Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or ...perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.
Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, ...making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer.
Murine 4T1 (metastatic) and 4T07 (poorly/non-metastatic) orthotopic triple negative breast cancer tumours were collected after 7, 14, or 21 days. The tumours were analysed via flow cytometry for the simultaneous expression of six CAF markers: alpha smooth muscle actin (αSMA), fibroblast activation protein alpha (FAPα), platelet derived growth factor receptor alpha and beta (PDGFRα and PDGFRβ), CD26/DPP4 and podoplanin (PDPN). All non-CAFs were excluded from the analysis using a lineage marker cocktail (CD24, CD31, CD45, CD49f, EpCAM, LYVE-1, and TER-119). In total 128 murine tumours and 12 healthy mammary fat pads were analysed.
We have developed a multicolour flow cytometry strategy based on exclusion of non-CAFs and successfully employed this to explore the temporal heterogeneity of freshly isolated CAFs in the 4T1 and 4T07 mouse models of triple-negative breast cancer. Analysing 128 murine tumours, we identified 5-6 main CAF populations and numerous minor ones based on the analysis of αSMA, FAPα, PDGFRα, PDGFRβ, CD26, and PDPN. All markers showed temporal changes with a distinct switch from primarily PDGFRα+ fibroblasts in healthy mammary tissue to predominantly PDGFRβ+ CAFs in tumours. CD26+ CAFs emerged as a large novel subpopulation, only matched by FAPα+ CAFs in abundance.
We demonstrate that multiple subpopulations of CAFs co-exist in murine triple negative breast cancer, and that the abundance and dynamics for each marker differ depending on tumour type and time. Our results form the foundation needed to isolate and characterise specific CAF populations, and ultimately provide an opportunity to therapeutically target specific CAF subpopulations.
Metastatic complications are responsible for more than 90% of cancer-related deaths. The progression from an isolated tumor to disseminated metastatic disease is a multistep process, with each step ...involving intricate cross talk between the cancer cells and their non-cellular surroundings, the extracellular matrix (ECM). Many ECM proteins are significantly deregulated during the progression of cancer, causing both biochemical and biomechanical changes that together promote the metastatic cascade. In this review, the influence of several ECM proteins on these multiple steps of cancer spread is summarized. In addition, we highlight the promising (pre-)clinical data showing benefits of targeting these ECM macromolecules to prevent cancer progression.
Increased tissue stiffness is a classic characteristic of solid tumors. One of the major contributing factors is increased density of collagen fibers in the extracellular matrix (ECM). Here, we ...investigate how cancer cells biomechanically interact with and respond to the stiffness of the ECM. Probing the adaptability of cancer cells to altered ECM stiffness using optical tweezers-based microrheology and deformability cytometry, we find that only malignant cancer cells have the ability to adjust to collagen matrices of different densities. Employing microrheology on the biologically relevant spheroid invasion assay, we can furthermore demonstrate that, even within a cluster of cells of similar origin, there are differences in the intracellular biomechanical properties dependent on the cells' invasive behavior. We reveal a consistent increase of viscosity in cancer cells leading the invasion into the collagen matrices in comparison with cancer cells following in the stalk or remaining in the center of the spheroid. We hypothesize that this differential viscoelasticity might facilitate spheroid tip invasion through a dense matrix. These findings highlight the importance of the biomechanical interplay between cells and their microenvironment for tumor progression.
The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is ...a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.
Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development ...and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform.
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