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BackgroundGastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival.MethodsTwo patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue.ResultsM5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800.ConclusionsHumanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.

IntroductionGastric cancer poses a major therapeutic challenge. Improved visualization of tumor margins at the time of gastrectomy with fluorescent tumor-specific antibodies could improve outcomes. The present report demonstrates the potential of targeting gastric cancer with a humanized anti-carcinoembryonic antigen (CEA) antibody in orthotopic mouse models.MethodsMKN45 cells were injected subcutaneously into nude mice to establish xenograft models. Tumor fragments collected from subcutaneous models were then implanted into the greater curvature of the stomach to establish orthotopic models. For tumor labeling, a humanized anti-CEA antibody (M5A) and IgG as a control, were conjugated with the near-infrared dye IRDye800CW. Time (24-72 h) and dose (50-100 μg) response curves were performed in subcutaneous models. Orthotopic models received 50 μg of M5A-IR800 or 50 μg IgG-IR800 as a control and were imaged after 72 h. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System.ResultsIn subcutaneous models, tumor to background ratios (TBRs) reached 8.85 at 72 h. Median TBRs of orthotopic model primary tumors were 6.25 (interquartile range [IQR] 6.03-7.12) for M5A-IR800 compared to 0.42 (IQR 0.38-0.54) for control. Abdominal wall metastasis median TBRs were 13.52 (IQR 12.79-13.76) for M5A-IR800 and 3.19 (IQR 2.65-3.73) for the control. Immunohistochemistry confirmed CEA expression within tumors.ConclusionsHumanized anti-CEA antibodies conjugated to near-infrared dyes provide specific labeling of gastric cancers in mouse models. Orthotopic models demonstrated bright and specific labeling with TBRs greater than ten times that of control. This tumor-specific fluorescent antibody is a promising potential clinical tool for improving visualization of gastric cancer margins at time of surgical resection.

Pancreatic cancer is a recalcitrant disease in which R0 resection is often not achieved owing to difficulty in visualization of the tumor margins and proximity of adjacent vessels. To improve outcomes, we have developed fluorescence-guided surgery (FGS) and photoimmunotherapy (PIT) using a fluorescent tumor-specific antibody. Nude mice received surgical orthotopic implantation (SOI) of the human pancreatic cancer cell line BxPC-3 expressing green fluorescent protein. An anti-carcinoembryonic antigen-related cell adhesion molecule (CEACAM) monoclonal antibody (6G5j) was conjugated to the 700-nm fluorescent dye IR700DyeDX (6G5j-IR700DX). Three weeks after SOI, 16 mice received 50 μg 6G5j-IR700DX via the tail vein 24 h before surgery and were randomized to two groups: FGS-only (n = 8) and FGS + PIT (n = 8). All tumors were imaged with the Pearl Trilogy imaging system and resected under the guidance of the FLARE imaging system. The FGS + PIT group received PIT of the post-surgical bed at an intensity of 150 mW/cm2 for 30 min. Mice were sacrificed 4 weeks after initial surgery, and tumors were imaged with a Dino-Lite digital microscope, excised, and weighed. The 6G5j-IR700DX dye illuminated the orthotopic pancreatic tumors for FGS and PIT. The metastatic recurrence rate was 100.0% for FGS-only and 25.0% for FGS + PIT (p = 0.007). The average total recurrent tumor weight was 2370.3 ± 1907.8 mg for FGS-only and 705.5 ± 1200.0 mg for FGS + PIT (p = 0.039). FGS and adjuvant PIT can be combined by using a single antibody-fluorophore conjugate to significantly reduce the frequency of pancreatic cancer recurrence.

BackgroundThere is a high rate of positive surgical margins with resection of liver metastases in colorectal cancer (CRC). The present study reports using a fluorescent anti-mucin 4 (MUC4) antibodies to label primary CRC and liver metastases to better visualize tumor margins in mouse models.MethodsWestern blotting for MUC4 protein expression of normal colon and CRC tumor lysates was performed. Orthotopic primary and liver metastatic CRC mouse models received anti-MUC4 antibody conjugated to IR800 (MUC4-IR800). Mice were sacrificed and imaged after 48 hours.ResultsWestern blotting demonstrated increased MUC4 expression in a human CRC cell line and patient-derived primary and liver-metastatic CRCs. The LS174T orthotopic primary CRC model tumor to background ratio (TBR) was 2.04 (±0.35). The patient-derived orthotopic xenograft (PDOX) primary CRC model TBR was 2.17 (±0.35). The PDOX liver metastasis model TBR was 1.56 (±0.53).ConclusionMUC4-IR800 provided bright labeling of primary and liver tumors in CRC orthotopic mouse models, demonstrating their future clinical potential for margin visualization in fluorescence guided surgery.

BackgroundOverexpression of mucin-5AC (MUC5AC) makes it a targetable biomarker in pancreatic cancer. The present study evaluated tumor targeting with a MUC5AC antibody conjugated to a near-infrared dye in a patient-derived orthotopic xenograft (PDOX) mouse model.Materials and methodsMUC5AC monoclonal antibody was conjugated to the near-infrared dye IRDye800CW to synthesize MUC5AC-IR800. PDOX models were established by implanting a high-MUC5AC-expressing patient-derived pancreatic tumor on the pancreas of nude mice. After 4 weeks of PDOX tumor growth, mice were imaged after receiving MUC5AC-IR800 (75 μg) intravenously.ResultsIn the PDOX models, MUC5AC-IR800 selectively and brightly targeted the pancreatic tumor (tumor to background ratio: 2.46±0.465).ConclusionMUC5AC-IR800 provides distinct visualization of pancreatic tumors. MUC5AC-IR800 may be used clinically in the future to improve pancreatic cancer resection. This novel fluorescent probe is also promising for targeting of pre-malignant pancreatic lesions with subsequent resection under fluorescence guidance.

Tumor-specific targeting with fluorescent probes can enhance contrast for identification of cancer during surgical resection and visualize otherwise invisible tumor margins. Nanobodies are the smallest naturally-occurring antigen-binding molecules with rapid pharmacokinetics. The present work demonstrates the efficacy of a fluorescent anti-CEA nanobody conjugated to an IR800 dye to target and label patient derived pancreatic cancer xenografts. After intravenous administration, the probe rapidly localized to the pancreatic cancer tumors within an hour and had a tumor-to-background ratio of 2.0 by 3 h. The fluorescence signal was durable over a prolonged period of time. With the rapid kinetics afforded by fluorescent nanobodies, both targeting and imaging can be performed on the same day as surgery.

