In this work, we constructed a Collagen I-Matrigel composite extracellular matrix (ECM). The composite ECM was used to determine the influence of the local collagen fiber orientation on the collective intravasation ability of tumor cells. We found that the local fiber alignment enhanced cell-ECM interactions. Specifically, metastatic MDA-MB-231 breast cancer cells followed the local fiber alignment direction during the intravasation into rigid Matrigel (∼10 mg/mL protein concentration).M etastasis is a lethal milestone in cancer: Cells escape from the confinement of primary tumor sites (intravasation), invade tissues as well as the lymphatic and vascular systems, and finally colonize (extravasation) distant sites. It has been estimated that less than 1% of tumor cells undergo this process, but metastasis contributes to more than 90% of cancerrelated deaths (1, 2). Metastasis involves both genetic and epigenetic alternation of tumor cells, as well as external biochemical and biophysical microenvironments (3-5). Pathology studies suggest that metastatic tumor cells exhibit highly branched morphologies and distinct aligned registration with aligned extracellular matrix (ECM) during metastatic tumor progression (4, 5).We address three important questions concerning metastasis. (i) Can we build in vitro complex ECM structures with heterogeneously oriented collagen fibers and basement membrane components to mimic the cancer cell intravasation process? (ii) How does aligned collagen influence cell intravasation into/ through the basement membrane before entering vessels? (iii) After cell detachment from the primary tumor site, how does a heterogeneous ECM with a varying degree of local fiber alignment influence cell intravasation and subsequent penetration into the basement membrane during their intravasation process? The major obstacle to addressing these questions is the difficulty in constructing both an in vitro 3D microenvironment to mimic the above process and flexible controls of the environmental parameters, such as fiber orientations in a complex collagen/Matrigel composite, nutrition, oxygen, drug concentrations, etc.In breast cancer metastasis, cancer cells are believed to reorganize and progress through the interstitial ECM matrix, break through the basement membrane, and enter blood vessels or lymphatic capillaries (6-10). Fig. 1C presents a schematic illustration of the intravasation process in metastasis. Tumor-associated collagen signatures (TACS), basically environmentally elevated collagen density and collagen fiber reorganization, are used to stage mammary carcinoma tumor progression levels (6, 11-13). Fig. 1 presents hematoxylin/eosin (H&E)-stained biopsy slices of breast cancer imaged by second harmonic generation (SHG) under a two-photon confocal microscopy (A1R MP; Nikon) (detailed information provided in SI Appendix, SI Text) (6, 14, 15). Fig. 1 A, 1-3 shows the stained human invasive ductal carcinoma tumor at grade I. In the enlarged figures (Fig. 1 A, 2 and 3), the cells have well-defined bord...

Clinical pulmonary surfactant is routinely used to treat premature newborns with respiratory distress syndrome, and has shown great potential in alleviating a number of neonatal and adult respiratory diseases. Despite extensive study of chemical composition, surface activity, and clinical performance of various surfactant preparations, a direct comparison of surfactant films is still lacking. In this study, we use atomic force microscopy to characterize and compare four animal-derived clinical surfactants currently used throughout the world, i.e., Survanta, Curosurf, Infasurf and BLES. These modified-natural surfactants are further compared to dipalmitoyl phosphatidylcholine (DPPC), a synthetic model surfactant of DPPC:palmitoyl-oleoyl phosphatidylglycerol (POPG) (7:3), and endogenous bovine natural surfactant. Atomic force microscopy reveals significant differences in the lateral structure and molecular organization of these surfactant preparations. These differences are discussed in terms of DPPC and cholesterol contents. We conclude that all animal-derived clinical surfactants assume a similar structure of multilayers of fluid phospholipids closely attached to an interfacial monolayer enriched in DPPC, at physiologically relevant surface pressures. This study provides the first comprehensive survey of the lateral structure of clinical surfactants at various surface pressures. It may have clinical implications on future application and development of surfactant preparations.

Interaction with the pulmonary surfactant film, being the first line of host defense, represents the initial bio-nano interaction in the lungs. Such interaction determines the fate of the inhaled nanoparticles and their potential therapeutic or toxicological effect. Despite considerable progress in optimizing physicochemical properties of nanoparticles for improved delivery and targeting, the mechanisms by which inhaled nanoparticles interact with the pulmonary surfactant film are still largely unknown. Here, using combined in vitro and in silico methods, we show how hydrophobicity and surface charge of nanoparticles differentially regulate the translocation and interaction with the pulmonary surfactant film. While hydrophilic nanoparticles generally translocate quickly across the pulmonary surfactant film, a significant portion of hydrophobic nanoparticles are trapped by the surfactant film and encapsulated in lipid protrusions upon film compression. Our results support a novel model of pulmonary surfactant lipoprotein corona associated with inhaled nanoparticles of different physicochemical properties. Our data suggest that the study of pulmonary nanotoxicology and nanoparticle-based pulmonary drug delivery should consider this lipoprotein corona.

Inhaled nanoparticles (NPs) must first interact with the pulmonary surfactant (PS) lining layer that covers the entire internal surface of the respiratory tract and plays an important role in surface tension reduction and host defense. Interactions with the PS film determine the subsequent clearance, retention, and translocation of the inhaled NPs and hence their potential toxicity. To date, little is known how NPs interact with PS, and whether or not NPs have adverse effects on the biophysical function of PS. We found a time-dependent toxicological effect of hydroxyapatite NPs (HA-NPs) on a natural PS, Infasurf, and the time scale of surfactant inhibition after particle exposure was comparable to the turnover period of surfactant metabolism. Using a variety of in vitro biophysicochemical characterization techniques, we have determined the inhibition mechanism to be due to protein adsorption onto the HA-NPs. Consequently, depletion of surfactant proteins from phospholipid vesicles caused conversion of original large vesicles into much smaller vesicles with poor surface activity. These small vesicles, in turn, inhibited biophysical function of surfactant films after adsorption at the air–water interface. Cytotoxicity study found that the HA-NPs at the studied concentration were benign to human bronchial epithelial cells, thereby highlighting the importance of evaluating biophysical effect of NPs on PS. The NP–PS interaction mechanism revealed by this study may not only provide new insight into the toxicological study of nanoparticles but also shed light on the feasibility of NP-based pulmonary drug delivery.

Natural lung surfactant contains less than 40% disaturated phospholipids, mainly dipalmitoylphosphatidylcholine (DPPC). The mechanism by which lung surfactant achieves very low near-zero surface tensions, well below its equilibrium value, is not fully understood. To date, the low surface tension of lung surfactant is usually explained by a squeeze-out model which predicts that upon film compression non-DPPC components are gradually excluded from the air–water interface into a surface-associated surfactant reservoir. However, detailed experimental evidence of the squeeze-out within the physiologically relevant high surface pressure range is still lacking. In the present work, we studied four animal-derived clinical surfactant preparations, including Survanta, Curosurf, Infasurf, and BLES. By comparing compression isotherms and lateral structures of these surfactant films obtained by atomic force microscopy within the physiologically relevant high surface pressure range, we have derived an updated squeeze-out model. Our model suggests that the squeeze-out originates from fluid phases of a phase-separated monolayer. The squeeze-out process follows a nucleation–growth model and only occurs within a narrow surface pressure range around the equilibrium spreading pressure of lung surfactant. After the squeeze-out, three-dimensional nuclei stop growing, thereby resulting in a DPPC-enriched interfacial monolayer to reduce the air–water surface tension to very low values.