Somatic Embryogenesis and Agrobacterium-Mediated Gene Transfer Procedures in Chilean Temperate Japonica Rice Varieties for Precision Breeding.

Rice (Oryza sativa) varieties are generated through breeding programs focused on local requirements. In Chile, the southernmost rice producer, rice productivity relies on the use and generation of temperate japonica germplasms, which need to be adapted to the intensifying effects of climate change. Advanced biotechnological tools can contribute to these breeding programs; new technologies associated with precision breeding, including gene editing, rely on procedures such as regeneration and gene transfer. In this study, the local rice varieties Platino, Cuarzo, Esmeralda, and Zafiro were evaluated for somatic embryogenesis potential using a process that involved the combined use of auxins and cytokinins. An auxin-based (2,4-D) general medium (2N6) allowed for the induction of embryogenic masses in all the genotypes. After induction, masses required culturing either in N6R (kinetin; Platino) or N6RN (BAP, kinetin, IBA, and 2,4-D; Cuarzo, Esmeralda, and Zafiro) to yield whole plants using regeneration medium (N6F, no hormone). The sprouting rates indicated Platino as the most responsive genotype; for this reason, this variety was evaluated for gene transfer. Fifteen-day-old embryo masses were assayed for Agrobacterium-mediated transformation using the bacterial strain EHA105 harboring pFLC-Myb/HPT/GFP, a modified T-DNA vector harboring a geminivirus-derived replicon. The vector included the green fluorescent protein reporter gene, allowing for continuous traceability. Reporter mRNA was produced as early as 3 d after agroinfiltration, and stable expression of the protein was observed along the complete process. These achievements enable further biotechnological steps in these and other genotypes from our breeding program.

Isolation and Analysis of Plasma-Derived Exosomes in Patients With Glioma.

Gliomas including glioblastoma (GBM) are the most common primary malignant brain tumors. Glioma extracellular vesicles (EVs) including exosomes have biological effects (e.g., immunosuppression) and contain tumor-specific cargo that could facilitate liquid biopsies. We aimed to develop a simple, reproducible technique to isolate plasma exosomes in glioma patients. Glioma patients' and normal donors' plasma exosomes underwent brief centrifugation to remove cells/debris followed by serial density gradient ultracentrifugation (DGU). EV size/concentration was determined by nanoparticle tracking. Protein cargo was screened by array, western blot, and ELISA. Nanoscale flow cytometry analysis quantified exosome and microvesicle populations pre- and post-DGU. One-step DGU efficiently isolates exosomes for nanoparticle tracking. Wild type isocitrate dehydrogenase glioma patients' (i.e., more aggressive tumors) plasma exosomes are smaller but higher concentration than normal donors. A second DGU efficiently concentrates exosomes for subsequent cargo analysis but results in vesicle aggregation that skews nanoparticle tracking. Cytokines and co-stimulatory molecules are readily detected but appeared globally reduced in GBM patients' exosomes. Surprisingly, immunosuppressive programmed death-ligand 1 (PD-L1) is present in both patients' and normal donors' exosomes. Nanoscale flow cytometry confirms efficient exosome (<100 nm) isolation post-DGU but also demonstrates increase in microvesicles (>100 nm) in GBM patients' plasma pre-DGU. Serial DGU efficiently isolates plasma exosomes with distinct differences between GBM patients and normal donors, suggesting utility for non-invasive biomarker assessment. Initial results suggest global immunosuppression rather than increased circulating tumor-derived immunosuppressive exosomes, though further assessment is needed. Increased glioma patients' plasma microvesicles suggest these may also be a key source for biomarkers.

Targeting FER Kinase Inhibits Melanoma Growth and Metastasis.

