Pooled screening for CAR function identifies novel IL13Rα2-targeted CARs for treatment of glioblastoma.

Chimeric antigen receptor therapies have demonstrated potent efficacy in treating B cell malignancies, but have yet to meaningfully translate to solid tumors. Here, we utilize our pooled screening platform, CARPOOL, to expedite the discovery of CARs with anti-tumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.3×10 6 3 rd generation CARs targeting IL13Rα2, a cancer testis antigen commonly expressed in glioblastoma. Selections were performed for cytotoxicity, proliferation, memory formation, and persistence upon repeated antigen challenge. Each enriched CAR robustly produced the phenotype for which it was selected, and one enriched CAR triggered potent cytotoxicity and long-term proliferation upon in vitro tumor rechallenge. It also showed significantly improved persistence and comparable antigen-specific tumor control in a microphysiological human in vitro model and a xenograft model of human glioblastoma. Taken together, this work demonstrates the utility of extending CARPOOL to diseases beyond hematological malignancies and represents the largest exploration of signaling combinations in human primary cells to date.

Unveiling the Influence of Tumor Microenvironment and Spatial Heterogeneity on Temozolomide Resistance in Glioblastoma Using an Advanced Human In Vitro Model of the Blood-Brain Barrier and Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain cancer in adults with a dismal prognosis. Temozolomide (TMZ) is the first-in-line chemotherapeutic; however, resistance is frequent and multifactorial. While many molecular and genetic factors have been linked to TMZ resistance, the role of the solid tumor morphology and the tumor microenvironment, particularly the blood-brain barrier (BBB), is unknown. Here, the authors investigate these using a complex in vitro model for GBM and its surrounding BBB. The model recapitulates important clinical features such as a dense tumor core with tumor cells that invade along the perivascular space; and a perfusable BBB with a physiological permeability and morphology that is altered in the presence of a tumor spheroid. It is demonstrated that TMZ sensitivity decreases with increasing cancer cell spatial organization, and that the BBB can contribute to TMZ resistance. Proteomic analysis with next-generation low volume sample workflows of these cultured microtissues revealed potential clinically relevant proteins involved in tumor aggressiveness and TMZ resistance, demonstrating the utility of complex in vitro models for interrogating the tumor microenvironment and therapy validation.

G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma.

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we find that short-term inhibition of G9a/GLP increases T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increases granzyme expression without terminal T cell differentiation or exhaustion and results in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

In vitro 3D liver tumor microenvironment models for immune cell therapy optimization.

Despite diagnostic and therapeutic advances, liver cancer kills more than 18 million people every year worldwide, urging new strategies to model the disease and to improve the current therapeutic options. In vitro tumor models of human cancer continue to evolve, and they represent an important screening tool. However, there is a tremendous need to improve the physiological relevance and reliability of these in vitro models to fulfill today's research requirements for better understanding of cancer progression and treatment options at different stages of the disease. This review describes the hepatocellular carcinoma microenvironmental characteristics and illustrates the current immunotherapy strategy to fight the disease. Moreover, we present a recent collection of 2D and 3D in vitro liver cancer models and address the next generation of in vitro systems recapitulating the tumor microenvironment complexity in more detail.

Isotropic myosin-generated tissue tension is required for the dynamic orientation of the mitotic spindle.

The ability of cells to divide along their longest axis has been proposed to play an important role in maintaining epithelial tissue homeostasis in many systems. Because the division plane is largely set by the position of the anaphase spindle, it is important to understand how spindles become oriented. While several molecules have been identified that play key roles in spindle orientation across systems, most notably Mud/NuMA and cortical dynein, the precise mechanism by which spindles detect and align with the long cell axis remain poorly understood. Here, in exploring the dynamics of spindle orientation in mechanically distinct regions of the fly notum, we find that the ability of cells to properly reorient their divisions depends on local tissue tension. Thus, spindles reorient to align with the long cell axis in regions where isotropic tension is elevated, but fail to do so in elongated cells within the crowded midline, where tension is low, or in regions that have been mechanically isolated from the rest of the tissue via laser ablation. Importantly, these differences in spindle behavior outside and inside the midline can be recapitulated by corresponding changes in tension induced by perturbations that alter nonmuscle myosin II activity. These data lead us to propose that isotropic tension within an epithelium provides cells with a mechanically stable substrate upon which localized cortical motor complexes can act on astral microtubules to orient the spindle.

Dickkopf-3 links HSF1 and YAP/TAZ signalling to control aggressive behaviours in cancer-associated fibroblasts.

Aggressive behaviours of solid tumours are highly influenced by the tumour microenvironment. Multiple signalling pathways can affect the normal function of stromal fibroblasts in tumours, but how these events are coordinated to generate tumour-promoting cancer-associated fibroblasts (CAFs) is not well understood. Here we show that stromal expression of Dickkopf-3 (DKK3) is associated with aggressive breast, colorectal and ovarian cancers. We demonstrate that DKK3 is a HSF1 effector that modulates the pro-tumorigenic behaviour of CAFs in vitro and in vivo. DKK3 orchestrates a concomitant activation of ß-catenin and YAP/TAZ. Whereas ß-catenin is dispensable for CAF-mediated ECM remodelling, cancer cell growth and invasion, DKK3-driven YAP/TAZ activation is required to induce tumour-promoting phenotypes. Mechanistically, DKK3 in CAFs acts via canonical Wnt signalling by interfering with the negative regulator Kremen and increasing cell-surface levels of LRP6. This work reveals an unpredicted link between HSF1, Wnt signalling and YAP/TAZ relevant for the generation of tumour-promoting CAFs.

