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Background A mouse model of metastatic osteosarcoma is imperative to identify effective agents for metastatic osteosarcoma, which is a recalcitrant disease. In the present study, we established osteosarcoma patient-derived cells (OS-PDCs) and transfected them with green fluorescent protein (GFP). Materials and methods The OS-PDCs were transfected with GFP-lentivirus. GFP-expressing OS-PDCs (2.0×105) were then injected into the tibia of nude mice to establish the patient-derived orthotopic cell (PDOC) model (n=3). Six weeks after injection, the primary tumor and each organ were resected and imaged. Results Primary orthotopic tumors were established in two out of three mice. The GFP-expressing OS-PDCs in the PDOC model were visualized. Multiple GFP-expressing lung metastases were detected in one of the two mice with primary tumor. Conclusion The present study proves the concept that a GFP-expressing PDOC model can mimic clinical lung-metastatic osteosarcoma. This model can serve as a paradigm to screen for effective drugs for osteosarcoma lung metastasis.

Background Osteosarcoma is a recalcitrant disease treated with surgery and intensive chemotherapy as standard. The 5-year survival rate of patients with relapsed and lung metastatic osteosarcoma is as low as 20%. Materials and methods A 16-year-old patient developed left distal femoral high-grade osteosarcoma and underwent cisplatinum-based neoadjuvant chemotherapy and surgery. From the resected tumor, a patient-derived orthotopic xenograft (PDOX) model was established in the femur of nude mice. PDOX models were randomized into the following groups: untreated control, or treatment with doxorubicin (3 mg/kg, i.p., weekly for 14 days), sunitinib (40 mg/kg, oral gavage, daily for 14 days), pazopanib (100 mg/kg, oral gavage, daily for 14 days), temozolomide(25 mg/kg, oral gavage, daily for 14 days), and eribulin (1.5 mg/kg, i.p., daily for 14 days). Tumor volume and body weight were monitored twice a week. Results The osteosarcoma PDOX was resistant to doxorubicin, sunitinib, and pazopanib. In contrast, eribulin and temozolomide arrested tumor growth. Conclusion This study demonstrated the utility of the PDOX model in allowing effective from non-effective drugs to be distinguished in a model in which the tumor was growing on the organ corresponding to that of the patient.

BACKGROUND/AIM Liver metastasis in colorectal-cancer is a recalcitrant disease. To develop precision individualized therapy of this disease, we developed a patient-derived orthotopic xenograft (PDOX) model of colorectal-cancer liver metastasis. In the present report, we evaluated the efficacy of oral recombinant methioninase (o-rMETase) in combination with 5-fluorouracil (5-FU) and oxaliplatinum (OXA) on the colorectal-cancer liver metastasis PDOX mouse model. MATERIALS AND METHODS Colorectal-cancer liver metastasis PDOX models were randomized into three groups of seven mice. Group 1, untreated control with phosphate buffered saline (PBS); Group 2, treated with 5-FU + OXA; and Group 3, treated with 5-FU + OXA + o-rMETase. RESULTS The colorectal-cancer liver metastasis PDOX model was resistant to 5-FU + OXA (p=0.83 at day 15 of treatment, Group 2). In contrast, the colorectal-cancer liver metastasis PDOX model was arrested by o-rMETase combined with 5-FU + OXA (p

Background Osteosarcoma is the most frequent malignant bone neoplasm. The efficacy of combination therapy of a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor and a mammalian-target-of-rapamycin (mTOR) inhibitor was previously reported in several cancer types. In the present study, we evaluated the efficacy of a combination of palbociclib (CDK 4/6 inhibitor) and everolimus (mTOR inhibitor) on an osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model. Materials and methods osteosarcoma PDOX mouse models were randomized into five treatment groups of seven mice each: Group 1, untreated control; group 2, doxorubicin treatment; group 3, palbociclib treatment; group 4, everolimus treatment; group 5, palbociclib-everolimus combination treatment. Treatment duration was 2 weeks. Results The palbociclib-everolimus combination reduced the tumor-volume ratio in the osteosarcoma PDOX mouse model compared with the control and doxorubicin (p=0.018). Everolimus alone also inhibited osteosarcoma PDOX growth compared to the control (p=0.04), but less than the combination. Palbociclib alone and doxorubicin were ineffective. There were no significant body-weight losses in any group. Only the palbociclib-everolimus combination induced extensive tumor necrosis observed histopathologically. Conclusion The present study demonstrated that the combination of CDK4/6 and mTOR inhibitors can be a translatable approach for doxorubicin-resistant osteosarcoma in the clinic.

