Release Of Chemoattractant Research Articles

Abstract 3516: Reversing extracellular vesicle induced tumor immune suppression at the sentinel lymph node: Role of secretory autophagy and mitophagy

Abstract The goal is to reverse cancer immune evasion at the level of the sentinel lymph node (SLN). Reduced expansion of CD8+ T cells and other innate immune effector cells in the cancer draining SLN is associated with progression and resistance to checkpoint inhibitors. Cancer-derived extracellular vesicles (EVs) that are PD-L1+ suppress immune recognition at the level of the SLN. The experimental goal is to remodel the SLN to overcome cancer EV-associated immune suppression and induce immune rejection of the tumor. We developed three methodologies for this project: a) Collection of draining lymph fluid to characterize EVs shed by 4T1 syngeneic breast tumors growing in the mammary fat pad. b) Chromatographic separation and characterization of the repertoire of EVs shed by tumors into the tumor microenvironment interstitial space using western blotting, mass spectrometry, and electron microscopy. c) Nanoparticle (NP) delivery of purified populations of EVs to the tumor draining SLN in combination with cytokine chemoattractants for innate immune cells recruitment. We characterized the in vivo interstitial fluid (IF) content of a GFP-4T1 syngeneic murine cancer model to study resident IF EVs transit to the draining lymph node. GFP labeling confirmed the IF EV tumor cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; including PINK1 and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial pinching), and VPS35, SEC22b, and Rab33b (vacuolar sorting). SLN immune cell populations could be massively remodeled by introducing hydrogel NPs which have a controlled release of T-cell and dendritic cell chemoattractant to the subcapsular sinus. NPs were successfully used to deliver concentrated packages of EVs subpopulations to the SLN. Introduction of the large CD81-/VEGF+/PD-L1- EV subpopulation (amphisome characteristics) to the SLN augmented tumor growth, angiogenesis, and metastasis, even when cytokine induction was used to remodel the SLN. In marked contrast, introduction of the CD81+/PD-L1+ EV subpopulation (containing mitophagy components) to the SLN in combination with NP release of chemoattractants, induced immune rejection of the syngeneic breast cancer, reducing tumor growth, and blocking metastasis. These findings demonstrate that different populations of EVs have opposite effects on cancer immune evasion at the level of the SLN and that EV-mediated immune suppression can be reversed by SLN remodeling to augment dendritic and CD8+ T cells. Citation Format: Marissa Howard, James Erickson, Amanda Haymond, Alessandra Luchini, Fatah Kashanchi, Lance Liotta. Reversing extracellular vesicle induced tumor immune suppression at the sentinel lymph node: Role of secretory autophagy and mitophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3516.

Nucleic Acid-Based Dual Cross-Linked Hydrogels for in Situ Tissue Repair via Directional Stem Cell Migration.

In situ tissue repair holds great potential as a cell-free regenerative strategy. A critical aspect of this approach is the selection of cell instructive materials that can efficiently regulate the defect microenvironment via the release of chemoattractant factors to mobilize and recruit endogenous stem cells toward the site of implantation. Here we report the design of a DNA-based hydrogel as a drug delivery platform for the sustained release of a promising chemoattractant, SDF-1α. The hydrogel is composed of chemically cross-linked DNA strands, which are bridged via silicate nanodisks (nSi). Silicate nanodisks electrostatically interact with the negatively charged DNA backbone resulting in the formation of a dual cross-linked nanocomposite hydrogel with a combination of chemical and physical cross-link points. The formulated nanocomposites display enhanced elasticity and mechanical toughness as compared to their nonsilicate containing counterparts. Moreover, the electrostatic interaction between nSi and SDF-1α leads to sustained release of the chemokine from the hydrogels. The in vitro bioactivity assays confirm the retention of chemotactic properties of the protein after its release. Overall, the dual cross-linked DNA-based hydrogel platform could be potentially used as a cell-instructive material for the recruitment of host stem cells to guide the process of in situ tissue repair.