Regulation of mechanotransduction: Emerging roles for septins.

Cells exist in dynamic three-dimensional environments where they experience variable mechanical forces due to their interaction with the extracellular matrix, neighbouring cells and physical stresses. The ability to constantly and rapidly alter cellular behaviour in response to the mechanical environment is therefore crucial for cell viability, tissue development and homeostasis. Mechanotransduction is the process whereby cells translate mechanical inputs into biochemical signals. These signals in turn adjust cell morphology and cellular functions as diverse as proliferation, differentiation, migration and apoptosis. Here, we provide an overview of the current understanding of mechanotransduction and how septins may participate in it, drawing on their architecture and localization, their ability to directly bind and modify actomyosin networks and membranes, and their associations with the nuclear envelope.

A national trauma database analysis of alcohol- associated injuries.

INTRODUCTION: Knowledge of the pattern of alcohol-associated injury (AAI) is lacking in Singapore. We aimed to determine the local demographic pattern, injury mechanism, injury severity and outcomes of AAI. METHODS: Data on trauma cases presenting to emergency departments in 2012-2013 was extracted from the National Trauma Registry. Cases with missing data fields and those aged 1-15 years were excluded. Patients were classified as alcohol positive (A+) or negative (A-) based on clinical assessment. The two groups' demographics, injury mechanism, injury severity, mortality and disposition were compared. Logistic regression analysis was used to determine independent associations with mortality. RESULTS: 105,468 trauma cases met the inclusion criteria. 3.9% were A+ and their peak age range was 25-44 years. The A+ group had more Indian males (p < 0.001), and significantly more assaults, self-harm and falls (p < 0.001). Injuries in the A+ group were more common in public areas and less common in homes, recreational facilities and workplaces. Outcomes in the A+ group showed higher mean Injury Severity Score and mortality (p < 0.001). Significantly more A+ patients were admitted to hospital but had shorter mean length of stay (p < 0.001). Multivariate logistic regression revealed age > 44 years and male gender as independent predictors of mortality. CONCLUSION: AAI in Singapore is associated with more severe injuries and resource utilisation. Using data from the registry, 'at risk' demographic groups are identified for targeted injury prevention. However, alcohol use is not an independent predictor of mortality in trauma cases.

Oriented Division: Using T-Junctions to Determine Direction.

Cell shape has long been thought to be the main cue for spindle positioning in mitotic cells, but new evidence suggests that, in the context of an epithelium, tricellular junctions encode positional information that helps orient mitotic spindles.

Emergence of homeostatic epithelial packing and stress dissipation through divisions oriented along the long cell axis.

Cell division plays an important role in animal tissue morphogenesis, which depends, critically, on the orientation of divisions. In isolated adherent cells, the orientation of mitotic spindles is sensitive to interphase cell shape and the direction of extrinsic mechanical forces. In epithelia, the relative importance of these two factors is challenging to assess. To do this, we used suspended monolayers devoid of ECM, where divisions become oriented following a stretch, allowing the regulation and function of epithelial division orientation in stress relaxation to be characterized. Using this system, we found that divisions align better with the long, interphase cell axis than with the monolayer stress axis. Nevertheless, because the application of stretch induces a global realignment of interphase long axes along the direction of extension, this is sufficient to bias the orientation of divisions in the direction of stretch. Each division redistributes the mother cell mass along the axis of division. Thus, the global bias in division orientation enables cells to act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state. Further, this behavior could be quantitatively reproduced using a model designed to assess the impact of autonomous changes in mitotic cell mechanics within a stretched monolayer. In summary, the propensity of cells to divide along their long axis preserves epithelial homeostasis by facilitating both stress relaxation and isotropic growth without the need for cells to read or transduce mechanical signals.

7,8-dihydroxyflavone exhibits therapeutic efficacy in a mouse model of Rett syndrome.

Rett syndrome (RTT), caused by mutations in the methyl-CpG binding protein 2 gene (MECP2), is a debilitating autism spectrum developmental disorder predominantly affecting females. Mecp2 mutant mice have reduced levels of brain-derived neurotrophic factor (BDNF) in the brain; conditional deletion and overexpression of BDNF in the brain accelerates and slows, respectively, disease progression in Mecp2 mutant mice. Thus we tested the hypothesis that 7,8-dihydroxyflavone (7,8-DHF), a small molecule reported to activate the high affinity BDNF receptor (TrkB) in the CNS, would attenuate disease progression in Mecp2 mutant mice. Following weaning, 7,8-DHF was administered in drinking water throughout life. Treated mutant mice lived significantly longer compared with untreated mutant littermates (80 ± 4 and 66 ± 2 days, respectively). 7,8-DHF delayed body weight loss, increased neuronal nuclei size and enhanced voluntary locomotor (running wheel) distance in Mecp2 mutant mice. In addition, administration of 7,8-DHF partially improved breathing pattern irregularities and returned tidal volumes to near wild-type levels. Thus although the specific mechanisms are not completely known, 7,8-DHF appears to reduce disease symptoms in Mecp2 mutant mice and may have potential as a therapeutic treatment for RTT patients.