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Microenvironmental changes in the early metastatic niche may be exploited to identify therapeutic targets to inhibit secondary tumor formation and improve disease outcomes. We dissected the developing lung metastatic niche in a model of metastatic, triple-negative breast cancer using single-cell RNA-sequencing. Lungs were extracted from mice at 7-, 14-, or 21 days after tumor inoculation corresponding to the pre-metastatic, micro-metastatic, and metastatic niche, respectively. The progression of the metastatic niche was marked by an increase in neutrophil infiltration (5% of cells at day 0 to 81% of cells at day 21) and signaling pathways corresponding to the hallmarks of cancer. Importantly, the pre-metastatic and early metastatic niche were composed of immune cells with an anti-cancer phenotype not traditionally associated with metastatic disease. As expected, the metastatic niche exhibited pro-cancer phenotypes. The transition from anti-cancer to pro-cancer phenotypes was directly associated with neutrophil and monocyte behaviors at these time points. Predicted metabolic, transcription factor, and receptor-ligand signaling suggested that changes in the neutrophils likely induced the transitions in the other immune cells. Conditioned medium generated by cells extracted from the pre-metastatic niche successfully inhibited tumor cell proliferation and migration in vitro and the in vivo depletion of pre-metastatic neutrophils and monocytes worsened survival outcomes, thus validating the anti-cancer phenotype of the developing niche. Genes associated with the early anti-cancer response could act as biomarkers that could serve as targets for the treatment of early metastatic disease. Such therapies have the potential to revolutionize clinical outcomes in metastatic breast cancer.

NCBI GEO formatted submission for OpenArray{trade mark, serif} RT-qPCR data including: ID_REF, Gene Symbol, Reference/Target use, Catalog Number, Assay Type, Gene Name, Alias, RefSeq Accession Number, GenBank mRNA Numbers, Target Species, Amplicon Length, Platform, Raw Non-Normalized Cq, Reference Gene Normalized Î"Cq and Î"Î"Cq Fold Changes.

Kaplan-Meier survival curves correlated with high and low gene expression from breast cancer patient samples. (a) Microarray (GEO via KMPlot) and prognosis data were queried for genes selected for the 10-gene panel derived from tissue within implants to determine the expression level relevance in primary tumors on the clinical outcome of systemically untreated breast cancer patients (n=818). Kaplan-Meier plots indicate as separation of two profiles separating high and low gene expression that are automatically divided by the median expression of the genes for all samples. Plots indicate the Hazard Ratio (HR) which is highest for S100a9 and logrank significance comparing high and low expression as a function of recurrence-free survival. FDR 10% corrected significance (p

Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease.Significance:Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression.

Supplementary Materials and Methods, Figures, and Descriptions of Supplementary Data

Monitoring metastatic events in distal tissues is challenged by their sporadic occurrence in obscure and inaccessible locations within these vital organs. A synthetic biomaterial scaffold can function as a synthetic metastatic niche to reveal the nature of these distal sites. These implanted scaffolds promote tissue ingrowth, which upon cancer initiation is transformed into a metastatic niche that captures aggressive circulating tumor cells. We hypothesized that immune cell phenotypes at synthetic niches reflect the immunosuppressive conditioning within a host that contributes to metastatic cell recruitment and can identify disease progression and response to therapy. We analyzed the expression of 632 immune-centric genes in tissue biopsied from implants at weekly intervals following inoculation. Specific immune populations within implants were then analyzed by single-cell RNA-seq. Dynamic gene expression profiles in innate cells, such as myeloid-derived suppressor cells, macrophages, and dendritic cells, suggest the development of an immunosuppressive microenvironment. These dynamics in immune phenotypes at implants was analogous to that in the diseased lung and had distinct dynamics compared with blood leukocytes. Following a therapeutic excision of the primary tumor, longitudinal tracking of immune phenotypes at the implant in individual mice showed an initial response to therapy, which over time differentiated recurrence versus survival. Collectively, the microenvironment at the synthetic niche acts as a sentinel by reflecting both progression and regression of disease. Significance: Immune dynamics at biomaterial implants, functioning as a synthetic metastatic niche, provides unique information that correlates with disease progression.

