The DISC1-Girdin complex - a missing link in signaling to the T cell cytoskeleton.

In this study, using Jurkat cells, we show that DISC1 (disrupted in schizophrenia 1) and Girdin (girders of actin filament) are essential for typical actin accumulation at the immunological synapse. Furthermore, DISC1, Girdin and dynein are bound in a complex. Although this complex initially forms as a central patch at the synapse, it relocates to a peripheral ring corresponding to the peripheral supramolecular activation cluster (pSMAC). In the absence of DISC1, the classic actin ring does not form, cell spreading is blocked, and the dynein complex fails to relocate to the pSMAC. A similar effect is seen when Girdin is deleted. When cells are treated with inhibitors of actin polymerization, the dynein-NDE1 complex is lost from the synapse and the microtubule-organizing center fails to translocate, suggesting that actin and dynein might be linked. Upon stimulation of T cell receptors, DISC1 becomes associated with talin, which likely explains why the dynein complex colocalizes with the pSMAC. These results show that the DISC1-Girdin complex regulates actin accumulation, cell spreading and distribution of the dynein complex at the synapse.This article has an associated First Person interview with the first author of the paper.

Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.

Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150(Glued)) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-enhanced GFP fusion) were activated by Staphylococcus enterotoxin E-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150(Glued), which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B-containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150(Glued) in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell-focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively.

A Perfusion Model to Evaluate Response to Photodynamic Therapy in 3D Tumors.

Numerous cancer models have been developed to investigate the effects of mechanical stress on the biology of cells. Here we describe a protocol to fabricate a perfusion model to culture 3-dimensional (3D) ovarian cancer nodules under constant flow. The modular design of this model allows for a wide range of treatment regimens and combinations, including PDT and chemotherapy. Finally, methods for a number of readouts are detailed, allowing researchers to investigate a variety of biological and cytotoxic parameters related to mechanical stress and therapeutic modalities.

Emerging Roles of Mast Cells in the Regulation of Lymphatic Immuno-Physiology.

Mast cells (MCs) are abundant in almost all vascularized tissues. Furthermore, their anatomical proximity to lymphatic vessels and their ability to synthesize, store and release a large array of inflammatory and vasoactive mediators emphasize their significance in the regulation of the lymphatic vascular functions. As a major secretory cell of the innate immune system, MCs maintain their steady-state granule release under normal physiological conditions; however, the inflammatory response potentiates their ability to synthesize and secrete these mediators. Activation of MCs in response to inflammatory signals can trigger adaptive immune responses by dendritic cell-directed T cell activation. In addition, through the secretion of various mediators, cytokines and growth factors, MCs not only facilitate interaction and migration of immune cells, but also influence lymphatic permeability, contractility, and vascular remodeling as well as immune cell trafficking through the lymphatic vessels. In summary, the consequences of these events directly affect the lymphatic niche, influencing inflammation at multiple levels. In this review, we have summarized the recent advancements in our understanding of the MC biology in the context of the lymphatic vascular system. We have further highlighted the MC-lymphatic interaction axis from the standpoint of the tumor microenvironment.

Cancer Cell-targeted and Activatable Photoimmunotherapy Spares T Cells in a 3D Coculture Model.

Photodynamic therapy (PDT) is an established therapeutic modality that uses nonionizing near-infrared light to activate photocytotoxicity of endogenous or exogenous photosensitizers (PSs). An ongoing avenue of cancer research involves leveraging PDT to stimulate antitumor immune responses; however, these effects appear to be best elicited in low-dose regimens that do not provide significant tumor reduction using conventional, nonspecific PSs. The loss of immune enhancement at higher PDT doses may arise in part from indiscriminate damage to local immune cell populations, including tumor-infiltrating T cells. We previously introduced "tumor-targeted, activatable photoimmunotherapy" (taPIT) using molecular-targeted and cell-activatable antibody-PS conjugates to realize precision tumor photodamage with microscale fidelity. Here, we investigate the immune cell sparing effect provided by taPIT in a 3D model of the tumor immune microenvironment. We report that high-dose taPIT spares 25% of the local immune cell population, five times more than the conventional PDT regimen, in a 3D coculture model incorporating epithelial ovarian cancer cells and T cells. These findings suggest that the enhanced selectivity of taPIT may be utilized to achieve local tumor reduction with sparing of intratumor effector immune cells that would otherwise be lost if treated with conventional PDT.

