Mika Kaakinen

Blue Rubber Bleb Nevus (BRBN) Syndrome Is Caused by Somatic TEK (TIE2) Mutations.

Blue rubber bleb nevus syndrome (Bean syndrome) is a rare, severe disorder of unknown cause, characterized by numerous cutaneous and internal venous malformations; gastrointestinal lesions are pathognomonic. We discovered somatic mutations in TEK, the gene encoding TIE2, in 15 of 17 individuals with blue rubber bleb nevus syndrome. Somatic mutations were also identified in five of six individuals with sporadically occurring multifocal venous malformations. In contrast to common unifocal venous malformation, which is most often caused by the somatic L914F TIE2 mutation, multifocal forms are predominantly caused by double (cis) mutations, that is, two somatic mutations on the same allele of the gene. Mutations are identical in all lesions from a given individual. T1105N-T1106P is recurrent in blue rubber bleb nevus, whereas Y897C-R915C is recurrent in sporadically occurring multifocal venous malformation: both cause ligand-independent activation of TIE2, and increase survival, invasion, and colony formation when expressed in human umbilical vein endothelial cells.

Evidence for γ-actin as a Z disc component in skeletal myofibers

We investigated the targeting of the gamma-actin isoform in skeletal myofibers. For this purpose we used expression vectors to produce green fluorescent protein (GFP-) as well as myc-tagged gamma-actin in rat flexor digitorum brevis myofibers. We found that the gamma-actin fusion proteins accumulated into Z discs but not beneath the sarcolemma. Instead, the GFP-tagged skeletal muscle-specific alpha-actin isoform was preferentially incorporated into the pointed ends of thin contractile filaments. The localization pattern of the gamma-actin fusion proteins was completely different from that of the dystrophin glycoprotein complex on the sarcolemma. The results emphasize the role of gamma-actin as a Z disc component but fail to reveal an actin-based sub-sarcolemmal cytoskeleton in skeletal muscle cells.

Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention

Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro.

DNA from dead cancer cells induces TLR9-mediated invasion and inflammation in living cancer cells

AbstractTLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple-negative, human MDA-MB-231 breast cancer cells stably expressing control, or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy.

Tumor tissue inhibitor of metalloproteinases-1 (TIMP-1) in hormone-independent breast cancer might originate in stromal cells, and improves stratification of prognosis together with nodal status

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is shown to be a potential marker for poor prognosis in breast cancer, but the biology of TIMP-1 is only partially understood. In this study, TIMP-1 production was studied in a co-culture model of hormone-independent breast cancer cell lines and mesenchymal stem cells mimicking the stromal components of the tumor. In addition, the prognostic value of TIMP-1 was histologically evaluated in a clinical material of 168 patients with hormone-independent breast tumors. The hormone-independent breast cancer (BC) cell lines MDA-MB-231, M4A4 and NM2C5 did not produce TIMP-1 protein in measureable quantities. Six tested primary mesenchymal stem cell lines all produced TIMP-1. Co-culturing of mesenchymal stem cells and breast cancer cells resulted in positive immunocytochemical diffuse staining for TIMP-1 for both cell types. Culturing breast cancer cells with MSC-conditioned media resulted in a positive cytoplasmic immunoreactivity for TIMP-1, and TIMP-1 protein concentration in cell lysates increased 2.7-fold (range 1.1-4.7). The TIMP-1 mRNA levels remained unaffected in BC cells. This might suggest that breast cancer cells can take up TIMP-1 produced by stromal cells and are thus displaying cellular immunoreactivity. In addition, TIMP-1 was shown to improve stratification of prognosis in clinical material.

