Pushing the Frontiers of Cancer Research: Highlights from the Frontiers in Cancer Science Conference 2023.

The 15th annual Frontiers in Cancer Science (FCS) conference gathered scientific experts who shared the latest research converging upon several themes of cancer biology. These themes included the dysregulation of metabolism, cell death, and other signaling processes in cancer cells; using patient "omics" datasets and single-cell and spatial approaches to investigate heterogeneity, understand therapy resistance, and identify targets; innovative strategies for inhibiting tumors, including rational drug combinations and improved drug delivery mechanisms; and advances in models that can facilitate screening for cancer vulnerabilities and drug testing. We hope the insights from this meeting will stimulate further progress in the field.

ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells.

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

Single-cell transcriptomics reveals maturation of transplanted stem cell-derived retinal pigment epithelial cells toward native state.

Transplantation of stem cell-derived retinal pigment epithelial (RPE) cells is considered a viable therapeutic option for age-related macular degeneration (AMD). Several landmark Phase I/II clinical trials have demonstrated safety and tolerability of RPE transplants in AMD patients, albeit with limited efficacy. Currently, there is limited understanding of how the recipient retina regulates the survival, maturation, and fate specification of transplanted RPE cells. To address this, we transplanted stem cell-derived RPE into the subretinal space of immunocompetent rabbits for 1 mo and conducted single-cell RNA sequencing analyses on the explanted RPE monolayers, compared to their age-matched in vitro counterparts. We observed an unequivocal retention of RPE identity, and a trajectory-inferred survival of all in vitro RPE populations after transplantation. Furthermore, there was a unidirectional maturation toward the native adult human RPE state in all transplanted RPE, regardless of stem cell resource. Gene regulatory network analysis suggests that tripartite transcription factors (FOS, JUND, and MAFF) may be specifically activated in posttransplanted RPE cells, to regulate canonical RPE signature gene expression crucial for supporting host photoreceptor function, and to regulate prosurvival genes required for transplanted RPE's adaptation to the host subretinal microenvironment. These findings shed insights into the transcriptional landscape of RPE cells after subretinal transplantation, with important implications for cell-based therapy for AMD.

Transglutaminase-2 is critical for corneal epithelial barrier function via positive regulation of Claudin-1.

PURPOSE: Transglutaminase (TG)-2 is a ubiquitous multi-functional protein expressed in all living cells. The purpose of the current study was to investigate the role of TG-2 in corneal barrier function and its potential regulation of epithelial junctional proteins and transcription factors. METHODS: Corneal barrier function to ions in TG-2-/- and TG-2+/+ mice was assessed by Ussing chamber assay. Hypo-osmolar water or FITC-dextran was applied on top of mouse eyes to evaluate the corneal barrier function to water and macromolecules. Western blots, qPCR and immunofluorescent staining were used to investigate the expression of tight junction proteins in TG-2-/- and TG-2+/+ mouse corneas, and also in TG-2 knockdown human corneal epithelial cells. RESULTS: Corneal explants from TG-2-/- mice had a lower trans-epithelial electrical resistance compared to TG-2+/+ mice. When challenged by hypo-osmolar water, the central corneal thickness of TG-2-/- mice increased faster, and these mice had a faster rise of fluorescence in the anterior chamber after ocular exposure to FITC-dextran, compared to TG-2+/+. Claudin-1 protein and transcript levels were reduced in the cornea of TG-2-/- mice and in TG-2 knockdown human corneal epithelial cells. Slug which previously reported suppressing Claudin-1 transcription, was increased at both protein and transcript level in TG-2 knockdown cells. TG-2 and Claudin-1 protein levels were unchanged in shRNA and shTG cells after MG132 treatment, while Slug accumulated in treated cells. CONCLUSION: TG-2 may positively regulate Claudin-1 through repressing Slug at transcript level, and thus it is critical for normal corneal barrier function.

ZO-2/Tjp2 suppresses Yap and Wwtr1/Taz-mediated hepatocyte to cholangiocyte transdifferentiation in the mouse liver.

