c-Met activation promotes extravasation of hepatocellular carcinoma cells into 3D-cultured hepatocyte cells in lab-on-a-chip device.

Activation of c-Met signaling is associated with an aggressive phenotype and poor prognosis in hepatocellular carcinoma (HCC); however, its contribution to organ preference in metastasis remains unclear. In this study, using a Lab on a Chip device, we defined the role of aberrant c-Met activation in regulating the extravasation and homing capacity of HCC cells. Our studies showed that (i) c-Met overexpression and activation direct HCC cells preferentially towards the hepatocytes-enriched microenvironment, and (ii) blockage of c-Met phosphorylation by a small molecule inhibitor attenuated extravasation and homing capacity of HCC cells. These results, thus, demonstrate the role of c-Met signaling in regulating the colonization of HCC cells preferentially in the liver.

FLI1 and FRA1 transcription factors drive the transcriptional regulatory networks characterizing muscle invasive bladder cancer.

Bladder cancer is mostly present in the form of urothelium carcinoma, causing over 150,000 deaths each year. Its histopathological classification as muscle invasive (MIBC) and non-muscle invasive (NMIBC) is the most prominent aspect, affecting the prognosis and progression of this disease. In this study, we defined the active regulatory landscape of MIBC and NMIBC cell lines using H3K27ac ChIP-seq and used an integrative approach to combine our findings with existing data. Our analysis revealed FRA1 and FLI1 as two critical transcription factors differentially regulating MIBC regulatory landscape. We show that FRA1 and FLI1 regulate the genes involved in epithelial cell migration and cell junction organization. Knock-down of FRA1 and FLI1 in MIBC revealed the downregulation of several EMT-related genes such as MAP4K4 and FLOT1. Further, ChIP-SICAP performed for FRA1 and FLI1 enabled us to infer chromatin binding partners of these transcription factors and link this information with their target genes. Finally, we show that knock-down of FRA1 and FLI1 result in significant reduction of invasion capacity of MIBC cells towards muscle microenvironment using IC-CHIP assays. Our results collectively highlight the role of these transcription factors in selection and design of targeted options for treatment of MIBC.

Cancer Stem Cells in Tumor Modeling: Challenges and Future Directions.

Microfluidic tumors-on-chips models have revolutionized anticancer therapeutic research by creating an ideal microenvironment for cancer cells. The tumor microenvironment (TME) includes various cell types and cancer stem cells (CSCs), which are postulated to regulate the growth, invasion, and migratory behavior of tumor cells. In this review, the biological niches of the TME and cancer cell behavior focusing on the behavior of CSCs are summarized. Conventional cancer models such as three-dimensional cultures and organoid models are reviewed. Opportunities for the incorporation of CSCs with tumors-on-chips are then discussed for creating tumor invasion models. Such models will represent a paradigm shift in the cancer community by allowing oncologists and clinicians to predict better which cancer patients will benefit from chemotherapy treatments.

On-chip determination of tissue-specific metastatic potential of breast cancer cells.

Metastasis is one of the major obstacles for breast cancer patients. Limitations of current models demand the development of custom platforms to predict metastatic potential and homing choices of cancer cells. Here, two organ-on-chip platforms, invasion/chemotaxis (IC-chip) and extravasation (EX-chip) were used for the quantitative assessment of invasion and extravasation towards specific tissues. Lung, liver and breast microenvironments were simulated in the chips using tissue-specific cells embedded in matrigel. In the IC-chip, invasive MDA-MB-231, but not noninvasive MCF-7 breast cancer cells invaded into lung and liver microenvironments. In the EX-chip, MDA-MB-231 cells extravasated more into the lung compared to the liver and breast microenvironments. In addition, lung-specific MDA-MB-231 clone invaded and extravasated into the lung microenvironment more efficiently than the bone-specific clone. Both invasion/chemotaxis and extravasation results were in agreement with published clinical data. Collectively, our results show that IC-chip and EX-chip, simulating tissue-specific microenvironments, can distinguish different in vivo metastatic phenotypes, in vitro. Determination of tissue-specific metastatic potential of breast cancer cells is expected to improve diagnosis and help select the ideal therapy.

Breast cancer cells and macrophages in a paracrine-juxtacrine loop.

Breast cancer cells (BCC) and macrophages are known to interact via epidermal growth factor (EGF) produced by macrophages and colony stimulating factor-1 (CSF-1) produced by BCC. Despite contradictory findings, this interaction is perceived as a paracrine loop. Further, the underlying mechanism of interaction remains unclear. Here, we investigated interactions of BCC with macrophages in 2D and 3D. While both BCC and macrophages showed invasion/chemotaxis to fetal bovine serum, only macrophages showed chemotaxis to BCC in custom designed 3D cell-on-a-chip devices. These results were in agreement with gradient simulation results and ELISA results showing that macrophage-derived-EGF was not secreted into macrophage-conditioned-medium. Live cell imaging of BCC in the presence and absence of iressa showed that macrophages but not macrophage-derived-matrix modulated adhesion and motility of BCC in 2D. 3D co-culture experiments in collagen and matrigel showed that BCC changed their multicellular organization in the presence of macrophages. In custom designed 3D co-culture cell-on-a-chip devices, macrophages promoted and reduced migration of BCC in collagen and matrigel, respectively. Furthermore, adherent but not suspended BCC endocytosed EGFR when in contact with macrophages. Collectively, our data revealed that macrophages showed chemotaxis towards BCC whereas BCC required direct contact to interact with macrophage-derived-EGF. Therefore, we propose that the interaction between cancer cells and macrophages is a paracrine-juxtacrine loop of CSF-1 and EGF, respectively.

