Snap-frozen Tissue Research Articles

Alternative splicing of Apoptosis Stimulating Protein of TP53-2 (ASPP2) results in an oncogenic isoform promoting migration and therapy resistance in soft tissue sarcoma (STS)

BackgroundMetastatic soft tissue sarcoma (STS) are a heterogeneous group of malignancies which are not curable with chemotherapy alone. Therefore, understanding the molecular mechanisms of sarcomagenesis and therapy resistance remains a critical clinical need. ASPP2 is a tumor suppressor, that functions through both p53-dependent and p53-independent mechanisms. We recently described a dominant-negative ASPP2 isoform (ASPP2κ), that is overexpressed in human leukemias to promote therapy resistance. However, ASPP2κ has never been studied in STS. Materials and methodsExpression of ASPP2κ was quantified in human rhabdomyosarcoma tumors using immunohistochemistry and qRT-PCR from formalin-fixed paraffin-embedded (FFPE) and snap-frozen tissue. To study the functional role of ASPP2κ in rhabdomyosarcoma, isogenic cell lines were generated by lentiviral transduction with short RNA hairpins to silence ASPP2κ expression. These engineered cell lines were used to assess the consequences of ASPP2κ silencing on cellular proliferation, migration and sensitivity to damage-induced apoptosis. Statistical analyses were performed using Student’s t-test and 2-way ANOVA.ResultsWe found elevated ASPP2κ mRNA in different soft tissue sarcoma cell lines, representing five different sarcoma sub-entities. We found that ASSP2κ mRNA expression levels were induced in these cell lines by cell-stress. Importantly, we found that the median ASPP2κ expression level was higher in human rhabdomyosarcoma in comparison to a pool of tumor-free tissue. Moreover, ASPP2κ levels were elevated in patient tumor samples versus adjacent tumor-free tissue within individual patients. Using isogenic cell line models with silenced ASPP2κ expression, we found that suppression of ASPP2κ enhanced chemotherapy-induced apoptosis and attenuated cellular proliferation.ConclusionDetection of oncogenic ASPP2κ in human sarcoma provides new insights into sarcoma tumor biology. Our data supports the notion that ASPP2κ promotes sarcomagenesis and resistance to therapy. These observations provide the rationale for further evaluation of ASPP2κ as an oncogenic driver as well as a prognostic tool and potential therapeutic target in STS.

Dissecting B/Plasma Cells in Periodontitis at Single-Cell/Bulk Resolution

In recent decades, our understanding of periodontitis has evolved from that based on a gross/histologic level to one on a cellular/molecular level. Previous landscape studies have explored molecular subtyping, diagnosis, and gingival tissue cell decomposition in periodontitis, and meaningful results have been obtained at a transcriptomic level. However, current periodontitis transcriptomic studies lack a finer dissection of the intercommunication between immune cells and the biological processes of specific immune cell subtypes. In this study, we classified 15 immune cell types in periodontitis at a single-cell level and conducted a cell communication analysis based on a multicenter integrated single-cell transcriptome profile, in which plasma cell–generated macrophage migration inhibitory factor can communicate with most other immune cells in periodontitis. A pseudotime analysis focusing on B/plasma cell infiltration in periodontitis revealed 2 distinct cell fates (CFs) for B/plasma cells. In addition, at a bulk tissue level, a single-sample gene set enrichment analysis showed a similar immune cell infiltration trend, and a weighted gene coexpression network analysis identified an immune-related gene module. Combined with the above findings, we used machine learning methods to further narrow down potential gene candidates for developing and validating molecular diagnostic models of periodontitis. Multivariable logistic regression of a large public cohort (68 healthy vs. 235 periodontitis) and an independent validation cohort (12 healthy vs. 7 periodontitis) showed the CF1 signature provides a good discrimination and calibration performance with clinical benefits at a proper threshold probability. Furthermore, quantitative real-time polymerase chain reaction validation of the gene candidates was performed in both snap-frozen gingival tissues and gingival crevicular fluids. Our transcriptomic landscape analysis at both single-cell and bulk tissue resolutions thereby illustrates the B/plasma cell infiltration process in periodontitis and reveals a gene signature that may assist in molecular diagnosis of the disease.

