Impact of soluble tumor necrosis factor-related apoptosis-inducing ligand released by engineered adipose mesenchymal stromal cells on white blood cells.

BACKGROUND AIMS: The proapoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is physiologically expressed by immune cells and performs regulatory functions in infections, autoimmune diseases and cancer, where it acts as a tumor suppressor. Adipose-derived mesenchymal stromal cells (AD-MSCs) also may play immunomodulatory roles in both primary and acquired immune responses. We have previously demonstrated the efficacy of an anticancer gene therapy based on AD-MSC engineered to secrete a soluble TRAIL variant (sTRAIL) against pancreatic cancer. However, the impact of AD-MSC sTRAIL on leukocyte subsets has been not yet considered also to predict a possible immunotoxicity profile in the clinical translation of this cell-based anticancer strategy. METHODS: Monocytes, polymorphonuclear cells and T lymphocytes were freshly isolated from the peripheral blood of healthy donors. Immunophenotype and functional (DR4 and DR5) and decoy (DcR1 and DcR2) TRAIL receptors were tested by flow cytometry. The viability of white blood cells treated with sTRAIL released by gene-modified AD-MSC or co-cultured with AD-MSC sTRAIL was then evaluated by both metabolic assays and flow cytometry. In addition, cytokine profile in co-cultures was analyzed by multiplex enzyme-linked immunosorbent assay. RESULTS: Monocytes and polymorphonuclear cells showed high positivity for DR5 and DcR2, respectively, whereas T cells revealed negligible expression of all TRAIL receptors. Irrespective of TRAIL receptors' presence on the cell membrane, white blood cells were refractory to the proapoptotic effect displayed by sTRAIL secreted by gene-modified AD-MSC, and direct cell-to-cell contact with AD-MSC sTRAIL had negligible impact on T-cell and monocyte viability. Cytokine crosstalk involving interleukin 10, tumor necrosis factor alpha, and interferon gamma secreted by T lymphocytes and vascular endothelial growth factor A and interleukin 6 released by AD-MSC was highlighted in T-cell and AD-MSC sTRAIL co-cultures. CONCLUSIONS: In summary, this study demonstrates the immunological safety and thus the clinical feasibility of an anticancer approach based on AD-MSC expressing the proapoptotic molecule sTRAIL.

Noninvasive diffusion magnetic resonance imaging of brain tumour cell size for the early detection of therapeutic response.

Cancer cells differ in size from those of their host tissue and are known to change in size during the processes of cell death. A noninvasive method for monitoring cell size would be highly advantageous as a potential biomarker of malignancy and early therapeutic response. This need is particularly acute in brain tumours where biopsy is a highly invasive procedure. Here, diffusion MRI data were acquired in a GL261 glioma mouse model before and during treatment with Temozolomide. The biophysical model VERDICT (Vascular Extracellular and Restricted Diffusion for Cytometry in Tumours) was applied to the MRI data to quantify multi-compartmental parameters connected to the underlying tissue microstructure, which could potentially be useful clinical biomarkers. These parameters were compared to ADC and kurtosis diffusion models, and, measures from histology and optical projection tomography. MRI data was also acquired in patients to assess the feasibility of applying VERDICT in a range of different glioma subtypes. In the GL261 gliomas, cellular changes were detected according to the VERDICT model in advance of gross tumour volume changes as well as ADC and kurtosis models. VERDICT parameters in glioblastoma patients were most consistent with the GL261 mouse model, whilst displaying additional regions of localised tissue heterogeneity. The present VERDICT model was less appropriate for modelling more diffuse astrocytomas and oligodendrogliomas, but could be tuned to improve the representation of these tumour types. Biophysical modelling of the diffusion MRI signal permits monitoring of brain tumours without invasive intervention. VERDICT responds to microstructural changes induced by chemotherapy, is feasible within clinical scan times and could provide useful biomarkers of treatment response.

Modelling the transport of fluid through heterogeneous, whole tumours in silico.

