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.

The Traumatic Inoculation Process Affects TSPO Radioligand Uptake in Experimental Orthotopic Glioblastoma.

BACKGROUND: The translocator protein (TSPO) has been proven to have great potential as a target for the positron emission tomography (PET) imaging of glioblastoma. However, there is an ongoing debate about the potential various sources of the TSPO PET signal. This work investigates the impact of the inoculation-driven immune response on the PET signal in experimental orthotopic glioblastoma. METHODS: Serial [18F]GE-180 and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) PET scans were performed at day 7/8 and day 14/15 after the inoculation of GL261 mouse glioblastoma cells (n = 24) or saline (sham, n = 6) into the right striatum of immunocompetent C57BL/6 mice. An additional n = 25 sham mice underwent [18F]GE-180 PET and/or autoradiography (ARG) at days 7, 14, 21, 28, 35, 50 and 90 in order to monitor potential reactive processes that were solely related to the inoculation procedure. In vivo imaging results were directly compared to tissue-based analyses including ARG and immunohistochemistry. RESULTS: We found that the inoculation process represents an immunogenic event, which significantly contributes to TSPO radioligand uptake. [18F]GE-180 uptake in GL261-bearing mice surpassed [18F]FET uptake both in the extent and the intensity, e.g., mean target-to-background ratio (TBRmean) in PET at day 7/8: 1.22 for [18F]GE-180 vs. 1.04 for [18F]FET, p < 0.001. Sham mice showed increased [18F]GE-180 uptake at the inoculation channel, which, however, continuously decreased over time (e.g., TBRmean in PET: 1.20 at day 7 vs. 1.09 at day 35, p = 0.04). At the inoculation channel, the percentage of TSPO/IBA1 co-staining decreased, whereas TSPO/GFAP (glial fibrillary acidic protein) co-staining increased over time (p < 0.001). CONCLUSION: We identify the inoculation-driven immune response to be a relevant contributor to the PET signal and add a new aspect to consider for planning PET imaging studies in orthotopic glioblastoma models.

PEGylated Fluorescent Anti-carcinoembryonic Antigen Antibody Labels Colorectal Cancer Tumors in Orthotopic Mouse Models.

INTRODUCTION: Colorectal cancer (CRC) patients often develop liver metastasis. However, curative resection of liver metastasis is not always possible due to poor visualization of tumor margins. The present study reports the characterization of a humanized anti-carcinoembryonic antigen monoclonal antibody conjugated to a PEGylated near-infrared dye, that targets and brightly labels human CRC tumors in metastatic orthotopic mouse models. METHODS: The hT84.66-M5A (M5A) monoclonal antibody was conjugated with a polyethylene glycol (PEG) chain that incorporated a near infrared (NIR) IR800 dye to establish M5A-IR800 Sidewinder (M5A-IR800-SW). Nude mice with CRC orthotopic primary tumors and liver metastasis both developed from a human CRC cell line, were injected with M5A-IR800-SW and imaged with the Pearl Trilogy Imaging System. RESULTS: M5A-IR800-SW targeted and brightly labeled CRC tumors, both in primary-tumor and liver-metastasis models. M5A-IR800-SW at 75 µg exhibited highly-specific tumor labeling in a primary-tumor orthotopic model with a median tumor-to-background ratio of 9.77 and in a liver-metastasis orthotopic model with a median tumor-to-background ratio of 7.23 at 96 h. The precise labeling of the liver metastasis was due to lack of hepatic accumulation of M5A-IR800-SW in the liver. CONCLUSIONS: M5A-IR800-SW provided bright and targeted NIR images of human CRC in orthotopic primary-tumor and liver-metastasis mouse models. The results of the present study suggest the clinical potential of M5A-IR800-SW for fluorescence-guided surgery including metastasectomies for CRC. The lack of hepatic NIR signal is of critical importance to allow for precise labeling of liver tumors.

[Corrigendum] The novel anti­neuroblastoma agent PF403, inhibits proliferation and invasion in vitro and in brain xenografts.

Subsequently to the publication of the above article, the authors have realized that Fig. 5D on p. 183 was published containing an error; essentially, the images chosen for the data panels representing the Fig. 5D, CAT3 Low and 5D, CAT3 High experiments were inadvertently selected from the same slide. However, the authors had retained access to their original data, and the revised version of Fig. 5 is shown on the next page, now showing the correct data for the Fig. 5D, CAT3 High panel. All the authors agree to the publication of this corrigendum, and they confirm that these data continue to support the main conclusions presented in their paper. Furthermore, the authors are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish this Corrigendum, and they also apologize to the readership for any inconvenience caused. [International Journal of Oncology 47: 179­187, 2015; DOI: 10.3892/ijo.2015.2977].