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer

BackgroundTumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance.MethodsStage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma.ResultsTYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09–2.56; p < 0.05).ConclusionsTYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Robust Acquisition of Spatial Transcriptional Programs in Tissues With Immunofluorescence-Guided Laser Capture Microdissection.

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue, as local interactions precipitate intra-cellular events that often lead to changes in expression. However, our understanding of these processes in tissues, whether healthy or diseased, is limited at present owing to the difficulty in acquiring comprehensive transcriptional programs of spatially- and phenotypically-defined cells in situ. Here we present a robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved transcriptional programs with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues sufficient to resolve even isoforms. The protocol is optimized to protect the RNA with a small molecule inhibitor, the ribonucleoside vanadyl complex (RVC), which thereby enables the typical time-consuming immunostaining and laser capture steps of this procedure during which RNA is usually severely degraded in existing approaches. The efficacy of this approach is exemplified by the characterization of differentially expressed genes between the mouse small intestine lacteal cells at the tip versus the main capillary body, including those that function in sensing and responding to local environmental cues to stimulate intra-cellular signalling. With the extensive repertoire of specific antibodies that are presently available, our method provides an unprecedented capability for the analysis of transcriptional networks and signalling pathways during development, pathogenesis, and aging of specific cell types within native tissues.

Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

We describe a low-input RNase footprinting approach for the rapid quantification of ribosome-protected fragments with as few as 1000 cultured cells. The assay uses a simplified procedure to selectively capture ribosome footprints based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution. We applied it to reveal selective functions of the elongation factor TUFM in mitochondrial translation, as well as synchronized repression of cytosolic translation after TUFM perturbation. We show the assay is applicable to small amounts of primary tissue samples with low protein synthesis rates, including snap-frozen tissues and immune cells from an individual's blood draw. We showed its feasibility to characterize the personalized immuno-translatome. Our analyses revealed that thousands of genes show lower translation efficiency in monocytes compared with lymphocytes, and identified thousands of translated noncanonical open reading frames (ORFs). Altogether, our RNase footprinting approach opens an avenue to assay transcriptome-wide translation using low-input samples from a wide range of physiological conditions.

Effects of the IDH1 R132H Mutation on the Energy Metabolism: A Comparison between Tissue and Corresponding Primary Glioma Cell Cultures.

The R132H mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) is the most important prognostic factor for the survival of glioma patients. Subsequent studies led to the discovery of a panel of enzymes mainly involved in glutamate anaplerosis and aerobic glycolysis that change in abundance as a result of the IDH1 mutation. To further study these changes, appropriate glioma models are required that accurately mimic in vivo metabolism. To investigate how metabolism is affected by in vitro cell culture, we here compared surgically obtained snap-frozen glioma tissues with their corresponding primary glioma cell culture models with a previously developed targeted mass spectrometry proteomic assay. We determined the relative abundance of a panel of metabolic enzymes. Results confirmed increased glutamate use and decreased aerobic glycolysis in resected IDH1 R132H glioma tissue samples. However, these metabolic profiles were not reflected in the paired glioma primary cell cultures. We suggest that culture conditions and tumor microenvironment play a crucial role in maintaining the in vivo metabolic situation in cell culture models. For this reason, new models that more closely resemble the in vivo microenvironment, such as three-dimensional cell co-cultures or organotypic multicellular spheroid models, need to be developed and investigated.

Decreased Expression of Estrogen Receptors Is Associated with Tumorigenesis in Papillary Thyroid Carcinoma.

Papillary thyroid carcinomas (PTC), which is derived from thyroid follicular cells, is the most commonly differentiated thyroid cancer with sex disparity. However, the role of estrogen receptors (ERs) in the pathogenesis of PTC remains unclear. The present study aimed to determine the association of ER mRNA expression levels with clinicopathologic features in PTC. To that aim, the mRNA levels of ESR1 (ERα66), ESR1 (ERα36), ESR2, and G-protein-coupled estrogen receptor 1 (GPER1) in snap-frozen tissue samples from PTCs and adjacent normal thyroid tissues were determined using quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the correlation between ER mRNA expression levels and clinicopathologic features was analyzed. The expression of ERα66, ERα36, ERβ, and GPER1 was lower in PTC specimens than in adjacent normal thyroid tissues. Moreover, low GPER1 expression was associated with extrathyroidal extension. There was no obvious difference in expression of ERs between PTC specimens from male and female patients. In conclusion, our findings highlight the importance of ERs in PTC tumorigenesis.

