Accumulation of senescent cells contributes to age-related diseases including idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding proteins (IGFBPs) regulate many biological processes; however, the functional contributions of IGFBP2 in lung fibrosis remain largely unclear. Here, we report that intranasal delivery of recombinant IGFBP2 protects aged mice from weight loss and demonstrated antifibrotic effects after bleomycin lung injury. Notably, aged human-Igfbp2 transgenic mice reveal reduced senescence and senescent-associated secretory phenotype factors in alveolar epithelial type 2 (AEC2) cells and they ameliorated bleomycin-induced lung fibrosis. Finally, we demonstrate that IGFBP2 expression is significantly suppressed in AEC2 cells isolated from fibrotic lung regions of patients with IPF and/or pulmonary hypertension compared with patients with hypersensitivity pneumonitis and/or chronic obstructive pulmonary disease. Altogether, our study provides insights into how IGFBP2 regulates AEC2-cell-specific senescence and that restoring IGFBP2 levels in fibrotic lungs can prove effective for patients with IPF.

Accumulation of senescent cells contributes to age related diseases including idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding proteins (IGFBPs) are evolutionarily conserved proteins that play a vital role in many biological processes. Overall, little is known about the functions of IGFBP2 in the epigenetic regulation of cellular senescence and pulmonary fibrosis. Here, we show that Igfbp2 expression was significantly downregulated at both mRNA and protein levels in a low-dose bleomycin-induced pulmonary fibrosis model of aged mice. Using the reduced representation of bisulfite sequencing technique, we demonstrated Igfbp2 downregulation is attributed to DNA methylation of CpG islands in fibrotic lungs of aged mice. Furthermore, Igfbp2 siRNA knockdown increased both P53 and P21 protein levels in mouse lung epithelial cells exposed to hypoxia treatment. Lentiviral mediated expression of Igfb2 decreased P21 protein levels and significantly reduced beta galactosidase activity in mouse lung epithelial cells challenged with a senescent drug (atazanavir) and hypoxia treatments. Using the RT2 Profiler PCR Array, we found that P21, PAI-1, IRF-5 and IRF-7, important regulators of senescence pathway, were significantly downregulated specifically in type-II alveolar epithelial cells (AECs) of aged human-Igfbp2 transgenic mice after bleomycin challenge. Finally, transgenic expression of human-Igfbp2 in type-II AECs from aged bleomycin challenged mice significantly decreased senescent associated secretory phenotype factors and also reduced extracellular matrix markers compared to aged wild-type mice challenged with bleomycin injury. Collectively, these findings reveal that epigenetic repression of Igfbp2 promotes pulmonary fibrosis and that restoring IGFBP2 in fibrotic lungs could prove effective in IPF treatment.

The αvβ6 integrin plays a key role in the activation of transforming growth factor-β (TGFβ), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvβ6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvβ6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFβ signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvβ6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvβ6, induces prolonged inhibition of TGFβ signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

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Performing drug screening of tissue derived from cancer patient biopsies using physiologically relevant 3D tumour models presents challenges due to the limited amount of available cell material. Here, we present a microfluidic platform that enables drug screening of cancer cell-enriched multicellular spheroids derived from tumour biopsies, allowing extensive anticancer compound screening prior to treatment. This technology was validated using cell lines and then used to screen primary human prostate cancer cells, grown in 3D as a heterogeneous culture from biopsy-derived tissue. The technology enabled the formation of repeatable drug concentration gradients across an array of spheroids without external fluid actuation, delivering simultaneously a range of drug concentrations to multiple sized spheroids, as well as replicates for each concentration. As proof-of-concept screening, spheroids were generated from two patient biopsies and a panel of standard-of-care compounds for prostate cancer were tested. Brightfield and fluorescence images were analysed to provide readouts of spheroid growth and health, as well as drug efficacy over time. Overall, this technology could prove a useful tool for personalised medicine and future drug development, with the potential to provide cost- and time-reduction in the healthcare delivery.

