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.

Towards systems tissue engineering: Elucidating the dynamics, spatial coordination, and individual cells driving emergent behaviors.

Biomaterial systems have enabled the in vitro production of complex, emergent tissue behaviors that were not possible with conventional two-dimensional culture systems, allowing for analysis of both normal development and disease processes. We propose that the path towards developing the design parameters for biomaterial systems lies with identifying the molecular drivers of emergent behavior through leveraging technological advances in systems biology, including single cell omics, genetic engineering, and high content imaging. This growing research opportunity at the intersection of the fields of tissue engineering and systems biology - systems tissue engineering - can uniquely interrogate the mechanisms by which complex tissue behaviors emerge with the potential to capture the contribution of i) dynamic regulation of tissue development and dysregulation, ii) single cell heterogeneity and the function of rare cell types, and iii) the spatial distribution and structure of individual cells and cell types within a tissue. By leveraging advances in both biological and materials data science, systems tissue engineering can facilitate the identification of biomaterial design parameters that will accelerate basic science discovery and translation.

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.

Physical confinement induces malignant transformation in mammary epithelial cells.

The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.

Glycation of collagen matrices promotes breast tumor cell invasion.

Cancer metastasis is a physical process in which tumor cells break away from the primary tumor, enter, and then exit the blood or lymph vessels, and establish secondary tumors in distant organs. Current clinical studies report a higher risk of cancer metastasis for diabetics than non-diabetics. However, due to complex overlapping risk factors between diabetes and cancer, the mechanism underlying this correlation is largely unknown. Elevated lifetime blood sugar levels in diabetics are known to increase glycation of collagen, causing stiffening of the ECM and connective tissue. In this study, we explored the roles of glycation of 3D collagen matrices in tumor cell invasion and migration. Using time-lapse images, we quantitatively compared the motility behavior of malignant breast tumor cells (MDA-MB-231) and co-culture spheroids (1:1 ratio of MDA-MB-231 cells with normal epithelial MCF-10A cells) embedded in glycated and non-glycated collagen matrices of various concentrations. Experimental results demonstrated that glycation increased tumor invasion within collagen matrices. More specifically, the average speed of MDA-MB-231 cells was higher in glycated collagen gels than in non-glycated collagen gels for all three gel concentrations tested. Cell spreading characterized by its diffusion coefficient or the effective spheroid radii at various time points was significantly greater in glycated collagen than in non-glycated collagen at a concentration of 3.5 mg/mL. This enhancement was moderate and less evident at lower collagen concentrations of 1.0 and 2.0 mg/mL. These results suggest a possible biomechanical link that relates to the high blood sugar level in diabetic patients and the cancer metastatic outcome.

Design of Large-Scale Reporter Construct Arrays for Dynamic, Live Cell Systems Biology.

Dynamic systems biology aims to identify the molecular mechanisms governing cell fate decisions through the analysis of living cells. Large scale molecular information from living cells can be obtained from reporter constructs that provide activities for either individual transcription factors or multiple factors binding to the full promoter following CRISPR/Cas9 directed insertion of luciferase. In this report, we investigated the design criteria to obtain reporters that are specific and responsive to transcription factor (TF) binding and the integration of TF binding activity with genetic reporter activity. The design of TF reporters was investigated for the impact of consensus binding site spacing sequence and off-target binding on the reporter sensitivity using a library of 25 SMAD3 activity reporters with spacers of random composition and length. A spacer was necessary to quantify activity changes after TGFß stimulation. TF binding site prediction algorithms (BEEML, FIMO and DeepBind) were used to predict off-target binding, and nonresponsiveness to a SMAD3 reporter was correlated with a predicted competitive binding of constitutively active p53. The network of activity of the SMAD3 reporter was inferred from measurements of TF reporter library, and connected with large-scale genetic reporter activity measurements. The integration of TF and genetic reporters identified the major hubs directing responses to TGFß, and this method provided a systems-level algorithm to investigate cell signaling.

Dynamic microRNA activity identifies therapeutic targets in trastuzumab-resistant HER2+ breast cancer.

MicroRNAs (miRNAs) are implicated in numerous physiologic and pathologic processes, such as the development of resistance to chemotherapy. Determining the role of miRNAs in these processes is often accomplished through measuring miRNA abundance by polymerase chain reaction, sequencing, or microarrays. We have developed a system for the large-scale monitoring of dynamic miRNA activity and have applied this system to identify the contribution miRNA activity to the development of trastuzumab resistance in a cell model of HER2+ breast cancer. MiRNA activity measurements identified significantly different activity levels between BT474 cells (HER2 + breast cancer) and BT474R cells (HER2 + breast cancer cells selected for resistance to trastuzumab). We created a library of 32 miRNA reporter constructs, which were delivered by lentiviral transduction into cells, and miRNA activity was quantified by bioluminescence imaging. Upon treatment with the bioimmune therapy, trastuzumab, the activity of 11 miRNAs were significantly altered in parental BT474 cells, and 20 miRNAs had significantly altered activity in the therapy-resistant BT474R cell line. A combination of statistical, network and classification analysis was applied to the dynamic data, which identified miR-21 as a controlling factor in trastuzumab response. Our data suggested downregulation of miR-21 activity was associated with resistance, which was confirmed in an additional HER2 + breast cancer cell line, SKBR3. Collectively, the dynamic miRNA activity measurements and analysis provided a system to identify new potential therapeutic targets in treatment-resistant cancers.