Background/aim Osteosarcoma is the most frequent malignant bone tumor. Failure of first-line therapy results in poor prognosis of osteosarcoma. In the present report, we examined the efficacy of the combination of oral recombinant methioninase (o-rMETase) and docetaxel (DOC) on an osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model. Materials and methods Osteosarcoma-PDOX models were established by tumor insertion within the tibia of nude mice. The osteosarcoma PDOX models were randomized into four groups (4-5 mice per group): control; o-rMETae alone; DOC alone; o- rMETase combined with DOC. The treatment period was 3 weeks. Results The combination of o-rMETase and DOC showed significant efficacy compared to the control group (p=0.03). In contrast, there was no significant efficacy of o-rMETase alone or DOC alone (p=0.65, 0.60, respectively). Conclusion o-rMETase converted an osteosarcoma PDOX from DOC-resistant to -sensitive. This combination therapy may be effective against recalcitrant osteosarcoma and other recalcitrant cancers.

Background Soft tissue defects following wide excision of malignant soft tissue tumors (STTs) are sometimes too large for primary closure, especially in the lower legs where available soft tissue is limited. This study aimed to determine the clinical outcomes of reconstruction of a defect after wide excision of an STT with a veno-accompanying artery fasciocutaneous (VAF) flap in the lower leg. Methods This study comprised 9 patients with malignant STTs who had undergone reconstructive surgeries using VAF flaps after wide excisions, between October 2010 and September 2017. We retrospectively reviewed and collected data involving age, sex, follow-up period, histological diagnosis, surgical procedures, size and location of defects, size and location of the flaps, venous source of the flaps, direction of the pedicles, closing of donor sites, perioperative chemotherapies, postoperative complications, and the presence of postoperative local recurrence and metastasis. Results The median follow-up period was 91.5 (range, 15.5–189.0) months. Four patients had defects located around the knee, 3 patients had defects located on the calf, and 2 patients had defects located around the ankle. The mean flap size was 95.6 × 119.4 (range, 50 × 100–130 × 140) mm. Six patients had venous sources from the small saphenous vein and 3 patients had venous sources from the great saphenous vein. The pedicles were proximally based in 4 patients and distally based in 5 patients. All flaps remained viable without any complications. Conclusions Our findings showed that the VAF flap was easily elevated and reliable. Furthermore, it was effective in reconstructing soft tissue defects following wide excisions of STTs in the lower leg.

A 35-year-old man presented with a four-year history of a growing mass on the anterior aspect of his left elbow. Magnetic resonance imaging revealed a soft tissue tumor in the brachialis muscle extending to the cubital fossa. Following an open biopsy, the tumor was diagnosed as a monophasic fibrous synovial sarcoma. After neoadjuvant chemotherapy, the patient underwent wide excision and reconstruction of the elbow joint with a pedicle frozen autograft. At the final follow-up four years after surgery, the elbow range of motion was 0-120˚. Although there were signs of osteoarthritis, there was no narrowing of the joint -, and the patient experienced only mild pain. To the best of our knowledge, the present case report is the first to describe wide tumor excision and reconstruction using a pedicle frozen autograft of the elbow. This method should be considered after excision of malignant bone and soft tissue tumors, especially in non-weight-bearing joints. Further cases have to be evaluated to understand the complications and long-term prognosis of this procedure.

