Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples.

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX. Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients' blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses. Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression. Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes.

Th1, Th17, and Th1Th17 Lymphocytes during Tuberculosis: Th1 Lymphocytes Predominate and Appear as Low-Differentiated CXCR3+CCR6+ Cells in the Blood and Highly Differentiated CXCR3+/-CCR6- Cells in the Lungs.

Th1 lymphocytes are considered the main mediators of protection against tuberculosis (TB); however, their phenotypic characteristics and relationship with Th17 and Th1Th17 populations during TB are poorly understood. We have analyzed Th1, Th17, and Th1Th17 lymphocytes in the blood and pulmonary lesions of TB patients. The populations were identified based on the production of IFN-γ and/or IL-17 and the coexpression of CXCR3 (X3) and CCR6 (R6). In the blood, IL-17+ and IFN-γ+IL-17+ lymphocytes were barely detectable (median, <0.01% of CD4+ lymphocytes), whereas IFN-γ+ lymphocytes predominated (median, 0.45%). Most IFN-γ+ lymphocytes (52%) were X3+R6+, suggesting their "nonclassical" (ex-Th17) nature. In the lungs, IL-17+ and IFN-γ+IL-17+ lymphocytes were more frequent (0.3%, p < 0.005), yet IFN-γ+ cells predominated (11%). Phenotypically, lung CD4+ cells were X3+/loR6- The degree of differentiation of blood effector CD4+ lymphocytes (evaluated based on CD62L/CD27/CD28 coexpression) increased as follows: X3+R6+ < X3+R6- < X3-R6-, with X3-R6- cells being largely terminally differentiated CD62L-CD27-CD28- cells. Lung CD4+ lymphocytes were highly differentiated, recalling blood X3+/-R6- populations. Following in vitro stimulation with anti-CD3/anti-CD28 Abs, X3+R6+CD4+ lymphocytes converted into X3+R6- and X3-R6- cells. The results demonstrate that, during active TB, Th1 lymphocytes predominate in blood and lungs, document differences in X3/R6 expression by blood and lung CD4+ cells, and link the pattern of X3/R6 expression with the degree of cell differentiation. These findings add to the understanding of immune mechanisms operating during TB and are relevant for the development of better strategies to control it.

Ruthenibacterium lactatiformans gen. nov., sp. nov., an anaerobic, lactate-producing member of the family Ruminococcaceae isolated from human faeces.

Two novel strains of Gram-stain-negative, rod-shaped, obligately anaerobic, non-spore-forming, non-motile bacteria were isolated from the faeces of healthy human subjects. The strains, designated as 585-1T and 668, were characterized by mesophilic fermentative metabolism, production of d-lactic acid, succinic acid and acetic acid as end products of d-glucose fermentation, prevalence of C18 : 1ω9, C18 : 1ω9 aldehyde, C16 : 0 and C16 : 1ω7c fatty acids, presence of glycine, glutamic acid, lysine, alanine and aspartic acid in the petidoglycan peptide moiety and lack of respiratory quinones. Whole genome sequencing revealed the DNA G+C content was 56.4-56.6 mol%. The complete 16S rRNA gene sequences of the two strains shared 91.7/91.6 % similarity with Anaerofilum pentosovorans FaeT, 91.3/91.2 % with Gemmiger formicilis ATCC 27749T and 88.9/88.8 % with Faecalibacterium prausnitzii ATCC 27768T. On the basis of chemotaxonomic and genomic properties it was concluded that the strains represent a novel species in a new genus within the family Ruminococcaceae, for which the name Ruthenibacterium lactatiformans gen. nov., sp. nov. is proposed. The type strain of Ruthenibacterium lactatiformans is 585-1T (=DSM 100348T=VKM B-2901T).

Alistipes inops sp. nov. and Coprobacter secundus sp. nov., isolated from human faeces.

Culture-based study of the faecal microbiome in two adult female subjects revealed the presence of two obligately anaerobic, non-spore-forming, rod-shaped, non-motile, Gram-negative bacterial strains that represent novel species. The first strain, designated 627T, was a fastidious, slow-growing, indole-positive bacterium with a non-fermentative type of metabolism.The strain was characterized by the production of acetic and succinic acids as metabolic end products, the prevalence of iso-C15 : 0 fatty acid and the presence of menaquinones MK-10 and MK-11. The DNA G+C content was found to be 56.6 mol%. The second strain, designated 177T, was capable of fermenting a rich collection of carbohydrate substrates, producing acetic acid as a terminal product. The strain was indole-negative and resistant to bile. The major cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0 (in a 1 : 1 ratio) and the predominant menaquinone was MK-11.The DNA G+C content was 37.8 mol%. A phylogenomic analysis of the draft genomes of strains 627T and 177T placed these bacteria in the genera Alistipes(family Rikenellaceae) and Coprobacter (family Porphyromonadaceae), respectively.On the basis of the phenotypic and genotypic properties of strains 627T and 177T, we conclude that these strains from human faeces represent two novel bacterial species, for which the names Alistipes inops sp. nov. (type strain 627T5DSM 28863T5VKM B-2859T) and Coprobacter secundus sp. nov. (type strain 177T=DSM 28864T=VKM B-2857T) are proposed.