Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma.

BACKGROUND: Ex vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults. METHODS: To compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells. RESULTS: Dose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient's tumor. CONCLUSIONS: All the tested ex vivo drug screening methods captured the patient's tumor cells' sensitivity to drugs that could be associated with the oncogenic KRASG12V mutation found in the patient's tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes.

Image-based ex vivo drug screen to assess targeted therapies in recurrent thymoma.

OBJECTIVES: Thymoma is a rare malignancy derived from the thymic epithelial cells. No standard salvage treatments are available for recurrent thymoma and due to the low number of cases, alternative treatment regimens have been assessed only in small case series with varying success. The aim of this study was to use an image-based ex vivo drug screening strategy to assess efficacy of a large panel of anti-cancer agents for thymoma using patient derived tumor cells. MATERIALS AND METHODS: Vital tumor and tumor associated cells were used to assess the efficacy of 147 anti-cancer drugs including approved and experimental agents. Drug efficacy was analyzed at single cell resolution using image-based high content drug screening to assess tumor cell specific responses. Molecular profiling and histopathology was used to confirm the drug targets identified by the screen. RESULTS: The ex vivo drug screen identified selective sensitivity of the cancerous epithelial thymoma cells to EGFR-, HDAC- and mTOR-inhibition. Histopathology confirmed high protein level expression of EGFR in the patient's tumor. Patient was initiated treatment with Cetuximab resulting in stable disease after relapse on five different chemotherapy regimens. CONCLUSION: The results show that the image-based ex vivo therapy efficacy screening strategy can be used to identify patient and tumor relevant drug sensitivity patterns in thymoma. The results also warrant continued research on EGFR as a biomarker and therapy target in recurrent thymomas.

Clonal Evolution of MEK/MAPK Pathway Activating Mutations in a Metastatic Colorectal Cancer Case.

BACKGROUND/AIM: The aim of this study was to examine clonal heterogeneity, to test the utility of liquid biopsy in monitoring disease progression and to evaluate the usefulness of ex vivo drug screening in a BRAF L597Q-mutated colorectal cancer (CRC) patient developing metastases during adjuvant therapy. MATERIALS AND METHODS: Next generation sequencing (NGS) and droplet digital PCR (ddPCR) were performed in samples from tumor tissues and liquid biopsies. Live cancer cells from a metastatic lesion were used in ex vivo drug sensitivity assays. RESULTS: We found evidence of continued dependence of MEK/MAPK pathway activation, but different activating mutations in primary tumor and metastases. Liquid biopsy based BRAF L597Q ddPCR testing was a sensitive personalized biomarker predicting the rise of clinically aggressive metastatic disease. Ex vivo drug sensitivity assays with BRAF L597Q mutated cells showed response to MEK/MAPK targeted therapies. CONCLUSION: The rare BRAF L597Q mutation may be associated with aggressive tumor behavior in CRC. Liquid biopsy can be used to capture clinically relevant tumor features.

Evidence for the mechanosensor function of filamin in tissue development.

Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. Here we tested, how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, our results show that an appropriate level of mechanical sensitivity is required for filamins' function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components.

Permeability changes of integrin-containing multivesicular structures triggered by picornavirus entry.

Cellular uptake of clustered α2ß1-integrin induces the formation of membrane compartments that subsequently mature into a multivesicular body (MVB). Enhanced internalization mediated by clustered integrins was observed upon infection by the picornavirus echovirus 1 (EVI). We elucidated the structural features of virus-induced MVBs (vMVBs) in comparison to antibody-induced control MVBs (mock infection) by means of high-pressure cryo fixation of cells followed by immuno electron tomography during early entry of the virus. Three-dimensional tomograms revealed a marked increase in the size and complexity of these vMVBs and the intraluminal vesicles (ILVs) at 2 and 3.5 hours post infection (p.i.), in contrast to the control MVBs without virus. Breakages in the membranes of vMVBs were detected from tomograms after 2 and especially after 3.5 h suggesting that these breakages could facilitate the genome release to the cytoplasm. The in situ neutral-red labeling of viral genome showed that virus uncoating starts as early as 30 min p.i., while an increase of permeability was detected in the vMVBs between 1 and 3 hours p.i., based on a confocal microscopy assay. Altogether, the data show marked morphological changes in size and permeability of the endosomes in the infectious entry pathway of this non-enveloped enterovirus and suggest that the formed breakages facilitate the transfer of the genome to the cytoplasm for replication.

The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features.