Mathematics of Experimentally Generated Chemoattractant Gradients.

Many eukaryotic cells move in the direction of a chemical gradient. Several assays have been developed to measure this chemotactic response, but no complete mathematical models of the spatial and temporal gradients are available to describe the fundamental principles of chemotaxis. Here we provide analytical solutions for the gradients formed by release of chemoattractant from a point source by passive diffusion or forced flow (micropipettes) and gradients formed by laminar diffusion in a Zigmond chamber. The results show that gradients delivered with a micropipette are formed nearly instantaneously, are very steep close to the pipette, and have a steepness that is strongly dependent on the distance from the pipette. In contrast, gradients in a Zigmond chamber are formed more slowly, are nearly independent of the distance from the source, and resemble the temporal and spatial properties of the natural cAMP wave that Dictyostelium cells experience during cell aggregation.

Promotion of neuronal migration towards the injured mouse cerebral cortex using sustained release of chemoattractant from gelatin hydrogel microspheres

Promotion of neuronal migration towards the injured mouse cerebral cortex using sustained release of chemoattractant from gelatin hydrogel microspheres

ERK1/2 and p38 MAP kinases control prion protein fragment 90–231‐induced astrocyte proliferation and microglia activation

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

Release of a potent polymorphonuclear leukocyte chemoattractant is regulated by white-opaque switching in Candida albicans.

Previous studies employing transmembrane assays suggested that Candida albicans and related species, as well as Saccharomyces cerevisiae, release chemoattractants for human polymorphonuclear leukocytes (PMNs). Because transmembrane assays do not definitively distinguish between chemokinesis and chemotaxis, single-cell chemotaxis assays were used to confirm these findings and test whether mating-type or white-opaque switching affects the release of attractant. Our results demonstrate that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata release bona fide chemoattractants for PMNs. S. cerevisiae, however, releases a chemokinetic factor but not a chemoattractant. Characterization of the C. albicans chemoattractant revealed that it is a peptide of approximately 1 kDa. Whereas the mating type of C. albicans did not affect the release of chemoattractant, switching did. White-phase cells released chemoattractant, but opaque-phase cells did not. Since the opaque phase of C. albicans represents the mating-competent phenotype, it may be that opaque-phase cells selectively suppress the release of chemoattractant to facilitate mating.

Chronic Alcohol Intoxication Enhances the Expression of CD18 Adhesion Molecules on Rat Neutrophils and Release of a Chemotactic Factor by Kupffer Cells

Chronic alcohol intoxication has been associated with increased migration of inflammatory leukocytes to the liver that may contribute to the development of alcoholic hepatitis in susceptible individuals. Thus, this work was performed to examine the mechanism by which neutrophils [polymorphonuclear neutrophils (PMNS)] are sequestered in the liver during prolonged consumption of alcohol. Male Sprague-Dawley rats were fed with Sustacal supplemented by 36% alcohol, or isocaloric diet for 16 weeks. Circulating blood PMNs were collected and examined for CD18 (beta 2-integrin) adhesion molecule expression. Monoclonal antibody 1F12, an anti-CD18 antibody and potent neutropenic agent, was used to detect CD18 on PMNs. More than 97% of neutrophils obtained from pair and ethanol-fed rats were positive for the antibody. Fluorescence intensity of fluorescein isothiocyanate-1F12 binding to PMNs from ethanol-fed rat was significantly enhanced 2-fold compared with the pair-fed controls. The release of chemoattractant and free radical-generating activity in culture supernatants of Kupffer cells was also examined. Twenty-four hr culture supernatants of Kupffer cells from chronic alcoholic rats enhanced the migration and superoxide anion generation by normal PMNs, compared with those of the pair-fed rats. Antirat interleukin-8 antiserum inhibited chemotactic activity and superoxide generating capacity of culture supernatants. These results suggest that upregulation of adhesion molecules on PMNs and chemotactic factor release from Kupffer cells may contribute, at least in part, to enhanced migration of inflammatory leukocytes to the liver during chronic alcohol intoxication.