Jose Reynoso

Nestin-Linked Green Fluorescent Protein Transgenic Nude Mouse for Imaging Human Tumor Angiogenesis

We report here a novel transgenic nude mouse for the visualization of human tumor angiogenesis. We have recently shown that the neural stem cell marker nestin is expressed in hair follicle stem cells and blood vessel networks in the skin of C57/B6 transgenic mice with nestin regulatory element-driven green fluorescent protein (ND-GFP). Others have shown ND-GFP is expressed in the brain, pancreas, and testes in these mice. In the present study, the nestin ND-GFP gene was crossed into nude mice on the C57/B6 background to obtain ND-GFP nude mice. ND-GFP was expressed in the brain, spinal cord, pancreas, stomach, esophagus, heart, lung, blood vessels of glomeruli, blood vessels of skeletal muscle, testes, hair follicles, and blood vessel network in the skin of ND-GFP nude mice. Human lung cancer, pancreatic cancer, and colon cancer cell lines as well as a murine melanoma cell line and breast cancer tumor cell line expressing red fluorescent protein were implanted orthotopically, and a red fluorescent protein-expressing human fibrosarcoma was implanted s.c. in the ND-GFP nude mice. These tumors grew extensively in the ND-GFP mice. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumors, visualized by dual-color fluorescence imaging. Results of immunohistochemical staining showed that CD31 was expressed in the ND-GFP-expressing nascent blood vessels. The ND-GFP transgenic nude mouse model enables the visualization of nascent angiogenesis in human and mouse tumor progression. These results suggest that this model is useful for the imaging of the angiogenesis of human as well as rodent tumors and visualization of the efficacy of angiogenetic inhibitors.

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 β-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.

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, 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 RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009.

Tumor-targeting Salmonella typhimurium A1-R in combination with doxorubicin eradicate soft tissue sarcoma in a patient-derived orthotopic xenograft (PDOX) model

A patient with high grade undifferentiated pleomorphic soft-tissue sarcoma from a striated muscle was grown orthotopically in the right biceps femoris muscle of mice to establish a patient-derived orthotopic xenograft (PDOX) model. Twenty PDOX mice were divided into 4 groups: G1, control without treatment; G2, Salmonella typhimurium (S. typhimurium)A1-R administered by intratumoral (i.t.) injection once a week for 4 weeks; G3, doxorubicin (DOX) administered by intraperitoneal (i.p.) injection once a week for 4 weeks; G4, S. typhimurium A1-R (i.t.) administered once a week for 2 weeks followed by i.p. doxorubicin once a week for 2 weeks. On day 25 from the initiation of treatment, tumor volume in G2, G3, and G4 was significantly lower than G1. Mice found without gross tumor included one mouse (20%) in G2; one mouse (20%) in G3; and 3 mice (60%) in G4. Body weight loss did not significantly differ between the 3 treated groups or from the untreated control. Histological examination revealed eradication of tumor only in G4 where mice were treated with S. typhimurium A1-R followed by DOX. Our present study indicates future clinical potential of combining S. typhimurium A1-R with chemotherapy such as DOX for soft tissue sarcoma patients.

Development of the Transgenic Cyan Fluorescent Protein (CFP)-Expressing Nude Mouse for ''Technicolor'' Cancer Imaging

A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non‐transgenic nude mice with the transgenic CK/ECFP mouse in which the β‐actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA‐1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color‐coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color‐coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment. J. Cell. Biochem. 107: 328–334, 2009.

Abstract 438: The CFP nude mouse as a host for color-coded imaging of the tumor microenvironment

We have developed a color-coded model to study the tumor microenvironment using cancer cells expressing RFP or GFP implanted in nude mice expressing cyan fluorescent protein (CFP). When RFP or GFP HCT-116 were injected subcutaneously in CFP expressing nude mice, CFP expressing cancer-associated fibroblasts migrated to the tumor site. CFP-expressing tumor blood vessels were observed in the tumor. Other CFP host stromal cells were observed throughout the tumor mass. When RFP HCT-116 cells were transplanted in the spleen of CFP nude mice, they formed tumors in the liver. When RFP HCT-116 cancer cells were transplanted in the tail vein of CFP nude mice and they formed tumors in the lung. CFP-expressing host cells were observed in these tumors. When the RFP mouse mammary cancer cell line (RFP MMT) was injected subcutaneously in the CFP nude mouse, numerous CFP-expressing fibroblasts and CFP-expressing blood vessels was observed in the resulting tumor. A large inflammatory population of different CFP-expressing white blood cells surrounded the necrotic area of the tumor suggesting a relationship of these cells to tumor necrosis. GFP and RFP HCT-116 cancer cells were co-injected subcutaneously in CFP-nude mice and tumor growth was followed for 21 days. This three-color model enabled visualization of the tumor mass and its neovasculature in addition to a detailed visualization of other host reactions. GFP HCT-116 cancer cells were initially injected subcutaneously in RFP-nude mice and tumors formed within 14 days. At this point, the tumor mass which consisted of GFP cancer cells and RFP stroma derived from the RFP nude mouse, was harvested and transplanted to a CFP-nude mouse. CFP host cells invaded the growing transplanted tumor containing dual-color cancer cells and RFP stroma. Thus the CFP-nude mouse adds another color to the pallet to study the human tumor microenvironment allowing 4 types of cells to be simultaneously imaged in tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 438. doi:10.1158/1538-7445.AM2011-438

Abstract 579: Chinese herbal mixture LQ inhibits tumor growth and metastasis and increases survival in orthotopic and other mouse models of human and mouse cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Natural products have been used as sources of first-line cancer chemotherapeutic agents, including vinca alkaloids, taxanes, podophyllotoxin and camptothecin. However, Traditional Chinese Medicine (TCM) combinations (Fufang), which have been widely used in China for over 5000 years, are still poorly understood with regard to the types of cancer they are effective against as well as their mechanism of action. We report here on the use of patient-like orthotopic nude-mouse models of metastatic cancer and other mouse tumor models to obtain proof-of-concept that TCM (Fufang) combinations have anti-tumor and anti-metastatic activity. In the present study, human and mouse lung, pancreatic and prostate cancer cell lines (H460, Lewis lung carcinoma [LLC) MIA-PaCa-2 and PC-3, respectively), labeled with red fluorescent protein (RFP) or green fluorescent protein (GFP), were orthotopically implanted or injected subcutaneously, or injected in the tail vein in nude mice to evaluate anti-tumor and anti-metastatic efficacy of the Fufang LQ, which contains a mixture of herbs. Results showed that LQ (gavage, 600 mg/kg/day) significantly inhibited prostate, pancreatic and lung cancer tumor growth as measured by imaging, tumor size and weight, in both subcutaneous and metastatic models. LQ was efficacious against primary and metastatic cancer without weight loss in contrast to drugs such as doxorubicin and cisplatin, which caused significant weight loss. Survival of tumor-bearing mice was also prolonged by LQ treatment. Furthermore, in vitro experiments demonstrated that LQ inhibited proliferation of tumor cells (LLC, H460, PC-3 and MIA-PaCa-2) in a dose-dependent manner. Those results indicate that LQ has efficacy against metastatic pancreatic and lung and prostate cancer which demonstrates its clinical potential. The results also suggest that GFP- and RFP-expressing tumors in orthotopic and other nude mouse models can be widely used for in vivo evaluation of TCM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 579.