Oligodendrocyte precursor cell transplantation into organotypic cerebellar shiverer slices: a model to study myelination and myelin maintenance

Current in vitro models to investigate the consequence of oligodendrocyte-specific loss-of-function mutations on myelination are primarily limited to co-culture experiments, which do not accurately recapitulate the complex in vivo environment. Here, we describe the development of an in vitro model of myelination and myelin maintenance in which oligodendrocyte precursor cells are transplanted into organotypic cerebellar slice cultures derived from dysmyelinated shiverer mice. Compared to neuron-oligodendrocyte co-cultures, organotypic slices more closely mimic the environment in vivo, while utilizing a genetic background that allows for straight-forward identification of myelin generated by transplanted cells. We show at the ultrastructural level that the myelin generated by wild-type transplanted oligodendrocytes is compact and terminates in cytoplasmic loops that form paranodal junctions with the axon. This myelination results in the appropriate sequestering of axonal proteins into specialized domains surrounding the nodes of Ranvier. We also demonstrate the applicability of this approach for xenograft transplantation of oligodendrocyte precursor cells derived from rat or human sources. This method provides a time-efficient and cost-effective adjunct to conditional knockout mouse lines or in vivo transplantation models to study oligodendrocyte-specific loss-of-function mutations. Furthermore, the approach can be readily used to assess the effect of pharmacological manipulations on myelin, providing a tool to better understand myelination and develop effective therapeutic strategies to treat myelin-related diseases.     

Mammary transmission of caprine arthritis encephalitis virus: a 3D model for in vitro study

Transmission of Caprine Arthritis Encephalitis virus (CAEV) from the mother to offspring is principally mediated by infected cells from colostrum and milk. The infection of the dam is often sub-clinical, and results in increased cellularity of milk, sometimes exacerbated by bacterial co-infections. Although monocytes are the major viral host cells, several other cell types, including epithelial mammary cells, fibroblasts and endothelial cells show low levels of in vivo infection. In vitro, however, all phenotypes of mammary gland cells are individually highly sensitive to CAEV infection. This suggests that local mechanisms act to control viral expression. Our goal is to analyse the mechanisms regulating local virus infection, including the physiological status of the mammary gland and bacterial co-infections. In this work, we present the development of a model for the in vitro reconstitution of mammary gland tissue using 3D cultures in Matrigel. Mononuclear cells from the blood are added to the 3D cultures in vitro. In these experimental conditions, the mammary cells spontaneously organize into mammospheres. Blood leucocytes migrate into the culture gel, and localize particularly at the periphery of the mammospheres. Mammospheres were susceptible to infection in vitro by CAEV, as shown by a cytopathic effect and expression of late CAEV antigen p30. This model will allow the in vitro study of virus expression, transfer of infection to mammary gland cells and interactions between the mammary gland cells, infected monocytes and immunocompetent cells. It will allow the study of mechanisms participating in the control of passage of pathogens into milk, according to the physiological and CAEV-infection status of the animal, microenvironment and the presence of bacterial co-infections.     

Integrating pharmacogenetics into society: in search of a model

There has been considerable scientific, corporate and policy interest in the more effective use of genetics in both drug development and delivery. Pharmacogenetics - the study of the relationship between an individual's genetic makeup and response to medicinal drugs - has attracted global interest, but will it live up to its promise? Looking beyond the hype that has accompanied much of the commentary in the area, the future of pharmacogenetics will depend on how competing interests and options are resolved.     

Rodent macrophages metabolize 25-hydroxyvitamin D3in,vitro

Rodent macrophages metabolized 25-hydroxyvitamin D₃ to an unidentified metabolite during $\text{in}\text{,}\text{vitro}$ incubations. The production of this macrophage-derived metabolite of 25-hydroxyvitamin D₃ increased as the substrate concentration was raised. A two step high pressure liquid chromatography system revealed a unique elution position of this macrophage-derived metabolite that did not match the elution positions of any of the vitamin D₃ metabolites available in this laboratory. This unique metabolite was formed $\text{in}\text{,}\text{vitro}$ within one minute by incubated macrophages although its formation increased gradually up to 60 minutes of incubation.     

