Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune pathways in antigen-presenting cells.

Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS-STING (for cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses.

Radiation Synergizes with IL2/IL15 Stimulation to Enhance Innate Immune Activation and Antitumor Immunity.

Ionizing radiation is known to possess immune modulatory properties. However, how radiotherapy (RT) may complement with different types of immunotherapies to boost antitumor responses is unclear. In mice implanted with EO771 syngeneic tumors, NL-201 a stable, highly potent CD25-independent agonist to IL2 and IL15 receptors with enhanced affinity for IL2Rßγ was given with or without RT. Flow analysis and Western blot analysis was performed to determine the mechanisms involved. STING (-/-) and CD11c+ knockout mice were implanted with EO771 tumors to confirm the essential signaling and cell types required to mediate the effects seen. Combination of RT and NL-201 to enhance systemic immunotherapy with an anti-PD-1 checkpoint inhibitor was utilized to determine tumor growth inhibition and survival, along characterization of tumor microenvironment as compared with all other treatment groups. Here, we showed that RT, synergizing with NL-201 produced enhanced antitumor immune responses in murine breast cancer models. When given together, RT and NL-201 enhanced activation of the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway, resulting in increased type I IFN production in dendritic cells (DC), and consequently greater tumor infiltration and more efficient priming of antigen-specific T cells. The immune stimulatory mechanisms triggered by NL-201 and RT resulted in superior tumor growth inhibition and survival benefit in both localized and metastatic cancers. Our results support further preclinical and clinical investigation of this novel synergism regimen in locally advanced and metastatic settings.

Age-associated disparity in phagocytic clearance affects the efficacy of cancer nanotherapeutics.

Nanomedicines have been approved to treat multiple human diseases. However, clinical adoption of nanoformulated agents is often hindered by concerns about hepatic uptake and clearance, a process that is not fully understood. Here we show that the antitumour efficacy of cancer nanomedicine exhibits an age-associated disparity. Tumour delivery and treatment outcomes are superior in old versus young mice, probably due to an age-related decline in the ability of hepatic phagocytes to take up and remove nanoparticles. Transcriptomic- and protein-level analysis at the single-cell and bulk levels reveals an age-associated decrease in the numbers of hepatic macrophages that express the scavenger receptor MARCO in mice, non-human primates and humans. Therapeutic blockade of MARCO is shown to decrease the phagocytic uptake of nanoparticles and improve the antitumour effect of clinically approved cancer nanotherapeutics in young but not aged mice. Together, these results reveal an age-associated disparity in the phagocytic clearance of nanotherapeutics that affects their antitumour response, thus providing a strong rationale for an age-appropriate approach to cancer nanomedicine.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

Adaptive design of mRNA-loaded extracellular vesicles for targeted immunotherapy of cancer.

The recent success of mRNA therapeutics against pathogenic infections has increased interest in their use for other human diseases including cancer. However, the precise delivery of the genetic cargo to cells and tissues of interest remains challenging. Here, we show an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded small extracellular vesicles (sEVs). This is achieved by using a microfluidic electroporation approach in which a combination of nano- and milli-second pulses produces large amounts of IFN-γ mRNA-loaded sEVs with CD64 overexpressed on their surface. The CD64 molecule serves as an adaptor to dock targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. The resulting immunogenic sEVs (imsEV) preferentially target glioblastoma cells and generate potent antitumour activities in vivo, including against tumours intrinsically resistant to immunotherapy. Together, these results provide an adaptive approach to engineering mRNA-loaded sEVs with targeting functionality and pave the way for their adoption in cancer immunotherapy applications.

Targeting phagocytosis to enhance antitumor immunity.

Many patients with metastatic or treatment-resistant cancer have experienced improved outcomes after immunotherapy that targets adaptive immune checkpoints. However, innate immune checkpoints, which can hinder the detection and clearance of malignant cells, are also crucial in tumor-mediated immune escape and may also serve as targets in cancer immunotherapy. In this review, we discuss the current understanding of immune evasion by cancer cells via disruption of phagocytic clearance, and the potential effects of blocking phagocytosis checkpoints on the activation of antitumor immune responses. We propose that a more effective combination immunotherapy strategy could be to exploit tumor-intrinsic processes that inhibit key innate immune surveillance processes, such as phagocytosis, and incorporate both innate and adaptive immune responses for treating patients with cancer.

Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy.

The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.

COVID-19 and Family Distancing Efforts: Contextual Demographic and Family Conflict Correlates.

Since the COVID-19 outbreak, family members have spent more time together at home. This study introduces the concept of "family distancing"-the efforts to prevent the spread of the coronavirus to family members. We explore which demographic characteristics are associated with family distancing efforts and how the family distancing efforts are associated with family conflicts. Survey data were collected from adults (N = 324, M = 37 years; SD = 10.5 years; 65.1% female) in Korea. We found that gender, education, marital status, physical health status, and number of family members who live together were significantly associated with family distancing efforts. In addition, lower compliance with the request for family distancing was significantly associated with a higher degree of negative emotions (i.e., anger), which in turn was associated with more family conflict. The findings highlight the potential importance of family distancing efforts to maintain health but also their potential to increase family conflict.

Enhanced Immunogenic Cell Death by Apoptosis/Ferroptosis Hybrid Pathway Potentiates PD-L1 Blockade Cancer Immunotherapy.

Even though chemotherapy regimens for treating cancer by inducing apoptosis are extensively utilized, their therapeutic effect is hindered by multiple limitations. Thus, a combination of other types of anticancer modalities is urgently needed. Herein, a tannic acid (TA)-Fe3+-coated doxorubicin (DOX)-encapsulated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (ammonium salt) (DSPE-PEG) micelle (TFDD) for apoptosis/ferroptosis-mediated immunogenic cell death (ICD) is reported. By coating TA-Fe3+ on the surface of DOX-loaded micelles, an apoptotic agent and a ferroptotic agent are simultaneously delivered into the cancer cells and induce cell death. Furthermore, the intracellular oxidative environment generated by the apoptosis/ferroptosis hybrid pathway stimulates the endoplasmic reticulum (ER) and leads to ICD induction. The in vivo results show that the combination treatment of TFDD and anti-programmed death-ligand 1 antibodies (anti-PD-L1) considerably inhibits tumor growth and improves antitumor immunity by activating CD4+ and CD8+ T cells and decreasing the ratio of regulatory T cells (Treg) to CD4+ T cells. This study suggests that the apoptosis/ferroptosis-mediated ICD inducer may offer a potent strategy for enhanced cancer immunotherapy.

Immunological conversion of solid tumours using a bispecific nanobioconjugate for cancer immunotherapy.

Solid tumours display a limited response to immunotherapies. By contrast, haematological malignancies exhibit significantly higher response rates to immunotherapies as compared with solid tumours. Among several microenvironmental and biological disparities, the differential expression of unique immune regulatory molecules contributes significantly to the interaction of blood cancer cells with immune cells. The self-ligand receptor of the signalling lymphocytic activation molecule family member 7 (SLAMF7), a molecule that is critical in promoting the body's innate immune cells to detect and engulf cancer cells, is expressed nearly exclusively on the cell surface of haematologic tumours, but not on solid ones. Here we show that a bispecific nanobioconjugate that enables the decoration of SLAMF7 on the surface of solid tumours induces robust phagocytosis and activates the phagocyte cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway, sensitizing the tumours to immune checkpoint blockade. Our findings support an immunological conversion strategy that uses nano-adjuvants to improve the effectiveness of immunotherapies for solid tumours.

Cancer immunotherapy based on image-guided STING activation by nucleotide nanocomplex-decorated ultrasound microbubbles.

The cytosolic innate immune sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is crucial for priming adaptive antitumour immunity through antigen-presenting cells (APCs). Natural agonists, such as cyclic dinucleotides (CDNs), activate the cGAS-STING pathway, but their clinical translation is impeded by poor cytosolic entry and serum stability, low specificity and rapid tissue clearance. Here we developed an ultrasound (US)-guided cancer immunotherapy platform using nanocomplexes composed of 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) electrostatically bound to biocompatible branched cationic biopolymers that are conjugated onto APC-targeting microbubbles (MBs). The nanocomplex-conjugated MBs engaged with APCs and efficiently delivered cGAMP into the cytosol via sonoporation, resulting in activation of cGAS-STING and downstream proinflammatory pathways that efficiently prime antigen-specific T cells. This bridging of innate and adaptive immunity inhibited tumour growth in both localized and metastatic murine cancer models. Our findings demonstrate that targeted local activation of STING in APCs under spatiotemporal US stimulation results in systemic antitumour immunity and improves the therapeutic efficacy of checkpoint blockade, thus paving the way towards novel image-guided strategies for targeted immunotherapy of cancer.

