Makoto Toneri

Color-coding cancer and stromal cells with genetic reporters in a patient-derived orthotopic xenograft (PDOX) model of pancreatic cancer enhances fluorescence-guided surgery

Precise fluorescence-guided surgery (FGS) for pancreatic cancer has the potential to greatly improve the outcome in this recalcitrant disease. To achieve this goal, we have used genetic reporters to color code cancer and stroma cells in a patient-derived orthotopic xenograft (PDOX) model. The telomerase-dependent green fluorescent protein (GFP)-containing adenovirus OBP-401 was used to label the cancer cells of a pancreatic cancer PDOX. The PDOX was previously grown in a red fluorescent protein (RFP) transgenic mouse that stably labeled the PDOX stroma cells bright red. The color-coded PDOX model enabled FGS to completely resect the pancreatic tumors including stroma. Dual-colored FGS significantly prevented local recurrence, which bright-light surgery or single-color FGS could not. FGS, with color-coded cancer and stroma cells has important potential for improving the outcome of recalcitrant-cancer surgery.

Efficacy of Tumor‐Targeting Salmonella Typhimurium A1‐R on Nude Mouse Models of Metastatic and Disseminated Human Ovarian Cancer

We report here the efficacy of tumor‐targeting Salmonella typhimurium A1‐R (A1‐R) on mouse models of disseminated and metastatic ovarian cancer. The proliferation‐inhibitory efficacy of A1‐R on human ovarian cancer cell lines (SKOV‐3‐GFP, OVCAR‐3‐RFP) was initially demonstrated in vitro. Orthotopic and dissemination mouse models of ovarian cancer were made with the human ovarian cancer cell line SKOV‐3‐GFP. After tumor implantation, the mice were treated with A1‐R (5 × 107 colony‐forming units [CFU], i.v.), and there were no severe adverse events observed. In the orthotopic model, tumor volume after treatment was 276 ± 60.8 mm3, compared to 930 ± 342 mm3 in the untreated control group (P = 0.022). There was also a significant difference in survival between treated mice and untreated mice in a peritoneal dissemination model (P = 0.005). The results of this report demonstrate that A1‐R is effective for highly aggressive human ovarian cancer in metastatic and dissemination mouse models and suggest its clinical potential for this highly treatment‐resistant disease. J. Cell. Biochem. 115: 1996–2003, 2014.

Experimental Curative Fluorescence-guided Surgery of Highly Invasive Glioblastoma Multiforme Selectively Labeled With a Killer-reporter Adenovirus.

Fluorescence-guided surgery (FGS) of cancer is an area of intense current interest. However, although benefits have been demonstrated with FGS, curative strategies need to be developed. Glioblastoma multiforme (GBM) is one of the most invasive of cancers and is not totally resectable using standard bright-light surgery (BLS) or current FGS strategies. We report here a curative strategy for FGS of GBM. In this study, telomerase-dependent adenovirus OBP-401 infection brightly and selectively labeled GBM with green fluorescent protein (GFP) for FGS in orthotopic nude mouse models. OBP-401-based FGS enabled curative resection of GBM without recurrence for at least 150 days, compared to less than 30 days with BLS.

Cell-cycle fate-monitoring distinguishes individual chemosensitive and chemoresistant cancer cells in drug-treated heterogeneous populations demonstrated by real-time FUCCI imaging

Essentially every population of cancer cells within a tumor is heterogeneous, especially with regard to chemosensitivity and resistance. In the present study, we utilized the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging system to investigate the correlation between cell-cycle behavior and apoptosis after treatment of cancer cells with chemotherapeutic drugs. HeLa cells expressing FUCCI were treated with doxorubicin (DOX) (5 μM) or cisplatinum (CDDP) (5 μM) for 3 h. Cell-cycle progression and apoptosis were monitored by time-lapse FUCCI imaging for 72 h. Time-lapse FUCCI imaging demonstrated that both DOX and CDDP could induce cell cycle arrest in S/G2/M in almost all the cells, but a subpopulation of the cells could escape the block and undergo mitosis. The subpopulation which went through mitosis subsequently underwent apoptosis, while the cells arrested in S/G2/M survived. The present results demonstrate that chemoresistant cells can be readily identified in a heterogeneous population of cancer cells by S/G2/M arrest, which can serve in future studies as a visible target for novel agents that kill cell-cycle-arrested cells.

