Dysfunctional natural killer cells can be reprogrammed to regain anti-tumor activity.

Natural killer (NK) cells are critical to the innate immune system, as they recognize antigens without prior sensitization, and contribute to the control and clearance of viral infections and cancer. However, a significant proportion of NK cells in mice and humans do not express classical inhibitory receptors during their education process and are rendered naturally "anergic", i.e., exhibiting reduced effector functions. The molecular events leading to NK cell anergy as well as their relation to those underlying NK cell exhaustion that arises from overstimulation in chronic conditions, remain unknown. Here, we characterize the "anergic" phenotype and demonstrate functional, transcriptional, and phenotypic similarities to the "exhausted" state in tumor-infiltrating NK cells. Furthermore, we identify zinc finger transcription factor Egr2 and diacylglycerol kinase DGKα as common negative regulators controlling NK cell dysfunction. Finally, experiments in a 3D organotypic spheroid culture model and an in vivo tumor model suggest that a nanoparticle-based delivery platform can reprogram these dysfunctional natural killer cell populations in their native microenvironment. This approach may become clinically relevant for the development of novel anti-tumor immunotherapeutic strategies.

Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies.

Cancer cells depend on actin cytoskeleton rearrangement to carry out hallmark malignant functions including activation, proliferation, migration and invasiveness. Wiskott-Aldrich Syndrome protein (WASp) is an actin nucleation-promoting factor and is a key regulator of actin polymerization in hematopoietic cells. The involvement of WASp in malignancies is incompletely understood. Since WASp is exclusively expressed in hematopoietic cells, we performed in silico screening to identify small molecule compounds (SMCs) that bind WASp and promote its degradation. We describe here one such identified molecule; this WASp-targeting SMC inhibits key WASp-dependent actin processes in several types of hematopoietic malignancies in vitro and in vivo without affecting naïve healthy cells. This small molecule demonstrates limited toxicity and immunogenic effects, and thus, might serve as an effective strategy to treat specific hematopoietic malignancies in a safe and precisely targeted manner.

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress.

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H2 DCFDA-staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into 'low-ROS' and 'high-ROS' subpopulations. Pollen germination assays following fluorescence-activated cell sorting revealed that the high-ROS pollen germinated with a frequency that was 35-fold higher than the low-ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose-dependent alterations in DCF-fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry-based approaches to investigate metabolic changes during stress responses in pollen.

FRET energy transfer via Pdots improves the efficiency of photodynamic therapy and leads to rapid cell death.

Photodynamic therapy (PDT) is well established as a clinical treatment modality for various diseases, including cancer and especially for the treatment of superficial tumors. However, one of the disadvantages of the photoactivatable molecules is their low absorbance in the optical window for photosensitizer excitation. The use of nanoparticles in photodynamic therapy can address this deficiency and improve treatment efficiency. Pdots are nano-sized particles, composed of conjugated chromophoric polymers. By mixing them with PEGylated phospholipids they can become soluble and stable colloids. They exhibit a broad absorption band with a strong and narrow emission band. In this study, we examined two types of Pdots (MEH-PPV and CN-PPV) with two different lengths of the PEGylated lipids coating, 350 and 2000. When a photosensitizer, such as mTHPC, comes in close contact with the amphiphilic coating of the Pdots, a very efficient fluorescence resonance energy transfer (FRET) occurs between the donor, the Pdots and the acceptor, the sensitizer. This process, together with the significant uptake of the Pdots-sensitizer pair by MCF-7 cancerous cells causes irreversible damage to the cells. This damage is greater when the Pdots are comprised from the CN-PPV polymer and coated with the PEG2000-PE lipid. Altogether, we demonstrate that implementing FRET energy transfer in the PDT protocol leads to quicker and more aggressive cell death, thus improving the efficacy of the photodynamic therapy.

Purifying Cytokinetic Cells from an Asynchronous Population.

