\u3ci\u3eIn Vitro and In Vivo\u3c/i\u3e Correlation of Skin and Cellular Responses to Nucleic Acid Delivery

Skin, the largest organ in the body, provides a passive physical barrier against infection and contains elements of the innate and adaptive immune systems. Skin consists of various cells, including keratinocytes, fibroblasts, endothelial cells and immune cells. This diversity of cell types could be important to gene therapies because DNA transfection could elicit different responses in different cell types. Previously, we observed the upregulation and activation of cytosolic DNA sensing pathways in several non-tumor and tumor cell types as well in tumors after the electroporation (electrotransfer) of plasmid DNA (pDNA). Based on this research and the innate immunogenicity of skin, we correlated the effects of pDNA electrotransfer to fibroblasts and keratinocytes to mouse skin using reverse transcription real-time PCR (RT-qPCR) and several types of protein quantification. After pDNA electrotransfer, the mRNAs of the putative DNA sensors DEAD (AspGlu-Ala-Asp) box polypeptide 60 (Ddx60), absent in melanoma 2 (Aim2), Z-DNA binding protein 1 (Zbp1), interferon activated gene 202 (Ifi202), and interferon-inducible protein 204 (Ifi204) were upregulated in keratinocytes, while Ddx60, Zbp1 and Ifi204 were upregulated in fibroblasts. Increased levels of the mRNAs and proteins of several cytokines and chemokines were detected and varied based on cell type. Mouse skin experiments in vivo confirmed our in vitro results with increased expression of putative DNA sensor mRNAs and of the mRNAs and proteins of several cytokines and chemokines. Finally, with immunofluorescent staining, we demonstrated that skin keratinocytes, fibroblasts and macrophages contribute to the immune response observed after pDNA electrotransfer

Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis

Multiscale topology characterises dynamic tumour vascular networks

Advances in imaging techniques enable high-resolution three-dimensional (3D) visualization of vascular networks over time and reveal abnormal structural features such as twists and loops, and their quantification is an active area of research. Here, we showcase how topological data analysis, the mathematical field that studies the “shape” of data, can characterize the geometric, spatial, and temporal organization of vascular networks. We propose two topological lenses to study vasculature, which capture inherent multiscale features and vessel connectivity, and surpass the single-scale analysis of existing methods. We analyze images collected using intravital and ultramicroscopy modalities and quantify spatiotemporal variation of twists, loops, and avascular regions (voids) in 3D vascular networks. This topological approach validates and quantifies known qualitative trends such as dynamic changes in tortuosity and loops in response to antibodies that modulate vessel sprouting; furthermore, it quantifies the effect of radiotherapy on vessel architecture

Type I IFN protects cancer cells from CD8+ T cell-mediated cytotoxicity after radiation

Treatment of tumors with ionizing radiation stimulates an antitumor immune response partly dependent on induction of IFNs. These IFNs directly enhance dendritic cell and CD8+ T cell activity. Here we show that resistance to an effective antitumor immune response is also a result of IFN signaling in a different cellular compartment of the tumor, the cancer cells themselves. We abolished type I IFN signaling in cancer cells by genetic elimination of its receptor, IFNAR1. Pronounced immune responses were provoked after ionizing radiation of tumors from 4 mouse cancer cell lines with Ifnar1 knockout. This enhanced response depended on CD8+ T cells and was mediated by enhanced susceptibility to T cell-mediated killing. Induction of Serpinb9 proved to be the mechanism underlying control of susceptibility to T cell killing after radiation. Ifnar1-deficient tumors had an augmented response to anti-PD-L1 immunotherapy with or without radiation. We conclude that type I IFN can protect cancer cells from T cell-mediated cytotoxicity through regulation of Serpinb9. This result helps explain why radiation of tumors can stimulate antitumor immunity yet also result in resistance. It further suggests potential targets for intervention to improve therapy and to predict responses

Image-based artefact removal in laser scanning microscopy

Recent developments in laser scanning microscopy have greatly extended its applicability in cancer imaging beyond the visualisation of complex biology, and opened up the possibility of quantitative analysis of inherently dynamic biological processes. However, the physics of image acquisition intrinsically means that image quality is subject to a trade-off between a number of imaging parameters including resolution, signal-to-noise ratio, and acquisition speed. We address the problem of geometric distortion, in particular jaggedness artefacts that are caused by the variable motion of the microscope laser, by using a combination of image processing techniques. Image restoration methods have already shown great potential for post-acquisition image analysis. The performance of our proposed image restoration technique was first quantitatively evaluated using phantom data with different textures, and then qualitatively assessed using in vivo biological imaging data. In both cases, the presented method, comprising a combination of image registration and filtering, is demonstrated to have substantial improvement over state-of-the-art microscopy acquisition methods

Image-based artefact removal in laser scanning microscopy

Recent developments in laser scanning microscopy have greatly extended its applicability in cancer imaging beyond the visualisation of complex biology, and opened up the possibility of quantitative analysis of inherently dynamic biological processes. However, the physics of image acquisition intrinsically means that image quality is subject to a trade-off between a number of imaging parameters including resolution, signal-to-noise ratio, and acquisition speed. We address the problem of geometric distortion, in particular jaggedness artefacts that are caused by the variable motion of the microscope laser, by using a combination of image processing techniques. Image restoration methods have already shown great potential for post-acquisition image analysis. The performance of our proposed image restoration technique was first quantitatively evaluated using phantom data with different textures, and then qualitatively assessed using in vivo biological imaging data. In both cases, the presented method, comprising a combination of image registration and filtering, is demonstrated to have substantial improvement over state-of-the-art microscopy acquisition methods