In order to identify more effective therapy for recalcitrant osteosarcoma, we evaluated the efficacy of an mTOR-VEGFR inhibitor combination on tumor growth in a unique osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model derived from the lung metastasis of an osteosarcoma patient who failed doxorubicin therapy. We also determined the efficacy of this inhibitor combination on angiogenesis using an in vivo Gelfoam fluorescence angiogenesis mouse model implanted with osteosarcoma patient-derived cells (OS-PDCs). PDOX models were randomly divided into five groups of seven nude mice. Group 1, control; Group 2, doxorubicin (DOX); Group 3, everolimus (EVE, an mTOR and VEGF inhibitor); Group 4, pazopanib (PAZ, a VEGFR inhibitor); Group 5, EVE-PAZ combination. Tumor volume and body weight were monitored 2 times a week. The in vivo Gelfoam fluorescence angiogenesis assay was performed with implanted OS-PDCs. The nude mice with implanted Gelfoam and OSPDCs also were divided into the four therapeutic groups and vessel length was monitored once a week. The EVE-PAZ combination suppressed tumor growth in the osteosarcoma PDOX model and decreased the vessel length ratio in the in vivo Gelfoam fluorescent angiogenesis model, compared with all other groups (p

Background/aim Undifferentiated pleomorphic sarcoma (UPS) is a common soft tissue sarcoma and highly recalcitrant. We have previously developed patient-derived orthotopic xenograft (PDOX) mouse models of UPS and other major sarcoma types. Unlike PDOX models of other cancer types, it has been difficult to demonstrate metastasis in the sarcoma PDOX models. Materials and methods To visualize metastasis at high resolution in the UPS PDOX model, established tumor fragments were implanted in transgenic nude mice expressing red fluorescent protein (RFP) for one passage. The tumors acquired RFP-expressing stroma from transgenic host. UPS tumor with RFP stromal cells were harvested and implanted orthotopically in non-transgenic nude mice. After six weeks of UPS tumor growth in the PDOX model, the primary tumor was imaged non-invasively and lung, liver, and spleen were resected and imaged ex-vivo in order to visualize the presence of RFP, with a FluorVivo® imaging system and FV1000® confocal laser microscope, respectively. Results The primary tumor was imaged non-invasively. Confocal microscopy visualized the presence of RFP in the lung and liver indicating metastases in these organs. This is the first report of metastasis in a sarcoma PDOX model. Conclusion This study should prove very useful to screen for anti-metastatic drugs for the PDOX donor patients and to understand the metastatic process in sarcoma.

Myxofibrosarcoma (MFS) is the most common sarcomas in elderly patients and is either chemo-resistant or recurs with metastasis after chemotherapy. This recalcitrant cancer in need of improved treatment. We have established a patient-derived orthotopic xenograft (PDOX) of MFS. The MFS PDOX model was established in the biceps femoris of nude mice and randomized into 7 groups of 7 mice each: control; doxorubicin (DOX); pazopanib (PAZ); temozolomide (TEM); Irinotecan (IRN); IRN combined with TEM; IRN combined with cisplatinum (CDDP) and Salmonella typhimurium A1-R (S. typhimurium A1-R). Treatment was evaluated by relative tumor volume and relative body weight. The MFS PDOX models were DOX, PAZ, and TEM resistant. IRN combined with TEM and IRN combined with CDDP were most effective on the MFS PDOX. S. typhimurium A1-R arrested the MFS PDOX tumor. There was no significant body weight loss in any group. The present study suggests that the combination of IRN with either TEM or CDDP, and S. typhimurium have clinical potential for MFS.