Flow-induced Shear Stress Confers Resistance to Carboplatin in an Adherent Three-Dimensional Model for Ovarian Cancer: A Role for EGFR-Targeted Photoimmunotherapy Informed by Physical Stress.

A key reason for the persistently grim statistics associated with metastatic ovarian cancer is resistance to conventional agents, including platinum-based chemotherapies. A major source of treatment failure is the high degree of genetic and molecular heterogeneity, which results from significant underlying genomic instability, as well as stromal and physical cues in the microenvironment. Ovarian cancer commonly disseminates via transcoelomic routes to distant sites, which is associated with the frequent production of malignant ascites, as well as the poorest prognosis. In addition to providing a cell and protein-rich environment for cancer growth and progression, ascitic fluid also confers physical stress on tumors. An understudied area in ovarian cancer research is the impact of fluid shear stress on treatment failure. Here, we investigate the effect of fluid shear stress on response to platinum-based chemotherapy and the modulation of molecular pathways associated with aggressive disease in a perfusion model for adherent 3D ovarian cancer nodules. Resistance to carboplatin is observed under flow with a concomitant increase in the expression and activation of the epidermal growth factor receptor (EGFR) as well as downstream signaling members mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK). The uptake of platinum by the 3D ovarian cancer nodules was significantly higher in flow cultures compared to static cultures. A downregulation of phospho-focal adhesion kinase (p-FAK), vinculin, and phospho-paxillin was observed following carboplatin treatment in both flow and static cultures. Interestingly, low-dose anti-EGFR photoimmunotherapy (PIT), a targeted photochemical modality, was found to be equally effective in ovarian tumors grown under flow and static conditions. These findings highlight the need to further develop PIT-based combinations that target the EGFR, and sensitize ovarian cancers to chemotherapy in the context of flow-induced shear stress.

Photodynamic Therapy in a 3D Model of Ovarian Cancer.

Photodynamic therapy (PDT), is a clinically-approved light-based anti-cancer treatment modality in which a photoactivatable photosensitizer is irradiated with an appropriate wavelength of light to generate cytotoxic molecules to kill cancer cells. In this article, we describe an in vitro PDT protocol using a 3-dimensional (3D) model of ovarian cancer that was established on beds of Matrigel. PDT was performed using a liposomal formulation of verteporfin photosensitizer (Visudyne®). The cancer cells were genetically-labeled with the fluorescent protein mCherry to facilitate the evaluation of the treatment response. This protocol is advantageous because the mCherry fluorescence is an indicator of cell viability, eliminating the need for external dyes, which often exhibit limited penetration and diffusion into 3D organoids. Additionally, Visudyne PDT achieves significant tumor-killing efficacy in a 3D model for ovarian cancer.

The Course of Immune Stimulation by Photodynamic Therapy: Bridging Fundamentals of Photochemically Induced Immunogenic Cell Death to the Enrichment of T-Cell Repertoire.

Photodynamic therapy (PDT) is a potentially immunogenic and FDA-approved antitumor treatment modality that utilizes the spatiotemporal combination of a photosensitizer, light and oftentimes oxygen, to generate therapeutic cytotoxic molecules. Certain photosensitizers under specific conditions, including ones in clinical practice, have been shown to elicit an immune response following photoillumination. When localized within tumor tissue, photogenerated cytotoxic molecules can lead to immunogenic cell death (ICD) of tumor cells, which release damage-associated molecular patterns and tumor-specific antigens. Subsequently, the T-lymphocyte (T cell)-mediated adaptive immune system can become activated. Activated T cells then disseminate into systemic circulation and can eliminate primary and metastatic tumors. In this review, we will detail the multistage cascade of events following PDT of solid tumors that ultimately lead to the activation of an antitumor immune response. More specifically, we connect the fundamentals of photochemically induced ICD with a proposition on potential mechanisms for PDT enhancement of the adaptive antitumor response. We postulate a hypothesis that during the course of the immune stimulation process, PDT also enriches the T-cell repertoire with tumor-reactive activated T cells, diversifying their tumor-specific targets and eliciting a more expansive and rigorous antitumor response. The implications of such a process are likely to impact the outcomes of rational combinations with immune checkpoint blockade, warranting investigations into T-cell diversity as a previously understudied and potentially transformative paradigm in antitumor photodynamic immunotherapy.

