Effect of graphene-based nanomaterials on corneal wound healing in vitro.

Graphene-based nanomaterials (GBNs) are widely used due to their chemical and physical properties for multiple commercial and environmental applications. From an occupational health perspective, there is concern regarding the effects of inhalation on the respiratory system, and many studies have been conducted to study inhalation impacts on lung. Similar to the respiratory system, the eyes may also be exposed to GBNs and thus impacted. In this study, immortalized human corneal epithelial (hTCEpi) cells and rabbit corneal fibroblasts (RCFs) were used to investigate the toxicity of eight types of GBN: graphene oxide (GO; 400 nm), GO (1 µm), partially reduced graphene oxide (PRGO; 400 nm), reduced graphene oxide (RGO; 400 nm), RGO (2 µm), graphene (110 nm), graphene (140 nm), and graphene (1 µm). We next examined the effects of these GBNs on hTCEpi cell migration. We also determined whether the expression of α-smooth muscle actin (αSMA), a myofibroblast marker, is altered by the GBNs using RCFs. We found that RGO (400 nm) and RGO (2 µm) were highly toxic to hTCEPi cells and RCFs meanwhile, PRGO (400 nm) was toxic only to hTCEpi cells. In addition, PRGO (400 nm), RGO (400 nm), and RGO (2 µm) inhibited hTCEpi cell migration and significantly increased αSMA mRNA expression. Further study in vivo is required to determine if RGO nanomaterials delay corneal epithelial healing and induce scar formation.

CRIPTO antagonist ALK4L75A-Fc inhibits breast cancer cell plasticity and adaptation to stress.

BACKGROUND: CRIPTO is a multi-functional signaling protein that promotes stemness and oncogenesis. We previously developed a CRIPTO antagonist, ALK4L75A-Fc, and showed that it causes loss of the stem cell phenotype in normal mammary epithelia suggesting it may similarly inhibit CRIPTO-dependent plasticity in breast cancer cells. METHODS: We focused on two triple negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to measure the effects of ALK4L75A-Fc on cancer cell behavior under nutrient deprivation and endoplasmic reticulum stress. We characterized the proliferation and migration of these cells in vitro using time-lapse microscopy and characterized stress-dependent changes in the levels and distribution of CRIPTO signaling mediators and cancer stem cell markers. We also assessed the effects of ALK4L75A-Fc on proliferation, EMT, and stem cell markers in vivo as well as on tumor growth and metastasis using inducible lentiviral delivery or systemic administration of purified ALK4L75A-Fc, which represents a candidate therapeutic approach. RESULTS: ALK4L75A-Fc inhibited adaptive responses of breast cancer cells under conditions of nutrient and ER stress and reduced their proliferation, migration, clonogenicity, and expression of EMT and cancer stem cell markers. ALK4L75A-Fc also inhibited proliferation of human breast cancer cells in stressed tumor microenvironments in xenografts and reduced both primary tumor size and metastatic burden. CONCLUSIONS: Cancer cell adaptation to stresses such as nutrient deprivation, hypoxia, and chemotherapy can critically contribute to dormancy, metastasis, therapy resistance, and recurrence. Identifying mechanisms that govern cellular adaptation, plasticity, and the emergence of stem-like cancer cells may be key to effective anticancer therapies. Results presented here indicate that targeting CRIPTO with ALK4L75A-Fc may have potential as such a therapy since it inhibits breast cancer cell adaptation to microenvironmental challenges and associated stem-like and EMT phenotypes.

CRIPTO promotes extracellular vesicle uptake and activation of cancer associated fibroblasts.

Expression of CRIPTO, a factor involved in embryonic stem cells, fetal development, and wound healing, is tied to poor prognosis in multiple cancers. Prior studies in triple negative breast cancer (TNBC) models showed CRIPTO blockade inhibits tumor growth and dissemination. Here, we uncover a previously unidentified role for CRIPTO in orchestrating tumor-derived extracellular vesicle (TEV) uptake and fibroblast activation through discrete mechanisms. We found a novel mechanism by which CRIPTO drives aggressive TNBC phenotypes, involving CRIPTO-laden TEVs that program stromal fibroblasts, toward cancer associated fibroblast cell states, which in turn prompt tumor cell invasion. CRIPTO-bearing TEVs exhibited markedly elevated uptake in target fibroblasts and activated SMAD2/3 through NODAL-independent and - dependent mechanisms, respectively. Engineered expression of CRIPTO on EVs enhanced the delivery of bioactive molecules. In vivo , CRIPTO levels dictated TEV uptake in mouse lungs, a site of EV-regulated premetastatic niches important for breast cancer dissemination. These discoveries reveal a novel role for CRIPTO in coordinating heterotypic cellular crosstalk which offers novel insights into breast cancer progression, delivery of therapeutic molecules, and new, potentially targetable mechanisms of heterotypic cellular communication between tumor cells and the TME.

Mcam stabilizes luminal progenitor breast cancer phenotypes via Ck2 control and Src/Akt/Stat3 attenuation.

Breast cancers are categorized into subtypes with distinctive therapeutic vulnerabilities and prognoses based on their expression of clinically targetable receptors and gene expression patterns mimicking different cell types of the normal gland. Here, we tested the role of Mcam in breast cancer cell state control and tumorigenicity in a luminal progenitor-like murine tumor cell line (Py230) that exhibits lineage and tumor subtype plasticity. Mcam knockdown Py230 cells show augmented Stat3 and Pi3K/Akt activation associated with a lineage state switch away from a hormone-sensing/luminal progenitor state toward alveolar and basal cell related phenotypes that were refractory to growth inhibition by the anti-estrogen therapeutic, tamoxifen. Inhibition of Stat3, or the upstream activator Ck2, reversed these cell state changes. Mcam binds Ck2 and acts as a regulator of Ck2 substrate utilization across multiple mammary tumor cell lines. In Py230 cells this activity manifests as increased mesenchymal morphology, migration, and Src/Fak/Mapk/Paxillin adhesion complex signaling in vitro, in contrast to Mcam's reported roles in promoting mesenchymal phenotypes. In vivo, Mcam knockdown reduced tumor growth and take rate and inhibited cell state transition to Sox10+/neural crest like cells previously been associated with tumor aggressiveness. This contrasts with human luminal breast cancers where MCAM copy number loss is highly coupled to Cyclin D amplification, increased proliferation, and the more aggressive Luminal B subtype. Together these data indicate a critical role for Mcam and its regulation of Ck2 in control of breast cancer cell state plasticity with implications for progression, evasion of targeted therapies and combination therapy design.

Metallic Engineered Nanomaterials and Ocular Toxicity: A Current Perspective.

The ocular surface, comprised of the transparent cornea, conjunctiva, and protective tear film, forms a protective barrier defending deeper structures of the eye from particulate matter and mechanical trauma. This barrier is routinely exposed to a multitude of naturally occurring and engineered nanomaterials (ENM). Metallic ENMs are particularly ubiquitous in commercial products with a high risk of ocular exposure, such as cosmetics and sunscreens. Additionally, there are several therapeutic uses for metallic ENMs owing to their attractive magnetic, antimicrobial, and functionalization properties. The increasing commercial and therapeutic applications of metallic ENMs come with a high risk of ocular exposure with poorly understood consequences to the health of the eye. While the toxicity of metallic ENMs exposure has been rigorously studied in other tissues and organs, further studies are necessary to understand the potential for adverse effects and inform product usage for individuals whose ocular health may be compromised by injury, disease, or surgical intervention. This review provides an update of current literature on the ocular toxicity of metallic ENMs in vitro and in vivo, as well as the risks and benefits of therapeutic applications of metallic ENMs in ophthalmology.

CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing.

A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore's MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore's MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39-416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the 'hidden genome' underlying human disease.

Transcorneal delivery of topically applied silver nanoparticles does not delay epithelial wound healing.

