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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.

Mucins (MUC1-MUC24) are a family of glycoproteins involved in cell signaling and barrier protection. They have been implicated in the progression of numerous malignancies including gastric, pancreatic, ovarian, breast, and lung cancer. Mucins have also been extensively studied with respect to colorectal cancer. They have been found to have diverse expression profiles amongst the normal colon, benign hyperplastic polyps, pre-malignant polyps, and colon cancers. Those expressed in the normal colon include MUC2, MUC3, MUC4, MUC11, MUC12, MUC13, MUC15 (at low levels), and MUC21. Whereas MUC5, MUC6, MUC16, and MUC20 are absent from the normal colon and are expressed in colorectal cancers. MUC1, MUC2, MUC4, MUC5AC, and MUC6 are currently the most widely covered in the literature regarding their role in the progression from normal colonic tissue to cancer.

Poor visualization of polyps can limit colorectal cancer screening. Fluorescent antibodies to mucin5AC (MUC5AC), a glycoprotein upregulated in adenomas and colorectal cancer, could improve screening colonoscopy polyp detection rate. Adenomatous polyposis coli flox mice with a Cdx2-Cre transgene (CPC-APC) develop colonic polyps that contain both dysplastic and malignant tissue. Mice received MUC5AC-IR800 or IRdye800 as a control IV and were sacrificed after 48 h for near-infrared imaging of their colons. A polyp-to-background ratio (PBR) was calculated for each polyp by dividing the mean fluorescence intensity of the polyp by the mean fluorescence intensity of the background tissue. The mean 25 μg PBR was 1.70 (±0.56); the mean 50 μg PBR was 2.64 (±0.97); the mean 100 μg PBR was 3.32 (±1.33); and the mean 150 μg PBR was 3.38 (±0.87). The mean PBR of the dye-only control was 2.22 (±1.02), significantly less than the 150 μg arm (p-value 0.008). The present study demonstrates the ability of fluorescent anti-MUC5AC antibodies to specifically target and label colonic polyps containing high-grade dysplasia and intramucosal adenocarcinoma in CPC-APC mice. This technology can potentially improve the detection rate and decrease the miss rate of advanced colonic neoplasia and early cancer at colonoscopy.

Accurately identifying metastatic disease is critical to directing the appropriate treatment in pancreatic cancer. Mucin 5AC is overexpressed in pancreatic cancer but absent in normal pancreas tissue. The present proof-of-concept study demonstrates the efficacy of an anti-mucin 5AC antibody conjugated to an IR800 dye (MUC5AC-IR800) to preferentially label a liver metastasis of pancreatic cancer (Panc Met) in a unique patient-derived orthotopic xenograft (PDOX) model. In orthotopic models, the mean tumor to background ratio was 1.787 (SD ± 0.336), and immunohistochemistry confirmed the expression of MUC5AC within tumor cells. MUC5AC-IR800 provides distinct visualization of pancreatic cancer liver metastasis in a PDOX mouse model, demonstrating its potential utility in staging laparoscopy and fluorescence-guided surgery.

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.

Methionine addiction, a fundamental and general hallmark of cancer, known as the Hoffman Effect, is due to altered use of methionine for increased and aberrant transmethylation reactions. However, the linkage of methionine addiction and malignancy of cancer cells is incompletely understood. An isogenic pair of methionine-addicted parental osteosarcoma cells and their rare methionine-independent revertant cells enabled us to compare them for malignancy, their epithelial-mesenchymal phenotype, and pattern of histone-H3 lysine-methylation. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were selected from methionine-addicted parental cells (143B-P) by their chronic growth in low-methionine culture medium for 4 passages, which was depleted of methionine by recombinant methioninase (rMETase). Cell-migration capacity was compared with a wound-healing assay and invasion capability was compared with a transwell assay in 143B-P and 143B-R cells in vitro. Tumor growth and metastatic potential were compared after orthotopic cell-injection into the tibia bone of nude mice in vivo. Epithelial-mesenchymal phenotypic expression and the status of H3 lysine-methylation were determined with western immunoblotting. 143B-P cells had an IC50 of 0.20 U/ml and 143B-R cells had an IC50 of 0.68 U/ml for treatment with rMETase, demonstrating that 143B-R cells had regained the ability to grow in low methionine conditions. 143B-R cells had reduced cell migration and invasion capability in vitro, formed much smaller tumors than 143B-P cells and lost metastatic potential in vivo, indicating loss of malignancy in 143B-R cells. 143B-R cells showed gain of the epithelial marker, ZO-1 and loss of mesenchymal markers, vimentin, Snail, and Slug and, an increase of histone H3K9me3 and H3K27me3 methylation and a decrease of H3K4me3, H3K36me3, and H3K79me3 methylation, along with their loss of malignancy. These results suggest that shifting the balance in histone methylases might be a way to decrease the malignant potential of cells. The present results demonstrate the rationale to target methionine addiction for improved sarcoma therapy.