Pancreatic cancer has the worst prognosis of all cancers due to disease aggressiveness and paucity of early detection platforms. We developed biomaterial scaffolds that recruit metastatic tumor cells and reflect the immune dysregulation of native metastatic sites. While this platform has shown promise in orthotopic breast cancer models, its potential in other models is untested. Herein, we demonstrate that scaffolds recruit disseminated pancreatic cells in the KPCY model of spontaneous pancreatic cancer prior to adenocarcinoma formation (3-fold increase in scaffold YFP + cells). Furthermore, immune cells at the scaffolds differentiate early- and late-stage disease with greater accuracy (0.83) than the natural metastatic site (liver, 0.50). Early disease was identified by an approximately 2-fold increase in monocytes. Late-stage disease was marked by a 1.5-2-fold increase in T cells and natural killer cells. The differential immune response indicated that the scaffolds could distinguish spontaneous pancreatic cancer from spontaneous breast cancer. Collectively, our findings demonstrate the utility of scaffolds to reflect immunomodulation in two spontaneous models of tumorigenesis, and their particular utility for identifying early disease stages in the aggressive KPCY pancreatic cancer model. Such scaffolds may serve as a platform for early detection of pancreatic cancer to improve treatment and prognosis.

This study uses biomaterial scaffolds to identify a macrophage gene signature defined by high expression of C1qa, C1qb, and Trem2 that is elevated systemically in mouse and human pancreatic cancer., Pancreatic ductal adenocarcinoma (PDA) is accompanied by reprogramming of the local microenvironment, but changes at distal sites are poorly understood. We implanted biomaterial scaffolds, which act as an artificial premetastatic niche, into immunocompetent tumor-bearing and control mice, and identified a unique tumor-specific gene expression signature that includes high expression of C1qa, C1qb, Trem2, and Chil3. Single-cell RNA sequencing mapped these genes to two distinct macrophage populations in the scaffolds, one marked by elevated C1qa, C1qb, and Trem2, the other with high Chil3, Ly6c2 and Plac8. In mice, expression of these genes in the corresponding populations was elevated in tumor-associated macrophages compared with macrophages in the normal pancreas. We then analyzed single-cell RNA sequencing from patient samples, and determined expression of C1QA, C1QB, and TREM2 is elevated in human macrophages in primary tumors and liver metastases. Single-cell sequencing analysis of patient blood revealed a substantial enrichment of the same gene signature in monocytes. Taken together, our study identifies two distinct tumor-associated macrophage and monocyte populations that reflects systemic immune changes in pancreatic ductal adenocarcinoma patients.

OBJECTIVES/GOALS: Breast cancer metastases are stochastic and difficult to detect. Therapy is often ineffective due to phenotypic changes of tumor cells at these sites. We engineered a synthetic metastatic niche to study the role of phenotypic transitions in the microenvironment on tumor cell phenotype. METHODS/STUDY POPULATION: The engineered metastatic niche is composed of a porous polycaprolactone scaffold implanted subcutaneously in Balb/c mice. The mice received an orthotopic inoculation of 4T1 cells (murine triple negative breast cancer) in the fourth right mammary fat pad and the disease was allowed to progress for 7-21 days (pre-metastatic to overt metastatic disease). The scaffolds and lungs (native metastatic site) were explanted and analyzed by single cell RNA-seq via Drop-seq. Cell phenotypes were identified and tracked over time with the Seurat and Monocle3 pipelines. Assessment of the impact of these cell populations on tumor cell phenotype was conducted through Transwell co-cultures. RESULTS/ANTICIPATED RESULTS: Healthy scaffolds are primarily composed of macrophages, dendritic cells, and fibroblasts – consistent with a foreign body response. Despite differences in the lung and scaffold prior to tumor inoculation, both tissues were marked by >5-fold increase in neutrophils/MDSCs. Additionally, 79% of genes at the scaffold that significantly changed over time were also identified in the lung, indicating key similarities in niche maturation. However, many immune cells at the scaffold had distinct phenotypes, with pro-inflammatory/cytotoxic characteristics. These changes clearly impacted tumor cell phenotype, as cells from the scaffold increased tumor cell migration and apoptosis in vitro. DISCUSSION/SIGNIFICANCE OF IMPACT: Early phenotypic changes at the engineered metastatic niche can identify signs of metastasis prior to colonization of tumor cells. Furthermore, dynamics of immune and stromal cells change throughout niche maturation, influencing tumor cell phenotype and may suggest targeted therapies. CONFLICT OF INTEREST DESCRIPTION: Lonnie Shea, Jacqueline Jeruss, and Grace Bushnell are named inventors on patents or patent applications.

