An engineered niche delineates metastatic potential of breast cancer.

Metastatic breast cancer is often not diagnosed until secondary tumors have become macroscopically visible and millions of tumor cells have invaded distant tissues. Yet, metastasis is initiated by a cascade of events leading to formation of the pre-metastatic niche, which can precede tumor formation by a matter of years. We aimed to distinguish the potential for metastatic disease from nonmetastatic disease at early times in triple-negative breast cancer using sister cell lines 4T1 (metastatic), 4T07 (invasive, nonmetastatic), and 67NR (nonmetastatic). We used a porous, polycaprolactone scaffold, that serves as an engineered metastatic niche, to identify metastatic disease through the characteristics of the microenvironment. Analysis of the immune cell composition at the scaffold was able to distinguish noninvasive 67NR tumor-bearing mice from 4T07 and 4T1 tumor-bearing mice but could not delineate metastatic potential between the two invasive cell lines. Gene expression in the scaffolds correlated with the up-regulation of cancer hallmarks (e.g., angiogenesis, hypoxia) in the 4T1 mice relative to 4T07 mice. We developed a 9-gene signature (Dhx9, Dusp12, Fth1, Ifitm1, Ndufs1, Pja2, Slc1a3, Soga1, Spon2) that successfully distinguished 4T1 disease from 67NR or 4T07 disease throughout metastatic progression. Furthermore, this signature proved highly effective at distinguishing diseased lungs in publicly available datasets of mouse models of metastatic breast cancer and in human models of lung cancer. The early and accurate detection of metastatic disease that could lead to early treatment has the potential to improve patient outcomes and quality of life.

A synthetic metastatic niche reveals antitumor neutrophils drive breast cancer metastatic dormancy in the lungs.

Biomaterial scaffolds mimicking the environment in metastatic organs can deconstruct complex signals and facilitate the study of cancer progression and metastasis. Here we report that a subcutaneous scaffold implant in mouse models of metastatic breast cancer in female mice recruits lung-tropic circulating tumor cells yet suppresses their growth through potent in situ antitumor immunity. In contrast, the lung, the endogenous metastatic organ for these models, develops lethal metastases in aggressive breast cancer, with less aggressive tumor models developing dormant lungs suppressing tumor growth. Our study reveals multifaceted roles of neutrophils in regulating metastasis. Breast cancer-educated neutrophils infiltrate the scaffold implants and lungs, secreting the same signal to attract lung-tropic circulating tumor cells. Second, antitumor and pro-tumor neutrophils are selectively recruited to the dormant scaffolds and lungs, respectively, responding to distinct groups of chemoattractants to establish activated or suppressive immune environments that direct different fates of cancer cells.

Computational Construction of Toxicant Signaling Networks.

Humans and animals are regularly exposed to compounds that may have adverse effects on health. The Toxicity Forecaster (ToxCast) program was developed to use high throughput screening assays to quickly screen chemicals by measuring their effects on many biological end points. Many of these assays test for effects on cellular receptors and transcription factors (TFs), under the assumption that a toxicant may perturb normal signaling pathways in the cell. We hypothesized that we could reconstruct the intermediate proteins in these pathways that may be directly or indirectly affected by the toxicant, potentially revealing important physiological processes not yet tested for many chemicals. We integrate data from ToxCast with a human protein interactome to build toxicant signaling networks that contain physical and signaling protein interactions that may be affected as a result of toxicant exposure. To build these networks, we developed the EdgeLinker algorithm, which efficiently finds short paths in the interactome that connect the receptors to TFs for each toxicant. We performed multiple evaluations and found evidence suggesting that these signaling networks capture biologically relevant effects of toxicants. To aid in dissemination and interpretation, interactive visualizations of these networks are available at http://graphspace.org.

Myeloid cell reprogramming alleviates immunosuppression and promotes clearance of metastatic lesions.

Suppressive myeloid cells, including monocyte and neutrophil populations, play a vital role in the metastatic cascade and can inhibit the anti-tumor function of cytotoxic T-cells. Cargo-free polymeric nanoparticles (NPs) have been shown to modulate innate immune cell responses in multiple pathologies of aberrant inflammation. Here, we test the hypothesis that the intravenous administration of drug-free NPs in the 4T1 murine model of metastatic triple-negative breast cancer can reduce metastatic colonization of the lungs, the primary metastatic site, by targeting the pro-tumor immune cell mediators of metastatic progression. In vivo studies demonstrated that NP administration reprograms the immune milieu of the lungs and reduces pulmonary metastases. Single-cell RNA sequencing of the lungs revealed that intravenous NP administration alters myeloid cell phenotype and function, skewing populations toward inflammatory, anti-tumor phenotypes and away from pro-tumor phenotypes. Monocytes, neutrophils, and dendritic cells in the lungs of NP-treated mice upregulate gene pathways associated with IFN signaling, TNF signaling, and antigen presentation. In a T-cell deficient model, NP administration failed to abrogate pulmonary metastases, implicating the vital role of T-cells in the NP-mediated reduction of metastases. NPs delivered as an adjuvant therapy, following surgical resection of the primary tumor, led to clearance of established pulmonary metastases in all treated mice. Collectively, these results demonstrate that the in vivo administration of cargo-free NPs reprograms myeloid cell responses at the lungs and promotes the clearance of pulmonary metastases in a method of action dependent on functional T-cells.

Biodegradable nanoparticles induce cGAS/STING-dependent reprogramming of myeloid cells to promote tumor immunotherapy.

Cancer treatment utilizing infusion therapies to enhance the patient's own immune response against the tumor have shown significant functionality in a small subpopulation of patients. Additionally, advances have been made in the utilization of nanotechnology for the treatment of disease. We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

Single-cell RNA-sequencing identifies anti-cancer immune phenotypes in the early lung metastatic niche during breast cancer.

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.

Pancreatic cancer is marked by complement-high blood monocytes and tumor-associated macrophages.

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.

Disease-induced immunomodulation at biomaterial scaffolds detects early pancreatic cancer in a spontaneous model.

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.

Engineered Niches to Analyze Mechanisms of Metastasis and Guide Precision Medicine.

Cancer metastasis poses a challenging problem both clinically and scientifically, as the stochastic nature of metastatic lesion formation introduces complexity for both early detection and the study of metastasis in preclinical models. Engineered metastatic niches represent an emerging approach to address this stochasticity by creating bioengineered sites where cancer can preferentially metastasize. As the engineered niche captures the earliest metastatic cells at a nonvital location, both noninvasive and biopsy-based monitoring of these sites can be performed routinely to detect metastasis early and monitor alterations in the forming metastatic niche. The engineered metastatic niche also provides a new platform technology that serves as a tunable site to molecularly dissect metastatic disease mechanisms. Ultimately, linking the engineered niches with advances in sensor development and synthetic biology can provide enabling tools for preclinical cancer models and fosters the potential to impact the future of clinical cancer care.

Metastatic Conditioning of Myeloid Cells at a Subcutaneous Synthetic Niche Reflects Disease Progression and Predicts Therapeutic Outcomes.

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. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/602/F1.large.jpg.See related commentary by Wolf and Elisseeff, p. 377.

Fibroblasts stimulate macrophage migration in interconnected extracellular matrices through tunnel formation and fiber alignment.

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 α2ß1 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-ß secretion. This work unveils new roles for fibroblasts in forming tunnels in networked ECM to modulate macrophage migration.

Multi-cellular transitional organotypic models to investigate liver fibrosis.

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 toxicity testing of chemicals and mixtures in organotypic multi-cellular cultures of primary human hepatic cells.

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.

Investigating acetaminophen hepatotoxicity in multi-cellular organotypic liver models.

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.

In Vitro Intestinal and Liver Models for Toxicity Testing.

The human body is exposed to hundreds of chemicals every day. Many of these toxicants have unknown effects on the body that can be deleterious. Furthermore, chemicals can have a synergistic effect, resulting in toxic responses of cocktails at relatively low individual exposure levels. The gastrointestinal (GI) tract and the liver are the first organs to be exposed to ingested pharmaceuticals and environmental chemicals. As a result, these organs often experience extensive damage from xenobiotics and their metabolites. In vitro models offer a promising method for testing toxic effects. Many advanced in vitro models have been developed for GI and liver toxicity. These models strive to recapitulate the in vivo organ architecture to more accurately model chemical toxicity. In this review, we discuss many of these advances, in addition to recent efforts to integrate the GI and the liver in vitro for a more holistic toxicity model.

The design of antimicrobial LL37-modified collagen-hyaluronic acid detachable multilayers.

UNLABELLED: 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 16µM. 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 16µM. The COL/HA PEMs can serve as antimicrobial coatings and to investigate pathogen-cell interactions.

The hepatocyte proteome in organotypic rat liver models and the influence of the local microenvironment.

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 ß-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.