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.

Colorectal cancer (CRC) is a common cause of cancer and cancer-related death. Surgery is the only curative modality. Fluorescence-enhanced visualization of CRC with targeted fluorescent probes that can delineate boundaries and target tumor-specific biomarkers can increase rates of curative resection. Approaches to enhancing visualization of the tumor-to-normal tissue interface are active areas of investigation. Nonspecific dyes are the most-used approach, but tumor-specific targeting agents are progressing in clinical trials. The present narrative review describes the principles of fluorescence targeting of CRC for diagnosis and fluorescence-guided surgery with molecular biomarkers for preclinical or clinical evaluation.

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.

Methionine addiction is a fundamental and general hallmark of cancer cells, which require exogenous methionine, despite their ability to synthesize normal amounts of methionine from homocysteine. In contrast, methionine-independent normal cells do not require exogenous methionine in the presence of a methionine precursor. The methionine addiction of cancer cells is due to excess transmethylation reactions. We have previously shown that histone H3 lysine marks are over-methylated in cancer cells and the over-methylation is unstable when the cancer cells are restricted of methionine. In the present study, we show that methionine-addicted osteosarcoma cells are sensitive to both methotrexate (MTX) and recombinant methioninase (rMETase), but they affect histone H3 lysine-methylation in the opposite direction. Concentrations of MTX and rMETase, which inhibit osteosarcoma cells viability to 20%, had opposing effects on the status of histone methylation of H3K9me3 and H3K27me3. rMETase significantly decreased the amount of H3K9me3 and H3K27me3. In contrast, MTX significantly increased the amount of H3K9me and H3K27me3. The results suggest that increase or decrease in these methylated histone lysine marks is associated with proliferation arrest of methionine-addicted osteosarcoma.

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.

The present study reviews the use of tumor-specific antibodies conjugated to fluorescent dyes in preclinical and clinical studies to enhance visualization of primary tumors and metastases for fluorescence-guided surgery (FGS) in colorectal cancer (CRC). A search strategy was developed using the peer-reviewed National Center for Biotechnology Information (NCBI) database on PubMed. Studies using tumor-specific fluorescence imaging and FGS techniques on murine models of colorectal cell lines or patient-derived orthotopic xenograft (PDOX) colorectal cancer are reviewed. A total of 24 articles were identified that met the inclusion criteria, 21 preclinical and 3 clinical trials. The most widely used target antigen in preclinical and clinical trials was carcinoembryonic antigen (CEA). Mouse studies and clinical studies have demonstrated that the use of FGS in CRC can aid in decreased residual tumor and decreased rates of recurrence. As the mainstay of colorectal cancer treatment is surgery, the addition of intraoperative fluorescence imaging can help locate tumor margins, visualize occult micro-metastases, drive surgical decision making and improve patient outcomes.

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.

A major barrier to the diagnosis and effective treatment of solid-tumor cancers is the difficulty in detection and visualization of tumor margins in primary and metastatic disease. The use of fluorescence can augment the surgeon's ability to detect cancer and aid in its resection. Several cancer types express carcinoembryonic antigen (CEA) including colorectal, pancreatic and gastric cancer. Antibodies to CEA have been developed and tagged with near-infrared fluorescent dyes. This review article surveyed the use of CEA antibodies conjugated to fluorescent probes for in vivo studies since 1990. PubMed and Google Scholar databases were queried, and 900 titles and abstracts were screened. Fifty-nine entries were identified as possibly meeting inclusion/exclusion criteria and were reviewed in full. Forty articles were included in the review and their citations were screened for additional entries. A total of 44 articles were included in the final review. The use of fluorescent anti-CEA antibodies has been shown to improve detection and resection of tumors in both murine models and clinically. The cumulative results indicate that fluorescent-conjugated anti-CEA antibodies have important potential to improve cancer diagnosis and surgery. In an emerging technology, anti-CEA fluorescent antibodies have also been successfully used for photoimmunotherapy treatment for cancer.