Melanoma is one of the most aggressive types of tumors and exhibits high metastatic potential. Fes-related (FER) kinase is a non-receptor tyrosine kinase that has been implicated in growth and metastasis of various epithelial tumors. In this study, we have examined the role that FER kinase plays in melanoma at the molecular level. FER-depleted melanoma cells exhibit impaired Wnt/ß-catenin pathway activity, as well as multiple proteomic changes, which include decreased abundance of L1-cell adhesion molecule (L1-CAM). Consistent with the pro-metastatic functions of these pathways, we demonstrate that depletion of FER kinase decreases melanoma growth and formation of distant metastases in a xenograft model. These findings indicate that FER is an important positive regulator of melanoma metastasis and a potential target for innovative therapies.

Invadopodia are chemosensing protrusions that guide cancer cell extravasation to promote brain tropism in metastasis.

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

Inhibition of MT1-MMP proteolytic function and ERK1/2 signalling influences cell migration and invasion through changes in MMP-2 and MMP-9 levels.

Membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14) is a unique protease that cleaves extracellular proteins, activates proMMPs, and initiates intracellular signalling. MCF-7 cells are non-invasive and deficient in MT1-MMP, MMP-2, and MMP-9 expression. We created an MCF-7 cell line (C2) that stably produces active MT1-MMP and demonstrated increased ERK1/2 phosphorylation. MAPK inhibition in this cell line showed an inverse relationship in MMP-2 and MMP-9 transcripts where levels of these genes increased and decreased, respectively. Using invasive MDA-MB 231 cells that endogenously produce MT1-MMP and have naturally high pERK levels, we demonstrated the identical inverse relationship between MMP-2 and -9 transcript and protein levels, suggesting that this novel relationship is conserved amongst MT1-MMP positive breast cancer cells. To further analyze the relationship between MMP-2 and -9 levels, we chemically inhibited activation and catalytic activity of MT1-MMP using a furin and MMP inhibitor, respectively, to show that interference with the functions of MT1-MMP induced changes in MMP-2 and 9 transcript levels that were always inverse of each other, and likely mediated by differential transcriptional activity of the NF-κB transcription factor. Furthermore, we analyzed the functional consequences of these expression changes to show MMP, and in particular ERK, inhibition decreased migration and invasion using 2D culture, and inhibits the formation of an invasive phenotype in Matrigel 3D culture. This study demonstrated a novel inverse transcriptional relationship between MMP-2 and -9 levels and MT1-MMP activity that have functional consequences, and also showed that increases in the levels of MMPs does not necessarily correlate with an invasive phenotype.

The cytoplasmic domain of MT1-MMP is dispensable for migration augmentation but necessary to mediate viability of MCF-7 breast cancer cells.

Membrane-type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that regulates ECM degradation, proMMP-2 activation, and varied cellular processes including migration and viability. MT1-MMP is believed to be a central mediator of tumourigenesis whose role is dictated by its functionally distinct protein domains. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain, exemplifying diverse regulatory functions. To further our understanding of the multifunctional contributions of MT1-MMP to cellular processes, we overexpressed cytoplasmic domain altered constructs in MCF-7 breast cancer cells and analyzed migration and viability in 2D culture conditions, morphology in 3D Matrigel culture, and tumorigenic ability in vivo. We found that the cytoplasmic domain was not needed for MT1-MMP mediated migration promotion, but was necessary to maintain viability during serum depravation in 2D culture. Similarly, during 3D Matrigel culture the cytoplasmic domain of MT1-MMP was not needed to initiate a protrusive phenotype, but was necessary to prevent colony blebbing when cells were serum deprived. We also tested in vivo tumorigenic potential to show that cells expressing cytoplasmic domain altered constructs demonstrated a reduced ability to vascularize tumours. These results suggest that the cytoplasmic domain regulates MT1-MMP function in a manner required for cell survival, but is dispensable for cell migration.

Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells.