Lexical Diversity, Lexical Sophistication, and Predictability for Speech in Multiple Listening Conditions.

When talkers anticipate that a listener may have difficulty understanding their speech, they adopt a speaking style typically described as "clear speech." This speaking style includes a variety of acoustic modifications and has perceptual benefits for listeners. In the present study, we examine whether clear speaking styles also include modulation of lexical items selected and produced during naturalistic conversations. Our results demonstrate that talkers do, indeed, modulate their lexical selection, as measured by a variety of lexical diversity and lexical sophistication indices. Further, the results demonstrate that clear speech is not a monolithic construct. Talkers modulate their speech differently depending on the communication situation. We suggest that clear speech should be conceptualized as a set of speaking styles, in which talkers take the listener and communication situation into consideration.

Harnessing Innate Immunity Using Biomaterials for Cancer Immunotherapy.

The discovery of immune checkpoint blockade has revolutionized the field of immuno-oncology and established the foundation for developing various new therapies that can surpass conventional cancer treatments. Most recent immunotherapeutic strategies have focused on adaptive immune responses by targeting T cell-activating pathways, genetic engineering of T cells with chimeric antigen receptors, or bispecific antibodies. Despite the unprecedented clinical success, these T cell-based treatments have only benefited a small proportion of patients. Thus, the need for the next generation of cancer immunotherapy is driven by identifying novel therapeutic molecules or new immunoengineered cells. To maximize the therapeutic potency via innate immunogenicity, the convergence of innate immunity-based therapy and biomaterials is required to yield an efficient index in clinical trials. This review highlights how biomaterials can efficiently reprogram and recruit innate immune cells in tumors and ultimately initiate activation of T cell immunity against advanced cancers. Moreover, the design and specific biomaterials that improve innate immune cells' targeting ability to selectively activate immunogenicity with minimal adverse effects are discussed.

Immunogenic Cell Death Inducing Fluorinated Mitochondria-Disrupting Helical Polypeptide Synergizes with PD-L1 Immune Checkpoint Blockade.

Immunogenic cell death (ICD) is distinguished by the release of tumor-associated antigens (TAAs) and danger-associated molecular patterns (DAMPs). This cell death has been studied in the field of cancer immunotherapy due to the ability of ICD to induce antitumor immunity. Herein, endoplasmic reticulum (ER) stress-mediated ICD inducing fluorinated mitochondria-disrupting helical polypeptides (MDHPs) are reported. The fluorination of the polypeptide provides a high helical structure and potent anticancer ability. This helical polypeptide destabilizes the mitochondrial outer membrane, leading to the overproduction of intracellular reactive oxygen species (ROS) and apoptosis. In addition, this oxidative stress triggers ER stress-mediated ICD. The in vivo results show that cotreatment of fluorinated MDHP and antiprogrammed death-ligand 1 antibodies (αPD-L1) significantly regresses tumor growth and prevents metastasis to the lungs by activating the cytotoxic T cell response and alleviating the immunosuppressive tumor microenvironment. These results indicate that fluorinated MDHP synergizes with the immune checkpoint blockade therapy to eliminate established tumors and to elicit antitumor immune responses.

Non-native English listeners' adaptation to native English speakers.

Listeners often have difficulty understanding unfamiliar speech (e.g., non-native speech), but they are able to adapt to or improve their ability to understand unfamiliar speech. However, it is unclear whether non-native listeners demonstrate adaptation to novel native English speech broadly with relatively limited exposure. Thus, this study examines non-native English listeners' adaptation to native English speakers and whether talker variability affects adaptation. Results suggest that while greater variability initially disrupts non-native English listeners' perception of native English speakers, listeners are able to rapidly adapt to novel speakers and exposure to greater variability could result in cross-talker generalization.