A Unique Immune-Related Gene Signature Represents Advanced Liver Fibrosis and Reveals Potential Therapeutic Targets.

Innate and adaptive immune responses are critically associated with the progression of fibrosis in chronic liver diseases. In this study, we aim to identify a unique immune-related gene signature representing advanced liver fibrosis and to reveal potential therapeutic targets. Seventy-seven snap-frozen liver tissues with various chronic liver diseases at different fibrosis stages (1: n = 12, 2: n = 12, 3: n = 25, 4: n = 28) were subjected to expression analyses. Gene expression analysis was performed using the nCounter PanCancer Immune Profiling Panel (NanoString Technologies, Seattle, WA, USA). Biological meta-analysis was performed using the CBS Probe PINGSTM (CbsBioscience, Daejeon, Korea). Using non-tumor tissues from surgically resected specimens, we identified the immune-related, five-gene signature (CHIT1_FCER1G_OSM_VEGFA_ZAP70) that reliably differentiated patients with low- (F1 and F2) and high-grade fibrosis (F3 and F4; accuracy = 94.8%, specificity = 91.7%, sensitivity = 96.23%). The signature was independent of all pathological and clinical features and was independently associated with high-grade fibrosis using multivariate analysis. Among these genes, the expression of inflammation-associated FCER1G, OSM, VEGFA, and ZAP70 was lower in high-grade fibrosis than in low-grade fibrosis, whereas CHIT1 expression, which is associated with fibrogenic activity of macrophages, was higher in high-grade fibrosis. Meta-analysis revealed that STAT3, a potential druggable target, highly interacts with the five-gene signature. Overall, we identified an immune gene signature that reliably predicts advanced fibrosis in chronic liver disease. This signature revealed potential immune therapeutic targets to ameliorate liver fibrosis.

Lysine Acetylation Stoichiometry Analysis at the Proteome Level.

Lysine acetylation is a widespread posttranslational modification (PTM) in all kingdoms of live. A large number of proteins involved in most of biological pathways are targets of this PTM. The lysine acetylation is a reversible modification controlled by two main groups of enzymes, lysine acetyltransferases responsible for transferring the acetyl group of acetylCoA to the side chain of lysine residues and lysine deacetylases which effectively remove the acetyl tag. Dysregulation of enzymes that control acetylation and/or target proteins have been associated with a growing number of human pathologies. Lysine acetylation is largely a modification that occurs at low stoichiometry at its target sites. Here we describe a method to identify lysine acetylation sites and estimate their site occupancy at the proteome scale. The method relies on a high-resolution mass spectrometry-based proteomics approach, which includes a specific chemical acetylation reaction on unmodified lysine residues that carry heavy isotopes. The procedures described here have been applied to cell line cultures and to clinically relevant samples stored as both snap-frozen and formalin-fixed paraffin-embedded (FFPE) tissues.

