Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing.

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

Research Progress in Acute Hypertensive Renal Injury by "in Vivo Cryotechnique".

Arterial hypertension has a large prevalence in the general population and as a major hypertensive target organ, the involvement of kidney is usually hard to avoid and gradually develops into chronic kidney disease (CKD). Acute hypertension is defined as a blood pressure greater than 180/120, also known as hypertensive emergency (HE). In acute severe hypertension, the pathophysiology damage to the kidney tends to worsen on the basis of chronic damage, and accounts for more significant mortality. However, the mechanisms of renal injury induced by acute hypertension remain unclear. This review summarizes the clinical and histopathological features of hypertensive renal injury by using "in vivo cyrotechnique" and focusses on the interplay of distinct systemic signaling pathways, which drive glomerular podocyte injury. A thorough understanding of the cellular and molecular mechanisms of kidney damage and repair in hypertension will provide significant insight into the development of new research methods and therapeutic strategies for global CKD progression.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets.

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.

Induction of non-apoptotic programmed cell death by oncogenic RAS in human epithelial cells and its suppression by MYC overexpression.

Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers.

Inflammatory mediator ultra-low-molecular-weight hyaluronan triggers necrosis of B-precursor leukemia cells with high surface CD44 expression.

Acute lymphoblastic leukemia (ALL) with mixed lineage leukemia (MLL) gene rearrangements (MLL+ALL) has a dismal prognosis and is characterized by high surface CD44 expression. Known that CD44 has the specific binding sites for a natural ligand hyaluronan (HA), we investigated biological effects of HA with different molecular sizes on MLL+ALL cell lines, and found that the addition of ultra-low-molecular-weight (ULMW)-HA strongly suppressed their thymidine uptakes. The MLL+ALL cell line lacking surface CD44 expression established by genome editing showed no suppression of thymidine uptake. Surface CD44-high B-precursor ALL cell lines other than MLL+, but not T-ALL cell lines, were also suppressed in their thymidine uptakes. The inhibition of thymidine uptakes was because of induction of cell death, but dead cells lacked features of apoptosis on cytospin smears and flow cytometric analysis. The cell death was neither blocked by pan-caspase inhibitor nor autophagy inhibitor, but was completely blocked by necrosis inhibitor necrostatin-1. Necrotic cell death was further supported by a marked release of a high-mobility protein group B1 and morphological changes on transmission electron microscopy. Elevation of intracellular reactive oxygen species production suggested a role for inducing this necrotic cell death. ULMW-HA-triggered cell death was similarly demonstrated in surface CD44-high primary B-precursor leukemia cells. Assuming that ULMW-HA is abundantly secreted at the site of infection and inflammation, this study sheds light on understanding the mechanism of a transient inflammation-associated remission of leukemia. Further, the CD44-targeting may become an effective approach in future for the treatment of refractory B-precursor ALL by its capability of predominantly eradicating CD44-high leukemia-initiating cells.

Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes.

The high-risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ-domain-containing proteins through its carboxy-terminal motif. This PDZ-binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6-transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRASG12V ) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild-type E6. Interestingly, we found decreased expression of PAR3 in E6-expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ-domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRASG12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild-type E6. These data highlight the importance of the carboxy-terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.

Renaissance of morphological studies: the examination of functional structures in living animal organs using the in vivo cryotechnique.

Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living animal organs, as well as the locations of their molecular components. Recently, the live imaging of animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living animal organs are compatible with those obtained from live images of the same organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living animal organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components-such as rapidly changing signaling molecules, membrane channels, or receptors-in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.

Morphofunctional Merits of an In Vivo Cryotechnique for Living Animal Organs: Challenges of Clinical Applications from Basic Medical Research.