Background/objectivesNanobodies are the smallest biologic antigen-binding fragments derived from camelid-derived antibodies. Nanobodies effect a peak tumor signal within minutes of injection and present a novel opportunity for fluorescence-guided surgery (FGS). The present study demonstrates the efficacy of an anti-CEA nanobody conjugated to near-infrared fluorophore LICOR-IRDye800CW for rapid intraoperative tumor labeling of colon cancer.MethodsLS174T human colon cancer cells or fragments of patient-derived colon cancer were implanted subcutaneously or orthotopically in nude mice. Anti-CEA nanobodies were conjugated with IRDye800CW and 1-3 nmol were injected intravenously. Mice were serially imaged over time. Peak fluorescence signal and tumor-to-background ratio (TBR) were recorded.ResultsColon cancer tumors were detectable using fluorescent anti-CEA nanobody within 5 min of injection at all three doses. Maximal fluorescence intensity was observed within 15 min-3 h for all three doses with TBR values ranging from 1.3 to 2.3. In the patient-derived model of colon cancer, fluorescence was detectable with a TBR of 4.6 at 3 h.ConclusionsFluorescent anti-CEA nanobodies rapidly and specifically labeled colon cancer in cell-line-based and patient-derived orthotopic xenograft (PDOX) models. The kinetics of nanobodies allow for same day administration and imaging. Anti-CEA-nb-800 is a promising and practical molecule for FGS of colon cancer.

BackgroundIt is difficult to distinguish between a tumor and its liver segment with traditional use of indocyanine green (ICG) alone. In the present study, a method was used to limit ICG to the liver segment adjacent to a tumor. A spectrally-distinct fluorescently-labeled tumor-specific antibody against human carcinoembryonic antigen-related cell-adhesion molecules was used to label the metastatic tumor in a patient-derived orthotopic xenograft mouse model to enable color-coded visualization and distinction of a colon-cancer liver metastases and its adjacent liver segment.Materials and methodsNude mice received surgical orthotopic implantation in the liver of colon-cancer liver metastases derived from two patients. An anti- carcinoembryonic antigen-related cell-adhesion molecules monoclonal antibody (mAb 6G5j) was conjugated to a near-infrared dye IR700DX (6G5j-IR700DX). After three weeks, mice received 6G5j-IR700DX via tail-vein injection 48 hours before surgery. ICG was intravenously injected after ligation of the left or left lateral Glissonean pedicle resulting in labeling of the segment with preserved blood-flow in the liver. Imaging was performed with the Pearl Trilogy and FLARE Imaging Systems.ResultsThe metastatic liver tumor had a clear fluorescence signal due to selective tumor targeting by 6G5j-IR700DX, which was imaged on the 700 nm channel. The adjacent liver segment, with preserved blood-flow in the liver, had a clear fluorescence ICG 800 nm signal, while the left or left lateral segment had no fluorescence signal. Overlay of the images showed clear color-coded differentiation between the tumor fluorescing at 700 nm and the adjacent liver segment fluorescing at 800 nm.ConclusionsColor-coding of a liver tumor and uninvolved liver segment has the potential for improved liver resection.

Background/aimCancers are selectively sensitive to methionine (MET) restriction (MR) due to their addiction to MET which is overused for elevated methylation reactions. MET addiction of cancer was discovered by us 45 years ago. MR of cancer results in depletion of S-adenosylmethionine (SAM) for transmethylation reactions, resulting in selective cancer-growth arrest in the late S/G2-phase of the cell cycle. The aim of the present study was to determine if blockade of the MET-methylation axis is a highly-effective strategy for cancer chemotherapy.Materials and methodsIn the present study, we demonstrated the efficacy of MET-methylation-axis blockade using MR by oral-recombinant methioninase (o-rMETase) combined with decitabine (DAC), an inhibitor of DNA methylation, and an inhibitor of SAM synthesis, cycloleucine (CL). We determined a proof-of-concept of the efficacy of the MET-methylation-axis blockade on a recalcitrant undifferentiated/unclassified soft-tissue sarcoma (USTS) patient-derived orthotopic xenograft (PDOX) mouse model.ResultsThe o-rMETase-CL-DAC combination regressed the USTS PDOX with extensive cancer necrosis.ConclusionThe new concept of combination MET-methylation-axis blockade is effective and can now be tested on many types of recalcitrant cancer.

Background/aimDuring surgical resection of gastroesophageal-junction (GEJ) adenocarcinoma, the margin status is often difficult to visualize resulting in high recurrence rates. The aim of the present study was to develop a labelling technique that would allow improved visualization of GEJ tumor margins for surgeons to reduce recurrence rates in a patient-like model.Materials and methodsA patient GEJ tumor was obtained from an endoscopic biopsy and implanted subcutaneously in a nude mouse. A patient-derived orthotopic xenograft (PDOX) model was established by implanting tumor fragments grown from a subcutaneous model to the cardia of the stomach of nude mice. CC1/3/5-SAB, an antibody to carcinoembryonic-antigen-related cell-adhesion molecules, was conjugated with infrared dye IRDye800 to create SAB-IR800. Forty-eight hours after i.v. injection of SAB-IR800, GEJ-PDOX mice were imaged.ResultsFluorescence imaging with SAB-IR800 brightly visualized the GEJ adenocarcinoma demonstrating specific targeting. In the PDOX model, injection of SAB-IR800 (50 μg) resulted in a tumor to background ratio of 1.78 at 48 hours and 1.86 at 72 hours.ConclusionPDOX models of GEJ tumors can be established from patients by endoscopic biopsy without undergoing surgical resection. GEJ PDOX models should be useful for developing novel diagnostics and therapeutics for this recalcitrant disease.

Background/aimXihuang Wan (XHW), a traditional Chinese medicine (TCM), has been used in China for a variety of cancers including lung cancer. The present study evaluated the efficacy of XHW on a Lewis lung mouse model and explored the potential mechanism via transcriptomics.Materials and methodsThe mice were randomized into 6 groups: 1) untreated control (n=10); 2) low-dose XHW; 3) medium-dose XHW; 4) high-dose XHW; 5) cisplatin; and 6) untreated blank (n=4). Lewis lung carcinoma (LLC) cells were injected subcutaneously except for the 4 mice in the blank group. The body weight and tumor length and width were measured every 3 days. RNA-sequencing was performed on tumors in the high-dose XHW group and the control group.ResultsXHW inhibited the growth of LLC in a syngeneic mouse model, without toxicity, with equivalent efficacy to cisplatin. RNA-sequencing demonstrated that many signaling pathways were involved in XHW-mediated inhibition of LLC, including tumor necrosis factor, estrogen, cyclic guanosine 3', 5'-monophosphate-protein kinase G, apelin and the peroxisome proliferator-activated receptor signaling pathways.ConclusionXHW inhibited LLC carcinoma through different pathways and shows clinical promise for patients who cannot tolerate platinum-based drugs.