Abstract MP06: Insights Into The Role Of Anp In Hfpef During Ss Hypertension

Background: Atrial natriuretic peptide ( ANP) is a hormone produced primarily in the heart, which induces vasodilation and natriuresis. We showed previously that a knockout of ANP in Dahl SS rat results in hypertension and HFpEF phenotype. There is a gap in knowledge regarding the role of inflammation and mitochondrial function in HFpEF that accompanies SSH. We hypothesize that the ANP deficiency in SSH leads to pressure overload, which affects cardiac inflammation and mitochondrial function. Methods: At 8 weeks of age, male wild type (SS WT ) and Nppa knockout (ANP knockout, SS Nppa-/- ) Dahl SS rats were placed on normal salt ( NS , 0.4% NaCl) or a high salt ( HS , 4% NaCl) for 21 days. Tissues were harvested for histological and Western analysis. TEM was used to obtain electron micrographs. EPR spectroscopy was performed in snap-frozen tissues post injection of POBN. 2-way ANOVA was employed for statistical comparison. Results: Heart-to-body-weight ratio was higher in SS Nppa-/- rats (p&lt;0.001), on NS and HS diet ( p &lt;0.001). SS Nppa-/- rats displayed increases in cardiac fibrosis following HS diet ( p &lt;0.01). EPR demonstrated that SS Nppa-/- hearts exhibit a trend for increased lipid peroxidation compared to SS WT . Cardiac cytokine profile demonstrated an increase in IL-1α, IL-1β, L-6, IL-17, TNF-α, sICAM-1, MIP-1α, MIP-3α, in the SS WT rats fed a HS diet vs NS diet, and lower levels of these in the HS-diet fed SS Nppa-/- rats vs SS WT . HS diet was sufficient to induce increases in mitochondrial area ( p &lt;0.001), and decreases in density ( p 0.001), and overall health score ( p &lt;0.001) in both SS WT and SS Nppa-/- rats, but no genotype-specific differences were reported. We observed changes in fission/fusion proteins: Opa1 expression was elevated in SS Nppa-/- vs SS WT ( p &lt;0.01), and Parkin expression was lower in all rats on a HS diet ( p &lt;0.05). Conclusions: Although SS Nppa-/- rats develop a HFpEF phenotype, exacerbated cardiac fibrosis and higher lipid peroxidation vs SS WT rats, inflammation and overall mitochondrial damage are diminished in the hearts of these animals at day 21 of the HS diet. Further studies are needed to elucidate the mechanisms by which inflammation may affect the development of HFpEF during SS hypertension in a cell-specific and time-dependent manner.

Expression of Angiotensin II Receptor 2 in Microcystic Lymphatic Malformation

Objectives: To investigate the presence of components of the renin-angiotensin system (RAS) on the embryonic stem cell (ESC)–like population in microcystic lymphatic malformation (mLM). Methods: mLM tissue samples from 18 patients underwent immunohistochemical staining for RAS components including angiotensinogen, renin, prorenin receptor (PRR), angiotensin-converting enzyme (ACE), ACE2, and angiotensin II receptor 2 (AT2R). Snap-frozen mLM tissues from 6 of the patients were used to confirm protein expression by western blotting for angiotensinogen, PRR, ACE, ACE2, and AT2R. Reverse transcription quantitative polymerase chain reaction was used to detect transcript expression of angiotensinogen, renin, PRR, ACE, ACE2, AT1R, and AT2R in 5 of the mLM tissue samples. Results: Immunohistochemical staining demonstrated expression of AT2R in all, and PRR in 1, while angiotensinogen, renin, ACE, and ACE2 were not observed in any of the 18 mLM samples. Western blotting showed expression of angiotensinogen, PRR, and ACE, but not ACE2 or AT2R in all 6 mLM tissue samples. Reverse transcription quantitative polymerase chain reaction demonstrated transcript expression of angiotensinogen, PRR, ACE, and ACE2 in all, AT1R in 4, AT2R in 2, and renin in 1 of the 5 mLM tissue samples. Immunofluorescence dual-staining in 2 mLM tissue samples demonstrated expression of AT2R on the OCT4+ cells. Conclusion: This study shows expression of angiotensinogen, PRR, ACE. ACE2, AT1R, and AT2R transcripts and AT2R protein, in mLM tissue samples, with AT2R localizing to the OCT4+ ESC-like population. This suggests the ESC-like population may be a novel therapeutic target by modulation of the RAS.

Comprehensive Analysis of Differentially Expressed Genes in Clinically Diagnosed Irreversible Pulpitis by Multiplatform Data Integration Using a Robust Rank Aggregation Approach

