Dual-color, whole-body imaging in mice.
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Animals↗
Biomedical subjects
Meng Yang
Publications and source records attributed to Meng Yang.
Role of arachidonic acid in hyposmotic membrane stretch-induced increase in calcium-activated potassium currents in gastric myocytes.
AIM: To study effects of arachidonic acid (AA) and its metabolites on the hyposmotic membrane stretch-induced increase in calcium-activated potassium currents (I(KCa)) in gastric myocytes. METHODS: Membrane currents were recorded by using a conventional whole cell patch-clamp technique in gastric myocytes isolated with collagenase. RESULTS: Hyposmotic membrane stretch and AA increased both I(K(Ca))) and spontaneous transient outward currents significantly. Exogenous AA could potentiate the hyposmotic membrane stretch-induced increase in I(K(Ca)). The hyposmotic membrane stretch-induced increase in I(K(Ca)) was significantly suppressed by dimethyleicosadienoic acid (100 micromol/L in pipette solution), an inhibitor of phospholipase A2. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly suppressed AA and hyposmotic membrane stretch-induced increases in I(K(Ca)). External calcium-free or gadolinium chloride, a blocker of stretch-activated channels, blocked the AA-induced increase in I(K(Ca)) significantly, but it was not blocked by nicardipine, an L-type calcium channel blocker. Ryanodine, a calcium-induced calcium release agonist, completely blocked the AA-induced increase in I(K(Ca)); however, heparin, a potent inhibitor of inositol triphosphate receptor, did not block the AA-induced increase in I(K(Ca)). CONCLUSION: Hyposmotic membrane stretch may activate phospholipase A2, which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca(2+) influx, which triggers Ca(2+)-induced Ca(2+) release and elicits activation of I(K(Ca)) in gastric antral circular myocytes of the guinea pig.
Animals↗
Facile whole-body imaging of internal fluorescent tumors in mice with an LED flashlight.
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Animals↗
Transgenic nude mouse with ubiquitous green fluorescent protein expression as a host for human tumors.
We report here the development of the transgenic green fluorescent protein (GFP) nude mouse with ubiquitous GFP expression. The GFP nude mouse was obtained by crossing nontransgenic nude mice with the transgenic C57/B6 mouse in which the beta-actin promoter drives GFP expression in essentially all tissues. In crosses between nu/nu GFP male mice and nu/+ GFP female mice, the embryos fluoresced green. Approximately 50% of the offspring of these mice were GFP nude mice. Newborn mice and adult mice fluoresced very bright green and could be detected with a simple blue-light-emitting diode flashlight with a central peak of 470 nm and a bypass emission filter. In the adult mice, the organs all brightly expressed GFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, and duodenum. The following systems were dissected out and shown to have brilliant GFP fluorescence: the entire digestive system from tongue to anus; the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart and major arteries and veins. The skinned skeleton highly expressed GFP. Pancreatic islets showed GFP fluorescence. The spleen cells were also GFP positive. Red fluorescent protein (RFP)-expressing human cancer cell lines, including PC-3-RFP prostate cancer, HCT-116-RFP colon cancer, MDA-MB-435-RFP breast cancer, and HT1080-RFP fibrosarcoma were transplanted to the transgenic GFP nude mice. All of these human tumors grew extensively in the transgenic GFP nude mouse. Dual-color fluorescence imaging enabled visualization of human tumor-host interaction by whole-body imaging and at the cellular level in fresh and frozen tissues. The GFP mouse model should greatly expand our knowledge of human tumor-host interaction.
Actins↗
Effect of actin microfilament on potassium current in guinea pig gastric myocytes.