Real-Time GFP Intravital Imaging of the Differences in Cellular and Angiogenic Behavior of Subcutaneous and Orthotopic Nude-Mouse Models of Human PC-3 Prostate Cancer

There are two major types of mouse xenograft models of cancer: subcutaneous implantation and orthotopic implantation. Subcutaneous transplant models are widely used with both cancer cell lines and human‐tumor specimens. Recently, subcutaneous models of patient tumors, termed patient‐derived xenographs (PDX) have become highly popular and have acquired such names as “Avatar” and “Xenopatients.” However, such s.c. models rarely metastasize and are therefore not patient‐like. In contrast, orthotopic models have the capability to metastasize. If intact fragments of tumor tissue are implanted by surgical orthotopic implantation (SOI), the metastatic potential can match that of the donor patient. The present study images in real time, using green fluorescent protein (GFP) expression, the very different tumor behavior at the orthotopic and subcutaneous sites of human prostate cancer PC‐3 in athymic nude mice. By day‐2 after tumor implantation, the orthotopic tumor is already highly vascularized and the cancer cells have begun to migrate out of the tumor. In contrast, the subcutaneous tumor only begins to be vascularized by day‐3 and cells do not migrate from the tumor. Angiogenesis is much more extensive in the orthotopic tumor throughout the 2‐week observation period. The orthotopic PC‐3‐GFP tumor progresses very rapidly and distinct metastasis have appeared in lymph nodes by day‐3 which rapidly appear in many areas of the abdominal cavity including portal lymph nodes by day‐7. At day‐14, no invasion or metastasis was observed with the s.c. tumor even when the animal was extensively explored. These results explain why orthotopic tumors mimimc clinical metastatic tumors in nude mice and why subcutaneous tumors do not. J. Cell. Biochem. 117: 2546–2551, 2016.

Fluorescence‐guided surgery of human prostate cancer experimental bone metastasis in nude mice using anti‐CEA dylight 650 for tumor illumination

The present report demonstrates efficacy of fluorescence‐guided surgery (FGS) to resect and prevent recurrence of experimental skeletal metastasis in a nude‐mouse model of human prostate cancer. Green fluorescent protein (GFP)‐expressing PC‐3 human prostate cancer cells were injected into the intramedullary cavity of the tibia in 25 nude mice. One week after implantation, monoclonal antibodies, specific for carcinoembryonic antigen (CEA), labeled with DyLight 650, were injected into the tail vein of 13 mice. Thirteen mice underwent FGS and 12 mice underwent bright‐light surgery (BLS). Weekly GFP fluorescence imaging of the mice was performed to observe tumor recurrence. The extent of residual tumor after BLS was 13‐fold greater than after FGS (p < 0.001). Time‐course imaging visualized rapid growth of the residual tumor in the BLS group, whereas the FGS group showed only slight tumor growth and significantly improved disease‐free survival of the treated mice. Our study demonstrated that FGS significantly reduced residual tumor as well as the recurrence of experimental prostate‐cancer bone metastasis. The present results suggest that FGS will be effective for resection of skeletal metastases in selected patients with prostate cancer.

Toxicology and efficacy of tumor-targeting Salmonella typhimurium A1-R compared to VNP 20009 in a syngeneic mouse tumor model in immunocompetent mice

Salmonella typhimurium A1-R (S. typhimurium A1-R) attenuated by leu and arg auxotrophy has been shown to target multiple types of cancer in mouse models. In the present study, toxicologic and biodistribution studies of tumor-targeting S. typhimurium A1-R and S. typhimurium VNP20009 (VNP 20009) were performed in a syngeneic tumor model growing in immunocompetent BALB/c mice. Single or multiple doses of S. typhimurium A1-R of 2.5 × 105 and 5 × 105 were tolerated. A single dose of 1 × 106 resulted in mouse death. S. typhimurium A1-R (5 × 105 CFU) was eliminated from the circulation, liver and spleen approximately 3-5 days after bacterial administration via the tail vein, but remained in the tumor in high amounts. S. typhimurium A1-R was cleared from other organs much more rapidly. S. typhimurium A1-R and VNP 20009 toxicity to the spleen and liver was minimal. S. typhimurium A1-R showed higher selective targeting to the necrotic areas of the tumors than VNP20009. S. typhimurium A1-R inhibited the growth of CT26 colon carcinoma to a greater extent at the same dose of VNP20009. In conclusion, we have determined a safe dose and schedule of S. typhimurium A1-R administration in BALB/c mice, which is also efficacious against tumor growth. The results of the present report indicate similar toxicity of S. typhimurium A1-R and VNP20009, but greater antitumor efficacy of S. typhimurium A1-R in an immunocompetent animal. Since VNP2009 has already proven safe in a Phase I clinical trial, the present results indicate the high clinical potential of S. typhimurium A1-R.

