Capturing complex tumour biology in vitro: Histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism

Pre-exposure Prophylaxis (PrEP) Awareness and Use Within High HIV Transmission Networks

Improved implementation of pre-exposure prophylaxis (PrEP) should be a valuable tool within communities experiencing high HIV incidence, such as black men who have sex with men (MSM). Using baseline data from the Chicago arm of the Transmission Reduction Intervention Project (TRIP), we examined awareness and use of PrEP within HIV potential transmission networks. Transmission Reduction Intervention Project recruited participants ages 18–69 (N = 218) during 2014–2016 from networks originating from recently and chronically HIV-infected MSM and transgender persons. In total, 53.2% of participants had heard of PrEP, while 8 (6.5%) HIV-negative participants reported ever using PrEP. In multivariable regression, PrEP awareness was associated with identifying as gay, attending some college or higher, having an HIV test in the previous 6 months, and experiencing HIV-related social support. PrEP awareness was not associated with experiencing or observing HIV-related stigma. PrEP use was associated with participants knowing two or more other PrEP-users. These findings demonstrate moderate awareness, but low uptake of PrEP within HIV potential transmission networks in Chicago. Future research should explore how to increase PrEP use in these networks and investigate the social dynamics behind our finding that PrEP users are more likely to know other PrEP users. © 2019, Springer Science+Business Media, LLC, part of Springer Nature

CD4+ T cell-mediated HER-2/neu-specific tumor rejection in the absence of B cells

HER-2/neu (HER-2) is a cell surface proto-oncogene that is often overexpressed in carcinomas. Passive administration of anti-HER-2 antibodies in breast cancer patients has achieved promising results, but less is known about the role of antibodies in active immunization. We asked whether B cells/antibodies are needed for tumor immunity induced by plasmid (HER-2 and GM-CSF) immunization. HER-2 specific tumor immunity relied completely on both CD4+ and CD8+ T cells. IFN-γ, and to a lesser extent IL-4, seemed to be crucial cytokines during tumor rejection. Protection was associated with production of anti-HER-2 IgG antibodies in B cell competent mice. After immunization, however, B cell-deficient mice rejected HER-2-expressing tumors as efficiently as control littermates. We conclude that T cells are the main effector cells in DNA vaccine induced immunity against HER-2 and that anti HER-2 antibodies are not necessary to elicit a protective anti tumor immune response in this model