Assembly and disassembly dynamics of nonmuscle myosin II control endosomal fission.

Endocytic vesicular trafficking requires merging of two lipid bilayers, but how the two lipid bilayers can come close together during fusion and fission in endocytic trafficking is not well explored. Here, we establish that knocking down nonmuscle myosin IIs (NM IIs) by small interfering RNA (siRNA) or inhibition of their activities by (-) blebbistatin causes the formation of a ring-like assembly of early endosomes (raEE). Inhibition of NM II assembly by an inhibitor of regulatory light-chain (RLC) kinase results in the formation of raEE, whereas inhibition of NM II disassembly by inhibitors of heavy chain kinases, protein kinase C (PKC) and casein kinase 2 (CK2), causes the dispersion of early endosomes. The raEEs retain EEA1, Rab7, and LAMP2 markers. Overexpression of an assembly incompetent form, RLC-AA, and disassembly incompetent form, NMHCIIB-S6A or NMHCIIA-1916A, induces such defects, respectively. Altogether, these data support that NM II assembly and disassembly dynamics participate in endocytic trafficking by regulating fission to maintain the size of early endosomes.

Label-free discrimination of tumorigenesis stages using in vitro prostate cancer bone metastasis model by Raman imaging.

Metastatic prostate cancer colonizes the bone to pave the way for bone metastasis, leading to skeletal complications associated with poor prognosis and morbidity. This study demonstrates the feasibility of Raman imaging to differentiate between cancer cells at different stages of tumorigenesis using a nanoclay-based three-dimensional (3D) bone mimetic in vitro model that mimics prostate cancer bone metastasis. A comprehensive study comparing the classification of as received prostate cancer cells in a two-dimensional (2D) model and cancer cells in a 3D bone mimetic environment was performed over various time intervals using principal component analysis (PCA). Our results showed distinctive spectral differences in Raman imaging between prostate cancer cells and the cells cultured in 3D bone mimetic scaffolds, particularly at 1002, 1261, 1444, and 1654 cm-1, which primarily contain proteins and lipids signals. Raman maps capture sub-cellular responses with the progression of tumor cells into metastasis. Raman feature extraction via cluster analysis allows for the identification of specific cellular constituents in the images. For the first time, this work demonstrates a promising potential of Raman imaging, PCA, and cluster analysis to discriminate between cancer cells at different stages of metastatic tumorigenesis.

Copper dependent ERK1/2 phosphorylation is essential for the viability of neurons and not glia.

Intracellular copper [Cu(I)] has been hypothesized to play role in the differentiation of the neurons. This necessitates understanding the role of Cu(I) not only in the neurons but also in the glia considering their anatomical proximity, contribution towards ion homeostasis, and neurodegeneration. In this study, we did a systematic investigation of the changes in the cellular copper homeostasis during neuronal and glial differentiation and the pathways triggered by them. Our study demonstrates increased mRNA for the plasma membrane copper transporter CTR1 leading to increased Cu(I) during the neuronal (PC-12) differentiation. ATP7A is retained in the trans-Golgi network (TGN) despite high Cu(I) demonstrating its utilization towards the neuronal differentiation. Intracellular copper triggers pathways essential for neurite generation and ERK1/2 activation during the neuronal differentiation. ERK1/2 activation also accompanies the differentiation of the foetal brain derived neuronal progenitor cells. The study demonstrates that ERK1/2 phosphorylation is essential for the viability of the neurons. In contrast, differentiated C-6 (glia) cells contain low intracellular copper and significant downregulation of the ERK1/2 phosphorylation demonstrating that ERK1/2 activation does not regulate the viability of the glia. But ATP7A shows vesicular localization despite low copper in the glia. In addition to the TGN, ATP7A localizes into RAB11 positive recycling endosomes in the glial neurites. Our study demonstrates the role of copper dependent ERK1/2 phosphorylation in the neuronal viability. Whereas glial differentiation largely involves sequestration of Cu(I) into the endosomes potentially (i) for ready release and (ii) rendering cytosolic copper unavailable for pathways like the ERK1/2 activation.

Nanostructured Biomaterials for In Vitro Models of Bone Metastasis Cancer.

