In contrast to high CD49d, low CXCR4 expression indicates the dependency of chronic lymphocytic leukemia (CLL) cells on the microenvironment.

CD49d and CXCR4 are key determinants of interactions between chronic lymphocytic leukemia (CLL) tumor cells and their microenvironment. In this study, we investigated the effect of CD49d and CXCR4 expressions on survival of CLL cells. Primary CLL cells were cultured with CD49d ligand, VCAM-1, or bone marrow stromal cells (BMSCs); then, apoptosis and immunophenotype analyses were performed. VCAM-1 treatment could not induce direct apoptosis protection or immunophenotype change on the CD49d-expressing CLL cells, but resulted in actin reorganization. The BMSC-induced apoptosis protection was independent from the presence of CD49d expression of CLL cells, but showed an inverse correlation with their CXCR4 expression level. We suppose that CD49d contributes to enhanced survival of leukemic cells by mediating migration to the protective microenvironment, not by direct prevention of apoptosis. Moreover, CLL cells with low CXCR4 expression represent a subpopulation that is more dependent on the microenvironmental stimuli for survival, and show increased "death by neglect" when separated from the supportive niche.

Human liver regeneration in advanced cirrhosis is organized by the portal tree.

BACKGROUND & AIMS: In advanced cirrhosis new hepatocytic nodules are generated by budding of ductules in areas of parenchymal extinction. However, the vascular alterations in the areas of parenchymal extinction, the blood supply and the structure of the new hepatocytic nodules have not been analyzed in detail. METHODS: Explanted human cirrhotic livers of three different etiologies and two experimental rat models of cirrhosis were thoroughly examined. 3D reconstruction of the immunohistochemically stained serial sections and casting of human and experimental cirrhotic livers have been used to reveal the structural organization of the regenerative buds. RESULTS: In areas of parenchymal extinction the skeleton of the liver, the portal tree is preserved. The developing regenerative nodules are positioned along the portal tree and are directly supplied by terminal portal venules. The expanding nodules grow along the trunks of the portal vein. Casting of human and experimental cirrhotic livers by colored resin confirms that nodules are supplied by portal blood. The two other members of the portal triads become separated from the portal veins. CONCLUSIONS: As the structure of the hepatocyte nodules (centrally located portal vein branches, bile ducts at the periphery, hepatic veins and arteries in the connective tissue) impedes the restoration of normal liver structure, the basic architecture of hepatic tissue suffers permanent damage. We suggest that "budding" may initiate the second, irreversible stage of cirrhosis. LAY SUMMARY: Cirrhosis is the final common outcome of long lasting hepatic injury defined as the destruction of the normal liver architecture by scar tissue. In the late phase of cirrhosis stem cells-derived hepatocyte nodules appear along the branches of the portal vein suggesting an important role of this specially composed blood vessels (containing digestive end-products from the stomach and intestines) in liver regeneration. Our results contribute to a better understanding of this serious liver disease.

Imatinib accelerates progenitor cell-mediated liver regeneration in choline-deficient ethionine-supplemented diet-fed mice.

Severe chronic hepatic injury can induce complex reparative processes. Ductular reaction and the appearance of small hepatocytes are standard components of this response, which is thought to have both adverse (e.g. fibrosis, carcinogenesis) and beneficial (regeneration) consequences. This complex tissue reaction is regulated by orchestrated cytokine action. We have investigated the influence of the tyrosine kinase inhibitor imatinib on a regenerative process. Ductular reaction was induced in mice by the widely used choline-deficient ethionine-supplemented diet (CDE). Test animals were treated daily with imatinib. After 6 weeks of treatment, imatinib successfully reduced the extent of ductular reaction and fibrosis in the CDE model. Furthermore, the number of small hepatocytes increased, and these cells had high proliferative activity, were positive for hepatocyte nuclear factor 4 and expressed high levels of albumin and peroxisome proliferator-activated receptor alpha. The overall functional zonality of the hepatic parenchyma (cytochrome P450 2E1 and glucose 6 phosphatase activity; endogenous biotin content) was maintained. The expression of platelet-derived growth factor receptor beta, which is the major target of imatinib, was downregulated. The anti-fibrotic activity of imatinib has already been reported in several experimental models. Additionally, in the CDE model imatinib was able to enhance regeneration and preserve the functional arrangement of hepatic lobules. These results suggest that imatinib might promote the recovery of the liver following parenchymal injury through the inhibition of platelet-derived growth factor receptor beta.

