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We compared the DNA damage repair response of cancer cells after UVB or UVC irradiation. The DNA-damage repair response to UV irradiation was imaged in tumors growing in 3D Gelfoam® histoculture and in superficial tumors grown in mice. UV-induced DNA damage repair was imaged with GFP fused to the DNA damage response (DDR)-related chromatin-binding protein 53BP1 in MiaPaCa-2 human pancreatic cancer cells. 53BP1 forms foci during DNA damage repair. A clonogenic assay initially showed that UVC and UVB inhibited MiaPaCa-2 cell proliferation in a dose-dependent manner, with UVC having more efficacy. Gelfoam® histocultures and confocal imaging enabled 53BP1-GFP nuclear foci to be observed within 1 h after UV irradiation, indicating the onset of DNA damage repair response. Induction of UVB-induced 53BP1-GFP focus formation was observed up to a depth of 120 µm in MiaPaCa-2 cells on Gelfoam®, while UVC induced foci only to a depth of 40 µm. The MiaPaCa-2 cells irradiated by both UVB and UVC light in a skin-flap mouse model had a significant decrease in tumor growth compared to untreated controls. Moreover, UVB-treatment had greater inhibition of tumor growth compared to UVC. Our results demonstrate that both UVB and UVC are useful tools for the treatment of residual cancer, and UVB was more effective than UVC in vivo, possibly due to greater tissue penetration of UVB because of its longer wavelength. Citation Format: Fuminari Uehara, Shinji Miwa, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Yukihiko Hiroshima, Shuya Yano, Mako Yamamoto, Yasunori Matsumoto, Elena V. Efimova, Robert M. Hoffman. Imaging of UVB and UVC-induced DNA damage repair in cancer cells in Gelfoam histoculture and minimal cancer in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2403. doi:10.1158/1538-7445.AM2014-2403

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. The present report describes technology to monitor real-time cell cycle dynamics of cancer cells throughout a live tumor in vivo using a fluorescence ubiquitination cell cycle indicator (FUCCI). In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor are in G0/G1 and 70% in S/G2/M. In contrast in an established tumor, approximately 90% of cancer cells in the center are in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 µm) tumor blood vessels are in G0/G1. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface of tumors and, in contrast, had little effect on quiescent cancer cells that are the vast majority of an established tumor. Moreover resistant quiescent cancer cells that moved to the tumor surface restarted cycling after the cessation of chemotherapy. Most drugs currently in clinical use target cancer cells in S/G2/M which our results show are a small minority in established tumors, thereby accounting, at least in part, for poor efficacy of cytotoxic drugs in solid cancer. Our results also suggest that drugs that target quiescent cancer cells are urgently needed. Citation Format: Shuya Yano, Yasunori Tome, Yong Zhang, Yukihiko Hiroshima, Shinji Miwa, Fuminari Uehara, Atsushi Suetsugu, Hiroyuki Kishimoto, Hiroshi Tazawa, Ming Zhao, Toshiyoshi Fujiwara, Robert M. Hoffman. Real-time FUCCI imaging of cell cycle phase in tumors demonstrates why cancer chemotherapy is not effective in solid cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5095. doi:10.1158/1538-7445.AM2014-5095

We report here real-time color-coded imaging of osteosarcoma-induced angiogenesis in vivo. Gelfoam® (5×5 mm) treated with β fibroblast growth factor (bFGF) was initially transplanted subcutaneously in the flank of transgenic nestin-driven green fluorescent protein (ND-GFP) nude mice (n=12). In ND-GFP mice, nascent blood vessels are labeled with GFP, including those growing in Gelfoam®. Seven days after transplantation of Gelfoam®, skin flaps were made and human 143B osteosarcoma cells expressing GFP in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected into the transplanted Gelfoam®. The control group had only Gelfoam® transplanted without subsequent cancer-cell inoculation. Skin flaps were made at days 14, 21, 28 after transplantation of the Gelfoam® to allow observation of vascularization of the Gelfoam® using fluorescence imaging after both with and without cell inoculation. ND-GFP expressing nascent blood vessels penetrated and spread into the Gelfoam® in both the non-cellular control and after cancer-cell inoculation. The mean length of ND-GFP expressing blood vessels of mice with osteosarcoma cells seeded on the Gelfoam® increased compared to the Gelfoam®-only control at days 14, 21, 28 after transplantation of the Gelfoam® (at day 14, 21 ; p Citation Format: Fuminari Uehara, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Shinji Miwa, Yukihiko Hiroshima, Shuya Yano, Mako Yamamoto, Yasunori Matsumoto, Robert M. Hoffman. Real-time in vivo imaging of osteosarcoma angiogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5. doi:10.1158/1538-7445.AM2014-5

We previously report the development of a color-coded imaging model that can visualize the anastomosis between blood vessels of red fluorescent protein (RFP)-expressing vessels in Gelfoam® previously vascularized in RFP transgenic mice and blood vessels of nestin-driven green fluorescent protein (ND-GFP) mice that express GFP in nascent vessels (Anticancer Res. 33: 3041-3046, 2013). In the present study, fourteen days after transplantation into RFP transgenic nude mice, the Gelfoam® was removed and re-transplanted into the subcutis on the flank of cyan fluorescent protein (CFP) transgenic mice. Skin flaps were made and anastomosis between the CFP-expressing vessels and RFP blood vessels in the Gelfoam® was imaged 14 and 21 days after re-transplantation. The results presented in this and our previous report suggest a new paradigm of visualizing blood vessel anastomosis and its implications for tumor angiogenesis. Citation Format: Fuminari Uehara, Yasunori Tome, Hiroki Maehara, Kazuhiro Tanaka, Fuminori Kanaya, Shinji Miwa, Yukihiko Hiroshima, Shuya Yano, Mako Yamamoto, Yasunori Matsumoto, Robert M. Hoffman. Color-coded imaging of vessel anastomosis in vivo using RFP and CFP transgenic mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 13. doi:10.1158/1538-7445.AM2014-13

