Stephanie Alexander

Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force

The physical limits of cell migration in dense porous environments are dependent upon the available space and the deformability of the nucleus and are modulated by matrix metalloproteinases, integrins and actomyosin function.

Collagen-based cell migration models in vitro and in vivo

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.

Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model.

The metastatic invasion of cancer cells from the primary lesion into the adjacent stroma is a key step in cancer progression, and is associated with poor outcome. The principles of cancer invasion have been experimentally addressed in various in vitro models; however, key steps and mechanisms in vivo remain unclear. Here, we establish a modified skin-fold chamber model for orthotopic implantation, growth and invasion of human HT-1080 fibrosarcoma cells, dynamically reconstructed by epifluorescence and multiphoton microscopy. This strategy allows repeated imaging of tumor growth, tumor-induced angiogenesis and invasion, as either individual cells, or collective strands and cell masses that move along collagen-rich extracellular matrix and coopt host tissue including striated muscle strands and lymph vessels. This modified window model will be suited to address mechanisms of cancer invasion and metastasis, and related experimental therapy.

Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging.

Multiphoton microscopy (MPM) is the method of choice for investigating cells and cellular functions in deep tissue sections and organs. Here we present the setup and applications of infrared-(IR-)MPM using excitation wavelengths above 1080 nm. IR-MPM enables the use of red fluorophores and fluorescent proteins, doubles imaging depth, improves second harmonic generation of tissue structures, and strongly reduces phototoxicity and photobleaching, compared with conventional MPM. Furthermore, it still provides subcellular resolution at depths of several hundred micrometers and thus will enhance long-term live cell and deep tissue microscopy.

Cancer invasion and resistance: interconnected processes of disease progression and therapy failure

Cancer progression and outcome depend upon two key functions executed by tumor cells: the growth and survival capability leading to resistance to therapy and the invasion into host tissues resulting in local and metastatic dissemination. Although both processes are widely studied separately, the underlying cell-intrinsic and microenvironmentally controlled signaling pathways reveal substantial overlap in mechanism. Candidate signaling hubs that serve both tumor invasion and resistance include growth factor and chemokine signaling, integrin engagement, and components of the Ras/MAPKs, PI3K, and mTOR signaling pathways. In this review, we summarize these and other mechanisms controlled by the microenvironment that jointly support cancer cell survival and resistance, as well as the invasion machinery. We also discuss their interdependencies and the implications for therapeutic dual- or multi-pathway targeting.

Examination of the foreign body response to biomaterials by nonlinear intravital microscopy

Implanted biomaterials often fail because they elicit a foreign body response (FBR) and concomitant fibrotic encapsulation. To design clinically relevant interference approaches, it is crucial to first examine the FBR mechanisms. Here, we report the development and validation of infrared-excited nonlinear microscopy to resolve the three-dimensional (3D) organization and fate of 3D-electrospun scaffolds implanted deep into the skin of mice, and the following step-wise FBR process. We observed that immigrating myeloid cells (predominantly macrophages of the M1 type) engaged and became immobilized along the scaffold/tissue interface, before forming multinucleated giant cells. Both macrophages and giant cells locally produced vascular endothelial growth factor (VEGF), which initiated and maintained an immature neovessel network, followed by formation of a dense collagen capsule 2–4 weeks post-implantation. Elimination of the macrophage/giant-cell compartment by clodronate and/or neutralization of VEGF by VEGF Trap significantly diminished giant-cell accumulation, neovascularization and fibrosis. Our findings identify macrophages and giant cells as incendiaries of the fibrotic encapsulation of engrafted biomaterials via VEGF release and neovascularization, and therefore as targets for therapy.

Genomic instability of micronucleated cells revealed by single-cell comparative genomic hybridization.

Nuclear variation in size and shape and genomic instability are hallmarks of dedifferentiated cancer cells. Although micronuclei are a typical long‐term consequence of DNA damage, their contribution to chromosomal instability and clonal diversity in cancer disease is unclear. We isolated cancer cells with or without micronuclei to perform genomic analysis. Cell suspensions of HT1080 fibrosarcoma cells from either 2D culture or after isolation from 3D collagen matrix culture were stained with Hoechst 33342 and after classification for presence or absence of a micronucleus via bright‐field and epifluorescence microscopy, cells were individually aspirated with a micropipette. Subsequently, whole‐genome amplification and single‐cell comparative genomic hybridization (CGH) were applied to detect genomic aberrations. The data show a high‐fidelity isolation and genome amplification that lacks adverse effects by prior Hoechst 33342 staining. HT1080 cells showed a high degree of divergent amplifications, but neither location nor frequency of aberrations was dependent on 2D or 3D culture conditions or micronucleation. Thus, single‐cell selection of defined nuclear states is amenable to single‐cell CGH and here provides first insight into the aberration drift and genomic diversity in cancer cells with and without micronuclei.

