Cell-Surface Programmed Death Ligand-1 Expression Identifies a Sub-Population of Distal Epithelial Cells Enriched in Idiopathic Pulmonary Fibrosis.

Idiopathic lung fibrosis (IPF) is a fatal lung disease characterized by chronic epithelial injury and exhausted repair capacity of the alveolar compartment, associated with the expansion of cells with intermediate alveolar epithelial cell (AT2) characteristics. Using SftpcCreERT2/+: tdTomatoflox/flox mice, we previously identified a lung population of quiescent injury-activated alveolar epithelial progenitors (IAAPs), marked by low expression of the AT2 lineage trace marker tdTomato (Tomlow) and characterized by high levels of Pd-l1 (Cd274) expression. This led us to hypothesize that a population with similar properties exists in the human lung. To that end, we used flow cytometry to characterize the CD274 cell-surface expression in lung epithelial cells isolated from donor and end-stage IPF lungs. The identity and functional behavior of these cells were further characterized by qPCR analysis, in vitro organoid formation, and ex vivo precision-cut lung slices (PCLSs). Our analysis led to the identification of a population of CD274pos cells expressing intermediate levels of SFTPC, which was expanded in IPF lungs. While donor CD274pos cells initiated clone formation, they did not expand significantly in 3D organoids in AT2-supportive conditions. However, an increased number of CD274pos cells was found in cultured PCLS. In conclusion, we demonstrate that, similar to IAAPs in the mouse lung, a population of CD274-expressing cells exists in the normal human lung, and this population is expanded in the IPF lung and in an ex vivo PCLS assay, suggestive of progenitor cell behavior. CD274 function in these cells as a checkpoint inhibitor may be crucial for their progenitor function, suggesting that CD274 inhibition, unless specifically targeted, might further injure the already precarious lung epithelial compartment in IPF.

Identification of a novel subset of alveolar type 2 cells enriched in PD-L1 and expanded following pneumonectomy.

Alveolar type 2 (AT2) cells are heterogeneous cells, with specialised AT2 subpopulations within this lineage exhibiting stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage that are activated during lung regeneration is unknown.SftpcCreERT2/+;tdTomatoflox/flox mice were used for the labelling of AT2 cells and labelled subpopulations were analysed by flow cytometry, quantitative PCR, assay for transposase-accessible chromatin using sequencing (ATAC-seq), gene arrays, pneumonectomy and culture of precision-cut lung slices. Single-cell RNA-sequencing (scRNA-seq) data from human lungs were analysed.In mice, we detected two distinct AT2 subpopulations, with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow cells express lower levels of the AT2 differentiation markers Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, show higher levels of Sftpc, Sftpb, Sftpa1, Fgfr2b and Etv5 expression. ATAC-seq analysis indicates that TomLow and TomHigh cells constitute two distinct cell populations, with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in the TomLow population. Upon pneumonectomy, the number of TomLow but not TomHigh cells increases and TomLow cells show upregulated expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to Sham. TomLow cells overexpress programmed cell death 1 ligand 1 (PD-L1), an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two subpopulations. In the human lung, data mining of a recent scRNA-seq AT2 data set demonstrates the existence of a PD-L1 Pos population. Therefore, we have identified a novel population of AT2 quiescent, immature progenitor cells in mouse that expand upon pneumonectomy and we have provided evidence for the existence of such cells in human.

Potential biomarkers for testicular germ cell tumour: Risk assessment, diagnostic, prognostic and monitoring of recurrence.

Testicular germ cell tumour (TGCT) is considered a relatively rare malignancy usually occurring in young men between 15 and 35 years of age, and both genetic and environmental factors contribute to its development. The majority of patients are diagnosed in an early-stage of TGCTs with an elevated 5-year survival rate after therapy. However, approximately 25% of patients show an incomplete response to chemotherapy or tumours relapse. The current therapies are accompanied by several adverse effects, including infertility. Aside from classical serum biomarker, many studies reported novel biomarkers for TGCTs, but without proper validation. Cancer cells share many similarities with embryonic stem cells (ESCs), and since ESC genes are not transcribed in most adult tissues, they could be considered ideal candidate targets for cancer-specific diagnosis and treatment. Added to this, several microRNAs (miRNA) including miRNA-371-3p can be further investigated as a molecular biomarker for diagnosis and monitoring of TGCTs. In this review, we will illustrate the findings of recent investigations in novel TGCTs biomarkers applicable for risk assessment, screening, diagnosis, prognosis, prediction and monitoring of the relapse.

