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Supplementary Figure S2. Correlation between plasma RTK changes with MAPKi, PFS, and initial tumor shrinkage (using RECIST) in melanoma patients.

Supplementary Figure 3. Corresponds to Fig. 3C, showing U0126 and PD325901 elicit AXL upregulation to a degree that correlates with AXLi/MEKi synergy across a panel of cell lines.

Supplementary Figure S4. Corresponding to Ab-microarray results (Fig. 4A), the enrichment score trace for the bottom-ranked gene-set in the GSEA shows an increase in secreted (not shed) cytokines with MEKi.

Supplementary Figure S1. U0126 treatment reduces p-Erk1/2 while increasing p-cJUN in a panel of cell lines, measured by bead-based immunoassay.

Supplementary Figure S6. Validation of ADAM10 and ADAM17 siRNA knockdown in MDA-MB157 and MDA-MB231, assessed by live-cell immunostaining and confirmed at right by western blot in MDA-MB231 (*p

Supplementary Figure S7. Live-cell immunostaining shows relatively minor changes in ADAM10 and ADAM17 surface levels following MEKi. Measurements were taken 1, 2, and 3h following inhibitor treatment and averaged (*p=0.02, n=3).

Supplementary Methods, Figure Legends

Supplementary Figures 1-2 from Three-kinase inhibitor combination recreates multipathway effects of a geldanamycin analogue on hepatocellular carcinoma cell death

Multitarget compounds that act on a diverse set of regulatory pathways are emerging as a therapeutic approach for a variety of cancers. Toward a more specified use of this approach, we hypothesize that the desired efficacy can be recreated in terms of a particular combination of relatively more specific (i.e., ostensibly single target) compounds. We test this hypothesis for the geldanamycin analogue 17-Allylamino-17-demethoxygeldanamycin (17AAG) in hepatocellular carcinoma cells, measuring critical phosphorylation levels that indicate the kinase pathway effects correlating with apoptotic responsiveness of the Hep3B cell line in contrast to the apoptotic resistance of the Huh7 cell line. A principal components analysis (PCA) constructed from time course measurements of seven phosphoprotein signaling levels identified modulation of the AKT, IκB kinase, and signal transducer and activator of transcription 3 pathways by 17AAG treatment as most important for distinguishing these cell-specific death responses. The analysis correctly suggested from 17AAG-induced effects on these phosphoprotein levels that the FOCUS cell line would show apoptotic responsiveness similarly to Hep3B. The PCA also guided the inhibition of three critical pathways and rendered Huh7 cells responsive to 17AAG. Strikingly, in all three hepatocellular carcinoma lines, the three-inhibitor combination alone exhibited similar or greater efficacy to 17AAG. We conclude that (a) the PCA captures and clusters the multipathway phosphoprotein time courses with respect to their 17AAG-induced apoptotic responsiveness and (b) we can recreate, in a more specified manner, the cellular responses of a prospective multitarget cancer therapeutic. [Mol Cancer Ther 2009;8(8):2183–92]

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influence phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHH) cultured in a commercially available perfused bioreactor. A 3D printing-based alginate microwell system was designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver were engineered to replace polystyrene scaffolds, and the biochemical microenvironment was modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increased tissue density, albumin secretion, and CYP3A4 activity but also upregulated inflammatory markers. Basal inflammatory markers were lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibited the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA-seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

On-platform pumping systems are a potentially critical technology for microphysiological systems (MPS) to control the pressure and flow of growth media. Supporting sufficient physiological tissue quantity in culture requires fluid flow rates on the order of microliters per second, which is larger than for typical microfluidic systems. Thus, a need exists for new types of pumping systems operating in this flow range while maintaining stringent sterility of the culture as well as enabling tight temperature control at ${37}^{\circ } {\rm C}$ . Flow rates and pressure also need to be readily computer-controlled, with a manageable set of connections into the culture incubator environment. This article describes a novel mesoscale electromagnetically driven pumping system designed to meet all these requirements. Our design achieves a very low energy dissipation of ${0.65}\,{\rm mJ}$ per switching event, which allows one pumping channel to operate at ${0.45}\,{{\rm {\mu } L} /{\rm s}}$ with an average power dissipation of 1.3 mW and ${0.04}\,{{^\circ } {\rm C}}$ temperature rise in each actuator. The actuator operates in a bistable, teeter-totter configuration with a latching force of ${4.5}\,{\rm N}$ , and a relatively large stroke of ${400}\,{{\mu } {\rm m}}$ at the actuator pole face. Preliminary operational pumping test results show the potential of this type of electromagnetic actuator for fluidic pumping. Due to their compact configuration and very high energy efficiency, these pumps can provide the foundation for multichannel, on-platform pumping for MPS platforms, as well as for a range of sterile, temperature-sensitive microflow devices such as portable, battery-operated insulin pumps.

Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin’s role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.

Background Pediatric gliomas comprise a diverse set of brain tumor entities that have substantial long-term ramifications for patient survival and quality of life. However, the study of these tumors is currently limited due to a lack of authentic models. Additionally, many aspects of pediatric brain tumor biology, such as tumor cell invasiveness, have been difficult to study with currently available tools. To address these issues, we developed a synthetic extracellular matrix (sECM)-based culture system to grow and study primary pediatric brain tumor cells. Methods We developed a brain-like sECM material as a supportive scaffold for the culture of primary, patient-derived pediatric glioma cells and established patient-derived cell lines. Primary juvenile brainstem-derived murine astrocytes were used as a feeder layer to support the growth of primary human tumor cells. Results We found that our culture system facilitated the proliferation of various primary pediatric brain tumors, including low-grade gliomas, and enabled ex vivo testing of investigational therapeutics. Additionally, we found that tuning this sECM material allowed us to assess high-grade pediatric glioma cell invasion and evaluate therapeutic interventions targeting invasive behavior. Conclusion Our sECM culture platform provides a multipurpose tool for pediatric brain tumor researchers that enables both a wide breadth of biological assays and the cultivation of diverse tumor types.