AIM: To investigate the effect of actin microfilament on potassium current and hyposmotic membrane stretch-induced increase of potassium current in gastric antral circular myocytes of guinea pig. METHODS: Whole-cell patch clamp technique was used to record potassium current in isolated gastric myocyes. RESULTS: When the membrane potential was clamped at -60 mV, an actin microfilament disruptor, cytochanlasin-B(Cyt-B, 20 micromol/L in pipette) increased calcium-activated potassium current (I(K(Ca))) and delayed rectifier potassium current (I(K(V))) to 138.4+/-14.3% and 142.1+/-13.1% respectively at +60 mV. In the same condition, an actin microfilament stabilizer phalloidin (20 micromol/L in pipette) inhibited I(K(Ca)) and I(K(V)) to 74.2+/-7.1% and 75.4+/-9.9% respectively. At the holding potential of -60 mV, hyposmotic membrane stretch increased I(K(Ca)) and I(K(V)) by 50.6+/-9.7% and 24.9+/-3.3% at +60 mV respectively. In the presence of cytochalasin-B and phalloidin (20 micromol/L, in the pipette) condition, hyposmotic membrane stretch also increased I(K(Ca)) by 44.5+/-7.9% and 55.7+/-9.8% at +60 mV respectively. In the same condition, cytochalasin-B and phalloidin also increased I(K(V)) by 23.0+/-5.5% and 30.3+/-4.5% respectively. However, Cyt-B and phalloidin did not affect the amplitude of hyposmotic membrane stretch-induced increase of I(K(Ca)) and I(K(V)). CONCLUSION: Actin microfilaments regulate the activities of potassium channels, but they are not involved in the process of hyposmotic membrane stretch-induced increase of potassium currents in gastric antral circular myocytes of guinea pig.
Actin Cytoskeleton↗
Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells.
Besides forming hair shafts, the highly organized, metabolically vigorous hair follicle plays several crucial roles in skin architecture. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located principally in the follicular bulge region. Nestin-driven GFP (ND-GFP), transfected into mice, principally labels cells in the bulge region, which is consistent with the cells' being the stem cells of the hair follicle. We report here that ND-GFP also labels developing skin blood vessels that appear to originate from hair follicles and form a follicle-linking network. This is seen most clearly by transplanting ND-GFP-labeled vibrissa (whisker) hair follicles to unlabeled nude mice. New vessels grow from the transplanted follicle, and these vessels increase when the local recipient skin is wounded. The ND-GFP-expressing structures are blood vessels, because they display the characteristic endothelial-cell-specific markers CD31 and von Willebrand factor. This model displays very early events in skin angiogenesis and can serve for rapid antiangiogenesis drug screening.
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Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-color nuclear-cytoplasmic fluorescent-protein expression.
We report here the genetic engineering of dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Nuclear GFP expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic RFP expression enabled visualization of nuclear cytoplasmic ratios as well as simultaneous cell and nuclear shape changes. Thus, total cellular dynamics can be visualized in the living dual-color cells in real time. The parental HT-1080 and the derived dual-color clones had similar cell proliferation rates, suggesting that expression of GFP and/or RFP does not affect cell cycle progression. The cell cycle position of individual living cells was readily visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse brain where extreme elongation of the cell body as well as the nucleus occurred. Dual-color cells in various positions of the cell cycle were visualized in excised mouse lungs after tail-vein injection of the dual-color cells. In the lung, the dual-color cells were observed frequently juxtaposing their nuclei, suggesting a potential novel form of cell-cell communication. The dual-color cells thus are a useful tool for visualizing living-cell dynamics in vivo as well as in vitro. Drugs that could specifically perturb these processes can now be readily screened in real time in vivo.
Animals↗
Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells.
LyP-1 is a peptide selected from a phage-displayed peptide library that specifically binds to tumor and endothelial cells of tumor lymphatics in certain tumors. Fluorescein-conjugated LyP-1 and a related peptide, LyP-1b, strongly accumulated in primary MDA-MB-435 breast cancer xenografts and their metastases from i.v. peptide injections, allowing visualization of orthotopic tumors in intact mice. The LyP peptide accumulation coincided with hypoxic areas in tumors. LyP-1 induced cell death in cultured human breast carcinoma cells that bind and internalize the peptide. Melanoma cells that do not bind LyP-1 were unaffected. Systemic LyP-1 peptide treatment of mice with xenografted tumors induced with the breast cancer cells inhibited tumor growth. The treated tumors contained foci of apoptotic cells and were essentially devoid of lymphatics. These results reveal an unexpected antitumor effect by the LyP-1 peptide that seems to be dependent on a proapoptotic/cytotoxic activity of the peptide. As LyP-1 affects the poorly vascularized tumor compartment, it may complement treatments directed at tumor blood vessels.
Angiogenesis Inhibitors↗
Syngeneic lymph-node-targeting model of green fluorescent protein-expressing Lewis lung carcinoma.
