The Role of Microsurgery and Fluorescent-reporter Genes in Establishing Mouse Models for Real-Time Imaging of Metastatic Cancer-Cell Trafficking and Colony Formation: A Revolutionary and Disruptive Technology for Metastasis Research.

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research.

Imaging Transgenic Nude Mice Expressing Spectrally-distinct Fluorescent Reporters Emitting From Blue to Far Red Light With One Instrument With Single-nanometer-tuning of Laser-excitation Fluorescence.

Background/Aim: Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument. Materials and Methods: Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP. Results: For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system. Conclusion: Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment.

Loss of Malignancy of Super-Methotrexate-resistant Osteosarcoma Cells Is Associated With an Increase of Methylated Histone Marks H3K9me3 and H3K27me3.

BACKGROUND/AIM: Methotrexate (MTX) resistance in osteosarcoma results in a very poor patient prognosis. We previously reported that super MTX-resistant osteosarcoma (143B-MTXSR) cells, selected from parental 143B osteosarcoma (143B-P) cells by culturing them with increasing concentrations of MTX, exhibited reduced malignancy, despite the over-expression of oncogenes. The present study explored the mechanism of reduced malignancy in the super MTX-resistant osteosarcoma cells. MATERIALS AND METHODS: Previously selected 143B-MTXSR cells which are 5,500 times more MTX resistant than parental cells, were used for this study. The status of methylated histone H3K9me3 and H3K27me3 marks was examined with western immunoblotting and compared between 143B-MTXSR and parental 143B-P cells. RESULTS: Histone H3K9me3 and H3K27me3 marks were over-expressed in 143B-MTXSR compared to 143B-P (p<0.05, p<0.01, respectively). CONCLUSION: Over-expression of histone H3K9me3 and H3K27me3 marks may be related to super-MTX resistance and to the loss of malignancy of super MTX-resistant osteosarcoma cells due to the fundamental relationship of methylation and cancer.

EL4 Murine-Lymphoma-Stromal-Cell Fusion Hybrids Observed With Multiple Distinct Morphologies in the Primary Tumor and Metastatic Organs of a Syngeneic Mouse Model.

BACKGROUND/AIM: We and others have previously shown that cell fusion plays an important role in cancer metastasis. Color coding of cancer and stromal cells with spectrally-distinct fluorescent proteins is a powerful tool, as pioneered by our laboratory to detect cell fusion. We have previously reported color-coded cell fusion between cancer cells and stromal cells in metastatic sites by using color-coded EL4 murine lymphoma cells and host mice expressing spectrally-distinct fluorescent proteins. Cell fusion occurred between cancer cells or, between cancer cells and normal cells, such as macrophages, fibroblasts, and mesenchymal stem cells. In the present study, the aim was to morphologically classify the fusion-hybrid cells observed in the primary tumor and multiple metastases EL4 formed from cells expressing red fluorescent protein (RFP) in transgenic mice expressing green fluorescent protein (GFP), in a syngeneic model. MATERIALS AND METHODS: RFP-expressing EL4 murine lymphoma cells were cultured in vitro. EL4-RFP cells were harvested and injected intraperitoneally into immunocompetent transgenic C57/BL6-GFP mice to establish a syngeneic model. Two weeks later, mice were sacrificed and each organ was harvested, cultured, and observed using confocal microscopy. RESULTS: EL4 intraperitoneal tumors (primary) and metastases in the lung, liver, blood, and bone marrow were formed. All tumors were harvested and cultured. In all specimens, RFP-EL4 cells, GFP-stromal cells, and fused yellow-fluorescent hybrid cells were observed. The fused hybrid cells showed various morphologies. Immune cell-like round-shaped yellow-fluorescent fused cells had a tendency to decrease with time in liver metastases and circulating blood. In contrast fibroblast-like spindle-shaped yellow-fluorescent fused cells increased in the intraperitoneal primary tumor, lung metastases, and bone marrow. CONCLUSION: Cell fusion between EL4-RFP cells and GFP stromal cells occurred in primary tumors and all metastatic sites. The morphology of the fused hybrid cells varied in the primary and metastatic sites. The present results suggest that fused cancer and stromal hybrid cells of varying morphology may play an important role in cancer progression.

