Astrocytic laminin-211 drives disseminated breast tumor cell dormancy in brain.

Although dormancy is thought to play a key role in the metastasis of breast tumor cells to the brain, our knowledge of the molecular mechanisms regulating disseminated tumor cell (DTC) dormancy in this organ is limited. Here using serial intravital imaging of dormant and metastatic triple-negative breast cancer lines, we identify escape from the single-cell or micrometastatic state as the rate-limiting step towards brain metastasis. We show that every DTC occupies a vascular niche, with quiescent DTCs residing on astrocyte endfeet. At these sites, astrocyte-deposited laminin-211 drives DTC quiescence by inducing the dystroglycan receptor to associate with yes-associated protein, thereby sequestering it from the nucleus and preventing its prometastatic functions. These findings identify a brain-specific mechanism of DTC dormancy and highlight the need for a more thorough understanding of tumor dormancy to develop therapeutic approaches that prevent brain metastasis.

The effect of normal, metaplastic, and neoplastic esophageal extracellular matrix upon macrophage activation.

INTRODUCTION: Macrophages are capable of extreme plasticity and their activation state has been strongly associated with solid tumor growth progression and regression. Although the macrophage response to extracellular matrix (ECM) isolated from normal tissue is reasonably well understood, there is a relative dearth of information regarding their response to ECM isolated from chronically inflamed tissues, pre-neoplastic tissues, and neoplastic tissues. Esophageal adenocarcinoma (EAC) is a type of neoplasia driven by chronic inflammation in the distal esophagus, and the length of the esophagus provides the opportunity to investigate macrophage behavior in the presence of ECM isolated from a range of disease states within the same organ. METHODS: Normal, metaplastic, and neoplastic ECM hydrogels were prepared from decellularized EAC tissue. The hydrogels were evaluated for their nanofibrous structure (SEM), biochemical profile (targeted and global proteomics), and direct effect upon macrophage (THP-1 cell) activation state (qPCR, ELISA, immunolabeling) and indirect effect upon epithelial cell (Het-1A) migration (Boyden chamber). RESULTS: Nanofibrous ECM hydrogels from the three tissue types could be formed, and normal and neoplastic ECM showed distinctive protein profiles by targeted and global mass spectroscopy. ECM proteins functionally related to cancer and tumorigenesis were identified in the neoplastic esophageal ECM including collagen alpha-1(VIII) chain (COL8A1), lumican, and elastin. Metaplastic and neoplastic esophageal ECM induce distinctive effects upon THP-1 macrophage signaling compared to normal esophageal ECM. These effects include activation of pro-inflammatory IFNγ and TNFα gene expression and anti-inflammatory IL1RN gene expression. Most notably, neoplastic ECM robustly increased macrophage TNFα protein expression. The secretome of macrophages pre-treated with metaplastic and neoplastic ECM increases the migration of normal esophageal epithelial cells, similar behavior to that shown by tumor cells. Metaplastic ECM shows similar but less pronounced effects than neoplastic ECM suggesting the abnormal signals also exist within the pre-cancerous state. CONCLUSION: A progressively diseased ECM, as exists within the esophagus exposed to chronic gastric reflux, can provide insights into novel biomarkers of early disease and identify potential therapeutic targets.

Esophageal extracellular matrix hydrogel mitigates metaplastic change in a dog model of Barrett's esophagus.

Chronic inflammatory gastric reflux alters the esophageal microenvironment and induces metaplastic transformation of the epithelium, a precancerous condition termed Barrett's esophagus (BE). The microenvironmental niche, which includes the extracellular matrix (ECM), substantially influences cell phenotype. ECM harvested from normal porcine esophageal mucosa (eECM) was formulated as a mucoadhesive hydrogel, and shown to largely retain basement membrane and matrix-cell adhesion proteins. Dogs with BE were treated orally with eECM hydrogel and omeprazole (n = 6) or omeprazole alone (n = 2) for 30 days. eECM treatment resolved esophagitis, reverted metaplasia to a normal, squamous epithelium in four of six animals, and downregulated the pro-inflammatory tumor necrosis factor-α+ cell infiltrate compared to control animals. The metaplastic tissue in control animals (n = 2) did not regress. The results suggest that in vivo alteration of the microenvironment with a site-appropriate, mucoadhesive ECM hydrogel can mitigate the inflammatory and metaplastic response in a dog model of BE.

Ultrasonic cavitation to prepare ECM hydrogels.

Hydrogels composed of extracellular matrix (ECM) have been used as a substrate for 3D organoid culture, and in numerous preclinical and clinical applications to facilitate repair and reconstruction of a variety of tissues. However, these ECM hydrogel materials are fabricated using lengthy methods that have focused on enzymatic digestion of the ECM with an acid protease in an acidic solution; or the use of chaotropic extraction buffers and dialysis procedures which can affect native protein structure and function. Herein we report a method to prepare hydrogels from ECM bioscaffolds using ultrasonic cavitation. The solubilized ECM can be induced to rapidly self-assemble into a gel by adjusting temperature, and the material properties of the gel can be tailored by adjusting ECM concentration and sonication parameters. The present study shows that ECM bioscaffolds can be successfully solubilized without enzymatic digestion and induced to repolymerize into a gel form capable of supporting cell growth. STATEMENT OF SIGNIFICANCE: ECM hydrogels have been used in numerous preclinical studies to facilitate repair of tissue following injury. However, there has been relatively little advancement in manufacturing techniques, thereby impeding progress in advancing this technology toward the clinic. Laboratory techniques for producing ECM hydrogels have focused on protease digestion methods, which require lengthy incubation times. The significance of this work lies in the development of a fundamentally different approach whereby an ECM hydrogel is rapidly formed without the need for acidic solutions or protease digestion. The ultrasonic cavitation method described herein represents a marked improvement in rheological properties and processing time over traditional enzymatic methods, and may lend itself as a platform for large-scale manufacturing of ECM hydrogels.