Fluorescence ubiquitination cell cycle indicator (FUCCI) imaging was used to identify the cell cycle phase of cancer cells in small and large tumors. In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor were in G0/G1 and 70% were in S/G2/M. In contrast in a large tumor, approximately 90% of cancer cells were in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 µm) tumor blood vessels were in G0/G1. Longitudinal real-time imaging demonstrated that cytotoxic drugs used in the clinic killed only proliferating cancer cells at the surface of the tumors and, in contrast, had little effect on quiescent cancer cells that are the vast majority of an established tumor. These results demonstrate why solid tumors are usually drug resistant in the clinic. The results also suggest that drugs that target quiescent cancer cells are urgently needed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B60. Citation Format: Shuya Yano, Yasunori Tome, Yong Zhang, Shinji Miwa, Atsushi Suetsugu, Hiroyuki Kishimoto, Hiroshi Tazawa, Michael Bouvet, Toshiyoshi Fujiwara, Robert M. Hoffman. FUCCI cell cycle imaging identifies drug-resistant quiescent cancer cells within tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B60.

e13577 Background: Dormant cancer cells are a significant problem in clinical cancer as they are usually chemoresistant and can give rise to recurrence years after treatment. Methods: Dormant cancer cells were identified with a fluorescence ubiquitination cell cycle indicator (FUCCI) and confocal microscopy. FUCCI-expressing xenografts were established from MKN45 gastric cancer cells. Results: Within 7 days after implantation in nude mice, FUCCI-expressing tumors contained a mixture of cancer cells in G0/G1 and S/G2/M phases. Cancer cells in G0/G1 phase survived and become dormant after treatment with cytotoxic agents. As the tumors grew, proliferating cells were only found at the surface. Chemotherapy killed only the proliferating cancer cells at the surface and had little effect on the majority of dormant cancer cells in the tumor center. Furthermore, we investigated tumor chemoresistance using nestin-driven green fluorescent protein (GFP) transgenic mice that have GFP-expressing nascent tumor vessels. Chemotherapy-treated tumors had much more and deeper tumor vessels than control tumors. Conclusions: These results suggest that dormant cancer cells may protect themselves by inducing angiogenesis.

Dormant cancer cells were visualized with a fluorescence ubiquitination cell cycle indicator (FUCCI). FUCCI-expressing xenografts were established from MKN45 gastric cancer cells. Within 7 days after inoculation, FUCCI-expressing nascent tumors contained a mixture of cancer cells in G0/G1 phase and in S/G2/M phases. Nascent tumors treated with cyto-toxic agents showed that cancer cells in G0/G1 phase can survive and become dormant. As tumors grew, the proliferating area was only at the surface and the dormant area predominated. Chemotherapy killed only the minority population of proliferating cancer cells at the surface and had little effect on the majority of dormant cancer cells in the tumor center. Furthermore, we investigated tumor chemoresistance using nestin-driven GFP transgenic mice that have GFP-expressing nascent tumor vessels. Chemotherapy-treated tumors had much more and deeper tumor vessels than control tumors, suggesting that dormant cancer cells may protect themselves by inducing tumor vessels. This report suggests that dormant cancer cells play a large role in drug resistance. Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Toshiyoshi Fujiwara, Robert M. Hoffman. Dormant cancer cells are resistant to conventional therapies and induce tumor vessels after chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1754. doi:10.1158/1538-7445.AM2013-1754

The cell cycle of cancer cells in the bone marrow was imaged using a fluorescence ubiquitination cell cycle indicator (FUCCI). With FUCCI, quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). FUCCI-expressing cancer cells were injected into the bone marrow of tibia of mice. At the early stage after injection (7 days), approximately 90% of the cells were in G0/G1. In contrast, at late stage after injection (28 days more), in the endosteal lesion, approximately 80% of the cancer cells were in S/G2/M phases and in the central zone, approximately 75% of cancer cells were in G0/G1. Thus, cancer-cell location and time in the bone marrow determines cell cycle position and possibly dormancy. Thus, quiescent (dormant) cancer cells in the bone marrow need to be targeted as well as proliferating cancer cells, which is a significant current challenge. Citation Format: Shuya Yano, Shinji Miwa, Yasunori Tome, Fuminari Uehara, Michelle Digman, Enrico Gratton, Robert M. Hoffman. Intravital imaging of cancer cell cycle in the bone marrow. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2013-1756

Human 143B osteosarcoma cells were stably transfected with an αv integrin-GFP vector. Confocal microscopy demonstrated that αv integrin immunofluorescence staining co-localized with αv integrin-GFP fluorescence. The αv integrin-GFP expressing tumors were established in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in non-colored nude mice, which maintained the RFP stroma. The interaction of αv integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in the tumors which persisted even 3 weeks after passage. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing αv integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing αv integrin-GFP could be identified. TAMs expressing RFP accompanied osteosarcoma cells expressing αv integrin-GFP in the lung. The present study demonstrates the importance of αv integrin interaction with stromal elements. Citation Format: Yasunori Tome, Fuminori Uehara, Hiroki Maehara, Shinji Miwa, Hiroyuki Tsuchiya, Katsuro Tomita, Fuminori Kanaya, Michael Bouvet, Robert M. Hoffman. Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor scaffold collagen in nude mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1638. doi:10.1158/1538-7445.AM2013-1638