Recent advances in molecular and genetic techniques have led to establishment of new biomedical fields; however, morphological techniques are still required for a more precise understanding of functioning cells and tissues. Conventional preparation procedures involve a series of chemical fixation, alcohol dehydration, paraffin or epoxy resin embedding, sectioning, and staining steps. In these steps, technical artifacts modify original morphologies of the cells being examined. Furthermore, difficulties are associated with capturing dynamic images in vivo using conventional chemical fixation. Therefore, a quick-freezing (QF) method was introduced for biological specimens in the 20th century. However, specimens have to be resected from living animal organs with blood supply, and their dynamical morphologies have not been investigated in detail using the QF method. In order to overcome these issues, the tissue resection step of organs had to be avoided and samples needed to be frozen under blood circulation. Our in vivo cryotechnique (IVCT) was an original technique to cryofix samples without resecting their tissues. The most significant merit of IVCT is that blood circulation into organs is preserved at the exact moment of freezing, which has been useful for arresting transient physiological processes of cells and tissues and maintaining their components in situ.

Immunohistochemical study of the membrane skeletal protein, membrane protein palmitoylated 6 (MPP6), in the mouse small intestine.

The membrane protein palmitoylated (MPP) family belongs to the membrane-associated guanylate kinase (MAGUK) family. MPP1 interacts with the protein 4.1 family member, 4.1R, as a membrane skeletal protein complex in erythrocytes. We previously described the interaction of another MPP family, MPP6, with 4.1G in the mouse peripheral nervous system. In the present study, the immunolocalization of MPP6 in the mouse small intestine was examined and compared with that of E-cadherin, zonula occludens (ZO)-1, and 4.1B, which we previously investigated in intestinal epithelial cells. The immunolocalization of MPP6 was also assessed in the small intestines of 4.1B-deficient (-/-) mice. In the small intestine, Western blotting revealed that the molecular weight of MPP6 was approximately 55-kDa, and MPP6 was immunostained under the cell membranes in the basolateral portions of almost all epithelial cells from the crypts to the villi. The immunostaining pattern of MPP6 in epithelial cells was similar to that of E-cadherin, but differed from that of ZO-1. In intestinal epithelial cells, the immunostained area of MPP6 was slightly different from that of 4.1B, which was restricted to the intestinal villi. The immunolocalization of MPP6 in small intestinal epithelial cells was similar between 4.1B(-/-) mice and 4.1B(+/+) mice. In the immunoprecipitation study, another MAGUK family protein, calcium/calmodulin-dependent serine protein kinase (CASK), was shown to molecularly interact with MPP6. Thus, we herein showed the immunolocalization and interaction proteins of MPP6 in the mouse small intestine, and also that 4.1B in epithelial cells was not essential for the sorting of MPP6.

Involvement of membrane skeletal molecules in the Schmidt-Lanterman incisure in Schwann cells.

Membrane skeletal networks form a two-dimensional lattice structure beneath erythrocyte membranes. 4.1R-MPP (membrane palmitoylated protein) 1-glycophorin C is one of the basic molecular complexes of the membrane skeleton. An analogous molecular complex, 4.1G-MPP6-cell adhesion molecule 4 (CADM4), is incorporated into the Schmidt-Lanterman incisure (SLI), a truncated cone shape in the myelin internode that is a specific feature of myelinated nerve fibers formed in Schwann cells in the peripheral nervous system. In this review, the dynamic structure of peripheral nerve fibers under stretching conditions is demonstrated using in vivo cryotechnique. The structures of nerve fibers had a beaded appearance, and the heights of SLI circular-truncated cones increased at the narrow sites of nerve fibers under the stretched condition. The height of SLI-truncated cones was lower in 4.1G-deficient nerve fibers than in wild-type nerve fibers. 4.1G was essential for the molecular targeting of MPP6 and CADM4 in SLI. The signal transduction protein, Src, was also involved in the 4.1G-MPP6-CADM4 molecular complex. The phosphorylation of Src was altered by the deletion of 4.1G. Thus, we herein demonstrate a membrane skeletal molecular complex in SLI that has potential roles in the regulation of adhesion and signal transduction as well as in structural stability in Schwann cells.

