Mako Yamamoto

Efficacy of Tumor-Targeting Salmonella A1-R on a Melanoma Patient-Derived Orthotopic Xenograft (PDOX) Nude-Mouse Model.

Tumor-targeting Salmonella enterica serovar Typhimurium A1-R (Salmonella A1-R) had strong efficacy on a melanoma patient-derived orthotopic xenograft (PDOX) nude-mouse model. GFP-expressing Salmonella A1-R highly and selectively colonized the PDOX melanoma and significantly suppressed tumor growth (p = 0.021). The combination of Salmonella A1-R and cisplatinum (CDDP), both at low-dose, also significantly suppressed the growth of the melanoma PDOX (P = 0.001). Salmonella A1-R has future clinical potential for combination chemotherapy with CDDP of melanoma, a highly-recalcitrant cancer.

Establishment of a patient-derived orthotopic Xenograft (PDOX) model of HER-2-positive cervical cancer expressing the clinical metastatic pattern.

Squamous cell carcinoma of the cervix, highly prevalent in the developing world, is often metastatic and treatment resistant with no standard treatment protocol. Our laboratory pioneered the patient-derived orthotopic xenograft (PDOX) nude mouse model with the technique of surgical orthotopic implantation (SOI). Unlike subcutaneous transplant patient-derived xenograft (PDX) models, PDOX models metastasize. Most importantly, the metastasis pattern correlates to the patient. In the present report, we describe the development of a PDOX model of HER-2-positive cervical cancer. Metastasis after SOI in nude mice included peritoneal dissemination, liver metastasis, lung metastasis as well as lymph node metastasis reflecting the metastatic pattern in the donor patient. Metastasis was detected in 4 of 6 nude mice with primary tumors. Primary tumors and metastases in the nude mice had histological structures similar to the original tumor and were stained by an anti-HER-2 antibody in the same pattern as the patient’s cancer. The metastatic pattern, histology and HER-2 tumor expression of the patient were thus preserved in the PDOX model. In contrast, subcutaneous transplantation of the patient’s cervical tumors resulted in primary growth but not metastasis.

Tumor-Targeting Salmonella typhimurium A1-R Arrests a Chemo-Resistant Patient Soft-Tissue Sarcoma in Nude Mice

A patient-derived nude-mouse model of soft-tissue sarcoma has been established and treated in the following groups: (1) untreated controls; (2) gemcitabine (GEM) (80 mg/kg, ip, weekly, 3 weeks); (3) Pazopanib (100 mg/kg, orally, daily, 3 weeks) and (4) Salmonella typhimurium A1-R (5 × 107 CFU/body, ip, weekly, 3 weeks). The sarcoma was resistant to GEM (p = 0.879). Pazopanib tended to reduce the tumor volume compared to the untreated mice, but there was no significant difference (p = 0.115). S. typhimurium A1-R significantly inhibited tumor growth compared to the untreated mice (p = 0.001). S. typhimurium A1-R was the only effective treatment for the soft-tissue sarcoma nude mouse model among all treatments including a newly approved multiple tyrosine kinase inhibitor; Pazopanib. These results suggest tumor-targeting S. typhimurium A1-R is a promising treatment for chemo-resistant soft-tissue sarcoma.

Spatial–temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We report here on the results of monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI) before, during, and after chemotherapy. 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, approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 µm) tumor blood vessels of an established tumor are in G0/G1. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Our results suggest why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. The results also suggest that drugs that target quiescent cancer cells are urgently needed.

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.

Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging.

Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G0/G1 cells express a red fluorescent protein and S/G2/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G0/G1 phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G2/M phases. Cancer cells ceased migrating when they entered S/G2/M phases and restarted migrating after cell division when the cells re-entered G0/G1. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G0/G1, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G0/G1 cancer cells should be developed to prevent metastasis.

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.

Tumor-Targeting Salmonella typhimurium A1-R in Combination with Trastuzumab Eradicates HER-2-Positive Cervical Cancer Cells in Patient-Derived Mouse Models

We have previously developed mouse models of HER-2-positive cervical cancer. Tumors in nude mice had histological structures similar to the original tumor and were stained by anti-HER-2 antibody in the same pattern as the patient’s cancer. We have also previously developed tumor-targeting Salmonella typhimurium A1-R and have demonstrated its efficacy against patient-derived tumor mouse models, both alone and in combination. In the current study, we determined the efficacy of S. typhimurium A1-R in combination with trastuzumab on a patient-cancer nude-mouse model of HER-2 positive cervical cancer. Mice were randomized to 5 groups and treated as follows: (1) no treatment; (2) carboplatinum (30 mg/kg, ip, weekly, 5 weeks); (3) trastuzumab (20 mg/kg, ip, weekly, 5 weeks); (4) S. typhimurium A1-R (5 × 107 CFU/body, ip, weekly, 5 weeks); (5) S. typhimurium A1-R (5 × 107 CFU/body, ip, weekly, 5 weeks) + trastuzumab (20 mg/kg, ip, weekly, 5 weeks). All regimens had significant efficacy compared to the untreated mice. The relative tumor volume of S. typhimurium A1-R + trastuzumab-treated mice was smaller compared to trastuzumab alone (p = 0.007) and S. typhimurium A1-R alone (p = 0.039). No significant body weight loss was found compared to the no treatment group except for carboplatinum-treated mice (p = 0.021). Upon histological examination, viable tumor cells were not detected, and replaced by stromal cells in the tumors treated with S. typhimurium A1-R + trastuzumab. The results of the present study suggest that S. typhimurium A1-R and trastuzumab in combination are highly effective against HER-2-expressing cervical cancer.

Fluorescence-Guided Surgery in Combination with UVC Irradiation Cures Metastatic Human Pancreatic Cancer in Orthotopic Mouse Models

The aim of this study is to determine if ultraviolet light (UVC) irradiation in combination with fluorescence-guided surgery (FGS) can eradicate metastatic human pancreatic cancer in orthotopic nude–mouse models. Two weeks after orthotopic implantation of human MiaPaCa-2 pancreatic cancer cells, expressing green fluorescent protein (GFP), in nude mice, bright-light surgery (BLS) was performed on all tumor-bearing mice (n = 24). After BLS, mice were randomized into 3 treatment groups; BLS-only (n = 8) or FGS (n = 8) or FGS-UVC (n = 8). The residual tumors were resected using a hand-held portable imaging system under fluorescence navigation in mice treated with FGS and FGS-UVC. The surgical resection bed was irradiated with 2700 J/m2 UVC (254 nm) in the mice treated with FGS-UVC. The average residual tumor area after FGS (n = 16) was significantly smaller than after BLS only (n = 24) (0.135±0.137 mm2 and 3.338±2.929 mm2, respectively; p = 0.007). The BLS treated mice had significantly reduced survival compared to FGS- and FGS-UVC-treated mice for both relapse-free survival (RFS) (p<0.001 and p<0.001, respectively) and overall survival (OS) (p<0.001 and p<0.001, respectively). FGS-UVC-treated mice had increased RFS and OS compared to FGS-only treated mice (p = 0.008 and p = 0.025, respectively); with RFS lasting at least 150 days indicating the animals were cured. The results of the present study suggest that UVC irradiation in combination with FGS has clinical potential to increase survival.