Marija Plodinec

The nanomechanical signature of breast cancer.

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.

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system.

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.

New concepts in basement membrane biology

Basement membranes (BMs) are thin sheets of extracellular matrix that outline epithelia, muscle fibers, blood vessels and peripheral nerves. The current view of BM structure and functions is based mainly on transmission electron microscopy imaging, in vitro protein binding assays, and phenotype analysis of human patients, mutant mice and invertebrata. Recently, MS‐based protein analysis, biomechanical testing and cell adhesion assays with in vivo derived BMs have led to new and unexpected insights. Proteomic analysis combined with ultrastructural studies showed that many BMs undergo compositional and structural changes with advancing age. Atomic force microscopy measurements in combination with phenotype analysis have revealed an altered mechanical stiffness that correlates with specific BM pathologies in mutant mice and human patients. Atomic force microscopy‐based height measurements strongly suggest that BMs are more than two‐fold thicker than previously estimated, providing greater freedom for modelling the large protein polymers within BMs. In addition, data gathered using BMs extracted from mutant mice showed that laminin has a crucial role in BM stability. Finally, recent evidence demonstrate that BMs are bi‐functionally organized, leading to the proposition that BM‐sidedness contributes to the alternating epithelial and stromal tissue arrangements that are found in all metazoan species. We propose that BMs are ancient structures with tissue‐organizing functions and were essential in the evolution of metazoan species.

Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane

At the stage of carcinoma in situ, the basement membrane (BM) segregates tumor cells from the stroma. This barrier must be breached to allow dissemination of the tumor cells to adjacent tissues. Cancer cells can perforate the BM using proteolysis; however, whether stromal cells play a role in this process remains unknown. Here we show that an abundant stromal cell population, cancer-associated fibroblasts (CAFs), promote cancer cell invasion through the BM. CAFs facilitate the breaching of the BM in a matrix metalloproteinase-independent manner. Instead, CAFs pull, stretch, and soften the BM leading to the formation of gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Blocking the ability of stromal cells to exert mechanical forces on the BM could therefore represent a new therapeutic strategy against aggressive tumors.Stromal cells play various roles in tumor establishment and metastasis. Here the authors, using an ex-vivo model, show that cancer-associated fibroblasts facilitate colon cancer cells invasion in a matrix metalloproteinase-independent manner, likely by pulling and stretching the basement membrane to form gaps.

Atomic Force Microscopy for Biological Imaging and Mechanical Testing across Length Scales

Atomic force microscopy (AFM) offers researchers a unique opportunity to visualize, manipulate, and quantitatively assess structural and mechanical aspects of native biological samples with nanometer resolution. An unparalleled advantage of AFM over other high-resolution microscopes is that biological specimens, ranging from tissues to cells to molecules, can be investigated in physiologically relevant aqueous environments. The AFM can be operated at 37°C, which makes it ideal for in situ cell or tissue studies. Combining an optical microscope with an AFM makes it possible to directly correlate structural/nanomechanical changes with optical/fluorescence images. This ability to simultaneously acquire structural and function information is unprecedented in biology. This article introduces the basics of AFM for imaging and investigating the properties of biological samples.

Spatial organization acts on cell signaling: how physical force contributes to the development of cancer

Cells constantly encounter physical forces and respond to neighbors and circulating factors by triggering intracellular signaling cascades that in turn affect their behavior. The mechanisms by which cells transduce mechanical signals to downstream biochemical changes are not well understood. In their work, Salaita and coworkers show that the spatial organization of cell surface receptors is crucial for mechanotransduction. Consequently, force modulation that disrupts the mechanochemical coupling may represent a critical step in cancerogenesis.

Nanomechanical characterization of living mammary tissues by atomic force microscopy.

The mechanical properties of living cells and tissues are important for a variety of functional processes in vivo, including cell adhesion, migration, proliferation and differentiation. Changes in mechano-cellular phenotype, for instance, are associated with cancer progression. Atomic force microscopy (AFM) is an enabling technique that topographically maps and quantifies the mechanical properties of complex biological matter in physiological aqueous environments at the nanometer length scale. Recently we applied AFM to spatially resolve the distribution of nanomechanical stiffness across human breast cancer biopsies in comparison to healthy tissue and benign tumors. This led to the finding that AFM provides quantitative mechano-markers that may have translational significance for the clinical diagnosis of cancer. Here, we provide a comprehensive description of sample preparation methodology, instrumentation, data acquisition and analysis that allows for the quantitative nanomechanical profiling of unadulterated tissue at submicron spatial resolution and nano-Newton (nN) force sensitivity in physiological conditions.

Structural centrosome aberrations promote non‐cell‐autonomous invasiveness

Centrosomes are the main microtubule‐organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein‐like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP‐induced structural centrosome aberrations trigger the escape (“budding”) of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP‐induced centrosome aberrations trigger a re‐organization of the cytoskeleton, which stabilizes microtubules and weakens E‐cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non‐cell‐autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes.

A tumorigenic actin mutant alters fibroblast morphology and multicellular assembly properties

Tumor initiation and progression are accompanied by complex changes in the cytoarchitecture that at the cellular level involve remodeling of the cytoskeleton. We report on the impact of a mutant β‐actin (G245D‐actin) on cell structure and multicellular assembly properties. To appreciate the effects of the Gly245Asp substitution on the organization of the actin cytoskeleton, we examined the polymerization properties of G245D‐actin in vitro by pyrene polymerization assays and total internal reflection fluorescence microscopy (TIRF). The mutant actin on its own has a significantly reduced polymerization efficiency compared to native actin but also modifies the polymerization of actin in copolymerization experiments. Comparison of the structure of Rat‐2 fibroblasts and a stably transfected derivate called Rat‐2‐sm9 revealed the effects of G245D‐actin in a cellular environment. The overall actin levels in Rat‐2‐sm9 show a 1.6‐fold increase with similar amounts of mutant and wild‐type actin. G245D‐actin expression renders Rat‐2‐sm9 cells highly tumorigenic in nude mice. In Rat‐2‐sm9 monolayers, G245D‐actin triggers the formation of extensive membrane ruffles, which is a characteristic feature of many transformed cells. To approximate complex cell–cell and cell‐matrix interactions that occur in tumors and might modulate the effects of G245D‐actin, we extended our studies to scaffold‐free 3D spheroid cultures. Bright field and scanning electron microscopy (SEM) show that Rat‐2‐sm9 and Rat‐2 cells share essential features of spheroid formation and compaction. However, the resulting spheroids exhibit distinct phenotypes that differ mainly in surface structure and size. The systematic comparison of transformed and normal spheroids by SEM provides new insights into scaffold‐free fibroblast spheroid formation.

HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.