Layered Microfluidics System to Study Cancer

Another course-related work from previous semester is presented here (I am not expert in this field, so, please read this as a blog post, not a scientific paper/project. If you are further interested in, I strongly recommend you to read main articles referred in the text.).

Main

Cancer is a disease caused by uncontrolled division of the cells in the body. Different regions in the body has different characteristics of cancer. If a specific type of cancer spreads from one tissue to another in the body, it is called “metastasis”. 130 years has passed since Stephan Paget claimed the hypothesis of “seed and soil” for cancer metastasis, which claimed that some cancer cells circulate in the body, also known as circulating cancer cells now (CTCs), and the survivors of those seed themselves to the appropriate locations to metastasize (Paget 1989).

However, cancer itself already a complex phenomenon having various factors involved (Hanahan & Weinberg 2000, 2011). It was previously hypothesized that several physical changes such as blood flow also contributes to the overall tumor system (Folkman 2000). Oxygen and nutrients are provided by blood flow to cancer, and flow patterns of vessels regulate the routes for these circulating cells (Folkman 2000, Carmeliot 2005). In fact, irregular blood flow was shown in tumor regions. Flow inside of the vascular network of cancer might determine differentiation, maturation and remodeling. In addition, it was previously shown that shear rate of blood flow and viscosity effects the functionality of vascular system (Kim & Sarelius 2003). In the vicinity of tumor-associated vasculature increases the resistance against blood flow due to highly torturous tumor associated vessels (Joyce & Pollard 2009). We now know that for metastasis to occur, intravasation, circulation, extravasation, and attachment of these tumor cells accompanied with physiological changes are required.

Exosomes are small vesicles secreted from various type of cells to blood carrying long range of cargo molecules and receptors including proteins, nucleic acids, and other regulatory molecules (Johnstone et al. 1987). In fact, they may support tumor microenvironment and mediate it through paracrine signalling (Rammal 2019). Tumor cell derived exosomes can also prepare metastatic niche and have a role in non-random metastasis of tumor in various sites (Hoshino 2015).

Micro/nano-fluidics is the field that investigates the fluid dynamics at macro/nano scales. Although the behavior of the fluid in such smaller level does not completely reflect the actual/real life model, it is still valid since ease of the controllability of the system (we can use Reynold numbers) and providing smaller space to test the real-world experiments in smaller scales.

Breast cancer the leading cancer responsible for death of lots of women across the world, and it can metastasize to the different tissues in the body such as lung. It is important to determine which patients’ tumor is prone to metastasize before applying the appropriate treatment. To this aim, Yankaras et al. (2019, Nature) proposed a solution with a Y-shaped microfluidic platform (MAqCI) (Figure 1), mimicking the mechanical environment in vivo, predicting the metastatic potential of the cancers. To understand the potential, they investigated the relative abundance of migratory cells in different cell lines and their proliferation potential with respect to less aggressive, not-metastasized counterparts. Advantages of their technique are the requirement of small volume of sample, delivery of rapid screenings, and physical separation and characterization of those high-migratory potential cells. However, the main drawbacks of this test are the number of cells required to derive the aggressiveness and metastasis potential of the tumor, false negatives in some of the cell types such as SUM149. Beside of these, it does not explain the mechanism of how metastasis mediated and can be controlled comprehensively.

On the other hand, very recently developed Stack microfluidic system by Jiaquan et al. (2019, Nature) provide spatiotemporal interaction between different cell types (Figure 2). Cell cultures of different cell types be layered up to more than 70 layers, unlayered, reconfigured to aim of the experiment without disturbing the cultures thanks to open microfluidic system via Laplace trap (Simon et al. 2012) mechanism. Open microfluidic platforms briefly depend on the surface tension instead of a pump system to flow to occur and retain the flow in the channels. The other advantage of this system is that it uses T-shaped pinning system to prevent leakage between the layers. In addition to providing 2D-3D spatial and temporarily controllable system, Stacks also allow for users to work with smaller volumes(4ul) compared to Transwell systems. Despite its advantages, the main disadvantage of this system is the limited layering and requirement of surface-tension pressures for different kind of fluid/culture system.

The solution in this project is to use Stack system with different rate of flows (if possible mimic Y-shaped design from the first paper), and combine with a system that we can capture exosomes.

References:

Hanahan D, Weinberg RA. 2000. The hallmarks of cancer. Cell 100:57–70

Hanahan D, Weinberg RA. 2011. Hallmarks of cancer: the next generation. Cell 144:646–74

Paget, S. The distribution of secondary growths in cancer of the breast. 1889. Cancer Metastasis Rev.8, 98–101 (1989)

Folkman J, Bach M, Rowe J, Davidoff F, Lambert P, et al. 1971. Tumor angiogenesis: therapeutic implications.

N. Engl. J. Med. 285:1182–86

Folkman J,Hahnfeldt P, Hlatky L. 2000. Cancer: looking outside the genome. Nat. Rev. Mol. Cell Biol. 1:76–79

Carmeliet P. 2005. Angiogenesis in life, disease and medicine. Nature 438:932–36

KimM, Sarelius I. 2003. Distributions of wall shear stress in venular convergences of mouse cremaster muscle. Microcirculation 10:167–78

M.G. Simon, R. Lin, J.S. Fisher, A.P. Lee. 2012. A Laplace pressure based microfluidic trap for passive droplet trapping and controlled release. Biomicrofluidics 6(1), 014110

Joyce JA, Pollard JW. 2009. Microenvironmental regulation of metastasis. Nat. Rev. Cancer 9:239–52

Johnstone R, Adam M, Hammond J, Orr L, Turbide C. Vesicle formation dur-ing reticulocyte maturation. Association of plasma membrane activities withreleased vesicles (exosomes). J Biol Chem 1987;262:9412–20.

Ghina Rammal, Assil Fahs, Firas Kobeissy, Yehia Mechref, Jingfu Zhao, Rui Zhu, Mona Diab-Assaf, Raya Saab, and Sandra E. Ghayad. 2019. Proteomic Profiling of Rhabdomyosarcoma-Derived Exosomes Yield Insights into Their Functional Role in Paracrine Signaling. Journal of Proteome Research. 18 (10), 3567–3579. DOI: 10.1021/acs.jproteome.9b00157

Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, et al. 2015.Tumour exosome integrins determine organotropic metastasis. Nature.527(7578):329–35

Yu, J., Berthier, E., Craig, A., de Groot, T. E., Sparks, S., Ingram, P. N., … Theberge, A. B. (2019). Reconfigurable open microfluidics for studying the spatiotemporal dynamics of paracrine signalling. Nature Biomedical Engineering, 3(10), 830–841. https://doi.org/10.1038/s41551-019-0421-4

A microfluidic assay for the quantification of the metastatic propensity of breast cancer specimens | Nature Biomedical Engineering. (n.d.). Retrieved October 15, 2019, from https://www-nature-com.libproxy1.nus.edu.sg/articles/s41551-019-0400-9

Figures are not posted due to copyright concerns.

*Two main articles discussed in this assay were highlighted.