A new drug testing platform based on 3D tri-culture in lab-on-a-chip devices.

Drug discovery has a 90% rate of failure because preclinical platforms for drug testing do not mimic the in vivo conditions. Doxorubicin (DOX) is a commonly used drug to treat breast cancer patients even though it has side effects. Lab-on-a-chip (LOC) devices provide spatial control at the micrometer scale and can thus emulate the cancer microenvironment. Here, using a multidisciplinary approach, a new drug testing platform based on 3D tri-culture in LOC devices was developed. Breast cancer cells alone or with normal mammary epithelial cells and macrophages were cultured in matrigel in LOC devices. The platform was used to test DOX and (R)-4'-methylklavuzon (KLA), which is a new anti-cancer drug candidate. Results showed that DOX and KLA were equally effective on breast cancer cells in 3D monoculture. KLA produced 26% less death for breast cancer cells than DOX in 3D tri-culture. More importantly, DOX was not selective between breast cancer cells and normal mammary epithelial cells in 3D tri- culture whereas KLA caused 56% less cell death than DOX for normal mammary epithelial cells. Results strongly recommend that 3D tri-culture in LOC devices be used for assessment of drug toxicity at the preclinical stage.

Cellular distribution of invadopodia is regulated by nanometer scale surface protein patterns.

Invadopodia are proteolytic structures formed by cancer cells. It is not known whether their cellular distribution can be regulated by the organization of the extracellular matrix or the organization of the golgi complex or whether they have an adhesion requirement. Here, we used electron beam lithography to fabricate fibronectin (FN) nanodots with isotropic and gradient micrometer scale spacings on K-casein and laminin backgrounds. Investigating cancer cells cultured on protein nanopatterns, we showed that (i) presence of FN nanodots on a K-casein background decreased percent of cells with neutral invadopodia polarization compared to FN control surfaces; (ii) presence of a gradient of FN nanodots on a K-casein background increased percent of cells with negative invadopodia polarization compared to FN control surfaces; (iii) polarization of the golgi complex was similar to that of invadopodia in agreement with a spatial link; (iv) local adhesion did not necessarily appear to be a prerequisite for invadopodia formation.

Differentiation of Normal and Cancer Cell Adhesion on Custom Designed Protein Nanopatterns.

Cell adhesion to the extracellular matrix is deregulated in metastasis. However, traditional surfaces used to study cell adhesion do not faithfully mimic the in vivo microenvironment. Electron beam lithography (EBL) is able to generate customized protein nanopatterns. Here, we used an EBL-based green lithography approach to fabricate homogeneous and gradient, single (fibronectin, K-casein) and double (fibronectin, laminin) active component protein nanopatterns with micrometer scale spacing to investigate differences in adhesion of breast cancer cells (BCC) and normal mammary epithelial cells (NMEC). Our results showed that as expected, in contrast to NMEC, BCC were plastic: they tolerated nonadhesion promoting regions, adapted to flow and exploited gradients better. In addition, the number of focal adhesions but not their area appeared to be the dominant parameter for regulation of cell adhesion. Our findings also demonstrated that custom designed protein nanopatterns, which can properly mimic the in vivo microenvironment, enable realistic distinction of normal and cancerous cell adhesion.

Step-by-step quantitative analysis of focal adhesions.

Focal adhesions (FAs) are specialized adhesive structures which serve as cellular communication units between cells and the surrounding extracellular matrix. FAs are involved in signal transduction and actin cytoskeleton organization. FAs mediate cell adhesion, which is a critical phenomenon in cancer research. Since cells can form many and micrometer scale FAs, their quantitative analysis demands well-optimized image analysis approaches [1-3]. Here, we have optimized the analysis of FAs of MDA-MB-231 breast cancer cells. The optimization is based on proper processing of immunofluorescence images of vinculin, which is one of the markers of FAs. All image processing steps are carried out using the ImageJ software, which is freely available and in the public domain. The advantages of our method are:•The analysis steps are simplified by combining different plugins of the ImageJ program.•FAs are better detected with minimal false negatives due to optimized processing of fluorescent images.•This approach can be applied to quantify a variety of fluorescent images comprising focal and/or localized signals within a high background such as FAs, one of the many complex signaling structures in a cell.

Fabrication of 3D Controlled in vitro Microenvironments.

Microfluidics-based lab-on-a-chips have many advantages, one of which is to provide physiologically relevant settings for cell biology experiments. Thus there is an ever increasing interest in their fabrication. Our goal is to construct three dimensional (3D) Controlled in vitro Microenvironments (CivMs) that mimic the in vivo microenvironments. Here, we present our optimized fabrication method that works for various lab-on-a-chip designs with a wide range of dimensions. The most crucial points are:•While using one type of SU-8 photoresist (SU-2075), fine tuning of ramp, dwell time, spin speed, durations of soft bake, UV exposure and development allows fabrication of SU-8 masters with various heights from 40 to 600 µm.•Molding PDMS (polydimethylsiloxane) at room temperature for at least two days instead of baking at higher temperatures prevents not only tears and bubbles in PDMS stamps but also cracks in the SU-8 master.•3D nature of the CivMs is ensured by keeping the devices inverted during gel polymerization.