Cytokinesis is an intensively studied process by which the cell cytoplasm divides to produce two daughter cells. Like any other aspect of cell cycle research, the study of cytokinesis relies heavily on cell synchronization. However, the synchronization of cells during cytokinesis is challenging due to the rapid nature of this process and the shortage of cell cycle blocking agents specifically targeting this phase. Here, we demonstrate the use of standard flow cytometry for directly isolating cytokinetic cells from an asynchronous population of normally proliferating cells. This approach is based on a cell cycle marker whose temporal proteolysis, in combination with DNA quantification or cell size approximation, distinguishes cells undergoing cytokinesis. Furthermore, by avoiding doublet discrimination, typically used in flow cytometry analyses, we were able to further increase selectivity, specifically purifying cells at late cytokinesis. Our method circumvents checkpoint activation, cell cycle arrest, and any other means of pre-synchronization. These qualities, as demonstrated for both unattached and adherent cells, enable high selectivity for cytokinetic cells despite their overall low abundance in an asynchronous population. The sorted cells can then be readily used for cell biological, biochemical, and genomic applications to facilitate cytokinesis and cell cycle research.

Pdots, a new type of nanoparticle, bind to mTHPC via their lipid modified surface and exhibit very high FRET efficiency between the core and the sensitizer.

Pdots are a new type of nanoparticle which exhibit strong potential for future applications in biophysics and cell biology. They are composed of organic chromophoric polymers, whose surfaces can be modified with different amphiphilic polymers, such as PEGylated lipids to make them very stable as colloids in water. We demonstrate in this manuscript that the lipid nano-coating around the Pdot can bind very efficiently to amphiphilic molecules, such as photosensitizers e.g. meso-tetrahydroxyphenylchlorin (mTHPC). As a result the sensitizer is brought into very close contact with the cores of the Pdots, and resonance energy transfer from the core to the sensitizer is very efficient; in some cases it is close to 1. We show the spectroscopic properties of two types of Pdots; their sizes, which are in the 13-47 nm range, depend on the kind of polymer and the length of the PEGylated lipid chains that wrap it. We measured the efficiency of FRET by investigating the decrease in donor intensity or its lifetime upon binding with mTHPC. We also show the relative yields of singlet oxygen that are obtained via two pathways: by exciting the Pdots which transfer the energy to the attached sensitizer, or by exciting the sensitizer directly. This methodology could be used to enhance the use of a photosensitizer by employing both pathways in parallel.

Using standard optical flow cytometry for synchronizing proliferating cells in the G1 phase.

Cell cycle research greatly relies on synchronization of proliferating cells. However, effective synchronization of mammalian cells is commonly achieved by long exposure to one or more cell cycle blocking agents. These chemicals are, by definition, hazardous (some more than others), pose uneven cell cycle arrest, thus introducing unwanted variables. The challenge of synchronizing proliferating cells in G1 is even greater; this process typically involves the release of drug-arrested cells into the cycle that follows, a heterogeneous process that can truly limit synchronization. Moreover, drug-based synchronization decouples the cell cycle from cell growth in ways that are understudied and intolerable for those who investigate the relationship between these two processes. In this study we showed that cell size, as approximated by a single light-scatter parameter available in all standard sorters, can be used for synchronizing proliferating mammalian cells in G1 with minimal or no risk to either the cell cycle or cell growth. The power and selectivity of our method are demonstrated for human HEK293 cells that, despite their many advantages, are suboptimal for synchronization, let alone in G1. Our approach is readily available, simple, fast, and inexpensive; it is independent of any drugs or dyes, and nonhazardous. These properties are relevant for the study of the mammalian cell cycle, specifically in the context of G1 and cell growth.

Extracellular matrix constituents interfere with Newcastle disease virus spread in solid tissue and diminish its potential oncolytic activity.

Advanced melanoma cells, characterized by resistance to chemotherapy, have been shown to be highly sensitive to oncolysis by Newcastle disease virus (NDV). In the present study, we investigated the capacity of NDV to specifically infect and spread into solid tissues of human melanoma and lung carcinoma, in vivo and ex vivo. For this purpose a new model of SCID-beige mice implanted with human melanoma was developed. Surprisingly, the replication competent NDV-MTH and the attenuated, single-cycle replication NDV-HUJ strains, demonstrated a similar oncolytic activity in the melanoma-implanted mice. Further, ex vivo analysis, using organ cultures derived from the melanoma tissues indicated a limited spread of the two NDV strains in the tissue. Extracellular matrix (ECM) molecules, notably heparin sulfate and collagen, were found to limit viral spread in the tissue. This observation was validated with yet another solid tumour of human lung carcinoma. Taken together, the results indicate that the ECM acts as a barrier to virus spread within solid tumour tissues and that this restriction must be overcome to achieve effective oncolysis with NDV.

