Shuya Yano

Successful Fluorescence-Guided Surgery on Human Colon Cancer Patient-Derived Orthotopic Xenograft Mouse Models Using a Fluorophore-Conjugated Anti-CEA Antibody and a Portable Imaging System

BACKGROUND Fluorescence-guided surgery (FGS) can enable successful cancer surgery where bright-light surgery often cannot. There are three important issues for FGS going forward toward the clinic: (a) proper tumor labeling, (b) a simple portable imaging system for the operating room, and (c) patient-like mouse models in which to develop the technology. The present report addresses all three. MATERIALS AND METHODS Patient colon tumors were initially established subcutaneously in nonobese diabetic (NOD)/severe combined immune deficiency (SCID) mice immediately after surgery. The tumors were then harvested from NOD/SCID mice and passed orthotopically in nude mice to make patient-derived orthotopic xenograft (PDOX) models. Eight weeks after orthotopic implantation, a monoclonal anti-carcinoembryonic antigen (CEA) antibody conjugated with AlexaFluor 488 (Molecular Probes Inc., Eugene, OR) was delivered to the PDOX models as a single intravenous dose 24 hours before laparotomy. A hand-held portable fluorescence imaging device was used. RESULTS The primary tumor was clearly visible at laparotomy with the portable fluorescence imaging system. Frozen section microscopy of the resected specimen demonstrated that the anti-CEA antibody selectively labeled cancer cells in the colon cancer PDOX. The tumor was completely resected under fluorescence navigation. Histologic evaluation of the resected specimen demonstrated that cancer cells were not present in the margins, indicating successful tumor resection. The FGS animals remained tumor free for over 6 months. CONCLUSIONS The results of the present report indicate that FGS using a fluorophore-conjugated anti-CEA antibody and portable imaging system improves efficacy of resection for CEA-positive colorectal cancer. These data provide the basis for clinical trials.

Efficacy of Salmonella typhimurium A1-R versus chemotherapy on a pancreatic cancer patient-derived orthotopic xenograft (PDOX)

The aim of this study is to determine the efficacy of tumor‐targeting Salmonella typhimurium A1‐R (A1‐R) on pancreatic cancer patient‐derived orthotopic xenografts (PDOX). The PDOX model was originally established from a pancreatic cancer patient in SCID‐NOD mice. The pancreatic cancer PDOX was subsequently transplanted by surgical orthotopic implantation (SOI) in transgenic nude red fluorescent protein (RFP) mice in order that the PDOX stably acquired red fluorescent protein (RFP)‐expressing stroma for the purpose of imaging the tumor after passage to non‐transgenic nude mice in order to visualize tumor growth and drug efficacy. The nude mice with human pancreatic PDOX were treated with A1‐R or standard chemotherapy, including gemcitabine (GEM), which is first‐line therapy for pancreatic cancer, for comparison of efficacy. A1‐R treatment significantly reduced tumor weight, as well as tumor fluorescence area, compared to untreated control (P = 0.011), with comparable efficacy of GEM, CDDP, and 5‐FU. Histopathological response to treatment was defined according to Evanss criteria and A1‐R had increased efficacy compared to standard chemotherapy. The present report is the first to show that A1‐R is effective against a very low‐passage patient tumor, in this case, pancreatic cancer. The data of the present report suggest A1‐1 will have clinical activity in pancreatic cancer, a highly lethal and treatment‐resistant disease and may be most effectively used in combination with other agents. J. Cell. Biochem. 115: 1254–1261, 2014.

Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells

The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC50 values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC50 values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.

Tumor-targeting Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle as visualized by FUCCI imaging and become sensitive to chemotherapy

Quiescent cancer cells are resistant to cytotoxic agents which target only proliferating cancer cells. Time-lapse imaging demonstrated that tumor-targeting Salmonella typhimurium A1-R (A1-R) decoyed cancer cells in monolayer culture and in tumor spheres to cycle from G0/G1 to S/G2/M, as demonstrated by fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging. A1-R infection of FUCCI-expressing subcutaneous tumors growing in nude mice also decoyed quiescent cancer cells, which were the majority of the cells in the tumors, to cycle from G0/G1 to S/G2/M, thereby making them sensitive to cytotoxic agents. The combination of A1-R and cisplatinum or paclitaxel reduced tumor size compared with A1-R monotherapy or cisplatinum or paclitaxel alone. The results of this study demonstrate that A1-R can decoy quiescent cancer cells to cycle to S/G2/M and sensitize them to cytotoxic chemotherapy. These results suggest a new paradigm of bacterial-decoy chemotherapy of 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.

Telomerase-Dependent Oncolytic Adenovirus Sensitizes Human Cancer Cells to Ionizing Radiation via Inhibition of DNA Repair Machinery

The inability to repair DNA double-strand breaks (DSB) leads to radiosensitization, such that ionizing radiation combined with molecular inhibition of cellular DSB processing may greatly affect treatment of human cancer. As a variety of viral products interact with the DNA repair machinery, oncolytic virotherapy may improve the therapeutic window of conventional radiotherapy. Here, we describe the mechanistic basis for synergy of irradiation and OBP-301 (Telomelysin), an attenuated type-5 adenovirus with oncolytic potency that contains the human telomerase reverse transcriptase promoter to regulate viral replication. OBP-301 infection led to E1B55kDa viral protein expression that degraded the complex formed by Mre11, Rad50, and NBS1, which senses DSBs. Subsequently, the phosphorylation of cellular ataxia-telangiectasia mutated protein was inhibited, disrupting the signaling pathway controlling DNA repair. Thus, tumor cells infected with OBP-301 could be rendered sensitive to ionizing radiation. Moreover, by using noninvasive whole-body imaging, we showed that intratumoral injection of OBP-301 followed by regional irradiation induces a substantial antitumor effect, resulting from tumor cell-specific radiosensitization, in an orthotopic human esophageal cancer xenograft model. These results illustrate the potential of combining oncolytic virotherapy and ionizing radiation as a promising strategy in the management of human cancer.

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.

Genetically engineered oncolytic adenovirus induces autophagic cell death through an E2F1-microRNA-7-epidermal growth factor receptor axis

Autophagy is known to have a cytoprotective role under various cellular stresses; however, it also results in robust cell death as an important safeguard mechanism that protects the organism against invading pathogens and unwanted cancer cells. Autophagy is regulated by cell signalling including microRNA (miRNA), a post‐transcriptional regulator of gene expression. Here, we show that genetically engineered telomerase‐specific oncolytic adenovirus induced miR‐7 expression, which is significantly associated with its cytopathic activity in human cancer cells. Virus‐mediated miR‐7 upregulation depended on enhanced expression of the E2F1 protein. Ectopic expression of miR‐7 suppressed cell viability and induced autophagy by inhibiting epidermal growth factor receptor (EGFR) expression. Our results suggest that oncolytic adenovirus induces autophagic cell death through an E2F1‐miR‐7‐EGFR pathway in human cancer cells, providing a novel insight into the molecular mechanism of an anticancer virotherapy.

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.