IntroductionMolecular diagnosis may overcome the limitations of clinical and histologic diagnosis in pulpitis, thereby benefiting many treatment techniques, such as vital pulp therapies. In this study, integrated microarray data on pulpitis were used to obtain a list of normalized differentially expressed (DE) genes for analyzing the molecular mechanisms underlying pulpitis and identifying potential diagnostic biomarkers. MethodsA systematic search of public microarray and sequencing databases was performed to obtain expression data of pulpitis. Robust rank aggregation (RRA) was used to obtain DE gene lists (RRA_DEmRNAs and RRA_DElncRNAs) between inflamed pulp and normal samples. DE genes were evaluated by functional enrichment analyses, correlation analyses for inflammation-related RRA_DEmRNAs, and protein-protein interaction and competing endogenous RNA network construction. Quantitative real-time polymerase chain reaction validation was applied in snap-frozen pulp tissues. ResultsUsing the GSE77459 and GSE92681 data sets, 280 RRA_DEmRNAs and 90 RRA_DElncRNAs were identified. RRA_DEmRNAs were significantly enriched in inflammation-related biological processes and osteoclast differentiation and tumor necrosis factor, chemokine, and B-cell receptor signaling pathways. The molecular complex detection and cytoHubba methods identified 2 clusters and 10 hub genes in the protein-protein interaction network. The competing endogenous RNA network was composed of 2 long noncoding RNAs (ADAMTS9-AS2 and LINC00290), 2 microRNAs (hsa-miR-30a-5p and hsa-miR-128-3p), and 3 messenger RNAs (ABCA1, FBLN5, and SOCS3). The expression between most top inflammation-related RRA_DEmRNAs in pulpitis showed positive correlations. Quantitative real-time polymerase chain reacation validated the expression trends of selected genes, including ITGAX, TREM1, CD86, FCGR2A, ADAMTS9-AS2, LINC00290, hsa-miR-30a-5p, hsa-miR-128-3p, RASGRP3, IL3RA, CCDC178, CRISPLD1, LINC01857, AC007991.2, ARHGEF26-AS1, and AL021408.1. ConclusionsThe identified biomarkers provide insight into the pathology and may aid in the molecular diagnosis of pulpitis.

Normothermic Ex Situ Liver Perfusion Enhances Mitochondrial Function of DCD Grafts as Evidenced by High-throughput Metabolomics.

Normothermic ex situ liver perfusion (NEsLP) reduces reperfusion injury of donation after circulatory death (DCD) grafts and optimizes graft function. The goal of our study was to elucidate how NEsLP impacts global metabolism in DCD grafts using high-throughput metabolomics. Pig livers were preserved by 2 different techniques: static cold storage and NEsLP. Grafts obtained from heart-beating donors were compared with donation after circulatory death (DCD) grafts with either 30 minutes (DCD30) or 60 minutes (DCD60) ischemia time. Liver tissues were collected at the end of preservation period (T0) with either cold storage or NEsLP (n = 5 per group). Grafts were transplanted into recipient pigs and a second liver biopsy was collected 2 hours following liver transplantation (T1). Snap-frozen tissue was processed and analyzed by Sciex 6600 Q-TOF high-resolution mass spectrometer. Data analysis was performed using MetaboAnalyst 4.0 software. Prolonged ischemia resulted in 38 out of 81 metabolites being differentially abundant over time. Mitochondrial metabolism was significantly affected, with disruption in oxidative phosphorylation capacity i.e the Warburg effect (P = 3.62E-03) and urea cycle (P = 7.95E-0.4). NEsLP resulted in improved mitochondrial metabolism and glycolysis (4.20E-02) oxidation of branched chain fatty acids (P = 4.07E-02). This unbiased, high-throughput metabolomics study reveals that mitochondrial function is globally rescued with NEsLP, associated with improvement in DCD graft function. NEsLP is able to rescue DCD grafts, improving their metabolic function to that of livers not exposed to DCD procurement.

Successful ATAC-Seq From Snap-Frozen Equine Tissues.

An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000–33,000 accessible chromatin regions in lamina and 22–61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments.

