Characterization of Breast Cancer Aggressiveness by Cell Mechanics.

In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.

Rolling Circle Amplification Tailored for Plasmonic Biosensors: From Ensemble to Single-Molecule Detection.

We report on the tailoring of rolling circle amplification (RCA) for affinity biosensors relying on the optical probing of their surface with confined surface plasmon field. Affinity capture of the target analyte at the metallic sensor surface (e.g., by using immunoassays) is followed by the RCA step for subsequent readout based on increased refractive index (surface plasmon resonance, SPR) or RCA-incorporated high number of fluorophores (in surface plasmon-enhanced fluorescence, PEF). By combining SPR and PEF methods, this work investigates the impact of the conformation of long RCA-generated single-stranded DNA (ssDNA) chains to the plasmonic sensor response enhancement. In order to confine the RCA reaction within the evanescent surface plasmon field and hence maximize the sensor response, an interface carrying analyte-capturing molecules and additional guiding ssDNA strands (complementary to the repeating segments of RCA-generated chains) is developed. When using the circular padlock probe as a model target analyte, the PEF readout shows that the reported RCA implementation improves the limit of detection (LOD) from 13 pM to high femtomolar concentration when compared to direct labeling. The respective enhancement factor is of about 2 orders of magnitude, which agrees with the maximum number of fluorophore emitters attached to the RCA chain that is folded in the evanescent surface plasmon field by the developed biointerface. Moreover, the RCA allows facile visualizing of individual binding events by fluorescence microscopy, which enables direct counting of captured molecules. This approach offers a versatile route toward a fast digital readout format of single-molecule detection with further reduced LOD.

Measuring Mechanical Properties of Breast Cancer Cells with Atomic Force Microscopy.

Using physical models to describe the response of cells to external stimuli has grown popular in the last three decades. The mechanical properties of cells are tightly linked to biochemical signaling pathways related to cell structure, proliferation, differentiation, motility, and cell fate. This chapter aims to describe how to perform mechanical experiments on MCF-7 breast cancer cells using the atomic force microscope. We present a stepwise procedure on sample preparation, force spectroscopy measurements and a guide on data evaluation using standard rheological models. We demonstrate how to derive all viscoelastic parameters of the cell by conducting stress relaxation and creep experiments. Additionally, the reader can find a sample dataset and the code required for data evaluation.

Substrate stiffness modulates the viscoelastic properties of MCF-7 cells.

Cells sense stiffness of surrounding tissues and adapt their activity, proliferation, motility and mechanical properties based on such interactions. Cells probe the stiffness of the substrate by anchoring and pulling to their surroundings, transmitting force to the extracellular matrix and other cells, and respond to the resistance they sense, mainly through changes in their cytoskeleton. Cancer and other diseases alter stiffness of tissues, and the response of cancer cells to this stiffness can also be affected. In the present study we show that MCF-7 breast cancer cells seeded on polyacrylamide gels have the ability to detect the stiffness of the substrate and alter their mechanical properties in response. MCF-7 cells plated on soft substrates display lower stiffness and viscosity when compared to those seeded on stiffer gels or glass. These differences can be associated with differences in the morphology and cytoskeleton organisation, since cells seeded on soft substrates have a round morphology, while cells seeded on stiffer substrates acquire a flat and spread morphology with formation of actin filaments, similar to that observed when seeded on glass. These findings show that MCF-7 cells can detect the stiffness of the surrounding microenvironment and thus, modify their mechanical properties.

Estrogen Modulates Epithelial Breast Cancer Cell Mechanics and Cell-to-Cell Contacts.

Excessive estrogen exposure is connected with increased risk of breast cancer and has been shown to promote epithelial-mesenchymal-transition. Malignant cancer cells accumulate changes in cell mechanical and biochemical properties, often leading to cell softening. In this work we have employed atomic force microscopy to probe the influence of estrogen on the viscoelastic properties of MCF-7 breast cancer cells cultured either in normal or hormone free-medium. Estrogen led to a significant softening of the cells in all studied cases, while growing cells in hormone free medium led to an increase in the studied elastic and viscoelastic moduli. In addition, fluorescence microscopy shows that E-cadherin distribution is changed in cells when culturing them under estrogenic conditions. Furthermore, cell-cell contacts seemed to be weakened. These results were supported by AFM imaging showing changes in surfaces roughness, cell-cell contacts and cell height as result of estrogen treatment. This study therefore provides further evidence for the role of estrogen signaling in breast cancer.

Measuring (biological) materials mechanics with atomic force microscopy. 2. Influence of the loading rate and applied force (colloidal particles).

Atomic force microscopy (AFM) is the most often used tool to study the mechanical properties of eukaryotic cells. Due to their complex assembly, cells show viscoelastic properties. When performing experiments, one has to consider the influence of both loading rate and maximum load on the measured mechanical properties. Here, we employed colloidal particles of various sizes (from 2 to 20 µm diameter) to perform force spectroscopy measurements on endothelial cells at loading rates varying from 0.1 to 50 µm/s, and maximum loads ranging from 1 to 25 nN. We were able to determine the non-linear dependence of cell viscoelastic properties on the loading rate which followed a weak power law. In addition, we show that previous loading at high forces leads to a stiffening of cells. Based on these results we discuss a road map for determining cell mechanical properties using AFM. Finally, this work provides an experimental framework for cell mechanical measurements using force-cycle experiments.

Organotypic human skin culture models constructed with senescent fibroblasts show hallmarks of skin aging.

Skin aging is driven by intrinsic and extrinsic factors impacting on skin functionality with progressive age. One factor of this multifaceted process is cellular senescence, as it has recently been identified to contribute to a declining tissue functionality in old age. In the skin, senescent cells have been found to markedly accumulate with age, and thus might impact directly on skin characteristics. Especially the switch from young, extracellular matrix-building fibroblasts to a senescence-associated secretory phenotype (SASP) could alter the microenvironment in the skin drastically and therefore promote skin aging. In order to study the influence of senescence in human skin, 3D organotypic cultures are a well-suited model system. However, only few "aged" skin- equivalent (SE) models are available, requiring complex and long-term experimental setups. Here, we adapted a previously published full-thickness SE model by seeding increasing ratios of stress-induced premature senescent versus normal fibroblasts into the collagen matrix, terming these SE "senoskin". Immunohistochemistry stainings revealed a shift in the balance between proliferation (Ki67) and differentiation (Keratin 10 and Filaggrin) of keratinocytes within our senoskin equivalents, as well as partial impairment of skin barrier function and changed surface properties. Monitoring of cytokine levels of known SASP factors confirmedly showed an upregulation in 2D cultures of senescent cells and at the time of seeding into the skin equivalent. Surprisingly, we find a blunted response of cytokines in the senoskin equivalent over time during 3D differentiation.