Methionine-dependence of cancer cells may be due to excessive methylation reactions in cancer cells. Deprivation of methionine α,γ lyase (methioninase or METase) selectively arrests cancer cells during late S-phase, where they are highly sensitive to DNA-damaging chemotherapy. Fluorescent ubiquitination-based cell cycle indicator (FUCCI)), was used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by METase. The S-phase-blocked cancer cells fluoresced yellow or green, in contrast to cancer cells in G1 which fluoresced red. Cancer cells, synchronously blocked in S-phase by METase and identified by their yellow-green fluorescence, were administered DNA-damaging chemotherapy drugs such as doxorubicin, cisplatin, or 5-fluorouracil. Treatment of cancer cells with drugs only without methioninase-effected S-phase synchrony, led to the majority of the cancer cell population being blocked in G0/G1 phase (red fluorescent) where they were resistant to the drugs. In contrast, METase treatment, followed by chemotherapy when FUCCI indicated the S/G2 block was highly effective for killing cancer cells. Color-coded chemotherapy, whereby the cell cycle of cancer cells is selectively and synchronously blocked in S-phase as identified by fluorescent reporters, may be a general approach to effective cancer treatment. Citation Format: Shuya Yano, Yasunori Tome, Michelle Digman, Masashi Momiyama, Atsushi Suetsugu, Enrico Gratton, Robert M. Hoffman. Imaging of methioninase-induced S/G2-phase-trapping for subsequent effective chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3412. doi:10.1158/1538-7445.AM2013-3412

Invasive cancer cells are a critical target for chemotherapy. However, the cell cycle phase of invasive cancer cells is poorly understood. Cancer cells were cultured in 3D Gelfoam® culture where they have in vivo-like cell cycle kinetics. Cancer cell-cycle kinetics were visualized using a fluorescence ubiquitination cell cycle indicator (FUCCI), whereby quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). Real-time imaging demonstrated that cancer cells in the G0/G1 phase are invasive and disseminate in Gelfoam histoculture. Moreover, in vivo, FUCCI-expressing cancer cells were in G0/G1 phase when they metastasized to the liver. Disseminated cancer cells in the peritoneal cavity were also in G0/G1 phase. Most drugs currently in use target cells in S/G2/M are therefore may not be able to target invasive cancer cells. Color-coded cell-cycle imaging of cancer cell invasion should provide a novel platform for the rapid screening of candidate therapeutic agents against quiescent, invasive cancer cells. Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Toshiyoshi Fujiwara, Robert M. Hoffman. Invasive cancer cells are mostly in the G0/G1 phase of the cell cycle. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1755. doi:10.1158/1538-7445.AM2013-1755

The S. typhimurium A1-R mutant (A1-R), which is auxotrophic for leu-arg, and selected for high antitumor virulence, has been previously shown to be effective as monotherapy against human breast, prostate, pancreatic, and fibrosarcoma tumors that have been orthotopically implanted in nude mice. In the present study, we generated an enhanced tumor-targeting S. typhimurium variant using in vivo selection. Dual-color murine mammary tumor (MMT) cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected (1 × 106 cells) in both flanks of 4-week-old female nude mice. Seven days after tumor implantation, A1-R bacteria were injected (1 × 107 cfu iv). Five days after injection, the tumors were harvested and minced. The bacteria which invaded the tumor were isolated and seeded on LB agar. After overnight incubation, colonies were picked and purified and termed A1-R1. The procedure was repeated with A1-R1 to obtain A1-R2. To determine the efficacy of the new bacterial variants, MMT-dual cells were seeded onto the 6-well plates. After 48 hours incubation, A1-R, A1-R1, and A1-R2 (1 × 108 cfu/well) were added to each plate. After 0, 24, 48, 72, 96 hours incubation, each plate was observed by confocal microscopy. Compared with A1-R, the infection rate of cancer cells by A1-R1 and A1-R2 was increased. The A1-R1 and A1-R2 variant bacteria generated in vivo selection show promise for improved bacterial targeting in vivo. Citation Format: Fuminori Uehara, Yong Zhang, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Robert M. Hoffman, Ming Zhao. In vivo selection of S. typhimurium A1-R enhanced tumor-targeting variants. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2013-3311

Caffeine has been shown to enhance the efficacy of chemotherapy, but the mechanism is not well understood. In this study, we investigated cell-cycle modulation induced by caffeine in combination with cisplatinum treatment of human cancer cells. Proliferation inhibition was determined with a clonogenic assay. Mitotic and apoptotic changes were observed by imaging of 143B dual-color cells, in which GFP is expressed in the nucleus and RFP in the cytoplasm. Modulation of the cell-cycle in combination with cisplatinum was observed using time-lapse imaging analyses of HeLa cells expressing a fluorescent cell-cycle indicator, FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). The clonogenic assay showed that caffeine increased the efficacy of cisplatin on inhibition of cell proliferation. Dual-color imaging demonstrated that cisplatin decreased mitosis and induced apoptosis in 143B cells. The combination of cisplatin and caffeine reduced the decrease of mitosis and increased apoptosis. Time-lapse FUCCI imaging showed that cisplatin strongly induced cell-cycle arrest at the S/G2/M phases in HeLa cells. Caffeine released the cell-cycle arrest induced by cisplatin, resulting in increased cell-killing efficacy. These data indicates that caffeine can effectively modulates the cell-cycle in cancer cells treated with cisplatin and possibly other drugs. Citation Format: Shinji Miwa, Shuya Yano, Yukihiko Hiroshima, Yasunori Tome, Fuminori Uehara, Sumiyuki Mii, Hiroaki Kimura, Katsuro Hayashi, Hiroyuki Tsuchiya, Michelle Digman, Enrico Gratton, Robert M. Hoffman. Caffeine modulates the cell cycle in cancer cells to enhance cisplatin efficacy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 703. doi:10.1158/1538-7445.AM2013-703

