MyCTC chip: microfluidic-based drug screen with patient-derived tumour cells from liquid biopsies

Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns, often leading to ineffective treatment. Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies, yet when disease progression accelerates, tissue biopsies are not performed on a regular basis. It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities, providing valuable information about previously underappreciated treatment opportunities. Here, we present MyCTC chip, a novel microfluidic device enabling the isolation, culture and drug susceptibility testing of cancer cells derived from liquid biopsies. Cancer cell capture is achieved through a label-free, antigen-agnostic enrichment method, and it is followed by cultivation in dedicated conditions, allowing on-chip expansion of captured cells. Upon growth, cancer cells are then transferred to drug screen chambers located within the same device, where multiple compounds can be tested simultaneously. We demonstrate MyCTC chip performance by means of spike-in experiments with patient-derived breast circulating tumour cells, enabling >95% capture rates, as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian, tubal and endometrial cancer, where sensitivity to specific chemotherapeutic agents was identified. Together, we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities

Partial replacement of corn by energy sources for beef cattle at pasture

O objetivo deste trabalho foi avaliar o efeito da substituição parcial do milho por fontes energéticas alternativas, em suplementos concentrados para bovinos de corte mantidos em pasto de Urochloa brizantha cultivar Marandu, durante o período das águas, no consumo e na digestibilidade aparente total dos alimentos. Foram utilizados cinco bovinos Nelore com peso médio corporal inicial de 367,8±4,31 kg. Utilizou-se o delineamento experimental quadrado latino 5x5, com cinco animais e cinco tratamentos. Foram avaliados: suplementos concentrados isoproteicos, com 30% de proteína bruta, formulados à base de grãos de milho, de milheto e de sorgo, e casca de soja; e um suplemento mineral (controle). Não foram observadas diferenças significativas no consumo de matéria seca (MS) total e de matéria orgânica (MO) entre os animais que receberam suplementação mineral e os que receberam suplementação concentrada; no entanto, estes últimos apresentaram menor (12,28%) consumo de MS de pasto. A suplementação concentrada aumentou o coeficiente de digestibilidade aparente total da MS, da MO, da proteína bruta e dos carboidratos não fibrosos que não foram influenciados pelas fontes de energia. Os grãos de milheto e de sorgo e a casca de soja podem ser utilizados em substituição parcial ao milho, na formulação de suplementos concentrados, sem prejudicar o consumo e a digestibilidade dos nutrientes da dieta.The objective of this work was to evaluate the effect of the partial replacement of corn by alternative energy sources in concentrate supplements for beef cattle kept on Urochloa brizantha cultivar Marandu pasture, during the rainy season, on feed intake and total apparent digestibility. Five Nellore cattle with initial average body weight of 367.8±4.31 kg were used. The experimental design was a 5x5 Latin square, with five animals and five treatments. The following were evaluated: isoproteic concentrate supplements, at 30% crude protein, formulated with corn, millet, and sorghum grains, and soybean hulls; and a mineral supplement (control). No significant differences were observed in total dry matter (DM) and organic matter (OM) intake between the animals fed mineral supplementation and those receiving concentrate supplementation; however, the latter showed the lowest (12.28%) pasture DM intake. Concentrate supplementation increased the coefficient of total apparent digestibility of DM, OM, crude protein, and of non‑fiber carbohydrates that were not affected by the energy sources. Millet and sorghum grains, and soybean hulls can be used to partially replace corn in the formulation of concentrate supplements without affecting intake and digestibility of dietary nutrients

Thermoplastic microfluidic devices for the analysis of cancer biomarkers in liquid biopsies

