Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain ...DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.
It is impossible to underplay the importance of fixation in histopathology. Whether the scientist is interested in the extraction of information on lipids, proteins, RNA or DNA, fixation is critical ...to this extraction. This review aims to give a brief overview of the current “state of play” in fixation and focus on the effect fixation, and particularly the effect of the newer brand of “molecular fixatives” have on morphology, histochemistry, immunohistochemistry and RNA/DNA analysis. A methodology incorporating the creation of a fixation tissue microarray for the study of the effect of fixation on histochemistry is detailed.
As RNA in situ hybridization (ISH) moves into the mainstream lab and increasingly into clinical adoption and additional multiplexing techniques are developed to enable further RNA ISH identification, ...a set of guidelines on the validation of ISH is required. These guidelines include choice of methods, appropriate controls, and protocol optimization as well as a central core message of understanding the target, understanding the ISH technique, and using the most appropriate controlling mechanisms to enable reproducible and trustworthy data to be obtained.
Senescence, a persistent form of cell-cycle arrest, is often associated with a diverse secretome, which provides complex functionality for senescent cells within the tissue microenvironment. We show ...that oncogene-induced senescence is accompanied by a dynamic fluctuation of NOTCH1 activity, which drives a TGF-β-rich secretome, while suppressing the senescence-associated pro-inflammatory secretome through inhibition of C/EBPβ. NOTCH1 and NOTCH1-driven TGF-β contribute to 'lateral induction of senescence' through a juxtacrine NOTCH-JAG1 pathway. In addition, NOTCH1 inhibition during senescence facilitates upregulation of pro-inflammatory cytokines, promoting lymphocyte recruitment and senescence surveillance in vivo. As enforced activation of NOTCH1 signalling confers a near mutually exclusive secretory profile compared with typical senescence, our data collectively indicate that the dynamic alteration of NOTCH1 activity during senescence dictates a functional balance between these two distinct secretomes: one representing TGF-β and the other pro-inflammatory cytokines, highlighting that NOTCH1 is a temporospatial controller of secretome composition.
The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation ...therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.
Tumor microenvironment influences the behavior of follicular lymphoma (FL), although the specific cell subsets involved are not well known. The aim of this study was to determine the impact of ...programmed cell death 1 (PD-1) -positive inhibitory immunoregulatory lymphoid cells in the clinicobiologic features and outcome of patients with FL.
We examined samples from 100 patients (53 men and 47 women; median age, 54 years) at diagnosis, as well as in 32 patients at first relapse, with a recently generated monoclonal antibody against PD-1. The cells were quantified using computerized image analysis. Additional analysis consisted of double immunofluorescence and flow cytometry.
PD-1 expression was alternative to FOXP3 in lymphoid cells from both reactive tonsils and FL. At diagnosis, the median percentage of PD-1-positive cells was 14% (range, 0.1% to 74%). Patients with grade 3 FL, poor performance status, and high serum lactate dehydrogenase showed lower numbers of PD-1-positive cells. After a median follow-up of 6.2 years, patients with PD-1-positive cells <or= 5% (n = 25), 6% to 33% (n = 50), and more than 33% (n = 25) had a 5-year progression-free survival rate of 20%, 46%, and 48% (P = .038) and overall survival (OS) of 50%, 77%, and 95% (P = .004), respectively. PD-1 and FL International Prognostic Index maintained prognostic value for OS in multivariate analysis. Patients with PD-1-positive cells <or= 5% showed a higher risk of histologic transformation. At that time, transformed diffuse large B-cell lymphomas had lower percentage of PD-1-positive cells than FL.
A high content of PD-1-positive cells predicted favorable outcome of FL patients, whereas a marked reduction is observed in transformation.
Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and ...metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials.
As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for “research use” continues ...to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1–3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.
Pharmacokinetic and pharmacodynamic parameters of cremophor-paclitaxel, nab-paclitaxel (human-albumin-bound paclitaxel, Abraxane) and a novel mouse-albumin-bound paclitaxel (m-nab-paclitaxel) were ...evaluated in genetically engineered mouse models (GEMMs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS), histological and biochemical analysis. Preclinical evaluation of m-nab-paclitaxel included assessment by three-dimensional high-resolution ultrasound and molecular analysis in a novel secreted protein acidic and rich in cysteine (SPARC)-deficient GEMM of pancreatic ductal adenocarcinoma (PDA).
nab-Paclitaxel exerted its antitumoural effects in a dose-dependent manner and was associated with less toxicity compared with cremophor-paclitaxel. SPARC nullizygosity in a GEMM of PDA, Kras(G12D);p53(flox/-);p48Cre (KPfC), resulted in desmoplastic ductal pancreas tumours with impaired collagen maturation. Paclitaxel concentrations were significantly decreased in SPARC null plasma samples and tissues when administered as low-dose m-nab-paclitaxel. At the maximally tolerated dose, SPARC deficiency did not affect the intratumoural paclitaxel concentration, stromal deposition and the immediate therapeutic response.
nab-Paclitaxel accumulates and acts in a dose-dependent manner. The interaction of plasma SPARC and albumin-bound drugs is observed at low doses of nab-paclitaxel but is saturated at therapeutic doses in murine tumours. Thus, this study provides important information for future preclinical and clinical trials in PDA using nab-paclitaxel in combination with novel experimental and targeted agents.
Pancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select ...patients for surgery. We investigated here whether pancreatic preneoplasia could be detected prior to the development of invasive cancers in genetically engineered mouse models of PCa using metabolic imaging.
The concentrations of alanine and lactate and the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were measured in extracts prepared from the pancreas of animals at different stages of disease progression; from pancreatitis, through tissue with predominantly low-grade and then high-grade pancreatic intraepithelial neoplasia and then tumour. (13)C magnetic resonance spectroscopic imaging ((13)C-MRSI) was used to measure non-invasively changes in (13)C labelling of alanine and lactate with disease progression, following injection of hyperpolarised 1-(13)Cpyruvate.
Progressive decreases in the alanine/lactate concentration ratio and ALT/LDH activity ratio with disease progression were accompanied by a corresponding decrease in the 1-(13)Calanine/1-(13)Clactate signal ratio observed in (13)C-MRSI images of the pancreas.
Metabolic imaging with hyperpolarised 1-(13)Cpyruvate enables detection and monitoring of the progression of PCa precursor lesions. Translation of this MRI technique to the clinic has the potential to improve the management of patients at high risk of developing PCa.
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