CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression.

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

Silencing CA1 pyramidal cells output reveals the role of feedback inhibition in hippocampal oscillations.

The precise temporal coordination of neural activity is crucial for brain function. In the hippocampus, this precision is reflected in the oscillatory rhythms observed in CA1. While it is known that a balance between excitatory and inhibitory activity is necessary to generate and maintain these oscillations, the differential contribution of feedforward and feedback inhibition remains ambiguous. Here we use conditional genetics to chronically silence CA1 pyramidal cell transmission, ablating the ability of these neurons to recruit feedback inhibition in the local circuit, while recording physiological activity in mice. We find that this intervention leads to local pathophysiological events, with ripple amplitude and intrinsic frequency becoming significantly larger and spatially triggered local population spikes locked to the trough of the theta oscillation appearing during movement. These phenotypes demonstrate that feedback inhibition is crucial in maintaining local sparsity of activation and reveal the key role of lateral inhibition in CA1 in shaping circuit function.

COVID-19 Outcomes in Inflammatory Bowel Disease Hospitalized Patients: A Comprehensive Analysis Using the National Inpatient Sample.

Background: There is no uniformity in the available literature concerning the effects of coronavirus disease 2019 (COVID-19) viral illness on people with inflammatory bowel disease (IBD). Methods: We conducted an analysis using the 2020 National Inpatient Sample (NIS) database to compare the outcomes of COVID-19 hospitalized patients with and without IBD. Results: Of 1,050,040 patients admitted with COVID-19, 5,750 (0.5%) also had IBD. The group with COVID-19 and IBD had higher percentages of females and White individuals and a greater prevalence of chronic lung disease, peripheral vascular disease, and liver disease. However, after accounting for confounding variables, there was no significant difference in mortality rates, length of hospital stays, or hospitalization costs between the two groups. Conclusion: According to our findings, the presence of IBD does not appear to elevate the risk of COVID-19 complications.

STING activation counters glioblastoma by vascular alteration and immune surveillance.

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease. In this study, we used two preclinical models of GBM: orthotopically delivering either GBM stem cells or employing CRISPR-mediated tumorigenesis by adeno-associated virus, to establish immunologically proficient and non-inflamed tumors, respectively. After tumor development, the innate immune system was activated through long-term STING activation by a pharmacological agonist, which reduced tumor progression and prolonged survival. Recruitment and activation of cytotoxic T-cells were detected in the tumors, and T-cell specificity towards the cancer cells was observed. Interestingly, prolonged STING activation altered the tumor vasculature, inducing hypoxia and activation of VEGFR, as measured by a kinome array and VEGF expression. Combination treatment with anti-PD1 did not provide a synergistic effect, indicating that STING activation alone is sufficient to activate immune surveillance and hinder tumor development through vascular disruption. These results guide future studies to refine innate immune activation as a treatment approach for GBM, in combination with anti-VEGF to impede tumor progression and induce an immunological response against the tumor.

GAP43-dependent mitochondria transfer from astrocytes enhances glioblastoma tumorigenicity.

The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.

The Impact of Complementary and Integrative Medicine Following Traumatic Brain Injury: A Scoping Review.

OBJECTIVE: To examine the evidence levels, study characteristics, and outcomes of nonpharmacologic complementary and integrative medicine (CIM) interventions in rehabilitation for individuals with traumatic brain injury (TBI). DATA SOURCES: MEDLINE (OvidSP), PubMed (NLM), EMBASE ( Embase.com ), CINAHL (EBSCO), PsycINFO (OvidSP), Cochrane Library (Wiley), and National Guidelines Clearinghouse databases were evaluated using PRISMA guidelines. The protocol was registered in INPLASY (protocol registration: INPLASY202160071). DATA EXTRACTION: Quantitative studies published between 1992 and 2020 investigating the efficacy of CIM for individuals with TBI of any severity, age, and outcome were included. Special diets, herbal and dietary supplements, and counseling/psychological interventions were excluded, as were studies with mixed samples if TBI data could not be extracted. A 2-level review comprised title/abstract screening, followed by full-text assessment by 2 independent reviewers. DATA SYNTHESIS: In total, 90 studies were included, with 57 001 patients in total. This total includes 2 retrospective studies with 17 475 and 37 045 patients. Of the 90 studies, 18 (20%) were randomized controlled trials (RCTs). The remainder included 20 quasi-experimental studies (2-group or 1-group pre/posttreatment comparison), 9 retrospective studies, 1 single-subject study design, 2 mixed-methods designs, and 40 case study/case reports. Guided by the American Academy of Neurology evidence levels, class II criteria were met by 61% of the RCTs. Included studies examined biofeedback/neurofeedback (40%), acupuncture (22%), yoga/tai chi (11%), meditation/mindfulness/relaxation (11%), and chiropractic/osteopathic manipulation (11%). The clinical outcomes evaluated across studies included physical impairments (62%), mental health (49%), cognitive impairments (39%), pain (31%), and activities of daily living/quality of life (28%). Additional descriptive statistics were summarized using narrative synthesis. Of the studies included for analyses, 97% reported overall positive benefits of CIM. CONCLUSION: Rigorous and well experimentally designed studies (including RCTs) are needed to confirm the initial evidence supporting the use of CIM found in the existing literature.

DTP-Net: A convolutional neural network model to predict threshold for localizing the lesions on dermatological macro-images.

Highly focused images of skin captured with ordinary cameras, called macro-images, are extensively used in dermatology. Being highly focused views, the macro-images contain only lesions and background regions. Hence, the localization of lesions on the macro-images is a simple thresholding problem. However, algorithms that offer an accurate estimate of threshold and retain consistent performance on different dermatological macro-images are rare. A deep learning model, termed 'Deep Threshold Prediction Network (DTP-Net)', is proposed in this paper to address this issue. For training the model, grayscale versions of the macro-images are fed as input to the model, and the corresponding gray-level threshold values at which the Dice similarity index (DSI) between the segmented and the ground-truth images are maximized are defined as the targets. The DTP-Net exhibited the least value of root mean square error for the predicted threshold, compared with 11 state-of-the-art threshold estimation algorithms (such as Otsu's thresholding, Valley emphasized otsu's thresholding, Isodata thresholding, Histogram slope difference distribution-based thresholding, Minimum error thresholding, Poisson's distribution-based minimum error thresholding, Kapur's maximum entropy thresholding, Entropy-weighted otsu's thresholding, Minimum cross-entropy thresholding, Type-2 fuzzy-based thresholding, and Fuzzy entropy thresholding). The DTP-Net could learn the difference between the lesion and background in the intensity space and accurately predict the threshold that separates the lesion from the background. The proposed DTP-Net can be integrated into the segmentation module in automated tools that detect skin cancer from dermatological macro-images.

An EfficientNet-based modified sigmoid transform for enhancing dermatological macro-images of melanoma and nevi skin lesions.

BACKGROUND AND OBJECTIVE: During the initial stages, skin lesions may not have sufficient intensity difference or contrast from the background region on dermatological macro-images. The lack of proper light exposure at the time of capturing the image also reduces the contrast. Low contrast between lesion and background regions adversely impacts segmentation. Enhancement techniques for improving the contrast between lesion and background skin on dermatological macro-images are limited in the literature. An EfficientNet-based modified sigmoid transform for enhancing the contrast on dermatological macro-images is proposed to address this issue. METHODS: A modified sigmoid transform is applied in the HSV color space. The crossover point in the modified sigmoid transform that divides the macro-image into lesion and background is predicted using a modified EfficientNet regressor to exclude manual intervention and subjectivity. The Modified EfficientNet regressor is constructed by replacing the classifier layer in the conventional EfficientNet with a regression layer. Transfer learning is employed to reduce the training time and size of the dataset required to train the modified EfficientNet regressor. For training the modified EfficientNet regressor, a set of value components extracted from the HSV color space representation of the macro-images in the training dataset is fed as input. The corresponding set of ideal crossover points at which the values of Dice similarity coefficient (DSC) between the ground-truth images and the segmented output images obtained from Otsu's thresholding are maximum, is defined as the target. RESULTS: On images enhanced with the proposed framework, the DSC of segmented results obtained by Otsu's thresholding increased from 0.68 ± 0.34 to 0.81 ± 0.17. CONCLUSIONS: The proposed algorithm could consistently improve the contrast between lesion and background on a comprehensive set of test images, justifying its applications in automated analysis of dermatological macro-images.

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in peach reproductive tissues.

