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1.
Anticancer Res ; 38(8): 4417-4423, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30061205

RESUMO

BACKGROUND/AIM: Our laboratory pioneered color-coded imaging of the tumor microenvironment (TME). We observed recruitment of cancer and stromal cells to the TME and recombination between cancer and stromal cells. The aim of the present study was to observe the dynamics of the TME by color-coded imaging during metastasis and in the formation of a pre-metastatic niche. MATERIALS AND METHODS: Red-fluorescent protein (RFP-expressing) mouse colon-cancer 26 cells were initially injected subcutaneously in green-fluorescent protein (GFP) nude mice. The resulting subcutaneous tumors were harvested and cultured. The cultured subcutaneous tumors contained RFP colon cancer cells, GFP stromal cells and recombinant cancer-stromal cells expressing yellow fluorescence. After 14 days culture, the cells were injected into the spleen. RESULTS: After splenic injection, colon-cancer 26 metastases were observed in the liver, ascites, and bone marrow. Using the Olympus FV1000 confocal microscope, the cells cultured from tumors and metastasis in each site were visualized. RFP colon-cancer cells, GFP stromal cells derived from host GFP nude mice, and recombinant yellow-fluorescent cells were observed in each organ. In addition, in the liver, areas with only GFP stromal cells were observed and assumed to be a pre-metastatic niche. CONCLUSION: Color-coded imaging demonstrated the dynamics of colon cancer and stromal cells at different metastatic sites including the formation of recombinant cancer-stromal cells.


Assuntos
Neoplasias do Colo/patologia , Metástase Neoplásica/patologia , Células Estromais/patologia , Microambiente Tumoral/fisiologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Cor , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Células Estromais/metabolismo , Proteína Vermelha Fluorescente
2.
Anticancer Res ; 38(4): 1927-1935, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29599308

RESUMO

BACKGROUND/AIM: The lethal characteristic of pancreatic cancer is metastasis which is recalcitrant to currently-used chemotherapy. Our aim was to understand metastasis at the cellular level. We previously reported that multi-nucleate cells or spindle cells were more prominent in pancreatic cancer metastasis than in the primary tumor. In the present report, we investigated four representative human pancreatic cell lines for differences in cell morphology between the primary tumor and various metastatic organ targets for each cell line. MATERIALS AND METHODS: Human pancreatic cancer cell lines AsPC-1, Panc-1, KP2 and KP3 were used. Pancreatic cancer cells were injected into spleen of nude mice resulting in experimental metastasis to various organs which were observed at the cellular level when the organs were placed into culture. RESULTS: AsPC-1 and KP2 pancreatic cells formed many experimental liver metastases, in contrast to Panc-1 and KP3. Lung metastasis was only observed for AsPC-1. In the cultures established from the primary and metastatic tumors, multi-nucleate cells were found to be more prominent in the metastasis of the pancreatic cell lines with frequent metastasis, AsPC-1 and KP2. Spindle-like cells were observed prominently in AsPC-1 lung metastasis. CONCLUSION: Human pancreatic cancer cell lines have differential metastatic characteristics with regard to target organs and cell-morphology changes. Multi-nucleate and spindle cells may play an important role in pancreatic cancer metastasis to the liver and lung, respectively.


Assuntos
Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Neoplasias Pancreáticas/patologia , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/diagnóstico por imagem
3.
Anticancer Res ; 37(7): 3429-3434, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28668831

RESUMO

BACKGROUND/AIM: Fatty liver disease is increasing in the developed and developing world. Liver metastasis from malignant lymphoma in the fatty liver is poorly understood. In a previous report, we developed color-coded imaging of the tumor microenvironment (TME) of the murine EL4-RFP malignant lymphoma during metastasis, including the lung. In the present report, we investigated the potential and microenvironment of the fatty liver induced by a choline-deficient diet as a metastatic site in this mouse lymphoma model. MATERIALS AND METHODS: C57BL/6-GFP transgenic mice were fed with a choline-deficient diet in order to establish a fatty liver model. EL4-RFP cells were injected in the spleen of normal mice and fatty-liver mice. Metastases in mice with fatty liver or normal liver were imaged with the Olympus SZX7 microscope and the Olympus FV1000 confocal microscope. RESULTS: Metastases of EL4-RFP were observed in the liver, ascites and bone marrow. Primary tumors were imaged in the spleen at the injection site. The fewest metastases were observed in the fatty liver. In addition, the fewest cancer-associated fibroblasts (CAFs) were observed in the fatty liver. CONCLUSION: The relative metastatic resistance of the fatty liver may be due to the reduced number of CAFs in the fatty livers. The mechanism of the effect of the choline-deficient diet is discussed.