An estimated 70% of individuals with type 2 diabetes also suffer from nonalcoholic fatty liver disease (NAFLD) . Insulin resistance is pathologically fundamental to both NAFLD and T2D, however the mechanisms of insulin resistance linking these complex diseases remains incompletely understood. Microphysiological systems provide an avenue to simulate disease parameters in the context of human biology, however current in vitro models of hepatic insulin resistance often include nutrient and insulin concentrations orders of magnitude above in vivo levels. Here, we established an in vitro 3D model culturing primary human hepatocytes and non-parenchymal cells in physiologically relevant media conditions simulating both baseline physiological and T2D conditions. Albumin secretion and cytochrome p450 assays indicate hepatocytes remain functional over two weeks in culture for both physiological and T2D conditions. Results further show hepatocytes cultured in physiological conditions maintain insulin responsiveness after 12 days in culture, measured by glucose output following dose response of insulin stimulation. Conversely, cells cultured in T2D media conditions have significantly attenuated response to insulin stimulation compared to the physiological condition. Measuring insulin clearance from the media also showed reduced clearance over time for the T2D conditions. Together, these results demonstrate the induction of hepatic insulin resistance through culture with insulin concentrations found in the portal vein of T2D patients. Studies further applying this in vitro model to understand insulin resistance mechanisms are ongoing. Disclosure E.Tevonian: Research Support; Novo Nordisk. J.F.Jeppesen: Employee; Novo Nordisk A/S. D.Lauffenburger: None. L.G.Griffith: Research Support; Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S, Novo Nordisk A/S.

Intestinal homeostasis is tightly regulated by the orchestrated actions of a multitude of cell types, including enterocytes, goblet cells and immune cells. Disruption of intestinal barrier function can increase susceptibility to pathogen invasion and destabilize commensal microbial-epithelial-immune interaction, manifesting in various intestinal and systemic pathologies. However, a quantitative understanding of how these cell types communicate and collectively contribute to tissue function in health and disease is lacking. Here, we utilized a human intestinal epithelial-dendritic cell model and multivariate analysis of secreted factors to investigate the cellular crosstalk in response to physiological and/or pathological cues (e.g., endotoxin, non-steroidal anti-inflammation drug (NSAID)). Specifically, we demonstrated that treatment with diclofenac (DCF), an NSAID commonly used to treat inflammation associated with acute infection and other conditions, globally suppressed cytokine secretion when dosed in isolation. However, the disruption of barrier function induced by DCF allowed for luminal lipopolysaccharide (LPS) translocation and activation of resident immune cells that overrode the anti-inflammatory influence of DCF. DCF-facilitated inflammation in the presence of LPS was in part mediated by upregulation of macrophage migration inhibitory factor (MIF), an important regulator of innate immunity. However, while neutralization of MIF activity normalized inflammation, it did not lead to intestinal healing. Our data suggest that systems-wide suppression of inflammation alone is insufficient to achieve mucosal healing, especially in the presence of DCF, the target of which, the COX-prostaglandin pathway, is central to mucosal homeostasis. Indeed, DCF removal post-injury enabled partial recovery of intestinal epithelium functions, and this recovery phase was associated with upregulation of a subset of cytokines and chemokines, implicating their potential contribution to intestinal healing. The results highlight the utility of an intestinal model capturing immune function, coupled with multivariate analysis, in understanding molecular mechanisms governing response to microbial factors, supporting application in studying host-pathogen interactions.

STUDY OBJECTIVE To assess the feasibility of a noncontact radio sensor as an objective measurement tool to study postoperative recovery from endometriosis surgery. DESIGN Prospective cohort pilot study. SETTING Center for minimally invasive gynecologic surgery at an academically affiliated community hospital in conjunction with in-home monitoring. PATIENTS Patients aged above 18 years who sleep independently and were scheduled to have laparoscopy for the diagnosis and treatment of suspected endometriosis. INTERVENTIONS A wireless, noncontact sensor, Emerald, was installed in the subjects' home and used to capture physiologic signals without body contact. The device captured objective data about the patients' movement and sleep in their home for 5 weeks before surgery and approximately 5 weeks postoperatively. The subjects were concurrently asked to complete a daily pain assessment using a numeric rating scale and a free text survey about their daily symptoms. MEASUREMENTS AND MAIN RESULTS Three women aged 23 years to 39 years and with mild to moderate endometriosis participated in the study. Emerald-derived sleep and wake times were contextualized and corroborated by select participant comments from retrospective surveys. In addition, self-reported pain levels and 1 sleep variable, sleep onset to deep sleep time, showed a significant (p

SummaryThe capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types.