Silver nanoparticles (AgNPs) are a common antimicrobial additive for a variety of applications, including wound care. However, AgNPs often undergo dissolution resulting in release of silver ions, with subsequent toxicity to mammalian cells. The cornea is a primary exposure site to topically administered AgNPs in and around the eye but their impact on corneal wound healing is understudied. Thus, the purpose of this study was to determine in vitro toxicity of AgNPs on corneal epithelial cells and fibroblasts as well as their effects on corneal epithelial wound healing utilizing an in vivo rabbit model. Non-coated 20 nm sized AgNP (AgNP-20) as well as 1% and 10% silver silica NPs (AgSiO2NPs) were tested at concentrations ranging from 0.05-250 µg/mL. Immortalized human corneal epithelial (hTCEpi) cells and primary rabbit corneal fibroblasts (RCFs) were incubated for 24 h with AgNPs and cell viability was tested. Additionally, a round wound healing assay was performed to determine hTCEpi cell migration. Quantitative real-time PCR and western blot analysis was performed to determine α-smooth muscle actin (α-SMA, a myofibroblast marker) mRNA and protein expression, respectively, in RCFs treated with 50 µg/mL of AgNPs. Corneal epithelial wound healing was evaluated with 1%-AgSiO2NPs (10 and 250 µg/mL) using an in vivo rabbit model. Rabbits were subsequently euthanized, and histologic sections of the enucleated globes were used to determine corneal penetration of 1%-AgSiO2NPs with autometallography and hyperspectral darkfield microscopy. Cell viability of both the hTCEpi cells and fibroblasts was significantly decreased by the three AgNPs in a dose dependent manner. Migration of hTCEpi cells was significantly inhibited by the three AgNPs. Alpha-SMA mRNA expression was significantly inhibited with three AgNPs, but only the 1%-AgSiO2NPs inhibited protein expression of α-SMA. In vivo epithelial wound closure did not significantly differ between groups treated with 10 or 250 µg/mL of 1%-AgSiO2NPs or vehicle control. The 1%-AgSiO2NPs penetrated throughout all corneal layers and into the anterior chamber in all treated eyes with no histopathological changes observed. In conclusion, the 1%-AgSiO2NPs are safe and have potential therapeutic applications through its efficacy of the corneal penetration and reduced scar formation during corneal wound healing.

Metal Oxide Engineered Nanomaterials Modulate Rabbit Corneal Fibroblast to Myofibroblast Transformation.

Purpose: Corneal keratocyte-fibroblast-myofibroblast (KFM) transformation plays a critical role in corneal stromal wound healing. However, the impact of engineered nanomaterials (ENMs), found in an increasing number of commercial products, on this process is poorly studied. This study investigates the effects of metal oxide ENMs on KFM transformation in vitro and in vivo. Methods: Cell viability of rabbit corneal fibroblasts (RCFs) was tested following treatment with 11 metal oxide ENMs at concentrations of 0.5 to 250 µg/ml for 24 hours. Messenger RNA (mRNA) and protein expression of αSMA, a marker of myofibroblast transformation, were measured using RCFs after exposure to 11 metal oxide ENMs at a concentration that did not affect cell viability, in media containing either 0 or 10 ng/ml of TGF-ß1. Additionally, the effect of topical Fe2O3 nanoparticles (NPs) (50 ng/ml) on corneal stromal wound healing following phototherapeutic keratectomy (PTK) was determined. Results: V2O5, Fe2O3, CuO, and ZnO ENMs were found to significantly reduce cell viability as compared to vehicle control and the other seven metal oxide ENMs tested. V2O5 nanoflakes significantly reduced mRNA and protein αSMA concentrations in the presence of TGF-ß1. Fe2O3 NPs significantly increased αSMA mRNA expression in the presence of TGF-ß1 but did not alter αSMA protein expression. Topically applied Fe2O3 NPs in an in vivo rabbit corneal stromal wound healing model did not delay healing. Conclusions: Fe2O3 NPs promote corneal myofibroblast induction in vitro but do not impair corneal stromal wound healing in vivo. Translational Relevance: These experimental results can apply to human nanomedical research.