First-line Chemotherapy in Combination With Oral Recombinant Methioninase and a Low-methionine Diet for a Stage IV Inoperable Pancreatic-Cancer Patient Resulted in 40% Tumor Reduction and an 86% CA19-9 Biomarker Decrease.

BACKGROUND/AIM: Pancreatic cancer has a very poor prognosis with a 5-year survival rate of less than 5% among patients with distant metastasis, a figure that has not improved over many decades. Only 10 to 20% patients are candidates for curative surgery at presentation due to the aggressive nature and asymptomatic progression of pancreatic cancer. Although first-line chemotherapy, such as FOLFIRINOX and gemcitabine + nab paclitaxel, improved the median survival from 8.5 to 11.1 months, more effective treatments are immediately needed. The aim of the present study was to evaluate the efficacy of methionine restriction with oral rMETase (o-rMETase) and a low-methionine diet combined with first-line chemotherapy on a patient with stage IV metastatic pancreatic cancer. CASE REPORT: A 63-year-old female was diagnosed with metastatic pancreatic cancer in October 2023. The patient started FOLFIRINOX as first-line chemotherapy in combination with methionine restriction, which comprised o-rMETase 250 units twice a day and a low-methionine diet. The patient was monitored using computed tomography and CA19-9 blood tests. After five months from the start of combination therapy, the size of the primary tumor decreased by 40% along with liver-metastasis regression. The CA19-9 blood marker decreased by 86%. The patient sustains a high performance status and continues the combination therapy without severe side effects. CONCLUSION: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with first-line chemotherapy, was highly effective in a patient with inoperable stage IV pancreatic cancer.

Overcoming High Trabectedin Resistance of Soft-tissue Sarcoma With Recombinant Methioninase: A Potential Solution of a Recalcitrant Clinical Problem.

BACKGROUND/AIM: Drug resistance has been a recalcitrant problem for sarcoma patients for many decades. Trabectedin is a second-line chemotherapy for soft-tissue sarcoma that often leads to resistance and death of the patients. The objective of the present study was to address the issue of trabectedin-chemoresistance in HT1080 fibrosarcoma cells by combining recombinant methioninase (rMETase) with trabectedin and examining their efficacy on trabectedin-resistant fibrosarcoma cells in vitro. MATERIALS AND METHODS: Trabectedin-resistant HT1080 (TR-HT1080) cells were generated by subjecting HT1080 human fibrosarcoma cells to increasing trabectedin concentrations (3.3-8 nM). IC50 values for trabectedin and rMETase were compared for HT1080 and TR-HT1080 cells. TR-HT 1080 cells were placed into four groups to determine synergy of rMETase and trabectedin on TR-HT1080 cells: a control group with no treatment; a group treated with trabectedin (3.3 nM); a group treated with rMETase (0.75 U/ml); and a group treated with both trabectedin (3.3 nM) and rMETase (0.75 U/ml). RESULTS: The IC50 value of trabectedin- on TR-HT1080 cells was 42.9 nM, whereas the IC50 value of trabectedin on the parental HT1080 cells was 3.3 nM, indicating a 13-fold increase. The combination of rMETase (0.75 U/ml) and trabectedin (3.3 nM) was synergistic on TR-HT1080 cells resulting in an inhibition of 64.2% compared to trabectedin alone (5.7%) or rMETase alone (50.5%) (p<0.05). rMETase increased the efficacy of trabectedin 11-fold on trabectedin-resistant fibrosarcoma cells. CONCLUSION: The combined administration of trabectedin and rMETase was synergistic on the viability of TR-HT1080 cells in vitro. The combination of rMETase and trabectedin has promising clinical potential for overcoming chemo-resistance of soft-tissue sarcoma.

[11C]Methionine PET vs. [18F]Fluorodeoxyglucose PET Whole-body Imaging to Determine the Extent of Methionine-addiction Compared to Glucose-addiction of Primary and Metastatic Cancer of the Trunk in Patients.

