Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade.

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Advanced patient-specific microglia cell models for pre-clinical studies in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is an incurable neurodegenerative disorder with a rapidly increasing prevalence worldwide. Current approaches targeting hallmark pathological features of AD have had no consistent clinical benefit. Neuroinflammation is a major contributor to neurodegeneration and hence, microglia, the brain's resident immune cells, are an attractive target for potentially more effective therapeutic strategies. However, there is no current in vitro model system that captures AD patient-specific microglial characteristics using physiologically relevant and experimentally flexible culture conditions. METHODS: To address this shortcoming, we developed novel 3D Matrigel-based monocyte-derived microglia-like cell (MDMi) mono-cultures and co-cultures with neuro-glial cells (ReNcell VM). We used single-cell RNA sequencing (scRNAseq) analysis to compare the transcriptomic signatures of MDMi between model systems (2D, 3D and 3D co-culture) and against published human microglia datasets. To demonstrate the potential of MDMi for use in personalized pre-clinical strategies, we generated and characterized MDMi models from sixteen AD patients and matched healthy controls, and profiled cytokine responses upon treatment with anti-inflammatory drugs (dasatinib and spiperone). RESULTS: MDMi in 3D exhibited a more branched morphology and longer survival in culture compared to 2D. scRNAseq uncovered distinct MDMi subpopulations that exhibit higher functional heterogeneity and best resemble human microglia in 3D co-culture. AD MDMi in 3D co-culture showed altered cell-to-cell interactions, growth factor and cytokine secretion profiles and responses to amyloid-ß. Drug testing assays revealed patient- and model-specific cytokine responses. CONCLUSION: Our study presents a novel, physiologically relevant and AD patient-specific 3D microglia cell model that opens avenues towards improving personalized drug development strategies in AD.

ALS monocyte-derived microglia-like cells reveal cytoplasmic TDP-43 accumulation, DNA damage, and cell-specific impairment of phagocytosis associated with disease progression.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterised by the loss of upper and lower motor neurons. Increasing evidence indicates that neuroinflammation mediated by microglia contributes to ALS pathogenesis. This microglial activation is evident in post-mortem brain tissues and neuroimaging data from patients with ALS. However, the role of microglia in the pathogenesis and progression of amyotrophic lateral sclerosis remains unclear, partly due to the lack of a model system that is able to faithfully recapitulate the clinical pathology of ALS. To address this shortcoming, we describe an approach that generates monocyte-derived microglia-like cells that are capable of expressing molecular markers, and functional characteristics similar to in vivo human brain microglia. METHODS: In this study, we have established monocyte-derived microglia-like cells from 30 sporadic patients with ALS, including 15 patients with slow disease progression, 6 with intermediate progression, and 9 with rapid progression, together with 20 non-affected healthy controls. RESULTS: We demonstrate that patient monocyte-derived microglia-like cells recapitulate canonical pathological features of ALS including non-phosphorylated and phosphorylated-TDP-43-positive inclusions. Moreover, ALS microglia-like cells showed significantly impaired phagocytosis, altered cytokine profiles, and abnormal morphologies consistent with a neuroinflammatory phenotype. Interestingly, all ALS microglia-like cells showed abnormal phagocytosis consistent with the progression of the disease. In-depth analysis of ALS microglia-like cells from the rapid disease progression cohort revealed significantly altered cell-specific variation in phagocytic function. In addition, DNA damage and NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome activity were also elevated in ALS patient monocyte-derived microglia-like cells, indicating a potential new pathway involved in driving disease progression. CONCLUSIONS: Taken together, our work demonstrates that the monocyte-derived microglia-like cell model recapitulates disease-specific hallmarks and characteristics that substantiate patient heterogeneity associated with disease subgroups. Thus, monocyte-derived microglia-like cells are highly applicable to monitor disease progression and can be applied as a functional readout in clinical trials for anti-neuroinflammatory agents, providing a basis for personalised treatment for patients with ALS.

