Not all (cells) who wander are lost: Upstream migration as a pervasive mode of amoeboid cell motility.

Leukocytes possess the ability to migrate upstream-against the direction of flow-on surfaces of specific chemistry. Upstream migration was first characterized in vitro for T-cells on surfaces comprised of intracellular adhesion molecule-1 (ICAM-1). Upstream migration occurs when the integrin receptor αLß2 (also known as lymphocyte function-associated antigen-1, or LFA-1) binds to ICAM-1. LFA-1/ICAM-1 interactions are ubiquitous and are widely found in leukocyte trafficking. Upstream migration would be employed after cells come to arrest on the apical surface of the endothelium and might confer an advantage for both trans-endothelial migration and tissue surveillance. It has now been shown that several other motile amoeboid cells which have the responsibility of trafficking from blood vessels into tissues, such as Marginal zone B cells, hematopoietic stem cells, and neutrophils (when macrophage-1 antigen, Mac-1, is blocked), can also migrate upstream on ICAM-1 surfaces. This review will summarize what is known about the basic mechanisms of upstream migration, which cells have displayed this phenomenon, and the possible role of upstream migration in physiology and tissue homeostasis.

Detergent-Free Decellularization Preserves the Mechanical and Biological Integrity of Murine Tendon.

Tissue decellularization has demonstrated widespread applications across numerous organ systems for tissue engineering and regenerative medicine applications. Decellularized tissues are expected to retain structural and/or compositional features of the natural extracellular matrix (ECM), enabling investigation of biochemical factors and cell-ECM interactions that drive tissue homeostasis, healing, and disease. However, the dense collagenous tendon matrix has limited the efficacy of traditional decellularization strategies without the aid of harsh chemical detergents and/or physical agitation that disrupt tissue integrity and denature proteins involved in regulating cell behavior. In this study, we adapted and established the advantages of a detergent-free decellularization method that relies on latrunculin B actin destabilization, alternating hypertonic-hypotonic salt and water incubations, nuclease-assisted elimination of cellular material, and protease inhibitor supplementation under aseptic conditions. Our method maintained the collagen molecular structure (i.e., minimal extent of denaturation), while adequately removing cells and preserving bulk mechanical properties. Furthermore, we demonstrated that decellularized tendon ECM-derived coatings isolated from different mouse strains, injury states (i.e., naive and acutely injured/"provisional"), and anatomical sites harness distinct biochemical cues and robustly maintain tendon cell viability in vitro. Together, our work provides a simple and scalable decellularization method to facilitate mechanistic studies that will expand our fundamental understanding of tendon ECM and cell biology. Impact statement In this study, we present a decellularization method for tendon that does not rely on any detergent or physical processing techniques. We assessed the impact of detergent-free decellularization using tissue, cellular, and molecular level analyses and validated the preservation of gross fiber architecture, collagen molecular structure, and extracellular matrix (ECM)-associated biological cues that are essential for studying physiological cell-ECM interactions. Finally, we demonstrated the applicability of this method for healthy and injured tendon environments, across mouse strains, and for different types of tendons, illustrating the utility of this approach for isolating the contributions of biochemical cues within unique tendon ECM microenvironments.

Pitfalls on sample preparation for ex vivo imaging of resected cancer tissue using enzyme-activatable fluorescent probes.

In vivo and ex vivo fluorescence imaging-assisted surgery can aid in determining the margins of tumors during surgical resection. While a variety of fluorescent probes have been proposed for this task, small molecule enzyme-activatable fluorescent probes are ideal for this application. They are quickly activated at tumor sites and result in bright signal with little background, resulting in high sensitivity. Testing in resected specimens, however, can be difficult. Enzymes are usually stable after freezing and thawing but catalytic reactions are generally temperature-dependent. Therefore, tissue sample temperature should be carefully considered. In this study two enzyme activatable probes, γ-glutamylhydroxymethyl rhodamine green (gGlu-HMRG) that reacted with γ-glutamyltransferase and SPiDER-ßGal that reacted with ß-galactosidase, were employed to determine the effects of temperature on fluorescence signal kinetics in both fresh and frozen and then thawed ex vivo experimental ovarian cancer tissue samples. The results suggest γ-glutamyltransferase was less sensitive to temperature than ß-galactosidase. Fresh samples showed higher fluorescence signals of gGlu-HMRG compared with thawed samples likely because the freeze-thaw cycle decreased the rate of internalization of the activated probe into the lysosome. In contrast, no significant difference of SPiDER-ßGal fluorescence signal was observed between fresh and frozen tissues. In conclusion, although imaging of fresh samples at 37°C is the best condition for both probes, successful imaging with gGlu-HMRG could be achieved even at room temperature with thawed samples. We demonstrate that temperature regulation and tissue handling of resected tissue are two pitfalls that may influence ex vivo imaging signals with enzyme-activatable fluorescent probes.