Development of a novel biomarker for the early detection of colorectal cancer

Colorectal cancer (CRC) is one of the most common cancer and a leading cause of cancer related deaths both in men and in women worldwide including US. A positive family history is strongly correlated with an increased risk of CRC diagnosis during lifetime. However, CRC is an indolent disease in its early stages and usually becomes symptomatic when it progresses to more advanced stages. Conventional screening methods have several limitations and currently, there is no screening procedure available to detect CRC at a very early stage. Under normal physiological condition, the epithelial lining of the gut gets breached by various factors including food, that creates local wound. The surviving epithelial cells around the wound lose their differentiated structure and undergoes epithelial mesenchymal transition (EMT) and migrate and proliferate to cover the injured area. Once the wound is covered, the cells start differentiating back into their normal cell type and become a part of the epithelium. If the cells fail to stop proliferating following wound closure by overriding the switch to differentiating that can lead to colonic polyp, or tumor, and if not detected in a timely manner than, it leads ultimately to CRC development. Previously, we reported that β-catenin; an adherence junction protein gets phosphorylated by Janus kinase 3 (Jak3), a non-receptor tyrosine kinase, where Jak3 directly phosphorylates three tyrosine residues, viz. Tyr30, Tyr64, and Tyr86 in the N-terminal domain (NTD) of β-catenin. Now, our data show that human biopsies from colonic polyps not only associate with EMT, but also with only those mutation in β-catenin where Jak3, fail to phosphorylate β-catenin thereby making the cells override the switch and undergo uncontrolled proliferation and polyp formation. Based on this mechanistic understanding, the present study demonstrates the development of probe that detects the transition of colonic cells from proliferative stage to a differentiated (non-proliferative) stage that can diagnose CRC at the pre-polyp stage. The probe can not only play an important role in the early detection and/or treatment of patients with colorectal cancer but also increase life expectancy post detection. For this study, we collectedde-identified and discarded specimen from normal and/or polyps (n=40 cases; 20 normal and 20 polyps) through our collaboration with clinicians from local hospitals. Briefly, snap-frozen tissues were used to isolate DNA with the QIAamp DNA mini kit (Qiagen) as reported before by our group. All DNA samples were quantified using UV spectrophotometer and analyzed through agarose gel electrophoresis. The DNA sample was used as template in PCR reactions using the β-catenin-probes to amplify the regions encompassing Y30, Y64, and Y86 followed by sequencing of PCR samples. This was followed by histological analysis of colonic polyp sections from each patient stained with H&E and images were taken with phase contrast microscope. Our results show strongly correlation between mutations and histological analysis indicating the aforementioned detection of intactness of EMT switch as potential novel biomarker for the early detection of colorectal cancer.

Non-selective Primary Human Tumor Cell Line Generation from Surgical Resections to be Paired With Flash Frozen and Paraffin Embedded Tissue: Advancements in Democratizing Translational Research Materials to Rural Institutions

Translational cancer research relies on the availability of human patient tissue demonstrating the specific disease process under investigation. Biobanks of human tissue have historically been and remain to date the primary access point for cancer research samples. Biorepositories routinely supply researchers with varying sample types for use in biomedical studies; most commonly formalin-fixed and paraffin-embedded (FFPE) tissue, or fresh snap-frozen tissue. In conjunction with preserved tissue samples, viable tumor cell lines derived from patient tissue have emerged to be a new gold standard in cancer research particularly in drug discovery and functional prognostic assays. Tissue banks providing these samples are being termed “next-generation” and are adapting to directly assist researchers by performing high throughput technical studies such as routine histology and immunostaining of donor tissue. These high quality, next-generation biorepositories are a relatively scarce resource in the broader research community in the United States and have traditionally been associated with large centralized and very well established university medical centers. This article describes the next-generation resources now available at the Edwards Comprehensive Cancer Center with its association with the Marshall University Joan C. Edwards School of Medicine in Huntington, West Virginia. This manuscript details the procedures, protocols, and success rates of the Tissue Procurement Program (TPP) to generate a growing cohort of viable primary human tumor cell lines representing varying malignancies to be used in conjunction with matched formalin-fixed and paraffin-embedded (FFPE) and snap-frozen tissue samples for comprehensive translational research.

Multimodal, label-free fluorescence and Raman imaging of amyloid deposits in snap-frozen Alzheimer\u2019s disease human brain tissue