The nicotinamide phosphoribosyltransferase antagonist FK866 inhibits growth of prostate tumour spheroids and increases doxorubicin retention without changes in drug transporter and cancer stem cell protein expression.

Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD) synthesis and is involved in cancer cell proliferation through regulation of energy production pathways. Therefore, NAMPT inhibitors are promising drugs for cancer therapy by limiting energy supply of tumours. Herein, we demonstrated that the NAMPT inhibitor FK866 ((E)-N-(4-(1-Benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) dose-dependently inhibited growth and cell motility of DU-145 prostate tumour spheroids and decreased the intracellular ATP concentration. The apoptosis marker cleaved caspase-3 remained unchanged, but the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) was upregulated. Growth inhibition was reversed upon co-administration of NAD to the cell culture medium. FK866 decreased calcein as well as pheophorbide A efflux from tumour spheroids and increased doxorubicin toxicity, indicating interference with function of drug efflux transporters. DU-145 multicellular tumour spheroids expressed the stem cell associated markers CD133, CD44, Oct4, Nanog, Sox2, and drug transporters ABCB1, ABCG2, and ABCC1 which are associated with stem cell properties in cancer cells. The ABCB1 inhibitor zosuquidar, the ABCG2 inhibitor Ko143, and the ABCC1 inhibitor MK571 increased calcein retention. Neither protein expression of stem cell markers, nor drug transporters was significantly changed upon FK866 treatment. In conclusion, our data suggest that FK866 inhibits prostate cancer cell proliferation by interference with the energy metabolism, and function of drug efflux transporters.

Ultrasensitive Label-Free Sensing of IL-6 Based on PASE Functionalized Carbon Nanotube Micro-Arrays with RNA-Aptamers as Molecular Recognition Elements.

This study demonstrates the rapid and label-free detection of Interleukin-6 (IL-6) using carbon nanotube micro-arrays with aptamer as the molecular recognition element. Single wall carbon nanotubes micro-arrays biosensors were manufactured using photo-lithography, metal deposition, and etching techniques. Nanotube biosensors were functionalized with 1-Pyrenebutanoic Acid Succinimidyl Ester (PASE) conjugated IL-6 aptamers. Real time response of the sensor conductance was monitored with increasing concentration of IL-6 (1 pg/mL to 10 ng/mL), exposure to the sensing surface in buffer solution, and clinically relevant spiked blood samples. Non-specific Bovine Serum Albumin (BSA), PBS samples, and anti-IgG functionalized devices gave similar signatures in the real time conductance versus time experiments with no significant change in sensor signal. Exposure of the aptamer functionalized nanotube surface to IL-6 decreased the conductance with increasing concentration of IL-6. Experiments based on field effect transistor arrays suggested shift in drain current versus gate voltage for 1 pg and 1 ng of IL-6 exposure. Non-specific BSA did not produce any appreciable shift in the Ids versus Vg suggesting specific interactions of IL-6 on PASE conjugated aptamer surface gave rise to the change in electrical signal. Both Z axis and phase image in an Atomic Force Microscope (AFM) suggested unambiguous molecular interaction of the IL-6 on the nanotube-aptamer surface at 1 pg/mL concentration. The concentration of 1 pg falls below the diagnostic gray zone for cancer (2.3 pg-4 ng/mL), which is an indicator of early stage cancer. Thus, nanotube micro-arrays could potentially be developed for creating multiplexed assays involving cancer biomarker proteins and possibly circulating tumor cells all in a single assay using PASE functionalization protocol.

Classification of biosensor time series using dynamic time warping: applications in screening cancer cells with characteristic biomarkers.