Pre-metastatic niches in distant tissue facilitate metastasis from the primary tumor. Cargo-free porous polymer scaffolds implanted in tumor-bearing mice act as synthetic metastatic niches recruiting metastasizing cancer cells. Herein, we investigated the mechanisms by which these implants attract cancer cells from circulation. Scaffolds attract cancer cells in part via S100A8/A9 secreted by Gr1+ myeloid cells in a mechanism that mimics lung metastasis. Further, cancer cells attracted to the scaffold have a lung-tropic gene expression signature regardless of their tissue of origin. The scaffold implant reduces metastasis to the lung suggesting otherwise lung-tropic cancer cells are diverted to the scaffold. The suppression of metastatic spread by the scaffold suggests this mechanism may be exploited for novel therapies, and may broadly influence the design of scaffold-based drug delivery system for anti-cancer therapy.

The design of antimicrobial membranes and thin films are critical for the design of biomaterials that can combat bacterial contamination. Since the long-term use of conventional antibiotics can result in bacterial resistance, there is a critical need to incorporate natural antimicrobial peptides (AMPs) that not only prevent a wide range of pathogens from causing infections but can also promote many beneficial outcomes in wounded tissues. We report the design and antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a naturally occurring human AMP. LL-37 was physically adsorbed and chemically immobilized on the surface of PEMs. The antimicrobial and cytotoxic properties of PEMs were tested with Gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. The ability to prevent bacterial adhesion and to neutralize an E. coli layer was investigated as a function of LL-37 concentration. An interesting trend was that even unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, bacterial adhesion was significantly lower on the surface of the films as well as in the surrounding broth. Immobilizing LL-37 resulted in less than 3% bacterial adhesion on the surface due to the presence of the peptide. LL-37 modified PEMs did not result in any cytotoxicity up to input concentrations of 16 μM. More importantly, urea and albumin secretion by hepatocytes were unaffected even at high LL-37 concentrations. The COL/HA PEMs can serve as antimicrobial coatings, biological membranes and as in vitro platforms to investigate pathogen-tissue interactions. Statement of Significance Antimicrobial peptides (AMPs) are emerging as an alternative to conventional antibiotics. We report the antimicrobial properties of detachable collagen (COL)/hyaluronic acid (HA) polyelectrolyte multilayers (PEMs) modified with LL-37, a human AMP. The antimicrobial and cytotoxic properties were tested with gram-negative Escherichia coli (E. coli, strain DH10B) and primary rat hepatocytes, respectively. Unmodified PEMs exhibited a 40% reduction in bacterial adhesion. When LL-37 was physically adsorbed on PEMs, the sustained release of the active peptide killed planktonic bacteria. Immobilizing LL-37 resulted in less than 3% bacterial adhesion. LL-37 modified PEMs did not result in cytotoxicity up to input concentrations of 16 μM. The COL/HA PEMs can serve as antimicrobial coatings and to investigate pathogen-cell interactions.