Superoxide dismutase activity in organotypic midbrain-striatum co-cultures is associated with resistance of dopaminergic neurons to excitotoxicity

We have previously demonstrated that dopaminergic neurons in midbrain-striatum slice co-cultures are more resistant to NMDA cytotoxicity than the same neuronal population in single midbrain slice cultures. Here, we show that dopaminergic neurons in midbrain-striatum co-cultures also exhibit resistance to the cytotoxicity of nitric oxide donors, 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18) and 3-morpholinosydnonimine (SIN-1). The cytotoxicity of NMDA (30 microM) in single cultures was significantly attenuated by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (100 microM), whereas the toxicity in co-cultures was not. The levels of tyrosine residue nitration of tyrosine hydroxylase, a hallmark of the occurence of peroxynitrite anion in dopaminergic neurons, were lower in co-cultures than those in single cultures. Single cultures and co-cultures did not show appreciable differences in the number or distribution of NOS-containing neurons as assessed by NADPH diaphorase histochemistry. On the other hand, midbrain slices cultured with striatal slices showed higher levels of superoxide dismutase (SOD) activity as well as increased protein levels of Cu,Zn-SOD, than midbrain slices cultured alone. These results suggested that the generation of NO is involved in NMDA cytotoxicity on dopaminergic neurons, and that increased activity of SOD in co-cultures renders dopaminergic neurons resistant to NMDA cytotoxicity by preventing the formation of peroxynitrite.     

Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.     

Integrating 2D and 3D shell morphology to disentangle the palaeobiology of ammonoids: a virtual approach

Based on data derived from computed tomography, we demonstrate that integrating 2D and 3D morphological data from ammonoid shells represents an important new approach for investigating the palaeobiology of ammonoids. Characterization of ammonite morphology has long been constrained to 2D data, with only a few studies collecting ontogenetic data in 180° steps. Here we combine this traditional approach with 3D data collected from high‐resolution nano‐computed tomography. Ontogenetic morphological data on the hollow shell of a juvenile ammonite Kosmoceras (Jurassic, Callovian) was collected. 2D data was collected in 10° steps and show significant changes in shell morphology. Preserved hollow spines show multiple mineralized membranes never reported before, representing temporal changes in the ammonoid mantle tissue. 3D data show that chamber volumes do not always increase exponentially, as was generally assumed, but may represent a proxy for life events, such as stress phases. Furthermore, chamber volume cannot be simply derived from septal spacing in forms comparable to Kosmoceras. Vogel numbers represent a 3D parameter for chamber shape, and those for Kosmoceras are similar to other ammonoids (Arnsbergites, Amauroceras) and modern cephalopods (Nautilus, Spirula). Two methods to virtually document the suture line ontogeny, used to document phylogenetic relationships of larger taxonomic entities, were applied for the first time and present a promising alternative to hand drawings. The curvature of the chamber surfaces increases during ontogeny due to increasing strength of ornamentation and septal complexity. As this may allow for faster handling of cameral liquid, it could compensate for decreasing SA/V ratios through ontogeny.     

Clotrimazole presents anticancer properties against a mouse melanoma model acting as a PI3K inhibitor and inducing repolarization of tumor-associated macrophages

The immune system is a key component of tumorigenesis, with the latter promoting the development of cancer, its progression and metastasis. In fact, abundant infiltration of tumor-associated macrophages (TAM), which are M2-like macrophages, has been associated with a poor outcome in most types of cancers. Here, we show that lactate produced by murine melanoma B16F10 cells induces an M2-like profile in cultured macrophages. Further, we demonstrate that clotrimazole (CTZ), an off-target anti-tumor drug, abolishes lactate effects on the activation of macrophages and induces the expression of M1-like markers. We show that clotrimazole has cytotoxic effects on tumor cells by negatively modulating PI3K, which inhibits glycolytic metabolism and leads to a diminishing lactate production by these cells. These effects are more pronounced in cancer cells exposed to conditioned media of M2-polarized macrophages. Moreover, clotrimazole inhibits tumor growth in a murine model of implanted melanoma, reduces lactate content in a tumor microenvironment and decreases vascular endothelial growth factor expression. Finally, clotrimazole drastically diminishes TAM infiltration in the tumors, thereby inducing M1 polarization. Collectively, these findings identify a new antitumor mechanism of clotrimazole by modulating the tumor microenvironment (TME), particularly the activation and viability of TAM.     