BACKGROUND: Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease implicated in metastatic progression ostensibly due to its ability to degrade extracellular matrix (ECM) components and allow migration of cells through the basement membrane. Despite in vitro studies demonstrating this principle, this knowledge has not translated into the use of MMP inhibitors (MMPi) as effective cancer therapeutics, or been corroborated by evidence of in vivo ECM degradation mediated by MT1-MMP, suggesting that our understanding of the role of MT1-MMP in cancer progression is incomplete. METHODS: MCF-7 and MDA-MB 231 breast cancer cell lines were created that stably overexpress different levels of MT1-MMP. Using 2D culture, we analyzed proMMP-2 activation (gelatin zymography), ECM degradation (fluorescent gelatin), ERK signaling (immunoblot), cell migration (transwell/scratch closure/time-lapse imaging), and viability (colorimetric substrate) to assess how different MT1-MMP levels affect these cellular parameters. We also utilized Matrigel 3D cell culture and avian embryos to examine how different levels of MT1-MMP expression affect morphological changes in 3D culture, and tumourigenecity and extravasation efficiency in vivo. RESULTS: In 2D culture, breast cancer cells expressing high levels of MT1-MMP were capable of widespread ECM degradation and TIMP-2-mediated proMMP-2 activation, but were not the most migratory. Instead, cells expressing low levels of MT1-MMP were the most migratory, and demonstrated increased viability and ERK activation. In 3D culture, MCF-7 breast cancer cells expressing low levels of MT1-MMP demonstrated an invasive protrusive phenotype, whereas cells expressing high levels of MT1-MMP demonstrated loss of colony structure and cell fragment release. Similarly, in vivo analysis demonstrated increased tumourigenecity and metastatic capability for cells expressing low levels of MT1-MMP, whereas cells expressing high levels were devoid of these qualities despite the production of functional MT1-MMP protein. CONCLUSIONS: This study demonstrates that excessive ECM degradation mediated by high levels of MT1-MMP is not associated with cell migration and tumourigenesis, while low levels of MT1-MMP promote invasion and vascularization in vivo.

Microwave ablation: state-of-the-art review.

This paper reviews state-of-the-art microwave ablation (MWA) of tumors. MWA is a novel method for treating inoperable tumors, ie, tumors that cannot be treated surgically. However, patients generally choose removal of the tumor by conventional techniques. A literature review of MWA for breast, liver, lung, and kidney tumors is reported here, with tabulation of our findings according to the type of technique used, with a detailed description of the time, type of microwave generator used, and number of patients treated with MWA. In some cases, the subjects were not human patients, but pig or bovine liver specimens. MWA is a technique that has proved to be promising and likely to be used increasingly in the ablation of cancerous tumors. However, MWA needs to be used more widely to establish itself as a common tool in the treatment of inoperable tumors.

Knockdown of Pex11ß reveals its pivotal role in regulating peroxisomal genes, numbers, and ROS levels in Xenopus laevis A6 cells.

Peroxisomes are organelles that are ubiquitously found in all eukaryotic cells. Enzymes within their lumen are responsible for a variety of processes including the metabolism of fatty acids and eradication (neutralization) of free radicals. Peroxisomes are dynamic organelles, able to alter their numbers in response to a variety of different metabolic and cell-specific cues. Changes in peroxisome numbers can occur through division of preexisting peroxisomes or through de novo biogenesis from the ER. Proteins such as the Pex11 family of peroxins have been implicated as regulatory factors involved in peroxisome division. Division of peroxisomes involves elongation and membrane constriction followed by fission, which requires Pex11ß. The regulation of peroxisome numbers in different cell types and tissues is variable and poorly understood. Here, we examine how knockdown of Pex11ß affects peroxisomal genes, proteins, and peroxisome numbers in A6 kidney epithelial cells derived from Xenopus laevis. Pex11ß morpholino use subsequently decreased mRNA levels of Pex1, PMP70, and PPARγ. Moreover, the Pex11ß morpholino decreased PMP70 protein levels and PMP70-positive structures. Furthermore, the marker GFP-SKL revealed fewer peroxisome-like structures. These decreases resulted in increased levels of H2O2 and cellular and mitochondrial reactive oxygen species as measured by Amplex Red, DCFDA, and MitoTracker assays, respectively.

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells.

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line.