Lung adenocarcinoma is the most common pathological type of lung cancer with poor patient outcomes; therefore, developing novel therapeutic agents is critically needed. Andrographolide (AD), a major active component derived from the traditional Chinese medicine (TCM) Andrographis paniculate, is a potential antitumor drug, but the role of AD in lung adenocarcinoma remains poorly understood. In the present study, we demonstrated that AD inhibited the proliferation of broad-spectrum lung cancer cell lines in a dose-dependent manner. Meanwhile, we found that a high dose of AD induced Noxa-dependent apoptosis in human lung adenocarcinoma cells (A549 and H1299). Further studies revealed that Noxa was transcriptionally activated by activating transcription factor 4 (ATF4) in AD-induced apoptosis. Knockdown of ATF4 by small interfering RNA (siRNA) significantly diminished the transactivation of Noxa as well as the apoptotic population induced by AD. These results of the present study indicated that AD induced apoptosis of human lung adenocarcinoma cells by activating the ATF4/Noxa axis and supporting the development of AD as a promising candidate for the new era of chemotherapy.

HopQ is an outer-membrane protein of Helicobacter pylori that binds to human carcinoembryonic antigen-related cell-adhesion molecules (CEACAMs) with high specificity. We aimed to investigate fluorescence targeting of CEACAM-expressing colorectal tumors in patient-derived orthotopic xenograft (PDOX) models with fluorescently labeled recombinant HopQ (rHopQ). Western blotting, flow cytometry and ELISA were performed to determine the efficiency of rHopQ binding to CEACAMs. rHopQ was conjugated to IR800DyeCW (rHopQ-IR800). Nude mice received orthotopic implantation of colon cancer tumors. Three weeks later, mice were administered 25 μg or 50 μg HopQ-IR800 and imaged 24 or 48 h later. Intravital images were analyzed for tumor-to-background ratio (TBR). Flow cytometry and ELISA demonstrated binding of HopQ to CEACAM1, 3 and 5. Dose-response intravital imaging in PDOX models demonstrated optimal results 48 h after administration of 50 μg rHopQ-IR800 (TBR = 3.576) in our protocol. Orthotopic models demonstrated clear tumor margins of primary tumors and small regional metastases with a mean TBR = 3.678 (SD ± 1.027). rHopQ showed specific binding to various CEACAMs in PDOX models. rHopQ may be useful for CEACAM-positive tumor and metastasis detection for pre-surgical diagnosis, intra-operative imaging and fluorescence-guided surgery.

HopQ is an outer-membrane protein of Helicobacter pylori that binds to human carcinoembryonic antigen-related cell-adhesion molecules (CEACAMs) with high specificity. We aimed to investigate fluorescence targeting of CEACAM-expressing colorectal tumors in patient-derived orthotopic xenograft (PDOX) models with fluorescently labeled recombinant HopQ (rHopQ). Western blotting, flow cytometry and ELISA were performed to determine the efficiency of rHopQ binding to CEACAMs. rHopQ was conjugated to IR800DyeCW (rHopQ-IR800). Nude mice received orthotopic implantation of colon cancer tumors. Three weeks later, mice were administered 25 μg or 50 μg HopQ-IR800 and imaged 24 or 48 h later. Intravital images were analyzed for tumor-to-background ratio (TBR). Flow cytometry and ELISA demonstrated binding of HopQ to CEACAM1, 3 and 5. Dose-response intravital imaging in PDOX models demonstrated optimal results 48 h after administration of 50 μg rHopQ-IR800 (TBR = 3.576) in our protocol. Orthotopic models demonstrated clear tumor margins of primary tumors and small regional metastases with a mean TBR = 3.678 (SD ± 1.027). rHopQ showed specific binding to various CEACAMs in PDOX models. rHopQ may be useful for CEACAM-positive tumor and metastasis detection for pre-surgical diagnosis, intra-operative imaging and fluorescence-guided surgery.

