Tissue Architectural Cues Drive Organ Targeting of Tumor Cells in Zebrafish.

Tumor cells encounter a myriad of physical cues upon arrest and extravasation in capillary beds. Here, we examined the role of physical factors in non-random organ colonization using a zebrafish xenograft model. We observed a two-step process by which mammalian mammary tumor cells showed non-random organ colonization. Initial homing was driven by vessel architecture, where greater numbers of cells became arrested in the topographically disordered blood vessels of the caudal vascular plexus (CVP) than in the linear vessels in the brain. Following arrest, bone-marrow- and brain-tropic clones exhibited organ-specific patterns of extravasation. Extravasation was mediated by ß1 integrin, where knockdown of ß1 integrin reduced extravasation in the CVP but did not affect extravasation of a brain-tropic clone in the brain. In contrast, silencing myosin 1B redirected early colonization from the brain to the CVP. Our results suggest that organ selectivity is driven by both vessel topography and cell-type-dependent extravasation.

Human macrophages survive and adopt activated genotypes in living zebrafish.

The inflammatory response, modulated both by tissue resident macrophages and recruited monocytes from peripheral blood, plays a critical role in human diseases such as cancer and neurodegenerative disorders. Here, we sought a model to interrogate human immune behavior in vivo. We determined that primary human monocytes and macrophages survive in zebrafish for up to two weeks. Flow cytometry revealed that human monocytes cultured at the physiological temperature of the zebrafish survive and differentiate comparable to cohorts cultured at human physiological temperature. Moreover, key genes that encode for proteins that play a role in tissue remodeling were also expressed. Human cells migrated within multiple tissues at speeds comparable to zebrafish macrophages. Analysis of gene expression of in vivo educated human macrophages confirmed expression of activated macrophage phenotypes. Here, human cells adopted phenotypes relevant to cancer progression, suggesting that we can define the real time immune modulation of human tumor cells during the establishment of a metastatic lesion in zebrafish.

Mechanical properties of the tumor stromal microenvironment probed in vitro and ex vivo by in situ-calibrated optical trap-based active microrheology.

One of the hallmarks of the malignant transformation of epithelial tissue is the modulation of stromal components of the microenvironment. In particular, aberrant extracellular matrix (ECM) remodeling and stiffening enhances tumor growth and survival and promotes metastasis. Type I collagen is one of the major ECM components. It serves as a scaffold protein in the stroma contributing to the tissue's mechanical properties, imparting tensile strength and rigidity to tissues such as those of the skin, tendons, and lungs. Here we investigate the effects of intrinsic spatial heterogeneities due to fibrillar architecture, pore size and ligand density on the microscale and bulk mechanical properties of the ECM. Type I collagen hydrogels with topologies tuned by polymerization temperature and concentration to mimic physico-chemical properties of a normal tissue and tumor microenvironment were measured by in situ-calibrated Active Microrheology by Optical Trapping revealing significantly different microscale complex shear moduli at Hz-kHz frequencies and two orders of magnitude of strain amplitude that we compared to data from bulk rheology measurements. Access to higher frequencies enabled observation of transitions from elastic to viscous behavior that occur at ~200Hz to 2750Hz, which largely was dependent on tissue architecture well outside the dynamic range of instrument acquisition possible with SAOS bulk rheology. We determined that mouse melanoma tumors and human breast tumors displayed complex moduli ~5-1000 Pa, increasing with frequency and displaying a nonlinear stress-strain response. Thus, we show the feasibility of a mechanical biopsy in efforts to provide a diagnostic tool to aid in the design of therapeutics complementary to those based on standard histopathology.

Mitogen-activated protein kinase signaling causes malignant melanoma cells to differentially alter extracellular matrix biosynthesis to promote cell survival.

BACKGROUND: Intrinsic and acquired resistance to drug therapies remains a challenge for malignant melanoma patients. Intratumoral heterogeneities within the tumor microenvironment contribute additional complexity to the determinants of drug efficacy and acquired resistance. METHODS: We use 3D biomimetic platforms to understand dynamics in extracellular matrix (ECM) biogenesis following pharmaceutical intervention against mitogen-activated protein kinases (MAPK) signaling. We further determined temporal evolution of secreted ECM components by isogenic melanoma cell clones. RESULTS: We found that the cell clones differentially secrete and assemble a myriad of ECM molecules into dense fibrillar and globular networks. We show that cells can modulate their ECM biosynthesis in response to external insults. Fibronectin (FN) is one of the key architectural components, modulating the efficacy of a broad spectrum of drug therapies. Stable cell lines engineered to secrete minimal levels of FN showed a concomitant increase in secretion of Tenascin-C and became sensitive to BRAF(V600E) and ERK inhibition as clonally- derived 3D tumor aggregates. These cells failed to assemble exogenous FN despite maintaining the integrin machinery to facilitate cell- ECM cross-talk. We determined that only clones that increased FN production via p38 MAPK and ß1 integrin survived drug treatment. CONCLUSIONS: These data suggest that tumor cells engineer drug resistance by altering their ECM biosynthesis. Therefore, drug treatment may induce ECM biosynthesis, contributing to de novo resistance.

In vivo tissue has non-linear rheological behavior distinct from 3D biomimetic hydrogels, as determined by AMOTIV microscopy.

Variation in matrix elasticity has been shown to determine cell fate in both differentiation and development of malignant phenotype. The tissue microenvironment provides complex biochemical and biophysical signals in part due to the architectural heterogeneities found in extracellular matrices (ECMs). Three dimensional cell cultures can partially mimic in vivo tissue architecture, but to truly understand the role of viscoelasticity on cell fate, we must first determine in vivo tissue mechanical properties to improve in vitro models. We employed Active Microrheology by Optical Trapping InVivo (AMOTIV), using in situ calibration to measure in vivo zebrafish tissue mechanics. Previously used trap calibration methods overestimate complex moduli by ∼ 2-20 fold compared to AMOTIV. Applying differential microscale stresses and strains showed that hyaluronic acid (HA) gels display semi-flexible polymer behavior, while laminin-rich ECM hydrogels display flexible polymer behavior. In contrast, zebrafish tissues displayed different moduli at different stresses, with higher power law exponents at lower stresses, indicating that living tissue has greater stress dependence than the 3D hydrogels examined. To our knowledge, this work is the first vertebrate tissue rheological characterization performed in vivo. Our fundamental observations are important for the development and refinement of in vitro platforms.

Antiproliferative activity of novel imidazopyridine derivatives on castration-resistant human prostate cancer cells.

Metastatic prostate cancer (mPCa) relapses after a short period of androgen deprivation therapy and becomes the castration-resistant prostate cancer (CR PCa); to which the treatment is limited. Hence, it is imperative to identify novel therapeutic agents towards this patient population. In the present study, antiproliferative activities of novel imidazopyridines were compared. Among three derivatives, PHE, AMD and AMN, examined, AMD showed the highest inhibitory activity on LNCaP C-81 cell proliferation, following dose- and time-dependent manner. Additionally, AMD exhibited significant antiproliferative effect against a panel of PCa cells, but not normal prostate epithelial cells. Further, when compared to AMD, its derivative DME showed higher inhibitory activities on PCa cell proliferation, clonogenic potential and in vitro tumorigenicity. The inhibitory activity was apparently in part due to the induction of apoptosis. Mechanistic studies indicate that AMD and DME treatments inhibited both AR and PI3K/Akt signaling. The results suggest that better understanding of inhibitory mechanisms of AMD and DME could help design novel therapeutic agents for improving the treatment of CR PCa.