BackgroundTumor-associated glycoprotein (TAG)-72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72-specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models.MethodsAnti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49-IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy.ResultsSubcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 μg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light.ConclusionsAnti-huCC49-IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

BACKGROUND/AIM:Cisplatinum (CDDP) is a first-line drug in osteosarcoma treatment and the acquisition of resistance to CDDP is associated with a poor prognosis. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that plays important roles in cell proliferation, differentiation, development, metabolism and cell death. Recently, PPARγ was reported to enhance the efficacy, overcome resistance, and decrease the toxicity of CDDP in various human cancers. In this study we tested whether pioglitazone (PIO), a PPARγ agonist, could overcome CDDP resistance in osteosarcoma. MATERIALS AND METHODS:In this study, we used a human osteosarcoma cell line and a patient-derived orthotopic xenograft (PDOX) models of osteosarcoma. We measured cell viability of 143B human osteosarcoma cells when treated with CDDP and PIO. We randomized PDOX models of osteosarcoma into four treatment groups: Group 1, Untreated control; Group 2, PIO alone; Group 3, CDDP alone; Group 4, a combination of CDDP and PIO. Each group comprised six mice. Mice were treated for 14 days and tumor size and body weight were measured. RESULTS:Cell viability of 143B human osteosarcoma cells was significantly reduced when PIO (50 μmol/l) was combined with CDDP compared to CDDP alone. PDOX osteosarcoma tumors treated with the CDDP-PIO combination showed the strongest tumor growth inhibition compared to other treatment groups. PDOX osteosarcoma tumors treated with the CDDP-PIO combination had the least cancer cells and the most necrosis in histological section. CONCLUSION:These results suggest that combining PIO along with CDDP could be an effective treatment strategy for osteosarcoma and has important clinical potential for patients.

Background/aimPazopanib (PAZ) can inhibit tumor progression, but whether PAZ inhibits lymph node metastasis and lymphangiogenesis in colorectal cancer is still unknown. The aim of the present study was to determine the efficacy of PAZ on tumor growth, lymph node metastasis and lymphangiogenesis in an orthotopic nude mouse model in colorectal cancer.Materials and methodsCT-26-green fluorescence protein (GFP)-expressing mouse colon cancer cells were injected into nude mice to establish a subcutaneous colorectal cancer model and were treated with saline and PAZ. Additionals subcutaneous tumors were harvested and cut into 5 mm3 fragments, then tumor fragments were implanted orthotopically in the cecum to establish an orthotopic colorectal-cancer nude mouse model. Orthotopic mice were randomized into two groups for the treatment with saline and PAZ, respectively. Tumor width, length and mouse body weight was measured twice a week. The Fluor Vivo imaging system was used to image the GFP. Hematoxylin & eosin staining and immunohistochemical staining was used for histological analysis.ResultsPAZ inhibited the growth of subcutaneous colorectal cancer, as wells as orthotopic transplanted colorectal cancer tumors. PAZ suppressed lymph node metastasis and lymphangiogenesis in the orthotopic colon cancer model. No significant changes were observed in the body weight between the control and the mice treated with PAZ.ConclusionPAZ can inhibit the growth of colorectal cancer and inhibit lymph node metastasis and lymphangiogenesis in orthotopic colon cancer nude mouse models.

Background/aimBrain metastases are found in approximately 30% of patients with epidermal-growth-factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC). We compared the efficacy of two EGFR-tyrosine kinase inhibitors (TKIs), erlotinib and osimertinib on a PC-9-GFP EGFR mutant NSCLC growing in the brain of nude mice.Materials and methodsThe brain metastasis models were randomized into five groups and treated for 15 days: Control; 5 mg/kg erlotinib; 50 mg/kg erlotinib; 0.5 mg/kg osimertinib; 5 mg/kg osimertinib. Tumor volume was evaluated by non-invasive fluorescence imaging.ResultsOnly 5 mg/kg osimertinib, a low-dose compared to the clinically-equivalent dose, showed significant tumor regression compared to the control.ConclusionThis study strongly supports the high activity of osimertinib for intracranial lesions of EGFR-mutant NSCLC.

BACKGROUND/AIM:We have previously developed a novel bone-targeting platinum compound, 3Pt, and showed that it has strong inhibitory activity against osteosarcoma cells and orthotopic cell-line xenograft mouse models. In the present report, we compared the efficacy of 3Pt to cisplatinum (CDDP) in a CDDP-resistant relapsed osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model. PATIENTS AND METHODS:The tumor of a patient with osteosarcoma of the distal femur was treated with CDDP-based chemotherapy followed by surgery. The surgical specimen was used to establish a PDOX model. An osteosarcoma cell line was also established from the original patient tumor. Osteosarcoma cell viability was assessed with the WST-8 assay and the IC50 values were calculated. The PDOX models were randomized into three groups: untreated control, CDDP-treated group, and 3Pt-treated group. Tumor size and body weight were measured twice a week. RESULTS:3Pt had a strong concentration-dependent cytocidal effect in vitro. The IC50 value of 3Pt was significantly lower than that of CDDP. On day 14 of the treatment, 3Pt caused a significantly greater tumor growth inhibition compared to the untreated control and CDDP-treated mice. CONCLUSION:3Pt is a promising clinical candidate for the treatment of recalcitrant osteosarcoma.

Background/aimDedifferentiated liposarcoma (DDLPS) is recalcitrant type of sarcoma. DDLPS has a low survival rate with high recurrence and metastasis. In the present study, we evaluated the efficacy of several drugs against doxorubicin-resistant DDLPS in a patient-derived orthotopic xenograft (PDOX) model for precision oncology. To establish the PDOX model, a tumor from a patient who had recurrent high-grade DDLPS from the retroperitoneum was previously grown orthotopically in the retroperitoneum of nude mice.Materials and methodsWe randomized DDLPS PDOX models into 8 treatment groups when tumor volume became approximately 100 mm3: control, no treatment; G2, doxorubicin (DOX); G3, pazopanib (PAZ); G4, gemcitabine (GEM) combined with docetaxel (DOC); G5, trabectedin (YON); G6, temozolomide (TEM); G7, palbociclib (PAL); G8, eribulin (ERB). Tumor length and width were measured both at the beginning and at the end of treatment.ResultsAt the end of treatment (day 14), all treatments significantly inhibited DDLPS PDOX tumor growth compared to the untreated control, except DOX. ERB was significantly more effective and regressed tumor volume compared to other treatments on day 14 after initiation of treatment. No significant differences were found in the relative body weight on day 14 compared to day 0 in any group.ConclusionThe clinical potential of ERB against DDLPS is herein presented in a PDOX model.

Background/aimFluorescence imaging has been shown to improve intra-operative detection of liver metastasis. The present study aimed to determine whether humanized anti-TAG-72 antibody (huCC49) conjugated to a near-infrared dye provides selective labeling of colorectal-cancer liver metastasis in orthotopic mouse models.Materials and methodsHumanized anti-TAG-72 (huCC49) was conjugated to IRDye800CW (huCC49-IR800). Orthotopic liver-metastasis nude-mouse models (n=5) were established with the human colon-cancer LS174T cell-line. Three weeks later, mice were administered huCC49-IR800 and intra-vital imaging was performed 48 h later. The mean tumor-to-liver ratio (TLR) was calculated.ResultsIntra-vital imaging demonstrated clear tumor margins with minimal liver fluorescence 48 h after administration of 50 μg huCC49-IR800 with mean TLR=7.53 (SD±2.76).ConclusionAnti-TAG-72 monoclonal antibody conjugated to IRDye800 provides distinct and bright labeling of colorectal tumors in orthotopic nude-mouse models of liver metastasis. TAG-72 may be a useful target for intra-operative imaging of colorectal cancer liver metastasis in the clinic.