© 2020 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Adenomyosis remains an enigmatic disease in the clinical and research communities. The high prevalence, diversity of morphological and symptomatic presentations, array of potential etiological explanations, and variable response to existing interventions suggest that different subgroups of patients with distinguishable mechanistic drivers of disease may exist. These factors, combined with the weak links to genetic predisposition, make the entire spectrum of the human condition challenging to model in animals. Here, after an overview of current approaches, a vision for applying physiomimetic modeling to adenomyosis is presented. Physiomimetics combines a system's biology analysis of patient populations to generate hypotheses about mechanistic bases for stratification with in vitro patient avatars to test these hypotheses. A substantial foundation for three-dimensional (3D) tissue engineering of adenomyosis lesions exists in several disparate areas: epithelial organoid technology; synthetic biomaterials matrices for epithelial-stromal coculture; smooth muscle 3D tissue engineering; and microvascular tissue engineering. These approaches can potentially be combined with microfluidic platform technologies to model the lesion microenvironment and can potentially be coupled to other microorgan systems to examine systemic effects. In vitro patient-derived models are constructed to answer specific questions leading to target identification and validation in a manner that informs preclinical research and ultimately clinical trial design.

Research question What is the association between endometrioma-affected ovaries, their follicular fluid inflammatory microenvironment, and ovary-specific oocyte and embryo yield and quality? Design Exposure-matched prospective cohort study conducted at a university-affiliated infertility clinic. Thirty-four women presenting for oocyte retrieval were enrolled between 2012 and 2013: women with unilateral endometrioma and no other observed peritoneal or deep lesions (n = 10) and women with no signs or symptoms of endometriosis (n = 24). Follicular fluid was aspirated at the time of oocyte retrieval. Samples from each ovary were analysed using a 27-plex immunoassay panel. The associations were evaluated by ovary-specific endometrioma exposure status (affected, unaffected, unexposed) with cytokine levels, oocyte yield and embryo quality. Results Levels of interleukin (IL)-8 and monocyte chemoattractant protein-1 were higher in fluid obtained from endometrioma-affected ovaries compared with the unexposed ovaries from women without endometriosis, with intermediate levels observed in the contralateral unaffected ovaries. More modest differences were observed for IL-1β and IL-6. The affected ovaries of women with endometriosis yielded fewer oocytes (mean ± SD = 4.6 ± 2.3) compared with both the unaffected (6.0 ± 3.8) and unexposed (7.9 ± 5.6) ovaries. After adjusting for potential confounders and variables generated in a cytokine principal components analysis, oocyte yield remained slightly lower for the endometrioma-affected ovaries compared with unexposed ovaries. No informative differences among ovary groups for embryo quality parameters were observed. Conclusions The results suggest that the inflammatory milieu of ovarian endometriosis is strongly localized and has a more modestly systemic effect. The effect of endometriomas on infertility, however, cannot be entirely explained by increased inflammation.

Endometriosis remains a challenge to understand and to diagnose. This is an observational cross-sectional pilot study to characterize the gut and vaginal microbiome profiles among endometriosis patients and control subjects without the disease and to explore their potential use as a less-invasive diagnostic tool for endometriosis. Overall, 59 women were included, n = 35 with endometriosis and n = 24 controls. Rectal and vaginal samples were collected in two different periods of the menstrual cycle from all subjects. Gut and vaginal microbiomes from patients with different rASRM (revised American Society for Reproductive Medicine) endometriosis stages and controls were analyzed. Illumina sequencing libraries were constructed using a two-step 16S rRNA gene PCR amplicon approach. Correlations of 16S rRNA gene amplicon data with clinical metadata were conducted using a random forest-based machine-learning classification analysis. Distribution of vaginal CSTs (community state types) significantly differed between follicular and menstrual phases of the menstrual cycle (p = 0.021, Fisher’s exact test). Vaginal and rectal microbiome profiles and their association to severity of endometriosis (according to rASRM stages) were evaluated. Classification models built with machine-learning methods on the microbiota composition during follicular and menstrual phases of the cycle were built, and it was possible to accurately predict rASRM stages 1–2 verses rASRM stages 3–4 endometriosis. The feature contributing the most to this prediction was an OTU (operational taxonomic unit) from the genus Anaerococcus. Gut and vaginal microbiomes of women with endometriosis have been investigated. Our findings suggest for the first time that vaginal microbiome may predict stage of disease when endometriosis is present. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s43032-019-00113-5) contains supplementary material, which is available to authorized users.

The human endometrium is a mucosal barrier that undergoes cycles of growth, differentiation, and breakdown in response to sex hormone fluctuations. Dynamic tissue responses to hormones are primarily driven by epithelial-stromal communication and its dysregulation is linked to myriad gynecological disorders. The lack of robust in vitro models for the long-term 3D co-culture of patient-derived endometrial epithelial and stromal cells hinders dissection of this crosstalk and thus impairs progress in disease treatment. Here, we describe a versatile synthetic extracellular matrix tailored to the endometrium that enables the in vitro modeling of human healthy and disease states across the menstrual cycle. We used a tissue-inspired approach to semi-empirically screen a parameter space that encompasses the biophysical and molecular features of the endometrial microenvironment. Leveraging cell-specific integrin expression profiles, we defined a modular polyethylene glycol (PEG)-based hydrogel that fosters hormone-driven expansion and differentiation of epithelial organoids co-cultured with stromal cells. Characteristic morphological and molecular responses of each cell type to hormone changes were observed when cells were co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Using transcriptomic and functional assays, we demonstrate the systems ability to recapitulate menstrual-cycle specific reproductive events and identified that inflammation-induced dysregulation of epithelial proliferation is mediated via the stromal compartment. Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand endometriotic diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/462577v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@132d3fborg.highwire.dtl.DTLVardef@1fb2841org.highwire.dtl.DTLVardef@ff18dorg.highwire.dtl.DTLVardef@bb9d8f_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Metastatic breast cancer remains a largely incurable and fatal disease with liver involvement bearing the worst prognosis. The danger is compounded by a subset of disseminated tumor cells that may lie dormant for years to decades before re-emerging as clinically detectable metastases. Pathophysiological signals can drive these tumor cells to emerge. Prior studies indicated CXCR3 ligands as being the predominant signals synergistically and significantly unregulated during inflammation in the gut-liver axis. Of the CXCR3 ligands, IP-10 (CXCL10) was the most abundant, correlated significantly with shortened survival of human breast cancer patients with metastatic disease and was highest in those with triple negative (TNBC) disease. Using a complex ex vivo all-human liver microphysiological (MPS) model of dormant-emergent metastatic progression, CXCR3 ligands were found to be elevated in actively growing populations of metastatic TNBC breast cancer cells whereas they remained similar to the tumor-free hepatic niche in those with dormant breast cancer cells. Subsequent stimulation of dormant breast cancer cells in the ex vivo metastatic liver MPS model with IP-10 triggered their emergence in a dose-dependent manner. Emergence was indicated to occur indirectly possibly via activation of the resident liver cells in the surrounding metastatic microenvironment, as stimulation of breast cancer cells with exogenous IP-10 did not significantly change their migratory, invasive or proliferative behavior. The findings reveal that IP-10 is capable of triggering the emergence of dormant breast cancer cells within the liver metastatic niche and identifies the IP-10/CXCR3 as a candidate targetable pathway for rational approaches aimed at maintaining dormancy.

