Effect of bovine milk fat-based infant formulae on microbiota, metabolites and stool parameters in healthy term infants in a randomized, crossover, placebo-controlled trial.

BACKGROUND: Natural enrichment of sn-2 palmitate content of infant formulae by using bovine milk fat is known to reduce formation of faecal fatty acid soaps and to improve stool consistency, but effects on gut microbiota composition are unknown. The purpose of this study was to test the influence of milk fat-based formula high in sn-2 palmitate on the infants' gut microbiota composition and to confirm the beneficial effects of the formula on formation of faecal fatty acid soaps and stool consistency. METHODS: Twenty-two healthy term, formula-fed infants were enrolled in a single-blinded randomized, crossover, placebo-controlled trial. After a 2-week run-in period, infants received either a 50% milk fat-based formula containing 39% sn-2 palmitate (MF) or a vegetable fat-based formula (VF) containing 10% sn-2 palmitate in a 2 × 2-week crossover design. Faecal microbiota composition was the primary outcome of the study. Other outcomes included faecal fatty acid soap excretion, calcium excretion, gut comfort parameters and faecal metabolites. RESULTS: Microbiota analysis showed that bifidobacteria dominated the gut microbiota of most infants. Neither alpha- nor beta-diversity was significantly influenced by the intervention. Also, abundance of metabolic pathways was independent of the intervention. The MF formula resulted in significantly lower faecal levels of palmitic acid soap (p = 0.0002) and total fatty acid soaps (p = 0.0001) than the VF formula. Additionally, calcium excretion and palmitic acid concentration were significantly (p = 0.0335) lower in stool samples after MF intervention. Furthermore, a significant physiological effect on softer stools was observed in the MF intervention compared to the VF intervention (p = 0.02). Of the 870 measured faecal metabolites, 190 were significantly different after MF and VF intervention (FDR corrected p < 0.05). Most of these were found at higher levels after MF intervention, potentially indicative of the complex structure of milk fat. Metabolites with more than twofold change between interventions were mostly lipid-derived and included several milk fat-specific fatty acids. CONCLUSIONS: Replacing part of the vegetable fat in infant formula with bovine milk fat with high sn-2 palmitate levels did not change the microbiota composition, although a reduction in faecal palmitate soaps, total fatty acid soaps and calcium excretion while improving stool consistency in the MF intervention was confirmed. In addition, 190 faecal metabolites were significantly different, many related to the fat source. TRIAL REGISTRATION: Netherlands Trial Registry Identifier: NL7815 19/06/2019.

Advanced biomaterials and microengineering technologies to recapitulate the stepwise process of cancer metastasis.

Cancer is one of the leading causes of death globally according to the World Health Organization. Although improved treatments and early diagnoses have reduced cancer related mortalities, metastatic disease remains a major clinical challenge. The local tumor microenvironment plays a significant role in cancer metastasis, where tumor cells respond and adapt to a plethora of biochemical and biophysical signals from stromal cells and extracellular matrix (ECM) proteins. Due to these complexities, there is a critical need to understand molecular mechanisms underlying cancer metastasis to facilitate the discovery of more effective therapies. In the past few years, the integration of advanced biomaterials and microengineering approaches has initiated the development of innovative platform technologies for cancer research. These technologies enable the creation of biomimetic in vitro models with physiologically relevant (i.e. in vivo-like) characteristics to conduct studies ranging from fundamental cancer biology to high-throughput drug screening. In this review article, we discuss the biological significance of each step of the metastatic cascade and provide a broad overview on recent progress to recapitulate these stages using advanced biomaterials and microengineered technologies. In each section, we will highlight the advantages and shortcomings of each approach and provide our perspectives on future directions.

Engineered Breast Cancer Cell Spheroids Reproduce Biologic Properties of Solid Tumors.

Solid tumors develop as 3D tissue constructs. As tumors grow larger, spatial gradients of nutrients and oxygen and inadequate diffusive supply to cells distant from vasculature develops. Hypoxia initiates signaling and transcriptional alterations to promote survival of cancer cells and generation of cancer stem cells (CSCs) that have self-renewal and tumor-initiation capabilities. Both hypoxia and CSCs are associated with resistance to therapies and tumor relapse. This study demonstrates that 3D cancer cell models, known as tumor spheroids, generated with a polymeric aqueous two-phase system (ATPS) technology capture these important biological processes. Similar to solid tumors, spheroids of triple negative breast cancer cells deposit major extracellular matrix proteins. The molecular analysis establishes presence of hypoxic cells in the core region and expression of CSC gene and protein markers including CD24, CD133, and Nanog. Importantly, these spheroids resist treatment with chemotherapy drugs. A combination treatment approach using a hypoxia-activated prodrug, TH-302, and a chemotherapy drug, doxorubicin, successfully targets drug resistant spheroids. This study demonstrates that ATPS spheroids recapitulate important biological and functional properties of solid tumors and provide a unique model for studies in cancer research.

Multiparametric Analysis of Oncology Drug Screening with Aqueous Two-Phase Tumor Spheroids.

