Optimized low-dose combinatorial drug treatment boosts selectivity and efficacy of colorectal carcinoma treatment.

The current standard of care for colorectal cancer (CRC) is a combination of chemotherapeutics, often supplemented with targeted biological drugs. An urgent need exists for improved drug efficacy and minimized side effects, especially at late-stage disease. We employed the phenotypically driven therapeutically guided multidrug optimization (TGMO) technology to identify optimized drug combinations (ODCs) in CRC. We identified low-dose synergistic and selective ODCs for a panel of six human CRC cell lines also active in heterotypic 3D co-culture models. Transcriptome sequencing and phosphoproteome analyses showed that the mechanisms of action of these ODCs converged toward MAP kinase signaling and cell cycle inhibition. Two cell-specific ODCs were translated to in vivo mouse models. The ODCs reduced tumor growth by ~80%, outperforming standard chemotherapy (FOLFOX). No toxicity was observed for the ODCs, while significant side effects were induced in the group treated with FOLFOX therapy. Identified ODCs demonstrated significantly enhanced bioavailability of the individual components. Finally, ODCs were also active in primary cells from CRC patient tumor tissues. Taken together, we show that the TGMO technology efficiently identifies selective and potent low-dose drug combinations, optimized regardless of tumor mutation status, outperforming conventional chemotherapy

Blockade of T cell contact-activation of human monocytes by high-density lipoproteins reveals a new pattern of cytokine and inflammatory genes.

BACKGROUND: Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNgamma, CCL2, CCL5, TNF, IL-1beta, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1beta, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E(2)-both involved in inflammatory response-were triggered by T cell contact and inhibited in the presence of HDL. CONCLUSIONS/SIGNIFICANCE: These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL

Genome-wide, high-resolution DNA methylation profiling using bisulfite-mediated cytosine conversion

Methylation of cytosines (mC) is essential for epigenetic gene regulation in plants and mammals. Aberrant mC patterns are associated with heritable developmental abnormalities in plants and with cancer in mammals. We have developed a genome-wide DNA methylation profiling technology employing a novel amplification step for DNA subjected to bisulfite-mediated cytosine conversion. The methylation patterns detected are not only consistent with previous results obtained with mC immunoprecipitation (mCIP) techniques, but also demonstrated improved resolution and sensitivity. The technology, named BiMP (for Bisulfite Methylation Profiling), is more cost-effective than mCIP and requires as little as 100 ng of Arabidopsis DNA

Orientation and expression of methicillin-resistant Staphylococcus aureus small RNAs by direct multiplexed measurements using the nCounter of NanoString technology

Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. Several genomes of this major human pathogen have been publicly available for almost 10 years, but comprehensive links between virulence or epidemicity and genome content of the bacterium are still missing. This project aims at characterizing a set of small transcribed molecules currently ignored by standard automated annotation algorithms. We assessed the NanoString's nCounter Analysis System for its ability to determine the orientation and quantity of the expressed small RNA (sRNA) molecules that we recently detected with RNA-Sequencing (RNA-Seq). The expression of approximately seventy small RNAs, including sRNA localized in pathogenic islands, was assessed at 5 time points during growth of the bacterium in a rich medium. In addition, two extraction strategies were tested: RNA was either purified on columns or simply prepared from crude lysates in the presence of a chaotropic buffer. The nCounter System allowed us to perform these 64 measurements in a single experiment, without any enzymatic reaction, thus avoiding well-known technical biases. We evaluated the reproducibility and reliability of the nCounter compared to quantitative RT-PCR (RT-qPCR). By using two different designs for the two coding strands, we were able to identify the coding strand of 61 small RNA molecules (95%). Overall, the nCounter System provided an identification of the coding strand in perfect concordance with RNA-Seq data. In addition, expression results were also comparable to those obtained with RT-qPCR. The sensitivity and minimal requirements of the nCounter system open new possibilities in the field of gene expression analysis, for assessing bacterial transcript profiles from complex media (i.e. during host-pathogen interactions) or when starting from poorly purified RNA or even directly from lysed infected tissues