Pleomorphic liposarcoma (PLPS) is a recalcitrant soft-tissue sarcoma (STS) subtype in need of transformative therapy. We have previously established a patient-derived orthotopic xenograft (PDOX) model, of PLPS with PDGFRA amplification, using surgical orthotopic implantation (SOI). In the present study, the PLPS PDOX model was randomized into three groups of seven mice each: untreated control; doxorubicin (DOX)-treated; and treated with Salmonella typhimurium A1-R (S. typhimurium A1-R) expressing green fluorescent protein (GFP). Tumor volume and body weight were monitored during the treatment period. The PLPS PDOX was resistant to DOX. In contrast, the PLPS PDOX was highly sensitive to S. typhimurium A1-R. There was no significant body-weight loss among these three groups. Fluorescence imaging demonstrated that S. typhimurium A1-R-GFP was very effective to target the PLPS PDOX tumor. The present study demonstrates that a PLPS PDOX, resistant to first-line therapy DOX, was highly sensitive to tumor targeting S. typhimurium A1-R.

Echistatin, a cyclic RGD peptide, which is an antagonist of α_vβ_3 integrin (disintegrin), inhibited human osteosarcoma in the chick chorioallontoic membrane (CAM) model and tumor growth and pulmonary metastases in a nude mouse orthotopic model. A high-metastatic variant of human osteosarcoma, 143B-LM4, overexpressing α_vβ_3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by echistatin (P, 論文

Follicular dendritic-cell sarcoma (FDCS) is a rare and recalcitrant disease. In the present study, a patient-derived orthotopic xenograft (PDOX) mouse model of FDCS was established in the biceps muscle of nude mice. The FDCS PDOX was resistant to both doxorubicin (DOX) and NVP-BEZ235, dactolisib (BEZ) an experimental agent which is a dual pan-phosphoinositide 3-kinase-mammalian target of rapamycin inhibitor. However, in contrast to DOX and BEZ, the FDCS PDOX was sensitive to the tumor-targeting bacterial strain, Salmonella typhimurium A1-R (S. typhimurium A1-R). The combination of S. typhimurium A1-R and either DOX or BEZ did not increase the antitumor efficacy of S. typhimurium A1-R, indicating that DOX and BEZ were not active in this PDOX model. The efficacy of S. typhimurium A1-R in this recalcitrant FDCS gives strong impetus to move bacterial therapy to clinical trials for this disease. The findings of the present study are of particular importance since it demonstrates that S. typhimurium A1-R is effective in a PDOX model of FDCS established from a patient who failed DOX therapy., 論文

Introduction: The efficacy of the combination therapy of mTOR inhibitor and VEGFR inhibitor was reported in advanced renal cell cancer with anti-angiogenesis. However, none of the reports have been published in osteosarcoma (OS). A tumor from 16-year-old patient who failed doxorubicin (DOX) was previously implanted orthotopically into the lateral condyle of distal femur of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model in our laboratory at AntiCancer Inc. We previously found that the OS PDOX model was resistant to DOX matching the clinical setting. We also previously established in vivo angiogenesis assay model using OS cell line with nestin green fluorescent protein (GFP) mouse and reported that OS cells have highly angiogenesis. In the present study, we evaluated the efficacy of everolimus (EVE, a mTOR inhibitor), pazopanib (PAZ, a VEGFR inhibitor) and the combination of EVE and PAZ on the DOX resistant OS PDOX model and in vivo angiogenesis assay model using OS patient derived cells (PDCs). Methods: The OS PDOX model was randomized into five groups of seven mice. An OS PDC was established from a patient-derived OS tumor fragment. After several passages, OS-PDCs were established and were transplanted into the back of nestin GFP mice to make in vivo angiogenesis assay model. For the angiogenesis study, In vivo angiogenesis assay models were randomized into five groups of eight fields: Group 1, Control (Ctrl) treated with PBS, i.p.; Group 2, DOX, 2.4 mg/kg, i.p., weekly; Group 3, EVE, 5 mg/kg, oral gavage, daily; Group 4, PAZ, 50 mg/kg, oral gavage, daily; Group 5,the combination of EVE and PAZ. Treatment was performed for 2 weeks in each model. In the OS PDOX model, treatment efficacy was evaluated on tumor volume ratio and histopathology. Adverse event was evaluated on body weight ratio. In the in vivo angiogenesis assay model, treatment efficacy was evaluated on vascular length ratio. Treatment efficacy and adverse event were evaluated after 2 weeks treatment. Results: Tumor volume ratios in OS PDOX model were the Ctrl group: 4.70 ± 0.58, DOX group: 3.97 ± 0.53, EVE group: 3.89 ± 0.80, PAZ group: 4.16 ± 1.01 and combination group: 1.70 ± 0.30, respectively. Vascular length ratios in vivo angiogenesis assay model were the Ctrl group: 1.44 ± 0.50, DOX group: 1.22 ± 0.30, EVE group: 0.97 ± 0.31, PAZ group: 0.80 ± 0.12 and combination group: 0.58 ± 0.08, respectively. EVE and PAZ combination group regress tumor volume ratio and vascular length ratio with significant difference compared to all groups. In H&E-staining, partial necrosis was detected only in OS PDOX tumors treated with combination of EVE and PAZ. Body weight ratio was no significant difference among all groups. Conclusion: The combination therapy of EVE and PAZ was the most effective in the OS PDOX mouse model and in vivo angiogenesis assay model due to suppress angiogenesis strongly. This study demonstrates that the combination therapy of mTOR inhibitor and VEGFR inhibitor has possibility to be therapeutic strategy for DOX resistant osteosarcoma. Citation Format: Hiromichi Oshiro, Yasunori Tome, Robert M. Hoffman, Kotaro Nishida. mTOR inhibitor and VEGFR inhibitor combination therapy regress a doxorubicin resistant osteosarcoma in a patient-derived orthotopic xenograft model and in vivo angiogenesis assay model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2791.