Polypeptide-Based K+ Ionophore as a Strong Immunogenic Cell Death Inducer for Cancer Immunotherapy.

Immunogenic cell death (ICD) is a key factor for generating antitumor immunity. Endoplasmic reticulum (ER) stress triggers the release of damage-associated molecular patterns (DAMPs), thus inducing immunogenicity. We developed a polypeptide-based K+ ionophore that perturbed ion homeostasis and elicited a prolonged ER stress. The ER stress not only fosters an oxidative environment that activates mitochondria-dependent apoptosis pathways but also drives immune responses by releasing DAMPs. The ionophore suppressed tumor proliferation in vitro and in vivo based on the pro-apoptotic activity and immunogenicity.

The maintenance of clear speech in naturalistic conversations.

Clear speech is a style that speakers adopt when talking with listeners whom these speakers anticipate may have a problem understanding speech. This study examines whether native English speakers use clear speech in conversations with non-native English speakers when native speakers are not explicitly asked to use clear speech (i.e., clear speech elicited with naturalistic methods). The results of the study suggest that native English speakers use clear speech in conversations with non-native English speakers even when native speakers are not explicitly asked to. Native English speakers' speech is more intelligible in the early portions of the conversations than in the late portions of each conversation. Further, the speakers "reset" to clearer speech at the start of each Diapix picture. Additionally, acoustic properties of the speech are examined to complement the intelligibility results. These findings suggest the instigation of clear speech may be listener-driven but the maintenance of clear speech is likely more speaker-driven.

Structure-inherent near-infrared bilayer nanovesicles for use as photoacoustic image-guided chemo-thermotherapy.

Image-guided therapy, combined with imaging and therapeutic action, forms an attractive system because it can induce outstanding effects at focused locations. However, the conventional liposomes-based system cannot figure in therapeutic or imaging roles themselves, thereby causing the disadvantage of their biological unavailability as a theragnosis tool. Herein, the structure-inherent near-infrared bilayer nanovesicles are fabricated with amphiphilic heptamethine cyanine dye, PEG conjugated heptamethine cyanine dye, and gemcitabine (NEPCG) is developed for the novel photoacoustic image-guided chemo-thermotherapy system. The organic structure-inherent near-infrared bilayer nanovesicles are self-assembled and exhibit a liposome-like bilayer structure. Furthermore, NEPCG showed the high photoacoustic signal (PA) due to the specific accumulation in the tumor site. Delivered NEPCG than displayed concurrent chemotherapy and photothermal therapy (PTT) effects against cancer, triggered by PA imaging with minimal side effects. In vitro and in vivo experiments show that NEPCG can be used as outstanding contrast agents and completely obliterate the tumor without reoccurrence under laser irradiation. Therefore, this work presents the potential for the realization of unprecedented structure-inherent near-infrared bilayer nanovesicles as highly accurate and effective theragnostic tools in clinical fields.

CD44-Mediated Methotrexate Delivery by Hyaluronan-Coated Nanoparticles Composed of a Branched Cell-Penetrating Peptide.

Branched polymers as drug delivery carriers have been widely attempted due to their outstanding drug loading capability and complex stability like branched polyethyleneimine (B-PEI). However, branched polymers without biodegradability may cause toxicity as they can accumulate in the body. Herein, we report branched modified nona-arginine (B-mR9) composed of redox-cleavable disulfide bonds to form stable complexes with methotrexate (MTX) as an anticancer agent, which is further coated with hyaluronic acid (HA). The HA-coated nanoparticles provide targetability for the CD44 cell surface receptor. The B-mR9-MTX/HA can effectively aid in intracellular MTX delivery to CD44 overexpressing cancer cells being degradable by the reducing environments of the cancer cells. The B-mR9-MTX/HA exhibits not only a glutathione-triggered degradability but also an outstanding CD44-mediated MTX delivery efficacy. In addition, its superior tumor inhibition capability was confirmed through an in vivo study. The results suggest that the HA-coated B-mR9 nanoparticle can be used as a drug delivery platform.