TJP2/ZO-2-inactivating mutations in humans cause progressive cholestatic liver disease. Liver-specific deletion of Tjp2 in the mouse (Tjp2 cKO mice) leads to mild progressive cholestasis without an overt degradation of the bile-blood barrier (BBB). These mice are more susceptible to cholic acid (CA) induced liver injury. Interestingly, while initially also more susceptible, Tjp2 cKO mice develop tolerance to a DDC-supplemented diet. The DDC diet induces an exacerbated ductular reaction in Tjp2 cKO mice, which arises from the transdifferentiation of hepatocytes to cholangiocytes. Consequently, this transdifferentiation is only observed if Tjp2 is inactivated in hepatocytes, but not if deleted in cholangiocytes. The DDC-diet-induced hepatocyte transdifferentiation in Tjp2 cKO mice requires Yap and Wwtr1/Taz, whose protein expression is upregulated in hepatocytes lacking Tjp2, but is independent of Notch2. Although inactivating Tjp2 is sufficient for the upregulation of Yap and Wwtr1/Taz protein, efficient transdifferentiation requires the DDC-diet insult. Thus, Tjp2 negatively regulates Yap/Taz-mediated transdifferentiation of hepatocytes to cholangiocytes in response to DDC-diet-induced liver injury. Furthermore, transdifferentiation is regulated at multiple levels and the type of injury inflicted on the Tjp2 deficient liver plays an important role in the resulting pathophysiology.

Customized strategies for high-yield purification of retinal pigment epithelial cells differentiated from different stem cell sources.

Retinal pigment epithelial (RPE) cell dysfunction and death are characteristics of age-related macular degeneration. A promising therapeutic option is RPE cell transplantation. Development of clinical grade stem-cell derived RPE requires efficient in vitro differentiation and purification methods. Enzymatic purification of RPE relies on the relative adherence of RPE and non-RPE cells to the culture plate. However, morphology and adherence of non-RPE cells differ for different stem cell sources. In cases whereby the non-RPE adhered as strongly as RPE cells to the culture plate, enzymatic method of purification is unsuitable. Thus, we hypothesized the need to customize purification strategies for RPE derived from different stem cell sources. We systematically compared five different RPE purification methods, including manual, enzymatic, flow cytometry-based sorting or combinations thereof for parameters including cell throughput, yield, purity and functionality. Flow cytometry-based approach was suitable for RPE isolation from heterogeneous cultures with highly adherent non-RPE cells, albeit with lower yield. Although all five purification methods generated pure and functional RPE, there were significant differences in yield and processing times. Based on the high purity of the resulting RPE and relatively short processing time, we conclude that a combination of enzymatic and manual purification is ideal for clinical applications.

A bio-functional polymer that prevents retinal scarring through modulation of NRF2 signalling pathway.

One common cause of vision loss after retinal detachment surgery is the formation of proliferative and contractile fibrocellular membranes. This aberrant wound healing process is mediated by epithelial-mesenchymal transition (EMT) and hyper-proliferation of retinal pigment epithelial (RPE) cells. Current treatment relies primarily on surgical removal of these membranes. Here, we demonstrate that a bio-functional polymer by itself is able to prevent retinal scarring in an experimental rabbit model of proliferative vitreoretinopathy. This is mediated primarily via clathrin-dependent internalisation of polymeric micelles, downstream suppression of canonical EMT transcription factors, reduction of RPE cell hyper-proliferation and migration. Nuclear factor erythroid 2-related factor 2 signalling pathway was identified in a genome-wide transcriptomic profiling as a key sensor and effector. This study highlights the potential of using synthetic bio-functional polymer to modulate RPE cellular behaviour and offers a potential therapy for retinal scarring prevention.

Antiangiogenic Nanomicelles for the Topical Delivery of Aflibercept to Treat Retinal Neovascular Disease.

The traditional intravitreal injection delivery of antivascular endothelial growth factor (anti-VEGF) to the posterior segment of the eye for treatment of retinal diseases is invasive and associated with sight-threatening complications. To avoid such complications, there has been significant interest in developing polymers for topical drug delivery to the retina. This study reports a nanomicelle drug delivery system made of a copolymer EPC (nEPCs), which is capable of delivering aflibercept to the posterior segment topically through corneal-scleral routes. EPC is composed of poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), and polycaprolactone (PCL) segments. In this study, aflibercept-loaded nEPCs (nEPCs + A) are capable of penetrating the cornea in ex vivo porcine eye models and deliver a clinically significant amount of aflibercept to the retina in laser-induced choroidal neovascularization (CNV) murine models, causing CNV regression. nEPCs + A also demonstrate biocompatibility in vitro and in vivo. Interestingly, this study also suggests that nEPCs have intrinsic antiangiogenic properties. The ability to deliver anti-VEGF drugs and the intrinsic antiangiogenic properties of nEPCs may result in synergistic effects, which can be harnessed for effective therapeutics. nEPCs may be a promising topical anti-VEGF delivery platform for the treatment of retinal diseases.