BACKGROUND: Perennial fruit trees display a growth behaviour characterized by annual cycling between growth and dormancy, with complex physiological features. Rosaceae fruit trees represent excellent models for studying not only the fruit growth/patterning but also the progression of the reproductive cycle depending upon the impact of climate conditions. Additionally, current developments in high-throughput technologies have impacted Rosaceae tree research while investigating genome structure and function as well as (epi)genetic mechanisms involved in important developmental and environmental response processes during fruit tree growth. Among epigenetic mechanisms, chromatin remodelling mediated by histone modifications and other chromatin-related processes play a crucial role in gene modulation, controlling gene expression. Chromatin immunoprecipitation is an effective technique to investigate chromatin dynamics in plants. This technique is generally applied for studies on chromatin states and enrichment of post-transcriptional modifications (PTMs) in histone proteins. RESULTS: Peach is considered a model organism among climacteric fruits in the Rosaceae family for studies on bud formation, dormancy, and organ differentiation. In our work, we have primarily established specific protocols for chromatin extraction and immunoprecipitation in reproductive tissues of peach (Prunus persica). Subsequently, we focused our investigations on the role of two chromatin marks, namely the trimethylation of histone H3 at lysine in position 4 (H3K4me3) and trimethylation of histone H3 at lysine 27 (H3K27me3) in modulating specific gene expression. Bud dormancy and fruit growth were investigated in a nectarine genotype called Fantasia as our model system. CONCLUSIONS: We present general strategies to optimize ChIP protocols for buds and mesocarp tissues of peach and analyze the correlation between gene expression and chromatin mark enrichment/depletion. The procedures proposed may be useful to evaluate any involvement of histone modifications in the regulation of gene expression during bud dormancy progression and core ripening in fruits.

A customized acutance metric for quality control applications in MRI.

Acutance is a subjective parameter which indicates the quality of edges in an image. Objective metrics for measuring image acutance are helpful for designing new imaging protocols and sequences in magnetic resonance imaging (MRI) studies. In addition to this, image acutance metrics have a significant role in the design and optimisation of post-processing algorithms used for restoration and sharpening of MR imagery. Most of the existing blur/sharpness metrics are specifically designed for natural-scene (panoramic) images. A blur/sharpness metric suitable for MR imaging applications is absent in the literature. To fill this gap, a computationally fast metric, 'largest local gradient-based sharpness metric (LLGSM)', for measuring sharpness and blur in MR imagery, is proposed in this paper. The LLGSM is the root mean square (RMS) of exponentially weighted elements in an array of lexicographically ordered largest local gradient (LLG) values in the image, sorted in descending order. In terms of overall agreement with subjective scores, and computational speed, the LLGSM is observed to be more efficient than its alternatives available in the literature.

A Noise-robust and Overshoot-free Alternative to Unsharp Masking for Enhancing the Acuity of MR Images.

Poor acutance of images (unsharpness) is one of the major concerns in magnetic resonance imaging (MRI). MRI-based diagnosis and clinical interventions become difficult due to the vague textural information and weak morphological margins on images. A novel image sharpening algorithm named as maximum local variation-based unsharp masking (MLVUM) to address the issue of 'unsharpness' in MRI is proposed in this paper. In the MLVUM, the sharpened image is the algebraic sum of the input image and the product of the user-defined scale and the difference between the output of a newly designed nonlinear spatial filter named maximum local variation-controlled edge smoothing Gaussian filter (MLVESGF) and the input image, weighted by the normalised MLV. The MLVESGF is a locally adaptive 2D Gaussian edge smoothing kernel whose standard deviation is directly proportional to the local value of the normalized MLV. The values of the acutance-to-noise ratio (ANR) and absolute mean brightness error (AMBE) shown by the MLVUM on 100 MRI slices are 0.6463 ± 0.1852 and 0.3323 ± 0.2200, respectively. Compared to 17 state-of-the-art image sharpening algorithms, the MLVUM exhibited a higher ANR and lower AMBE. The MLVUM selectively enhances the sharpness of edges in the MR images without amplifying the background noise without altering the mean brightness level.

Early Initiation of Voice Therapy After Microlaryngeal Surgery - A Randomized Control Study.

A prospective single blinded randomized control trial was conducted to study if early initiation of voice therapy after microlaryngeal produces a better voice outcome in patients with benign vocal fold lesions. METHODS: In this Patients undergoing microlaryngeal surgery for benign vocal fold lesions were recruited for the study and underwent voice evaluation and videostroboscopy preoperatively. Participants were randomised into two groups depending on the duration of voice rest-A (2 days voice rest) and B (5 days voice rest). Following the period of voice rest, voice therapy (tube phonation) was carried out for a month. Postoperative evaluation was done at 6 weeks (over telephone) and 3 months (in person visit) follow up. Outcome measures included the VHI-10, auditory-perceptual voice ratings, acoustic analysis and videostroboscopic vibratory ratings. RESULTS: Of the 50 subjects, 35 completed the follow up evaluation. The overall compliance to absolute voice rest was 43%. Among the various parameters used for voice evaluation, there was no statistically significant difference between the two groups except for jitter, where the improvement in 5 day voice rest group compared to the 2 day voice rest group was statistically significant. CONCLUSION: Prolonged voice rest after microlaryngeal surgery is difficult to comply with. As there was no significant difference between the two study groups, clinicians may prescribe a shorter duration of voice rest followed by early initiation of voice therapy after microlaryngeal surgery.