Background/aimThe visualization of hepatic segments with indocyanine green (ICG) fluorescence can aid in anatomic liver resection. The present study aimed to develop a method to specifically label an hepatic segment in a nude mouse model with liver metastasis.Materials and methodsAn orthotopic mouse model was established by surgical orthotopic implantation (SOI) of a patient-derived colon-cancer liver metastasis in the left lobe of the liver. Three weeks after SOI, the left Glissonean pedicle was ligated and 10 μg ICG was administrated intravenously. Images were obtained with the Pearl Trilogy Imaging System.ResultsAll mice expressed an 800 nm signal from ICG on the right lobe of the liver. The left lobe of the liver, in which the tumor was located, showed no fluorescence and had ischemia due to successful ligation of the Glissonean pedicle.ConclusionThe ligation of the Glissonean pedicle enables specific liver-segment labeling with ICG, which has potential clinical application for liver metastasectomy.

Background/aimSurgical orthotopic implantation (SOI) is used to establish patient-derived orthotopic xenograft (PDOX) and other orthotopic mouse models. Orthotopic liver models can be challenging, as the liver parenchyma is prone to bleeding. The present report describes a sutureless method to implant tumors in the liver that reduces bleeding and procedural time.Materials and methodsHuman HCC cell-line (Huh-7-GFP) and CM2, a patient-derived colon-cancer liver metastasis, were used for sutureless SOI of tumor fragments in the liver of nude mice. A small cavity was formed on the liver surface. A solitary tumor fragment was implanted in the cavity without suturing to create hemostasis.ResultsSix weeks after sutureless SOI, the tumor volume of Huh-7-GFP (n=5) was 584.41±147.64 mm3 and the tumor volume of CM2 (n=5) was 1336.54±1038.20 mm3 The engraftment rate was 100%.ConclusionThis novel method for establishing orthotopic liver-implantation mouse models is suitable for studies of liver cancer and liver metastases due to its simple procedure and potential high engraftment rate.

Ischemia reperfusion injury (IRI) during liver-metastasis resection for treatment of colon cancer may increase the risk of further metastasis. Peroxisome proliferator-activated receptor-γ (PPARγ) activation has been observed to exert a protective effect against IRI and IRI-induced metastasis of hepatocellular carcinoma. The present study aimed to investigate the effect of the PPARγ agonist pioglitazone on tumor metastasis and liver injury following IRI in a mouse model of colon cancer. Pioglitazone (30 mg/kg weight) was administered orally 1.5 h before and 2 h after the initiation of ischemia and was orally administrated daily to mice from day 0-21. SL4-cancer cells expressing red fluorescent protein (SL4-RFP) (1 × 106) were injected into the spleen. Fifteen minutes after injection, the hepatoduodenal ligament was clamped with a vessel clip, and released 5 min later. Liver, blood and tumor samples were taken from mice in order to determine if inflammation was induced by IRI. The effect of pioglitazone on liver metastasis was assessed. Furthermore, the effect of pioglitazone to control the inflammatory response during IRI progression was examined. Liver metastasis along with MMP-9 activation and the production of inflammatory cytokines were resistant to pioglitazone. Our results indicate that liver metastasis and associated inflammation in mice were resistant to pioglitazone.

Liver metastasis is a recalcitrant disease that usually leads to death of the patient. The present study established a unique patient-derived orthotopic xenograft (PDOX) nude mouse model of a highly aggressive liver metastasis of colon cancer. The aim of the present study was to demonstrate proof-of-concept that candidate drug combinations could significantly inhibit growth and re-metastasis of this recalcitrant tumor. The patient's liver metastasis was initially established subcutaneously in nude mice and the subcutaneous tumor tissue was then orthotopically implanted in the liver of nude mice to establish a PDOX model. Two studies were performed to test different drugs or drug combination, indicating that 5-fluorouracil (5-FU) + irinotecan (IRI) + bevacizumab (BEV) and regorafenib (REG) + selumetinib (SEL) had significantly inhibited liver metastasis growth (p = 0.013 and p = 0.035, respectively), and prevented liver satellite metastasis. This study is proof of concept that a PDOX model of highly aggressive colon-cancer metastasis can identify effective drug combinations and that the model has future clinical potential.

IntroductionClaudins are tight-junction proteins, which maintain an epithelial barrier in normal colon cells. Overexpression of Claudin-1 has been implicated for development of colon cancer. We postulated that Claudin-1 may be a useful target in near-infrared imaging and fluorescence-guided surgery.MethodsWe conjugated Claudin-1 antibody to LI-COR IR800DyeCW (Claudin-1-IRDye800CW). Western blotting of 9 human colon cancer cell lysates was performed. Animal imaging was performed with the LI-COR Pearl Trilogy Fluorescence Imaging System. A dose-response study was carried out with subcutaneous LS174T colon cancer cell line models. Increasing doses of Claudin-1-IRDye800CW via tail vein injection were administered to three groups of mice. Two groups of mice were used as controls (antibody alone, and dye alone). In vivo imaging was performed at 24, 48, and 72 h after administration of the conjugated dye. Orthotopic implantation of patient-derived tumors and cell lines was performed and peritoneal carcinomatosis models were created. After tumor growth, mice were administered Claudin-1-IRDye800CW and imaged in vivo 48 h later. The mice were euthanized and laparotomy was performed to assess internal organs and toxicity.ResultsWestern blotting revealed that all colon cancer cell lysates expressed varying amounts of Claudin-1. All tumors demonstrated strong and specific fluorescence labeling at 800 nm, even with the lowest dose of 12.5 μg of Claudin-1-IRDye800CW.ConclusionsClaudin-1 is a useful target for near-infrared antibody-based imaging for visualization of colorectal tumors for future use in fluorescence-guided surgery.

BackgroundPleomorphic 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.MethodsWe 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.ResultsThe PLPS PDOX showed resistance towards DOX. However, TRAB could arrest the PLPS (p

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. In the current study, the PLPS PDOX model was randomized into 3 groups of 7 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 3 groups. Fluorescence imaging demonstrated that S. typhimurium A1-R-GFP was very effective to target the PLPS PDOX tumor. The current study demonstrates that a PLPS PDOX, resistant to first-line therapy DOX, was highly sensitive to tumor targeting S. typhimurium A1-R.

Fluorescence-guided surgery can enhance the surgeon's ability to achieve a complete oncologic resection. There are a number of tumor-specific probes being developed with many preclinical mouse models to evaluate their efficacy. The current review discusses the different preclinical mouse models in the setting of probe evaluation and highlights the advantages of patient-derived orthotopic xenografts (PDOX) mouse models and genetic reporters to develop fluorescence-guided surgery.

