POST DOCTORAL SCHOLAR, UNIVERSITY OF CONNECTICUT
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.
Abstract: Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Young's modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 degrees C), and their apparent Young's modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis.
Pub.: 29 Jul '08, Pinned: 28 Jun '17
Abstract: Cancer initiation and progression follow complex molecular and structural changes in the extracellular matrix and cellular architecture of living tissue. However, it remains poorly understood how the transformation from health to malignancy alters the mechanical properties of cells within the tumour microenvironment. Here, we show using an indentation-type atomic force microscope (IT-AFM) that unadulterated human breast biopsies display distinct stiffness profiles. Correlative stiffness maps obtained on normal and benign tissues show uniform stiffness profiles that are characterized by a single distinct peak. In contrast, malignant tissues have a broad distribution resulting from tissue heterogeneity, with a prominent low-stiffness peak representative of cancer cells. Similar findings are seen in specific stages of breast cancer in MMTV-PyMT transgenic mice. Further evidence obtained from the lungs of mice with late-stage tumours shows that migration and metastatic spreading is correlated to the low stiffness of hypoxia-associated cancer cells. Overall, nanomechanical profiling by IT-AFM provides quantitative indicators in the clinical diagnostics of breast cancer with translational significance.
Pub.: 23 Oct '12, Pinned: 28 Jun '17
Abstract: Change in cell stiffness is a new characteristic of cancer cells that affects the way they spread. Despite several studies on architectural changes in cultured cell lines, no ex vivo mechanical analyses of cancer cells obtained from patients have been reported. Using atomic force microscopy, we report the stiffness of live metastatic cancer cells taken from the body (pleural) fluids of patients with suspected lung, breast and pancreas cancer. Within the same sample, we find that the cell stiffness of metastatic cancer cells is more than 70% softer, with a standard deviation over five times narrower, than the benign cells that line the body cavity. Different cancer types were found to display a common stiffness. Our work shows that mechanical analysis can distinguish cancerous cells from normal ones even when they show similar shapes. These results show that nanomechanical analysis correlates well with immunohistochemical testing currently used for detecting cancer.
Pub.: 26 Jul '08, Pinned: 05 Jul '17
Abstract: Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of beta1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells.
Pub.: 19 Aug '09, Pinned: 05 Jul '17
Abstract: The inefficiency of nanoparticle penetration in tissues limits the therapeutic efficacy of such formulations for cancer applications. Recent work has indicated that modulation of tissue architecture with enzymes such as collagenase significantly increases macromolecule delivery. In this study we developed a mathematical model of nanoparticle penetration into multicellular spheroids that accounts for radially dependent changes in tumor architecture, as represented by the volume fraction of tissue accessible to nanoparticle diffusion. Parameters such as nanoparticle binding, internalization rate constants, and accessible volume fraction were determined experimentally. Unknown parameters of nanoparticle binding sites per cell in the spheroid and pore shape factor were determined by fitting to experimental data. The model was correlated with experimental studies of the penetration of 40 nm nanoparticles in SiHa multicellular spheroids with and without collagenase treatment and was able to accurately predict concentration profiles of nanoparticles within spheroids. The model was also used to investigate the effects of nanoparticle size. This model contributes toward the understanding of the role of tumor architecture on nanoparticle delivery efficiency.
Pub.: 27 May '08, Pinned: 28 Jun '17
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
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