Mechanism of tumour vascularization in experimental lung metastases.

The appearance of lung metastases is associated with poor outcome and the management of patients with secondary pulmonary tumours remains a clinical challenge. We examined the vascularization process of lung metastasis in six different preclinical models and found that the tumours incorporated the pre-existing alveolar capillaries (ie vessel co-option). During the initial phase of vessel co-option, the incorporated capillaries were still sheathed by pneumocytes, but these incorporated vessels subsequently underwent different fates dependent on the model. In five of the models examined (B16, HT1080, HT25, C26, and MAT B-III), the tumour cells gradually stripped the pneumocytes from the vessels. These dissected pneumocytes underwent fragmentation, but the incorporated microvessels survived. In the sixth model (C38), the tumour cells failed to invade the alveolar walls. Instead, they induced the development of vascularized desmoplastic tissue columns. Finally, we examined the process of arterialization in lung metastases and found that they became arterialized when their diameter grew to exceed 5 mm. In conclusion, our data show that lung metastases can vascularize by co-opting the pulmonary microvasculature. This is likely to have important clinical implications, especially with respect to anti-angiogenic therapies.

[Role of tumour cell invasion/migration in the vascularisation of experimental lung metastases]. / Tumorsejt-invázió/migráció szerepe kísérletes tüdõmetasztázisok erezõdésében.

Treatment of patients with lung metastases remains a major challenge. A possible target for therapies is the inhibition of vascularization of metastases. Our study aimed to determine the possible mechanisms of the experimental lung metastasis vascularisation for tumours of various origins. We created lung metastases by intravenous injection of five tumour cell lines (HT1080, HT25, B16, C26 and MATB). Each cell line showed the same vascularisation type. Tumours gained vasculature by advancing through the alveolar spaces thereby incorporating the pre-existing alveolar capillaries (i.e. vessel co-option). From the alveolar spaces tumours entered into the alveolar walls. The tumour cells during the invasion/migration separated the pneumocytes from the capillaries. During this process the basement membrane was split into an epithelial and an endothelial layer. The heavily compressed pneumocytes inside the tumour became fragmented but the incorporated and stripped vessels remained functional, so they were able to provide blood supply for the metastases.

Structural analysis of oval-cell-mediated liver regeneration in rats.

UNLABELLED: We have analyzed the architectural aspects of progenitor-cell-driven regenerative growth in rat liver by applying the 2-acetaminofluorene/partial hepatectomy experimental model. The regeneration is initiated by the proliferation of so-called oval cells. The oval cells at the proximal tips of the ductules have a more differentiated phenotype and higher proliferative rate. This preferential growth results in the formation of a seemingly random collection of small hepatocytes, called foci. These foci have no clonal origin, but possess a highly organized structure, which shows similarities to normal hepatic parenchyma. Therefore, they can easily remodel into the lobular structure. Eventually, the regenerated liver is constructed by enlarged hepatic lobules; no new lobules are formed during this process. The foci of the Solt-Farber experimental hepatocarcinogenesis model have identical morphological features; accordingly, they also represent only regenerative, not neoplastic, growth. CONCLUSION: Progenitor-cell-driven liver regeneration is a well-designed, highly organized tissue reaction, and better comprehension of the architectural events may help us to recognize this process and understand its role in physiological and pathological reactions.

A new mechanism for pillar formation during tumor-induced intussusceptive angiogenesis: inverse sprouting.