Bone is one of the most frequent metastatic sites in non-small cell lung cancer (NSCLC). Osimertinib, with and without bevacizumab (BV), has been investigated on advanced NSCLC patients. However, the efficacy of those drugs on bone metastasis of NSCLC has not been investigated. The human NSCLC cell line H1975, expressing red fluorescent protein (H1975-RFP), was orthotopically injected to the tibia of nude mice. The established mouse models were randomized into four treatment groups of nine mice: Control; BV alone; osimertinib alone; osimertinib and BV combination. The tumors were observed by non-invasive fluorescence imaging. Osimertinib, with or without BV, caused tumor regression, increased mouse survival, and bone remodeling in the bone metastasis models. These results suggest that osimertinib is a promising clinical option for NSCLS patients with bone metastasis.

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.

BackgroundNanobodies, derived from camelid antibodies made of only heavy chains, are the smallest, biologic, antigen-binding fragments (~15kDa) with faster pharmacokinetics and better tumor penetration efficiency than standard antibodies. The present study evaluates the efficacy of a fluorescent, anti-carcinoembryonic antigen (CEA) nanobody for rapid tumor labeling in an orthotopic mouse model of pancreatic cancer.MethodsAnti-CEA or control nanobodies were conjugated with the near-infrared fluorophore IRDye 800CW. Fragments of BxPC-3 (high-CEA expressing) or MiaPACA-2 (low-CEA expressing) human pancreatic cancer cell lines were orthotopically implanted into the pancreatic tail of nude mice. After tumors reached 7 to 10 mm in size, 2 nmol anti-CEA or control nanobody-IRDye800CW were injected intravenously. Mice were imaged at various time points hours post-injection.ResultsAnti-CEA nanobodies clearly labeled BxPC3 orthotopic pancreatic tumors 3 hours after injection. The signal was present as early as 15 minutes after injection and was robust at 1 to 3 hours after injection with a tumor-to-background ratio of 2.66. In contrast, there was very low accumulation in the low CEA-expressing, MiaPACA2 pancreatic orthotopic tumors. The fluorophore-conjugated nanobody was specific for CEA-expressing tumors, while the control nanobody did not show any tumor-specific signal. Both nanobodies had strong kidney uptake as expected for small-molecule probes. The fluorescence signal was detectable using 2 clinical, Food and Drug Administration-approved, 800 nm imaging devices as well as small animal imaging systems.ConclusionThis anti-CEA, nanobody-based, fluorescent probe labeled pancreatic orthotopic tumors within 15 minutes of intravenous injection. Fluorescent anti-CEA nanobodies have labeling kinetics that approach the speed of nonspecific dyes such as indocyanine green but with the specificity of antibodies. The use of fluorescently-labeled, intact antibodies leads to a labeling delay of 48 to 96 hours between probe administration and the necessarily delayed time of operation, which can be avoided with nanobodies. The kinetics of a nanobody-based probe makes it a practical agent for same-day, patient administration and fluorescence-guided surgery.

PurposeThe goal of the present study was to determine the efficacy of temozolomide (TEM) and pazopanib (PAZ) combined with FOLFOX (oxaliplatin, leucovorin and 5-fluorouracil) on a colorectal cancer patient-derived orthotopic xenograft (PDOX) mouse model.Materials and methodsA colorectal cancer tumor from a patient previously established in non-transgenic nude mice was implanted subcutaneously in transgenic green fluorescence protein (GFP)-expressing nude mice in order to label the tumor stromal cells with GFP. Then labeled tumors were orthotopically implanted into the cecum of nude mice. Mice were randomized into four groups: Group 1, untreated control; group 2, TEM + PAZ; group 3, FOLFOX; group 4, TEM + PAZ plus FOLFOX. Tumor width, length, and mouse body weight were measured weekly. The Fluor Vivo imaging System was used to image the GFP-lableled tumor stromal cells in vivo. H&E staining and immunohistochemical staining were used for histological analysis.ResultsAll three treatments inhibited tumor growth as compared to the untreated control group. The combination of TEM + PAZ + FOLFOX regressed tumor growth significantly more effectively than TEM + PAZ or FOLFOX. Only the combination of TEM + PAZ + FOLFOX group caused a decrease in body weight. PAZ suppressed lymph vessels density in the colorectal cancer PDOX mouse model suggesting inhibition of lymphangiogenesis.ConclusionOur results suggest that the combination of TEM + PAZ + FOLFOX has clinical potential for colorectal cancer patient.