ZO-1 Intracellular Localization Organizes Immune Response in Non-Small Cell Lung Cancer.

Delocalization of zonula occludens-1 (ZO-1) from tight junctions plays a substantial role in epithelial cell plasticity observed during tumor progression. In vitro, we reported an impact of ZO-1 cyto-nuclear content in modulating the secretion of several pro-inflammatory chemokines. In vivo, we demonstrated that it promotes the recruitment of immune cells in mouse ear sponge assays. Examining lung cancers, we showed that a high density of CD8 cytotoxic T cells and Foxp3 immunosuppressive regulatory T cells in the tumor microenvironment correlated with a cyto-nuclear expression of ZO-1. Taken together, our results support that, by affecting tumor cell secretome, the cyto-nuclear ZO-1 pool may recruit immune cells, which could be permissive for tumor progression.

cGMP-grade human iPSC-derived retinal photoreceptor precursor cells rescue cone photoreceptor damage in non-human primates.

BACKGROUND: Retinal regenerative therapies hold great promise for the treatment of inherited retinal degenerations (IRDs). Studies in preclinical lower mammal models of IRDs have suggested visual improvement following retinal photoreceptor precursors transplantation, but there is limited evidence on the ability of these transplants to rescue retinal damage in higher mammals. The purpose of this study was to evaluate the therapeutic potential of photoreceptor precursors derived from clinically compliant induced pluripotent stem cells (iPSCs). METHODS: Photoreceptor precursors were sub-retinally transplanted into non-human primates (Macaca fascicularis). The cells were transplanted both in naïve and cobalt chloride-induced retinal degeneration models who had been receiving systemic immunosuppression for one week prior to the procedure. Optical coherence tomography, fundus autofluorescence imaging, electroretinography, ex vivo histology and immunofluorescence staining were used to evaluate retinal structure, function and survival of transplanted cells. RESULTS: There were no adverse effects of iPSC-derived photoreceptor precursors on retinal structure or function in naïve NHP models, indicating good biocompatibility. In addition, photoreceptor precursors injected into cobalt chloride-induced retinal degeneration NHP models demonstrated an ability both to survive and to mature into cone photoreceptors at 3 months post-transplant. Optical coherence tomography showed restoration of retinal ellipsoid zone post-transplantation. CONCLUSIONS: These findings demonstrate the safety and therapeutic potential of clinically compliant iPSC-derived photoreceptor precursors as a cell replacement source for future clinical trials.

Surgical Transplantation of Human RPE Stem Cell-Derived RPE Monolayers into Non-Human Primates with Immunosuppression.

Recent trials of retinal pigment epithelium (RPE) transplantation for the treatment of disorders such as age-related macular degeneration have been promising. However, limitations of existing strategies include the uncertain survival of RPE cells delivered by cell suspension and the inherent risk of uncontrolled cell proliferation in the vitreous cavity. Human RPE stem cell-derived RPE (hRPESC-RPE) transplantation can rescue vision in a rat model of retinal dystrophy and survive in the rabbit retina for at least 1 month. The present study placed hRPESC-RPE monolayers under the macula of a non-human primate model for 3 months. The transplant was able to recover in vivo and maintained healthy photoreceptors. Importantly, there was no evidence that subretinally transplanted monolayers underwent an epithelial-mesenchymal transition. Neither gliosis in adjacent retina nor epiretinal membranes were observed. These findings suggest that hRPESC-RPE monolayers are safe and may be a useful source for RPE cell replacement therapy.

Protective Functions of ZO-2/Tjp2 Expressed in Hepatocytes and Cholangiocytes Against Liver Injury and Cholestasis.