Experimental in vitro models that accurately capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix, specifically designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from murine genetically engineered models and human patients, essential adhesive cues were empirically defined and incorporated into the hydrogel scaffold, revealing a functional role of laminin – integrin a3/a6 signalling in adhesion and survival of pancreatic organoids. Altered tissue stiffness — a hallmark of pancreatic cancer — was recapitulated in culture by adjusting the hydrogel properties to engage mechano-signalling and alter organoid growth. Pancreatic stromal cells were readily incorporated and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro., This upload is compiled of 21 individual data packages which contain the raw data and R-scripts necessary to generate the outputs displayed in the paper. Each folder contains a README document which assists the reader through the files.

Summary Background The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. Methods Here, we investigate the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a gut microbiome (GuMI) physiome platform that we designed and fabricated. Findings Long-term continuous co-culture of F. prausnitzii for 2 days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, results in a strictly anaerobic apical environment fostering the growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identify elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrate the anti-inflammatory effects of F. prausnitzii through histone deacetylase (HDAC) and the Toll-like receptor-TLR-nuclear factor κB (NF-κB) axis. Finally, we identify that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. Conclusions Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies using this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria. Funding US National Institute of Biomedical Imaging and Bioengineering, Boehringer Ingelheim Strategic Hub for Innovative New Therapeutics Concept Exploration (SHINE) Program, US National Institute of Environmental Health Sciences.

PROBLEM To compare inflammatory- and immune-associated peritoneal cytokines of adolescents and adults with and without endometriosis. METHODS OF STUDY In a nested case-control study in multiple university-affiliated scientific centers, ten adolescents and thirteen adults with visually and histologically confirmed endometriosis (cases), thirteen adolescents with visually suspected endometriosis but indeterminate (seven patients) or negative (six patients) histology, and fifteen adults undergoing surgery for non-malignant gynecologic disease without endometriosis (controls) underwent laparoscopic aspiration of peritoneal fluid (PF), from which PF and conditioned medium (CM) cytokine levels were assayed. RESULTS Compared to adults with endometriosis, MCP-3, IL-12p40, MIP-1β, and IL-15 were significantly higher among adolescents with endometriosis, while TNF-β and CTACK were lower among adolescents. These differences were similar comparing adolescents with endometriosis to adult controls except for MIP-1β, which was not statistically different. MIP-1β was, however, the only cytokine observed to differ between adult cases and controls. There were no significant differences in CM cytokines among the three groups. Results were similar when analyses were restricted to samples collected (a) during menstrual cycle days 1-10, (b) from patients unexposed to exogenous hormones, or (c) from all adolescents despite presence or absence of histologic endometriosis. CONCLUSION Biologically relevant and statistically significant differences in six PF cytokines were observed and suggest a more pro-invasion cytokine profile among adolescents with endometriosis. Adolescents with endometriosis have unique peritoneal cytokine profiles and molecular behavior when compared to adults with and without endometriosis.

Engineered model of the human gut-liver-brain axis can be used to study the link between the microbiome and Parkinson’s disease., Slow progress in the fight against neurodegenerative diseases (NDs) motivates an urgent need for highly controlled in vitro systems to investigate organ-organ– and organ-immune–specific interactions relevant for disease pathophysiology. Of particular interest is the gut/microbiome-liver-brain axis for parsing out how genetic and environmental factors contribute to NDs. We have developed a mesofluidic platform technology to study gut-liver-cerebral interactions in the context of Parkinson’s disease (PD). It connects microphysiological systems (MPSs) of the primary human gut and liver with a human induced pluripotent stem cell–derived cerebral MPS in a systemically circulated common culture medium containing CD4+ regulatory T and T helper 17 cells. We demonstrate this approach using a patient-derived cerebral MPS carrying the PD-causing A53T mutation, gaining two important findings: (i) that systemic interaction enhances features of in vivo–like behavior of cerebral MPSs, and (ii) that microbiome-associated short-chain fatty acids increase expression of pathology-associated pathways in PD.

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.