Correlative live and super-resolution imaging reveals the dynamic structure of replication domains

Chromosome organization in higher eukaryotes controls gene expression, DNA replication, and DNA repair. Genome mapping has revealed the functional units of chromatin at the submegabase scale as self-interacting regions called topologically associating domains (TADs) and showed they correspond to replication domains (RDs). A quantitative structural and dynamic description of RD behavior in the nucleus is, however, missing because visualization of dynamic subdiffraction-sized RDs remains challenging. Using fluorescence labeling of RDs combined with correlative live and super-resolution microscopy in situ, we determined biophysical parameters to characterize the internal organization, spacing, and mechanical coupling of RDs. We found that RDs are typically 150 nm in size and contain four co-replicating regions spaced 60 nm apart. Spatially neighboring RDs are spaced 300 nm apart and connected by highly flexible linker regions that couple their motion only <550 nm. Our pipeline allows a robust quantitative characterization of chromosome structure in situ and provides important biophysical parameters to understand general principles of chromatin organization.

Abstract 4941: A humanized bone model for preclinical monitoring of prostate cancer lesions by intravital multiphoton microscopy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Bone metastases are the initial site of progression and account for many of the complications experienced by men with metastatic prostate cancer (PCa), including resistance to therapy. The purpose of our study is to determine whether intravital multiphoton microscopy (iMPM) integrated with a novel mouse model can better inform clinical development of molecular therapeutics targeting bone metastases. iMPM can overcome limitations of existing models by monitoring the dynamic reciprocal cell-cell interplay at the center of PCa bone metastasis. To study PCa-stromal cell interactions and therapy response in bone, we established a humanized neo-bone in the mouse dermis, using grafted electrospun polycaprolactone scaffolds functionalized with human mesenchymal stem cells differentiated to osteoblasts. After in vivo maturation, bone scaffolds served for co-implantation of human fluorescent PCa cells (PC3) followed by multi-parameter iMPM through a body window, including: collagen and bone matrix (second harmonic generation), mineralized matrix (calcein blue), osteoblasts (H2B/mCherry in human mesenchymal stem cells), adipocytes and bone surface (third harmonic generation), blood vessels, stromal phagocytes and osteoclasts (fluorescence-labeled dextran), and PC3 cells (nuclear H2B eGFP with cytoplasmatic DsRed2). Using multi-parameter 3D reconstruction, tumor growth and invasion, reactive remodeling of the stroma and neovessel organization were monitored longitudinally, providing a baseline for evaluating preclinical disease and therapy response. The model provides new insight into PCa bone metastases and will be used to more efficiently develop and prioritize biologically founded therapy for clinical development. Citation Format: Eleonora Dondossola, Stephanie Alexander, Steve Alexander, Boris M. Holzapfel, Christopher J. Logothetis, Dietmar W. Hutmacher, Peter Friedl. A humanized bone model for preclinical monitoring of prostate cancer lesions by intravital multiphoton microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4941. doi:10.1158/1538-7445.AM2014-4941

Cancer invasion and resistance

Preclinical microscopy has greatly enhanced our mechanistic understanding of cancer invasion and metastasis, the contribution of the tumour microenvironment to metastatic progression, and how invasion and the microenvironment jointly support cancer cell survival and resistance. Using organotypic models in vitro, live-cell imaging in three-dimensional (3D) tissue culture has identified how cytoskeletal, adhesion and protease systems drive invasion and metastasis [1]. When altered at the molecular level, these pathways underlie the unexpected diversity of the invasive process [2]. The recent use of intravital microscopy has further suggested that cancer invasion into interstitial stroma in vivo: (1) occurs mostly as collective invasion in which cells remain coupled to neighbouring cancer cells, (2) is guided by and responsive to signals delivered by connective tissue structures and (3) that invasion pathways cross-talk with pathways of cancer cell survival and resistance to anticancer therapy [3].