Immunohistochemical and morphofunctional studies of skeletal muscle tissues with electric nerve stimulation by in vivo cryotechnique.

In this study, morphological and immunohistochemical alterations of skeletal muscle tissues during persistent contraction were examined by in vivo cryotechnique (IVCT). Contraction of gastrocnemius muscles was induced by sciatic nerve stimulation. The IVCT was performed immediately, 3 min or 10 min after the stimulation start. Prominent ripples of muscle fibers or wavy deformation of sarcolemma were detected immediately after the stimulation, but they gradually diminished to normal levels during the stimulation. The relative ratio of sarcomere and A band lengths was the highest in the control group, but it immediately decreased to the lowest level and then gradually recovered at 3 min or 10 min. Although histochemical intensity of PAS reaction was almost homogeneous in muscle tissues of the control group or immediately after the stimulation, it decreased at 3 min or 10 min. Serum albumin was immunolocalized as dot-like patterns within some muscle fibers at 3 min stimulation. These patterns became more prominent at 10 min, and the dots got larger and saccular in some sarcoplasmic regions. However, IgG1 and IgM were immunolocalized in blood vessels under nerve stimulation conditions. Therefore, IVCT was useful to capture the morphofunctional and metabolic changes of heterogeneous muscle fibers during the persistent contraction.

Morphological and immunohistochemical analyses of soluble proteins in mucous membranes of living mouse intestines by cryotechniques.

We have performed immunohistochemical or ultrastructural analyses of living mouse small intestines using Epon blocks prepared by 'in vivo cryotechnique' (IVCT). By electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5-10 µm away from cryogen-contact surface tissues. Their microvilli contained dynamically waving actin filaments, and highly electron-dense organelles, such as mitochondria, were seen under the widely organized terminal web. By quick-freezing of fresh resected tissues (FT-QF), many erythrocytes were congested within blood vessels due to loss of blood pressure. By immersion-fixation (IM-DH) and perfusion-fixation (PF-DH), small vacuoles were often seen in the cytoplasm of epithelial cells, and their intercellular spaces were also dilated. Moreover, actin filament bundles were irregular in cross sections of microvilli, compared with those with IVCT. Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval and immunostained for immunoglobulin A (IgA), Ig kappa light chain (Igκ), J-chain and albumin. By cryotechniques, IgA immunoreactivity was detected as tiny dot-like patterns in cytoplasm of some epithelial cells. Both J-chain and Igκ immunoreactivities were detected in the same local areas as those of IgA. By FT-QF, however, the IgA immunoreactivity was more weakly detected, compared with that with IVCT. In thick sections prepared by IM-DH and PF-DH, it was rarely observed in both plasma and epithelial cells. Another albumin was diffusely immunolocalized in extracellular matrices of mucous membranes and also within blood vessels. Thus, IVCT was useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.

Activation of NF-κB by human papillomavirus 16 E1 limits E1-dependent viral replication through degradation of E1.

UNLABELLED: NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE: A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-κB mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-κB activity is involved in the establishment and maintenance of persistent HPV infection.

Three-dimensional volume imaging with electron microscopy toward connectome.

Ultrastructural analyses with electron microscopy have provided indispensable information to understand physiology and pathology of the nervous system. Recent advancement in imaging methodology paved the way for complete reconstruction of the neuronal connection map in the central nervous system, which is termed 'connectome' and would provide key insights to understand the functions of the brain. The critical advancement includes serial ultrastructural observation with scanning electron microscopy (SEM) instead of conventional serial sectioning transmission electron microscopy along with specific tissue preparation methods to increase heavy metal deposition for efficient SEM imaging. The advanced imaging methods using SEM have distinct advantages and disadvantages in multiple aspects, such as resolution and imaging speed, and should be selected depending on the observation conditions, such as target tissue sizes, required spatial resolution and necessity for re-observation. Dealing with the huge dataset remained to be a major obstacle, and automation in segmentation and 3D reconstruction would be critical to understand neuronal circuits in a larger volume of the brain. Future improvement in acquisition and analyses of the morphological data obtained with the advanced SEM imaging is awaited to elucidate the significance of whole connectome as the structural basis of the consciousness, intelligence and memory of a subject.

