Tumor Vascular Remodeling Affects Molecular Dissemination to Lymph Node and Systemic Leukocytes.

Angiogenic and lymphangiogenic remodeling has long been accepted as a hallmark of cancer development and progression; however, the impacts of this remodeling on immunological responses, which are paramount to the responses to immunotherapeutic treatments, are underexplored. As immunotherapies represent one of the most promising new classes of cancer therapy, in this study, we explore the effects of angiogenic and lymphangiogenic normalization on dissemination of molecules injected into the tumor microenvironment to immune cells in lymph nodes draining the tumor as well as in systemically distributed tissues. A system of fluorescent tracers, size-matched to biomolecules of interest, was implemented to track different mechanisms of tumor transport and access to immune cells. This revealed that the presence of a tumor, and either angiogenic or lymphangiogenic remodeling, altered local retention of model biomolecules, trended toward normalizing dissemination to systemic organs, and modified access to lymph node-resident immune cells in manners dependent on mechanism of transport. More specifically, active cell migration by skin-derived antigen presenting cells was enhanced by both the presence of a tumor and lymphangiogenic normalization, while both angiogenic and lymphangiogenic normalization restored patterns of immune cell access to passively draining species. As a whole, this work uncovers the potential ramifications of tumor-induced angiogenesis and lymphangiogenesis, along with impacts of interrogation into these pathways, on access of tumor-derived species to immune cells. Impact Statement Angiogenic and lymphangiogenic normalization strategies have been utilized clinically to interrogate tumor vasculature with some success. In the age of immunotherapy, the impacts of these therapeutic interventions on immune remodeling are unclear. This work utilizes mouse models of angiogenic and lymphangiogenic normalization, along with a system of fluorescently tagged tracers, to uncover the impacts of angiogenesis and lymphangiogenesis on access of tumor-derived species to immune cell subsets within various organs.

Publisher Correction: Programmable multistage drug delivery to lymph nodes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Programmable multistage drug delivery to lymph nodes.

Therapeutic delivery selectively to lymph nodes has the potential to address a variety of unmet clinical needs. However, owing to the unique structure of the lymphatics and the size-restrictive nature of the lymph node reticular network, delivering cargo to specific cells in the lymph node cortex and paracortex is difficult. Here, we describe a delivery system to overcome lymphatic and intra-lymph node transport barriers by combining nanoparticles that are rapidly conveyed to draining lymph nodes after administration in peripheral tissues with programmable degradable linkers. This platform enables the controlled release of intra-lymph-mobile small-molecular cargo, which can reach vastly more immune cells throughout the lymph node than either the particles or free compounds alone. The release rate can be programmed, allowing access to different lymph node structures and therefore specific lymphocyte subpopulations. We are thus able to alter the subtypes of drugged lymph node cells to improve immunotherapeutic effects.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in in vitro and in vivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

P-, but not E- or L-, selectin-mediated rolling adhesion persistence in hemodynamic flow diverges between metastatic and leukocytic cells.

The ability of leukocytic cells to engage selectins via rolling adhesion is critical to inflammation, but selectins are also implicated in mediating metastatic dissemination. Using a microfluidic- and flow-based cell adhesion chromatography experimental and analytical technique, we interrogated the cell-subtype differences in engagement and sustainment of rolling adhesion on P-, E-, and L-selectin-functionalized surfaces in physiological flow. Our results indicate that, particularly at low concentrations of P-selectin, metastatic but not leukocytic cells exhibit reduced rolling adhesion persistence, whereas both cell subtypes exhibited reduced persistence on L-selectin and high persistence on E-selectin, differences not revealed by flow cytometry analysis or reflected in the extent or velocity of rolling adhesion. Conditions under which adhesion persistence was found to be significantly reduced corresponded to those exhibiting the greatest sensitivity to a selectin-antagonist. Our results suggest that potentially therapeutically exploitable differences in metastatic and leukocytic cell subtype interactions with selectins in physiological flow are identifiable through implementation of functional assays of adhesion persistence in hemodynamic flow utilizing this integrated, flow-based cell adhesion chromatography analytical technique.

P-Selectin and ICAM-1 synergy in mediating THP-1 monocyte adhesion in hemodynamic flow is length dependent.

The tightly orchestrated recruitment of monocytes, whose progeny are critical to the progression and resolution of various physiological and pathophysiological processes, is implicated in the time course, severity, and resolution of pathology. Using a microfluidic-based cell adhesion assay integrating spatiotemporal analyses and micropatterning of adhesive proteins, we interrogated the effects of adhesive molecule presentation length, which varies in vivo with disease and stage, on THP-1 monocyte cell rolling versus firm adhesion mediated by P-selectin and/or ICAM-1 in hemodynamic flow. Our results indicate that co-presentation of P-selectin and ICAM-1 substantially decreases the length of adhesive substrate required to sustain adhesion in flow and that P-selectin functions synergistically with ICAM-1 to substantially enhance THP-1 firm adhesion. This synergy was found to furthermore correlate with diminished cell rolling velocities and length-enhanced secondary cell capture. Our results suggest pathophysiological ramifications for local remodeling of the inflamed microvascular microenvironment in directing the efficiency of monocyte trafficking.