Meta-analysis of targeted small-group reading interventions.

Small-group reading interventions are commonly used in schools but the components that make them effective are still debated or unknown. The current study meta-analyzed 26 small-group reading intervention studies that resulted in 27 effect sizes. Findings suggested a moderate overall effect for small-group reading interventions (weighted g=0.54). Interventions were more effective if they were targeted to a specific skill (g=0.65), then as part of a comprehensive intervention program that addressed multiple skills (g=0.35). There was a small correlation between intervention effects and group size (r=0.21) and duration (r=0.11). Small-group interventions led to a larger median effect size (g=0.64) for elementary-aged students than for those in middle or high school (g=0.20), but the two confidence intervals overlapped. Implications for research and practice are discussed.

Fibrous nonlinear elasticity enables positive mechanical feedback between cells and ECMs.

In native states, animal cells of many types are supported by a fibrous network that forms the main structural component of the ECM. Mechanical interactions between cells and the 3D ECM critically regulate cell function, including growth and migration. However, the physical mechanism that governs the cell interaction with fibrous 3D ECM is still not known. In this article, we present single-cell traction force measurements using breast tumor cells embedded within 3D collagen matrices. We recreate the breast tumor mechanical environment by controlling the microstructure and density of type I collagen matrices. Our results reveal a positive mechanical feedback loop: cells pulling on collagen locally align and stiffen the matrix, and stiffer matrices, in return, promote greater cell force generation and a stiffer cell body. Furthermore, cell force transmission distance increases with the degree of strain-induced fiber alignment and stiffening of the collagen matrices. These findings highlight the importance of the nonlinear elasticity of fibrous matrices in regulating cell-ECM interactions within a 3D context, and the cell force regulation principle that we uncover may contribute to the rapid mechanical tissue stiffening occurring in many diseases, including cancer and fibrosis.

Epidermal growth factor promotes a mesenchymal over an amoeboid motility of MDA-MB-231 cells embedded within a 3D collagen matrix.

The receptor of epidermal growth factor (EGFR) critically regulates tumor cell invasion and is a potent therapeutic target for treatment of many types of cancers, including carcinomas and glioblastomas. It is known that EGF regulates cell motility when tumor cells are embedded within a 3D biomatrix. However, roles of EGF in modulating tumor cell motility phenotype are largely unknown. In this article, we report that EGF promotes a mesenchymal over an amoeboid motility phenotype using a malignant breast tumor cell line, MDA-MB-231, embedded within a 3D collagen matrix. Amoeboid cells are rounded in shape, while mesenchymal cells are elongated, and their migrations are governed by a distinctly different set of biomolecules. Using single cell tracking analysis, we also show that EGF promotes cell dissemination through a significant increase in cell persistence along with a moderate increase of speed. The increase of persistence is correlated with the increase of the percentage of the mesenchymal cells within the population. Our work reveals a novel role of microenvironmental cue, EGF, in modulating heterogeneity and plasticity of tumor cell motility phenotype. In addition, it suggests a potential visual cue for diagnosing invasive states of breast cancer cells. This work can be easily extended beyond breast cancer cells.

An adaptive algorithm for tracking 3D bead displacements: application in biological experiments.

This paper presents a feature-vector-based relaxation method (FVRM) to track bead displacements within a three-dimensional (3D) volume. FVRM merges the feature vector method, a technique used in tracking bead displacements in biological gels, with the relaxation method, an algorithm employed successfully in tracking bead pairs in fluids. More specifically, FVRM evaluates the probability of a bead pairing event based on the quasi-rigidity condition between the feature vectors of a bead and its candidate positions within a searching domain. Computational efficiency is improved via the introduction of an adaptive searching domain size and mismatches are reduced via a two-directional matching strategy. The algorithm is validated using simulated 3D bead displacements caused by a force dipole within a linear elastic gel. Results demonstrate a consistently high recovery ratio (above 98%) and low mismatch ratio (below 0.1%) for tracking parameter (mean bead distance/maximum bead displacement) greater than 0.73.

Toward single cell traction microscopy within 3D collagen matrices.

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels.

Mapping three-dimensional stress and strain fields within a soft hydrogel using a fluorescence microscope.

Three-dimensional cell culture is becoming mainstream as it is recognized that many animal cell types require the biophysical and biochemical cues within the extracellular matrices to perform truly physiologically realistic functions. However, tools for characterizing cellular mechanical environment are largely limited to cell culture plated on a two-dimensional substrate. We present a three-dimensional traction microscopy that is capable of mapping three-dimensional stress and strain within a soft and transparent extracellular matrix using a fluorescence microscope and a simple forward data analysis algorithm. We validated this technique by mapping the strain and stress field within the bulk of a thin polyacrylamide gel layer indented by a millimeter-size glass ball, together with a finite-element analysis. The experimentally measured stress and strain fields are in excellent agreements with results of the finite-element simulation. The unique contributions of the presented three-dimensional traction microscopy technique are: 1), the use of a fluorescence microscope in contrast with the confocal microscope that is required for the current three-dimensional traction microscopes in the literature; 2), the determination of the pressure field of an incompressible gel from strains; and 3), the simple forward-data-analysis algorithm. Future application of this technique for mapping animal cell traction in three-dimensional nonlinear biological gels is discussed.

Peyronie's disease in teenagers.

INTRODUCTION: Peyronie's disease (PD) is commonly seen in middle-aged men, and little is known about this condition in teenagers. AIM: To investigate the characteristics of PD in teenagers. METHODS: The findings were compared between patients with the disease who were teenagers with those over 40 years of age. Statistical analyses were conducted to define differentiating features between these two groups. MAIN OUTCOME MEASURES: The demographics, clinical features, and associated comorbidities of patients with PD were reviewed. RESULTS: Thirty-two teenaged males were evaluated for PD in a single institution over a 10-year period. The median age for our cohort was 18 (15-19) years. Forty-five percent of patients had already been seen by another urologist, and 28% had been told they did not have PD. The mean duration of PD before seeking medical care in our cohort was 3 ± 1 months. Sixteen percent of patients reported antecedent penile trauma, half of which happened during coitus or masturbation, and 18% of patients had hemoglobin (Hb) A1c levels > 5%. Dupuytren's contracture was not seen in this population. Twenty-two percent of patients presented with penile pain. Subsequent ED was seen in 37% of patients. Multiple noncontiguous plaques were seen in 37% of patients. Twelve percent were previously treated with vitamin E, while another 12% had previous intralesional verapamil. High distress was reported by 94% of patients. Thirty-four percent sought medical attention for anxiety/mood disorder, and 28% had a negative encounter with a sexual partner related to PD. All of the 32 patients had penile curvature with a mean of 32 ± 12 degrees. Seventy-two percent of the patients had dorsal curvature while 22% had an associated deformity. Using duplex Doppler ultrasound, 12% had a calcified plaque, while none of the patients had abnormal hemodynamics. When compared with PD in adults, teenagers had greater than seven times the prevalence of multiple noncontiguous plaques (37% vs. 5%). Also, the prevalence of HbA1c level > 5% was higher in the teenagers as well (18% vs. 5%). CONCLUSIONS: PD does occur in teenagers often causing high distress levels. Compared to older adults, teenagers often present earlier, and more commonly have elevated HbA1c level and increased number of plaques at presentation.

Effects of gel thickness on microscopic indentation measurements of gel modulus.

In vitro, animal cells are mostly cultured on a gel substrate. It was recently shown that substrate stiffness affects cellular behaviors in a significant way, including adhesion, differentiation, and migration. Therefore, an accurate method is needed to characterize the modulus of the substrate. In situ microscopic measurements of the gel substrate modulus are based on Hertz contact mechanics, where Young's modulus is derived from the indentation force and displacement measurements. In Hertz theory, the substrate is modeled as a linear elastic half-space with an infinite depth, whereas in practice, the thickness of the substrate, h, can be comparable to the contact radius and other relevant dimensions such as the radius of the indenter or steel ball, R. As a result, measurements based on Hertz theory overestimate the Young's modulus. In this work, we discuss the limitations of Hertz theory and then modify it, taking into consideration the nonlinearity of the material and large deformation using a finite-element method. We present our results in a simple correction factor, ψ, the ratio of the corrected Young's modulus and the Hertz modulus in the parameter regime of δ/h ≤ min (0.6, R/h) and 0.3 ≤R/h ≤ 12.7. The ψ factor depends on two dimensionless parameters, R/h and δ/h (where δ is the indentation depth), both of which are easily accessible to experiments. This correction factor agrees with experimental observations obtained with the use of polyacrylamide gel and a microsphere indentation method in the parameter range of 0.1 ≤δ/h ≤ 0.4 and 0.3 ≤R/h ≤ 6.2. The effect of adhesion on the use of Hertz theory for small indentation depth is also discussed.