Performing drug screening, of physiologically relevant three-dimensional (3D) tumor models, for personalized treatment remains challenging, due to the small amount of tissue available from most biopsies. New microfluidic technologies, enabling greater control over cell positioning and fluid behavior at the micro-scale, allow extensive testing of anticancer agents on human tumor tissue preparations in 3D and offer new solutions for the development of anticancer compounds and personalized medicine. We have developed a microfluidic platform for extensive drug screening of tumor biopsies in a cost-effective manner and validated the system with tumor prostate patient samples. As a typical drug screening assay, up to 22 drug concentration-response curves could be generated from a single biopsy, within a time frame of up to 4 weeks. Biopsy tissue, grown as a heterogeneous co-culture from the primary sample, was prepared as cancer-cell enriched multicellular spheroids, cultured for 3 to 5 days prior to the application of a panel of standard-of-care drugs for prostate cancer. Readouts were obtained via bright-field and epifluorescence microscopy. The microfluidic platform was designed to be operated entirely without the need of external fluid actuation, with the microfluidic network capable of generating long-lasting, stable and repeatable drug concentration gradients across arrays of 240 spheroids. Outcomes were generated as 8-point drug concentration response curves per device, with each drug concentration tested on at least 24 spheroids. In-house developed software was used to analyze bright-field and fluorescent images to provide readouts of spheroid growth and viability, as well as information on drug penetration and drug efficacy over time. Following platform and assay validation using cancer cell lines, proof-of-concept screening was performed on prostate biopsies from 2 different patients. Results showed that biopsy-derived spheroids were more resistant to treatment than LNCaP spheroids, a prostate cancer cell line. For one biopsy, spheroids were sensitive to docetaxel, but resistant to enzalutamide, despite the presence of intact androgen receptors. This preliminary data outlines how this technology could become a useful tool to investigate patient-specific drug resistance and to test novel anticancer agents in a cost-effective manner, based on maximized screening of human tumor tissue in a 3D format. Citation Format: Theresa E. Mulholland, Milly McAllister, Samantha Patek, David Flint, Mark Underwood, Alexander Sim, Joanne Edwards, Michele Zagnoni. Drug screening of biopsy-derived multicellular spheroids using microfluidic technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5024.

Post-exposure nerve agent treatment usually includes administration of an oxime, which acts to restore function of the enzyme acetylcholinesterase (AChE). For immediate treatment of military personnel, this is usually administered with an autoinjector device, or devices containing the oxime such as pralidoxime, atropine and diazepam. In addition to the autoinjector, it is likely that personnel exposed to nerve agents, particularly by the percutaneous route, will require further treatment at medical facilities. As such, there is a need to understand the relationship between dose rate, plasma concentration, reactivation of AChE activity and efficacy, to provide supporting evidence for oxime infusions in nerve agent poisoning. Here, it has been demonstrated that intravenous infusion of HI-6, in combination with atropine, is efficacious against a percutaneous VX challenge in the conscious male Dunkin-Hartley guinea-pig. Inclusion of HI-6, in addition to atropine in the treatment, improved survival when compared to atropine alone. Additionally, erythrocyte AChE activity following poisoning was found to be dose dependent, with an increased dose rate of HI-6 (0.48 mg/kg/min) resulting in increased AChE activity. As far as we are aware, this is the first study to correlate the pharmacokinetic profile of HI-6 with both its pharmacodynamic action of reactivating nerve agent inhibited AChE and with its efficacy against a persistent nerve agent exposure challenge in the same conscious animal.

A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (αvβ6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvβ6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([3H]A20FMDV2), its high affinity (KD: 0.22 nmol/l) and selectivity (at least 85-fold) for αvβ6 over the other members of the RGD integrin family was confirmed. [3H]A20FMDV2 αvβ6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvβ6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t1/2: 1.5 and 3.1 min, respectively), concentration-dependent (EC50: values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvβ6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvβ6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvβ6 following RGD ligand engagement.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality in disorders that include IPF. The αvβ6 integrin has been identified as playing a key role in the activation of TGFβ that is hypothesized to be pivotal in …

A major drawback in the development of new cancer therapeutics is the use of two‐dimensional (2D) monolayer cultures as models for solid tumours. A solution lies in the use of three‐dimensional (3D) multicellular aggregate models, known as spheroids or organoids. These mimic the interactions between cells within a tumour, the morphology and the microenvironment of tumours, representing a simplified but more physiologically relevant in vitro system for drug screening than that provided by 2D bioassays. In this study, we developed a novel microfluidic platform that allows the formation and long‐term culture of hundreds of multicellular cancer spheroids, retaining complete control over the number and the size of spheroids grown, the ability to deliver chemical compounds in a customisable concentration gradient format and the ability to recover the spheroids for off‐chip post‐processing after treatment. UVW cells, a human brain high‐grade glioma cell line, were used to create spheroids in an array format and were exposed to a microfluidic concentration gradient of cisplatin. Results showed that the growth of 3D glioma spheroids after treatment is reduced in a concentration‐dependent manner and that a dose response curve can be obtained from a single device. These findings demonstrate the suitability of the system for high‐throughput drug screening, combination therapeutics and the potential for use with biopsy samples.