Spheroids present a biologically relevant three-dimensional model of avascular tumors and a unique tool for discovery of anticancer drugs. Despite being used in research laboratories for several decades, spheroids are not routinely used in the mainstream drug discovery pipeline primarily due to the difficulty of mass-producing uniformly sized spheroids and intense labor involved in handling, drug treatment, and analyzing spheroids. We overcome this barrier using a polymeric aqueous two-phase microtechnology to robotically microprint spheroids of well-defined size in standard 384-microwell plates. We use different cancer cells and show that resulting spheroids grow over time and display characteristic features of solid tumors. We demonstrate the feasibility of robotic, high-throughput screening of 25 standard chemotherapeutics and molecular inhibitors against tumor spheroids of three different cancer cell lines. This screening uses over 7000 spheroids to elicit high quality dose-dependent drug responses from spheroids. To quantitatively compare performance of different drugs, we employ a multiparametric scoring system using half-maximum inhibitory concentration (IC50), maximum inhibition (Emax), and area under the dose-response curve (AUC) to take into account both potency and efficacy parameters. This approach allows us to identify several compounds that effectively inhibit growth of spheroids and compromise cellular viability, and distinguish them from moderately effective and ineffective drugs. Using protein expression analysis, we demonstrate that spheroids generated with the aqueous two-phase microtechnology reliably resolve molecular targets of drug compounds. Incorporating this low-cost and convenient-to-use tumor spheroid technology in preclinical drug discovery will make compound screening with realistic tumor models a routine laboratory technique prior to expensive and tedious animal tests to dramatically improve testing throughput and efficiency and reduce costs of drug discovery.

Liquid-based three-dimensional tumor models for cancer research and drug discovery.

Tumors are three-dimensional tissues where close contacts between cancer cells, intercellular interactions between cancer and stromal cells, adhesion of cancer cells to the extracellular matrix, and signaling of soluble factors modulate functions of cancer cells and their response to therapeutics. Three-dimensional cultures of cancer cells overcome limitations of traditionally used monolayer cultures and recreate essential characteristics of tumors such as spatial gradients of oxygen, growth factors, and metabolites and presence of necrotic, hypoxic, quiescent, and proliferative cells. As such, three-dimensional tumor models provide a valuable tool for cancer research and oncology drug discovery. Here, we describe different tumor models and primarily focus on a model known as tumor spheroid. We summarize different technologies of spheroid formation, and discuss the use of spheroids to address the influence of stromal fibroblasts and immune cells on cancer cells in tumor microenvironment, study cancer stem cells, and facilitate compound screening in the drug discovery process. We review major techniques for quantification of cellular responses to drugs and discuss challenges ahead to enable broad utility of tumor spheroids in research laboratories, integrate spheroid models into drug development and discovery pipeline, and use primary tumor cells for drug screening studies to realize personalized cancer treatment.

Microprinted tumor spheroids enable anti-cancer drug screening.

Spheroids present a biologically relevant model of avascular tumors and a unique tool for discovery of anti-cancer drugs. Despite being used in research laboratories for several decades, spheroids are not routinely used for drug discovery primarily due to the difficulty of mass-producing uniformly-sized spheroids and intense labor involved in handling, drug treatment, and analyzing them. We overcome this barrier using a novel technology to robotically microprint spheroids in standard 384-well plates. An aqueous drop containing cancer cells is dispensed into a bath of a second, immiscible aqueous phase. The drop maintains cells in close proximity to aggregate into a single spheroid. Using U-87 MG brain cancer cells, we show that this approach produces spheroids of well-defined size with ~10% deviation from their mean diameter. We demonstrate the feasibility of robotic, high throughput compound screening against tumor spheroids using a collection of 25 standard chemotherapeutics and molecular inhibitors against U-87 MG spheroids. Each drug is used in a wide range of concentrations. Viability of cancer cells in drug-treated spheroids is measured using a PrestoBlue assay. Morphological changes are used as a secondary measure for analysis of drug effect. We identify several compounds that effectively inhibit growth of spheroids. To generate a scoring system for effectiveness of drugs, we use half-maximum inhibitory concentration (IC50), maximum inhibition (Emax), and area under the dose-response curve (AUC) to present a multi-parametric approach that takes into account both potency and efficacy of drugs. Our robotic technology offers a low cost and convenient platform for screening large collections of chemical compounds against realistic tumor models prior to expensive and tedious in vivo tests, dramatically improving testing throughput and efficiency, and reducing costs.

Robotic printing and drug testing of 384-well tumor spheroids.

A major impediment to anti-cancer drug development is the lack of a reliable and inexpensive tumor model to test the efficacy of candidate compounds. This need has emerged due to the insufficiency of widely-used monolayer cultures to predict drug efficacy in vivo. Spheroids, 3D compact clusters of cancer cells, mimic important characteristics of tumors and provide a tissue analog for drug testing. Here we present a novel spheroid formation microtechnology that is simple to use and allows high throughput drug screening in 384-microwell plates. This approach is based on a polymeric aqueous two-phase system. The denser aqueous phase is mixed with cancer cells at a desired density. Using a robotic liquid handler, a drop of this cell suspension is dispensed into each well of a 384-microwell plate containing the second, immersion aqueous phase. Cancer cells remain contained in the drop, which rests on the well bottom, and form a spheroid during incubation. The use of liquid handling robotics ensures precise dispensing of a single drop, resulting in a single spheroid per well and homogenously sized spheroids within each plate. We confirmed the consistency of production of spheroids and demonstrated their biological relevance to tumors. A proof of concept study with spheroids of triple negative breast cancer cells treated with a standard chemotherapeutic compound, doxorubicin, showed the potential of this method for drug testing. This spheroid culture microtechnology presents key advantages over existing methods such as the ease of drug and viability reagent addition, ability to analyze spheroids without transferring them to a new plate, and the elimination of the need for specialized plates or devices to form spheroids. Incorporating this technology in anti-cancer drug development pipeline will help examine the efficacy of drug candidates more effectively and expedite discovery of novel drugs.