The Lewis lung tumor has been extensively studied in both syngeneic and allogeneic mouse models. However, its metastatic potential and mechanism are poorly understood. The aim of the present study was to develop a highly metastatic lymph-node targeting, imageable model of the Lewis lung carcinoma in a syngeneic host. We report here a syngeneic model of the Lewis lung carcinoma in which the carcinoma cells are labeled with green fluorescent protein (GFP). The tumor cells were transplanted in the dorsal side of the ear of C57-B16 mice in order to give the tumor cells access to the lymphatic system. This model of the Lewis lung carcinoma extensively metastasized to numerous lymph nodes throughout the body of the animal as well as visceral organs, as visualized by fluorescence microscopy using the bright GFP signal. Twenty-one different metastatic sites, including lymph nodes throughout the body, were identified among the cohort of transplanted animals. The data demonstrate a predilection of the Lewis lung carcinoma for lymphatic pathways for metastasis throughout the animal body. The concomitant macrometastases to the visceral organs observed in this model may be remetastasis from the lymph nodes. This model of the Lewis lung carcinoma should be very useful in defining cellular trafficking and targeting mechanisms of metastasis, in particular those involving lymphatic pathways.
Animals↗
Development of a green fluorescent protein metastatic-cancer chick-embryo drug-screen model.
The chick-embryo model has been an important tool to study tumor growth, metastasis, and angiogenesis. However, an imageable model with a genetic fluorescent tag in the growing and spreading cancer cells that is stable over time has not been developed. We report here the development of such an imageable fluorescent chick-embryo metastatic cancer model with the use of green fluorescent protein (GFP). Lewis lung carcinoma cells, stably expressing GFP, were injected on the 12th day of incubation in the chick embryo. GFP-Lewis lung carcinoma metastases were visualized by fluorescence, after seven days additional incubation, in the brain, heart, and sternum of the developing chick embryo, with the most frequent site being the brain. The combination of streptokinase and gemcitabine was evaluated in this GFP metastatic model. Twelve-day-old chick embryos were injected intravenously with GFP-Lewis lung cancer cells, along with these two agents either alone or in combination. The streptokinase-gemcitabine combination inhibited metastases at all sites. The effective dose of gemcitabine was found to be 10 mg/kg and streptokinase 2000 IU per embryo. The data in this report suggest that this new stably fluorescent imageable metastatic-cancer chick-embryo model will enable rapid screening of new antimetastatic agents.
Animals↗
Efficacy of intra-tumor injection of Kang-Lai-Te in treating transplanted hepatoma in rats.
BACKGROUND: Non-operative therapy takes an important position in comprehensive therapy of liver cancer. Despite some effects by using ethanol, acetic acid and heat saline for intra-tumor injection in the treatment of liver cancer, it is difficult to attain a complete cure but bring about injury to the liver to some extent. Hence, searching for other drugs for the local treatment of liver tumor is an important option. This study was designed to set up rat models of transplanted liver cancer, intra-tumor injection of Kang-Lai-Te (KLT), and negative control (saline) and positive control (ethanol). The effect of intra-tumor injection of KLT in treating transplanted hepatoma in rats and its advantages and disadvantages were assessed and the possibility of its use in treating patients with liver cancer was evaluated. METHODS: Forty rats were divided into 4 groups (G1, G2, G3 and G4, 10 rats in each group). Different drugs were injected into their implanted hepatoma (G1 with 0.2 ml saline as control, G2 with 10 mg KLT, G3 with 20 mg KLT, G4 with 0.2 ml ethanol). After 3 and 8 days, the hepatoma volume (HV), the serum levels of albumin, alanine aminotransferase(ALT), aspartate aminotransferase alkaline phosphatase(ALP) and creatinine, as well as the expression of proliferation cell nuclear antigen(PCNA) in hepatoma were detected. RESULTS: After 3 days, the HVs were smaller in G3 and G4 than in G1 (P<0.05), the serum levels of albumin were higher in G2 and G3 than in G1 and G4 (P<0.05), the serum levels of ALT and AST were lower in G2 and G3 than in G4 (P<0.05), the serum levels of ALP was lower in G2 and G3 than in G1 and G4 (P<0.05),the PCNA labeling indexes (PCNA LI) were lower in G2 and G3 than in G1 and G4 (P<0.05). After 8 days, the HVs were smaller in G2, G3 and G4 than in G1 (P<0.05), and the differences of HVs among G2, G3 and G4 were not significant. The serum levels of ALP were lower in G1, G2 and G3 than in G4 (P<0.05), and the PCNA LI were lower in G3 than in G1 and G4 (P<0.05). CONCLUSION: Intra-tumor injection of KLT into implanted hepatoma is evidently effective, but it is less effective than ethanol. The effect of KLT on liver function is markedly lower than that of ethanol.