Assuntos
Deficiência de Colina/metabolismo , Deficiência de Colina/patologia , Colina/metabolismo , Dieta/efeitos adversos , Fígado Gorduroso/metabolismo , Microambiente Tumoral/fisiologia , Animais , Ascite/metabolismo , Ascite/patologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Cor , Modelos Animais de Doenças , Fígado Gorduroso/patologia , Comportamento Alimentar/fisiologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/metabolismo , Baço/patologia
4.
Anticancer Res ; 37(7): 3435-3440, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28668832

RESUMO

BACKGROUND/AIM: The interaction between pancreatic-cancer cells and stromal cells in the tumor microenvironment (TME) is of particular importance in cancer progression and metastasis. The present report demonstrates the role of cancer-associated fibroblasts (CAFs) and multinucleate pancreatic-cancer cells in peritoneal metastasis. MATERIALS AND METHODS: An orthotopic mouse model of pancreatic cancer was established with the human pancreatic cancer cell line BxPC3, which stably expresses green fluorescent protein (GFP). RESULTS: BxPC3-GFP cells formed peritoneal metastases by week 18 after orthotopic implantation. Using an Olympus FV1000 confocal microscope, multi-nucleated cancer cells were frequently observed in the peritoneal metastases. The primary pancreatic tumor and peritoneal-metastases were harvested, cultured and then transplanted subcutaneously. Subcutaneous tumors established from peritoneal-metastatic cells were larger than subcutaneous tumors established from primary-tumor cells. Subcutaneous tumors of each type were subsequently cultured in vitro. CAFs were observed growing out from the tumors established from peritoneal-metastatic cells, but not the tumors established from the primary cancer. CONCLUSION: The results of the present study suggest that multi-nucleated cancer cells and CAFs were related to peritoneal metastasis of pancreatic cancer.


Assuntos
Fibroblastos Associados a Câncer/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Peritoneais/patologia , Peritônio/patologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Peritoneais/metabolismo , Peritônio/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Microambiente Tumoral/fisiologia
5.
Anticancer Res ; 36(9): 4443-8, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27630280

RESUMO

BACKGROUND/AIM: Fluorescence-guided surgery (FGS) of cancer is an emerging technology. We have previously shown the importance of resecting both the tumor and the tumor microenvironment (TME) for curative FGS. We also previously developed a syngeneic model using the mouse lymphoma cell line EL-4, expressing red fluorescent protein (EL-4-RFP), growing in green fluorescent protein (GFP) transgenic mice, which we have used in the present report to develop FGS of the tumor microenvironment. MATERIALS AND METHODS: EL-4-RFP lymphoma cells were injected subcutaneously in C57/BL6 GFP transgenic mice. EL-4-RFP cells subsequently formed tumors by 35 days after cell transplantation. Using the portable hand-held Dino-Lite digital imaging system, subcutaneous tumors were resected by FGS. Resected tumor tissues were visualized with the Olympus FV1000 confocal microscope. RESULTS: Using the Dino-Lite, subcutaneous tumors and the tumor microenvironment were clearly visualized and resected. In the resected tumor, host stromal cells, including adipocyte-like cells and blood vessels with lymphocytes, were observed by confocal microscopy in addition to cancer cells by color-coded confocal imaging. The cancer cells and stromal cells in the TME were deeply intermingled in a highly-complex pattern. CONCLUSION: Color-coded FGS is an effective method to completely resect cancer cells along with the stromal cells in the TME which interact in a highly-complex pattern. Microscopically, cancer cells invade the TME and vice versa. To prevent tumor recurrence, it is necessary to resect the TME along with the tumor.


Assuntos
Cor , Linfoma/diagnóstico por imagem , Microscopia Confocal/métodos , Recidiva Local de Neoplasia , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Linfoma/cirurgia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Cirurgia Assistida por Computador/métodos , Proteína Vermelha Fluorescente
6.
Anticancer Res ; 36(9): 4483-7, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27630285

RESUMO

BACKGROUND: Fluorescence-guided surgery (FGS) of tumors is an area of intense development. Peritoneally-disseminated cancer, however, represents a difficult surgical challenge. MATERIALS AND METHODS: To help meet this challenge, EL4 mouse T-cell lymphoma cells expressing red fluorescent protein (RFP) were injected intraperitoneally in nude mice. RESULTS: Two weeks after injection of EL4-RFP cells, established peritoneally-disseminated tumors were observed. FGS was performed using a hand-held portable fluorescence imaging system (Dino-Lite). FGS enabled detection of very small peritoneal disseminated tumors and completely resected them in contrast to bright-light which only partially detected the tumors. CONCLUSION: The present report indicates the feasibility of FGS of peritoneally-disseminated cancer.