PurposeThe goal of the present study was to determine the efficacy of temozolomide (TEM) and pazopanib (PAZ) combined with FOLFOX (oxaliplatin, leucovorin and 5-fluorouracil) on a colorectal cancer patient-derived orthotopic xenograft (PDOX) mouse model.Materials and methodsA colorectal cancer tumor from a patient previously established in non-transgenic nude mice was implanted subcutaneously in transgenic green fluorescence protein (GFP)-expressing nude mice in order to label the tumor stromal cells with GFP. Then labeled tumors were orthotopically implanted into the cecum of nude mice. Mice were randomized into four groups: Group 1, untreated control; group 2, TEM + PAZ; group 3, FOLFOX; group 4, TEM + PAZ plus FOLFOX. Tumor width, length, and mouse body weight were measured weekly. The Fluor Vivo imaging System was used to image the GFP-lableled tumor stromal cells in vivo. H&E staining and immunohistochemical staining were used for histological analysis.ResultsAll three treatments inhibited tumor growth as compared to the untreated control group. The combination of TEM + PAZ + FOLFOX regressed tumor growth significantly more effectively than TEM + PAZ or FOLFOX. Only the combination of TEM + PAZ + FOLFOX group caused a decrease in body weight. PAZ suppressed lymph vessels density in the colorectal cancer PDOX mouse model suggesting inhibition of lymphangiogenesis.ConclusionOur results suggest that the combination of TEM + PAZ + FOLFOX has clinical potential for colorectal cancer patient.

Adult pleomorphic rhabdomyosarcoma (RMS) is a rare and malignant mesenchymal tumor. Recently, we developed a patient-derived orthotopic xenograft (PDOX) model of adult pleomorphic RMS. In the present study, we evaluated the efficacy of tumor-targeting Salmonella typhimurium (S. typhimurium) A1-R combined with caffeine (CAF) and valproic acid (VPA) on the adult RMS PDOX. An adult pleomorphic RMS cell line was established from the PDOX model. Cell survival after exposure to CAF and VPA was assessed, and the IC50 value was calculated for each drug. The RMS PDOX models were randomized into five groups: untreated control; tumor treated with cyclophosphamide (CPA); tumor treated with CAF + VPA; tumor treated with S. typhimurium A1-R; and tumor treated with S. typhimurium A1-R + CAF + VPA. Tumor size and body weight was measured twice a week. VPA caused a concentration-dependent cytocidal effect. A synergistic effect of combination treatment with CAF was observed against the RMS cell line. For the in vivo study, all treatments significantly inhibited tumor growth compared with the untreated control. S. typhimurium A1-R combined with VPA and CAF was significantly more effective than CPA, VPA combined with CAF, or S. typhimurium A1-R alone and significantly regressed the tumor volume compared with day 0. These results suggest that S. typhimurium A1-R together with VPA and CAF could regresses an adult pleomorphic RMS in a PDOX model and therefore has important future clinical potential.

Adult pleomorphic rhabdomyosarcoma (RMS) is a rare and malignant mesenchymal tumor. Recently, we developed a patient-derived orthotopic xenograft (PDOX) model of adult pleomorphic RMS. In the present study, we evaluated the efficacy of tumor-targeting Salmonella typhimurium (S. typhimurium) A1-R combined with caffeine (CAF) and valproic acid (VPA) on the adult RMS PDOX. An adult pleomorphic RMS cell line was established from the PDOX model. Cell survival after exposure to CAF and VPA was assessed, and the IC50 value was calculated for each drug. The RMS PDOX models were randomized into five groups: untreated control; tumor treated with cyclophosphamide (CPA); tumor treated with CAF + VPA; tumor treated with S. typhimurium A1-R; and tumor treated with S. typhimurium A1-R + CAF + VPA. Tumor size and body weight was measured twice a week. VPA caused a concentration-dependent cytocidal effect. A synergistic effect of combination treatment with CAF was observed against the RMS cell line. For the in vivo study, all treatments significantly inhibited tumor growth compared with the untreated control. S. typhimurium A1-R combined with VPA and CAF was significantly more effective than CPA, VPA combined with CAF, or S. typhimurium A1-R alone and significantly regressed the tumor volume compared with day 0. These results suggest that S. typhimurium A1-R together with VPA and CAF could regresses an adult pleomorphic RMS in a PDOX model and therefore has important future clinical potential.