Cancer is among the leading causes of death worldwide. Significant challenges for effective treatment are the required early diagnosis, the proteomic and genomic heterogeneity of the tumor, the formation and spread of metastases and the occurrence of drug resistance. If a tumor is detected, current decision-making for treatment are primarily based on tissue biopsies. Although they hold valuable genetic and proteomic information, tissue biopsies cannot be performed frequently throughout treatment. Hence, complications like genetic mutations or acquired drug resistances are often missed. The possibility of monitoring treatment success or relapse risk through the regular analysis of minimally-invasive liquid biopsies is beneficial. Cancer biomarkers found in blood samples allow for a better diagnosis and prognosis and could be crucial for an improved personalized and effective treatment. Microfluidic technology offers the tools for miniaturization and automation of liquid biopsy analysis that enables precision oncology. Here, we explored and advanced technologies for the analysis of proteins, extracellular vesicles or circulating tumor cells in blood samples. Existing microfluidic technologies are often not up-scalable, cumbersome and expensive, prohibiting translation to widespread use. In this work, we present the development of simple microfluidic thermoplastic devices for the analysis of cancer biomarkers to overcome these technical hurdles. We developed a rapid prototyping process for the replication of microchannel structures in thermoplastics and optimized a laser-assisted biomolecule micropatterning process. Based on these methods, we first implemented multiplexed competitive immunoassays for prostate cancer-associated protein markers that performed at clinically relevant levels. We successfully demonstrated the simultaneous detection of up to three markers from undiluted human plasma. The multiplexed detection of several cancer-associated proteins will improve the early diagnosis. Furthermore, we applied the developed tools for the enrichment and cargo analysis of tumor-derived extracellular vesicles (EVs). An antigen-dependent enrichment was optimized for EVs derived from different breast cancer cell lines. The enrichment was combined with a downstream immunoassay to detect EV-contained cargo proteins after lysis. We investigated a low-dose chemotherapeutic treatment of these different breast cancer cells. The developed platform allowed us to identify an increased release of the apoptosis inhibiting protein survivin via EVs. It showcases the potential of EVs for monitoring the treatment and the evolution of drug resistance early on. Lastly, a thermoplastic platform for the size-based isolation, culture and drug screening of circulating tumor cells was evaluated. The platform was used to isolate and analyze single circulating tumor cells (CTCs) and CTC clusters from full blood patient samples. Additionally, cancer cells from ascites samples of late-stage cancer patients were isolated, and the cell viability was screened during incubation with different anti-cancer drugs. The thermoplastic microfluidic devices for biomarker analysis presented in this work are potentially scalable for high-volume manufacturing, which fosters a more straightforward translation of the developed technologies into clinical practice. While further standardization, automation and simplification of workflows are required, the presented methods demonstrate the potential of analyzing blood-borne biomarkers for cancer diagnosis and prognosis and are a further step towards a personalized treatment

Méthodes actuelles D´expertises employées au laboratoire municipal de Paris et documents sur les matiéres relatives a l´alimentation

T. I. VIII, 326 p. 6 f. de lam col ; T. II. 328 p. ; T. III. 292 p. ; T. IV. 464 pContén:T. I Produits Animaux Conserves. Salaisons et produits conservés ; T. III. Boissons et dérivés immédiats ; T. IV. Produits végétaux et dérivés ; T. II. Matéres grasses. Beurre. Cires et paraffine. Essence de térébenthine. Huiles minérale

Capturing and Detecting of Extracellular Vesicles Derived from Single Escherichia coli Mother Cells

Cells have a phenotypic heterogeneity even in isogeneic populations. Differences in secretion of substances have been well-investigated with single mammalian cells. However, studies on the heterogeneity of secreted substances at the single-bacterial-cell level are challenging due to the small size, motility, and rapid proliferation of bacterial cells such as Escherichia coli. Here, we propose a microfluidic device to achieve an isolated culture of single bacterial cells and capture of extracellular vesicles (EVs) secreted from individuals. The device has winding channels to trap single rod-shaped E. coli cells at their entrances. Isolated single mother cells grew constantly up to 24 h, while their daughter cells were removed by flow. The flow carried EVs of the trapped cells along the channel, whose surface was rendered positively charged to electrostatically capture negatively charged EVs, followed by staining with a lipophilic dye to detect EVs by microscopy. Our results underline that the amounts of segregated EVs vary among cells. Moreover, individual responses to perturbation using a membrane-perturbing antibiotic were observed in growth dynamics and EV secretion of living-alone bacteria. The proposed method can be applied to detect other secreted substances of interest, possibly paving the way for elucidating unknown heterogeneities in bacteria