The clinical effect of the inhibitor of apopotosis protein livin in melanoma.

OBJECTIVE: The inhibitor of apoptosis protein (IAP) livin is frequently overexpressed in melanoma. Livin binds caspases and thereby inhibits apoptosis. We found that caspases cleave livin to produce a truncated form with a paradoxical proapoptotic activity. METHODS: We assessed the correlation of livin expression with survival among 114 melanoma patients treated with an autologous melanoma vaccine. In 52 patients, resection resulted in no evidence of disease (NED) and 62 remained with active disease (WAD). Protein levels were assessed using Western blot. RESULTS: We found livin protein expression in 44/114 samples (38.4%). Median overall survival was 1.4 years in NED patients with high levels of livin protein, 8.4 years in those with low-intermediate levels and not reached in patients who did not express livin (p = 0.025). The corresponding overall survival was 2.3 years among WAD patients with high levels of livin protein, 11.3 years in those with low-intermediate levels and, paradoxically, only 4.0 years in patients who did not express livin (p = 0.012). CONCLUSION: Livin protein expression may play a role in the progression of melanoma and correlates with survival. A high level of the protein is associated with a poor prognosis. However, in WAD patients low to intermediate level of livin, rather than absence of the protein, is associated with a favorable prognosis. This is probably due to the paradoxical proapoptotic activity of this important regulator of apoptosis.

The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin.

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.

Manipulation of NK cytotoxicity by the IAP family member Livin.

Natural killer (NK) cells are part of the innate immune system, capable of killing tumor and virally infected cells. NK cells induce apoptosis in the target cell by either granule- or receptor-mediated pathways. A set of inhibitory and activation ligands governs NK cell activation. As transformed cells often attempt to evade NK cell killing, up-regulation of a potential anti-apoptotic factor should provide a survival advantage. The inhibitor of apoptosis protein (IAP) family can inhibit apoptosis induced by a variety of stimuli. We have previously described a new IAP family member, termed Livin, which has two splice variants (alpha and beta) with differential anti-apoptotic activities. In this study, we explore the ability of Livin to inhibit NK cell-induced killing. We demonstrate that Livin beta moderately protects against NK cell killing whereas Livin alpha augments killing. We show that Livin beta inhibition in Jurkat cells is apparent upon concomitant activation of an inhibitory signal, suggesting that Livin augments an extrinsic inhibitory signal rather than functioning as an independent inhibitory mechanism. Finally, we demonstrate that detection of both Livin isoforms in melanoma cells correlates with a low killing rate. To date, this is the first evidence that directly demonstrates the ability of IAP to protect against NK cell-induced apoptosis.

Subcellular localization determines the delicate balance between the anti- and pro-apoptotic activity of Livin.

Livin is a member of the Inhibitor of Apoptosis Protein family which inhibits apoptosis induced by a variety of stimuli. We previously identified Livin and demonstrated that following apoptotic stimuli, Livin is cleaved by effector caspases to produce a truncated form with paradoxical pro-apoptotic activity. In the present study, we reveal that while full-length Livin shows diffuse cytoplasmic localization, truncated Livin (tLivin) is found in a peri-nuclear distribution with marked localization to the Golgi apparatus. Using mutation analysis, we identified two domains that are crucial for the pro-apoptotic activity of tLivin: the N-terminal region of tLivin which is exposed by cleavage, and the RING domain. We demonstrate that, of the N-terminal sequence, only the first N-terminal glycine residue dictates the peri-nuclear distribution of tLivin. However, while the perinuclear localization of tLivin is essential, it is not sufficient for tLivin to exert its pro-apoptotic function. Once tLivin is properly localized, an intact RING domain enables its pro-apoptotic function.