Pancreatic cancer is a recalcitrant disease. Gemcitabine (GEM) is the most widely-used first-line therapy for pancreatic cancer, but most patients eventually fail. Transformative therapy is necessary to significantly improve the outcome of pancreatic cancer patients. Tumors have an elevated requirement for methionine and are susceptible to methionine restriction. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer to determine the efficacy of recombinant methioninase (rMETase) to effect methionine restriction and thereby overcome GEM-resistance. A pancreatic cancer obtained from a patient was grown orthotopically in the pancreatic tail of nude mice to establish the PDOX model. Five weeks after implantation, 40 pancreatic cancer PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); GEM (100 mg/kg, i.p., once a week for 5 weeks, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); GEM+rMETase (GEM: 100 mg/kg, i.p., once a week for 5 weeks, rMETase: 100 units, i.p., 14 consecutive days, n = 10). Although GEM partially inhibited PDOX tumor growth, combination therapy (GEM+rMETase) was significantly more effective than mono therapy (GEM: p = 0.0025, rMETase: p = 0.0010). The present study is the first demonstrating the efficacy of rMETase combination therapy in a pancreatic cancer PDOX model to overcome first-line therapy resistance in this recalcitrant disease.

Background/purposeDevelopment of a humanized fluorophore-conjugated antibody that can improve contrast for fluorescence-guided oncologic surgeries.MethodsBxPC-3-GFP pancreatic cancer cells were injected into flanks of nude mice. Fragments of subcutaneous tumors were grafted onto the pancreatic tail of recipient mice to create orthotopic xenograft models of pancreatic cancer. After tumors developed for 4 weeks, a humanized anti-carcinoembryonic antigen antibody conjugated to an 800 nm near-infrared fluorescent dye (hM5A-IR800) was injected intravenously. Mice were imaged at 6, 12, 24, 48, and 72 h after injection.ResultsFluorescence imaging showed that hM5A-IR800 specifically localized to BxPC-3 human pancreatic cancer cells. The fluorescent probe localized to cell surfaces in vitro and specifically co-localized with green fluorescent protein-labeled tumors in an orthotopic pancreatic xenograft model in vivo. Serial imaging at specific time points showed peak signal intensity of the orthotopic pancreatic tumor at 48 h; this time point corresponded with a maximal tumor-to-background ratio (TBR) of 16.6 at 48 h.DiscussionhM5A-IR800 was successfully able to specifically label orthotopic pancreatic tumors in situ. The longer wavelength allowed deeper tissue penetration, particularly in tumor areas covered by normal pancreatic parenchyma. The probe had expected kinetics for an antibody-fluorophore conjugate, with the peak signal intensity reached at 48 h. A clear tumor signal was observed with a TBR > 5 at all time points, with high contrast (TBR of 16.6) at 48 h.ConclusionhM5A-IR800 demonstrated excellent tumor localization and a very bright signal. It is a promising agent for future clinical fluorescence-guided surgery applications.

Undifferentiated spindle-cell sarcoma (USCS) is a recalcitrant -cancer in need of individualized therapy. A high-grade USCS from a striated muscle of a patient was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In a previous study, we evaluated the efficacy of standard first-line chemotherapy of doxorubicin (DOX), gemcitabine (GEM) combined with docetaxel (DOC), compared to pazopanib (PAZ), a multi-targeting tyrosine-kinase inhibitor, in an USCS PDOX model. In the present study, mice-bearing the USCS PDOX tumors were randomized into the following groups when tumor volume reached 100 mm3 : G1, untreated control without treatment; G2, DOX (3 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase]) (100 U/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured with calipers and a digital balance twice a week. The methionine level of supernatants derived from sonicated tumors was also measured. rMETase inhibited tumor growth, measured by tumor volume, compared to untreated controls and the DOX-treated group on day 14 after initiation of treatment: control (G1): 347.6 ± 88 mm3 ; DOX (G2): 329.5 ± 79 mm3 , P = 0.670; rMETase (G3): 162.6 ± 51 mm3 , P = 0.0003. The mouse body weight of the treated mice was not significantly different from the untreated controls. Tumor L-methionine levels were reduced after the rMETase-treatment compared to untreated control and pre-rMETase treatment. We previously reported efficacy of rMETase against Ewing's sarcoma and melanoma in a PDOX models. These studies suggest clinical development of rMETase, especially in recalcitrant cancers such as sarcoma.

In the present study, a patient-derived orthotopic xenograft (PDOX) model of recurrent cisplatinum (CDDP)-resistant metastatic osteosarcoma was treated with Salmonella typhimurium A1-R (S. typhimurium A1-R), which decoys chemoresistant quiescent cancer cells to cycle, and recombinant methioninase (rMETase), which selectively traps cancer cells in late S/G2, and chemotherapy. The PDOX models were randomized into the following groups 14 days after implantation: G1, control without treatment; G2, CDDP (6 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks). G4, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., weekly, for 2 weeks); G5, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G6, S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks) and CDDP (6 mg/kg, i.p. injection, weekly, for 2 weeks). On day 14 after initiation, all treatments except CDDP alone, significantly inhibited tumor growth compared to untreated control: (CDDP: p = 0.586; rMETase: p = 0.002; S. typhimurium A1-R: p = 0.002; S. typhimurium A1-R combined with rMETase: p = 0.0004; rMETase combined with both S. typhimurium A1-R and CDDP: p = 0.0001). The decoy, trap and kill combination of S. typhimurium A1-R, rMETase and CDDP was the most effective of all therapies and was able to eradicate the metastatic osteosarcoma PDOX.

Doxorubicin (DOX) is often first-line treatment of undifferentiated/unclassified soft tissue sarcoma (USTS). However, the DOX response rate for USTS patients is low. Individualized precision-medicine technology that could identify DOX responders as well as non-responders would be of high value to cancer patients. In the present study, we established 5 patient-derived orthotopic xenograft (PDOX) nude mouse models from 5 USTS patients and evaluated the efficacy of DOX in each PDOX model. USTS's were grown orthotopically in the right thigh of nude mice to establish the PDOX models. Two weeks after implantation, the mouse models were randomized into two groups of 8 mice each: untreated control; and DOX (3 mg/kg, i.p., once a week for 2 weeks). DOX showed significant growth inhibition in only 2 USTS PDOX models out of 5 (p = 0.0054, p = 0.0055, respectively) on day 14 after initiation. DOX was ineffective in the other 3 PDOX models. However, even in the DOX-sensitive cases, DOX could not regress the PDOX tumors responding to treatment. The present study has important implications since this is the first in vivo study to compare the DOX sensitivity for USTS on multiple patient tumors. We showed that only two of five USTS were responsive to DOX, despite DOX being first line chemotherapy for USTS. The 3 resistant cases should not be treated with DOX clinically, in order to spare the patients' unnecessary toxicity. This PDOX model is useful for precise individualized drug sensitivity testing, especially for rare heterogeneous recalcitrant sarcomas such as USTS.