Cancers exhibit spatially heterogeneous, unique vascular architectures across individual samples, cell-lines and patients. This inherently disorganised collection of leaky blood vessels contribute significantly to suboptimal treatment efficacy. Preclinical tools are urgently required which incorporate the inherent variability and heterogeneity of tumours to optimise and engineer anti-cancer therapies. In this study, we present a novel computational framework which incorporates whole, realistic tumours extracted ex vivo to efficiently simulate vascular blood flow and interstitial fluid transport in silico for validation against in vivo biomedical imaging. Our model couples Poiseuille and Darcy descriptions of vascular and interstitial flow, respectively, and incorporates spatially heterogeneous blood vessel lumen and interstitial permeabilities to generate accurate predictions of tumour fluid dynamics. Our platform enables highly-controlled experiments to be performed which provide insight into how tumour vascular heterogeneity contributes to tumour fluid transport. We detail the application of our framework to an orthotopic murine glioma (GL261) and a human colorectal carcinoma (LS147T), and perform sensitivity analysis to gain an understanding of the key biological mechanisms which determine tumour fluid transport. Finally we mimic vascular normalization by modifying parameters, such as vascular and interstitial permeabilities, and show that incorporating realistic vasculatures is key to modelling the contrasting fluid dynamic response between tumour samples. Contrary to literature, we show that reducing tumour interstitial fluid pressure is not essential to increase interstitial perfusion and that therapies should seek to develop an interstitial fluid pressure gradient. We also hypothesise that stabilising vessel diameters and permeabilities are not key responses following vascular normalization and that therapy may alter interstitial hydraulic conductivity. Consequently, we suggest that normalizing the interstitial microenvironment may provide a more effective means to increase interstitial perfusion within tumours.

Computational fluid dynamics with imaging of cleared tissue and of in vivo perfusion predicts drug uptake and treatment responses in tumours.

Understanding the uptake of a drug by diseased tissue, and the drug's subsequent spatiotemporal distribution, are central factors in the development of effective targeted therapies. However, the interaction between the pathophysiology of diseased tissue and individual therapeutic agents can be complex, and can vary across tissue types and across subjects. Here, we show that the combination of mathematical modelling, high-resolution optical imaging of intact and optically cleared tumour tissue from animal models, and in vivo imaging of vascular perfusion predicts the heterogeneous uptake, by large tissue samples, of specific therapeutic agents, as well as their spatiotemporal distribution. In particular, by using murine models of colorectal cancer and glioma, we report and validate predictions of steady-state blood flow and intravascular and interstitial fluid pressure in tumours, of the spatially heterogeneous uptake of chelated gadolinium by tumours, and of the effect of a vascular disrupting agent on tumour vasculature.

Planar cell polarity genes Celsr1 and Vangl2 are necessary for kidney growth, differentiation, and rostrocaudal patterning.

The mammalian kidney contains nephrons comprising glomeruli and tubules joined to ureteric bud-derived collecting ducts. It has a characteristic bean-like shape, with near-complete rostrocaudal symmetry around the hilum. Here we show that Celsr1, a planar cell polarity (PCP) gene implicated in neural tube morphogenesis, is required for ureteric tree growth in early development and later in gestation prevents tubule overgrowth. We also found an interaction between Celsr1 and Vangl2 (another PCP gene) in ureteric tree growth, most marked in the caudal compartment of the kidneys from compound heterozygous mutant mice with a stunted rump. Furthermore, these genes together are required for the maturation of glomeruli. Interestingly, we demonstrated patients with CELSR1 mutations and spina bifida can have significant renal malformations. Thus, PCP genes are important in mammalian kidney development and have an unexpected role in rostrocaudal patterning during organogenesis.

Quantification of light attenuation in optically cleared mouse brains.

Optical clearing, in combination with recently developed optical imaging techniques, enables visualization and acquisition of high-resolution, three-dimensional images of biological structures deep within the tissue. Many different approaches can be used to reduce light absorption and scattering within the tissue, but there is a paucity of research on the quantification of clearing efficacy. With the use of a custom-made spectroscopy system, we developed a way to quantify the quality of clearing in biological tissue and applied it to the mouse brain. Three clearing techniques were compared: BABB (1:2 mixture of benzyl alcohol and benzyl benzoate, also known as Murray's clear), pBABB (peroxide BABB, a modification of BABB which includes the use of hydrogen peroxide), and passive CLARITY. We found that BABB and pBABB produced the highest degree of optical clearing. Furthermore, the approach allows regional measurement of light attenuation to be performed, and our results show that light is most attenuated in regions with high lipid content. We provide a way to choose between the multiple clearing protocols available, and it could prove useful for evaluating images that are acquired with cleared tissues.