© 2017 The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. Keywords: Microphysiologic systems; Organoids; 3D culture systems, National Institutes of Health (U.S.) (Grant UH3TR000496), National Institutes of Health (U.S.) (Grant UH3TR000503)

Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed., National Institutes of Health (U.S.) (R01 CA96504)

In vitro models of human liver functions are used across a diverse range of applications in preclinical drug development and disease modeling, with particular increasing interest in models that capture facets of liver inflammatory status. This study investigates how the interplay between biophysical and biochemical microenvironment cues influences phenotypic responses, including inflammation signatures, of primary human hepatocytes (PHHs) cultured in a commercially available perfused bioreactor. A 3D printing‐based alginate microwell system is designed to form thousands of hepatic spheroids in a scalable manner as a comparator 3D culture modality to the bioreactor. Soft, synthetic extracellular matrix (ECM) hydrogel scaffolds with biophysical properties mimicking features of liver are engineered to replace polystyrene scaffolds, and the biochemical microenvironment is modulated with a defined set of growth factors and signaling modulators. The supplemented media significantly increases tissue density, albumin secretion, and CYP3A4 activity but also upregulates inflammatory markers. Basal inflammatory markers are lower for cells maintained in ECM hydrogel scaffolds or spheroid formats than polystyrene scaffolds, while hydrogel scaffolds exhibit the most sensitive response to inflammation as assessed by multiplexed cytokine and RNA‐Seq analyses. Together, these engineered 3D liver microenvironments provide insights for probing human liver functions and inflammatory response in vitro.

Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 106 cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases

Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases., National Institutes of Health (U.S.) (R01EB010246), National Institutes of Health (U.S.) (UH2TR000496), Institute for Collaborative Biotechnologies (W911NF-09-0001), National Institutes of Health (U.S.) (T32GM008334), United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), John and Karinne Begg Fund, Begg New Horizon Fund for Undergraduate Research at MIT, Massachusetts Institute of Technology. Biophysical Instrumentation Facility, Manton Foundation, Ludwig Postdoctoral Fellowship for Cancer Research, Swiss National Science Foundation (Postdoctoral Fellowship)

Microphysiological systems (MPS) provide relevant physiological environments in vitro for studies of pharmacokinetics, pharmacodynamics and biological mechanisms for translational research. Designing multi-MPS platforms is essential to study multi-organ systems. Typical design approaches, including direct and allometric scaling, scale each MPS individually and are based on relative sizes not function. This study's aim was to develop a new multi-functional scaling approach for integrated multi-MPS platform design for specific applications. We developed an optimization approach using mechanistic modeling and specification of an objective that considered multiple MPS functions, e.g., drug absorption and metabolism, simultaneously to identify system design parameters. This approach informed the design of two hypothetical multi-MPS platforms consisting of gut and liver (multi-MPS platform I) and gut, liver and kidney (multi-MPS platform II) to recapitulate in vivo drug exposures in vitro. This allows establishment of clinically relevant drug exposure-response relationships, a prerequisite for efficacy and toxicology assessment. Design parameters resulting from multi-functional scaling were compared to designs based on direct and allometric scaling. Human plasma time-concentration profiles of eight drugs were used to inform the designs, and profiles of an additional five drugs were calculated to test the designed platforms on an independent set. Multi-functional scaling yielded exposure times in good agreement with in vivo data, while direct and allometric scaling approaches resulted in short exposure durations. Multi-functional scaling allows appropriate scaling from in vivo to in vitro of multi-MPS platforms, and in the cases studied provides designs that better mimic in vivo exposures than standard MPS scaling methods., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 μL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples., National Institutes of Health (Grant R01 EB010246)

An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host–pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

Bacteroides thetaiotaomicron is a human-associated bacterium that holds promise for delivery of therapies in the gut microbiome1. Therapeutic bacteria would benefit from the ability to turn on different programs of gene expression in response to conditions inside and outside of the gut; however, the availability of regulatory parts, and methods to combine them, have been limited in B. thetaiotaomicron2-5. We report implementation of Cello circuit design automation software6 for this species. First, we characterize a set of genome-integrated NOT/NOR gates based on single guide RNAs (CRISPR-dCas9) to inform a Bt user constraint file (UCF) for Cello. Then, logic circuits are designed to integrate sensors that respond to bile acid and anhydrotetracycline (aTc), including one created to distinguish between environments associated with bioproduction, the human gut, and after release. This circuit was found to be stable under laboratory conditions for at least 12 days and to function in bacteria associated with a primary colonic epithelial monolayer in an in vitro human gut model system.

Three-dimensional micro-physiological in vitro representations of human tissues and organs are emerging as important models of human pathophysiology and stand to make a significant impact on the process of drug discovery and development. An enduring need is to create microvascular networks within such 3D models, particularly for tissues with high metabolic demand such as the liver, pancreas, and the central nervous system. Here we report a facile approach to drive angiogenesis in nascent 3D culture models by embedding degradable hydrogel microbeads coated with induced pluripotent stem cell-derived endothelial cells (MB-iPSC-ECs) in a dense epithelial tissue. Specifically, we describe an approach to optimize microbead scaffold cues, independent of the external environment, by evaluating the iPSC-EC to microbead adhesion properties and how they influence the propensity of cells to both coat microbeads uniformly and undergo sprouting angiogenesis. We encapsulated MB-iPSC-ECs in PEG hydrogels, systematically varied the relative concentration of integrin-targeting peptide motifs in the microbead and surrounding environment, and found that an optimal microbead scaffold ligand regime of 0.1–0.25 mM promotes iPSC-EC monolayer formation and subsequent invasion into the synthetic matrix. We used these results to predict the regime of adhesion ligand required to promote angiogenesis of MB-iPSC-ECs in a co-culture hepatocarcinoma (HEPG2) microtissue model. This modular degradable microbead platform has the potential to promote angiogenic sprouting, which may ultimately support vascularization of a variety of cell-dense tissues.