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/AIM:Colorectal cancer (CRC) cells secrete inflammatory cytokines that affect CRC progression. The aim of the present study was to determine if micro-RNA-140(miR-140) regulates inflammatory cytokine secretion induced by lipopolysaccharide (LPS) in colorectal cancer cells by targeting tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). MATERIALS AND METHODS:Fifty fresh colon-cancer specimens and normal colorectal tissues were collected from patients with CRC and tested for the expression miR-140. Human CRC cell lines SW480 and HCT116 were treated with various concentrations and times with LPS. miR-140 and mRNA expression of potentially related genes were analyzed by qPCR. Protein expression was analyzed using western blot or ELISA. Overexpression plasmids with pcDNA3.1-TRAF6, pGL4.10-wtTRAF6 and pGL4.10-mutTRAF6 were constructed. miRNA target gene prediction and a dual luciferase assay were used to analyze miR-140-targeted TRAF6. RESULTS:miR-140 expression was up-regulated in CRC tissues. In CRC cells, LPS could increase miR-140 expression in a time- and concentration-dependent manner. LPS increased inflammatory cytokine mRNA expression levels in SW480 and HCT116 human colon-cancer cells. miRNA-140 suppressed TRAF6 expression via targeting the 3'UTR. TRAF6 affected miR-140-mediated inflammatory cytokine expression of SW480 and HCT116 cells under LPS treatment. CONCLUSION:miR-140 regulates inflammatory cytokine secretion of LPS-induced colorectal cancer cells by targeting TRAF6.

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.

The prognosis for patients with pancreatic cancer is extremely poor, as they are resistant to first line chemotherapy. The long-term goal of this study was to identify effective combination chemotherapy for pancreatic cancer using pancreatic cancer surgical specimens in the histoculture drug response assay (HDRA) based on three-dimensional culture of tumour fragments, which maintains nature tumour histology in vitro. From 2015 to 2017, the HDRA was performed with tumour specimens from 52 pancreatic cancer patients from Asan Medical Hospital. First, combination drug regimens showed higher drug efficacy and less patient variation than single drugs. Initially, 5-Fluorouracil(5-FU)/Belotecan/Oxaliplatinum and Tegafur/Gimeracil (TS-1)/Oxaliplatinum/Irinotecan were found to be effective. Second, we were able to correlate the efficacy of some drugs with tumour stage. Third, when designing new combination regimens containing 5-FU or gemcitabine, we could identify more effective drug combinations. This is the first study to demonstrate usefulness of the HDRA for pancreatic cancer. Using this technique, we could identify novel candidate combination drug regimens that should be effective in treating pancreatic cancer.

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.

The EphA2 tyrosine kinase receptor is highly expressed in several types of solid tumors. In our recent studies, we targeted EphA2 in pancreatic cancer with agonistic agents and demonstrated that suppression of EphA2 significantly reduced cancer-cell migration in cell-based assays. In the present study, we focused on targeting EphA2 in prostate cancer. While not all prostate cancers express EphA2, we showed that enzalutamide induced EphA2 expression in prostate cancer cells and in a patient-derived xenograft (PDX) animal model, which provides further impetus to target EphA2 in prostate cancer. Western blot studies showed that agonistic dimeric synthetic (135H12) and natural (ephrinA1-Fc) ligands effectively degraded EphA2 receptor in the prostate cancer cell line PC-3. The agents also delayed cell migration of prostate cancer (PC-3) cells, while an in vivo PC-3 orthotopic metastatic nude-mouse model also revealed that administration of ephrinA1-Fc or 135H12 strongly reduced metastases. The present study further validates EphA2 as an important target in metastatic prostate cancer treatment. Our results should incentivize further efforts aimed at developing potent and effective EphA2 synthetic agonistic agents for the treatment of EphA2-driven aggressive metastatic tumors including prostate, pancreatic, and breast cancer.

