The concept of using tissue density as a mechanism to diagnose a tumor has been around for centuries. However, this concept has not been sufficiently explored in a laboratory setting. Therefore, in this paper, we observed the effects of cell density and extracellular matrix (ECM) density on colon cancer invasion and proliferation using SW620 cells. We also attempted to inhibit ROCK-I to determine its effect on cell invasion and proliferation using standard molecular biology techniques and advanced imaging. Increasing cell seeding density resulted in a 2-fold increase in cell invasion as well as cell proliferation independent of treatment with Y-27632. Increasing collagen I scaffold density resulted in a 2.5-fold increase in cell proliferation while treatment with Y-27632 attenuated this effect although 1.5 fold increase in cell invasion was observed in ROCK inhibited samples. Intriguingly, ROCK inhibition also resulted in a 3.5-fold increase in cell invasion within 3D collagen scaffolds for cells seeded at lower densities. We show in this paper that ROCK-I inhibition leads to increased invasion within 3D collagen I microenvironments. This data suggests that although ROCK inhibitors have been used clinically to treat several medical conditions, its effect largely depends on the surrounding microenvironment. 1. Introduction Colon cancer is the third most commonly diagnosed cancer and the third leading cause of cancer death in both men and women in the USA [1, 2]. Today, there is a wide array of methods used to diagnose cancer including biopsy, endoscopy, and diagnostic imaging. Imaging techniques utilize the fact that tumorigenic tissue has a higher tissue density than the surrounding normal extracellular matrix (ECM). Thus, areas of increased tissue density are considered a warning sign of a potential malignancy [3–5]. With this strong link between tissue density and cancer, there has not been sufficient in vitro data, particularly for colon cancer, to fully understand this phenomenon. Two variables that affect mechanics of a tissue are cell and ECM density. Altering cell density induces cellular differentiation, proliferation, and even apoptosis [6, 7]; thus, cell density is one of the relevant parameters in cancer research. Previous studies have suggested that higher cell density environments significantly increase cell metastasis, especially colon 26, [8] and the initial seeding density affects differentiation of stem cells more than the cytokines and growth factors [9]. Similarly, mechanical induction done by altering the surrounding ECM alone
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