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro-in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (Grant W911NF-12-2-0039), National Institutes of Health (U.S.). Microphysiological Systems Program (Grant 4-UH3-TR000496-03)

The liver is a highly metastasis-permissive organ, tumor seeding of which usually portends mortality. Its unique and diverse architectural and cellular composition enable the liver to undertake numerous specialized functions, however, this distinctive biology, notably its hemodynamic features and unique microenvironment, renders the liver intrinsically hospitable to disseminated tumor cells. The particular focus for this perspective is the bidirectional interactions between the disseminated tumor cells and the unique resident cell populations of the liver; notably, parenchymal hepatocytes and non-parenchymal liver sinusoidal endothelial, Kupffer, and hepatic stellate cells. Understanding the early steps in the metastatic seeding, including the decision to undergo dormancy versus outgrowth, has been difficult to study in 2D culture systems and animals due to numerous limitations. In response, tissue-engineered biomimetic systems have emerged. At the cutting-edge of these developments are ex vivo ‘microphysiological systems’ (MPS) which are cellular constructs designed to faithfully recapitulate the structure and function of a human organ or organ regions on a milli- to micro-scale level and can be made all human to maintain species-specific interactions. Hepatic MPSs are particularly attractive for studying metastases as in addition to the liver being a main site of metastatic seeding, it is also the principal site of drug metabolism and therapy-limiting toxicities. Thus, using these hepatic MPSs will enable not only an enhanced understanding of the fundamental aspects of metastasis but also allow for therapeutic agents to be fully studied for efficacy while also monitoring pharmacologic aspects and predicting toxicities. The review discusses some of the hepatic MPS models currently available and although only one MPS has been validated to relevantly modeling metastasis, it is anticipated that the adaptation of the other hepatic models to include tumors will not be long in coming.

Stem cell use for tissue replacement is limited, owing to rapid death induced in the hostile wound environment. This study found that restricting epidermal growth factor (EGF) receptor signaling to the membrane provided a survival advantage. A method is proposed to tether EGF to bone induction material to improve the survival of mesenchymal stem cells/multipotent stromal cells in vivo., Mesenchymal stem cells/multipotent stromal cells (MSCs) are attractive candidates for cell therapies owing to their ability to differentiate into many lineages. However, these cells often fail to survive when implanted into a harsh wound environment, limiting efficacy in vivo. To improve MSC survival, we previously found that tethered epidermal growth factor (tEGF) molecules that restrict epidermal growth factor receptor (EGFR) signaling to the cell surface provide resistance to death signals. To adapt this system to wound healing, we tethered epidermal growth factor (EGF) to tricalcium phosphate (TCP) particle scaffolds, clinically used in bone healing. Human primary MSCs seeded on TCP and mixed into a collagen-based gel were injected in the perifascial space of immunocompetent mice with or without tEGF attached to the surface. We found that tethering EGF to the TCP scaffolds yielded approximately a fourfold increase in MSC survival compared with non-EGF scaffolds at 21 days, as well as significant improvements in survival in the short term at 2 and 7 days after implantation. Overall, our approach to sustaining EGFR signaling reduced MSC death in vivo and may be useful for future cell therapies where MSCs typically die on implantation. Significance Stem cells are limited as tissue replacements owing to rapid death induced in the hostile wound environment. It has been found that restricting epidermal growth factor (EGF) receptor signaling to the membrane provides a survival advantage. This report elucidates a method to tether EGF to bone induction material to improve the survival of mesenchymal stem cells/multipotent stromal cells in vivo.

Kinase inhibitor resistance often involves upregulation of poorly understood “bypass” signaling pathways. Here, we show that extracellular proteomic adaptation is one path to bypass signaling and drug resistance. Proteolytic shedding of surface receptors, which can provide negative feedback on signaling activity, is blocked by kinase inhibitor treatment and enhances bypass signaling. In particular, MEK inhibition broadly decreases shedding of multiple receptor tyrosine kinases (RTK), including HER4, MET, and most prominently AXL, an ADAM10 and ADAM17 substrate, thus increasing surface RTK levels and mitogenic signaling. Progression-free survival of patients with melanoma treated with clinical BRAF/MEK inhibitors inversely correlates with RTK shedding reduction following treatment, as measured noninvasively in blood plasma. Disrupting protease inhibition by neutralizing TIMP1 improves MAPK inhibitor efficacy, and combined MAPK/AXL inhibition synergistically reduces tumor growth and metastasis in xenograft models. Altogether, extracellular proteomic rewiring through reduced RTK shedding represents a surprising mechanism for bypass signaling in cancer drug resistance. Significance: Genetic, epigenetic, and gene expression alterations often fail to explain adaptive drug resistance in cancer. This work presents a novel post-translational mechanism of such resistance: Kinase inhibitors, particularly targeting MAPK signaling, increase tumor cell surface receptor levels due to widely reduced proteolysis, allowing tumor signaling to circumvent intended drug action. Cancer Discov; 6(4); 382–99. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 331