Immune contexture analysis in immuno-oncology: applications and challenges of multiplex fluorescent immunohistochemistry.

The tumor microenvironment is an integral player in cancer initiation, tumor progression, response and resistance to anti-cancer therapy. Understanding the complex interactions of tumor immune architecture (referred to as 'immune contexture') has therefore become increasingly desirable to guide our approach to patient selection, clinical trial design, combination therapies, and patient management. Quantitative image analysis based on multiplexed fluorescence immunohistochemistry and deep learning technologies are rapidly developing to enable researchers to interrogate complex information from the tumor microenvironment and find predictive insights into treatment response. Herein, we discuss current developments in multiplexed fluorescence immunohistochemistry for immune contexture analysis, and their application in immuno-oncology, and discuss challenges to effectively use this technology in clinical settings. We also present a multiplexed image analysis workflow to analyse fluorescence multiplexed stained tumor sections using the Vectra Automated Digital Pathology System together with FCS express flow cytometry software. The benefit of this strategy is that the spectral unmixing accurately generates and analyses complex arrays of multiple biomarkers, which can be helpful for diagnosis, risk stratification, and guiding clinical management of oncology patients.

Crohn's disease is facilitated by a disturbance of programmed death-1 ligand 2 on blood dendritic cells.

OBJECTIVE: Crohn's disease (CD) is characterised by inflammation, predominantly associated with ilea. To investigate the basis for this inflammation in patients with CD, we examined dendritic cells (DC) which are pivotal for maintenance of immunological tolerance in the gut. METHODS: Ileal biopsies and blood DCs from CD patients and controls were examined by microscopy and flow cytometry for PD-L1 and PD-L2 expression, as PD-L1 has been implicated in colitis but the contribution of PD-L2 is less clear. In vitro studies, of blood samples from CD patients, were used to demonstrate a functional role for PD-L2 in disease pathogenesis. RESULTS: Quantitative microscopy of CD11c+ DCs in inflamed and noninflamed ilea from CD patient showed > 75% loss of these cells from the villi, lamina propria and Peyer's patches compared with non-CD controls. Given this loss of DCs from ilia of CD patients, we hypothesised DCs may have migrated to the blood as these patients can have extra-intestinal symptoms. We thus examined blood DCs from CD patients by flow cytometry and found significant increases in PD-L1 and PD-L2 expression compared with control samples. Microscopy revealed an aggregated form of PD-L2 expression, known to drive Th1 immunity, in CD patients but not in controls. In vitro functional studies with PD-L2 blockade confirmed PD-L2 contributes significantly to the secretion of pro-inflammatory cytokines known to cause disease pathogenesis. CONCLUSION: Taken together, this study shows that PD-L2 can influence the progression of CD and blockade of PD-L2 may have therapeutic potential.

A Nationally-Representative Epidemiological and Risk Factor Assessment of Child Mental Health in Vietnam.

As part of the global mental health movement's focus on identifying and reducing international disparities, this study conducted the first nationally representative child mental health epidemiological survey in Vietnam. We assessed as risk/protective factors several family social structure characteristics (e.g., presence of grandparents, number of siblings in the home) of particular relevance to non-Western countries. Epidemiological data using the Child Behavior Checklist and the Strengths and Difficulties Questionnaire were collected at 60 sites in 10 of Vietnam's 63 provinces selected to provide a nationally representative sample, which included 1,314 adult informants of children 6-16 years of age, and 591 children aged 12-16. Vietnamese children's mental health functioning was reported overall to be better by approximately a third standard deviation than the international average; this international difference was particularly large for externalizing (behavior) problems as compared to internalizing (emotional) problems, suggesting that a cultural problem suppression model may be operating in Vietnam. Significant variability in mental health problems was found across provinces, emphasizing the need for nationally representative samples when conducting child mental health epidemiological surveys. Contrary to many other studies, in Vietnam higher SES was found to be a risk factor for attention/hyperactivity problems.