Tumor-targeting Salmonella typhimurium A1-R inhibits human prostate cancer experimental bone metastasis in mouse models

Bone metastasis is a frequent occurrence in prostate cancer patients and often is lethal. Zoledronic acid (ZOL) is often used for bone metastasis with limited efficacy. More effective models and treatment methods are required to improve the outcome of prostate cancer patients. In the present study, the effects of tumor-targeting Salmonella typhimurium A1-R were analyzed in vitro and in vivo on prostate cancer cells and experimental bone metastasis. Both ZOL and S. typhimurium A1-R inhibited the growth of PC-3 cells expressing red fluorescent protien in vitro. To investigate the efficacy of S. typhimurium A1-R on prostate cancer experimental bone metastasis, we established models of both early and advanced stage bone metastasis. The mice were treated with ZOL, S. typhimurium A1-R, and combination therapy of both ZOL and S. typhimurium A1-R. ZOL and S. typhimurium A1-R inhibited the growth of solitary bone metastases. S. typhimurium A1-R treatment significantly decreased bone metastasis and delayed the appearance of PC-3 bone metastases of multiple mouse models. Additionally, S. typhimurium A1-R treatment significantly improved the overall survival of the mice with multiple bone metastases. The results of the present study indicate that S. typhimurium A1-R is useful to prevent and inhibit prostate cancer bone metastasis and has potential for future clinical use in the adjuvant setting.

Heterogeneous cell-cycle behavior in response to UVB irradiation by a population of single cancer cells visualized by time-lapse FUCCI imaging

The present study analyzed the heterogeneous cell-cycle dependence and fate of single cancer cells in a population treated with UVB using a fluorescence ubiquitination-based cell-cycle (FUCCI) imaging system. HeLa cells expressing FUCCI were irradiated by 100 or 200 J/m2 UVB. Modulation of the cell-cycle and apoptosis were observed by time-lapse confocal microscopy imaging every 30 min for 72 h. Correlation between cell survival and factors including cell-cycle phase at the time of the irradiation of UVB, mitosis and the G1/S transition were analyzed using the Kaplan–Meier method along with the log rank test. Time-lapse FUCCI imaging of HeLa cells demonstrated that UVB irradiation induced cell-cycle arrest in S/G2/M phase in the majority of the cells. The cells irradiated by 100 or 200 J/m2 UVB during G0/G1 phase had a higher survival rate than the cells irradiated during S/G2/M phase. A minority of cells could escape S/G2/M arrest and undergo mitosis which significantly correlated with decreased survival of the cells. In contrast, G1/S transition significantly correlated with increased survival of the cells after UVB irradiation. UVB at 200 J/m2 resulted in a greater number of apoptotic cells.

Real-Time Fluorescence Imaging of the DNA Damage Repair Response During Mitosis

The response to DNA damage during mitosis was visualized using real‐time fluorescence imaging of focus formation by the DNA‐damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1‐GFP) in the MiaPaCa‐2Tet‐On pancreatic cancer cell line. To observe 53BP1‐GFP foci during mitosis, MiaPaCa‐2Tet‐On 53BP1‐GFP cells were imaged every 30 min by confocal microscopy. Time‐lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa‐2Tet‐On 53BP1‐GFP cells had increased focus formation over time. Non‐mitotic cells did not have an increase in 53BP1‐GFP focus formation over time. Some of the mitotic MiaPaCa‐2Tet‐On 53BP1‐GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe. J. Cell. Biochem. 116: 661–666, 2015.