Animals↗
Determination of clonality of metastasis by cell-specific color-coded fluorescent-protein imaging.
It has been thought that metastases are clonal and originate from rare cells in primary tumors that are heterogeneous in genotype and phenotype. Recent studies using DNA array analysis challenge this hypothesis and suggest the genetic background of the host is the important determinant of metastatic potential implying that metastases are not necessarily clonal. Previous methods to determine clonality of metastasis used karyotype or molecular analysis that were complicated, thereby limiting the number of metastatic colonies analyzed and the conclusions that could be drawn. We describe here the use of green fluorescent protein-labeled or red fluorescent protein-labeled HT-1080 human fibrosarcoma cells to determine clonality by simple fluorescence visualization of metastatic colonies after mixed implantation of the red and green fluorescent cells. Resulting pure red or pure green colonies were scored as clonal, whereas mixed yellow colonies were scored as nonclonal. In a spontaneous metastasis model originating from footpad injection in severe combined immunodeficient mice, 95% of the resulting lung colonies were either pure green or pure red, indicating monoclonal origin, whereas 5% were of mixed color, indicating polyclonal origin. In an experimental lung metastasis model established by tail vein injection in severe combined immunodeficient mice, clonality of lung metastasis was dependent on cell number. With a minimum cell number injected, almost all (96%) colonies were pure red or green and therefore monoclonal. When a large number of cells were injected, almost all (87%) colonies were mixed color and therefore heteroclonal. We conclude that spontaneous metastasis may be clonal because they are rare events, thereby supporting the rare-cell clonal origin of metastasis hypothesis. The clonality of the experimental metastasis model depended on the number of input cells. The simple fluorescence method of determining clonality of metastases described here can allow large-scale clonal analysis in numerous types of metastatic models.
Animals↗
Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells.
We have developed a simple yet powerful technique for delineating the morphological events of tumor-induced angiogenesis and other tumor-induced host processes with dual-color fluorescence. The method clearly images implanted tumors and adjacent stroma, distinguishing unambiguously the host and tumor-specific components of the malignancy. The dual-color fluorescence imaging is effected by using red fluorescent protein (RFP)-expressing tumors growing in GFP-expressing transgenic mice. This model shows with great clarity the details of the tumor-stroma interaction, especially tumor-induced angiogenesis and tumor-infiltrating lymphocytes. The GFP-expressing tumor vasculature, both nascent and mature, could be readily distinguished interacting with the RFP-expressing tumor cells. GFP-expressing dendritic cells were observed contacting RFP-expressing tumor cells with their dendrites. GFP-expressing macrophages were observed engulfing RFP-expressing cancer cells. GFP lymphocytes were seen surrounding cells of the RFP tumor, which eventually regressed. Dual-color fluorescence imaging visualizes the tumor-host interaction by whole-body imaging and at the cellular level in fresh tissues, dramatically expanding previous studies in fixed and stained preparations.
Animals↗
Prolonged dormancy and site-specific growth potential of cancer cells spontaneously disseminated from nonmetastatic breast tumors as revealed by labeling with green fluorescent protein.
This study used an isogenic pair of metastatic (M4A4) and nonmetastatic (NM2C5), green fluorescent protein-labeled human breast cancer cell lines derived from the same patient and inoculated into the mammary glands of nude mice to investigate the dissemination patterns and fate of cells that escaped spontaneously from the resulting tumors. After tumors appeared, fluorescing single tumor cells were regularly seen in the lungs, even in animals inoculated with NM2C5, which fails to form secondary tumors in other organs. The sensitivity of the technique confirmed the continuing presence of scattered NM2C5 cells after primary tumor resection, although they formed no metastases by 6 months. These self-disseminated human tumor cells were retrievable from the tissues and were still viable and malignant, manifested by indefinite proliferation in vitro and green fluorescence and local tumorigenicity in vivo. Therefore, these scattered tumor cells were still immortal but rendered indefinitely quiescent by the microenvironmental conditions in the lung tissue. This is the first unequivocal demonstration of spontaneous distant dissemination of human cancer cells by undisturbed nonmetastatic tumors and comprises a valuable system for the analysis of tumor dormancy. In contrast, although many of the cells disseminating from M4A4 tumors grew into fluorescing metastases in the lungs, others remained solitary and quiescent. Therefore, even in a clonally derived cell population with metastatic properties, many cells do not, or cannot, mobilize the organ-specific growth properties needed to generate metastases. This experimental approach, by using self-disseminating, green fluorescent protein-labeled, sister cell lines of opposing metastatic phenotypes, opens new avenues for investigating topics of clinical relevance, including tumor cell dormancy, anatomical distribution of metastases, and host factors influencing the metastatic process.