Identification of CHEK1, SLC26A4, c-KIT, TPO and TG as new biomarkers for human follicular thyroid carcinoma

The search for preoperative biomarkers for thyroid malignancies, in particular for follicular thyroid carcinoma (FTC) diagnostics, is of utmost clinical importance. We thus aimed at screening for potential biomarker candidates for FTC. To evaluate dynamic alterations in molecular patterns as a function of thyroid malignancy progression, a comparative analysis was conducted in clinically distinct subgroups of FTC and poorly differentiated thyroid carcinoma (PDTC) nodules. NanoString analysis of FFPE samples was performed in 22 follicular adenomas, 56 FTC and 25 PDTC nodules, including oncocytic and non-oncocytic subgroups. The expression levels of CHEK1, c-KIT, SLC26A4, TG and TPO were significantly altered in all types of thyroid carcinomas. Based on collective changes of these biomarkers which correlating among each other, a predictive score has been established, allowing for discrimination between benign and FTC samples with high sensitivity and specificity. Additional transcripts related to thyroid function, cell cycle, circadian clock, and apoptosis regulation were altered in the more aggressive oncocytic subgroups only, with expression levels correlating with disease progression. Distinct molecular patterns were observed for oncocytic and non-oncocytic FTCs and PDTCs. A predictive score correlation coefficient based on collective alterations of identified here biomarkers might help to improve the preoperative diagnosis of FTC nodules

Identification of new biomarkers for human papillary thyroid carcinoma employing NanoString analysis

We previously reported an upregulation of the clock transcript BMAL1, correlating with TIMP1 expression in fresh-frozen samples from papillary thyroid carcinoma (PTC). Since frozen postoperative biopsy samples are difficult to obtain, we aimed to validate the application of high-precision NanoString analysis for formalin-fixed paraffin-embedded (FFPE) thyroid nodule samples and to screen for potential biomarkers associated with PTC. No significant differences were detected between fresh-frozen and FFPE samples. NanoString analysis of 51 transcripts in 17 PTC and 17 benign nodule samples obtained from different donors and in 24 pairs of benign and PTC nodules, obtained from the same donor (multinodular goiters), confirmed significant alterations in the levels of BMAL1, c-MET, c-KIT, TIMP1, and other transcripts. Moreover, we identified for the first time alterations in CHEK1 and BCL2 levels in PTC. A predictive score was established for each sample, based on the combined expression levels of BMAL1, CHEK1, c-MET, c-KIT and TIMP1. In combination with BRAF mutation analysis, this predictive score closely correlated with the clinicopathological characteristics of the analyzed thyroid nodules. Our study identified new thyroid transcripts with altered levels in PTC using the NanoString approach. A predictive score correlation coefficient might contribute to improve the preoperative diagnosis of thyroid nodules

FOLFOXIRI Resistance Induction and Characterization in Human Colorectal Cancer Cells

FOLFOXIRI, i.e., the combination of folinic acid, 5-fluorouracil, oxaliplatin, and irinotecan, is a first-line treatment for colorectal carcinoma (CRC), yet non-personalized and aggressive. In this study, to mimic the clinical situation of patients diagnosed with advanced CRC and exposed to a chronic treatment with FOLFOXIRI, we have generated the CRC cell clones chronically treated with FOLFOXIRI. A significant loss in sensitivity to FOLFOXIRI was obtained in all four cell lines, compared to their treatment-naïve calls, as shown in 2D cultures and heterotypic 3D co-cultures. Acquired drug resistance induction was observed through morphometric changes in terms of the organization of the actin filament. Bulk RNA sequencing revealed important upregulation of glucose transporter family 5 (GLUT5) in SW620 resistant cell line, while in the LS174T-resistant cell line, a significant downregulation of protein tyrosine phosphatase receptor S (PTPRS) and oxoglutarate dehydrogenase-like gene (OGDHL). This acquired resistance to FOLFOXIRI was overcome with optimized low-dose synergistic drug combinations (ODCs) acting via the Ras-Raf-MEK-ERK pathway. The ODCs inhibited the cell metabolic activity in SW620 and LS174T 3Dcc, respectively by up to 82%

