Antmen, E. | Search bioRxiv

bioRxiv2019

Loss Of Nuclear Deformability Of Breast Cancer Cells By The Disruption Of Actin Filaments

It is well known that chemical and biomechanical interactions between the nucleus and cytoskeleton are involved in and critical for movement, migration and nuclear positioning of cancer cells. Through nucleo-cytoskeletal coupling, proteins of the LINC complex and the nuclear envelope are capable of transducing cytoplasmic mechanical input across the nuclear membrane; however, their functional importance in the behavior of cancer cells and their nuclei has never been directly tested. In this study, our assumption was that the differences in the malignancies of breast cancer cells are the result of the differences in their nuclear deformation and its expression can be amplified on micropatterned surfaces. Based on this, our hypothesis was that the level of completeness of polymerization of actin filaments can affect nuclear deformability, and as a result, the metastatic capability of the cancer cells. In order to prove this we disrupted the polymerization of the actin filaments by using two drugs, Cytochalasin D and Jasplakinolide, which caused impaired propagation of intracellular forces, prevented nuclear deformation and increased in the expression levels of Lamin A/C and Nesprin-2 in malignant breast cancer cells. Our findings suggest that activity of these two proteins is critical for nucleo-cytoskeletal force transmission. More importantly, actin filament disruption can prevent the distortions in nuclear morphology and as a result avoid the development of cancer metastasis.

bioengineering↗

bioRxiv2019

A 2-Compartment Bone Tumor Model for Testing the Efficacy of Cancer Drugs

We produced a three dimensional (3D) bone tumor model (BTM) to study the interactions between healthy and tumor cells in a tumor tissue microenvironment, migration of the tumor cells and the efficacy of an anticancer drug, Doxorubicin, for personalized medicine applications. The model consisted of two compartments: (a) a healthy bone tissue mimic, poly(lactic acid-co-glycolic acid) (PLGA)/beta-tricalcium phosphate ({beta}-TCP) sponge that was seeded with human fetal osteoblastic cells (hFOB) and human umbilical vein endothelial cells (HUVECs), and (b) a tumor mimic, a lyophilized collagen sponge that was seeded with human osteosarcoma cells (Saos-2). The tumor component was introduced to a central cavity created in the healthy bone mimic and together they constituted the total 3D model (3D-BTM). The scaffolds were characterized by determining their mechanical properties, studying their topography and stability with compression tests, microCT, SEM, confocal microscopy and gravimetry. Porosities of the sponges were determined from {micro}CT data as 96.7% and 86% for PLGA/TCP and collagen sponges, respectively. The average diameters of the pores were measured by using ImageJ (NIH, USA) as 199{+/-}52 {micro}m for PLGA/TCP and 50-150 m for collagen scaffolds. Young modulus of the PLGA/TCP and collagen sponges were determined as 4.76MPa and 140kPa, respectively. Cells seeded on the two sponges were studied independently and together on the BTM. Cell proliferation, morphology, calcium phosphate forming capacity and ALP production were studied on both healthy bone and tumor mimics. All types of cells showed cellular extensions and spread on and in the scaffolds indicating good cell-material interactions. Angiogenic developments in BTM were studied along with migration of cells between the components with immunocytochemistry, SEM, microCT, qRT-PCR and agarose gel electrophoresis. Confocal microscopy showed that a direct contact was established between the cells present in different parts of the BTM; and the HUVEC cells within the healthy bone mimic were observed to migrate into the tumor mimic. This was confirmed by the increase in the levels of angiogenic factors VEGF, bFGF, and IL-8 in the tumor component. The IC50 of Doxorubicin on Saos-2 cells was determined as 0.1876 {micro}g.mL-1. Doxorubicin was administered to the BTM at 2.7 {micro}g.mL-1 concentration and after allowing one day for interaction, the cell number was determined with Alamar Blue cell viability test as 7-fold lesser compared to 24 h earlier. Apoptosis of the osteosarcoma cells was measured by caspase-3 enzyme activity assay. These results demonstrate the suitability of the 3D BTM model for use in the investigation of activities and migrations of cells in a tumor tissue. These will be very useful in studying metastatic capabilities of cells in addition to personalized drug treatments.

bioengineering↗