Immune signaling within a metastatic niche drives recruitment and colonization, and the subsequent tumor cell fate within the niche. We employed a biomaterial scaffold as a synthetic metastatic niche to dissect the signals driving the responses within the niche relative to the native metastatic niche, which was the lung in these models. For both the natural and the synthetic niche, Gr-1+ cells secreting S100A8/A9 were the major factors contributing to tumor cell recruitment. Interestingly, upon recruitment, tumor cells in the synthetic niche were observed to undergo senescence and were cleared whereas cells within the native niche accumulated and ultimately proliferated to form macrometastases. The synthetic niche has underlying inflammation due to the foreign body responses (FBR) consisting of N1-polarized Gr1+ myeloid cells, M1-polarizedmacrophage, mature dendritic cells and highly cytotoxic CTLs and NK cells, which were not observed in the lung. Furthermore, we found two proteins (osteopontin and decorin) that are associated with developing FBR greatly improve the cytotoxicity of CTLs and NK cells in the lung from stage 3 breast cancers by increasing their production of Perforin/Granzyme, Fas/TRAIL and IFNr/TNFa. Our results highlight the potential of FBR-associated biomolecules to be used as immune-editing anticancer drugs and also inspire the development of therapeutic implants to actively catch and eradicate metastatic cells. Citation Format: Jing Wang, Matthew Hall, Grace Bushnell, Joseph Decker, Sophia Orbach, Ravi Raghani, Yining Zhang, Aaron Morris, Jeffery Ma, Pridvi Kandagatla, Jacqueline Jeruss, Lonnie Shea. Foreign body responses create a highly active anti-tumor environment to recruit and eradicate metastatic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-360.

Background: Current technology limits the detection of metastatic lesions until the patient becomes symptomatic or radiological evidence is identified, and thus invasive cancer cells are not detectable prior to their colonization in distant organs. We have developed a biomaterial implant that serves as a synthetic metastatic niche that recruits tumor cells prior to their accumulation in the lung, a natural metastatic niche. We aimed to dissect the heterogeneity of stromal and immune cells at the synthetic and lung metastatic niches over time, thereby identifying changes in cell populations, phenotypes, and gene expression that could be used for the molecular staging of metastatic disease. Methods: Porous polycaprolactone scaffolds (5 mm diameter x 2 mm height) were implanted subcutaneously into Balb/c mice two weeks before triple negative murine breast cancer cells (4T1) were inoculated into the mammary fat pad. We tracked disease progression in the lung from a pre-metastatic niche (7 days post-inoculation) to micrometastases (14 days) to overt metastatic disease (21 days). Ten genes were selected from the TaqManTM Open Array Mouse Inflammation Panel to develop a PCR-based signature and single value decomposition (SVD) score in the scaffold that successfully delineated metastatic staging. Furthermore, the scaffold and lung were analyzed through single cell RNA-sequencing (Drop-seq) to identify heterogeneity and contributions of each cell type. Results: Ten genes were identified from Open Array of the scaffolds that directly correlated to disease stage in the lungs – S100a8, S100a9, Pglyrp1, Ltf, Camp, Ela2, and Chi3l3 increased with metastatic progression, while Bmp15, Ccl22 and Ccr7 decreased. SVD was applied to establish a single score from the scaffold that could be used for accurate molecular staging. Scaffolds were scored on a scale from 0 to 1, where a value of 0.2 – 0.3 generally correlated to pre-metastatic disease, 0.4 - 0.8 indicated micrometastases and 0.8 – 1.0 demarked metastasis. This model successfully predicted disease stage with an area under the curve of 0.878. When the scaffold-derived gene signature was applied to the lung metastases (21 days), the output was comparable to the scaffold, further demonstrating the physiological relevance of the implants. Single cell analysis indicated that the genes in the signature were primarily expressed by macrophages and neutrophils. After 7 days, 22% of macrophages and 83% of neutrophils were classified as tumor associated cells in the scaffold. In contrast, only 16% of macrophages and 44% of neutrophils in the lung were tumor associated at this time point. These findings support the hypothesis that the scaffolds could be used as early surrogates for the pre-metastatic niche, becoming responsive prior to their biological metastatic counterpart. Moreover, this finding was demonstrated through changes in T cell phenotype, specifically loss of cytotoxic Cd8+ T cells. After 7 days, Cd8+ T cells in the scaffolds decreased by 35%. Whereas Cd8+ T cells in the lungs decreased by 35% after 14 days. Conclusions: A biomaterial scaffold was employed as a synthetic niche that recruits metastatic tumor cells. These scaffolds established a molecular signature of metastatic breast cancer using a 10-gene panel. Additionally, single cell analysis identified the cell types that induce this signature and the disease-associated changes that occur in the scaffold earlier than the lung. In summary, we have shown the potential of a biomaterial scaffold to act as a surrogate metastatic niche that can be used to delineate disease progression and understand the role of specific cell types in the formation of this niche. Mechanistic investigation into the synthetic niche may identify novel therapeutic targets that could be activated to inhibit the advent of metastasis. Citation Format: Sophia M Orbach, Robert S Oakes, Michael D Brooks, Grace G Bushnell, Pridvi Kandagatla, Max S Wicha, Jacqueline S Jeruss, Lonnie D Shea. Early detection of metastatic breast cancer through gene expression and cell heterogeneity at an engineered metastatic niche [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-07.