Assuntos
Linfoma/diagnóstico por imagem , Microscopia de Fluorescência , Neoplasias Peritoneais/diagnóstico por imagem , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteínas Luminescentes/metabolismo , Linfoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia Confocal , Imagem Óptica , Neoplasias Peritoneais/patologia , Cirurgia Assistida por Computador/métodos , Proteína Vermelha Fluorescente
7.
Anticancer Res ; 36(8): 3827-31, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27466484

RESUMO

BACKGROUND/AIM: Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. MATERIALS AND METHODS: The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. RESULTS: Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. CONCLUSION: The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy.


Assuntos
Núcleo Celular/ultraestrutura , Citoplasma/ultraestrutura , Fibrossarcoma/diagnóstico por imagem , Fusão Nuclear , Linhagem Celular Tumoral , Núcleo Celular/patologia , Citoplasma/patologia , Fibrossarcoma/ultraestrutura , Proteínas de Fluorescência Verde , Histonas/isolamento & purificação , Humanos , Microscopia Confocal
8.
Anticancer Res ; 36(4): 1473-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27069122

RESUMO

BACKGROUND: The EL4 cell line was previously derived from a lymphoma induced in a C57/BL6 mouse by 9,10-dimethyl-1,2-benzanthracene. In a previous study, EL4 lymphoma cells expressing red fluorescent protein (EL4-RFP) were established and injected into the tail vein of C57/BL6 green fluorescent protein (GFP) transgenic mice. Metastasis was observed at multiple sites which were also enriched with host GFP-expressing stromal cells. In the present study, our aim was to establish an orthotopic model of EL4-RFP. MATERIALS AND METHODS: In the present study, EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice as an orthotopic model of lymphoma. Resultant primary tumor and metastases were imaged with the Olympus FV1000 scanning laser confocal microscope. RESULTS: EL4-RFP metastasis was observed 21 days later. EL4-RFP tumors in the spleen (primary injection site), liver, supra-mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized by color-coded imaging. EL4-RFP metastases in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Small tumors were observed in the spleen, which were rich in host stromal cells. In the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. CONCLUSION: Color-coded imaging of the lymphoma microenvironment suggests an important role of stromal cells in lymphoma progression and metastasis.


Assuntos
Diagnóstico por Imagem/métodos , Proteínas de Fluorescência Verde/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Luminescentes/metabolismo , Linfoma/metabolismo , Células Estromais/metabolismo , Animais , Linhagem Celular Tumoral , Cor , Proteínas de Fluorescência Verde/genética , Neoplasias Hepáticas/secundário , Proteínas Luminescentes/genética , Linfoma/patologia , Camundongos Transgênicos , Proteína Vermelha Fluorescente
9.
Anticancer Res ; 36(5): 2113-7, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27127111

RESUMO

BACKGROUND: Colon cancer frequently results in metastasis to the liver, where it becomes the main cause of death. However, the cell cycle in primary tumors and metastases is poorly understood. MATERIALS AND METHODS: We developed a mouse model of liver metastasis using the human colon cancer cell line HCT-116, which expresses green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP cells were injected into the spleen of nu/nu nude mice. RESULTS: HCT-116-GFP-RFP cells subsequently formed primary tumors in the spleen, as well as metastatic colonies in the liver and retroperitoneum by 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, it was possible to clearly image mitosis of the dual-colored colon cancer cells in the primary tumor as well as liver and other metastases. Multi-nucleate cancer cells, in addition to mono-nucleate cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nucleate HCT-116-GFP-RFP cells were also observed after culture of the primary and metastatic tumors. A similar ratio of mono-nucleate, multi-nucleate, and mitotic cells grew from the primary and metastatic tumors in culture, suggesting similarity of the nuclear-cytoplasmic dynamics of primary and metastatic cancer cells, further emphasizing the stochastic nature of metastasis. CONCLUSION: Our results demonstrate a similar heterogeneity of nuclear-cytoplasmic dynamics within primary tumors and metastases, which may be an important factor in the stochastic nature of metastasis.


Assuntos
Núcleo Celular/patologia , Neoplasias do Colo/patologia , Citoplasma/patologia , Animais , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Metástase Neoplásica
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