Alzheimer’s disease (AD) neuropathology is characterized by hyperphosphorylated tau containing neurofibrillary tangles and amyloid-beta (Aβ) plaques. Normally these hallmarks are studied by (immuno-) histological techniques requiring chemical pretreatment and indirect labelling. Label-free imaging enables one to visualize normal tissue and pathology in its native form. Therefore, these techniques could contribute to a better understanding of the disease. Here, we present a comprehensive study of high-resolution fluorescence imaging (before and after staining) and spectroscopic modalities (Raman mapping under pre-resonance conditions and stimulated Raman scattering (SRS)) of amyloid deposits in snap-frozen AD human brain tissue. We performed fluorescence and spectroscopic imaging and subsequent thioflavin-S staining of the same tissue slices to provide direct confirmation of plaque location and correlation of spectroscopic biomarkers with plaque morphology; differences were observed between cored and fibrillar plaques. The SRS results showed a protein peak shift towards the β-sheet structure in cored amyloid deposits. In the Raman maps recorded with 532 nm excitation we identified the presence of carotenoids as a unique marker to differentiate between a cored amyloid plaque area versus a non-plaque area without prior knowledge of their location. The observed presence of carotenoids suggests a distinct neuroinflammatory response to misfolded protein accumulations.

Achieving the Best RNA Quality in Urologic Tumor Samples Intended for Transcriptome Analysis

Purpose: To conduct research on the molecular oncology, physiology, and immunology of urologic tumors requires dissociated viable samples. Improper collection compromises the quality of data attained in molecular and functional assays due to the increased quantities of degraded proteins and RNA. We sought to improve the methods for tissue collection which can avoid generating considerable loss in the viability of cells for further analyses. Materials and Methods: Fifty resected tumor samples from 35 patients were obtained with different surgical techniques and at various time points for viability and RNA quality evaluation. The degradation of RNA was evaluated by its Qubit IQ score, OD 260/280 ratio, total yield, and quantity of β-actin. Results: Snap-frozen tissue samples obtained within 30 min showed better cell viability (P &lt; 0.0001), RNA total yield (P = 0.0081), Qubit ratio (P = 0.003), OD 260/280 ratio (P = 0.4213), and quantity of β-actin (P = 0.0015). Moreover, the bladder tumor samples collected from transurethral biopsy presented more satisfied cell viability results than the ones resected by transurethral electroresection (P &lt; 0.0001). Conclusion: Tumor samples should be processed or frozen freshly within 30 min once removed from human body. Furthermore, transurethral biopsy of bladder tumor is considered a better method for collecting samples for further molecular oncology studies. The high-quality RNA produced enable researchers to conduct more reliable studies by avoiding the experimental artifacts due to the presence of cellular debris or dead cells.

Label-free vibrational imaging of different A\u03b2 plaque types in Alzheimer\u2019s disease reveals sequential events in plaque development

The neuropathology of Alzheimer’s disease (AD) is characterized by hyperphosphorylated tau neurofibrillary tangles (NFTs) and amyloid-beta (Aβ) plaques. Aβ plaques are hypothesized to follow a development sequence starting with diffuse plaques, which evolve into more compact plaques and finally mature into the classic cored plaque type. A better molecular understanding of Aβ pathology is crucial, as the role of Aβ plaques in AD pathogenesis is under debate. Here, we studied the deposition and fibrillation of Aβ in different plaque types with label-free infrared and Raman imaging. Fourier-transform infrared (FTIR) and Raman imaging was performed on native snap-frozen brain tissue sections from AD cases and non-demented control cases. Subsequently, the scanned tissue was stained against Aβ and annotated for the different plaque types by an AD neuropathology expert. In total, 160 plaques (68 diffuse, 32 compact, and 60 classic cored plaques) were imaged with FTIR and the results of selected plaques were verified with Raman imaging. In diffuse plaques, we detect evidence of short antiparallel β-sheets, suggesting the presence of Aβ oligomers. Aβ fibrillation significantly increases alongside the proposed plaque development sequence. In classic cored plaques, we spatially resolve cores containing predominantly large parallel β-sheets, indicating Aβ fibrils. Combining label-free vibrational imaging and immunohistochemistry on brain tissue samples of AD and non-demented cases provides novel insight into the spatial distribution of the Aβ conformations in different plaque types. This way, we reconstruct the development process of Aβ plaques in human brain tissue, provide insight into Aβ fibrillation in the brain, and support the plaque development hypothesis.

TCR Repertoires in Graft-Versus-Host-Disease (GVHD)-Target Tissues Reveals Tissue Specificity of the Alloimmune Response

TCR Repertoires in Graft-Versus-Host-Disease (GVHD)-Target Tissues Reveals Tissue Specificity of the Alloimmune Response