Pancreatic cancer is a lethal malignancy with a 5-year survival rate of less than 10%. This poor prognosis is, in part, due to patients most often presenting with already metastatic disease. Pancreatic cancer is characterized by an abundant, reactive fibroinflammatory stroma, consisting of cancer-associated fibroblasts (CAFs) and infiltrating immune cells that send and receive signals to and from other nearby cells, including the tumor cells. The crosstalk between tumor cells and elements of the stroma results in an immunosuppressive tumor microenvironment both at the primary tumor and the sites of metastases. A key limitation in studying metastatic disease in mouse models is the inability to predict when the cancer cells will ultimately metastasize. In this study biomaterial scaffolds that mimic the premetastatic niche in vivo, by attracting immune cells followed by tumor cells, were used to longitudinally follow the metastasis process in individual animals. Biomaterial scaffolds were implanted subcutaneously to mimic the premetastatic niche in both syngeneic and spontaneous mouse (iKras* p53*) models of pancreatic cancer. Scaffolds were harvested 21 days post implantation and taken for histologic analysis to characterize the cellular infiltrate in response to the primary tumor. Histologic analysis (H&E, Gomori’s trichrome, and multiplex IHC) shows tumor cell colonization in the scaffold along with a robust immune and fibrotic response. To further characterize the scaffold infiltrate in control versus tumor-bearing mice, mass cytometry (CyTOF) and single-cell RNA sequencing (scRNA seq) analysis was performed. CyTOF analysis revealed the most abundant cell population is heterogenous myeloid cells, which are immunosuppressive in nature. Scaffolds from tumor-bearing animals have an increase in fibroblasts, and subsequently have fewer cytotoxic, CD8+ T cells. scRNA seq analysis of the scaffold infiltrate reveals CD8+ T cells from tumor-bearing animals express higher levels of the immune checkpoint, programmed cell death protein 1 (PD-1) and lower levels of the functional markers, perforin 1 and granzyme B, suggesting the CD8+ T cells are unable to perform their cytotoxic functions. Further analysis revealed a chemokine signaling axis, where fibroblasts recruit distinct subsets of myeloid cells. Taken together, the premetastatic niche (recapitulated by the biomaterial scaffolds) in pancreatic cancer has a high abundance of immunosuppressive myeloid cells, an increase in fibroblasts, and fewer CD8+ T cells, creating an immunosuppressive niche that allows for tumor cell colonization and growth. Future directions include blocking myeloid cell chemotaxis to the scaffold to prevent tumor cell dissemination and reduce metastatic burden. Citation Format: Samantha Kemp, Nina Steele, Eileen Carpenter, Veerin Sirihorachai, Grace Bushnell, Aaron Morris, Carlos Espinoza, Fatima Lima, Zeribe Nwosu, Sophia Orbach, Lonnie Shea, Filip Bednar, Howard Crawford, Marina Pasca di Magliano. Using biomaterial scaffolds to study the genesis of the immunosuppressive premetastatic niche in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A25.

