Tumor-treating fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy.

Tumor-treating fields (TTFields) are alternating electric fields in a specific frequency range (100-300 kHz) delivered to the human body through transducer arrays. In this study, we evaluated whether TTFields-mediated cell death can elicit antitumoral immunity and hence would be effectively combined with anti-PD-1 therapy. We demonstrate that in TTFields-treated cancer cells, damage-associated molecular patterns including high-mobility group B1 and adenosine triphosphate are released and calreticulin is exposed on the cell surface. Moreover, we show that TTFields treatment promotes the engulfment of cancer cells by dendritic cells (DCs) and DCs maturation in vitro, as well as recruitment of immune cells in vivo. Additionally, our study demonstrates that the combination of TTFields with anti-PD-1 therapy results in a significant decline of tumor volume and increase in the percentage of tumor-infiltrating leukocytes in two tumor models. In orthotopic lung tumors, these infiltrating leukocytes, specifically macrophages and DCs, showed elevated expression of PD-L1. Compatibly, cytotoxic T-cells isolated from these tumors demonstrated increased production of IFN-γ. In colon cancer tumors, T-cells infiltration was significantly increased following long treatment duration with TTFields plus anti-PD-1. Collectively, our results suggest that TTFields therapy can induce anticancer immune response. Furthermore, we demonstrate robust efficacy of concomitant application of TTFields and anti-PD-1 therapy. These data suggest that integrating TTFields with anti-PD-1 therapy may further enhance antitumor immunity, hence achieve better tumor control.

The viral KSHV chemokine vMIP-II inhibits the migration of Naive and activated human NK cells by antagonizing two distinct chemokine receptors.

Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56(Dim) CD16(Pos)) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56(Bright) CD16(Neg)). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56(Dim) CD16(Pos) NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.

Tumor Treating Fields (TTFields) Hinder Cancer Cell Motility through Regulation of Microtubule and Acting Dynamics.

Tumor Treating Fields (TTFields) are noninvasive, alternating electric fields within the intermediate frequency range (100-300 kHz) that are utilized as an antimitotic cancer treatment. TTFields are loco-regionally delivered to the tumor region through 2 pairs of transducer arrays placed on the skin. This novel treatment modality has been FDA-approved for use in patients with glioblastoma and malignant pleural mesothelioma based on clinical trial data demonstrating efficacy and safety; and is currently under investigation in other types of solid tumors. TTFields were shown to induce an anti-mitotic effect by exerting bi-directional forces on highly polar intracellular elements, such as tubulin and septin molecules, eliciting abnormal microtubule polymerization during spindle formation as well as aberrant cleavage furrow formation. Previous studies have demonstrated that TTFields inhibit metastatic properties in cancer cells. However, the consequences of TTFields application on cytoskeleton dynamics remain undetermined. In this study, methods utilized in combination to study the effects of TTFields on cancer cell motility through regulation of microtubule and actin dynamics included confocal microscopy, computational tools, and biochemical analyses. Mechanisms by which TTFields treatment disrupted cellular polarity were (1) interference with microtubule assembly and directionality; (2) altered regulation of Guanine nucleotide exchange factor-H1 (GEF-H1), Ras homolog family member A (RhoA), and Rho-associated coiled-coil kinase (ROCK) activity; and (3) induced formation of radial protrusions of peripheral actin filaments and focal adhesions. Overall, these data identified discrete effects of TTFields that disrupt processes crucial for cancer cell motility.

Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion.

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.

AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields).

Tumor Treating Fields (TTFields), an approved treatment modality for glioblastoma, are delivered via non-invasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application leads to abnormal mitosis, aneuploidy, and increased cell granularity, which are often associated with enhancement of autophagy. In this work, we evaluated whether TTFields effected the regulation of autophagy in glioma cells. We found that autophagy is upregulated in glioma cells treated with TTFields as demonstrated by immunoblot analysis of the lipidated microtubule-associated protein light chain 3 (LC3-II). Fluorescence and transmission electron microscopy demonstrated the presence of LC3 puncta and typical autophagosome-like structures in TTFields-treated cells. Utilizing time-lapse microscopy, we found that the significant increase in the formation of LC3 puncta was specific to cells that divided during TTFields application. Evaluation of selected cell stress parameters revealed an increase in the expression of the endoplasmic reticulum (ER) stress marker GRP78 and decreased intracellular ATP levels, both of which are indicative of increased proteotoxic stress. Pathway analysis demonstrated that TTFields-induced upregulation of autophagy is dependent on AMP-activated protein kinase (AMPK) activation. Depletion of AMPK or autophagy-related protein 7 (ATG7) inhibited the upregulation of autophagy in response to TTFields, as well as sensitized cells to the treatment, suggesting that cancer cells utilize autophagy as a resistance mechanism to TTFields. Combining TTFields with the autophagy inhibitor chloroquine (CQ) resulted in a significant dose-dependent reduction in cell growth compared with either TTFields or CQ alone. These results suggest that dividing cells upregulate autophagy in response to aneuploidy and ER stress induced by TTFields, and that AMPK serves as a key regulator of this process.

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields).

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (Ɛ > 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.

Inflammatory monocytes and NK cells play a crucial role in DNAM-1-dependent control of cytomegalovirus infection.

The poliovirus receptor (PVR) is a ubiquitously expressed glycoprotein involved in cellular adhesion and immune response. It engages the activating receptor DNAX accessory molecule (DNAM)-1, the inhibitory receptor TIGIT, and the CD96 receptor with both activating and inhibitory functions. Human cytomegalovirus (HCMV) down-regulates PVR expression, but the significance of this viral function in vivo remains unknown. Here, we demonstrate that mouse CMV (MCMV) also down-regulates the surface PVR. The m20.1 protein of MCMV retains PVR in the endoplasmic reticulum and promotes its degradation. A MCMV mutant lacking the PVR inhibitor was attenuated in normal mice but not in mice lacking DNAM-1. This attenuation was partially reversed by NK cell depletion, whereas the simultaneous depletion of mononuclear phagocytes abolished the virus control. This effect was associated with the increased expression of DNAM-1, whereas TIGIT and CD96 were absent on these cells. An increased level of proinflammatory cytokines in sera of mice infected with the virus lacking the m20.1 and an increased production of iNOS by inflammatory monocytes was observed. Blocking of CCL2 or the inhibition of iNOS significantly increased titer of the virus lacking m20.1. In this study, we have demonstrated that inflammatory monocytes, together with NK cells, are essential in the early control of CMV through the DNAM-1-PVR pathway.

Cytokine secretion and NK cell activity in human ADAM17 deficiency.

Genetic deficiencies provide insights into gene function in humans. Here we describe a patient with a very rare genetic deficiency of ADAM17. We show that the patient's PBMCs had impaired cytokine secretion in response to LPS stimulation, correlating with the clinical picture of severe bacteremia from which the patient suffered. ADAM17 was shown to cleave CD16, a major NK killer receptor. Functional analysis of patient's NK cells demonstrated that his NK cells express normal levels of activating receptors and maintain high surface levels of CD16 following mAb stimulation. Activation of individual NK cell receptors showed that the patient's NK cells are more potent when activated directly by CD16, albeit no difference was observed in Antibody Depedent Cytotoxicity (ADCC) assays. Our data suggest that ADAM17 inhibitors currently considered for clinical use to boost CD16 activity should be cautiously applied, as they might have severe side effects resulting from impaired cytokine secretion.