Experimental and clinical studies on liver regeneration and hepatocellular carcinoma. Roles of redox proteins, iron homeostasis and multikinase inhibition.

Compensatory liver regeneration is triggered by chronic liver injury or surgery and is crucial to maintain tissue homeostasis. The underlying mechanisms which include a whole battery of complex signaling events have been thoroughly studied for decades. The majority of hepatocellular carcinomas develop in a highly proliferative environment caused by underlying chronic liver disease in which lost liver tissue must be restored to meet the needs of the organism. The chronic inflammatory condition with chronic liver repair enhances the presence of free radicals leading to an increased risk of cell alterations. This thesis includes four papers; the first three of which comprise studies regarding the importance of the regulation and endurance of cell proliferation, and also the sensitivity of the proliferating cells to compounds used in cancer prevention and treatment. In these studies we used a 2/3 partial hepatectomy (PH) rat model and also a chemically induced rat liver cancer model (The Solt and Farber Resistant hepatocyte model). The fourth paper is a human study in which we quantified immunohistochemical stainings for 6 different redox proteins in livers from patients resected for hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) liver metastases. The specific aims were: (I) to characterize gene expression of the different pathways involved in hepcidin regulation after PH, until liver regeneration is complete; (II) to study the effects of sodium selenite on regenerative versus neoplastic liver cell proliferation in rat, and to investigate if TrxR1 is a constitutive tumour marker or an unspecific marker for cell growth in rat liver; (III) to study the effect of the anticancer agent sorafenib, a multikinase inhibitor, on normal liver regeneration after PH in rat; and (IV) to evaluate if redox protein (thioredoxins and glutaredoxins) expressions correlate to clinical features in human hepatocellular carcinoma and if they can be used as prognostic markers after liver surgery. Our results showed that high serum levels of IL6 induced the levels of STAT3 and the expression of hepcidin mRNA during the acute phase after PH. The gene expressions of the iron sensing proteins HFE, hemojuvelin (HJV) and transferrin receptor 2 (TfR2) were decreased during the whole regeneration, gradually decreasing hepcidin gene expression and thereby mobilizing iron to the growing liver. The expression of genes involved in iron uptake; transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) were increased thereby facilitating iron uptake (paper I). After administration of sodium selenite in a tumour preventive, supranutritional dose followed by PH no effect on body weights or gain of liver mass was seen. In the hepatocarcinogenesis model the tumour volume was significantly decreased in animals supplied with selenium during the progression phase compared tumours in rats not treated with selenium. The expression of TrxR1 was exclusively seen in the neoplastic liver lesions but not in the remodelling preneoplastic lesions (paper II). Treatment with sorafenib transiently suppressed liver regeneration and the gain of relative liver mass, but was followed by a delayed compensatory increase of liver cell proliferation one week after resection with the result that after 14 days the treated animals reached the same relative liver weights as the controls did in five days (paper III). In the human study we saw an up-regulation of Trx1, Trx2 and Grx5 in HCC compared to its respective surrounding non-tumorous tissue. The same was observed in the CRC metastases where also the staining of Grx1 and Grx3 was significantly higher compared to non-tumorous tissue. Trx1 expression correlated well to cell proliferation but not to tumour differentiation, micro-vascular invasion or tumour recurrence. A relative down regulation of Trx1 was seen in tumours compared to the surrounding liver in males, smokers and in patients with high alcohol consumption. We concluded that the peak of hepcidin expression during the acute phase was eventually overruled by the downregulation of the iron sensing pathway in order to promote iron mobilization to the regenerating liver. We also concluded that selenium in a supranutritional dose impaired tumour growth without impairing the normal liver cell proliferation, and that the selenoprotein TrxR1 is a constituent of the neoplastic phenotype. Sorafenib prolonged liver regeneration in proportion to the length of treatment but the liver adapted to the early inhibitory effects of the drug. Thioredoxins and glutaredoxins were ubiquitously expressed in livers exposed to oxidative stress and various malignancies and can therefore not be used as diagnostic markers for HCC. Smoking and high alcohol consumption increased the Trx1 expression in tissue surrounding the HCCs, whereas expression of Trx1 in the HCCs correlated to cell proliferation. Redox protein expression in HCCs cannot be used as predictive markers for tumour recurrence after liver resection

Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics

Periodontitis is a highly prevalent chronic inflammatory disease of the periodontium, leading ultimately to tooth loss. In order to characterize the gene expression of periodontitis-affected gingival tissue, we have here simultaneously quantified and localized gene expression in periodontal tissue using spatial transcriptomics, combining RNA sequencing with histological analysis. Our analyses revealed distinct clusters of gene expression, which were identified to correspond to epithelium, inflamed areas of connective tissue, and non-inflamed areas of connective tissue. Moreover, 92 genes were identified as significantly up-regulated in inflamed areas of the gingival connective tissue compared to non-inflamed tissue. Among these, immunoglobulin lambda-like polypeptide 5 (IGLL5), signal sequence receptor subunit 4 (SSR4), marginal zone B and B1 cell specific protein (MZB1), and X-box binding protein 1 (XBP1) were the four most highly up-regulated genes. These genes were also verified as significantly higher expressed in gingival tissue of patients with periodontitis compared to healthy controls, using reverse transcription quantitative polymerase chain reaction. Moreover, the protein expressions of up-regulated genes were verified in gingival biopsies by immunohistochemistry. In summary, in this study, we report distinct gene expression signatures within periodontitis-affected gingival tissue, as well as specific genes that are up-regulated in inflamed areas compared to non-inflamed areas of gingival tissue. The results obtained from this study may add novel information on the genes and cell types contributing to pathogenesis of the chronic inflammatory disease periodontitis

An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries.

Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.QC 20161124</p

Deconvolution of spatial sequencing provides accurate characterization of hESC-derived DA transplants in vivo

Cell therapy for Parkinson’s disease has experienced substantial growth in the past decades with several ongoing clinical trials. Despite increasing refinement of differentiation protocols and standardization of the transplanted neural precursors, the transcriptomic analysis of cells in the transplant after its full maturation in vivo has not been thoroughly investigated. Here, we present spatial transcriptomics analysis of fully differentiated grafts in their host tissue. Unlike earlier transcriptomics analyses using single-cell technologies, we observe that cells derived from human embryonic stem cells (hESCs) in the grafts adopt mature dopaminergic signatures. We show that the presence of phenotypic dopaminergic genes, which were found to be differentially expressed in the transplants, is concentrated toward the edges of the grafts, in agreement with the immunohistochemical analyses. Deconvolution shows dopamine neurons being the dominating cell type in many features beneath the graft area. These findings further support the preferred environmental niche of TH-positive cells and confirm their dopaminergic phenotype through the presence of multiple dopaminergic markers

Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver

Reconstruction of heterogeneity through single cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we apply Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology. Global transcriptional differences across lobular units in the liver remain unknown. Here the authors perform spatial transcriptomics of liver tissue to delineate transcriptional differences in physical space, confirm lobular zonation along transcriptional gradients and suggest the presence of previously uncharacterized structures within liver tissue

Massive and parallel expression profiling using microarrayed single-cell sequencing

Single-cell transcriptome analysis overcomes problems inherently associated with averaging gene expression measurements in bulk analysis. However, single-cell analysis is currently challenging in terms of cost, throughput and robustness. Here, we present a method enabling massive microarray-based barcoding of expression patterns in single cells, termed MASC-seq. This technology enables both imaging and high-throughput single-cell analysis, characterizing thousands of single-cell transcriptomes per day at a low cost (0.13 USD/cell), which is two orders of magnitude less than commercially available systems. Our novel approach provides data in a rapid and simple way. Therefore, MASC-seq has the potential to accelerate the study of subtle clonal dynamics and help provide critical insights into disease development and other biological processes

The spatial RNA integrity number assay for in situ evaluation of transcriptome quality

The RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows

Visualization and analysis of gene expression in tissue sections by spatial transcriptomics

Analysis of the pattern of proteins or messenger RNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics.This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics