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Anticancer drugs have the lowest success rate of approval in drug development programs. Thus, preclinical assays that closely predict the clinical responses to drugs are of utmost importance in both clinical oncology and pharmaceutical research. 3D tumour models preserve the tumoral architecture and are cost- and time-efficient. However, the short-term longevity, limited throughput, and limitations of live imaging of these models have so far driven researchers towards less realistic tumour models such as monolayer cell cultures. Here, we present an open-space microfluidic drug screening platform that enables the formation, culture, and multiplexed delivery of several reagents to various 3D tumour models, namely cancer cell line spheroids and ex vivo primary tumour fragments. Our platform utilizes a microfluidic pixelated chemical display that creates isolated adjacent flow sub-units of reagents, which we refer to as fluidic 'pixels', over tumour models in a contact-free fashion. Up to nine different treatment conditions can be tested over 144 samples in a single experiment. We provide a proof-of-concept application by staining fixed and live tumour models with multiple cellular dyes. Furthermore, we demonstrate that the response of the tumour models to biological stimuli can be assessed using the platform. Upscaling the microfluidic platform to larger areas can lead to higher throughputs, and thus will have a significant impact on developing treatments for cancer.

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BACKGROUND: L-asparaginase (L-ASNase) is an essential component of chemotherapy strategies due to its differential action between normal and leukemic cells. Recently, concerns about the efficiency of commercial formulations administered in developing countries have been reported, and available methods have limitations for directly determining the quality of the formulation of the medications. PROCEDURE: We developed a cell-based protocol to analyze the activity of different L-ASNase formulations used in Colombia to induce apoptosis of the NALM-6 cell line after 24, 48, and 72 hours, using flow cytometry. Then we compared results and determined the statistically significant differences. RESULTS: Three statistically different groups, ranging from full to no activity against leukemic cells, using 0.05, 0.5, and 5.0 IU/ml concentrations, were identified. Group 1 (asparaginase codified [ASA]2-4) exhibited very low to no activity against B-cell acute lymphoblastic leukemia (B-ALL) cells. Group 2 (ASA6) exhibited intermediate-level activity, and group 3 (ASA1 and ASA5) exhibited high activity. CONCLUSIONS: Differences found between the therapeutic formulations of L-ASNase distributed in Colombia raise concerns about the quality of the treatment administered to patients in low- and middle-income countries. Therefore, we recommend a preclinical evaluation of formulations of L-ASNase in order to prevent therapeutical impacts on the outcome of ALL patients.

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INTRODUCTION: Clostridioides difficile (C. difficile) infection is the main cause of nosocomial diarrhea. First-line treatment is oral vancomycin, but that presentation is not commercially available in Latin America. Our aim was to determine the fecal concentration of the oral administration of the conventional dose of an intravenous vancomycin preparation (VCM), in an experimental model. METHODS: A preclinical trial was conducted on 18 male mice (Balb/c strain), in three batches. The following doses of VCM were administered: 125 mg in batch A; 500 mg in batch B; and VCM-placebo in batch C. After receiving the doses, the mice were placed in metabolic cages, by batch. Feces were collected and the fecal concentration of VCM was analyzed through high pressure liquid chromatography 2, 4 and 6 h after drug administration. RESULTS: The 125 mg dose of VCM reached the minimum inhibitory concentration (MIC) for C. difficile, without reaching the minimum bactericidal concentration (MBC90), at 2, 4, and 6 h (521, 688, and 280 mg/L, respectively). Likewise, the 500 mg dose of VCM reached the MIC at 2 h, increased gradually, and reached MBC90 between 4 and 6 h, in feces (1,062 and 1,779 mg/L, respectively), ANOVA, p = 0.0005. CONCLUSION: The fecal concentration of vancomycin was dependent on the intragastric dose administered. Only the 500 mg dose of VCM reached therapeutic concentration for C. difficile (MIC and MBC90), in the mice. We suggest starting a dose of 500 mg QID for achieving therapeutic concentration against C. difficile, as soon as 4 h after the first dose.

