Optical Projection Tomography (OPT), the optical equivalent of x-ray computed tomography, reconstructs the 3D structure of a sample from a series of wide-field 2D projections acquired at different angles [1]. OPT is used to map the optical attenuation and/or fluorescence distributions of intact transparent samples without the need for mechanical sectioning. While it is typically applied to chemically cleared samples, it can also be used to image inherently transparent or weakly scattering live organisms including adult zebrafish up to ~1cm in diameter [2]. When applying OPT to live samples it is important to minimise the data acquisition time while maximising the image quality in the presence of scattering. The former issue can be addressed using compressive sensing to reduce the number of projections required [3]. Scattered light can be rejected using structured illumination [4], but this removes emission from regions the excitation modulation does not reach and reduces the available dynamic range. To address this, we have explored the rejection of scattered light by acquiring projections with parallel semi-confocal line illumination and detection in an approach we describe as slice-OPT (sl-OPT). The impact of optical scattering can also be reduced by imaging at longer wavelengths [5]. We are exploring OPT in the NIR 1&2 spectral windows. However, exotic array detectors, e.g. for short wave infrared light, are costly and so we are also developing a single pixel camera [6] approach. We will present our progress applying these techniques to 3D imaging of vasculature and tumour burden in live adult zebrafish. [1] Sharpe et al, Science, vol. 296, Issue 5567, pp. 541-545, 2002. [2] Kumar et al, Oncotarget, vol. 7, no.28, pp. 43939-43948, 2016. [3] Correia et al, PloS one, vol. 10, no. 8, p. e0136213, 2015. [4] Kristensson et al, Optics express, vol. 20, no. 13, pp. 14437-14450, 2012. [5] Shi et al., Journal of Biophotonics, vol. 9, no. 1-2, pp. 38-43, 2016. [6] Duarte et al., IEEE Signal Processing Magazine, vol. 25, no. 2, pp. 83-91, 2008.
<div>Abstract<p>Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA-damaging therapy–induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing, or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared with gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated patients with PDAC, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome.</p>Significance:<p>These findings establish the potential utility of combinatorial endothelial cell FAK targeting together with gemcitabine in future clinical applications to control metastasis in patients with pancreatic ductal adenocarcinoma.</p></div>
Abstract Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA-damaging therapy–induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing, or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared with gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated patients with PDAC, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome. Significance: These findings establish the potential utility of combinatorial endothelial cell FAK targeting together with gemcitabine in future clinical applications to control metastasis in patients with pancreatic ductal adenocarcinoma.
Abstract Unlike adult mammals, zebrafish are able to naturally regenerate their heart. A key mechanism in zebrafish heart regeneration is the activation of the epicardium, leading to the establishment of a supporting scaffold for newly formed cardiomyocytes, angiogenesis and cytokine secretion. Neuropilins (NRPs) are cell surface co-receptors mediating functional signaling of kinase receptors for cytokines known to play critical roles in zebrafish heart regeneration, including Platelet-Derived growth factor (PDGF), Vascular Endothelial growth factor (VEGF), and Fibroblast growth factor (FGF). Herein, we investigated the role of neuropilins in the response of the zebrafish heart to injury and its subsequent regeneration. All four zebrafish neuropilin isoforms, nrp 1a , 1b , 2a , and 2b , were upregulated following cardiac cryoinjury and were strongly expressed by the activated epicardium. A nrp1a mutant, coding for a truncated, non-functional protein, showed a significant delay in heart regeneration in comparison to Wild-Type fish and displayed persistent collagen deposition. The regenerating hearts of nrp1a mutants were less vascularized and epicardial-derived cell migration and re-expression of the developmental gene Wilms’ tumor 1 was severely impaired in nrp1a mutants. Moreover, cryoinjury-induced activation and migration of epicardial cells in heart explants was strongly reduced in nrp1a mutant zebrafish. These results identify a key role for Nrp1 in zebrafish heart regeneration, mediated through epicardial activation, migration and revascularization.
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