STING-dependent interferon-λ1 induction in HT29 cells, a human colorectal cancer cell line, after gamma-radiation

To investigate the induction of type III interferons (IFNs) in human cancer cells by gamma-rays.Type III IFN expression in human cancer cell lines after gamma-ray irradiation in vitro was assessed by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Signaling pathways mediating type III IFN induction were examined by a variety of means, including immunoblotting, flow cytometry, confocal imaging, and reverse transcription-quantitative polymerase chain reaction. Key mediators in these pathways were further explored and validated using gene CRISPR knockout or short hairpin RNA knockdown.Exposure to gamma-rays directly induced type III IFNs (mainly IFNL1) in human cancer cell lines in dose- and time-dependent fashions. The induction of IFNL1 was primarily mediated by the cytosolic DNA sensors-STING-TBK1-IRF1 signaling axis, with a lesser contribution from the nuclear factor kappa b signaling in HT29 cells. In addition, type III IFN signaling through its receptors serves as a positive feedback loop, further enhancing IFN expression via up-regulation of the kinases in the STING-TBK1 signaling axis.Our results suggest that IFNL1 can be up-regulated in human cancer cell lines after gamma-ray treatment. In HT29 cells this induction occurs via the STING pathway, adding another layer of complexity to the understanding of radiation-induced antitumor immunity, and may provide novel insights into IFN-based cancer treatment

Predicting the influence of microvascular structure on tumour response to radiotherapy

Objective: The purpose of this study is to investigate how theoretical predictions of tumour response to radiotherapy depend on the morphology and spatial representation of the microvascular network. Methods: A hybrid multiscale model, that couples a cellular automaton model of tumour growth with a model for oxygen transport from blood vessels, is used to predict the viable fraction of cells following one week of simulated radiotherapy. Both artificial and biologically derived three-dimensional vessel networks of well vascularized tumours are considered and predictions compared with two-dimensional descriptions. Results: For literature-derived values of the cellular oxygen consumption rate there is little difference in predicted viable fraction when three-dimensional network representations of biological or artificial vessel networks are employed. Different two-dimensional representations are shown to either over- or under-estimate viable fractions relative to the three-dimensional cases, with predictions based on point-wise descriptions shown to have greater sensitivity to vessel network morphology. Conclusion: The predicted radiotherapy response is relatively insensitive to the morphology of the microvessel network when threedimensional representations are adopted, however sensitivity is greater in certain two-dimensional representations. Significance: By using realistic three-dimensional vessel network geometries this study shows that real and artificial network descriptions and assumptions of spatially uniform oxygen distributions lead to similar radiotherapy response predictions in relatively small tissue volumes. This suggests that either a more detailed description of oxygen transport in the microvasculature is required or that the oxygen enhancement ratio used in the well known linearquadratic radiotherapy response model is relatively insensitive to microvascular structure

Reciprocal interactions between tumour cell populations enhance growth and reduce radiation sensitivity in prostate cancer

Intratumoural heterogeneity (ITH) contributes to local recurrence following radiotherapy in prostate cancer. Recent studies also show that ecological interactions between heterogeneous tumour cell populations can lead to resistance in chemotherapy. Here, we evaluated whether interactions between heterogenous populations could impact growth and response to radiotherapy in prostate cancer. Using mixed 3D cultures of parental and radioresistant populations from two prostate cancer cell lines and a predator-prey mathematical model to investigate various types of ecological interactions, we show that reciprocal interactions between heterogeneous populations enhance overall growth and reduce radiation sensitivity. The type of interaction influences the time of regrowth after radiation, and, at the population level, alters the survival and cell cycle of each population without eliminating either one. These interactions can arise from oxygen constraints and from cellular cross-talk that alter the tumour microenvironment. These findings suggest that ecological-type interactions are important in radiation response and could be targeted to reduce local recurrence

Estimating oxygen distribution from vasculature in three-dimensional tumour tissue

Regions of tissue which are well oxygenated respond better to radiotherapy than hypoxic regions by up to a factor of three. If these volumes could be accurately estimated, it might be possible to selectively boost dose to radio-resistent regions, a concept known as dose-painting. While imaging modalities such as 18F-Miso PET allow identification of hypoxic regions, they are intrinsically limited by the physics of such systems to the millimeter domain whereas tumour oxygenation is known to vary over a micron scale. Mathematical modeling of microscopic tumour oxygen distribution therefore has the potential to complement and enhance macroscopic information derived from PET. In this work, we develop a general method of estimating oxygen distribution in three-dimensions from a source vessel map. The method is applied analytically to line sources and quasilinear idealized line source maps, and also applied to full 3D vessel distributions through a kernel method and compared with oxygen distribution in tumour sections. The model outlined is flexible and stable, and can readily be applied to estimating likely microscopic oxygen distribution from any source geometry. We also investigate the problem of reconstructing 3D oxygen maps from histological and confocal 2D sections, concluding that 2D histological sections are generally inadequate representations of the 3D oxygen distribution