Breast cancer (BrCa) mortality continues to result predominately from distant metastases that can emerge years after successful treatment of the primary disease. Metastatic resistance to agents that eradicate the primary mass is likely due to protection from the metastatic microenvironment and the quiescent state of dormant BrCa cells. Advancements for the treatment of metastatic tumors have been made, but significant progress has been hampered by the lack of relevant model systems, particularly for dormancy. We address this gap with an innovative all-human 3D liver microphysiological system (MPS). The liver is both a major site for BrCa metastasis (and other solid tumors) and the primary site of drug metabolism and limiting toxicities, an important consideration in evaluating cancer therapy efficacy and availability. Primary hepatocytes and non-parenchymal cells (NPC) from human liver resections were seeded into the MPS. Following tissue formation on day 3, tagged BrCa cells were seeded and allowed a minimum of 4 days to integrate into the tissue before interventions were initiated. On day 7, chemotherapy treatment of micrometastases was initiated for 72h. Cultures were allowed 3 days to recover before the MPS was challenged with inflammatory factors (LPS/EGF) for 48h. BrCa cells were then re-treated with chemotherapy (either the same or alternate therapy) on day 21 for 72h. Hepatocyte function and injury were measured by urea, AST, ALT, A1AT, fibrinogen and CYP P450 assays. BC proliferation was monitored by quantification, Ki67 staining, and EdU incorporation. Communication networks within the metastatic microenvironment during different stages of metastatic BrCa progression were identified using Luminex assays (55 analytes). The metastatically aggressive MDA-MB-231 BrCa cells demonstrated growth attenuation after 12d of culture in a subpopulation of cells (Ki67-/EdU-). Treatment of BrCa cells with doxorubicin for 72h eradicates the cycling cells, leaving behind a dormant cell population (Ki67-/EdU-) that can be subsequently stimulated to cycle by addition of inflammatory stimuli. A second dose of doxorubicin or cisplatin reduced the BrCa load but did not eradicate the BrCa. Luminex analysis of culture supernatants identified signaling molecules potentially involved in metastatic progression. In addition, we present the use of adjuvant therapy in the MPS to prevent this outgrowth of the dormant tumor cells. In parallel, we have piloted hydrogel scaffolds that better support tissue formation and produce signals consistent with a healthier liver physiology. Hydrogels enhanced MDA-MB-231 cell entry into dormancy, resulting in reduced efficacy of doxorubicin treatment with greater persistence of tumor load. The MPS provides a mechanism to close the gap in understanding metastatic dormancy. We demonstrate spontaneous dormancy for the first time in an all-human system and mimicked the dormancy and outgrowth observed in patients. Namely, that dormant BrCa are resistant to chemotherapy and can be stimulated to reemerge following an inflammatory insult. The completion of these studies will provide insights into the tumor biology of metastatic seeding, dormancy, and re-emergence and provide an accessible tool for testing therapeutics against metastatic BrCa in a metabolically competent system capable of evaluating dose-limiting toxicity. Citation Format: Beckwitt C, Wheeler SE, Clark AM, Pillai VC, Young CL, Stolz DB, Lauffenburger DA, Venkataramanan R, Griffith LG, Wells A. Breast cancer dormancy, re-emergence, and treatment. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-19.

In vitro tissue engineered models are poised to have significant impact on disease modeling and preclinical drug development. Reliable methods to induce microvascular networks in such microphysiological systems are needed to improve the size and physiological function of these models. By systematically engineering several physical and biomolecular properties of the cellular microenvironment (including crosslinking density, polymer density, adhesion ligand concentration, and degradability), we establish design principles that describe how synthetic matrix properties influence vascular morphogenesis in modular and tunable hydrogels based on commercial 8-arm poly (ethylene glycol) (PEG8a) macromers. We apply these design principles to generate endothelial networks that exhibit consistent morphology throughout depths of hydrogel greater than 1 mm. These PEG8a-based hydrogels have relatively high volumetric swelling ratios (>1.5), which limits their utility in confined environments such as microfluidic devices. To overcome this limitation, we mitigate swelling by incorporating a highly functional PEG-grafted alpha-helical poly (propargyl- l -glutamate) (PPLGgPEG) macromer along with the canonical 8-arm PEG8a macromer in gel formation. This hydrogel platform supports enhanced endothelial morphogenesis in neutral-swelling environments. Finally, we incorporate PEG8a-PPLGgPEG gels into microfluidic devices and demonstrate improved diffusion kinetics and microvascular network formation in situ compared to PEG8a-based gels.

Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self-assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label-free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell–derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)-based hydrogels. Two custom-built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label-free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label-free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues., National Institutes of Health (U.S.) (Grant R01 HL093282-01A1), National Institutes of Health (U.S.) (Grant 1U H2TR000506-01), National Institutes of Health (U.S.) (Grant 3UH2TR000506-02S1), National Institutes of Health (U.S.) (Grant 4U H3TR000506-03), National Institutes of Health (U.S.) (Grant R01CA205101), National Institutes of Health (U.S.) (Grant R01CA185747), National Institutes of Health (U.S.) (Grant R01CA211082), National Institutes of Health (U.S.) (Grant R01CA226526), National Science Foundation (U.S.) (Grant (CBET-1642287), Entertainment Industry Foundation. Stand Up to Cancer Colorectal Cancer Dream Team (Grant SU2C-AACR-IG-08-16), Stand Up To Cancer (Grant SU2C-AACR-IG-08-16)