Antiangiogenic cancer drug drives lymphangiogenic metastasis.

Tumor blood vessel-destroying cancer drug induces development of tumor lymphatics that contribute to treatment failure.

Flexible Macromolecule versus Rigid Particle Retention in the Injected Skin and Accumulation in Draining Lymph Nodes Are Differentially Influenced by Hydrodynamic Size.

Therapeutic immunomodulation in the skin, its draining lymph nodes, or both tissues simultaneously using an intradermal administration scheme is desirable for a variety of therapeutic scenarios. To inform how drug carriers comprising engineered biomaterials can be leveraged to improve treatment efficacy by enhancing the selective accumulation or retention of payload within these target tissues, we analyzed the influence of particle versus macromolecule hydrodynamic size on profiles of retention in the site of dermal injection as well as the corresponding extent of accumulation in draining lymph nodes and systemic off-target tissues. Using a panel of fluorescently labeled tracers comprising inert polymers that are resistant to hydrolysis and proteolytic degradation that span a size range of widely used drug carrier systems, we find that macromolecule but not rigid particle retention within the skin is size-dependent, whereas the relative dermal enrichment compared to systemic tissues increases with size for both tracer types. Additionally, macromolecules 10 nm in hydrodynamic size and greater accumulate in draining lymph nodes more extensively and selectively than particles, suggesting that intra- versus extracellular availability of delivered payload within draining lymph nodes may be influenced by both the size and form of engineered drug carriers. Our results inform how biomaterial-based drug carriers can be designed to enhance the selective exposure of formulated drug in target tissues to improve the therapeutic efficacy as well as minimize off-target effects of locoregional immunotherapy.

Melanoma growth effects on molecular clearance from tumors and biodistribution into systemic tissues versus draining lymph nodes.

Factors produced within or administered directly into the tumor interstitium, such as cytokines, chemokines, proteases, exosomes, microvesicles, or therapeutic agents, play important and multifaceted roles in the regulation of malignant disease progression. Their bioavailability to mediate signaling in distributed tissues outside of the tumor microenvironment, however, has not been well described. We therefore sought to elucidate the relative extent to which factors from within the primary tumor disseminate to systemic tissues as well as how these distribution profiles are influenced by both hydrodynamic size and the remodeling tumor vasculature. To accomplish this goal, we intratumorally co-infused into the dermal lesions of B16F10 melanoma-bearing mice at prescribed times post tumor implantation a near infrared fluorescent tracer panel ranging from 5 to 500nm in hydrodynamic diameter and compared the in vivo clearance and biodistribution profiles to that of naïve animals. Our results indicate that tumor growth reduces tumor-draining lymph node accumulation and alters the distribution of tumor-derived factors amongst systemic tissues. Despite these changes, previously developed principles of size-dependent lymph node drug targeting are conserved in melanomas, suggesting their applicability to sentinel lymph node-targeted drug delivery. Tumor progression was also found to result in a significant increase in the hydrodynamic size of factors originating from the tumor that accumulated within systemic tissues. This suggests that tumor vascular remodeling may redirect the organism-wide signaling activity of tumor-derived factors and may negatively contribute to disease progression by altering the bioavailability of molecules important to the regulation of pre-metastatic niche formation and the induction of anti-tumor immunity.

Lymph node biophysical remodeling is associated with melanoma lymphatic drainage.

Tissue remodeling is a characteristic of many solid tumor malignancies including melanoma. By virtue of tumor lymphatic transport, remodeling pathways active within the local tumor microenvironment have the potential to be operational within lymph nodes (LNs) draining the tumor interstitium. Here, we show that lymphatic drainage from murine B16 melanomas in syngeneic, immune-competent C57Bl/6 mice is associated with LN enlargement as well as nonuniform increases in bulk tissue elasticity and viscoelasticity, as measured by the response of whole LNs to compression. These remodeling responses, which quickly manifest in tumor-draining lymph nodes (TDLNs) after tumor inoculation and before apparent metastasis, were accompanied by changes in matrix composition, including up to 3-fold increases in the abundance of soluble collagen and hyaluronic acid. Intranodal pressures were also significantly increased in TDLNs (+1 cmH2O) relative to both non-tumor-draining LNs (-1 cmH2O) and LNs from naive animals (-1 to 2 cmH2O). These data suggest that the reorganization of matrix structure, composition, and fluid microenvironment within LNs associated with tumor lymphatic drainage parallels remodeling seen in primary malignancies and has the potential to regulate the adhesion, proliferation, and signaling function of LN-resident cells involved in directing melanoma disease progression.