In vivo, macrophages and fibroblasts navigate through and remodel the three-dimensional (3D) extra-cellular matrix (ECM). The orientation of fibers, the porosity, and degree of cross-linking can change the interconnectivity of the ECM and affect cell migration. In turn, migrating cells can alter their microenvironment. To study the relationships between ECM interconnectivity and migration of cells, we assembled collagen hydrogels with dense (DCN) or with loosely interconnected networks (LCN). We find that in DCNs, RAW 264.7 macrophages in monocultures were virtually stationary. In DCN co-cultures, Balb/c 3T3 fibroblasts created tunnels that provided conduits for macrophage migration. In LCNs, fibroblasts aligned fibers up to a distance of 100 μm, which provided tracks for macrophages. Intra-cellular and extra-cellular fluorescent fragments of internalized and degraded collagen were detected inside both cell types as well as around their cell peripheries. Macrophages expressed higher levels of urokinase-type plasminogen activator receptor associated protein (uPARAP)/mannose receptor 1 (CD206) compared to α2β1 indicating that collagen internalization in these cells occurred primarily via integrin-independent mechanisms. Network remodeling indicated by higher Young's modulus was observed in fibroblast monocultures as a result of TGF-β secretion. This work unveils new roles for fibroblasts in forming tunnels in networked ECM to modulate macrophage migration.

Hepatic fibrosis is the result of wound healing and inflammation resulting in organ dysfunction. Hepatocytes, liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs) play critical roles in fibrogenesis. As the liver undergoes fibrosis, there are populations of cells that are healthy, fibrotic as well as those undergoing fibrosis. We investigated how a varying mechanical environment could induce changes in hepatic cells. In this study, a gradient in the mechanical properties of the microenvironment resulted in transitioning phenotypes in hepatic cells. We have designed detachable polyelectrolyte multilayers (PEMs) whose elastic moduli ranged from 21 to 43 kPa to serve as Space of Disse mimics. We assembled novel 3D organotypic liver models comprised of hepatocytes, LSECs, HSCs, KCs, and the Space of Disse mimic. We demonstrate how cells in contact with a mechanical gradient exhibit different properties compared to cells cultured using non-gradient PEMs. Significant differences were observed in HSC and KC proliferation between 3D cultures assembled with gradient and non-gradient PEMs. While HSCs on the stiffer regions of the gradient PEMs expressed both GFAP and α-SMA, cells in cultures assembled with homogeneous 43 kPa multilayers primarily expressed α-SMA. Over an 8-day culture, the elastic modulus in the 21 and 43 kPa regions of the gradient PEMs increased by 1.6 and 3.7-fold, respectively. This was accompanied by a 4-fold increase in hydroxyproline. Such in vitro tissues can be used to investigate the effects of liver fibrosis. Statement of Significance We have assembled a liver model assembled with four major primary hepatic cell types to investigate how a varying mechanical environment induces changes in hepatic cells. In this study, a gradient in the mechanical properties of the microenvironment results in transitioning phenotypes in hepatic cells. Our goal was to investigate the interplay between mechanical properties and a multi-cellular engineered liver tissue. In these models, Kupffer cell proliferation and hepatic stellate cell activation occurred due to mechanical cues and inter-cellular signaling across a distance of 2000 μm. These models are unique, in that, fibrosis was initiated purely through changes to the microenvironment. These models were not exposed to fibrogenic factors nor were the models assembled with cells from fibrotic rats. To the best of our knowledge, these are the first liver models that capture how a gradient microenvironment can result in transitioning cellular phenotypes.