TGF-ß promotes microtube formation in glioblastoma through thrombospondin 1.

BACKGROUND: Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular, glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. METHODS: Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate MT formation under transforming growth factor-beta (TGF-ß) stimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-ß. RESULTS: Analysis of TCGA data showed that the TGF-ß pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-ß1 stimulation of GBM cell lines promotes enhanced MT formation and communication via calcium signaling. Inhibition of the TGF-ß pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-ß, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-ß stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. CONCLUSION: TGF-ß and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT-driven invasion/resistance network.

Three-dimensional culture models to study glioblastoma - current trends and future perspectives.

Glioblastoma (GBM) is the most prevalent form of primary malignant brain tumor in adults and remains almost invariably lethal owing to its aggressive and invasive nature. There have only been marginal improvements in its bleak survival rate of 12-15 months over the last four decades. The lack of preclinical models that efficiently recapitulate tumor biology and the tumor microenvironment is also in part responsible for the slow phase of translational GBM research. Emerging three-dimensional (3D) organoids and cell culture systems offer new and innovative possibilities for GBM modelling. These 3D models find their application to engineer the disease, screen drugs, establishing live biobank, and explore personalized therapy. Furthermore, these models can also be genetically modified by using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, which would allow one to study the specific role of key genes associated with gliomagenesis. Establishment of a coculture system with GBM cells to understand its invasive behavior is yet another major application of this model. Despite these merits, the organoid models also have certain limitations, including the absence of immune responses and vascular systems. In recent years, major progress has been made in the development and refinement of 3D models of GBM. In this review, we intend to highlight these recent advances and the potential future implications of this rapidly evolving field, which should facilitate a better understanding of GBM biology.

Chick fetal organ spheroids as a model to study development and disease.

BACKGROUND: Organ culture models have been used over the past few decades to study development and disease. The in vitro three-dimensional (3D) culture system of organoids is well known, however, these 3D systems are both costly and difficult to culture and maintain. As such, less expensive, faster and less complex methods to maintain 3D cell culture models would complement the use of organoids. Chick embryos have been used as a model to study human biology for centuries, with many fundamental discoveries as a result. These include cell type induction, cell competence, plasticity and contact inhibition, which indicates the relevance of using chick embryos when studying developmental biology and disease mechanisms. RESULTS: Here, we present an updated protocol that enables time efficient, cost effective and long-term expansion of fetal organ spheroids (FOSs) from chick embryos. Utilizing this protocol, we generated FOSs in an anchorage-independent growth pattern from seven different organs, including brain, lung, heart, liver, stomach, intestine and epidermis. These three-dimensional (3D) structures recapitulate many cellular and structural aspects of their in vivo counterpart organs and serve as a useful developmental model. In addition, we show a functional application of FOSs to analyze cell-cell interaction and cell invasion patterns as observed in cancer. CONCLUSION: The establishment of a broad ranging and highly effective method to generate FOSs from different organs was successful in terms of the formation of healthy, proliferating 3D organ spheroids that exhibited organ-like characteristics. Potential applications of chick FOSs are their use in studies of cell-to-cell contact, cell fusion and tumor invasion under defined conditions. Future studies will reveal whether chick FOSs also can be applicable in scientific areas such as viral infections, drug screening, cancer diagnostics and/or tissue engineering.

An investigation of coping in response to different race-related stressor experiences in school among racially diverse participants.

The landmark Brown versus Board of Education decision led to the desegregation of public schools in the United States. Consequently, Black/African American, Latinx, Asian, and Native American (BALANA) students experienced multiple race-related stressors. Not surprisingly, BALANA students still report experiences with racial discrimination, exclusion, and harassment in public schools. Encounters with race-related stressors in school can lead to maladaptive outcomes; however, for some young people, such stressors may induce a more adaptive coping response. The coping response may depend on the experience in school and the degree to which young people perceive they have control over the stressor. This study focused on the transactional nature of coping concerning different race-related stressors in school. The study investigates whether emotion-focused coping use prevailed across a sample of 165 ethnically and racially diverse participants. Results suggest that emotion-focused coping use is high among participants; however, the unique race-related stressor experienced in school can also lead to social-focused and other coping strategies. Coping with race-related stressors in school allows BALANA students to survive racism; reducing the presence of race-related stressors in school allows BALANA students to thrive. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Comparative Analysis of Stk11/Lkb1 versus Pten Deficiency in Lung Adenocarcinoma Induced by CRISPR/Cas9.