Photoimmunotherapy of Ovarian Cancer: A Unique Niche in the Management of Advanced Disease.

Ovarian cancer (OvCa) is the leading cause of gynecological cancer-related deaths in the United States, with five-year survival rates of 15-20% for stage III cancers and 5% for stage IV cancers. The standard of care for advanced OvCa involves surgical debulking of disseminated disease in the peritoneum followed by chemotherapy. Despite advances in treatment efficacy, the prognosis for advanced stage OvCa patients remains poor and the emergence of chemoresistant disease localized to the peritoneum is the primary cause of death. Therefore, a complementary modality that is agnostic to typical chemo- and radio-resistance mechanisms is urgently needed. Photodynamic therapy (PDT), a photochemistry-based process, is an ideal complement to standard treatments for residual disease. The confinement of the disease in the peritoneal cavity makes it amenable for regionally localized treatment with PDT. PDT involves photochemical generation of cytotoxic reactive molecular species (RMS) by non-toxic photosensitizers (PSs) following exposure to non-harmful visible light, leading to localized cell death. However, due to the complex topology of sensitive organs in the peritoneum, diffuse intra-abdominal PDT induces dose-limiting toxicities due to non-selective accumulation of PSs in both healthy and diseased tissue. In an effort to achieve selective damage to tumorous nodules, targeted PS formulations have shown promise to make PDT a feasible treatment modality in this setting. This targeted strategy involves chemical conjugation of PSs to antibodies, referred to as photoimmunoconjugates (PICs), to target OvCa specific molecular markers leading to enhanced therapeutic outcomes while reducing off-target toxicity. In light of promising results of pilot clinical studies and recent preclinical advances, this review provides the rationale and methodologies for PIC-based PDT, or photo-immunotherapy (PIT), in the context of OvCa management.

A Combination of Visudyne and a Lipid-anchored Liposomal Formulation of Benzoporphyrin Derivative Enhances Photodynamic Therapy Efficacy in a 3D Model for Ovarian Cancer.

A major objective in developing new treatment approaches for lethal tumors is to reduce toxicity to normal tissues while maintaining therapeutic efficacy. Photodynamic therapy (PDT) provides a mechanistically distinct approach to treat tumors without the systemic toxicity of chemotherapy drugs. PDT involves the light-based activation of a small molecule, a photosensitizer (PS), to generate reactive molecular species (RMS) that are toxic to target tissue. Depending on the PS localization, various cellular and subcellular components can be targeted, causing selective photodamage. It has been shown that targeted lysosomal photodamage followed by, or simultaneous with, mitochondrial photodamage using two different PS results in a considerable enhancement in PDT efficacy. Here, two liposomal formulations of benzoporphyrin derivative (BPD): (1) Visudyne (clinically approved) and (2) an in-house formulation entrapping a lipid conjugate of BPD are used in combination with direct PS localization to mitochondria, endoplasmic reticulum and lysosomes, enabling simultaneous photodamage to all three organelles using a single wavelength of light. Building on findings by our group, and others, this study demonstrates, for the first time in a 3D model for ovarian cancer, that BPD-mediated photodestruction of lysosomes and mitochondria/ER significantly enhances PDT efficacy at lower light doses than treatment with either PS formulation alone.

Potential for largemouth bass virus to associate with and gain protection from bacterial biofilms.

Quantitative PCR (qPCR) was used to investigate whether largemouth bass virus (LMBV) can exist within biofilms. Suspended LMBV was partitioned into either laboratory-grown Pseudomonas fluorescens biofilms or pond-grown, mixed-population biofilms. Biofilm-entrapped LMBV retained infectivity when tested on epithelioma papillosum cyprini tissue culture cells. The LMBV associated with P. fluorescens biofilms were resistant to disinfection by sodium hypochlorite and an iodine-based compound (betadine) but were susceptible to ethanol. Largemouth bass virus was not detected in biofilms or water from ponds that had previously contained LMBV-positive fish, suggesting either that the viral concentrations were below the detection limit of qPCR or that the fish represented the main LMBV reservoir. This study illustrates the potential for LMBV to associate with bacterial biofilms and thereby gain protection from some chemical disinfectants.