Approaches to Determine Nuclear Shape in Cells During Migration Through Collagen Matrices.

Fibrillar collagen is an abundant extracellular matrix (ECM) component of interstitial tissues which supports the structure of many organs, including the skin and breast. Many different physiological processes, but also pathological processes such as metastatic cancer invasion, involve interstitial cell migration. Often, cell movement takes place through small ECM gaps and pores and depends upon the ability of the cell and its stiff nucleus to deform. Such nuclear deformation during cell migration may impact nuclear integrity, such as of chromatin or the nuclear envelope, and therefore the morphometric analysis of nuclear shapes can provide valuable insight into a broad variety of biological processes. Here, we describe a protocol on how to generate a cell-collagen model in vitro and how to use confocal microscopy for the static and dynamic visualization of labeled nuclei in single migratory cells. We developed, and here provide, two scripts that (Fidler, Nat Rev Cancer 3(6):453-458, 2003) enable the semi-automated and fast quantification of static single nuclear shape descriptors, such as aspect ratio or circularity, and the nuclear irregularity index that forms a combination of four distinct shape descriptors, as well as (Frantz et al., J Cell Sci 123 (Pt 24):4195-4200, 2010) a quantification of their changes over time. Finally, we provide quantitative measurements on nuclear shapes from cells that migrated through collagen either in the presence or the absence of an inhibitor of collagen degradation, showing the distinctive power of this approach. This pipeline can also be applied to cell migration studied in different assays, ranging from 3D microfluidics to migration in the living organism.

Actomyosin contractility requirements and reciprocal cell-tissue mechanics for cancer cell invasion through collagen-based channels.

The interstitial tumor microenvironment is composed of heterogeneously organized collagen-rich porous networks as well as channel-like structures and interfaces which provide both barriers and guidance for invading cells. Tumor cells invading 3D random porous collagen networks depend upon actomyosin contractility to deform and translocate the nucleus, whereas Rho/Rho-associated kinase-dependent contractility is largely dispensable for migration in stiff capillary-like confining microtracks. To investigate whether this dichotomy of actomyosin contractility dependence also applies to physiological, deformable linear collagen environments, we developed nearly barrier-free collagen-scaffold microtracks of varying cross section using two-photon laser ablation. Both very narrow and wide tracks supported single-cell migration by either outward pushing of collagen up to four times when tracks were narrow, or cell pulling on collagen walls down to 50% of the original diameter by traction forces of up to 40 nN when tracks were wide, resulting in track widths optimized to single-cell diameter. Targeting actomyosin contractility by synthetic inhibitors increased cell elongation and nuclear shape change in narrow tracks and abolished cell-mediated deformation of both wide and narrow tracks. Accordingly, migration speeds in all channel widths reduced, with migration rates of around 45-65% of the original speed persisting. Together, the data suggest that cells engage actomyosin contraction to reciprocally adjust both own morphology and linear track width to optimal size for effective cellular locomotion.

ABCB1 as a potential beneficial target of midostaurin in acute myeloid leukemia.

Low curability of patients diagnosed with acute myeloid leukemia (AML) must be seen as a call for better understanding the disease's mechanisms and improving the treatment strategy. Therapeutic outcome of the crucial anthracycline-based induction therapy often can be compromised by a resistant phenotype associated with overexpression of ABCB1 transporters. Here, we evaluated clinical relevance of ABCB1 in a context of the FMS-like tyrosine kinase 3 (FLT3) inhibitor midostaurin in a set of 28 primary AML samples. ABCB1 gene expression was absolutely quantified, confirming its association with CD34 positivity, adverse cytogenetic risk, and unachieved complete remission (CR). Midostaurin, identified as an ABCB1 inhibitor, increased anthracycline accumulation in peripheral blood mononuclear cells (PBMC) of CD34+ AML patients and those not achieving CR. This effect was independent of FLT3 mutation, indicating even FLT3- AML patients might benefit from midostaurin therapy. In line with these data, midostaurin potentiated proapoptotic processes in ABCB1-overexpressing leukemic cells when combined with anthracyclines. Furthermore, we report a direct linkage of miR-9 to ABCB1 efflux activity in the PBMC and propose miR-9 as a useful prognostic marker in AML. Overall, we highlight the therapeutic value of midostaurin as more than just a FLT3 inhibitor, suggesting its maximal therapeutic outcomes might be very sensitive to proper timing and well-optimized dosage schemes based upon patient's characteristics, such as CD34 positivity and ABCB1 activity. Moreover, we suggest miR-9 as a predictive ABCB1-related biomarker that could be immensely helpful in identifying ABCB1-resistant AML phenotype to enable optimized therapeutic regimen and improved treatment outcome.