Supervisor: Dr. M. N. Rylander

As a final project for my biomechanics technical elective, Tissue Microenvironments, I was tasked with 
drafting a sample NIH proposal to outline a new approach to the development of novel technologies and model systems for the study of the tumor microenvironment. 

I decided to focus on developing a novel in vitro 3-dimensional matrix reconstitution of the breast tumor microenvironment in order to study the invasion phase of metastasis.

Mechanisms of metastasis are not fully understood, yet secondary tumors cause most cancer deaths [1]. If we understand the characteristics and functions of the tumor microenvironment, then we could better understand the mechanism of metastasis so that we can develop novel targeted therapies. To better understand propensity for metastasis, it is relevant to study the cellular characteristics of heterogeneous tumors [2]. This heterogeneity will help us better identify tumor microenvironments whose characteristics are connected with metastatic activity, so that we can design target therapies more effectively [2]. One aspect of the tumor microenvironment that reflects both intrapatient heterogeneity and interpatient heterogeneity is the topography of the tumor. Invasion can occur when cancer cells migrate out of the tumor and into the stroma through channels in the ECM where collagen fibers are parallel or aligned [3]. Recent studies have investigated how tumor topography influences metastatic invasion by fabricating pillars/grooves on the tumor surfaces and monitoring cancer cell migration [3]. This research suggested 3D topography in the form of pillars or gratings inhibits metastatic progression of cancer epithelial cells, except in breast cancer epithelial cells (See Figure 1)[3]. Understanding the extent by which topography relates to metastatic activity in breast cancer cells, therefore, is necessary, and the development of a model that more accurately represents the topography of breast tumors will facilitate this research.

We hypothesize by using a biomimetic approach to bioprint 3D scaffolds of breast tumors using an extrusion method, we will be able to more accurately simulate the tumor microenvironment for the purpose of studying the extent to which ECM topography influences metastatic invasion progression.  

The goal of this research study will be to study the effect of tumor topography on cancer cell invasion in metastasis. To do this, it is imperative that we make accurate models of human breast tumors, so that we can replicate the breast tumor microenvironment. Understanding the tumor microenvironment more thoroughly will help us realize more about malignant breast tumor progression by tracking the cellular responses to topographical heterogeneity. 3D bioprinting is an adept method to replicate the topography of the tumor because it has high resolution and design control capabilities that give the user the autonomy to create as many tumor models, each with differing topographies, as needed.

Specific Aim 1: Analyze MRI scans of human breast tumors to identify commonly occuring ECM topographical patterns, and convert tumor scans into printable STL files for 3D modeling.

      Aim 1 Milestone: Collect a large dataset of 3D scans of breast tumors that includes varying tumor topographical
        patterns and spatial orientations. Code each tumor into an STL file and store information on the topographical
        patterns that each tumor has for later comparison with metastatic activity.

Specific Aim 2: Use 3D bioprinting and cell co-culture to recreate various tumors in 3D models that represent varying topographical patterns.

      Aim 2 Milestone: 3D-bioprint the collagen ECM scaffold and then seed breast epithelial cells on the scaffold. Test
        cell viability to ensure model efficacy [4],[5].

Specific Aim 3: Test the metastatic invasion progression of breast cancer cells using the 3D bioprinted models by comparing the spread of proliferating breast cancer cells on varying ECM topographies.

      Aim 3 Milestone: Measure how the extent of invasion differs based on tumor topography by using real-time video
        imaging to see to what extent cells are migrating across the ECM. Investigate whether there is a  correlation
        between common topographical patterns and the likelihood of metastasis.

This research will mimic the actual spatial orientation and topography of of real breast tumors, so that understanding metastasis is done so using an accurate model to mimic cellular responses appropriately. Not only might 3D-bioprinting be a novel way to mimic ECM topography in the tumor microenvironment because it is a very new fabrication method, but also, because the reproducibility and high design control of the method allow users to create highly specific, high resolution objects. Additionally, because 3D-bioprinting is a new technology, there are not many pre-existing models that monitor metastasis, specifically by studying the relationship between topography and invasion behavior. The high resolution of 3D-bioprinting, we hypothesize, is particularly adept for printing topography, which needs to reflect tumor heterogeneity on a micrometer scale.

1. Xie, H.-Y., Shao, Z.-M., & Li, D.-Q. (2017). Tumor microenvironment: driving forces and potential therapeutic targets for breast cancer metastasis. Chinese Journal of Cancer, 36(1), 36. 
2. Belgodere, J. A., King, C. T., Bursavich, J. B., Burow, M. E., Martin, E. C., & Jung, J. P. (2018). Engineering Breast Cancer Microenvironments and 3D Bioprinting. Frontiers in bioengineering and biotechnology, 6, 66. doi:10.3389/fbioe.2018.00066
3. Chaudhuri, P. K., Pan, C. Q., Low, B. C., & Lim, C. T. (2016). Topography induces differential sensitivity on cancer cell proliferation via Rho-ROCK-Myosin contractility. Scientific reports, 6, 19672. doi:10.1038/srep19672
4. Bishop, E. S., Mostafa, S., Pakvasa, M., Luu, H. H., Lee, M. J., Wolf, J. M., … Reid, R. R. (2017). 3-D bioprinting technologies in tissue engineering and regenerative medicine: Current and future trends. Genes & diseases, 4(4), 185–195. doi:10.1016/j.gendis.2017.10.002
5. Pati, F. et al. Printing three-dimensional tissue analogues with decellularized extracellular matrix bioink. Nat. Commun. 5:3935 doi: 10.1038/ncomms4935 (2014).
6. Sleeboom, J., Eslami Amirabadi, H., Nair, P., Sahlgren, C. M., & den Toonder, J. (2018). Metastasis in context: modeling the tumor microenvironment with cancer-on-a-chip approaches. Disease models & mechanisms, 11(3), dmm033100. doi:10.1242/dmm.033100
7. Nabavizadeh, N., Klifa, C., Newitt, D., Lu, Y., Chen, Y. Y., Hsu, H., … Park, C. C. (2011). Topographic enhancement mapping of the cancer-associated breast stroma using breast MRI. Integrative biology : quantitative biosciences from nano to macro, 3(4), 490–496. doi:10.1039/c0ib00089b
8. Albritton, J. L., & Miller, J. S. (2017). 3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments. Disease models & mechanisms, 10(1), 3–14. doi:10.1242/dmm.025049
9. Kushito, K., Yaginuma, T., Ryo, A., & Takai, M. (2017). Difference in Three-dimensional geometric recognition by non-cancerous and cancerous epithelial cells on microgroove-based topography. Scientific reports, 7(1), 4244.