The DNA repair protein 53BP1 is re-localized in foci after DNA damage. In this study, we imaged the real-time response of 53BP1 linked to GFP to DNA damage induced by UV irradiation. Inhibition of proliferation by UV irradiation was analyzed by a clonogenic assay. The relocalization of GFP fused to the chromatin-binding domain of 53BP1 was imaged after UV irradiation of MiaPaca-2 53BP1-GFP human pancreatic cancer cells. The clonogenic assay demonstrated that UVC dose-dependently inhibited cell proliferation, while UVA and UVB showed little effect. During live-cell imaging, 53BP1-GFP foci formation was observed within 30 min after UVC irradiation. High-dose UVC also induced apoptosis within 24 h after irradiation. The cells treated with UVC irradiation appear to have two different fates: DNA repair or apoptosis. A fraction of the cells underwent apoptosis, and the other fraction repaired their DNA as evidenced by 53BP1 focus formation. The present study demonstrates that some cells may repair their DNA successfully and others can not. This has important implication for UV-based photodynamic therapy. Citation Format: Shinji Miwa, Shuya Yano, Yukihiko Hiroshima, Yasunori Tome, Fuminori Uehara, Sumiyuki Mii, Hiroaki Kimura, Katsuro Hayashi, Hiroyuki Tsuchiya, Elena Efimova, Michelle Digman, Enrico Gratton, Robert M. Hoffman. Real-time imaging demonstrates cancer cells exposed to UV irradiation either repair their DNA or die. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1769. doi:10.1158/1538-7445.AM2013-1769

We report here imaging the interaction between αv integrin tagged to GFP in human osteosarcoma cells and blood vessels growing on Gelfoam@ implanted subcutaneously in mice. 143B osteosarcoma cells expressing αv integrin-GFP were generated by transfection of an αv integrin-GFP vector. Gelfoam@ (5×5 mm) was treated with basic fibroblast growth factor (bFGF) and transplanted subcutaneously in RFP transgenic nude mice. Skin-flap imaging windows were made at days 7, 14, 21, 28 after transplantation of Gelfoam@. Angiogenesis was observed in the Gelfoam@ using fluorescence imaging via the skin flap windows. Frozen sections were made at day 28 after Gelfoam@ transplantation. RFP-expressing blood vessels were imaged growing into the Gelfoam@. Immunohistochemical (IHC) staining showed that CD31 and RFP fluorescence co-localized in the newly formed RFP-expressing blood vessels in the Gelfoam@. To observe the interaction of tumor cells and RFP blood vessels, 143B cells (5×105),expressing αv integrin-GFP, were injected into the Gelfoam@ 7 days after transplantation. Seven days after cancer-cell injection, the cancer cells were observed in the Gelfoam@ by confocal microscopy via skin-flap windows. 143B cells expressing αv integrin-GFP proliferated into the Gelfoam@ as observed by skin-flap-window imaging. Strong expression of αv integrin-GFP cells was observed near RFP vessels in the Gelfoam@ in the RFP mice 28 days after cancer cell injection. The results show that Gelfoam@ is a useful tool to observe angiogensis in vivo and the interactions of αv integrin-GFP with blood vessels which will allow further understanding of its role in angiogenesis and tumor progression. Citation Format: Fuminori Uehara, Yasunori Tome, Hiroki Maehara, Fuminori Kanaya, Robert M. Hoffman. Color-coded imaging of interaction between RFP-expressing blood vessels and αv integrin-GFP expressing osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1641. doi:10.1158/1538-7445.AM2013-1641

The phase of the cell cycle can determine whether a cancer cell can respond to most drugs. The fluorescence ubiquitination cell cycle indicator (FUCCI) was used to image the cell cycle phase within tumors. In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor were in G0/G1 and 70% in S/G2/M. In contrast, approximately 90% of cancer cells in the center of an established tumor were in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 μm) tumor vessels were in G0/G1. Although 60-70% of cancer cells near the surface of an established tumor (< 100 μm) or near blood vessels were in S/G2/M, these cells are a small minority of an established tumor. Moreover, tumors that are stable in size for more than six months in nude mice consisted of mostly G0/G1 cells, suggesting they are dormant. The results presented here have profound implications for cancer therapy, since most drugs currently in use target cells in S/G2/M, which are a minor fraction in an established tumor. With the developments reported here, drugs that target non-dividing and dormant cancer cells can be readily identified. Citation Format: Shuya Yano, Yasunori Tome, Michelle Digman, Masashi Momiyama, Atsushi Suetsugu, Enrico Gratton, Robert M. Hoffman. Cancer cell location in tumor determines cell cycle position and dormancy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1758. doi:10.1158/1538-7445.AM2013-1758