The development of biosensors that produce time series data will facilitate improvements in biomedical diagnostics and in personalized medicine. The time series produced by these devices often contains characteristic features arising from biochemical interactions between the sample and the sensor. To use such characteristic features for determining sample class, similarity-based classifiers can be utilized. However, the construction of such classifiers is complicated by the variability in the time domains of such series that renders the traditional distance metrics such as Euclidean distance ineffective in distinguishing between biological variance and time domain variance. The dynamic time warping (DTW) algorithm is a sequence alignment algorithm that can be used to align two or more series to facilitate quantifying similarity. In this article, we evaluated the performance of DTW distance-based similarity classifiers for classifying time series that mimics electrical signals produced by nanotube biosensors. Simulation studies demonstrated the positive performance of such classifiers in discriminating between time series containing characteristic features that are obscured by noise in the intensity and time domains. We then applied a DTW distance-based k-nearest neighbors classifier to distinguish the presence/absence of mesenchymal biomarker in cancer cells in buffy coats in a blinded test. Using a train-test approach, we find that the classifier had high sensitivity (90.9%) and specificity (81.8%) in differentiating between EpCAM-positive MCF7 cells spiked in buffy coats and those in plain buffy coats.

Static micro-array isolation, dynamic time series classification, capture and enumeration of spiked breast cancer cells in blood: the nanotube-CTC chip.

We demonstrate the rapid and label-free capture of breast cancer cells spiked in blood using nanotube-antibody micro-arrays. 76-element single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (anti-EpCAM), Anti-human epithelial growth factor receptor 2 (anti-Her2) and non-specific IgG antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester. Following device functionalization, blood spiked with SKBR3, MCF7 and MCF10A cells (100/1000 cells per 5 µl per device, 170 elements totaling 0.85 ml of whole blood) were adsorbed on to the nanotube device arrays. Electrical signatures were recorded from each device to screen the samples for differences in interaction (specific or non-specific) between samples and devices. A zone classification scheme enabled the classification of all 170 elements in a single map. A kernel-based statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping series to classify device electrical signals that corresponded to plain blood (control) or SKBR3 spiked blood (case) on anti-Her2 functionalized devices with ∼90% sensitivity, and 90% specificity in capture of 1000 SKBR3 breast cancer cells in blood using anti-Her2 functionalized devices. Screened devices that gave positive electrical signatures were confirmed using optical/confocal microscopy to hold spiked cancer cells. Confocal microscopic analysis of devices that were classified to hold spiked blood based on their electrical signatures confirmed the presence of cancer cells through staining for DAPI (nuclei), cytokeratin (cancer cells) and CD45 (hematologic cells) with single cell sensitivity. We report 55%-100% cancer cell capture yield depending on the active device area for blood adsorption with mean of 62% (∼12 500 captured off 20 000 spiked cells in 0.1 ml blood) in this first nanotube-CTC chip study.

Label-free capture of breast cancer cells spiked in buffy coats using carbon nanotube antibody micro-arrays.

We demonstrate the rapid and label-free capture of breast cancer cells spiked in buffy coats using nanotube-antibody micro-arrays. Single wall carbon nanotube arrays were manufactured using photo-lithography, metal deposition, and etching techniques. Anti-epithelial cell adhesion molecule (EpCAM) antibodies were functionalized to the surface of the nanotube devices using 1-pyrene-butanoic acid succinimidyl ester functionalization method. Following functionalization, plain buffy coat and MCF7 cell spiked buffy coats were adsorbed on to the nanotube device and electrical signatures were recorded for differences in interaction between samples. A statistical classifier for the 'liquid biopsy' was developed to create a predictive model based on dynamic time warping to classify device electrical signals that corresponded to plain (control) or spiked buffy coats (case). In training test, the device electrical signals originating from buffy versus spiked buffy samples were classified with ∼100% sensitivity, ∼91% specificity and ∼96% accuracy. In the blinded test, the signals were classified with ∼91% sensitivity, ∼82% specificity and ∼86% accuracy. A heatmap was generated to visually capture the relationship between electrical signatures and the sample condition. Confocal microscopic analysis of devices that were classified as spiked buffy coats based on their electrical signatures confirmed the presence of cancer cells, their attachment to the device and overexpression of EpCAM receptors. The cell numbers were counted to be ∼1-17 cells per 5 µl per device suggesting single cell sensitivity in spiked buffy coats that is scalable to higher volumes using the micro-arrays.