This study focused on STK11, PTEN, KRAS, and TP53, which are often found to be mutated in lung cancer. We compared Stk11 and Pten implication in lung cancer in combination with loss of Trp53 and gain of function of Kras in a CRISPR/Cas9 mouse model. Mice with loss of Stk11, Trp53, and KrasG12D mutation (SKT) reached human endpoint at around four months post-initiation. In comparison, mice with loss of Pten, Trp53, and KrasG12D mutation (PKT) survived six months or longer post-initiation. Pathological examination revealed an increase in proliferation in SKT deficient lung epithelia compared to PKT. This difference was independent of Pten loss, indicating that loss of Pten is dispensable for cell proliferation in lung adenocarcinoma. Furthermore, tumors with loss of Stk11, Trp53, and KrasG12D mutation had a significantly higher progression rate, monitored by PET/MRI scanning, compared to mice with loss of Pten, Trp53, and KrasG12D mutation, revealing that mutations in Stk11 are essential for adenocarcinoma progression. Overall, by using the CRISPR/Cas9 mouse model of lung adenocarcinoma, we showed that mutations in Stk11 are a key driver, whereas loss of Pten is dispensable for adenocarcinoma progression.

A Rare Case of Pericardial Decompression Syndrome in a Filipino Female Patient With Suspected Malignant Pericardial Effusion.

Pericardial decompression syndrome (PDS) is a rare, under-reported and potentially fatal complication of pericardial drainage characterized by paradoxical hemodynamic deterioration. The onset ranges from immediate to as long as 48 h post drainage. We present a case of a 51-year-old woman admitted due to progressive dyspnea. She was hemodynamically stable but with signs of cardiac tamponade. On two-dimensional echocardiography (2D-echo) there was a massive pericardial effusion in tamponade physiology. Immediate surgical drainage was done but intra-operatively there was depressed cardiac contractility necessitating inotropic support. Post-operative 2D-echo showed right ventricular (RV) and left ventricular (LV) systolic dysfunction. She was admitted in the intensive care unit (ICU) for 72 h. Repeat 2D-echo showed marked improvement in RV and LV systolic function. She was then discharged improved on the fifth hospital day. The pathophysiology of PDS is still not very clear. The simplest mechanism is that sudden removal of compressing pericardial fluid causes increased venous return with expansion of the RV at the expense of the LV leading to acute heart failure. There are no published studies to propose preventive measures and treatment remains supportive. There has been only one published case reported here in our country. We report this case of a patient who successfully recovered from PDS.

The cGAS-STING pathway is a therapeutic target in a preclinical model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, and the incidence of HCC is increasing. Recently, cancer immunotherapy has emerged as an efficient treatment against some cancers. Here we have used a mouse model of mutagen-induced HCC to explore the therapeutic usefulness of targeting the DNA-activated STING pathway in HCC. STING-deficient mice exhibited unaltered initial development of HCC, but had higher number of large tumors at late stages of disease. In the liver of STING-deficient HCC mice, we observed reduced levels of phospho-STAT1, autophagy, and cleaved caspase3. These responses were activated in the liver by treatment with a cyclic dinucleotide (CDN) STING agonist. Importantly, CDN treatment of mice after HCC development efficiently reduced tumor size. Initiation of CDN treatment at an even later stage of disease to allow HCC detection by MR scanning revealed that the majority of tumors regressed in response to CDN, but new tumors were also detected, which were unresponsive to CDN treatment. Overall, the modulation of the STING pathway affects the development of HCC, and holds promise for a use as a treatment of this disease, most likely in combination with other immunomodulatory treatments such as PD1 inhibitors or with standard of care.

Pretreatment of Glioblastoma with Bortezomib Potentiates Natural Killer Cell Cytotoxicity through TRAIL/DR5 Mediated Apoptosis and Prolongs Animal Survival.

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC-MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24-46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33-76% through augmented IFNγ release (p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252-0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295-0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood-brain barrier, attenuated proteasomal activity in vivo (p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284-0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen.