Mara Gilardi

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

Significance The cancer biology seed-and-soil paradigm recognizes the existence of organ-specific patterns of metastasization that drive the spread of selected primary tumors toward specific secondary loci. However, despite efforts to model the organotypic microenvironment, the organ specificity of cancer metastases needs to be elucidated. The relevance of this study lies in the generation of a human vascularized organ-specific microenvironment, which can be used to investigate and tune the extravasation process of metastatic tumor cells. Furthermore, beyond mimicking the pro- or antimetastatic signatures of different microenvironments, our microfluidic model provides insights into different properties of organ-specific endothelia. This study paves the way toward advanced in vitro models to screen for highly tailored organ-specific therapeutics and investigate key molecular pathways involved in organ-specific metastases. A key aspect of cancer metastases is the tendency for specific cancer cells to home to defined subsets of secondary organs. Despite these known tendencies, the underlying mechanisms remain poorly understood. Here we develop a microfluidic 3D in vitro model to analyze organ-specific human breast cancer cell extravasation into bone- and muscle-mimicking microenvironments through a microvascular network concentrically wrapped with mural cells. Extravasation rates and microvasculature permeabilities were significantly different in the bone-mimicking microenvironment compared with unconditioned or myoblast containing matrices. Blocking breast cancer cell A3 adenosine receptors resulted in higher extravasation rates of cancer cells into the myoblast-containing matrices compared with untreated cells, suggesting a role for adenosine in reducing extravasation. These results demonstrate the efficacy of our model as a drug screening platform and a promising tool to investigate specific molecular pathways involved in cancer biology, with potential applications to personalized medicine.

A 3D vascularized bone remodeling model combining osteoblasts and osteoclasts in a CaP nanoparticle-enriched matrix

AIM We aimed to establish a 3D vascularized in vitro bone remodeling model. MATERIALS & METHODS Human umbilical endothelial cells (HUVECs), bone marrow mesenchymal stem cells (BMSCs), and osteoblast (OBs) and osteoclast (OCs) precursors were embedded in collagen/fibrin hydrogels enriched with calcium phosphate nanoparticles (CaPn). We assessed vasculogenesis in HUVEC-BMSC coculture, osteogenesis with OBs, osteoclastogenesis with OCs, and, ultimately, cell interplay in tetraculture. RESULTS HUVECs developed a robust microvascular network and BMSCs differentiated into mural cells. Noteworthy, OB and OC differentiation was increased by their reciprocal coculture and by CaPn, and even more by the combination of the tetraculture and CaPn. CONCLUSION We successfully developed a vascularized 3D bone remodeling model, whereby cells interacted and exerted their specific function.

Direct but not indirect co-culture with osteogenically differentiated human bone marrow stromal cells increases RANKL/OPG ratio in human breast cancer cells generating bone metastases

BackgroundBone metastases arise in nearly 70% of patients with advanced breast cancer, but the complex metastatic process has not been completely clarified yet. RANKL/RANK/OPG pathway modifications and the crosstalk between metastatic cells and bone have been indicated as potential drivers of the process. Interactions between tumor and bone cells have been studied in vivo and in vitro, but specific effects of the direct contact between human metastatic cells and human bone cells on RANKL/RANK/OPG pathway have not been investigated.FindingsWe directly co-cultured bone metastatic human breast cancer cells (BOKL) with osteo-differentiated human mesenchymal cells (BMSCs) from 3 different donors. BMSCs and BOKL were then enzymatically separated and FACS sorted. We found a significant increase in the RANKL/OPG ratio as compared to control, which was not observed in BOKL cultured in medium conditioned by BMSCs, neither in BOKL directly cultured with fibroblasts or medium conditioned by fibroblasts. Direct co-culture with osteo-differentiated BMSCs caused BOKL aggregation while proliferation was not affected by co-culture. To more specifically associate RANKL expression to osteogenic differentiation degree of BMSCs, we determined their osteogenic markers expression and matrix calcification relative to osteoblasts and fibroblasts.ConclusionsIn conclusion, our co-culture model allowed to demonstrate for the first time that direct contact but not paracrine interactions between human metastatic breast cancer cells and bone cells has a significant effect on RANKL/OPG expression in bone metastatic cells. Furthermore, only direct contact with the bone microenvironment induced BOKL clustering without however significantly influencing their proliferation and migration.

In Vitro Co-Culture Models of Breast Cancer Metastatic Progression towards Bone

Advanced breast cancer frequently metastasizes to bone through a multistep process involving the detachment of cells from the primary tumor, their intravasation into the bloodstream, adhesion to the endothelium and extravasation into the bone, culminating with the establishment of a vicious cycle causing extensive bone lysis. In recent years, the crosstalk between tumor cells and secondary organs microenvironment is gaining much attention, being indicated as a crucial aspect in all metastatic steps. To investigate the complex interrelation between the tumor and the microenvironment, both in vitro and in vivo models have been exploited. In vitro models have some advantages over in vivo, mainly the possibility to thoroughly dissect in controlled conditions and with only human cells the cellular and molecular mechanisms underlying the metastatic progression. In this article we will review the main results deriving from in vitro co-culture models, describing mechanisms activated in the crosstalk between breast cancer and bone cells which drive the different metastatic steps.

Cardiac Meets Skeletal: What's New in Microfluidic Models for Muscle Tissue Engineering.

In the last few years microfluidics and microfabrication technique principles have been extensively exploited for biomedical applications. In this framework, organs-on-a-chip represent promising tools to reproduce key features of functional tissue units within microscale culture chambers. These systems offer the possibility to investigate the effects of biochemical, mechanical, and electrical stimulations, which are usually applied to enhance the functionality of the engineered tissues. Since the functionality of muscle tissues relies on the 3D organization and on the perfect coupling between electrochemical stimulation and mechanical contraction, great efforts have been devoted to generate biomimetic skeletal and cardiac systems to allow high-throughput pathophysiological studies and drug screening. This review critically analyzes microfluidic platforms that were designed for skeletal and cardiac muscle tissue engineering. Our aim is to highlight which specific features of the engineered systems promoted a typical reorganization of the engineered construct and to discuss how promising design solutions exploited for skeletal muscle models could be applied to improve cardiac tissue models and vice versa.

Bioprinting and Organ-on-Chip Applications Towards Personalized Medicine for Bone Diseases

The skeleton supports and confers structure to the whole body but several pathological and traumatic conditions affect the bone tissue. Most of those pathological conditions are specific and different among different patients, such as bone defects due to traumatic injuries or bone remodeling alterations due to congenital diseases. In this context, the development of personalized therapies would be highly desirable. In recent years the advent of innovative techniques like bioprinting and microfluidic organ-on-chip raised hopes of achieving key tools helping the application of personalized therapies for bone diseases. In this review we will illustrate the latest progresses in the bioprinting of personalized bone grafts and generation of patient-specific bone-on-chip devices, describing current approaches and limitations and possible future improvements for more effective personalized bone grafts and disease models.

PO-12 - The key role of talin-1 in cancer cell extravasation dissected through human vascularized 3D microfluidic model

INTRODUCTION Metastases are responsible for more than 90% of cancer related mortality. The hematogenous metastatic invasion is a complex process in which the endothelium plays a key role. Extravasation is a dynamic process involving remodeling and change in cell shape and in cytoskeleton whereby a series of strongly dependent interactions between CTCs and endothelium occurs [1]. Talins are proteins regulating focal adhesions and cytoskeleton remodeling. Talin-1 seems to be involved in the aggressiveness, motility, survival and invadopodia formation of cancer cells throughout the entire metastatic cascade [2], being up-regulated in breast cancer cells and mutated in sarcomas. Understand the implication of talin-1 in extravasation could facilitate the design of new therapies and finally fight cancer. AIM We hypothesized that Talin-1 could be specifically involved in extravasation driving each of its steps. MATERIALS AND METHODS We developed a human 3D microfluidic model that enables the study of human cancer cell extravasation within a perfusable human microvascularized organ specific environment[3]. For the study of extravasation we applied microfluidic approach through the development of a microfluidic device in which endothelial cells and fibroblasts generated a 3D human functional vascular networks. Microvessel characterization was performed with immunofluorescence and permeability assays. We knocked-down talin-1 in triple negative breast cancer cell line MDA-MB231 and metastatic fibro-sarcoma cell line HT1080 with SiRNA and verified by Western-blot. Cancer cells were then perfused in the vessels and extravasation monitored through confocal imaging. RESULTS We developed a human vascularized 3D microfluidic device with human perfusable capillary-like structures embedded in fibrin matrix, characterized by mature endothelium markers and physiological permeability (1.5±0.76)×10(-6) cm/s. We focused on the role of Talin-1 in adhesion to endothelium, trans-endothelial migration (TEM) and early invasion. Adhesion to the endothelium, TEM and migration within the ECM were monitored through confocal analyses. We demonstrated that Talin-1 KD significantly reduced the adhesion efficiency and TEM in both cell lines. Early invasion was also strongly and statistically reduced by the SiRNA treatment in both cell lines. CONCLUSIONS We proved Talin-1 function in driving the extravasation mechanism in a human 3D vascularized environment. We demonstrated that Talin-1 is involved in each part of extravasation significantly affecting adhesion, TEM and the invasion stages. Targeting this protein could thus be an effective strategy to block metastasis.

Abstract PR07: Dissection of cancer cells extravasation through human vascularized 3D microfluidic model: The major role of talin-1

Cancer cells spread from a primary tumor to secondary loci is responsible for more than 90% of cancer related mortality. Hematogenous metastasis is a complex process [1]. It includes a chain of events that can be summarized as follows: migration from primary tumor site and intravasation of the primary tumor cancer cells into the blood flow, dissemination through the circulation, extravasation in different organs, survival in the new microenvironment and colonization with generation of a new tumor. Recently our group presented a microfluidic 3D model reproducing the effects of the CXCL5-CXCR2 interaction between bone cells and metastatic breast cancer cells observed in vivo [2]. We further developed a human 3D microfluidic model that enables the study of human metastatic breast cancer cell extravasation within a perfusable human microvascularized bone-mimicking microenvironment [3]. Understanding the cellular and molecular events implicated in extravasation could facilitate the design of new therapeutic strategies targeting cancer cells only in order to couple these new developed therapies with already existing treatments and finally fight cancer. Talin-1 is a cytoplasmic adaptor essential for integrin-mediated adhesion to the ECM. Talin-1 links the actin cytoskeleton to integrin at the plasma membrane, regulates the focal adhesion pathway in normal cells and it is up-regulated in triple negative breast cancer cells such as MDA-MB231 and it is mutated in sarcomas. Based on the above described models, we developed a human vascularized 3D microfluidic device where human perfusable capillary like structures were embedded in fibrin matrix, characterized by mature endothelium markers (VE-cadherin, ZO-1) and physiological permeability (1.5±0.76)*10-6cm/s. Since these models can provide quantitative data on cancer cell extravasation, we focused on the role of Talin-1 in extravasation through blood vessels and in early invasion in two cell lines whose Talin-1 expression was silenced by SiRNA: triple negative metastatic breast cancer cells (MDA-MB231) and the metastatic fibro-sarcoma cell line (HT1080). Talin-1 silencing was confirmed by western blotting with specific antibodies. Adhesion to the endothelial wall, extravasation and ensuing migration out from the wall within the extracellular matrix was monitored by means of confocal high resolution real time imaging analyses. We demonstrated that silencing of Talin-1 expression dramatically and in a statistically significant way reduced both the adhesion efficiency and extravasation in both MDA-MB 231 and HT1080. Cell migration was also strongly and statistically reduced by the SiRNA treatment in both analyzed cell lines. In summary, we are the first to dissect the role of Talin-1 in each step of extravasation, demonstrating that targeting this protein could be an effective strategy to block metastasis. These data identify Talin-1 as a promising target for the development of new anti-metastatic therapies based on Talin-1 inhibition. References: [1] Cell 147:275-92 (2011) [2] Biomaterials 35:2454-2461 (2013) [3] Proc Natl Acad Sci U S A 112:214-9 (2015) Citation Format: Mara Gilardi, Simone Bersini, Roger Dale Kamm, Matteo Moretti, Marco Vanoni. Dissection of cancer cells extravasation through human vascularized 3D microfluidic model: The major role of talin-1. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR07.

Abstract 5814: Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration

In extravasation cancer cells and vascular niche are involved in a tight cross-talk which has been defined as the rate-regulating event for metastases establishment [1]. Recent, animal studies support the hypothesis that metastatic deficiency lies in focal adhesion complex alterations, however, it still needs to be elucidated which are the specific regulators of each event composing extravasation. Focal adhesion proteins Talin-1 (TLN-1) and Focal Adhesion Kinase (FAK) are up-regulated in breast cancer. Both targets due to their structural and functional role, dramatically influence cancer mechanotransduction leading to endothelial junction disruption, critical in extravasation process [2]. Here, we generated ad hoc engineered models for each extravasation step allowing single cell behavior analyses through high resolution real time imaging in a reliable and quantitative way in a physiological environment. Through this novel approach we analyzed the effect of TLN-1 and FAK and their genetic and chemical inhibition in breast cancer extravasation. The 3D-microfluidic vasculature displayed maturation markers and physiological permeability (1.5±0.76*10-6cm/s) and allowed cancer cell injection through the hollow vessels. Western blot confirmed TLN-1 and FAK knock down (KD) in MDA-MB231. Both targets significantly affected morphology and proliferation (p Citation Format: Mara Gilardi, Simone Bersini, Rosa Maria Moresco, Marco Vanoni, Roger D. Kamm, Matteo Moretti. Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5814. doi:10.1158/1538-7445.AM2017-5814