Animals↗
Nestin expression in hair follicle sheath progenitor cells.
The intermediate filament protein, nestin, marks progenitor cells of the CNS. Such CNS stem cells are selectively labeled by placing GFP under the control of the nestin regulatory sequences. During early anagen or growth phase of the hair follicle, nestin-expressing cells, marked by GFP fluorescence in nestin-GFP transgenic mice, appear in the permanent upper hair follicle immediately below the sebaceous glands in the follicle bulge. This is where stem cells for the hair follicle outer-root sheath are thought to be located. The relatively small, oval-shaped, nestin-expressing cells in the bulge area surround the hair shaft and are interconnected by short dendrites. The precise locations of the nestin-expressing cells in the hair follicle vary with the hair cycle. During telogen or resting phase and in early anagen, the GFP-positive cells are mainly in the bulge area. However, in mid- and late anagen, the GFP-expressing cells are located in the upper outer-root sheath as well as in the bulge area but not in the hair matrix bulb. These observations show that the nestin-expressing cells form the outer-root sheath. Results of the immunohistochemical staining showed that nestin, GFP, keratin 5/8, and keratin 15 colocalize in the hair follicle bulge cells, outer-root sheath cells, and basal cells of the sebaceous glands. These data indicate that nestin-expressing cells, marked by GFP, in the hair follicle bulge are indeed progenitors of the follicle outer-root sheath. The expression of the unique protein, nestin, in both neural stem cells and hair follicle stem cells suggests their possible relation.
Animals↗
Viable circulating metastatic cells produced in orthotopic but not ectopic prostate cancer models.
Elucidation of the mechanisms of hormone-independent metastatic prostate cancer remains a significant and highly relevant challenge. We report here that hormone-refractory human prostate carcinoma growing orthotopically efficiently deliver viable metastatic cells in the host circulation. This is in contrast to the ectopic tumors of the same lineage, which do not deliver live cells into the circulation. To investigate the malignant potential of viable circulating carcinoma cells, we developed a novel dual-color orthotopic coimplantation model of human prostate cancer metastasis in nude mice. This model is comprised of coinjection of an equivalent mixture of isolated and cultured circulating green fluorescent protein-expressing clones and parental red fluorescent protein-expressing human prostate carcinoma cells. In the dual-color model, the selected green fluorescent protein-labeled viable circulating cells have an increased metastatic propensity relative to the red fluorescent protein-labeled parental cells. The identification and isolation of highly malignant viable circulating human prostate carcinoma cells from orthotopic but not ectopic models will enable important new insights into the metastatic process including the role of the tumor microenvironment.
Animals↗
Real-time GFP imaging of spontaneous HT-1080 fibrosarcoma lung metastases.
Metastasis to the lung is often a lethal event in sarcoma as well as other cancers. We report here a new animal model of sarcoma enabling the external real-time fluorescence imaging of spontaneous lung metastasis. The human fibrosarcoma cell line HT-1080 was transduced with the green fluorescent protein (GFP) gene. HT-1080-GFP cells were injected into the right hind footpad of severe combined immunodeficient (SCID) mice. The lung metastases were evaluated by whole-body fluorescence imaging as well as direct-view imaging in live animals through a skin-flap window over the chest wall. Spontaneous lung metastases were observed on the lungs of 11 of 12 mice. SCID mice well tolerated the skin-flap procedure enabling real-time imaging of spontaneous lung metastases with a resolution of approximately 50-100 microm. This procedure enabled external imaging at the micrometastasis level. Real-time evaluation of spontaneous lung metastasis in the same animals should allow drug evaluation and mechanistic studies not previously possible.
Animals↗
Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo.
We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 microl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3 x 10(6) cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest.
Animals↗