Integration of hyper-compliant microparticles into a 3D melanoma tumor model

Multicellular spheroids provide a physiologically relevant platform to study the microenvironment of tumors and therapeutic applications, such as microparticle-based drug delivery. The goal of this study was to investigate the incorporation/penetration of compliant polyacrylamide microparticles (MPs), into either cancer or normal human cell spheroids. Incorporation of collagen-1-coated MPs (stiffness: 0.1 and 9 kPa; diameter: 15–30 µm) into spheroids (diameter ∼100 µm) was tracked for up to 22 h. Results indicated that cells within melanoma spheroids were more influenced by MP mechanical properties than cells within normal cell spheroids. Melanoma spheroids had a greater propensity to incorporate and displace the more compliant MPs over time. Mature spheroids composed of either cell type were able to recognize and integrate MPs. While many tumor models exist to study drug delivery and efficacy, the study of uptake and incorporation of cell-sized MPs into established spheroids/tissues or tumors has been limited. The ability of hyper-compliant MPs to successfully penetrate 3D tumor models with natural extracellular matrix deposition provides a novel platform for potential delivery of drugs and other therapeutics into the core of tumors and micrometastases.     

A physiological model to study iron recycling in macrophages

Following erythrophagocytosis (EP) of senescent red blood cells (RBCs), heme iron is recycled to the plasma by tissue macrophages. This process is critical for mammalian iron homeostasis but remains elusive. We characterized a cellular model using artificially-aged murine RBCs and murine bone marrow-derived macrophages (BMDMs) and study mRNA and protein expression of HO-1, ferroportin and ferritin after EP. In vitro ageing of RBCs was obtained by raising intracellular calcium concentration. These RBCs exhibit several features of erythrocyte senescence including externalization of phosphatidyl-serine, specific binding and phagocytosis by BMDMs. During the first hours of EP, we observed a rapid increase of HO-1 and ferroportin mRNAs and proteins, whereas ferritin protein expression was progressively induced with no major changes in RNA levels. At later stages after EP, a different pattern of expression was observed with a net decrease of ferroportin, a sustained high level of HO-1, and a strong increase in ferritins. Taken together, these results suggest that after EP, iron is rapidly extracted from heme and exported by ferroportin. Surprisingly, the gene expression profile at late stages after EP, which is indicative of iron storage, is reminiscent of what is observed in inflammation. However, phagocytosis of artificially-aged red blood cells seems to repress the proinflammatory response of macrophages.     

Differentiation in vitro of a leukemia virus-induced B-cell lymphoma into macrophages.

Cells of the hemopoietic system arise by proliferation and differentiation of progenitor cells. This process begins with multipotential stem cells which can self-renew and also undergo progressive differentiation to progenitor cells committed to particular lineages, ultimately yielding mature blood cells (D. Metcalf and M. A. S. Moore, Haematopoietic Cells, 1971). Early commitment of lymphoid progenitors is generally believed to separate the lymphoid lineage from the myeloid and erythroid lineages, whose progenitors are separated late in differentiation (Metcalf and Moore, 1971). We recently developed a derivative of Moloney murine leukemia virus (M-MuLV) in which the enhancer sequences from simian virus 40 were substituted into the M-MuLV long terminal repeat. This recombinant virus (delta Mo + SV M-MuLV) induces pre-B and B lymphoid leukemia with long latency after inoculation of 2-day-old NIH Swiss mice (R. Hanecak, P. K. Pattengale, and H. Fan, J. Virol. 62:2427-2436, 1988). In this report, we describe the derivation of a permanent, virus-producing cell line with the phenotypic characteristics of mature macrophages from a B-cell-derived lymphoblastic lymphoma induced by delta Mo + SV M-MuLV. Comparison studies of immunoglobulin heavy-chain gene rearrangements and also delta Mo + SV M-MuLV proviral integration sites confirmed that the macrophage cell line was derived from the original B-lymphoblastic lymphoma. Moreover, inoculation of the macrophage cell line into animals resulted in histiocytic sarcomas of the macrophage type, thus reflecting stable conversion of B-lymphoid tumor cells to the macrophage phenotype. These results suggest a closer relationship between lymphoid and myeloid cells than previously believed.     