Extracellular Matrix Degradation Products Downregulate Neoplastic Esophageal Cell Phenotype.

IMPACT STATEMENT: Extracellular matrix (ECM) biomaterials were used to treat esophageal cancer patients after cancer resection and promoted regrowth of normal mucosa without recurrence of cancer. The present study investigates the mechanisms by which these materials were successful to prevent the cancerous phenotype. ECM downregulated neoplastic esophageal cell function (proliferation, metabolism), but normal esophageal epithelial cells were unaffected in vitro, and suggests a molecular basis (downregulation of PI3K-Akt, cell cycle) for the promising clinical results. The therapeutic effect appeared to be enhanced using homologous esophageal ECM. This study suggests that ECM can be further investigated to treat cancer patients after resection or in combination with targeted therapy.

The impact of detergents on the tissue decellularization process: A ToF-SIMS study.

Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy. STATEMENT OF SIGNIFICANCE: We report here on the use of a highly sensitive analytical technique, time of flight secondary ion mass spectroscopy (ToF-SIMS) to characterize detergent decellularized scaffolds. ToF-SIMS detected cellular remnants and residual detergent fragments; increased intensity of the detergent fragments correlated with adverse cell matrix interactions. This study demonstrates the importance of maintaining a balance between cell removal and detergent disruption of matrix architecture and matrix surface ligand landscape. This study also demonstrates the power of ToF-SIMS for the characterization of decellularized scaffolds and capability for assessment of decellularization efficacy. Future use of biologic scaffolds in clinical tissue reconstruction will benefit from the fundamental results described in this work.

Extracellular matrix hydrogels from decellularized tissues: Structure and function.

Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discussed. STATEMENT OF SIGNIFICANCE: More than 70 papers have been published on extracellular matrix (ECM) hydrogels created from source tissue in almost every organ system. The present manuscript represents a review of ECM hydrogels and attempts to identify structure-function relationships that influence the tissue remodeling outcomes and gaps in the understanding thereof. There is a Phase 1 clinical trial now in progress for an ECM hydrogel.

Primary tumor microRNA signature predicts recurrence and survival in patients with locally advanced esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is an aggressive cancer necessitating the development of improved risk stratification tools for personalized care. Previously, microRNAs have been shown to correlate with the progression and prognosis of various cancer types; however, the value in EAC remains largely unexplored. We performed global microRNA profiling on 32 formalin-fixed, paraffin-embedded EAC specimens to identify microRNAs associated with progression. Literature search and pathway analysis further refined output to five significantly deregulated candidate biomarkers. Four of the five microRNAs (miR-652-5p, miR-7-2-3p, miR-3925-3p, and miR-219-3p) were validated by qRT-PCR. Survival outcomes were evaluated in testing set of 26 stage II/III EAC patients to determine the prognostic relevance of the selected microRNAs. In the testing set, miR-652-5p and miR-7-2-3p expressions were significantly associated with progression-free survival (p-value = .00771 and p-value = .00293). The highest area under receiver operating characteristic (ROC) curve was 0.8212 for the combination of miR-652-5p and miR-7-2-3p. Collectively, our findings demonstrated that the miR-652-5p/miR-7-2-3p signature may serve as a promising prognostic marker in patients with locally advanced EAC.

Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity.

Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar tissue formation in many anatomical sites. However, the optimal shape and form of ECM scaffold for each clinical application can vary markedly. ECM hydrogels have been shown to promote chemotaxis and differentiation of neuronal stem cells, but minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form. These ECM materials can be manufactured to exist in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. Implantation into the lesion cavity after a stroke could hence provide a means to support endogenous repair mechanisms. Herein, we characterize the rheological properties of an ECM hydrogel composed of urinary bladder matrix (UBM) that influence its delivery and in vivo interaction with host tissue. There was a notable concentration-dependence in viscosity, stiffness, and elasticity; all characteristics important for minimally invasive intracerebral delivery. An efficient MRI-guided injection with drainage of fluid from the cavity is described to assess in situ hydrogel formation and ECM retention at different concentrations (0, 1, 2, 3, 4, and 8mg/mL). Only ECM concentrations >3mg/mL gelled within the stroke cavity. Lower concentrations were not retained within the cavity, but extensive permeation of the liquid phase ECM into the peri-infarct area was evident. The concentration of ECM hydrogel is hence an important factor affecting gelation, host-biomaterial interface, as well intra-lesion distribution. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) hydrogel promotes constructive tissue remodeling in many tissues. Minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form that exists in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. We here report the rheological characterization of an injectable ECM hydrogel and its concentration-dependent delivery into a lesion cavity formed after a stroke based on MRI-guidance. The concentration of ECM determined its retention within the cavity or permeation into tissue and hence influenced its interaction with the host brain. This study demonstrates the importance of understanding the structure-function relationship of biomaterials to guide particular clinical applications.

Tissue-Specific Effects of Esophageal Extracellular Matrix.

Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified.

MicroRNA signature characterizes primary tumors that metastasize in an esophageal adenocarcinoma rat model.

OBJECTIVE: To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC). BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies. METHODS: The modified Levrat's surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA) along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5) and metastasis negative (n=28) primary tumors. RESULTS: The epithelial origin of distant metastasis was established by IF using villin (VIL1) and mucin 5AC (MUC5AC) antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001), miR-141-3p (p=0.0022), miR-451-1a (p=0.0181) and miR133a-3p (p=0.0304). Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2. CONCLUSION: In vivo metastasis was confirmed in the modified Levrat's model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.