One of the hallmarks of intussusceptive angiogenesis is the development of intraluminal connective tissue pillars. The exact mechanism of pillar formation has not yet been elucidated. By using electron and confocal microscopy, we observed intraluminal nascent pillars that contain a collagen bundle covered by endothelial cells (ECs) in the vasculature of experimental tumors. We proposed a new mechanism for the development of these pillars. First, intraluminal endothelial bridges are formed. Second, localized dissolution of the basement membrane occurs and a bridging EC attaches to a collagen bundle in the underlying connective tissue. A pulling force is then exerted by the actin cytoskeleton of the ECs via specific attachment points, which contain vinculin, to the collagen bundle, resulting in suction and subsequent transport of the collagen bundle into and through the vessel lumen. Third, the pillar matures through the immigration of connective tissue cells and the deposition of new collagenous connective tissue. The proposed simple mechanism generates a connection between the processes of endothelial bridging and intussusceptive angiogenesis and identifies the source of the force behind pillar formation. Moreover, it ensures the rapid formation of pillars from pre-existing building blocks and the maintenance of EC polarity. To describe it, we coined the term inverse sprouting.

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene induces substantial hyperplasia in fibrotic mouse liver.

The proliferative response of hepatocytes in vivo can be induced by two mechanisms: severe damage to hepatic tissue results in regenerative growth and so-called primary hepatocyte mitogens can initiate liver cell proliferation without preceding loss of parenchyma. The regulation of the two responses is quite different. The decreased regenerative response of cirrhotic/fibrotic liver is well known, and is a severe obstacle to surgery of the diseased liver. In the present experiments we investigated the efficiency of a primary hepatocyte mitogen 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOB) on two different liver cirrhosis/fibrosis models in mice induced by chronic administration of CCl(4) and thioacetamide respectively. BrdU incorporation and cyclin A expression established clearly that there is a reduced but still powerful mitogenic response of the fibrotic livers. Therefore, primary hepatocyte mitogens appear to be suitable to be used to rescue the regenerative response of cirrhotic livers.

Malignant pleural mesothelioma nodules remodel their surroundings to vascularize and grow.

Background: The microanatomical steps of malignant pleural mesothelioma (MPM) vascularization and the resistance mechanisms to anti-angiogenic drugs in MPM are unclear. Methods: We investigated the vascularization of intrapleurally implanted human P31 and SPC111 MPM cells. We also assessed MPM cell's motility, invasion and interaction with endothelial cells in vitro. Results: P31 cells exhibited significantly higher two-dimensional (2D) motility and three-dimensional (3D) invasion than SPC111 cells in vitro. In co-cultures of MPM and endothelial cells, P31 spheroids permitted endothelial sprouting (ES) with minimal spatial distortion, whereas SPC111 spheroids repealed endothelial sprouts. Both MPM lines induced the early onset of submesothelial microvascular plexuses covering large pleural areas including regions distant from tumor colonies. The development of these microvascular networks occurred due to both intussusceptive angiogenesis (IA) and ES and was accelerated by vascular endothelial growth factor A (VEGF-A)-overexpression. Notably, SPC111 colonies showed different behavior to P31 cells. P31 nodules incorporated tumor-induced capillary plexuses from the earliest stages of tumor formation. P31 cells deposited a collagenous matrix of human origin which provided "space" for further intratumoral angiogenesis. In contrast, SPC111 colonies pushed the capillary plexuses away and thus remained avascular for weeks. The key event in SPC111 vascularization was the development of a desmoplastic matrix of mouse origin. Continuously invaded by SPC111 cells, this matrix transformed into intratumoral connective tissue trunks, providing a route for ES from the diaphragm. Conclusions: Here, we report two distinct growth patterns of orthotopically implanted human MPM xenografts. In the invasive pattern, MPM cells invade and thus co-opt peritumoral capillary plexuses. In the pushing/desmoplastic pattern, MPM cells induce a desmoplastic response within the underlying tissue which allows the ingrowth of a nutritive vasculature from the pleura.

In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BACKGROUND: Small-cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype-specific proteins of diagnostic and therapeutic relevance. METHODS: Pellets and cell media of 26 human SCLC cell lines were subjected to label-free shotgun proteomics for large-scale protein identification and quantitation, followed by in-depth bioinformatic analyses. Proteomic data were correlated with the cell lines' phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues. RESULTS: Our quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial-mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up- or downregulation in one subtype, including known druggable proteins and potential blood-based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC-A, DNA replication in SCLC-N, neurotrophin signalling in SCLC-P and epithelial-mesenchymal transition in SCLC-Y. Importantly, we identified the YAP1-driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC-Y (e.g. GPX8, PKD2 and UFO). CONCLUSIONS: We report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype-specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

Development and Evaluation of a Human Skin Equivalent in a Semiautomatic Microfluidic Diffusion Chamber.