BACKGROUND: Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2). METHODS: Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability. RESULTS: Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stimulated by different VEGFR2 ligands was dependent on their ability to bind to co-receptor Neuropilin (Nrp), which was shown to form complexes with VEGFR2. The ability of these VEGFR2 ligands to induce vascular permeability was dependent on their concentration and VEGFR2 affinity, but not on Nrp binding. CONCLUSIONS: VEGFR2 activation alone is sufficient for inducing endothelial cell proliferation, formation of tube-like structures and vascular permeability. The level of VEGFR2 activation is dependent on the binding properties of the ligand used. However, closely similar activation pattern of the receptor kinase domain is seen with all VEGFR2 ligands. Nrp binding strengthens the angiogenic potency without increasing vascular permeability. GENERAL SIGNIFICANCE: This study sheds light on how different structurally closely related VEGFR2 ligands bind to and signal via VEGFR2/Nrp complex to induce angiogenesis and vascular permeability. The knowledge of this study could be used for designing VEGFR2/Nrp ligands with improved therapeutic properties.

Cholesterol dependence of collagen and echovirus 1 trafficking along the novel α2ß1 integrin internalization pathway.

We have previously shown that soluble collagen and a human pathogen, echovirus 1 (EV1) cluster α2ß1 integrin on the plasma membrane and cause their internalization into cytoplasmic endosomes. Here we show that cholesterol plays a major role not only in the uptake of α2ß1 integrin and its ligands but also in the formation of α2 integrin-specific multivesicular bodies (α2-MVBs) and virus infection. EV1 infection and α2ß1 integrin internalization were totally halted by low amounts of the cholesterol-aggregating drugs filipin or nystatin. Inhibition of cholesterol synthesis and accumulation of lanosterol after ketoconazole treatment inhibited uptake of collagen, virus and clustered integrin, and prevented formation of multivesicular bodies and virus infection. Loading of lipid starved cells with cholesterol increased infection to some extent but could not completely restore EV1 infection to control levels. Cold Triton X-100 treatment did not solubilize the α2-MVBs suggesting, together with cholesterol labeling, that the cytoplasmic endosomes were enriched in detergent-resistant lipids in contrast to αV integrin labeled control endosomes in the clathrin pathway. Cholesterol aggregation leading to increased ion permeability caused a significant reduction in EV1 uncoating in endosomes as judged by sucrose gradient centrifugation and by neutral red-based uncoating assay. In contrast, the replication step was not dependent on cholesterol in contrast to the reports on several other viruses. In conclusion, our results showed that the integrin internalization pathway is dependent on cholesterol for uptake of collagen, EV1 and integrin, for maturation of endosomal structures and for promoting EV1 uncoating. The results thus provide novel information for developing anti-viral strategies and more insight into collagen and integrin trafficking.

Calpains promote α2ß1 integrin turnover in nonrecycling integrin pathway.

Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2ß1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2ß1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in α2-MVBs, internalized clustered α2ß1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade α2ß1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts α2ß1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route.

Echovirus 1 infection depends on biogenesis of novel multivesicular bodies.

Non-enveloped picornavirus echovirus 1 (EV1) clusters its receptor α2ß1 integrin and causes their internalization and accumulation in α2ß1 integrin enriched multivesicular bodies (α2-MVBs). Our results here show that these α2-MVBs are distinct from acidic late endosomes/lysosomes by several criteria: (i) live intra-endosomal pH measurements show that α2-MVBs are not acidic, (ii) they are not positive for the late endosomal marker LBPA or Dil-LDL internalized to lysosomes, and (iii) simultaneous stimulation of epidermal growth factor receptor (EGFR) and α2ß1 integrin clustering leads to their accumulation in separate endosomes. EGFR showed downregulation between 15 min and 2 h, whereas accumulation of α2ß1 integrin/EV1 led to an increase of integrin fluorescence in cytoplasmic vesicles further suggesting that EV1 pathway is separate from the lysosomal downregulation pathway. In addition, the results demonstrate the involvement of ESCRTs in the biogenesis of α2-MVBs. Overexpression of dominant-negative form of VPS4 inhibited biogenesis of α2-MVBs and efficiently prevented EV1 infection. Furthermore, α2-MVBs were positive for some members of ESCRTs such as Hrs, VPS37A and VPS24 and the siRNA treatment of TSG101, VPS37A and VPS24 inhibited EV1 infection. Our results show that the non-enveloped EV1 depends on biogenesis of novel multivesicular structures for successful infection.

Parvovirus capsid disorders cholesterol-rich membranes.

In this study canine parvovirus, CPV, was found to induce disorder in DPPC:cholesterol membranes in acidic conditions. This acidicity-induced fluidizing effect is suggested to originate from the N-terminus of the viral capsid protein VP1. In accordance with the model membrane studies, a fluidizing effect was seen also in the endosomal membranes during CPV infection implying an important functional role of the fluidization in the endocytic entry of the virus.