BACKGROUND/AIM: Positron emission tomography (PET) is an important imaging modality, especially in oncology. [18F]fluorodeoxyglucose PET (FDG-PET) is the most used cancer PET imaging. However, since the elevated glucose use by cancers, termed the Warburg effect, is usually only moderate, FDG often does not provide a strong or well-delineated signal. Malignancies have a stronger addiction to methionine, known as the Hoffman effect, and thus [11C]methionine PET (MET-PET) has demonstrated superiority over FDG-PET in gliomas and other brain tumors. Our team is pioneering the use of MET-PET for tumors of the trunk for both better detection of cancer and to determine candidates for methionine-restriction therapy. The present study provides examples of cancers of organs in the trunk in which MET-PET outperforms FDG-PET in detecting and delineating primary and metastatic cancer. PATIENTS AND METHODS: In all cases, MET-PET and FDG-PET were performed simultaneously. An evaluation of the images was conducted by a nuclear medicine physician. RESULTS: Four cases, including prostate, bladder, esophageal, and breast cancer demonstrated the superiority of MET-PET compared to FDG-PET. CONCLUSION: MET-PET can out-perform FDG PET for accurate detection of primary and metastatic cancer in the trunk and can determine the extent of methionine addiction of cancer, thereby indicating whether cancer patients can benefit from methionine-restriction therapy.

Non-Invasive Direct Visualization of Luciferase-Luciferin Emitted Light Producing True Images from an Orthotopic Lung Cancer Xenograft Model in Nude Mice Using an Ultra-sensitive Low-light Camera and Optics.

BACKGROUND/AIM: In vivo imaging with luciferase-luciferin has been limited by the inability to visualize the low emitted light, with the signal quantified only by photon counting using a cumbersome highly-cooled CCD camera in a dark room. In the present study, we demonstrate direct visualization of the luciferase-luciferin signal from an orthotopic lung cancer in a nude-mouse xenograft model with a sensitive low-light camera and optics. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells expressing luciferase (LL/2-Luc2) were injected transcutaneously into the lung of a nude mouse. One week later after cell injection, luciferase imaging for emission at 560 nm was performed using the UVP Biospectrum Advanced system after i.v. injection of D-luciferin potassium salt. The intensity of the visualized light was measured and quantified with the instrument. RESULTS: A week following the implantation of LL/2-Luc2 cells in nude mice, the luciferase-luciferin signal from LL/2-Luc2 tumors in the lung was sufficiently visible through the skin to produce true images. At fifteen minutes, the intensity peaked and then progressively dropped due to clearance of luciferin from the tumor. CONCLUSION: Using the UVP Biospectrum Advanced system we demonstrated non-invasive visualization of true images from luciferase-luciferin signals from an orthotopic lung-cancer mouse model. The luciferase-luciferin emitted light was directly visible through the skin which is a major improvement over previous photon counting to detect the luciferase-luciferin signal.

Recombinant Methioninase Increases Eribulin Efficacy 16-fold in Highly Eribulin-resistant HT1080 Fibrosarcoma Cells, Demonstrating Potential to Overcome the Clinical Challenge of Drug-resistant Soft-tissue Sarcoma.

BACKGROUND/AIM: A major challenge in treating soft-tissue sarcoma is the development of drug resistance. Eribulin, an anti-tubulin agent, is used as a second-line chemotherapy for patients with unresectable or metastatic soft-tissue sarcoma. However, most patients with advanced soft-tissue sarcoma are resistant to eribulin and do not survive. Recombinant methioninase (rMETase) targets the fundamental and general hallmark of cancer, methionine addiction, termed the Hoffman Effect. The present study aimed to show how much rMETase could increase the efficacy of eribulin on eribulin-resistant fibrosarcoma cells in vitro. MATERIALS AND METHODS: HT1080 human fibrosarcoma cells were exposed to step-wise increasing concentrations of eribulin from 0.15-0.4 nM to establish eribulin-resistant HT1080 (ER-HT1080). ER-HT1080 cells were cultured in vitro and divided into four groups: untreated control; eribulin treated (0.15 nM); rMETase treated (0.75 U/ml); and eribulin (0.15 nM) plus rMETase (0.75 U/ml) treated. RESULTS: The IC50 of eribulin on ER-HT1080 cells was 0.95 nM compared to the IC50 of 0.15 nM on HT1080 cells, a 6-fold increase. The IC50 of rMETase on ER-HT1080 and HT1080 was 0.87 U/ml and 0.75 U/ml, respectively. The combination of rMETase (0.75 U/ml) and eribulin (0.15 nM) was synergistic on ER-HT1080 cells resulting in an inhibition of 80.1% compared to eribulin alone (5.0%) or rMETase alone (47.1%) (p<0.05). rMETase thus increased the efficacy of eribulin 16-fold on eribulin-resistant fibrosarcoma cells. CONCLUSION: The present study showed that the combination of eribulin and rMETase can overcome high eribulin resistance of fibrosarcoma. The present results demonstrate that combining rMETase with first- or second-line therapy for soft-tissue sarcoma has the potential to overcome the intractable clinical problem of drug-resistant soft-tissue sarcoma.