Introduction: Follicular dendritic cell sarcoma (FDCS) is a very rare and highly refractory soft tissue sarcoma. The CHOP chemotherapy has been used as systemic therapy for FDCS, which includes cyclophosphamide, doxorubicin (DOX), vincristine, and prednisolone. However, FDCS has poor clinical outcome even though CHOP therapy was administered. A FDCS patient tumor was previously implanted orthotopically in the biceps femoris of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model in our laboratory at AntiCancer Inc. The FDCS patient had resistance to DOX in clinical setting. We previously found the FDCS PDOX model had resistance to DOX as well as clinical settings. In the present study, we evaluated the efficacy of temozolomide (TMZ), trabectedin (TRAB), and pazopanib (PAZ) on the DOX resistant FDCS PDOX model. Methods: The FDCS PDOX mouse model was established and randomized into five groups of eight mice, respectively. Group 1, Control (Ctrl) treated with PBS, i.p.; Group 2, DOX, 2.4 mg/kg, i.p., weekly; Group 3, PAZ, 50 mg/kg, oral gavage, daily; Group 4, TRAB, 0.15 mg/kg, i.v., weekly; Group 5, TMZ, 25 mg/kg, oral gavage, daily. Treatment term was for 2 weeks. Treatment efficacy was evaluated based on tumor volume ratio (tumor volume after treatment relative to tumor volume at the beginning of treatment) and histopathology. Adverse event was evaluated based on body weight ratio. Tumor volume and body weight were assessed 2 times per week. Results: Tumor volume ratio was the Ctrl group: 11.35±4.50, DOX: 8.24±1.87, PAZ: 9.67±4.66, TRAB: 6.97±3.23, TMZ: 4.28±1.41, respectively. TMZ significantly arrested the FDCS PDOX model compared to the control group (p Conclusion: The FDCS PDOX model was resistant to DOX as well as in the clinical settings demonstrating concordance of the FDCS model and clinic. TMZ was the most effective for the FDCS PDOX mouse model established from a patient who failed DOX treatment. There is possibility that TMZ has effective to FDCS patients who has resistant to the first line chemotherapy. Citation Format: Hiromichi Oshiro, Yasunori Tome, Robert M. Hoffman, Kotaro Nishida. Temozolomide suppresses a doxorubicin resistant follicular dendritic cell sarcoma in a patient derived orthotopic xenograft mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1661.