The elevated methionine (MET) use by cancer cells is termed MET dependence and may be the only known general metabolic defect in cancer. Targeting MET by recombinant methioninase (rMETase) can arrest the growth of cancer cells in vitro and in vivo. We previously reported that rMETase, administrated by intra-peritoneal injection (ip-rMETase), could inhibit tumor growth in a patient-derived orthotopic xenograft (PDOX) model of a BRAF-V600E mutant melanoma. In the present study, we compared ip-rMETase and oral rMETase (o-rMETase) for efficacy on the melanoma PDOX. Melanoma PDOX nude mice were randomized into four groups of 5 mice each: untreated control; ip-rMETase (100 units, i.p., 14 consecutive days); o-rMETase (100 units, p.o., 14 consecutive days); o-rMETase+ip-rMETase (100 units, p.o.+100 units, i.p., 14 consecutive days). All treatments inhibited tumor growth on day 14 after treatment initiation, compared to untreated control (ip-rMETase, p

Gastrointestinal stromal tumor (GIST) with a mutation in exons 11 and 17 of c-kit is a rare type of sarcoma. The aim of this study was to determine drug sensitivity for a regionally-recurrent case of GIST using a patient-derived orthotopic xenograft (PDOX) model. The PDOX model was established in the anterior wall of the stomach. GIST PDOX models were randomized into 5 groups of 6 mice each when the tumor volume reached 60 mm3: G1, control group; G2, imatinib group (oral administration (p.o.), daily, for 3 weeks); G3, sunitinib group (p.o., daily, for 3 weeks); G4, regorafenib (p.o., daily, for 3 weeks); G5, pazopanib (p.o., daily, for 3 weeks). All mice were sacrificed on day 22. Tumor volume was evaluated on day 0 and day 22 by laparotomy. Body weight were measured 2 times per week. Though regorafenib is third-line therapy for GIST, it was the most effective drug and regressed the tumor significantly (p < 0.001). Sunitinib suppressed tumor growth compared to the control group (p = 0.002). Imatinib, first-line therapy for GIST, and pazopanib did not have significant efficacy compared to the control group (p = 0.886, p = 0.766). The implications of this result is discussed for GIST patients.

BackgroundThe success of a curative surgery for cancer is dependent on the complete removal of all cancer cells. Tumor visualization by the surgeon can be enhanced through fluorescent-antibody targeting. To further develop such technology, we selected humanized anti-carcinoembryonic antigen (CEA) conjugated to a near-infrared dye to target orthotopically-implanted human colon cancer in nude mice.Materials and methodsThe HT-29 human colon cancer cell line was grown in culture and subcutaneously injected in mice. After 3 wk of growth, tumors were resected and cut into 2 mm3 fragments that were sutured to the cecum of five additional nude mice for orthotopic implantation. The tumors were allowed to grow for 4 wk at which point 3 had successful orthotopic tumor growth and were selected for injection of the humanized anti-CEA antibody conjugated to the near-infrared dye IRDye800CW (anti-CEA-IRDye800CW). The antibody-dye conjugate (75 μg) was administered via tail vein injection. Images were obtained with the Pearl Trilogy Small Animal Imaging System with both 700 and 800 nm channels and evaluated using Image Studio.ResultsLaparotomy was performed 24 h after labeling the tumors. When imaged through the 800 nm channel, the tumors were observed to be strongly labeled with anti-CEA-IRDye800. At 48 h, laparotomy was repeated which again demonstrated strong labeling of the tumors through the 800 nm channel, but with a lower absolute intensity (in relative units), than at 24 h.ConclusionsHumanized anti-CEA-IRDye800CW can rapidly and effectively label CEA-expressing human colon cancer in an orthotopic nude mouse model. Given the ability of this technology to target and label tumors with great specificity, the anti-CEA-IRDye800CW is currently being developed for clinical use in fluorescence-guided surgery.

A patient-derived orthotopic xenograft (PDOX) model of undifferentiated pleomorphic sarcoma (UPS) was previously established that acquired red fluorescent protein (RFP)-expressing stroma by growth in an RFP transgenic nude mouse. In the present study, an imageable PDOX model (iPDOX) of UPS was established by orthotopic implantation in the biceps femoris of transgenic RFP nude mice. After the tumors grew to a diameter of 10 mm, they were harvested and the brightest portion of the tumors were subsequently orthotopically transplanted to both RFP and non-colored nude mice. The UPS PDOX tumor was again transplanted to RFP transgenic and non-colored nude mice, and finally a 3rd passage was made in the same manner. Five UPS tumors from each passage in both RFP and non-colored mouse models were harvested. The FV1,000 confocal microscope was used to visualize and quantitate the RFP area of the resected tumors. The average percent fluorescent area in the first passage of RFP mice was 34 ± 22%; in the second passage, 34 ± 20%; and 36 ± 11% in the third passage of RFP transgenic nude mice. The average tumor RFP area in the first passage from RFP mice to non-colored mice was 20 ± 7%; in the second passage, 28 ± 11%; in the third passage was 27 ± 13%. The present results demonstrate the extensive and stable acquisition of stroma by the UPS-tumor growing orthotopically in transgenic RFP nude mice (iPDOX). This model can be used for screening for effective drugs for individual patients and drug discovery. J. Cell. Biochem. 118: 3367-3371, 2017. © 2017 Wiley Periodicals, Inc.

A metastatic melanoma obtained from the right chest wall of a patient was previously established orthotopically in the right chest wall of nude mice as a patient-derived orthotopic xenograft (PDOX) model. We previously showed that the combination of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and chemotherapy was highly effective against the melanoma PDOX. In the present study, we investigated the mechanism of the high efficacy of this combination. Two weeks after implantation, 40 PDOX mouse models were randomized into 4 groups of 10 mice each: untreated control (n = 10); treated with S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with temozolomide (TEM) (25 mg/kg, p.o. for 14 consecutive days) combined with S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks, n = 10); treated with vemurafenib (VEM) (30 mg/kg, p.o., for 14 consecutive days) combined with S. typhimurium A1-R (5 × 107 CFU/100 μl, i.v., once a week for 2 weeks) (n = 10). On day 14 from initiation, all treatments significantly inhibited tumor growth compared with untreated control (S. typhimurium A1-R: p < 0.01; TEM combined with S. typhimurium A1-R: p < 0.01; VEM combined with S. typhimurium A1-R: p < 0.01). Combination therapy with S. typhimurium A1-R was significantly more effective on tumor growth than S. typhimurium A1-R alone (with TEM: p < 0.01; with VEM: p < 0.01). Combination therapy significantly increased S. typhimurium A1-R tumor targeting alone (S. typhimurium A1-R + TEM: p < 0.01, S. typhimurium A1-R + VEM: p < 0.01), relative to S. typhimurium A1-R alone, respectively. In conclusion, chemotherapy drugs promoted targeting of S. typhimurium A1-R of melanoma, thereby enhancing efficacy against the melanoma PDOX.