BACKGROUND & AIMS: Liver tight junctions (TJs) establish tissue barriers that isolate bile from the blood circulation. TJP2/ZO-2-inactivating mutations cause progressive cholestatic liver disease in humans. Because the underlying mechanisms remain elusive, we characterized mice with liver-specific inactivation of Tjp2. METHODS: Tjp2 was deleted in hepatocytes, cholangiocytes, or both. Effects on the liver were assessed by biochemical analyses of plasma, liver, and bile and by electron microscopy, histology, and immunostaining. TJ barrier permeability was evaluated using fluorescein isothiocyanate-dextran (4 kDa). Cholic acid (CA) diet was used to assess susceptibility to liver injury. RESULTS: Liver-specific deletion of Tjp2 resulted in lower Cldn1 protein levels, minor changes to the TJ, dilated canaliculi, lower microvilli density, and aberrant radixin and bile salt export pump (BSEP) distribution, without an overt increase in TJ permeability. Hepatic Tjp2-defcient mice presented with mild progressive cholestasis with lower expression levels of bile acid transporter Abcb11/Bsep and detoxification enzyme Cyp2b10. A CA diet tolerated by control mice caused severe cholestasis and liver necrosis in Tjp2-deficient animals. 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene ameliorated CA-induced injury by enhancing Cyp2b10 expression, and ursodeoxycholic acid provided partial improvement. Inactivating Tjp2 separately in hepatocytes or cholangiocytes showed only mild CA-induced liver injury. CONCLUSION: Tjp2 is required for normal cortical distribution of radixin, canalicular volume regulation, and microvilli density. Its inactivation deregulated expression of Cldn1 and key bile acid transporters and detoxification enzymes. The mice provide a novel animal model for cholestatic liver disease caused by TJP2-inactivating mutations in humans.

An Optimized Protocol for Proximity Biotinylation in Confluent Epithelial Cell Cultures Using the Peroxidase APEX2.

The peroxidase APEX2 has been used widely for proximity biotinylation and subsequent proteomics analyses. However, the poor membrane permeability of the biotin phenol substrate and the inhibitory effect of peroxide on the enzyme's activity has hampered proximity labeling in certain cell culture systems and tissues. Here, we describe an APEX2 protocol that uses alternative peroxide and biotin phenol concentrations. The protocol permits robust proximity biotinylation in confluent epithelial cell cultures and may be applicable to other cell cultures and tissues. For complete details on the use and execution of this protocol, please refer to Tan et al. (2020).

A Pilot Study on MicroRNA Profile in Tear Fluid to Predict Response to Anti-VEGF Treatments for Diabetic Macular Edema.

(1) Background: Intravitreal anti-vascular endothelial growth factor (anti-VEGF) is an established treatment for center-involving diabetic macular edema (ci-DME). However, the clinical response is heterogeneous. This study investigated miRNAs as a biomarker to predict treatment response to anti-VEGF in DME. (2) Methods: Tear fluid, aqueous, and blood were collected from patients with treatment-naïve DME for miRNA expression profiling with quantitative polymerase chain reaction. Differentially expressed miRNAs between good and poor responders were identified from tear fluid. Bioinformatics analysis with the miEAA tool, miRTarBase Annotations, Gene Ontology categories, KEGG, and miRWalk pathways identified interactions between enriched miRNAs and biological pathways. (3) Results: Of 24 participants, 28 eyes received bevacizumab (15 eyes) or aflibercept (13 eyes). Tear fluid had the most detectable miRNA species (N = 315), followed by serum (N = 309), then aqueous humor (N = 134). MiRNAs that correlated with change in macular thickness were miR-214-3p, miR-320d, and hsa-miR-874-3p in good responders; and miR-98-5p, miR-196b-5p, and miR-454-3p in poor responders. VEGF-related pathways and the angiogenin-PRI complex were enriched in good responders, while transforming growth factor-ß and insulin-like growth factor pathways were enriched in poor responders. (4) Conclusions: We reported a panel of novel miRNAs that provide insight into biological pathways in DME. Validation in larger independent cohorts is needed to determine the predictive performance of these miRNA candidate biomarkers.

The Mammalian Crumbs Complex Defines a Distinct Polarity Domain Apical of Epithelial Tight Junctions.