High-throughput screening (HTS) of liver toxicants can bridge the gap in understanding adverse effects of chemicals on humans. Toxicity testing of mixtures is time consuming and expensive, since the number of possible combinations increases exponentially with the number of chemicals. The combination of organotypic culture models (OCMs) and HTS assays can lead to the rapidly evaluation of chemical toxicity in a cost and time-effective manner while prioritizing chemicals that warrant additional investigation. We describe the design, assembly and toxicant response of multi-cellular hepatic organotypic culture models comprised of primary human or rat cells assembled in 96-well plates (denoted as μOCMs). These models were assembled using automated procedures that did not affect hepatocyte function or viability, rendering them ideal for large-scale toxicity evaluations. Rat μOCMs were assembled to obtain insights into deviations from human toxicity. Four test chemicals (acetaminophen, ethanol, isoniazid, and perfluorooctanoic acid) were added to the μOCMs individually or in mixtures. HTS assays were utilized to measure cell death, apoptosis, glutathione depletion, mitochondrial membrane damage, and cytochrome P450 2E1 activity. The μOCMs exhibited increased toxicant sensitivity compared to hepatocyte sandwich cultures. Synergistic and non-synergistic interactions were observed when the toxicants were added as mixtures. Specifically, chemical interactions in the μOCMs were manifested by changes in apoptosis and decreased glutathione. The μOCMs accurately predicted hepatotoxicity for individual and mixtures of toxicants, demonstrating their potential for large-scale toxicity evaluations in the future.

Background Liver models that closely mimic the in vivo microenvironment are useful for understanding liver functions, capabilities, and intercellular communication processes. Three-dimensional (3D) liver models assembled using hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM) provide a functional system while also permitting isolation of individual cell types for proteomic analyses. Methods To better understand the mechanisms and processes that underlie liver model function, hepatocytes were maintained as monolayers and 3D PEM-based formats in the presence or absence of primary LSECs. The resulting hepatocyte proteomes, the proteins in the PEM, and extracellular levels of urea, albumin and glucose after three days of culture were compared. Results All systems were ketogenic and found to release glucose. The presence of the PEM led to increases in proteins associated with both mitochondrial and peroxisomal-based β-oxidation. The PEMs also limited production of structural and migratory proteins associated with dedifferentiation. The presence of LSECs increased levels of Phase I and Phase II biotransformation enzymes as well as several proteins associated with the endoplasmic reticulum and extracellular matrix remodeling. The proteomic analysis of the PEMs indicated that there was no significant change after three days of culture. These results are discussed in relation to liver model function. Conclusions Heterotypic cell-cell and cell-ECM interactions exert different effects on hepatocyte functions and phenotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12953-017-0120-6) contains supplementary material, which is available to authorized users.

In vivo studies clearly demonstrate the participation and subsequent death of non-parenchymal liver cells (NPCs) with corresponding hepatocyte effects. This results in a critical need to investigate how major liver cell types function cohesively during hepatotoxicity. However, virtually no studies replicate these phenomena in vitro. We report the design of multi-cellular three-dimensional (3D) organotypic liver models of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). LSECs and KCs were separated from hepatocytes by a detachable membrane that emulates the physical and chemical properties of the Space of Disse. Acetaminophen (APAP)-induced changes to cellular function and phenotype were investigated. LSECs exhibited approximately 40% cell death at 20mM APAP. KCs exhibited decreased interleukin-10 and increased tumor necrosis factor-alpha and interferon-gamma secretion. The secretion of these proteins altered hepatocyte function and signaling. Both LSECs and KCs maintained phenotypic markers. At 20mM APAP, the 3D models exhibited aspartate aminotransferase to alanine aminotransferase ratios from 2.1-2.5 and 45% glutathione depletion, corresponding to what is seen in vivo. At 10 and 20mM APAP, the 3D models exhibited cell death, primarily through necrosis. Therefore, the 3D cultures described in this report demonstrate significant potential as realistic models for hepatotoxicity studies.