BackgroundOver the past four decades, outcomes for osteosarcoma patients have plateaued as there have been few emerging therapies showing clinical results. Thus, the identification of novel biomarkers and therapeutic strategies are urgently needed to address these primary obstacles in patient care. Although the Myc-oncogene has known roles in oncogenesis and cancer cell growth, its expression and function in osteosarcoma are largely unknown.MethodsExpression of Myc was determined by Western blotting of osteosarcoma cell lines and patient tissues, and by immunohistochemistry of a unique osteosarcoma tissue microarray (TMA) constructed from 70 patient samples with extensive follow-up data. Myc specific siRNA and inhibitor 10058-F4 were applied to examine the effect of Myc inhibition on osteosarcoma cell proliferation. The clonogenicity and migration activity was determined by clonogenic and wound-healing assays. A mimic in vivo assay, three-dimensional (3D) cell culture model, was performed to further validate the effect of Myc inhibition on osteosarcoma cell tumorigenic markers.ResultsMyc was significantly overexpressed in human osteosarcoma cell lines compared with normal human osteoblasts, and also highly expressed in fresh osteosarcoma tissues. Higher Myc expression correlated significantly with metastasis and poor prognosis. Through the addition of Myc specific siRNA and inhibitor, we significantly reduced Myc protein expression, resulting in decreased osteosarcoma cell proliferation. Inhibition of Myc also suppressed the migration, clonogenicity, and spheroid growth of osteosarcoma cells.ConclusionOur results support Myc as an emerging prognostic biomarker and therapeutic target in osteosarcoma therapy.

Background:Over the past four decades, outcomes for osteosarcoma patients have plateaued as there have been few emerging therapies showing clinical results. Thus, the identification of novel biomarkers and therapeutic strategies are urgently needed to address these primary obstacles in patient care. Although the Myc-oncogene has known roles in oncogenesis and cancer cell growth, its expression and function in osteosarcoma are largely unknown. Methods:Expression of Myc was determined by Western blotting of osteosarcoma cell lines and patient tissues, and by immunohistochemistry of a unique osteosarcoma tissue microarray (TMA) constructed from 70 patient samples with extensive follow-up data. Myc specific siRNA and inhibitor 10058-F4 were applied to examine the effect of Myc inhibition on osteosarcoma cell proliferation. The clonogenicity and migration activity was determined by clonogenic and wound-healing assays. A mimic in vivo assay, three-dimensional (3D) cell culture model, was performed to further validate the effect of Myc inhibition on osteosarcoma cell tumorigenic markers. Results:Myc was significantly overexpressed in human osteosarcoma cell lines compared with normal human osteoblasts, and also highly expressed in fresh osteosarcoma tissues. Higher Myc expression correlated significantly with metastasis and poor prognosis. Through the addition of Myc specific siRNA and inhibitor, we significantly reduced Myc protein expression, resulting in decreased osteosarcoma cell proliferation. Inhibition of Myc also suppressed the migration, clonogenicity, and spheroid growth of osteosarcoma cells. Conclusion:Our results support Myc as an emerging prognostic biomarker and therapeutic target in osteosarcoma therapy.

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.

Chemotherapy-resistant osteosarcoma is a recalcitrant disease. It is a frequent cause of death to the patients who are usually adolescent or young adults. The goal of the present study was to determine the efficacy of the combination of olaratumab (OLA), doxorubicin (DOX), and cisplatinum (CDDP) on osteosarcoma, which is resistant to first-line therapy, in a patient-derived orthotopic xenograft (PDOX) model. The osteosarcoma PDOX model was randomized into six treatment groups of six mice: control; CDDP alone; DOX and CDDP; OLA + DOX; OLA + CDDP; and OLA + DOX and CDDP. Tumor size and body weight were measured during 14 days of treatment. Tumor growth was regressed only by the treatment with a combination of OLA + DOX and CDDP. Tumors treated with this three-drug combination had the most tumor necrosis and the lowest Ki-67 index. The present study demonstrates the power of the PDOX model to identify novel effective treatment strategy for chemotherapy-resistant osteosarcoma.