Tumors in vivo are well known to behave quite differently from cancer cells cultured on plastic or glass plates (monolayer culture). Therefore, cancer cell-cycle kinetics were visualized in Gelfoam® 3D-histoculture using HeLa cells and MKN45 cells expressing fluorescence ubiquitination cell cycle indicator (FUCCI), whereby quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). Gelfoam® histoculture of FUCCI-expressing cancer cells results in tumor-like structures with quiescent cancer cells in the center and proliferating cells at the surface, similar to what occurs in vivo as visualized by FUCCI confocal microscopy. These results have profound implications for cell cycle studies, since most cellular and molecular studies of the cancer cell cycle are performed on plastic monolayer culture where characteristics of the cells may be quite different from the tumor in vivo. Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Robert M. Hoffman. Gelfoam® histoculture enables cancer cells to mimic in vivo cell cycle behavior. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1757. doi:10.1158/1538-7445.AM2013-1757

Tumors in vivo are well known to behave quite differently from cancer cells cultured on plastic or glass plates (monolayer culture). Cell culture on agar (sphere culture) is used for three-dimensional culture, however tumor spheres on agar rarely invade the substrate. It has been previously demonstrated that 3-D Gelfoam histoculture of tumors enables the cancer cells to maintain in vivo properties, in contrast to monolayer or sphere culture. Cancer cell-cycle kinetics were visualized in Gelfoam histoculture using HeLa cells and MKN45 cells expressing fluorescence ubiquitination cell cycle indicator (FUCCI), whereby quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). Gelfoam histoculture of FUCCI-expressing cancer cells results in tumor-like structures with quiescent cancer cells in the center and proliferating cells at the surface, similar to what occurs in vivo as visualized by confocal microscopy. These results have profound implications for cancer biology, since most cellular and molecular studies of cancer cells were performed on plastic monolayer culture where characteristics of the cells may be quite different from the tumor in vivo. Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Robert M. Hoffman. Cancer cells form in vivo-like tumors in Gelfoam histoculture. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A40.

Invasive cancer cells are a critical target for chemotherapy. However most chemotherapeutic agents in clinical use target dividing cancer cells. Cancer cell-cycle kinetics were visualized in Gelfoam histoculture using a fluorescence ubiquitination cell cycle indicator (FUCCI), whereby quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). Real-time imaging demonstrated that cancer cells in G0/G1 phase are invasive and disseminate in Gelfoam histoculture. In vivo, FUCCI-expressing cancer cells were in G0/G1 phase when they metastasized to the liver. Disseminated cancer cells in the peritoneal cavity were also in G0/G1 phase. Most drugs currently in use target cells in S/G 2/M, which may not be capable of invasion. Color-coded cell-cycle imaging in Gelfoam histoculture should provide a novel platform for the rapid screening of candidate therapeutic agents against quiescent invasive cancer cells. Citation Format: Shuya Yano, Sumiyuki Mii, Yasunori Tome, Yukihiko Hiroshima, Fuminari Uehara, Shinji Miwa, Robert M. Hoffman. Cancer cells invade or divide, not both. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A41.

10072 Background: Integrins play a role in tumor growth and metastasis (Int. J. Cancer 129, 2905-2915, 2011). However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody affects seeding and growth of osteosarcoma cells on the lung. Methods: The 143B human osteosarcoma cell line expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus was established. Using the double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged in live mice. Results: The anti-b1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung while a control antibody had no effect. To image the efficacy of the anti-integrin antibody on spontaneous metastasis, mice with orthotopically-growing 143B-RFP cells in the tibia were also treated with AIIB2 or control anti-rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth. In a separate experiment, the anti-β1 integrin antibody increased survival in the orthothopic osteosarcoma model. Conclusions: The efficacy of the anti-β1 integrin antibody against metastasis may be due to inhibition of lung seeding of the cancer cells. The increased survival of mice with orthotopically-growing 143B-RFP treated with AIIB2 may be due to inhibition of metastasis, which in turn may be inhibited by effect of the anti-β1 integrin on cancer-cell seeding in the lung.

The S. typhimurium A1-R mutant, which is auxotrophic for leu-arg to attenuate growth in normal tissue, and selected for high antitumor virulence, has been shown to be effective as monotherapy against human prostate, breast, pancreatic, and fibrosarcoma tumors. In the present study, we first administered A1-R to nude with subcutaneous murine colon cancer Colon-26 which expressed red fluorescent protein (RFP). S. typhimurium A1-R (1×107 cfu) was administered intravenously (i.v.) or intratumorally (i.t.) to the subcutaneous tumor as a positive control. The size of tumor was measured by weekly fluorescence imaging. Both i.v.- and i.t.-treated mice had smaller tumors and survived longer (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5547. doi:1538-7445.AM2012-5547

We report here real-time imaging of αv integrin-GFP expression during osteosarcoma cell trafficking in blood vessels in nude mice. 143B osteosarcoma cells were transfected with an αv integrin-GFP vector. Confocal microscopy demonstrated that αv integrin immunofluorescent staining colocalized with αv integrin-GFP. Osteosarcoma 143B cells expressing αv integrin-GFP (1×106) were injected in the epigastric cranialis vein. Subsequently, round osteosarcoma cells diffusely expressing αv integrin-GFP were observed in larger veins. In contrast, punctuated αv integrin-GFP was imaged in stretched cells attached to narrow vessel walls. Osteosarcoma cells, arrested in venous valves, were observed to strongly express αv integrin-GFP. αv integrin-GFP allows the imaging of αv integrin expression during cancer cell trafficking and arrest in blood vessels which will enable the further understanding of its role in this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5553. doi:1538-7445.AM2012-5553

