer and altered ECM, ignores how changes in ECM properties can influence therapeutic efficacy. In breast cancer (BC), pronounced expression of sialoglycans correlates with the aggressiveness of a tumor, its ability to survive drug treatments, and its capacity to invade neighboring tissue.³ Aging of a tumor is also related to its fibrotic nature and directly correlates with the stiffness of the ECM around the cancer cells.⁴ The present study is aimed to understand the correlation between ECM stiffness and the degree of cellular sialylation. Although the sialylation of BC cells has been strongly studied, the role that a substrate with higher stiffness presents remains unknown. By tracking the sialylation expressed on the cell membranes, we are able to assess the chemotherapeutic response of these cells and correlate that to ECM stiffness.

Materials and Methods::

Considering collagen composes the vast majority of the ECM, here we measured glycocalyx sialylation of MCF-10A cells seeded on collagen-agarose hydrogels with varying elastic moduli. Briefly, Agarose T1 was dissolved in cell culture media at a concentration of 2% w/v by heating to 95 ℃. Separately, a stock solution of 5 mg/ml of collagen I from rat tail was diluted to 0.5 mg/mL and kept on ice before its pH was brought to 7.4 by using 1 M NaOH. The neutralized collagen solution was quickly mixed with the required volume of hot agarose solution and dispensed onto glass bottom dishes to form a thin layer, followed by incubation at 37° for 30 minutes after which a superlayer of media was added to them. The agarose was maintained at varying concentrations in increments of 0.125% ranging from 0 to 0.5%.

(a) MCF-10A and MDA-MB-231 cells were seeded on the surface of freshly prepared gels (10⁵ cells per well in a 12-well plate). 24 h later, Doxorubicin (0.5 µM) was added to the adhered cells and MTT assay was performed 72 h later to determine cell viability.

(b) MCF 10A and MDA-MB-231 cells were seeded on the surface of freshly prepared gels (2 x 10⁴ cells per well) in a 30 mm dish with a glass bottom center. ManNAz (100 μM) was added to allow for metabolic incorporation over a period of 72 h. Click chemistry using DBCO-FITC followed by confocal microscopy was used to image sialic acid on the cellular glycocalyx.

Results, Conclusions, and Discussions::

Results and Discussion

Measurement of cell viability on the collagen-agarose gels revealed that the collagen surface proved to be slightly protective to the cells towards Doxorubicin treatment. However, an increased agarose concentration in the mixture appeared to sensitize the cells toward chemotherapeutic application (Figure 1B). An additional observation hinted at the inherent toxicity of the gels with higher agarose concentration (0.5%) (Figure 1A). Analysis of the degree of cellular sialylation revealed an inverse relationship with increasing the stiffness of collagen-agarose hydrogels. Compared to the tissue-culture glass bottom dish, the degree of glycocalyx sialylation showed a lowering in the presence of higher agarose content (Figure 2). This observation is in agreement with the established role of sialic acid as a promoter of chemoresistance.

 

Conclusion

Collagen-agarose gels were used as a model system to mimic ECM of varying stiffness and sialylation was measured via (a) bioorthogonal labeling (b) chemotherapeutic response. The data collected shows a decrease in the expression of sialic acid on the MCF-10A glycocalyx when growing on stiffer substrates. This decrease is also manifested in a higher sensitivity to chemotherapeutic (DOX) applications. Further studies in triple-negative breast cancer cell lines like MDA-MB-231 will broaden our understanding of how ECM stiffness affects highly invasive tumor cells.

Acknowledgements (Optional): : Research presented in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers Rl5GM118969, TL4GM118971, and UL1GM118970. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References (Optional): :

References

1. Teoh, S. T., Ogrodzinski, M. P., Ross, C., Hunter, K. W., & Lunt, S. Y., Frontiers in Oncology, 8, (2018).


2. Chaudhuri, O., Cooper-White, J., Janmey, P. A., Mooney, D. J., & Shenoy, V. B., Nature, 584(7822), (2020).


3. Julien S, Ivetic A, Grigoriadis A, QiZe D, Burford B, Sproviero D, et al., Cancer Research, ( 2011).


4. Tung, J. C., Barnes, J. M., Desai, S. R., Sistrunk, C., Conklin, M. W., Schedin, P., Eliceiri, K. W., Keely, P. J., Seewaldt, V. L., & Weaver, V. M., Free Radical Biology and Medicine, (2015).