Microphysiological systems (MPS), consisting of tissue constructs, biomaterials, and culture media, aim to recapitulate relevant organ functions in vitro. MPS components are housed in fluidic hardware with operational protocols, such as periodic complete media replacement. Such batch-like operations provide relevant nutrients and remove waste products but also reset cell-secreted mediators (e.g. cytokines, hormones) and potentially limit exposure to drugs (and metabolites). While each component plays an essential role for tissue functionality, MPS-specific nutrient needs are not yet well-characterized nor utilized to operate MPSs at more physiologically-relevant conditions. MPS-specific nutrient needs for gut (immortalized cancer cells), liver (human primary hepatocytes) and cardiac (iPSC-derived cardiomyocytes) MPSs were experimentally quantified. In a long-term study of the gut MPS (10 days), this knowledge was used to design operational protocols to maintain glucose and lactate at desired levels. This quasi-steady state operation was experimentally validated by monitoring glucose and lactate as well as MPS functionality. In a theoretical study, nutrient needs of an integrated multi-MPS platform (gut, liver, cardiac MPSs) were computationally simulated to identify long-term quasi-steady state operations. This integrative experimental and computational approach demonstrates the utilization of quantitative multi-scale characterization of MPSs and incorporating MPS-specific information to establish more physiologically-relevant experimental operations., United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039), National Institutes of Health (U.S.) (U24TR001951), National Institutes of Health (U.S.). Microphysiological Systems Program (4-UH3-TR000496-03)

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs – “4-way”, “7-way”, and “10-way” – each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS “physiome-on-a-chip” approaches in drug discovery.

Engineered tissues offer great promise as engrafted therapies and in vitro models, but these tissues require a vascular network to retain viability at large scales. Significant efforts are focused on optimizing these in vitro vascular constructs, yet current evaluation methods require fixation and immunostaining. These destructive evaluation methods alter vascular network morphology, and cannot non-invasively monitor vascular assembly over time. Here, we demonstrate that autofluorescence multiphoton microscopy (MPM) can quantitatively assess the morphology of living 3D vascular networks without fixation, labels, or dyes. Autofluorescence MPM was used to non-invasively monitor the effect of culture conditions on 3D vascular network formation. Human embryonic stem (ES) cell-derived endothelial cells and primary human pericytes cultured in polyethylene glycol (PEG) hydrogels self-assembled into 3D vascular networks. Autofluorescence MPM of the metabolic co-enzyme NAD(P)H (excitation/emission wavelengths of 750 nm/400-460 nm) was used to quantify morphological parameters at day 6 of culture. Specifically, vessel diameter, vascular density, branch point density, and integration of endothelial cells into the network were quantified. Dynamic culture conditions (flow at 1μL/sec) led to vascular networks with higher mean vessel diameter compared to static culture (p

Most breast cancer (BrCa) mortality results from distant metastases. Current evidence strongly suggests that in some instances these disseminated cells remain dormant for long periods of time. Both the non-proliferative state and protective microenvironment of the metastatic niche likely contribute to the observed resistance of metastases to chemotherapies that are otherwise effective against the primary tumor. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model systems for metastases has hindered the development of therapies against this stage. To address this gap, we developed an innovative all-human 3D ex vivo hepatic microphysiological system (MPS) to faithfully reproduce human physiology and thereby facilitate the investigation of BrCa behavior in a micrometastatic niche. The liver is a major site of metastasis for carcinomas and is also the primary site of drug metabolism (activation and/or detoxification), which is a significant factor in determining efficacy and limiting toxicities of cancer therapies. The MPS incorporates hepatocytes and nonparenchymal cells (NPC) isolated from fresh human liver resections. BrCa cells (RFP+) are seeded on day 3 and afforded time to intercalate into the hepatic tissue until treatment with chemotherapy on day 7 for 72h. Surviving BrCa cells are stimulated on day 13 with LPS/EGF and cultured through day 15. Proliferation is monitored by RFP quantification, Ki67 staining and EdU incorporation. Physiological function of the hepatic tissue is monitored throughout the experiment by protein catabolism (urea), active metabolism (glucose, CYP P450) and injury markers (AST, ALT, A1AT, fibrinogen). Luminex assays (55 analytes) were used to provide insights into the communication networks in the hepatic metastatic milieu during different stages of dormancy and progression, and identify potential metastatic biomarkers via computational approaches. The MPS maintains the physiologic function of the hepatic niche through 15 days and BrCa cells effectively integrate into the established niche. Spontaneous dormancy is observed amongst a subpopulation of BrCa cells, indicated by the absence of Ki67 staining and EdU incorporation after 12 days of culture. Further, we demonstrate that the BrCa cells surviving chemotherapy (doxorubicin) are non-proliferating (Ki67-/EdU-). Notably, ‘re-awakening’ of the surviving non-proliferating cancer cells is observed in the presence of physiological inflammatory stressors (LPS/EGF). Luminex analyses of the milieu effluent identified signaling molecules from NPC influenced the metastatic cell fraction entering dormancy. This MPS provides unprecedented insights into the tumor biology of dormant micrometastases. We demonstrate the recreation of spontaneous, rather than engineered, BrCa dormancy in an all-human ex vivo hepatic MPS. Mimicking the dormancy and outgrowth observed in patients, we found that dormant breast cancer cells that are resistant to chemotherapy can be stimulated to re-emerge following an inflammatory insult. Ultimately, this MPS provides an accessible tool to identify new therapeutic strategies for metastasis during initial seeding, dormancy and re-emergence, while concurrently evaluating agent efficacy for metastasis, metabolism and dose-limiting toxicity. Citation Format: Amanda M Clark, Sarah E Wheeler, Donald P Taylor, Carissa L Young, Venkateswaran C Pillai, Donna B Stolz, Raman Venkataramanan, Douglas A Lauffenburger, Linda G Griffith, Alan Wells. Modeling breast cancer dormancy and re-emergence [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-01.