Activatable fluorescent probes in fluorescence-guided surgery: Practical considerations.

Fluorescence-guided imaging during surgery is a promising technique that is increasingly used to aid surgeons in identifying sites of tumor and surgical margins. Of the two types of fluorescent probes, always-on and activatable, activatable probes are preferred because they produce higher target-to-background ratios, thus improving sensitivity compared with always-on probes that must contend with considerable background signal. There are two types of activatable probes: 1) enzyme-reactive probes that are normally quenched but can be activated after cleavage by cancer-specific enzymes (activity-based probes) and 2) molecular-binding probes which use cancer targeting moieties such as monoclonal antibodies to target receptors found in abundance on cancers and are activated after internalization and lysosomal processing (binding-based probes). For fluorescence-guided intraoperative surgery, enzyme-reactive probes are superior because they can react quickly, require smaller dosages especially for topical applications, have limited side effects, and have favorable pharmacokinetics. Enzyme-reactive probes are easier to use, fit better into existing work flows in the operating room and have minimal toxicity. Although difficult to prove, it is assumed that the guidance provided to surgeons by these probes results in more effective surgeries with better outcomes for patients. In this review, we compare these two types of activatable fluorescent probes for their ease of use and efficacy.

A topically-sprayable, activatable fluorescent and retaining probe, SPiDER-ßGal for detecting cancer: Advantages of anchoring to cellular proteins after activation.

SPiDER-ßGal is a newly-developed probe that is activated by ß-galactosidase and is then retained within cells by anchoring to intracellular proteins. Previous work has focused on gGlu-HMRG, a probe activated by γ-glutamyltranspeptidase, which demonstrated high sensitivity for the detection of peritoneal ovarian cancer metastases in an animal model. However, its fluorescence, after activation by γ-glutamyltranspeptidase, rapidly declines over time, limiting the actual imaging window and the ability to define the border of lesions. The purpose of this study is to compare the fluorescence signal kinetics of SPiDER-ßGal with that of gGlu-HMRG using ovarian cancer cell lines in vitro and ex vivo tissue imaging. In vitro removal of gGlu-HMRG resulted in a rapid decrease of fluorescence intensity followed by a more gradual decrease up to 60 min while there was a gradual increase in fluorescence up to 60 min after removal of SPiDER-ßGal. This is most likely due to internalization and retention of the dye within cells. This was also confirmed ex vivo tissue imaging using a red fluorescence protein (RFP)-labeled tumor model in which the intensity of fluorescence increased gradually after activation of SPiDER-ßGal. Additionally, SPiDER-ßGal resulted in intense enhancement within the tumor due to the high target-to-background ratio, which extended up to 60 min after activation. In contrast, gGlu-HMRG fluorescence resulted in decreasing fluorescence over time in extracted tumors. Thus, SPiDER-ßGal has the advantages of higher signal with more signal retention, resulting in improved contrast of the tumor margin and suggesting it may be an alternative to existing activatable probes.

Nanodrug Delivery: Is the Enhanced Permeability and Retention Effect Sufficient for Curing Cancer?

Nanotechnology offers several attractive design features that have prompted its exploration for cancer diagnosis and treatment. Nanosized drugs have a large loading capacity, the ability to protect the payload from degradation, a large surface on which to conjugate targeting ligands, and controlled or sustained release. Nanosized drugs also leak preferentially into tumor tissue through permeable tumor vessels and are then retained in the tumor bed due to reduced lymphatic drainage. This process is known as the enhanced permeability and retention (EPR) effect. However, while the EPR effect is widely held to improve delivery of nanodrugs to tumors, it in fact offers less than a 2-fold increase in nanodrug delivery compared with critical normal organs, resulting in drug concentrations that are not sufficient for curing most cancers. In this Review, we first overview various barriers for nanosized drug delivery with an emphasis on the capillary wall's resistance, the main obstacle to delivering drugs. Then, we discuss current regulatory issues facing nanomedicine. Finally, we discuss how to make the delivery of nanosized drugs to tumors more effective by building on the EPR effect.