Mechanical mapping of tumor cell extravasation in brain parenchyma in vivo

Introduction:: Although efficiency of metastasis process is less than 0.1%, it is still responsible for almost 90% of cancer patient death. Especially, the median survival of patients with metastatic brain lesions is approximately 7-16 months from diagnosis. About 15-25% of brain metastasis is from primary breast cancer. Therefore, understanding factors crucial to cancer cell survival in the brain following dissemination is critical for developing optimal treatments for metastatic and resistant tumors. Here, we use a human xenograft model in zebrafish with MDA-MB-231 Parental breast cancer cells (231P) to understand the mechanical adaptability of cancer cells, focusing on early brain metastasis. The zebrafish has rapidly become a model for studying tumor behavior at different stages of the metastatic cascade due to its optical transparency for single cell imaging and homology between human and zebrafish proteins. Recent studies have demonstrated that multiple brain-homing human breast cancer cell lines also target the zebrafish brain, suggesting that key events in metastasis are sufficiently conserved in the model to gain insights relevant to human disease.¹ Moreover, a subset of zebrafish capillary endothelium and blood flow are comparable to mammalian systems.^1,2 During circulation, cancer cells must resist mechanical stress of blood fluid shear stress before crossing the luminal interface of the endothelium. Thus, cancer cells must overcome these mechanical barriers even before adapting to a new microenvironment.


Materials and Methods:: To visualize and perform real time mechanical mapping of tumor cells in vivo as they move from the luminal spaces into the tissue and as they adapt to the brain parenchyma, polystyrene beads were introduced to cancer cells by spontaneous phagocytosis and cancer cells/beads were injected to the circulation of 2.5 days post-fertilization (dpf) zebrafish (Fig. 1a). After 12 hours of dissemination as cells become occluded in the brain tissue, broad band optical tweezer-based active microrheology was used to assess the mechanical phenotypes of cells in terms of intravascular cells and extravasated cells (Fig. 1a-d).



Additionally, microfluidic-based real-time deformability cytometry (RT-DC) was also used to study the effect of fluid speed on the physical traits of cancer cells (Fig. 1e-g) at low laminar flow in order to avoid destruction of cancer cells. RT-DC is a high throughput technique as it can measure thousands of cells in short amount of time.³ Furthermore, we developed in vitro extravasation mimicking microfluidics device to enforce cell deformation while passing narrow channel to investigate the mechanics of cancer cells in terms of in-channel and post-channel for comparison with in vivo extravasation process (Fig. 1h-i).



We also tested the role of mechanopathway on extravasation focusing on yes-associated protein (YAP), using small molecule drug, verteporfin. We treated the cancer cells with the drug overnight before seeding into extravasation device or injecting into zebrafish. The drug efficacy was validated by immunofluorescence and the fluorescence of drug after seeding into new plate without drug for a day.


Results, Conclusions, and Discussions:: Here, we show that human breast cancer cells resist blood flow in zebrafish before extravasating into zebrafish brain. Our intravital movie shows that extravasation is a fast process finishing in 30 minutes (movie not shown here). Then, our mechanical mapping of human breast cancer show that cancer cells soften after extravasation into the brain parenchyma in comparison to intravascular cells (Fig. 1c). After fitting the complex modulus into power law (frequency-dependent mechanics; lG*l = Aω^b) at higher frequency from 401Hz to 15kHz, extravasated cancer cells exhibit more semi-flexibility characteristics due to increasing power law exponent b value (Fig. 1c). Moreover, cancer cells become more liquid-like based on crossover frequency (Fig. 1e). With real-time deformability cytometry (RT-DC), cancer cells show increasing stiffness with more deformation upon elevating fluid speed (Fig. 1f-g). Based on extravasation mimicking device, cancer cell softens after migrating via a narrow channel with huge deformation (Fig. 1h-i). Furthermore, we show that breast cancer cells can tune their mechanical properties during extravasation process to colonize brain in vivo as extravasated cancer show increasing stiffness after 1 day post extravasation.



Then, next question arises if softer cancer cells would extravasate better as our in vivo data indicates that cancer cells soften upon extravasation. As mechanics are altered during this process, we focused on mechanopathway, especially on yes-associated protein (YAP). YAP also has been known to play an important role in metastasis process.⁴ Thus, we inhibited YAP activity by using small molecule drug, verteporfin, to show that the YAP inhibited cancer cells are softer than control/DMSO based on RT-DC. Additionally, the drug treated cancer cells couldn’t extravasate in in vitro extravasation mimicking device. Our in vivo data using zebrafish also confirmed that YAP inhibited cancer cells had less trafficking at brain, showing that softer cancer cells are not the prerequisite for extravasation. Instead, genetic regulation also plays important role in this mechanical adaptation of cancer cells. We also show that melanoma also exhibit the same phenomena of mechanical adaptability at early in vivo extravasation steps.


Acknowledgements (Optional): : All schemes are created with BioRender.com

References (Optional): : 1. Paul, C. D.; et. al; Tanner, K. Cell Systems. 2019, 9(2): 187-206.e16.

2. Follain, G.; et. al; Timpson, P.; Goetz, J. G. Nat. Rev. Cancer. 2020, 20: 107-124.

3. Mokbel, M., et. al, Aland, S. ACS Biomater. Sci. Eng. 2017, 3(11): 2962-2973


4. Benjamin, D. C., et. al, Hynes, R. O. Cancer Research. 2020, 80(18): 3867-3879