The Combination of Methionine Restriction and Docetaxel Synergistically Arrests Androgen-independent Prostate Cancer But Not Normal Cells.

Background/Aim: Androgen-independent prostate cancer (AIPC) is resistant to androgen-depletion therapy and is a recalcitrant disease. Docetaxel is the first-line treatment for AIPC, but has limited efficacy and severe side-effects. All cancers are methionine-addicted, which is termed the Hoffman effect. Recombinant methioninase (rMETase) targets methionine addiction. The purpose of the present study was to determine if the combination of docetaxel and rMETase is effective for AIPC. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of docetaxel and rMETase alone were determined for the human AIPC cell line PC-3 and Hs27 normal human fibroblasts in vitro. The synergistic efficacy for PC-3 and Hs27 using the combination of docetaxel and rMETase at their IC50s for PC-3 was determined. Results: The IC50 of docetaxel for PC-3 and for Hs27 was 0.72 nM and 0.94 nM, respectively. The IC50 of rMETase for PC-3 and for Hs27 was 0.67 U/ml and 0.76 U/ml, respectively. The combination of docetaxel and rMETase was synergistic for PC-3 but not Hs27 cells. Conclusion: The combination of a relatively low concentration of docetaxel and rMETase was synergistic and effective for AIPC. The present results also suggest that the effective concentration of docetaxel can be reduced by using rMETase, which may reduce toxicity. The present results also suggest the future clinical potential of the combination of docetaxel and rMETase for AIPC.

Synergy of Rapamycin and Methioninase on Colorectal Cancer Cells Requires Simultaneous and Not Sequential Administration: Implications for mTOR Inhibition.

Background/Aim: Rapamycin inhibits the mTOR protein kinase. Methioninase (rMETase), by degrading methionine, targets the methionine addiction of cancer cells and has been shown to improve the efficacy of chemotherapy drugs, reducing their effective doses. Our previous study demonstrated that rapamycin and rMETase work synergistically against colorectal-cancer cells, but not on normal cells, when administered simultaneously in vitro. In the present study, we aimed to further our previous findings by exploring whether  synergy exists between rapamycin and rMETase when used sequentially against HCT-116 colorectal-carcinoma cells, compared to simultaneous administration, in vitro. Materials and Methods: The half-maximal inhibitory concentrations (IC50) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line were previously determined using the CCK-8 cell viability assay (11). We then examined the efficacy of rapamycin and rMETase, both at their IC50, administered simultaneously or sequentially on the HCT-116 cell line, with rapamycin administered before rMETase and vice versa. Results: The IC50 for rapamycin and rMETase, determined from previous experiments (11), was 1.38 nM and 0.39 U/ml, respectively, of HCT-116 cells. When rMETase was administered four days before rapamycin, both at the IC50, there was a 30.46% inhibition of HCT-116 cells. When rapamycin was administered four days before rMETase, both at the IC50, there was an inhibition of 41.13%. When both rapamycin and rMETase were simultaneously administered, both at the IC50, there was a 71.03% inhibition. Conclusion: Rapamycin and rMETase have synergistic efficacy against colorectal-cancer cells in vitro when administered simultaneously, but not sequentially.

Recombinant Methioninase Is Selectively Synergistic With Doxorubicin Against Wild-type Fibrosarcoma Cells Compared to Normal Cells and Overcomes Highly-Doxorubicin-resistant Fibrosarcoma.