Reducing Cx43 expression stimulates skin wound healing. This is mimicked in models when Cx43 function is blocked by the connexin mimetic peptide Gap27. IGF-I also stimulates wound healing with IGFBP-5 attenuating its actions. Further, the IGF-I to IGFBP-5 ratio is altered in diabetic skin, where wound closure is impaired. We investigated whether Gap27 remains effective in augmenting scrape-wound closure in human skin wound models simulating diabetes-induced changes, using culture conditions with raised glucose, insulin and IGFBP-5. Gap27 increased scrape-wound closure in normal glucose and insulin (NGI) and to a lesser extent in high glucose and insulin (HGI). IGF-I enhanced scrape-wound closure in keratinocytes whereas IGFBP-5 inhibited this response. Gap27 overcame the inhibitory effects of IGFBP-5 on IGF-I activity. Connexin-mediated communication (CMC) was reduced in HGI, despite raised Cx43, and Gap27 significantly decreased CMC in NGI and HGI. IGF-I and IGFBP-5 did not affect CMC. IGF-I increased keratinocyte proliferation in NGI, and Gap27 increased proliferation in NGI to a greater extent than in HGI. We conclude that IGF-I and Gap27 stimulate scrape-wound closure by independent mechanisms with Gap27 inhibiting Cx43 function. Gap27 can enhance wound closure in diabetic conditions, irrespective of the IGF-I:IGFBP-5 balance.

TGFβ1 is a major fibrotic factor and its actions involve induction of epithelial cell death, together with the stimulation and transdifferentiation of fibroblasts into collagen- and fibronectin-secreting myofibroblasts. These actions of TGFβ1 are also consistent with a pro-metastatic role, by aiding epithelial cell escape through mesenchymal tissues. Recently IGFBP-5 has been described as a pro-fibrotic (pro-metastatic?) agent and the aim of this study was to compare and contrast the actions of IGFBP-5 with TGFβ1. We used NMuMG cells and cloned stable epithelial and mesenchymal lines from the parent cells. TGFβ1 induced apoptosis and/or EMT in the epithelial cells, whereas it enhanced mesenchymal cell survival and migration. IGFBP-5, in contrast, enhanced both cell–cell and cell–ECM adhesion and also improved wound closure in epithelial cells whereas, in mesenchymal cells, IGFBP-5 decreased adhesion and migration. Furthermore, IGFBP-5 was able to antagonise the actions of TGFβ1. In a co-culture model simulating epithelial–mesenchymal boundaries, IGFBP-5 was able to antagonise the disruptive transgressions induced by TGFβ1. Overall, these findings suggest that IGFBP-5 is important in maintaining epithelial–mesenchymal boundaries and thus may limit metastasis and fibrosis by inducing an orderly repair mechanism, very distinct from the fibrotic disruption induced by TGFβ1. A role for IGFBP-5 in the inhibition of metastasis is supported by immunohistochemical studies of breast cancer microarrays, where we show that elevated IGFBP-5 expression is associated with increased disease-free survival.

Maintenance of tissue boundaries is crucial for control of metastasis. We describe a novel signalling pathway in which epithelial cell disruption can be minimised and thereby restricts epithelial-mesenchymal transgressions. This involves IGFBP-5 release from apoptotic cells, which increases epithelial cell adhesion on mesenchymal but not epithelial ECM and involves direct interaction of IGFBP-5 with α2β1 integrins. IGFBP-5 also induced adhesion to vitronectin in the absence of αVβ3 integrin, the vitronectin receptor, again via an α2β1 integrin-dependent action, suggesting that IGFBP-5 can induce spreading on matrices, even in the absence of the integrins normally used. Using IGFBP-5 mutants we demonstrate that the effect is IGF-independent but requires the heparin-binding domain in the c-terminus of IGFBP-5. A truncated c-terminal mutant of IGFBP-5 also induced adhesion. Adhesion induced by IGFBP-5 was cdc42-dependent and resulted in activation of ILK and Akt. Consistent with these changes, IGFBP-5 facilitated prolonged cell survival in nutrient-poor conditions and decreased phosphorylation of the stress-activated kinase p38MAPK. Whilst IGFBP-5 enhanced adhesion, it inhibited cell migration although this was not evident using the truncated c-terminal mutant, suggesting that effects of IGFBP-5 on adhesion and migration involve different mechanisms. These responses to IGFBP-5 would be anticipated to reduce metastatic potential.