Temozolomide and UVC light were used for induction of cancer cell apoptosis of cancer cells which was imaged in real time at the subcellular level in the brain. Temozolomide inhibited brain tumor growth (p < 0.001). UVC irradiation inhibited superficial brain tumor growth (4.7-fold) (p < 0.0001) and improved survival (39.0%) (p = 0.002). This model can be used to evaluate new treatment modalities of brain metastasis or brain cancer at the subcellular level. In order to visualize single cancer cell nuclear-cytoplasmic dynamics of cancer cells in the brain in real time, including mitosis and apoptosis, Lewis lung cancer cells or human U87 glioma cells were injected into the brain either stereotactically or via the carotid artery of nude mice. The cancer cells were labeled with GFP in the nucleus and RFP in the cytoplasm, such that mitosis and apoptosis could be clearly imaged. A craniotomy open window was made for real-time fluorescence imaging of the cancer cells growing in the brain. Mitosis as well as treatment-induced apoptosis of the individual cancer cells were imaged in real time. UVC irradiation was initiated 1 day after the cancer cell inoculation and tumors were irradiated for 120 seconds on days 1, 4, 7, and 10. The tumor volume and survival were compared with untreated mice. UVC irradiation inhibited 4.7-fold changes in the brain tumor growth (p < 0.0001) and improved 39% higher survival (p = 0.002). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5546. doi:1538-7445.AM2012-5546

This study examines the effects of types of liver resection on the growth of liver and lung metastasis. Experimental liver metastases were established by spleen injection of the Colon 26 murine adenocarcinoma cell line expressing GFP into transgenic nude mice expressing RFP. Experimental lung metastases were established by tail vein injection with Colon 26-GFP. Three days after cell injection, groups of mice underwent liver resection (35%+35% [repeated minor resection] vs. 70% [major resection]). Metastatic tumor growth was measured by color-coded fluorescence imaging of the GFP-expressing cancer cells and RFP-expressing stroma. Although major and repeated minor resection removed the same volume of liver parenchyma, the two procedures had very different effects on metastatic tumor growth: major resection, stimulated liver and lung metastatic growth as well as recruitment of host-derived stroma compared to repeated minor resection. Repeated minor resection did not stimulate metastasis or stromal recruitment. There was no significant difference in liver regeneration between the two groups. Host-derived stroma density, which is stimulated by major resection compared to repeated minor resection, may stimulate growth in the liver-metastatic tumor. TGF-β is also preferentially stimulated by major resection and may play a role in stroma and metastasis stimulation. The results of this study indicate that when liver resection is necessary, repeated minor liver resection is superior to major liver resection, since major resection, in contrast to repeated minor resection, stimulates metastasis, which should be taken into consideration in clinical situations indicating liver resection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4583. doi:1538-7445.AM2012-4583

Expression of αvβ3 integrin is associated with tumor progression and metastasis in several types of cancer, including metastatic osteosarcoma. We tested echistatin, an antagonist of αvβ3 integrin, as a molecular targeting drug, in an imageable orthotopic mouse model of human metastatic osteosarcoma. αvβ3 integrin expression was measured in a series of human osteosarcoma cell lines (143B parental and LM1-LM4) with increasing metastatic potential, using RT-PCR and immunoprecipitation. The cell lines expressed GFP in the nucleus and RFP in the cytoplasm and therefore could be imaged down to the subcellular level in vitro and in vivo. αvβ3 integrin expression correlated with the metastatic potential of the cells, with the LM4 cells showing the highest expression and highest metastatic capability, 6-fold and 18-fold, respectively, compared to parental 143B cells. In vitro cell proliferation of LM4 was inhibited by echistatin (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5177. doi:1538-7445.AM2012-5177

Malignant glioma tumors are the most common primary central nervous system tumors. Despite the multidisciplinary approach to treatment, prognosis remains poor. In this study, we demonstrated that the Salmonella typhimurium A1-R tumor-targeting strain can inhibit and eradicate human glioma in an orthotopic nude mouse model. S. typhimuirum A1-R was administered by injection through a craniotomy open-window or intravenously in nude mice. To establish the model, 2×105 U87-RFP human glioma cells were injected stereotactically into the mouse brain through the craniotomy open window. Two weeks after glioma-cell implantation, mice were treated with S. typhimurium A1-R (2×107 CFU / 200 αl intravenous injection [i.v.] or 1×106 CFU / 1 αl intracranial injection [i.c.]) once a week for 3 weeks. Brain tumors were observed by fluorescence imaging through the craniotomy open window over time. S. typhimurium A1-R, administered i.c., inhibited brain tumor growth 7.6-fold compared with untreated mice (p = 0.009) and improved survival 73% (p = 0.001). Two of ten mice appeared to have their tumors eradicated. In contrast, intravenous administration of S. typhimurium A1-R was not effective. The craniotomy open window enabled observation of tumor growth in the brain in real time in both treated and untreated mice. The results of the present study demonstrate that bacterial therapy of brain cancer is a novel, effective and safe treatment strategy in a highly treatment-resistance cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4563. doi:1538-7445.AM2012-4563