BACKGROUND/AIM: Doxorubicin is first-line therapy for soft-tissue sarcoma, but patients can develop resistance which is usually fatal. As a novel therapeutic strategy, the present study aimed to determine the synergy of recombinant methioninase (rMETase) and doxorubicin against HT1080 fibrosarcoma cells compared to Hs27 normal fibroblasts, and rMETase efficacy against doxorubicin-resistant HT1080 cells in vitro. MATERIALS AND METHODS: The 50% inhibitory concentrations (IC50) of doxorubicin and rMETase, as well as their combination efficacy, against HT1080 human fibrosarcoma cells, Hs27 normal human fibroblasts and doxorubicin-resistant HT1080 (DR-HT1080) cells were determined. Dual-color HT1080 cells which expressed red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize nuclear fragmentation during treatment. Nuclear fragmentation was observed with an IX71 fluorescence microscope. RESULTS: The IC50 for doxorubicin was 3.3 µM for HT1080 cells, 12.4 µM for DR-HT1080 cells, and 7.25 µM for Hs27 cells. The IC50 for rMETase was 0.75 U/ml for HT1080 cells, 0.42 U/ml for DR-HT1080 cells, and 0.93 U/ml for Hs27 cells. The combination of rMETase and doxorubicin was synergistic against fibrosarcoma cells but not against normal fibroblasts. The combination of doxorubicin plus rMETase also caused more fragmented nuclei than either treatment alone in HT1080 cells. rMETase alone was highly effective against the DR-HT1080 cells as well as the parental HT1080 cells. CONCLUSION: The present results indicate the future clinical potential of rMETase in combination with doxorubicin for fibrosarcoma, including doxorubicin-resistant fibrosarcoma.

Exosome Transfer Between Different Co-implanted Human Pancreatic Cancer Cell Lines Occurs in the Primary Tumor and Multiple Metastatic Sites of Nude Mice But Not in Co-Culture In Vitro.

BACKGROUND/AIM: Exosome exchange between cancer cells or between cancer and stromal cells is involved in cancer metastasis. We have previously developed in vivo color-coded labeling of cancer cells and stromal cells with spectrally-distinct fluorescent genetic reporters to demonstrate the role of exosomes in metastasis. In the present study, we studied exosome transfer between different pancreatic-cancer cell lines in vivo and in vitro and its potential role in metastasis. MATERIALS AND METHODS: Human pancreatic-cancer cell lines AsPC-1 and MiaPaCa-2 were used in the present study. AsPC-1 cells contain a genetic exosome reporter gene labeled with green fluorescent protein (pCT-CD63-GFP) and MiaPaCa-2 cells express red fluorescent protein (RFP). Both cell lines were co-injected into the spleen of nude mice (n=5) to further study the role of exosome exchange in metastasis. Three weeks later mice were sacrificed and tumors at the primary and metastatic sites were cultured and observed by confocal fluorescence microscopy for exosome transfer. RESULTS: The primary tumor formed in the spleen and metastasized to the liver, as observed macroscopically. Cells were cultured from the spleen, liver, lung, bone marrow and ascites. Transfer of exosomes from AsPC-1 to MiaPaCa-2 was demonstrated in the cultured cells by confocal fluorescence microscopy. Moreover, cell fusion was also observed along with exosome transfer. Exosome transfer did not occur during in vitro co-culture between the two pancreatic-cancer cell lines, suggesting a role of the tumor microenvironment (TME) in exosome transfer. CONCLUSION: The transfer of exosomes between different pancreatic-cancer cell lines was observed during primary-tumor and metastatic growth in nude mice. This cell-cell communication might be a trigger of cell fusion and promotion of cancer metastasis. Exosome transfer between the two pancreatic-cancer cell lines appears to be facilitated by the TME, as it did not occur during in vitro co-culture.

Maximum Efficacy of Immune Checkpoint Inhibitors Occurs in Esophageal Cancer Patients With a Low Neutrophil-to-Lymphocyte Ratio and Good Performance Status Prior to Treatment.

BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs) play an important role in the treatment of esophageal cancer (EC). However, few patients achieve long-term survival, and some patients develop serious immune-related adverse events (irAEs). Reliable predictive biomarkers of efficacy and safety need to be established in order to improve efficacy. We retrospectively analyzed the outcomes of nivolumab monotherapy on EC at Showa University, Department of Medicine, to identify biomarkers and characteristics of patients who benefit from ICI monotherapy. PATIENTS AND METHODS: Eighty-six patients with EC who received nivolumab monotherapy were included in the present study. Patient characteristics, efficacy, and safety were analyzed. A multivariable analysis evaluated the correlation among overall survival (OS), progression-free survival (PFS), best overall response (BOR), irAEs, and the following variables: sex, age, performance status (PS), neutrophil-to-lymphocyte ratio (NLR), C-reactive protein (CRP) level, albumin level, and body-mass index before treatment. RESULTS: Median PFS was 3.1 months, and median OS was 9.0 months. In multivariable analysis, pretreatment PS, NLR, and sex were significantly correlated with OS and PFS. NLR <3.3 predicted longer survival (median OS 17.5 vs. 6.4 months for NLR ≥3.3; p<0.001). Median OS was 10.6 months for PS 0-1 and 1.3 months for PS 2-3 (p<0.001). NLR remained significantly predictive in the PS 0-1 group. The development of irAEs was significantly associated with increased OS and PFS. CONCLUSION: Patients with low NLR and good PS before treatment may maximize the benefits of ICIs. A low NLR may be an indicator of higher immunocompetence for anti-tumor immunity, suggesting that NLR may be a convenient predictive biomarker in daily practice.

Targeting Patient-Derived Orthotopic Gastric Cancers with a Fluorescent Humanized Anti-CEA Antibody.

BACKGROUND: Gastric 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. METHODS: Two 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. RESULTS: M5A-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. CONCLUSIONS: Humanized 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.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

BACKGROUND/AIM: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. RESULTS: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. CONCLUSION: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db mice.

Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-ß1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-ß1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.

Expression of PD-L1 Is Increased by Methionine Restriction Using Recombinant Methioninase in Human Colorectal Cancer Cells.

BACKGROUND/AIM: It has been recently demonstrated that a methionine-restricted diet increases the response to immune checkpoint inhibitors (ICIs) via an increase in PD-L1 in a syngeneic mouse colorectal-cancer model. Our laboratory has developed recombinant methioninase (rMETase) to restrict methionine. The aim of the present study was to determine if rMETase can increase PD-L1 expression in a human colorectal cancer cell line in vitro. MATERIALS AND METHODS: We evaluated the half-maximal inhibitory concentration (IC50) value of rMETase on HCT-116 human colorectal cancer cells. HCT-116 cells were treated with rMETase at the IC50 Western immunoblotting was used to compare PD-L1 expression in HCT-116 cells treated with and without rMETase. RESULTS: The IC50 value of rMETase on HCT-116 was 0.79 U/ml. Methionine restriction using rMETase increased PD-L1 expression compared to the untreated control (p<0.05). CONCLUSION: Methionine restriction with rMETase up-regulates PD-L1 expression in human colorectal cancer cells and the combination of rMETase and ICIs may have the potential to improve immunotherapy in human colorectal cancer.

Induction of the DNA-Repair Gene POLQ only in BRCA1-mutant Breast-Cancer Cells by Methionine Restriction.

BACKGROUND/AIM: BRCA1/2 mutations in breast cancer cells impair homologous recombination and promote alternative end joining (Alt-EJ) for DNA-damage repair. DNA polymerase theta, encoded by POLQ, plays a crucial role in Alt-EJ, making it a potential therapeutic target, particularly in BRCA1/2-mutant cancers. Methionine restriction is a promising approach to target cancer cells due to their addiction to this amino acid. The present study investigated the expression of POLQ in BRCA1/2 wild-type and BRCA1-mutant breast cancer cells under methionine restriction. MATERIALS AND METHODS: POLQ mRNA expression was measured using qRT-PCR in BRCA1/2 wild-type (MDA-MB-231) and BRCA1- mutant (HCC1937 and MDA-MB-436) breast-cancer cells under normal, or serum-restricted, or serum- and methionine-restricted conditions. RESULTS: Compared to BRCA1/2 wild-type cells, BRCA1-mutant cells displayed significantly higher basal POLQ expression in normal medium. Methionine restriction further increased POLQ expression in the BRCA1-mutant cells but decreased it in the BRCA1/2 wild-type cells. CONCLUSION: The present findings suggest that methionine restriction showed differential effects on POLQ expression, potentially impacting Alt-EJ activity, in BRCA1/2 wild-type and BRCA1-mutant breast-cancer cells. Further investigation is needed to explore the potential of combining methionine restriction with DNA-repair inhibitors, such as PARP inhibitors, to overcome drug resistance in BRCA1/2 mutant cancers.