Gene expression profiling provides insights into pathways of oxaliplatin-related sinusoidal obstruction syndrome in humans

Sinusoidal obstruction syndrome (SOS; formerly veno-occlusive disease) is a well-established complication of hematopoietic stem cell transplantation, pyrrolizidine alkaloid intoxication, and widely used chemotherapeutic agents such as oxaliplatin. It is associated with substantial morbidity and mortality. Pathogenesis of SOS in humans is poorly understood. To explore its molecular mechanisms, we used Affymetrix U133 Plus 2.0 microarrays to investigate the gene expression profile of 11 human livers with oxaliplatin-related SOS and compared it to 12 matched controls. Hierarchical clustering analysis showed that profiles from SOS and controls formed distinct clusters. To identify functional networks and gene ontologies, data were analyzed by the Ingenuity Pathway Analysis Tool. A total of 913 genes were differentially expressed in SOS: 613 being upregulated and 300 downregulated. Reverse transcriptase-PCR results showed excellent concordance with microarray data. Pathway analysis showed major gene upregulation in six pathways in SOS compared with controls: acute phase response (notably interleukin 6), coagulation system (Serpine1, THBD, and VWF), hepatic fibrosis/hepatic stellate cell activation (COL3a1, COL3a2, PDGF-A, TIMP1, and MMP2), and oxidative stress. Angiogenic factors (VEGF-C) and hypoxic factors (HIF1A) were upregulated. The most significant increase was seen in CCL20 mRNA. In conclusion, oxaliplatin-related SOS can be readily distinguished according to morphologic characteristics but also by a molecular signature. Global gene analysis provides new insights into mechanisms underlying chemotherapy-related hepatotoxicity in humans and potential targets relating to its diagnosis, prevention, and treatment. Activation of VEGF and coagulation (vWF) pathways could partially explain at a molecular level the clinical observations that bevacizumab and aspirin have a preventive effect in SOS

Identification of Differential Transcriptional Patterns in Primary and Secondary Hyperparathyroidism

Abstract Context Hyperparathyroidism is associated with hypercalcemia and the excess of parathyroid hormone secretion; however, the alterations in molecular pattern of functional genes during parathyroid tumorigenesis have not been unraveled. We aimed at establishing transcriptional patterns of normal and pathological parathyroid glands (PGs) in sporadic primary (HPT1) and secondary hyperparathyroidism (HPT2). Objective To evaluate dynamic alterations in molecular patterns as a function of the type of PG pathology, a comparative transcript analysis was conducted in subgroups of healthy samples, sporadic HPT1 adenoma and hyperplasia, and HPT2. Design Normal, adenomatous, HPT1, and HPT2 hyperplastic PG formalin-fixed paraffin-embedded samples were subjected to NanoString analysis. In silico microRNA (miRNA) analyses and messenger RNA-miRNA network in PG pathologies were conducted. Individual messenger RNA and miRNA levels were assessed in snap-frozen PG samples. Results The expression levels of c-MET, MYC, TIMP1, and clock genes NFIL3 and PER1 were significantly altered in HPT1 adenoma compared with normal PG tissue when assessed by NanoString and quantitative reverse transcription polymerase chain reaction. RET was affected in HPT1 hyperplasia, whereas CaSR and VDR transcripts were downregulated in HPT2 hyperplastic PG tissue. CDH1, c-MET, MYC, and CaSR were altered in adenoma compared with hyperplasia. Correlation analyses suggest that c-MET, MYC, and NFIL3 exhibit collective expression level changes associated with HPT1 adenoma development. miRNAs, predicted in silico to target these genes, did not exhibit a clear tendency upon experimental validation. Conclusions The presented gene expression analysis provides a differential molecular characterization of PG adenoma and hyperplasia pathologies, advancing our understanding of their etiology