An Orthotopic Murine Model of Peritoneal Carcinomatosis of Ovarian Origin for Intraoperative PDT.

Advanced ovarian cancer is the most serious among gynecological malignancies and is associated with 35% five-year overall survival. Surgery is the first therapeutic indication, and the absence of remaining macroscopic lesions is the most important prognostic factor. However, tumor dissemination over the whole abdominal cavity largely contributes to the difficulty of complete surgical resection. Consequently, any therapeutic approach that may complete surgical resection should improve patient survival. Considering that some sites are not suitable for surgery because of their close location to vital organs, intraoperative photodynamic therapy (ioPDT) appears to be a complementary therapeutic approach to surgery to obtain the lowest residual disease.Relevant in vivo cancer models that closely resemble human ovarian cancer are essential for preclinical research of alternative antitumor therapeutic strategies. Thus, we propose a comprehensive protocol to set up an orthotopic ovarian xenograft in mice leading to peritoneal carcinomatosis that could be harnessed for antitumor therapeutic application and evaluation.

A Comparative Study of Patient-Derived Tumor Models of Pancreatic Ductal Adenocarcinoma Involving Orthotopic Implantation.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is associated with very poor prognoses. Therefore, new therapies and preclinical models are urgently needed. In the present study, we sought to develop more realistic experimental models for use in PDA research. METHODS: We developed patient-derived xenografts (PDXs), established PDX-derived cell lines (PDCLs), and generated cell line-derived xenografts (CDXs), which we integrated to create 13 matched "trios" - i.e., patient-derived tumor models of PDA. We then compared and contrasted histological and molecular alterations between these three model systems. RESULTS: Orthotopic implantation (OI) of the PDCLs resulted in tumorigenesis and metastases to the liver and peritoneum. Morphological comparisons of OI-CDXs and OI-PDXs with passaged tumors revealed that the histopathological features of the original tumor were maintained in both models. Molecular alterations in PDX tumors (including those to KRAS, TP53, SMAD4, and CDKN2A) were similar to those in the respective PDCLs and CDX tumors. When gene expression levels in the PDCLs, ectopic tumors, and OI tumors were compared, the distant metastasis-promoting gene CXCR4 was specifically upregulated in OI tumors, whose immunohistochemical profiles suggested epithelial-mesenchymal transition and adeno-squamous trans-differentiation. CONCLUSION: These patient-derived tumor models provide useful tools for monitoring responses to antineoplastic agents and for studying PDA biology.

Brain penetration and efficacy of tepotinib in orthotopic patient-derived xenograft models of MET-driven non-small cell lung cancer brain metastases.

Central nervous system-penetrant therapies with intracranial efficacy against non-small cell lung cancer (NSCLC) brain metastases are urgently needed. We report preclinical studies investigating brain penetration and intracranial activity of the MET inhibitor tepotinib. After intravenous infusion of tepotinib in Wistar rats (n = 3), mean (±standard deviation) total tepotinib concentration was 2.87-fold higher in brain (505 ± 22 ng/g) than plasma (177 ± 20 ng/mL). In equilibrium dialysis experiments performed in triplicate, mean tepotinib unbound fraction was 0.35% at 0.3 and 3.0 µM tepotinib in rat brain tissue, and 4.0% at 0.3 and 1.0 µM tepotinib in rat plasma. The calculated unbound brain-to-plasma ratio was 0.25, indicating brain penetration sufficient for intracranial target inhibition. Of 20 screened subcutaneous patient-derived xenograft (PDX) models from lung cancer brain metastases (n = 1), two NSCLC brain metastases models (LU5349 and LU5406) were sensitive to the suboptimal dose of tepotinib of 30 mg/kg/qd (tumor volume change [%TV]: -12% and -88%, respectively). Molecular profiling (nCounter®; NanoString) revealed high-level MET amplification in both tumors (mean MET gene copy number: 11.2 and 24.2, respectively). Tepotinib sensitivity was confirmed for both subcutaneous models at a clinically relevant dose (125 mg/kg/qd; n = 5). LU5349 and LU5406 were orthotopically implanted into brains of mice and monitored by magnetic resonance imaging (MRI). Tepotinib 125 mg/kg/qd induced pronounced tumor regression, including complete or near-complete regressions, compared with vehicle in both orthotopic models (n = 10; median %TV: LU5349, -84%; LU5406, -63%). Intracranial antitumor activity of tepotinib did not appear to correlate with blood-brain barrier leakiness assessed in T1-weighted gadolinium contrast-enhanced MRI.

Preclinical Testing of Living Tissue-Engineered Heart Valves for Pediatric Patients, Challenges and Opportunities.