Purpose: The aim of this study was to investigate the histopathological changes in the humeral head in cuff tear arthropathy (CTA) compared with those in glenohumeral osteoarthritis (OA) and humeral neck fracture, which served as non-cuff tear controls.\nMethods: Twenty-three humeral heads extracted at the time of shoulder prosthesis arthroplasty between June 2014 and July 2015 were evaluated in the present study. The diagnoses included four-part humeral neck fracture (n = 4; average age, 85.0 years), glenohumeral OA (n = 4; average age, 71.0 years), and CTA (n = 15; average age, 73.0 years). The humeral heads were evaluated pathologically by hematoxylin and eosin and Safranin-O staining, and the thickness of the articular cartilage was measured.\nResults: Fibrillation, thinning, and tearing of the cartilage were observed in the superior area of the humeral heads in CTA and glenohumeral OA. In CTA cases, clusters of chondrocytes in the cartilage were observed. Moreover, the thickness of the cartilage layer in the middle of the humeral head was 1.54 ± 0.07, 0.32 ± 0.46, and 2.19 ± 0.50 mm in humeral neck fracture, glenohumeral OA, and CTA, respectively. The cartilage layer in CTA was thicker than that in glenohumeral OA (CTA vs. OA: p < 0.05).\nConclusion: OA changes in the superior area of the humeral heads and thickening of the cartilage layer from the middle to the inferior of the humeral heads were confirmed histopathologically, suggesting that simultaneous mechanical and nutritional factors might be contributing to CTA pathogenesis. The current study provided the better understanding of cartilage damage and thickening in CTA. This will help guide treatment options in the setting of CTA., 論文

Background: Pleomorphic liposarcoma (PLPS) is a rare, heterogeneous and an aggressive variant of liposarcoma. Therefore, individualized therapy is urgently needed. Our recent reports suggest that trabectedin (TRAB) is effective against several patient-derived orthotopic xenograft (PDOX) mouse models. Here, we compared the efficacy of first-line therapy, doxorubicin (DOX), and TRAB in a platelet-derived growth factor receptor-α (PDGFRA)-amplified PLPS.\nMethods: We used a fresh sample of PLPS tumor derived from a 68-year-old male patient diagnosed with a recurrent PLPS. Subcutaneous implantation of tumor tissue was performed in a nude mouse. After three weeks of implantation, tumor tissues were isolated and cut into small pieces. To match the patient a PDGFRA-amplified PLPS PDOX was created in the biceps femoris of nude mice. Mice were randomized into three groups: Group 1 (G1), control (untreated); Group 2 (G2), DOX-treated; Group 3 (G3), TRAB-treated. Measurement was done twice a week for tumor width, length, and mouse body weight.\nResults: The PLPS PDOX showed resistance towards DOX. However, TRAB could arrest the PLPS (p, 論文

左大腿骨遠位部の骨肉腫に対して，術前化学療法後に広範切除術および液体窒素処理自家骨を用いた再建術を施行し，膝関節を温存し得た症例を経験したので報告する．症例は9歳男児，主訴は左膝痛であった．2週間前より特に誘因なく左膝痛が出現した．近医にて悪性骨腫瘍を疑われ，当科へ紹介された．単純X線像で左大腿骨骨幹端外側に不整な骨硬化像とCodman三角を認めた．MRIでは，左大腿骨遠位に骨端線を越えてT1強調像で低信号，T2強調像で高信号を呈し，強い造影効果を示す骨外病変を伴う腫瘍性病変を認めた．切開生検術を施行し，骨形成型の骨肉腫と診断した．術前化学療法を行い，広範切除術および液体窒素処理自家骨を用いた再建術を施行した．術後6ヵ月で再発・転移なく，坐骨支持免荷装具を用いて独歩可能である．骨接合部には仮骨を認め，液体窒素処理骨の圧潰，骨折は認めず，良好な患肢機能が得られている．