We describe here real-time imaging of αv integrin-GFP expression in osteosarcoma cells (143B) in vitro and in vivo. 143B cells were stably transfected with an αv integrin-GFP vector. Confocal microscopy demonstrated that αv integrin immunofluorescent staining colocalized with αv integrin-GFP fluorescence. In vitro, when αv integrin-GFP osteosarcoma cells were seeded on a dish coated with fibronectin, which bonds αv integrin. αv integrin adhesion plaques were observed by confocal microscopy using the Olympus FV1000. When αv integrin-GFP-expressing 143B cells (1×106) were transplanted in the tibia or injected subcutaneously in nude mice, the cells aligned along collagen orthotopic fibers. In the orthotopic model, osteosarcoma cells punctate expression of αv integrin-GFP was imaged in the osteosarcoma cells as they invaded muscular tissue. In the subcutaneous model, osteosarcoma cells, strongly expressing αv integrin-GFP, were observed around the blood vessels in the tumor. These results show that αv integrin-GFP allows the real-time imaging of the role of αv integrin during cancer cell invasion allowing the further understanding of its role in the process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5182. doi:1538-7445.AM2012-5182

In this study, we describe imaging of αv integrin-GFP in cancer cells interacting with blood vessels. Osteosarcoma cells (143B) (5×106) expressing αv integrin-GFP were injected in the 10-day old chick chorioallontoic membrane (CAM). In vivo imaging with the Olympus OV-100, visualized tumor-induced angiogenesis one week after transplantation. Osteosarcoma cells expressing αv integrin-GFP were aligned along the tumor-induced blood vessels. High resolution ex vivo imaging with the Olympus FV1000 confocal microscope showed narrow capillary vessels surrounded by osteosarcoma cells which strongly expressing αv integrin-GFP compared to those cells far from vessels. This result imply that expressed of osteosarcoma αv integrin is related to the interaction of the osteosarcoma cells and blood vessels. The ability to image αv integrin during interaction of cancer cells and blood vessels will enable the further understanding of its role in this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5176. doi:1538-7445.AM2012-5176

The S. typhimurium A1-R mutant, which is auxotrophic for leu-arg, and selected for high antitumor virulence, has been shown to be effective as monotherapy against human prostate, breast, pancreatic, and fibrosarcoma tumors. In the present study, we developed a strategy to maximize efficacy and minimize toxicity for bacterial tumor treatment in immunocompetent mice with subcutaneous Lewis lung carcinoma expressing red fluorescent protein (RFP). A small dose of S. typhimurium A1-R bacteria was first administered to tumor-bearing animals (the primer dose, 1×106 cfu i.v.) as pre-treatment followed by a high dose (1×107 cfu i.v.) 4 hours later. Compared with non-primer-dose bacteria treatment, the primer-dose strategy resulted in smaller tumors and no observable side effects. The serum lever of TNF-α was elevated in the primer-dose arms compared to non-primer dose arms. Vessel destruction by S. typhimurium A1-R was observed in immunocompetent mice expressing nestin-driven green fluorescent protein (ND-GFP), which is selectively expressed in nascent blood vessels. Vessel destruction was enhanced in the primer-dose arms compared to non-primer-dose arms. The primer dose may activate the immune system and induce cytokines in the mouse necessary for invasion of the tumor by the bacteria allowing a relatively small subsequent dose to be effective and safe. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1691. doi:1538-7445.AM2012-1691

The ability to image DNA repair in cancer cells after irradiation as well as its inhibition by potential therapeutic agents is important for the further development of radiotherapy of cancer. The relocalization of GFP fused to the chromatin-binding domain of a check-point-adapter protein 53BP1 was imaged after UV irradiation of breast cancer and MiaPaca-2 human pancreatic cancer cells. 53BP1 is a DNA repair protein that is over-expressed when double-stranded breaks occur in DNA. During live-cell imaging, 53BP1-GFP foci formation was observed within 15 minutes after UV irradiation. Most 53BP1 foci resolved by 90 min. To block UV-induced double stranded break (DSB) repair in cancer cells, we targeted poly (ADP-ribose) polymerase (PARP) with ABT-888 (veliparib). PAPR inhibition markedly enhanced UV-irradiation-induced foci persistence, even more than 90 min after UV irradiation, and decreased cell proliferation of breast and pancreatic cancer cells. The results suggest UV-irradiation and PAPR inhibition are a promising therapeutic combination for breast and pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4232. doi:1538-7445.AM2012-4232

Malignant glioma tumors are the most common primary central nervous system tumors and they are very difficult to completely resect due to their invasive margins. In this study, we compared fluorescence-guided and standard bright-light resection. U87-RFP human glioma cells (2 x 105) were injected stereotactically into the nude mouse brain through a craniotomy open window. Two weeks after tumor-cell inoculation, brain tumors were resected under fluorescence guidance or under bright light. U87-RFP tumors were clearly visualized under fluorescent microscopy with long working distance at the single cell level. Almost all cancer cells were removed using fluorescence-guided navigation without damage to the brain tissue. However, brain tumors were difficult to visualize under bright light and many residual cancer cells remained in the brain after surgery. These results suggest that fluorescence-guided surgery has significant potential for treatment of brain cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5529. doi:1538-7445.AM2012-5529