In recent years, tissue engineering approaches have attracted substantial attention owing to their ability to create physiologically relevant in vitro disease models that closely mimic in vivo conditions. Here, we review nanocomposite materials and scaffolds used for the design of in vitro models of cancer, including metastatic sites. We discuss the role of material properties in modulating cellular phenotype in 3D disease models. Also, we highlight the application of tissue-engineered bone as a tool for faithful recapitulation of the microenvironment of metastatic prostate and breast cancer, since these two types of cancer have the propensity to metastasize to bone. Overall, we summarize recent efforts on developing 3D in vitro models of bone metastatic cancers that provide a platform to study tumor progression and facilitate high-throughput drug screening.

Evaluation of quasi-static and dynamic nanomechanical properties of bone-metastatic breast cancer cells using a nanoclay cancer testbed.

In recent years, there has been increasing interest in investigating the mechanical properties of individual cells to delineate disease mechanisms. Reorganization of cytoskeleton facilitates the colonization of metastatic breast cancer at bone marrow space, leading to bone metastasis. Here, we report evaluation of mechanical properties of two breast cancer cells with different metastatic ability at the site of bone metastases, using quasi-static and dynamic nanoindentation methods. Our results showed that the significant reduction in elastic modulus along with increased liquid-like behavior of bone metastasized MCF-7 cells was induced by depolymerization and reorganization of F-actin to the adherens junctions, whereas bone metastasized MDA-MB-231 cells showed insignificant changes in elastic modulus and F-actin reorganization over time, compared to their respective as-received counterparts. Taken together, our data demonstrate evolution of breast cancer cell mechanics at bone metastases.

Perfusion bioreactor enabled fluid-derived shear stress conditions for novel bone metastatic prostate cancer testbed.

Metastatic prostate cancer spreads preferentially to the bone, causing skeletal complications associated with significant morbidity and a poor prognosis, despite current therapeutic approaches. Hence, it is imperative to understand the complex metastatic cascade to develop therapeutic interventions for treating metastatic prostate cancer. Increasing evidence suggests the synergistic role of biochemical and biophysical cues in cancer progression at metastases. However, the mechanism underlying the crosstalk between interstitial flow-induced mechanical stimuli and prostate cancer progression at the bone microenvironment remains poorly understood. To this end, we have developed a three-dimensional (3D)in vitrodynamic model of prostate cancer bone metastasis using perfusion bioreactor and compared our results with static conditions to delineate the role of flow-induced shear stress on prostate cancer progression at metastases. We observed an increase in human mesenchymal stem cell (hMSCs) proliferation and differentiation rate under the dynamic culture. The hMSCs form cell agglutinates under static culture, whereas the hMSCs exhibited a directional alignment with broad and flattened morphology under dynamic culture. Further, the expression of mesenchymal to epithelial transition biomarkers is increased in bone metastasized prostate cancer models, and large changes are observed in the cellular and tumoroid morphologies under dynamic culture. Evaluation of cell adhesion proteins indicated that the altered cancer cell morphologies resulted from the constant force pulling due to increased E-cadherin and phosphorylated focal adhesion kinase proteins under shear stress. Overall, we report a successful 3Din vitrodynamic model to recapitulate bone metastatic prostate cancer behavior under dynamic conditions.

Bone interface modulates drug resistance in breast cancer bone metastasis.

Metastatic breast cancer cells on arriving at bone site interact with the bone cells to influence their growth, proliferation, and chemoresistance. There are currently no effective therapeutics available in the clinic for bone metastases. Many existing anti-cancer therapeutics are ineffective at the metastatic bone site due to a lack of accurate models of breast cancer bone metastasis for drug screening. Here, we report the development of an effective in vitro model using osteogenically differentiated human mesenchymal stem cells (MSCs) and human breast cancer cells on 3D nanoclay scaffolds as a testbed for screening drugs. Our results demonstrate that breast cancer cells grown in 3D bone-mimetic scaffolds exhibited altered physiological and biochemical properties, including tumoroids formation, elevated levels of cytokine such as IL-6, and its downstream effector-mediated inhibition of apoptosis and upregulation of multidrug transporters proteins, leading to drug resistance against paclitaxel. Most importantly, Signal Transducer and Activator of Transcription 3 (STAT3), a potential biomarker for chemoresistance in many cancers, was activated in the 3D breast cancer bone metastasis model. Thus, our data suggest that 3D bone-mimetic nanoclay scaffolds-based in vitro tumor model is a promising testbed for screening new therapeutics for breast cancer bone metastasis where bone interface governs drug resistance in breast cancer cells.