There is an increasing demand for transdermal transport measurements to optimize topical drug formulations and to achieve proper penetration profile of cosmetic ingredients. Reflecting ethical concerns the use of both human and animal tissues is becoming more restricted. Therefore, the focus of dermal research is shifting towards in vitro assays. In the current proof-of-concept study a three-layer skin equivalent using human HaCaT keratinocytes, an electrospun polycaprolactone mesh and a collagen-I gel was compared to human excised skin samples. We measured the permeability of the samples for 2% caffeine cream using a miniaturized dynamic diffusion cell ("skin-on-a-chip" microfluidic device). Caffeine delivery exhibits similar transport kinetics through the artificial skin and the human tissue: after a rapid rise, a long-lasting high concentration steady state develops. This is markedly distinct from the kinetics measured when using cell-free constructs, where a shorter release was observable. These results imply that both the established skin equivalent and the microfluidic diffusion chamber can serve as a suitable base for further development of more complex tissue substitutes.

Molecular profiles of small cell lung cancer subtypes: therapeutic implications.

Small cell lung cancer (SCLC; accounting for approximately 13%-15% of all lung cancers) is an exceptionally lethal malignancy characterized by rapid doubling time and high propensity to metastasize. In contrast to the increasingly personalized therapies in other types of lung cancer, SCLC is still regarded as a homogeneous disease and the prognosis of SCLC patients remains poor. Recently, however, substantial progress has been made in our understanding of SCLC biology. Advances in genomics and development of new preclinical models have facilitated insights into the intratumoral heterogeneity and specific genetic alterations of this disease. This worldwide resurgence of studies on SCLC has ultimately led to the development of novel subtype-specific classifications primarily based on the neuroendocrine features and distinct molecular profiles of SCLC. Importantly, these biologically distinct subtypes might define unique therapeutic vulnerabilities. Herein, we summarize the current knowledge on the molecular profiles of SCLC subtypes with a focus on their potential clinical implications.

Development of arterial blood supply in experimental liver metastases.

In this study, we present a mechanism for the development of arterial blood supply in experimental liver metastases. To analyze the arterialization process of experimental liver metastases, we elucidated a few key questions regarding the blood supply of hepatic lobules in mice. The microvasculature of the mouse liver is characterized by numerous arterioportal anastomoses and arterial terminations at the base of the lobules. These terminations supply one hepatic microcirculatory subunit per lobule, which we call an arterial hepatic microcirculatory subunit (aHMS). The process of arterialization can be divided into the following steps: 1) distortion of the aHMS by metastasis; 2) initial fusion of the sinusoids of the aHMS at the tumor parenchyma interface; 3) fusion of the sinusoids located at the base of the aHMSs, which leads to the disruption of the vascular sphincter (burst pipe); 4) incorporation of the dilated artery and the fused sinusoids into the tumor; and 5) further development of the tumor vasculature (arterial tree) by proliferation, remodeling, and continuous incorporation of fused sinusoids at the tumor-parenchyma interface. This process leads to the inevitable arterialization of liver metastases above the 2000- to 2500-mum size, regardless of the origin and growth pattern of the tumor.

The primary mitogen (TCPOBOP)-induced hepatocyte proliferation is resistant to transforming growth factor- ß-1 inhibition.

BACKGROUND: Transforming growth factor (TGF)-ß-1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens. AIMS: In this study, we compared the proliferative response in the liver between wild-type and transgenic mice, overexpressing active TGF-ß-1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene). METHODS: The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real-time polymerase chain reaction analysis of cell cycle-related genes. RESULTS: Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF-ß-1 expression following the mitogenic treatment. CONCLUSIONS: TGF-ß-1 does not inhibit the primary mitogen-induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment.

Author Correction: Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Multicellular contractility contributes to the emergence of mesothelioma nodules.