A pancreatic ductal adenocarcinoma (PDAC), obtained from a patient, was grown orthotopically in the pancreatic tail of nude mice to establish a patient-derived orthotopic (PDOX) model. Seven weeks after implantation, PDOX nude mice were divided into the following groups: untreated control (n = 7); gemcitabine (100 mg/kg, i.p., once a week for 2 weeks, n = 7); cobimetinib (5 mg/kg, p.o., 14 consecutive days, n = 7); trametinib (0.3 mg/kg, p.o., 14 consecutive days, n = 7); trabectedin (0.15 mg/kg, i.v., once a week for 2 weeks, n = 7); temozolomide (25 mg/kg, p.o., 14 consecutive days, n = 7); carfilzomib (2 mg/kg, i.v., twice a week for 2 weeks, n = 7); bortezomib (1 mg/kg, i.v., twice a week for 2 weeks, n = 7); MK-1775 (20 mg/kg, p.o., 14 consecutive days, n = 7); BEZ-235 (45 mg/kg, p.o., 14 consecutive days, n = 7); vorinostat (50 mg/kg, i.p., 14 consecutive days, n = 7). Only the MEK inhibitors, cobimetinib and trametinib, regressed tumor growth, and they were more significantly effective than other therapies (p < 0.0001, respectively), thereby demonstrating the precision of the PDOX models of PDAC and its potential for individualizing pancreatic-cancer therapy.

To obtain improved efficacy against pancreatic cancer, we investigated the efficacy and safety of a locally-applied 5-fluorouracil (5-FU)-loaded polymeric patch on pancreatic tumors in an orthotopic nude-mouse model. The 5-FU-releasing polymeric patch was produced by 3D printing. After application of the patch, it released the drug slowly for 4 weeks, and suppressed BxPC-3 pancreas cancer growth. Luciferase imaging of BxPC3-Luc cells implanted in the pancreas was performed longitudinally. The drug patch delivered a 30.2 times higher level of 5-FU than an intra-peritoneal (i.p.) bolus injection on day-1. High 5-FU levels were accumulated within one week by the patch. Four groups were compared for efficacy of 5-FU. Drug-free patch as a negative control (Group I); 30% 5-FU-loaded patch (4.8 mg) (Group II); 5-FU i.p. once (4.8 mg) (Group III); 5-FU i.p. once a week (1.2 mg), three times (Group IV). The tumor growth rate was significantly faster in Group I than Group II, III, IV (p=0.047 at day-8, p=0.022 at day-12, p=0.002 at day-18 and p=0.034 at day-21). All mice in Group III died of drug toxicity within two weeks after injection. Group II showed more effective suppression of tumor growth than Group IV (p=0.018 at day-12 and p=0.017 at day-21). Histological analysis showed extensive apoptosis in the TUNEL assay and by Ki -67 staining. Western blotting confirmed strong expression of cleaved caspase-3 in Group II. No significant changes were found hematologically and histologically in the liver, kidney and spleen in Groups I, II, IV but were found in Group III.

William B. Coley developed bacterial therapy of cancer more than 100 years ago and had clinical success. James Ewing, a very famous cancer pathologist for whom the Ewing sarcoma is named, was Coley's boss at Memorial Hospital in New York and terminated Coley's bacterial therapy of cancer. A tumor from a patient with soft-tissue Ewing's sarcoma, who failed doxorubicin (DOX) therapy, was previously implanted in nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In the present study, the Ewing's sarcoma PDOX was treated with tumor-targeting S. typhimurium A1-R expressing green fluorescent (GFP), alone and in combination with DOX. S. typhimurium A1-R-GFP was detected in the tumors after intratumor (i.t.) or intravenous (i.v.) injection. The combination of S. typhimurium A1-R and DOX significantly reduced tumor weight (37.8 ± 15.6 mg) compared to the untreated control (73.8 ± 10.1 mg, P < 0.01). S. typhimurium A1-R monotherapy-treated tumors tended to be smaller (50.9 ± 17.8 mg, P = 0.051). DOX monotherapy did not show efficacy (66.3 ± 26.4 mg, P = 0.82), as was the case with the patient. The PDOX model faithfully replicated the DOX resistance the Ewing's sarcoma had in the patient. S. typhimurium A1-R converted the Ewing's sarcoma from DOX resistant to sensitive. One can only wonder how bacterial therapy and immunotherapy of cancer would have developed over the past 80 years if Ewing did not stop Coley.

Gastric leiomyosarcoma is a recalcitrant cancer and the chemotherapy strategy is controversial. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of gastric leiomyosarcoma to identify an effective therapeutic regimen to develop individualized precision medicine for this disease. The gastric leiomyosarcoma obtained from a patient was first grown in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP) to stably label the tumor stroma. The RFP-expressing tumor was then passaged orthotopically in the gastric wall of non-transgenic nude mice to establish an imageable PDOX (iPDOX) model. The bright fluorescent tumor was readily imaged over time to determine drug efficacy. Four weeks after implantation, 70 PDOX nude mice were divided into 7 groups: control without treatment (n = 10); doxorubicin (DOX) (2.4 mg/kg, intraperitoneally (i.p.), once a week for 2 weeks, n = 10); gemcitabine (GEM)/ docetaxel (DOC) (GEM: 100 mg/kg, DOC: 20 mg/kg, i.p., once a week for 2 weeks, n = 10); cyclophosphamide (CPA) (140 mg/kg, i.p., once a week for 2 weeks, n = 10); temozolomide (TEM) (25 mg/kg, orally, daily for 14 consecutive days, n = 10); yondelis (YON) (0.15 mg/kg, i.v., once a week for 2 weeks, n = 10); pazopanib (PAZ) (100 mg/kg, orally, daily for 14 consecutive days, n = 10). On day 14 from initiation of treatment, all treatments except PAZ significantly inhibited tumor growth compared with untreated control (DOX: p < 0.01, GEM/DOC: p < 0.01, CPA: p < 0.01, TEM: p < 0.01, YON: p < 0.01) on day 14 after initiation. In addition, only GEM/DOC was more significantly effective than DOX (p < 0.05). GEM/DOC could regress the leimyosarcoma in the PDOX model and has important clinical potential for precision individual treatment of leiomyosarcoma patients.