Wnt/ß-Catenin Signaling Pathway Regulates Osteogenesis for Breast Cancer Bone Metastasis: Experiments in an In Vitro Nanoclay Scaffold Cancer Testbed.

Breast cancer shows a high affinity toward bone, causing bone-related complications, leading to a poor clinical prognosis. The Wnt/ß-catenin signaling pathway has been well-documented for the bone regenerative process; however, the regulation of the Wnt/ß-catenin pathway in breast cancer bone metastasis is poorly explored. Here, we report that the Wnt/ß-catenin signaling pathway has a significant effect on osteogenesis during breast cancer bone metastasis. In this study, we have created a 3D in vitro breast cancer bone metastatic microenvironment using nanoclay-based scaffolds along with osteogenically differentiated human mesenchymal stem cells (MSCs) and human breast cancer cells (MCF-7 and MDA-MB-231). The results showed upregulation in expressions of Wnt-related factors (Wnt-5a, ß-catenin, AXIN2, and LRP5) in sequential cultures of MSCs with MCF-7 as compared to sequential cultures of MSCs with MDA-MB-231. Sequential cultures of MSCs with MCF-7 also showed higher ß-catenin expression on the protein levels than sequential cultures of MSCs with MDA-MB-231. Stimulation of Wnt/ß-catenin signaling in sequential cultures of MSCs with MCF-7 by ET-1 resulted in increased bone formation, whereas inactivation of Wnt/ß-catenin signaling by DKK-1 displayed a significant decrease in bone formation, mimicking bone lesions in breast cancer patients. These data collectively demonstrate that Wnt/ß-catenin signaling governs osteogenesis within the tumor-harboring bone microenvironment, leading to bone metastasis. The nanoclay scaffold provides a unique testbed approach for analysis of the pathways of cancer metastasis.

Fourier transform infrared spectroscopy based spectral biomarkers of metastasized breast cancer progression.

Breast cancer is a global health issue and the second leading cause of cancer death in women. Breast cancer tends to migrate to bone and causes bone metastases which is ultimately the cause of death. Here, we report the use of FTIR to identify spectral biomarkers of cancer progression on 3D in vitro model of breast cancer bone metastasis. Our results indicate that the following spectral biomarkers can monitor cancer progression, for example, lipids (CH2 asymmetric/CH2 symmetric stretch), Amide I/Amide II, and RNA/DNA. Principal component analysis also confirmed the involvement of protein, lipids and nucleic acids in cancer progression on sequential culture. The collective observations from this study suggest successful application of FTIR as a non-invasive and accurate method to identify biochemical changes in cancer cells during the progression of breast cancer bone metastasis.

Tissue-engineered nanoclay-based 3D in vitro breast cancer model for studying breast cancer metastasis to bone.

Breast cancer (BrCa) preferentially spreads to bone and colonises within the bone marrow to cause bone metastases. To improve the outcome of patients with BrCa bone metastasis, we need to understand better the mechanisms underlying bone metastasis. Researchers have relied heavily upon in vivo xenografts due to limited availability of human bone metastasis samples. A significant limitation of these is that they do not have a human bone microenvironment. To address this issue, we have developed a nanoclay-based 3D in vitro model of BrCa bone metastasis using human mesenchymal stem cells (MSCs) and human BrCa cells mimicking late stage of BrCa pathogenesis at the metastatic site. This 3D model can provide a microenvironment suitable for cell-cell and cell-matrix interactions whilst retaining the behaviour of BrCa cells with different metastasis potential (i.e., highly metastatic MDA-MB-231 and low metastatic MCF-7) as shown by the production of alkaline phosphatase and matrix metalloproteinase-9. The sequential culture of MSCs with MCF-7 exhibited 3D tumouroids formation and also occurrence of mesenchymal to epithelial transition of cancer metastasis as evidenced by gene expression and immunocytochemistry. The unique and distinct behaviour of highly metastatic MDA-MB-231 and the low metastatic MCF-7 was observed at the bone metastasis site. The changes to migratory capabilities and invasiveness in MDA-MB-231 in comparison with tumour growth with MCF-7 was observed. Together, a novel bone-mimetic 3D in vitro BrCa model has been developed that could be used to study mechanisms governing the later stage of cancer pathogenesis in bone.