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Conduct mechanical characterization of in vitro 3D tumor models and study diffusion

Mechanical properties have been considered as an early biomarker to distinguish between cancerous and normal cell. In this study, a novel micro-tweezer device developed for mechanical characterization of spheroids and hydrogels was designed and fabricated. The device consisted of two force sensing cantilevers made from soft polymer held in position by a fixed and moving arm. The chop-stick like action of the arm facilitated easy sample handling and microscopic observation for mechanical characterization. Cantilever bending was tracked from optical images using a custom build optical tracking software. The cantilevers were calibrated and the efficacy of the method was demonstrated by using agarose pillars of known concentration. The method was also evaluated by confirming the agarose Young’s modulus with the established micro-indentation technique. After the initial evaluation, three cancerous (MCF7, T47D and BT474) and one normal epithelial (MCF10A) breast cell lines were used make multi-cellular spheroid whose Young’s moduli was measured using the microtweezers, developed in this work. Since micro-cantilevers are replaceable, this method successfully characterized samples with wide range of Young’s modulus including agarose (25-100 kPa), spheroids of cancerous and non-malignant cells (190-200 µm, 250- 1350 Pa), collagenase-treated spheroids (215 µm, 180 Pa) and overgrown spheroids (410µm, day 20, 820 Pa). Correlation between the factors that affect mechanical properties and the penetration of drugs carrying molecules to the spheroid has been also studied. Diffusion of liposomes (50nm) was measured for day 5 (200 µm), day 20 (420µm) and day 5 (size matched with day 20, 400 µm) spheroids made from BT474 and T474D cell lines. The diffusion study showed ~1.5 times more total fluorescence for day 5 (size matched) spheroids compared to day 20 spheroids for both the cell lines. Similarly, 3 hour collagenase (0.1%) treated spheroids showed 1.4 times more total fluorescence than control spheroids. The diffusion results were related to mechanical characteristics of the spheroids, measured using micro-cantilevers, which showed comparatively more liposome uptake for softer spheroids than stiffer spheroids. In conclusion, a novel method was developed for mechanical characterization of cancer spheroids and the results were correlated with nanoparticle diffusion.


A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing.

Abstract: Two-dimensional (2D) monolayer cultures are the standard in vitro model for cancer research. However, they fail to recapitulate the three-dimensional (3D) environment and quickly lose their function. In this study, we developed a new 3D multicellular heterospheroid tumor model in a collagen hydrogel culture system that more closely mimics the in vivo tumor microenvironment for anti-cancer drug testing. Three aspects of cancer were chosen to be modeled based on their ability to resist anti-cancer drugs: 3D, multicellularity, and extracellular matrix (ECM) barrier. The hanging drop method and co-culture of liver carcinoma with stromal fibroblasts were used to form controlled and uniform heterospheroids. These heterospheroids were then encapsulated in collagen gel in order to create a 3D model of liver cancer that would act more similarly to in vivo ECM conditions. The 3D heterospheroid tumor model was tested with an anti-cancer drug to determine how each of the above aspects affects drug resistance. The results demonstrate that the 3D heterospheroid model is more resistant to drug over 2D monolayer and homospheroid cultures, indicating stromal fibroblasts and collagen hydrogel culture system provides more resistance to anti-cancer drug. This study will provide useful information toward the development of improved biomimetic tumor models in vitro for cancer research in pre-clinical drug development.

Pub.: 19 Mar '13, Pinned: 05 Jul '17