円形細胞骨肉腫は骨肉腫の亜型で，発生頻度は少ない．今回，橈骨骨幹部に発生した円形細胞骨肉腫の1例を報告する．症例は16歳女性，主訴は左前腕の腫瘤．2カ月前より左前腕のしこりを自覚し，疼痛が出現したため近医を受診し左橈骨骨幹部の骨腫瘍が疑われ，当科へ紹介された．単純X線像で左橈骨骨幹部に骨透亮像および皮質骨の不正な肥厚を認めた．MRIでは橈骨骨幹部の皮質骨にT1強調像で低信号，T2強調像で高信号を呈し，強い造影効果を示す9×11mm大の腫瘍性病変を認めた．切開生検術を施行し，円形細胞骨肉腫と診断された．術前に化学療法を行い，広範切除術と腓骨移植（non-vascularized），腱移行を用いた再建術を施行した．術後1年2カ月で再発はなく，良好な患肢機能が得られている．

KLS systemの術後成績，腫瘍学的転帰，術後合併症，人工関節累積生存率，術後患肢機能評価（MSTS score）について報告する．対象は，大腿骨悪性骨腫瘍9症例（男2例，女7例），手術時年齢は16～83歳，経過観察期間は3～97ヵ月であった．原疾患は骨肉腫3例, Ewing肉腫3例，転移性骨腫瘍3例，再建部位は大腿骨全長1例，近位部3例，遠位部5例であった．腫瘍学的転帰はCDF 4例，NED 3例，DOD 2例であった．術後合併症は深部感染を2例に認めた．その他の合併症として人工関節の緩みや折損は認めなかった．人工関節の3年，5年累積生存率は各々87.5％，58.3％であった．MSTS scoreは平均81％であった．KLS systemの治療成績は短期では良好であった．今後症例数の蓄積と長期の観察が必要であり，感染症の克服が今後の課題と考えられた．

MIPO法を用いて脛骨骨折に対して手術治療を行ったので，その有用性を検討した．2003年10月より2005年12月までにMIPO法を用いて手術治療を行った脛骨骨折の症例は20例で，内訳は近位端骨折7例，近位～骨幹部骨折1例，骨幹部骨折2例，遠位～骨幹部骨折1例，遠位端骨折9例であった．また，骨端線閉鎖前の症例は3例（11歳1例，14歳2例）であった．全例，MIPO法を併用してLCPを用いて固定を行った．plateやscrewのlooseningや折損例はなかった．また感染例もなかった．脛骨遠位端骨折の1例に骨癒合の遷延を認めたが，全例で骨癒合が得られた．経過中，術直後の固定状態の矯正損失例はなかった．MIPO法は骨幹端部骨折がよい適応で，髄内釘の適応とならない骨幹部骨折に対して有用であると考えられた．

To clarify which factors influence the results of operative treatment of ankle malleolar fracture, the outcome was assessed clinically and radiographically in 76 joints of 76 patients who consisted of 49 males and 27 females with an average age of 36 years. After an average follow-up of 11.3 months, clinical and radiographic results were evaluated according to Burwells clinical and radiographic criteria. Clinical results were good in 63 joints, fair and poor in 13 joints. Postoperative reduction was anatomical in 65 joints, fair and poor in 11 joints.There were no significant differences between clinically good and fair/poor groups with regard to sex, age, fracture type according to the Lauge-Hansen classification, number of malleolar fractures, and injury-operation interval. However, clinical results were significantly lower in patients injured by traffic accidents compared to other causes such as fall and sport injury. Although there was a tendency for anatomical reduction to be obtained in patients with good clinical results, it was not significant in this study.