The goal of the present study was to determine the efficacy of osimertinib (AZD9291), a third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor for the treatment of aggressive EGFR-mutant non-small cell lung cancer (NSCLC), compared to cisplatinum (CDDP) + pemetrexed (PEM). The NSCLC cell line PC-9 expressing green fluorescence protein (PC-9-GFP) was implanted in the brain of nude mice and was treated with CDDP + PEM or AZD9291. Tumors were observed by non-invasive fluorescence imaging. AZD9291 treatment caused tumor regression in contrast to CDDP + PEM which had only a slight inhibitory effect. These results suggest that AZD9291 is a promising clinical option for NSCLC patients with brain metastasis.

Background/aimInflammation may play a role in cancer initiation and progression. The molecular mechanisms by which inflammation causes colorectal cancer, remains unclear. The present study investigated a signaling pathway that affects inflammation in colorectal cancer.Materials and methodsSW480 cells, HCT116 cells, and cells with knockdown of myeloid differentiation 88 (MyD88), and forced expression of MyD88 were treated with lipopolysaccharide (LPS; 1 μg/ml). Inflammation-related mRNA expression was analyzed by the quantitative reverse transcription polymerase chain reaction and inflammatory cytokines were detected by western blotting. The enzyme-linked immunosorbent assay (ELISA) was used to quantify inflammation-related cytokines in colorectal cancer cells. Cancer cell properties were evaluated using the wound-healing assay, transwell migration assay, transwell invasion assay, colony-formation assay, and CCK-8 assay.ResultsLPS up-regulated mRNA and protein levels of inflammatory factors in colorectal cancer cells. Knockdown of MyD88 inhibited LPS-induced mRNA expression and inflammatory protein expression in colorectal cancer cells. Similarly, silencing of MyD88 expression suppressed LPS-induced changes in the biological behavior of colorectal cancer cells. Silencing of MyD88 expression down-regulated expression of proteins of the LPS/nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-ĸB)/mitogen-activated protein kinase (MAPK) signaling pathway. Restoration of the expression of MyD88 reversed the effects in LPS-treated HCT116 cells.ConclusionMyD88-regulated LPS/NF-ĸB/MAPK signaling pathway affects the inflammatory and biological behavior of LPS-induced colorectal cancer cells.

The bulge area of the hair follicle contains hair-follicle-associated pluripotent (HAP) stem cells. Here, we present effective cryopreservation procedures of the human hair follicle that preserve the differentiation potential of HAP stem cells. Whole hair follicles isolated from human scalp were cryopreserved by a slow-rate cooling medium and stored in liquid nitrogen. A careful thawing method was used to collect the upper parts of the human hair follicles which were cultured for four weeks in a Dulbecco's Modified Eagle's Medium with fetal bovine serum (FBS). Proliferating hair follicle cells were then shifted to DMEM/Ham's Nutrient Mixture F-12 medium without FBS and allowed to grow for one week. These proliferating cells were able to produce HAP stem cell colonies with multilineage differentiation capacity. They produced keratinocytes, smooth muscle cells, cardiac muscle cells, neurons and glial cells. Interestingly, these cryopreserved hair follicles produced pluripotent HAP stem cell colonies similar to fresh follicles. These findings suggest that the cryopreserved whole human hair follicle preserves the ability to produce HAP stem cells, which will enable any individual to preserve a bank of these stem cells for personalized regenerative medicine.

Pancreatic cancer is a highly lethal disease in part due to incomplete tumor resection. Targeting by tumor-specific antibodies conjugated with a fluorescent label can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we describe a patient-derived orthotopic xenograft model of pancreatic cancer that recapitulated the disease on a gross and microscopic level, along with physiologic clinical manifestations. We additionally show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively highlight the pancreatic cancer and its metastases in this model with a tumor-to-background ratio of 3.5 (SEM 0.9). The present results demonstrate the clinical potential of this labeling technique for fluorescence-guided surgery of pancreatic cancer.