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

Influenza virus infection in multinucleated skeletal myofibers

We examined the progression of the WSN influenza virus infection in isolated, multinucleated rat skeletal myofibers. Contrary to mononucleated cells, the adsorbed virions showed markedly delayed entry kinetics. Viral budding occurred on the sarcolemma, but the hemagglutinin envelope glycoprotein matured inefficiently and was poorly cleaved. Compatible with this, plaque assays indicated that infective viral particles were not formed. In situ hybridization studies showed that at low-dose infection, viral RNA production was restricted to one or a few nuclei within a myofiber. Dual in situ hybridization indicated that two different viral RNAs usually co-localized in the same nucleus or nuclei, suggesting that different viral genome segments replicated in the same nucleus. Newly synthesized viral ribonucleoprotein particles (vRNPs) did not re-enter virgin nuclei. Therefore, a single infected nucleus was able to support viral protein production, and notably, these proteins could reach hundreds of micrometers from the nucleus of origin. These results suggest that after viral disassembly in the endosome, the genome segments remained glued together and entered a myonucleus as a package. Spreading of the infection into virgin nuclei either by vRNPs or newly made virions did not occur, and thus the infection was abortive.

Caveolin 3, Flotillin 1 and Influenza Virus Hemagglutinin Reside in Distinct Domains on the Sarcolemma of Skeletal Myofibers

We examined the distribution of selected raft proteins on the sarcolemma of skeletal myofibers and the role of cholesterol environment in the distribution. Immunofluorescence staining showed that flotillin-1 and influenza hemagglutinin exhibited rafts that located in the domains deficient of the dystrophin glycoprotein complex, but the distribution patterns of the two proteins were different. Cholesterol depletion from the sarcolemma by means of methyl-β-cyclodextrin resulted in distorted caveolar morphology and redistribution of the caveolin 3 protein. Concomitantly, the water permeability of the sarcolemma increased significantly. However, cholesterol depletion did not reshuffle flotillin 1 or hemagglutinin. Furthermore, a hemagglutinin variant that lacked a raft-targeting signals exhibited a similar distribution pattern as the native raft protein. These findings indicate that each raft protein exhibits a strictly defined distribution in the sarcolemma. Only the distribution of caveolin 3 that binds cholesterol was exclusively dependent on cholesterol environment.

New immunohistochemical method for improved myotonia and chloride channel mutation diagnostics

ABSTRACT Objective: The objective of this study was to validate the immunohistochemical assay for the diagnosis of nondystrophic myotonia and to provide full clarification of clinical disease to patients in whom basic genetic testing has failed to do so. Methods: An immunohistochemical assay of sarcolemmal chloride channel abundance using 2 different ClC1-specific antibodies. Results: This method led to the identification of new mutations, to the reclassification of W118G in CLCN1 as a moderately pathogenic mutation, and to confirmation of recessive (Becker) myotonia congenita in cases when only one recessive CLCN1 mutation had been identified by genetic testing. Conclusions: We have developed a robust immunohistochemical assay that can detect loss of sarcolemmal ClC-1 protein on muscle sections. This in combination with gene sequencing is a powerful approach to achieving a final diagnosis of nondystrophic myotonia.

Detection of Tumor Cell Spheroids from Co-cultures Using Phase Contrast Images and Machine Learning Approach

Automated image analysis is demanded in cell biology and drug development research. The type of microscopy is one of the considerations in the trade-offs between experimental setup, image acquisition speed, molecular labelling, resolution and quality of images. In many cases, phase contrast imaging gets higher weights in this optimization. And it comes at the price of reduced image quality in imaging 3D cell cultures. For such data, the existing state-of-the-art computer vision methods perform poorly in segmenting specific cell type. Low SNR, clutter and occlusions are basic challenges for blind segmentation approaches. In this study we propose an automated method, based on a learning framework, for detecting particular cell type in cluttered 2D phase contrast images of 3D cell cultures that overcomes those challenges. It depends on local features defined over super pixels. The method learns appearance based features, statistical features, textural features and their combinations. Also, the importance of each feature is measured by employing Random Forest classifier. Experiments show that our approach does not depend on training data and the parameters.