Yukihiko Hiroshima

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.

Tumor-targeting Salmonella typhimurium A1-R in combination with doxorubicin eradicate soft tissue sarcoma in a patient-derived orthotopic xenograft (PDOX) model

A patient with high grade undifferentiated pleomorphic soft-tissue sarcoma from a striated muscle was grown orthotopically in the right biceps femoris muscle of mice to establish a patient-derived orthotopic xenograft (PDOX) model. Twenty PDOX mice were divided into 4 groups: G1, control without treatment; G2, Salmonella typhimurium (S. typhimurium)A1-R administered by intratumoral (i.t.) injection once a week for 4 weeks; G3, doxorubicin (DOX) administered by intraperitoneal (i.p.) injection once a week for 4 weeks; G4, S. typhimurium A1-R (i.t.) administered once a week for 2 weeks followed by i.p. doxorubicin once a week for 2 weeks. On day 25 from the initiation of treatment, tumor volume in G2, G3, and G4 was significantly lower than G1. Mice found without gross tumor included one mouse (20%) in G2; one mouse (20%) in G3; and 3 mice (60%) in G4. Body weight loss did not significantly differ between the 3 treated groups or from the untreated control. Histological examination revealed eradication of tumor only in G4 where mice were treated with S. typhimurium A1-R followed by DOX. Our present study indicates future clinical potential of combining S. typhimurium A1-R with chemotherapy such as DOX for soft tissue sarcoma patients.

Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence.

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.

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.

Effective molecular targeting of CDK4/6 and IGF-1R in a rare FUS-ERG fusion CDKN2A -deletion doxorubicin-resistant Ewing's sarcoma patient-derived orthotopic xenograft (PDOX) nude-mouse model

Ewings sarcoma is a rare and aggressive malignancy. In the present study, tumor from a patient with a Ewings sarcoma with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion was implanted in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present study was to determine efficacy of cyclin-dependent kinase 4/6 (CDK4/6) and insulin-like growth factor-1 receptor (IGF-1R) inhibitors on the Ewings sarcoma PDOX. The PDOX models were randomized into the following groups when tumor volume reached 50 mm3: G1, untreated control; G2, doxorubicin (DOX) (intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, CDK4/6 inhibitor (palbociclib, PD0332991, per oral (p.o.), daily, for 14 days); G4, IGF-1R inhibitor (linsitinib, OSI-906, p.o., daily, for 14 days). Tumor growth was significantly suppressed both in G3 (palbociclib) and in G4 (linsitinib) compared to G1 (untreated control) at all measured time points. In contrast, DOX did not inhibit tumor growth at any time point, which is consistent with the failure of DOX to control tumor growth in the patient. The results of the present study demonstrate the power of the PDOX model to identify effective targeted molecular therapy of a recalcitrant DOX-resistant Ewings sarcoma with specific genetic alterations. The results of this study suggest the potential of PDOX models for individually-tailored, effective targeted therapy for recalcitrant cancer.

Selective efficacy of zoledronic acid on metastasis in a patient-derived orthotopic xenograph (PDOX) nude-mouse model of human pancreatic cancer

Patient‐derived orthotopic xenograft (PDOX) nude‐mouse models replicate the behavior of clinical cancer, including metastasis. The objective of the study was to determine the efficacy of zoledronic acid (ZA) on metastasis of a patient‐derived orthotopic xenograft (PDOX) nude‐mouse model of pancreatic cancer.