In order to visualize single cancer cell nuclear dynamics in the brain, U87 human glioma cells were injected into the brain stereotactically, or Lewis lung mouse carcinoma (LLC) cells were injected into the carotid artery of nude mice. Both cancer cells types express GFP in the nucleus and RFP in the cytoplasm. A craniotomy open window was made over the right parietal bone for real-time imaging of cancer cells on the brain. Mitotic and apoptotic cancer cells in the brain were imaged in real time. Temozolomide (TMZ) and UVC light were used for induction of cancer cell apoptosis. Imaging showed that approximately 86% of Lewis lung carcinoma cells irradiated with UVC light underwent apoptosis and died in contrast to U87 cells which were resistant to UVC. Real-time subcellular imaging in the brain showed TMZ-induced apoptosis of both U87 and LLC cells. Imaging could be carried out for at least two weeks through the craniotomy window enabling treatment to be evaluated on cancer cells in the brain at the subcellular level. The subcellular real-time imaging model of cancer cells in the brain will be of important use for discovery and evaluation novel drugs active against brain cancer and brain metastasis. 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 4282. doi:10.1158/1538-7445.AM2011-4282

Liver resection has traditionally been the first choice treatment for patients presenting with primary and metastatic liver tumor. Many of the factors up-regulated during liver regeneration after a liver resection have been implicated in tumor growth and the extent of the up-regulation of these factors correlate with the degree of liver resection. However, the effects of a major liver resection compared to repeated minor resections on the growth of malignant tumor are unknown. This study examines the effects of liver regeneration on the growth of the lung metastatic tumors in an established mouse model, after major (70%) and repeated minor (35%+35%) liver resection. Experimental lung metastases were established by tail vein injection with the Colon 26 murine adenocarcinoma cell line expressing green fluorescent protein (GFP). Three days after cancer cell injection, groups of mice underwent liver resection (35%+35% vs. 70%). Lung tumor growth was measured by fluorescence imaging with the Olympus OV100 Small Animal Imaging System. Although, major and repeated minor resection removed the same volume of liver parenchyma, the two procedures had very different effects on lung metastases growth: Major resection stimulated lung metastatic growth. In contrast to major resection, repeated minor resection significantly inhibited the growth of metastatic lung tumors. There was no significant difference in liver regeneration between the two groups. These results show that repeated minor resection of liver is much safer regarding tumor stimulation than major resection. The results of this study indicate that when liver resection is necessary, repeated minor liver resection is superior to major liver resection, since repeated minor resection inhibits lung metastasis and major resection stimulates lung metastasis. 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 2371. doi:10.1158/1538-7445.AM2011-2371

In order to investigate the molecular pathogenesis of human osteosarcoma, a reliable metastatic osteosarcoma animal model is required. Here we report the development of a series of metastatic sublines (LM1-LM4) derived from a human osteosarcoma 143B parental cell line expressing GFP in the nucleus and RFP in the cytoplasm. The sublines have increasing ability to form pulmonary metastases in nude mice after intravenous injection. In vitro proliferation rates of all sublines were similar. When the 143B parental and the metastatic sublines were transplanted orthotopically in the tibia of nude mice, all cell lines were tumorigenic. Primary tumor growth and metastases were imaged with the OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan). LM2 and LM3 had higher metastatic capability than the parental 143B. LM1 had higher metastatic capability than LM2 or LM3. LM4 cells resulted in the most pulmonary micrometastases. The number of pulmonary metastases was 8-fold higher in LM4 cells than that with 143B parental cells. This series of metastatic sublines should be a powerful tool to investigate genetic and other factors that promote or inhibit osteosarcoma metastasis and for evaluation of novel anti-metastatic drugs. 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 5230. doi:10.1158/1538-7445.AM2011-5230

Expression of integrin αvβ3 has been shown to be associated with tumor progression and metastasis in several types of cancer. To investigate the specific role of integrin αvβ3 expression in human osteosarcoma, we compared integrin αvβ3 expression in a series of osteosarcoma cell sublines (143B parental and LM1-LM4) with increasing metastatic potential. We quantified integrin αvβ3 expression using RT-PCR and Western-blotting. Integrin αvβ3 expression correlated with the metastatic potential of the cells, with the LM4 cells showing the highest expression and highest metastatic capability. LM4 in vitro cell proliferation decreased after treatment with echistatin, which is an antagonist of integrin αvβ3. In vitro migration and invasion of LM4 were also inhibited by echistatin. Tumor-induced angiogenesis by LM4 in the chick chorioallantoic membrane was also inhibited by echistatin. These data indicated that integrin αvβ3 overexpression may play a role in metastasis and that echistatin has potential as an inhibitor of cancer cell migration and invasion. 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 1686. doi:10.1158/1538-7445.AM2011-1686

In order to visualize the tumor microenvironment (TME) in pancreatic cancer, the MIA PaCa-2 human pancreatic cancer cell line, expressing red fluorescent protein (RFP), was orthotopically transplanted in nude mice or transgenic mice expressing nestin-GFP. In the nestin-GFP mouse, nascent blood vessels which vascularize the growing tumor are labeled with GFP. A peritoneal flap was made on the left flank of nude mice. The tail of the pancreas was fixed on the peritoneal flap in order to image tumors on the pancreas directly and repeatedly. Color-coded imaging was observed with the Olympus OV100 Small Animal Imaging System under high magnification. The growing tumor expressing RFP, nascent blood vessels expressing GFP, and the interaction between RFP cancer cells and GFP macrophages were observed in real time. Imaging could be carried out until the tumor exceeded the size of the flap which occurred at approximately 21 days after tumor implantation. This mouse model for imaging in the pancreas TME will enable a deeper understanding of tumor-host interaction pancreatic cancer and make it possible to study new therapeutics for pancreatic cancer. 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 437. doi:10.1158/1538-7445.AM2011-437