母指CM関節不安定性による障害に対して長橈側手根伸筋腱（以下ECRL腱）を用いてIntermetacarpal lig.（以下IML）再建術を施行したので報告する．対象は靭帯再建術を施行した7例7手（母指CM関節症5例5手，外傷性不安定症2例2手）．性別は男性2例，女性5例，年齢は15～67歳，経過観察期間は6カ月～6年3カ月であった．Eaton分類はstage Iが4手，stage IIが3手であった．手術は琉球大法に準じて停止部を茎とするECRL半切腱を用いてIMLを再建した．手術成績（Eatonの評価），母指CM関節可動域，ピンチ力，握力，X線学的評価を検討した．手術成績はexcellent 5例，good 1例，failure 1例であった．Eaton分類でstageが進行した例は認めなかった．関節症変化が軽度の母指CM関節症と外傷性不安定症に対して本法は有効と考えられた．

Three cases of femoral fracture and one case of femoral lengthening were treated surgically by mimimally invasive plate osteosynthesis. All cases except one achieved good bone union. One case of distal femoral fracture needed re-operation because of re-fracture. Mimimally invasive plate osteosynthesis is useful for femoral fractures for which intramedullary nailing is not suitable.

The aim of this study is to determine if fluorescence-guided surgery (FGS) can eradicate human fibrosarcoma growing in the retroperitoneum of nude mice. One week after retroperitoneal implantation of human HT1080 fibrosarcoma cells, expressing green fluorescent protein (GFP) (HT-1080-GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n = 22). After BLS, mice were randomized into 2 treatment groups; BLS-only (n = 11) or the combination of BLS + FGS (n = 11). The residual tumors remaining after BLS were resected with FGS using a hand-held portable imaging system under fluorescence navigation. The average residual tumor area after BLS + FGS was significantly smaller than after BLS-only (0.4 ± 0.4 mm^2 and 10.5 ± 2.4 mm^2, respectively; p = 0.006). Five weeks after surgery, the fluorescent-tumor areas of BLS- and BLS + FGS-treated mice were 379 ± 147 mm^2 and 11.7 ± 6.9 mm^2, respectively, indicating that FGS greatly inhibited tumor recurrence compared to BLS. The combination of BLS + FGS significantly decreased fibrosarcoma recurrence compared to BLS-only treated mice (p < 0.001). Mice treated with BLS+FGS had a significantly higher disease-free survival rate than mice treated with BLS-only at five weeks after surgery. These results suggest that combination of BLS + FGS significantly reduced the residual fibrosarcoma volume after BLS and improved disease-free survival., 論文

Color-coded chemotherapy: S/G2-phase-trapping by methioninase pre-treatment, indicated by FUCCI imaging, enables highly effective cancer chemotherapy (923.11) Shuya Yano1, Yasunori Tome 1 , Michelle Digman 2 , Masashi Momiyama 1 , Atsushi Suetsugu 1 , Enrico Gratton 2 and Robert Hoffman 1 April 2014 The FASEB Journal vol. 28 no. 1 Supplement 923.11 - Author Affiliations AntiCancer Inc. San Diego CA United States Department of Biomedical Engineering University of California Irvine Irvine CA United States Abstract Methionine deprivation by methionine α,γ lyase (methioninase or METase) selectively arrests cancer cells during late S/G2-phase of the cell cycle, where the cancer cells are highly sensitive to DNA- damaging chemotherapy. Fluorescent ubiquitination-based cell cycle indicator (FUCCI) (Cell 132, 487- 498, 2008), was used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by METase. The S/G2-phase-blocked cancer cells fluoresced yellow or green, in contrast to cancer cells in G1/G0 which fluoresced red due to FUCCI. Cancer cells, synchronously blocked in S/G2- phase by METase fluorescing yellow-green, were treated with doxorubicin, cisplatin, or 5-fluorouracil. As a control, cancer cells treated with drugs only without rMETAse, were resistant to the drugs. rMETase treatment, followed by chemotherapy, when FUCCI indicated the onset of S/G2 block, was highly effective. Color-coded chemotherapy, whereby the cell cycle of cancer cells is blocked in S/G2-phase, as identified by fluorescent reporters, may be a general approach to effective cancer treatment.

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

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

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

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

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