Malignant pleural mesothelioma (MPM) has an overall poor prognosis and unsatisfactory treatment options. MPM nodules, protruding into the pleural cavity may have growth and spreading dynamics distinct that of other solid tumors. We demonstrate that multicellular aggregates can develop spontaneously in the majority of tested MPM cell lines when cultured at high cell density. Surprisingly, the nodule-like aggregates do not arise by excessive local cell proliferation, but by myosin II-driven cell contractility. Prominent actin cables, spanning several cells, are abundant both in cultured aggregates and in MPM surgical specimens. We propose a computational model for in vitro MPM nodule development. Such a self-tensioned Maxwell fluid exhibits a pattern-forming instability that was studied by analytical tools and computer simulations. Altogether, our findings may underline a rational for targeting the actomyosin system in MPM.

[Heterogeneity of small cell lung cancer: biological and clinicopathological implications]. / A kissejtes tüdorák heterogenitásának biológiai és klinikopatológiai jelentosége.

Small cell lung cancer (SCLC; comprising approximately 14% of all lung cancer cases in Hungary) is an aggressive tumor type characterized by rapid growth and early metastasis. Although SCLC is a particularly malignant form of cancer, targeted therapies in its treatment have remained largely unsuccessful and thus there were no major therapeutic advances in the last three decades. SCLC was once considered a molecularly homogeneous malignancy. However, recent analyses led to the classification of neuroendocrine and molecular subtypes, based on the dominant expression of one of the following four transcriptional regulator genes: ASCL1, NEUROD1, YAP1 and POU2F3. Because these genetically and biologically distinct subtypes might contribute to therapeutic resistance, the better understanding of their biological and clinicopathological characteristics may help in the development of more effective SCLC therapies.

Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4.

Endothelial cell motility has fundamental role in vasculogenesis and angiogenesis during developmental or pathological processes. Tks4 is a scaffold protein known to organize the cytoskeleton of lamellipodia and podosomes, and thus modulating cell motility and invasion. In particular, Tks4 is required for the localization and activity of membrane type 1-matrix metalloproteinase, a key factor for extracellular matrix (ECM) cleavage during cell migration. While its role in transformed cells is well established, little is known about the function of Tks4 under physiological conditions. In this study we examined the impact of Tks4 gene silencing on the functional activity of primary human umbilical vein endothelial cells (HUVEC) and used time-lapse videomicrosopy and quantitative image analysis to characterize cell motility phenotypes in culture. We demonstrate that the absence of Tks4 in endothelial cells leads to impaired ECM cleavage and decreased motility within a 3-dimensional ECM environment. Furthermore, absence of Tks4 also decreases the ability of HUVEC cells to form multicellular sprouts, a key requirement for angiogenesis. To establish the involvement of Tks4 in vascular development in vivo, we show that loss of Tks4 leads sparser vasculature in the fetal chorion in the Tks4-deficient 'nee' mouse strain.

Origin and Distribution of Connective Tissue and Pericytes Impacting Vascularization in Brain Metastases With Different Growth Patterns.

The impact of growth pattern on the distribution of connective tissue and on the vascularization of brain metastases (40 colon, lung and breast carcinoma samples) was analyzed. Most of the cases showed either a "pushing-type" (18/40, mostly colon and lung carcinomas) or a "papillary-type" (19/40, mostly breast carcinomas) growth pattern. There was a striking difference in the growth pattern and vascularization of colon/lung versus breast carcinoma metastases. Pushing-type brain metastases incorporated fewer vessels and accumulated more collagen in the adjacent brain parenchyma, whereas papillary-type brain metastases incorporated more vessels and accumulated collagen in the center of the tumor. We observed duplication of the PDGFRß-positive pericyte layer accompanied by an increase in the amount of collagen within the vessel walls. The outer layer of pericytes and the collagen was removed from the vessel by invasive activity of the tumors, which occurred either peri- or intratumorally, depending on the growth pattern of the metastasis. Our findings suggest that pericytes are the main source of the connective tissue in brain metastases. Vascularization and connective tissue accumulation of the brain metastases largely depend on the growth pattern of the tumors.