A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation

The objective of our study was to establish spheroid cocultures as a valid 3-D in vitro model mimicking tumor-fibroblast interactions in scirrhous breast tumors. The experimental setup was designed to verify if in cocultures (a) adherence and migration reflect the invasive potential of breast tumor cells, (b) breast tumor cells induce tumor-associated fibroblast differentiation, and (c) tumor-derived fibroblasts better reflect the in vivo situation than normal skin fibroblasts. Only one (SK-BR-3) out of five tumor cell types showed extensive fibroblast infiltration, MCF-7 cells frequently invaded fibroblast spheroids; BT474, T47D, and ZR-75-1 were noninvasive. While tumor cell invasion was independent of fibroblast origin, tumor-associated myofibroblast differentiation defined by alpha-SMA expression was demonstrated for tumor-derived but not normal skin fibroblasts in coculture indicating that (a) tumor cell invasion and myofibroblast differentiation are autonomous processes and (b) cocultures with tumor-derived fibroblasts resemble advanced stages of desmoplastic carcinomas while cocultures with normal skin fibroblasts rather reflect the early tumor development. The latter is also implied by fibroblast-associated alterations in tumor cell morphology and ECM distribution in the system. By using RNA arbitrarily primed PCR and cells isolated from cocultures by fluorescence-activated and magnetic cell separation, peripheral myelin protein PMP22/SR13 has been identified as a novel candidate with potential relevance in the interaction between tumor cell and normal fibroblast since PMP22 mRNA was significantly reduced in normal skin fibroblasts in coculture with BT474 cells.     

Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion

Several lines of evidence suggest that dysregulated lipid metabolism may participate in the pathogenesis of Alzheimer’s disease (AD). Epidemiologic studies suggest that elevated mid-life plasma cholesterol levels may be associated with an increased risk of AD and that statin use may reduce the prevalence of AD. Cellular studies have shown that the levels and distribution of intracellular cholesterol markedly affect the processing of amyloid precursor protein into Aβ peptides, which are the toxic species that accumulate as amyloid plaques in the AD brain. Most importantly, genetic evidence identifies apolipoprotein E, the major cholesterol carrier in the central nervous system, as the primary genetic risk factor for sporadic AD. In humans, apoE exists as three major alleles (apoE2, apoE3, and apoE4), and inheritance of the apoE4 allele increases the risk of developing AD at an earlier age. However, exactly how apoE functions in the pathogenesis of AD remains to be fully determined. Our studies have identified that the cholesterol transporter ABCA1 is a crucial regulator of apoE levels and lipidation in the brain. Deficiency of ABCA1 leads to the loss of approximately 80% of apoE in the brain, and the residual 20% that remains is poorly lipidated. Several independent studies have shown this poorly lipidated apoE increases amyloid burden in mouse models of AD, demonstrating that apoE lipidation by ABCA1 affects key steps in amyloid deposition or clearance. Conversely, robust overexpression of ABCA1 in the brain promotes apoE lipidation and nearly eliminates the formation of mature amyloid plaques. These studies show that the lipid binding capacity of apoE is a major mechanism of its function in the pathogenesis of AD, and suggest that increasing apoE lipidation may be of therapeutic importance for this devastating disease.     

IL-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo

Tumor-associated macrophages (TAMs) play a major role in promoting tumor growth and metastasis and in suppressing the antitumor immune response. Despite the immunosuppressive environment created by the tumor and enforced by tumor-associated macrophages, treatment of tumor-bearing mice with IL-12 induces tumor regression associated with appearance of activated NK cells and activated tumor-specific CTLs. We therefore tested the hypothesis that IL-12 treatment could alter the function of these tumor-associated suppressor macrophages. Analysis of tumor-infiltrating macrophages and distal TAMs revealed that IL-12, both in vivo and in vitro, induced a rapid (<90 min) reduction of tumor supportive macrophage activities (IL-10, MCP-1, migration inhibitory factor, and TGFbeta production) and a concomitant increase in proinflammatory and proimmunogenic activities (TNF-alpha, IL-15, and IL-18 production). Similar shifts in functional phenotype were induced by IL-12 in tumor-infiltrating macrophages isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen, and peritoneal cavity. Therefore, although TAMs display a strongly polarized immunosuppressive functional profile, they retain the ability to change their functional profile to proinflammatory activities given the appropriate stimulus. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive antitumor immune response.