Cost-Efficient Transcriptomic-Based Drug Screening.

Transcriptomics allows to obtain comprehensive insights into cellular programs and their responses to perturbations. Despite a significant decrease in the costs of library production and sequencing in the last decade, applying these technologies at the scale necessary for drug screening remains prohibitively expensive, obstructing the immense potential of these methods. Our study presents a cost-effective system for transcriptome-based drug screening, combining miniaturized perturbation cultures with mini-bulk transcriptomics. The optimized mini-bulk protocol provides informative biological signals at cost-effective sequencing depth, enabling extensive screening of known drugs and new molecules. Depending on the chosen treatment and incubation time, this protocol will result in sequencing libraries within approximately 2 days. Due to several stopping points within this protocol, the library preparation, as well as the sequencing, can be performed time-independently. Processing simultaneously a high number of samples is possible; measurement of up to 384 samples was tested without loss of data quality. There are also no known limitations to the number of conditions and/or drugs, despite considering variability in optimal drug incubation times.

Bioinformatic Assessment of Macrophage Activation by the Innate Immune System.

Weighted gene co-expression network analysis (WGCNA) allows for the identification and characterization of cell type-specific gene modules in complex transcriptome datasets. Here, we use a microarray dataset of human macrophages comprising 29 conditions and 299 samples generated by differentiation of CD14+ monocytes into macrophages followed by in vitro stimulations to identify stimulation-specific gene modules. These gene modules can be used for experimental validation, as well as further bioinformatic analysis to determine key pathways or upstream transcription factors.

Comparative approach to define increased regulatory T cells in different cancer subtypes by combined assessment of CD127 and FOXP3.

In recent years an increase of functional CD4(+)CD25(+) regulatory T cells (T(reg) cells) has been established for patients with solid tumors, acute leukemias, and lymphomas. We have reported an expanded pool of CD4(+)CD25(high) T(reg) cells in patients with chronic lymphatic leukemia (CLL), multiple myeloma (MM) as well as its premalignant precursor monoclonal gammopathy of undetermined significance (MGUS). In healthy individuals, low-level expression of CD127 on T cells in addition to the expression of FOXP3 has been associated with T(reg) cells. Here, we demonstrate that the expanded FOXP3(+) T-cell population in patients with colorectal cancer, CLL, MGUS, MM, follicular lymphoma, and Hodgkin's disease are exclusively CD127(low) T(reg) cells and were strongly suppressive. A significant portion of CD127(low)FOXP3(+) T(reg) cells expressed only low levels of CD25 suggesting that the previously reported expansion of CD25(+) T(reg) cells underestimates the true expansion. The assessment of CCR7 and CD45RA expression on the expanded CD4(+)CD127(low)FOXP3(+) T(reg) cells revealed an increase of both naïve as well as central and effector memory T(reg) cells in peripheral blood. Our data strongly support superiority of combined CD127 and FOXP3 analysis in comparison to CD25 and FOXP3 assessment for further quantification of T(reg) cells in malignant diseases.