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The recent trend in 3D cell modeling has fostered the emergence of a wide range of models, addressing very distinct goals ranging from the fundamental exploration of cell-cell interactions to preclinical assays for personalized medicine. It is clear that no single model will recapitulate the complexity and dynamics of in vivo situations. The key is to define the critical points, achieve a specific goal and design a model where they can be validated. In this report, we focused on cancer progression. We describe our model which is designed to emulate breast carcinoma progression during the invasive phase. We chose to provide topological clues to the target cells by growing them on microsupports, favoring a polarized epithelial organization before they are embedded in a 3D matrix. We then watched for cell organization and differentiation for these models, adding stroma cells then immune cells to follow and quantify cell responses to drug treatment, including quantifying cell death and viability, as well as morphogenic and invasive properties. We used model cell lines including Comma Dß, MCF7 and MCF10A mammary epithelial cells as well as primary breast cancer cells from patient-derived xenografts (PDX). We found that fibroblasts impacted cell response to Docetaxel and Palbociclib. We also found that NK92 immune cells could target breast cancer cells within the 3D configuration, providing quantitative monitoring of cell cytotoxicity. We also tested several sources for the extracellular matrix and selected a hyaluronan-based matrix as a promising alternative to mouse tumor basement membrane extracts for primary human cancer cells. Overall, we validated a new 3D model designed for breast cancer for preclinical use in personalized medicine.

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ETHNOPHARMACOLOGICAL RELEVANCE: Lantana canescens is popularly known in Brazil as "cidreirinha" or "chumbinho-branco". It is found in Pantanal biome and its flowers and leaves are used in traditional medicine to treat pain and inflammation. Information about this species is limited to the activity of isolated essential oils. Studies with different extracts, composition, and biological properties are still scarce. AIM OF THIS STUDY: The objective of this study was to evaluate the anti-inflammatory and anti-hyperalgesic activity of the hydroethanolic extract of L. canescens aerial parts. MATERIALS AND METHODS: The hydroethanolic extract L. canescens aerial parts (HELc) was analyzed using HPLC-DAD-EM. Male and female Swiss mice weighing 18-25 g were used in the in vivo assays. Acute toxicity was assessed (2000 mg/kg); anti-inflammatory activity through paw edema, mast cell degranulation and peritonitis, and anti-hyperalgesic activity through abdominal writhing assays induced by acetic acid and formalin sensitization, were evaluated using the doses of 3, 30 and 300 mg/kg. RESULTS: The phytochemical characterization of HELc confirmed the presence of glycosylated iridoids (theveside, theviridoside), verbascosides and flavonoids. The HELc did not present toxicity in the evaluated dose. HELc reduced formation of paw edema, degranulation of peritoneal mast cells and infiltration of polymorphonuclear cells into the animals peritoneal cavity. In addition, HELc decreased the number of abdominal writhing induced by acetic acid and the time of paw licking in the evaluation of formalin sensitization. CONCLUSIONS: These results confirm the anti-inflammatory and anti-hyperalgesic effects of hydroethanolic extract of L. canescens, validating the use of this plant in folk medicine.

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Tumour models mimicking in vivo three-dimensional (3D) microenvironments are of increasing interest in drug discovery because of the limitations inherent to current models. For example, preclinical assays that rely on monolayer or spheroid cell cultures cannot easily predict 3D cancer behaviours because they have no vasculature. Furthermore, there are major differences in cancer behaviour between human and animal experiments. Here, we show the construction of 3D blood/lymph-vascularized human stromal tissues that can be combined with cancer cells to mimic dynamic metastasis for real-time throughput screening of secreted proteinases. We validated this tool using three human carcinoma cell types that are known to invade blood/lymph vessels and promote angiogenesis. These cell types exhibited characteristic haematogenous/lymphogenous metastasis and tumour angiogenesis properties. Importantly, these carcinoma cells selectively secreted different matrix metalloproteinases depending on their metastasis stage and target vasculature, suggesting the possibility of developing drugs that can target each secreted proteinase. We conclude that the 3D tissue tool will be a powerful throughput system for predicting cancer cell responses and time-dependent secretion of molecules in preclinical assays.

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