Epithelial apico-basal polarity is established through the asymmetric cortical distribution of the Par, Crumbs and Scribble polarity modules. Apical (Par and Crumbs) and basolateral (Scribble) polarity modules overlap at the apical-lateral border, which, in mammals, is defined by the apical junctional complex (AJC). The AJC is composed of tight junctions (TJ) and adherens junctions (AJ) and plays fundamental roles in epithelial morphogenesis and plasticity. However, the molecular composition and precise sub-junctional organization of the AJC and its associated polarity regulators are not well defined. Here, we used the peroxidase APEX2 for quantitative proximity proteomics (QPP) and electron microscopy (EM) imaging to dissect the architecture of the AJC in fully polarized MDCK-II cells. We present a high-confidence proteome of the apical-lateral border in which TJ and AJ components and apical and lateral compartment markers are spatially resolved. We further demonstrate that the Crumbs complex (Pals1, PatJ, Lin7c, and Crumbs3) defines a hitherto unidentified membrane compartment apical of TJ, which we coin the vertebrate marginal zone (VMZ). QPP, imaging, and immunoprecipitation assays showed that the HOMER scaffolding proteins, PKN2 and PTPN13, and the membrane-proximal HIPPO pathway proteins ARHGAP29 and STXBP4 are recruited to the VMZ via the PDZ domains of PatJ. Taken together, our work defines the spatial and molecular organization of the apical-lateral border in mammalian epithelial cells, reveals an intriguing molecular and spatial conservation of invertebrate and vertebrate cell polarity protein domains, and identifies a VMZ-associated protein network implicated in HIPPO signaling and the control of the cortical actin cytoskeleton.

Correction: Use of biomaterials for sustained delivery for anti-VEGF to treat retinal diseases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Use of biomaterials for sustained delivery of anti-VEGF to treat retinal diseases.

Anti-vascular endothelial growth factors (anti-VEGF) have become the most common treatment modality for many retinal diseases. These include neovascular age-related macular degeneration (n-AMD), proliferative diabetic retinopathy (PDR) and retinal vein occlusions (RVO). However, these drugs are administered via intravitreal injections that are associated with sight-threatening complications. The most feared of these complications is endophthalmitis, a severe infection of the eye with extremely poor visual outcomes. Patients with retinal diseases typically have to undergo multiple injections before achieving the desired therapeutic effect. Each injection incurs the risk of the sight-threatening complications. As such, there has been great interest in developing sustained delivery platforms for anti-VEGF agents to the posterior segment of the eye. In recent years, there have been various strategies that have been conceptualised. These include non-biodegradable implants, nano-formulations and hydrogels. In this review, the barriers of drug delivery to the posterior segment of the eye will be explained. The characteristics of an ideal sustained delivery platform will then be discussed. Finally, the current available strategies will be analysed with the above-mentioned characteristics in mind to determine the advantages and disadvantages of each sustained drug delivery modality. Through the above, this review attempts to provide an overview of the sustained delivery platforms in their various phases of development.

A pump-free tricellular blood-brain barrier on-a-chip model to understand barrier property and evaluate drug response.

Disruption of the blood-brain barrier (BBB) leads to various neurovascular diseases. Development of therapeutics required to cross the BBB is difficult due to a lack of relevant in vitro models. We have developed a three-dimensional (3D) microfluidic BBB chip (BBBC) to study cell interactions in the brain microvasculature and to test drug candidates of neurovascular diseases. We isolated primary brain microvascular endothelial cells (ECs), pericytes, and astrocytes from neonatal rats and cocultured them in the BBBC. To mimic the 3D in vivo BBB structure, we used type I collagen hydrogel to pattern the microchannel via viscous finger patterning technique to create a matrix. ECs, astrocytes, and pericytes were cocultured in the collagen matrix. The fluid flow in the BBBC was controlled by a pump-free strategy utilizing gravity as driving force and resistance in a paper-based flow resistor. The primary cells cultured in the BBBC expressed high levels of junction proteins and formed a tight endothelial barrier layer. Addition of tumor necrosis factor alpha to recapitulate neuroinflammatory conditions compromised the BBB functionality. To mitigate the neuroinflammatory stimulus, we treated the BBB model with the glucocorticoid drug dexamethasone, and observed protection of the BBB. This BBBC represents a new simple, cost-effective, and scalable in vitro platform for validating therapeutic drugs targeting neuroinflammatory conditions.