Previously, a patient-derived orthotopic xenograft (PDOX) model was established with a lung metastasis from an osteosarcoma patient which developed after adjuvant cisplatinum (CDDP) treatment. In this model, we previously demonstrated the efficacy of trabectedin (TRAB) and temozolomide (TEM) compared with CDDP. In the present report, osteosarcoma tissue was implanted orthotopically in the distal femur of mice which were randomized into the following groups when tumor volume reached approximately 100 mm3; On day 14 after initiation of treatment, all but CDDP significantly inhibited tumor volume growth compared with untreated controls. Control (G1): 793.7 ± 215.0 mm3; CDDP (G2): 588.1 ± 176.9 mm3; Salmonella typhimurium A1-R (S. typhimurium A1-R) intravenous (i.v.) (G3): 269.7 ± 72.7 mm3; S. typhimurium A1-R intra-arterial (i.a.) (G4): 70.2 ± 18.9 mm3 (CDDP: p = 0.056; S. typhimurium A1-R i.v.: p = 0.0001; S. typhimurium A1-R i.a.: p = 0.00003, all vs. untreated controls). i.a. administration of S. typhimurium A1-R was significantly more effective than either CDDP (p = 0.00007), or i.v. administration of S. typhimurium A1-R (p = 0.00007) and significantly regressed the tumor volume compared with day 0 (p = 0.001). The new model of i.a. administration of S. typhimurium A1-R has great promise for the treatment of recalcitrant osteosarcoma.

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.

Methionine dependence is due to the overuse of methionine for aberrant transmethylation reactions in cancer. Methionine dependence may be the only general metabolic defect in cancer. In order to exploit methionine dependence for therapy, our laboratory previously cloned L-methionine α-deamino-γ-mercaptomethane lyase [EC 4.4.1.11]). The cloned methioninase, termed recombinant methioninase, or rMETase, has been tested in mouse models of human cancer cell lines. Ewing's sarcoma is recalcitrant disease even though development of multimodal therapy has improved patients'outcome. Here we report efficacy of rMETase against Ewing's sarcoma in a patient-derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma was implanted in the right chest wall of nude mice to establish a PDOX model. Eight Ewing's sarcoma PDOX mice were randomized into untreated control group (n = 4) and rMETase treatment group (n = 4). rMETase (100 units) was injected intraperitoneally (i.p.) every 24 hours for 14 consecutive days. All mice were sacrificed on day-15, 24 hours after the last rMETase administration. rMETase effectively reduced tumor growth compared to untreated control. The methionine level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase group. Body weight did not significantly differ at any time points between the 2 groups. The present study is the first demonstrating rMETase efficacy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as Ewing's sarcoma.

Liver metastasis is the main cause of colon cancer-related death and is a recalcitrant disease. We report here the efficacy and safety of intra-portal-vein (iPV) targeting of Salmonella typhimurium A1-R on colon cancer liver metastasis in a nude-mouse orthotopic model. Nude mice with HT29 human colon cancer cells, expressing red fluorescent protein (RFP) (HT29-RFP), growing in the liver were administered S. typhimurium A1-R by either iPV (1×104 colony forming units (CFU)/100 μl) or, for comparison, intra-venous injection (iv; 5×107 CFU/100 μl). Similar amounts of bacteria were delivered to the liver with the two doses, indicating that iPV delivery is 5×103 times more efficient than iv delivery. Treatment efficacy was evaluated by tumor fluorescent area (mm2) and total fluorescence intensity. Tumor fluorescent area and fluorescence intensity highly correlated (p

Our laboratory pioneered patient-derived orthotopic xenograft (PDOX) mouse models using surgical orthotopic implantation (SOI). PDOX models are patient-like, in contrast to the ectopic subcutaneous-transplant cancer models. In the present study, we demonstrate that an undifferentiated pleomorphic soft-tissue sarcoma (UPS-STS) PDOX model acquired bright RFP-expressing stroma through one passage in red fluorescent protein (RFP) transgenic mice, which upon passage to non-colored nude mice was non-invasively imageable. A PDOX nude mouse model of UPS-STS was established in the biceps femoris of nude mice. After the tumors grew to a diameter of 10 mm, the tumors were subsequently passaged to RFP transgenic mice, and after tumor growth were then passaged to non-transgenic nude mice. Tumors were divided into small fragments and transplanted in the biceps femoris at each passage. The OV100 Small Animal Fluorescence Imaging System and FV1000 laser scanning confocal microscope were used to image RFP fluorescence in the UPS-STS PDOX models. UPS-STS PDOX tumors, previously grown in RFP transgenic nude mice for only one passage, had very bright fluorescence and after passage to non-transgenic nude mice maintained the bright fluorescence and were non-invasively imageable. FV1000 confocal imaging revealed diffusely distributed bright RFP stromal cells in the PDOX tumor, both in RFP transgenic mice and after passage to non-transgenic mice. These results demonstrate a powerful method to make the PDOX UPS-STS model brightly fluorescent for non-invasive imaging, as well as for confocal microscopy of individual stromal cells associated with the tumor. The RFP-labeled UPS PDOX has the potential to rapidly screen for novel effective agents for individual patients, including stroma-targeting drugs, whereby the stromal cells are a visual target. J. Cell. Biochem. 118: 361-365, 2017. © 2016 Wiley Periodicals, Inc.

Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies.

Rhabdomyosarcoma (RMS) is a rare mesenchymal tumor. The aim of the present study was to develop a patient-derived orthotopic xenograft (PDOX) mouse model of RMS and compare the PDOX model to a subcutaneous (s.c.)-transplant model. A patient RMS from a striated muscle was grown orthotopically in the right biceps femoris muscle and right quadriceps muscle of nude mice to establish a PDOX model, as well as under the skin to establish an s.c.ModelPDOX tumors grew at a statistically-significant faster rate compared to the s.c. tumors. Recurrence after surgical resection occurred only in PDOX tumors, not in the s.c.ModelHistologically, only the PDOX model was shown to be invasive. In conclusion, these results indicate that the PDOX model of adult RMS is malignant and the subcutaneous model is benign. These results emphasize that a proper tumor microenvironment is necessary for patient-like behavior of a tumor in a mouse model.

BackgroundCervical cancer is a world-wide problem that requires transformative therapeutic strategies. We have previously developed patient-derived orthotopic xenograft (PDOX) nude-mouse models of this disease. In the present report, we demonstrate that the standard drug, cisplatinum (CDDP), is highly-effective while the new, highly-touted agent, nab-paclitaxel (NAB-PTX) is ineffective.Materials and methodsCervical PDOX tumors were grown on the cervix of nude mice for 4 weeks after surgical orthotopic implantation (SOI). Tumors were treated with CDDP or NAB-PTX.ResultsH&E staining demonstrated that the PDOX tumor recapitulated the original patient tumor. CDDP was highly-effective. One tumor that was treated with CDDP completely regressed. CDDP-treated tumors were smaller (tumor volume ratio: 0.42±0.36) than the control group (tumor volume ratio: 3.47±1.66) (p

Osteosarcoma occurs mostly in children and young adults, who are treated with multiple agents in combination with limb-salvage surgery. However, the overall 5-year survival rate for patients with recurrent or metastatic osteosarcoma is 20-30% which has not improved significantly over 30 years. Refractory patients would benefit from precise individualized therapy. We report here that a patient-derived osteosarcoma growing in a subcutaneous nude-mouse model was regressed by tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R, p

Echistatin, a cyclic RGD peptide, which is an antagonist of αvβ3 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 αvβ3 integrin was used. Tumor angiogenesis by high-metastatic variant 143B-LM4 cells in the CAM was significantly inhibited by echistatin (P