A human cancer xenograft model utilizing normal pancreatic duct epithelial cells conditionally transformed with defined oncogenes.

Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS(G12V), MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Reexpression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three-dimensional culture. In combination with p53 silencing, KRAS(G12V) alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasinormal human tissues preformed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites.

Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG) protein in fulminant type 1 diabetes.

BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.

Immunohistochemical study of mouse sciatic nerves under various stretching conditions with "in vivo cryotechnique".

BACKGROUND: In living animal bodies, some morphological changes of nerve fibers will probably occur when peripheral nerves are stretched or not stretched during various joint exercises. We aimed to capture the dynamic structures of nerves under various stretching conditions and to keep soluble serum proteins in their tissue sections. NEW METHOD: Morphological changes of stretched or non-stretched sciatic nerve fibers were examined with "in vivo cryotechnique" (IVCT). Fibers were directly frozen with liquid isopentane-propane cryogen (-193°C). Immunolocalizations of protein 4.1G and albumin were also examined in the fibers. RESULTS: The structures of IVCT-prepared sciatic nerves under the stretched condition showed a beaded appearance. By immunostaining for membrane skeletal protein 4.1G, Schmidt-Lanterman incisures (SLIs) were clearly identified, and the heights of their circular truncated cones were increased at narrow sites of the nerve fibers under the stretched condition, compared to those of non-stretched nerve fibers. Albumin was immunolocalized in blood vessels and also along endoneurium including regions near the node of Ranvier. COMPARISON WITH EXISTING METHODS: With the conventional perfusion-fixation method (PF), it was difficult to keep stable postures of living mouse limbs for tissue preparation. In nerve fibers after PF, the structures of SLI were easily modified, and albumin was heterogeneously immunolocalized due to diffusion artifacts. CONCLUSIONS: IVCT revealed (1) the structures of peripheral nerve fibers under dynamically different conditions, indicating that the morphological changes of SLIs play a functional role as a bumper structure against mechanical forces, and (2) accurate immunolocalization of serum albumin in the sciatic nerve fibers.

Bioimaging of fluorescence-labeled mitochondria in subcutaneously grafted murine melanoma cells by the "in vivo cryotechnique".

The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes.

Imaging of thrombosis and microcirculation in mouse lungs of initial melanoma metastasis with in vivo cryotechnique.

Microscopic bioimaging of blood flow and distribution of cancer cells in lungs is essential to analyze mechanism of lung metastasis. Such cancer metastasis has been well known to induce hypercoagulable states and thrombosis. In histopathological tissue sections, however, it has been difficult to capture rapid phenomenon of thrombus formation due to technical problems associated with much less retention of soluble serum components as well as dynamic histological features reflecting their living states. In this study, to achieve bioimaging of both hypercoagulable states and thrombosis induced by early metastasis of mouse B16-BL6 melanoma, "in vivo cryotechnique" (IVCT) was used, which retained soluble components at their original sites. Glutathione-coated quantum dots (QDs) were subsequently injected after melanoma cells via right ventricles to examine plasma flow with fluorescence emission. At 5s after the melanoma injection, melanoma cells were mostly stacked and intruded in alveolar capillaries with changing their shapes. Assembly of platelets initially appeared at 1min, and they aggregated around the stacked melanoma cells at 5min. Such aggregated platelets were immunopositive for both phospho-tyrosine 418 and 527 of Src, indicating their partial signal activation. Fibrin monomers were also immunolocalized around both melanoma cells and platelet aggregates, and massive immunoreaction deposits of fibrinogen were also detected near the same areas, but more strongly detected around the melanoma cells, indicating initial thrombus formation. In those areas, QDs were rarely detected, probably because of the lack of blood supply. Thus, IVCT revealed histopathological features of initial thrombosis under their circulatory conditions.