Introduction: Pediatric patients with cardiac congenital diseases require heart valve implants that can grow with their natural somatic increase in size. Current artificial valves perform poorly in children and cannot grow; thus, living-tissue-engineered valves capable of sustaining matrix homeostasis could overcome the current drawbacks of artificial prostheses and minimize the need for repeat surgeries. Materials and Methods: To prepare living-tissue-engineered valves, we produced completely acellular ovine pulmonary valves by perfusion. We then collected autologous adipose tissue, isolated stem cells, and differentiated them into fibroblasts and separately into endothelial cells. We seeded the fibroblasts in the cusp interstitium and onto the root adventitia and the endothelial cells inside the lumen, conditioned the living valves in dedicated pulmonary heart valve bioreactors, and pursued orthotopic implantation of autologous cell-seeded valves with 6 months follow-up. Unseeded valves served as controls. Results: Perfusion decellularization yielded acellular pulmonary valves that were stable, no degradable in vivo, cell friendly and biocompatible, had excellent hemodynamics, were not immunogenic or inflammatory, non thrombogenic, did not calcify in juvenile sheep, and served as substrates for cell repopulation. Autologous adipose-derived stem cells were easy to isolate and differentiate into fibroblasts and endothelial-like cells. Cell-seeded valves exhibited preserved viability after progressive bioreactor conditioning and functioned well in vivo for 6 months. At explantation, the implants and anastomoses were intact, and the valve root was well integrated into host tissues; valve leaflets were unchanged in size, non fibrotic, supple, and functional. Numerous cells positive for a-smooth muscle cell actin were found mostly in the sinus, base, and the fibrosa of the leaflets, and most surfaces were covered by endothelial cells, indicating a strong potential for repopulation of the scaffold. Conclusions: Tissue-engineered living valves can be generated in vitro using the approach described here. The technology is not trivial and can provide numerous challenges and opportunities, which are discussed in detail in this paper. Overall, we concluded that cell seeding did not negatively affect tissue-engineered heart valve (TEHV) performance as they exhibited as good hemodynamic performance as acellular valves in this model. Further understanding of cell fate after implantation and the timeline of repopulation of acellular scaffolds will help us evaluate the translational potential of this technology.

CR13626: a novel oral brain penetrant tyrosine kinase inhibitor that reduces tumor growth and prolongs survival in a mouse model of glioblastoma.

Glioblastoma multiforme (GBM) is the most malignant primary brain cancer. Despite aggressive treatments currently there is no cure for GBM. Many challenges should be considered for the development of new therapeutical agents for glioblastoma, including appropriate target selectivity and pharmacokinetics. Several mutations and alterations of key cellular pathways including tyrosine kinases (TKs) are involved in malignant transformation and tumor progression. Thus, the targeting of multiple pathways and the development of innovative combination drug regimens is expected to yield improved therapies. Moreover, the abilities to cross the blood-brain barrier (BBB) reaching effective concentrations in brain and to remain into this tissue avoiding the effects of efflux transporters are also critical issues in the development of new therapeutics for GBM. CR13626 is a novel brain penetrant small molecule able to potently inhibit in vitro the activity of EGFR, VEGFR2 (aka KDR), Fyn, Yes, Lck, HGK (aka MAP4K4) and RET kinases relevant for GBM development. CR13626 shows good oral bioavailability (72%) and relevant brain penetration (brain/plasma ratio of 1.4). In an orthotopic xenograft glioblastoma mouse model, oral treatment with CR13626 results in a time-dependent reduction of tumor growth, leading to a significant increase of animal survival. The unique properties of CR13626 warrant its further investigation as a potential new drug candidate in glioblastoma.

An Animal Model of Colorectal Cancer Liver Metastasis With a High Metastasis Rate and Clonal Dynamics.

BACKGROUND: Various animal models have been introduced into the study of liver metastasis of colorectal cancer, but they have not been compared under the same conditions. The aim of this study was to identify an optimized mouse model that showed a high rate of hepatic metastasis and expression of clonal dynamics. MATERIALS AND METHODS: Athymic nude mice (n=30) were divided into two equal groups for the creation of a splenic injection model (SIM) and surgically orthotopic implantation model (SOIM) of liver metastasis of colorectal cancer using HCT116 cells. Hepatic metastasis was confirmed by gross and microscopic examinations. Expression of MET transcriptional regulator MACC1 (MACC1) in colon cancer cell lines and metastatic tumors in the group with a higher liver metastasis rate was confirmed by quantitative reverse-transcription-polymerase chain reaction. RESULTS: The observation time was significantly shorter for SIM than for SOIM (33.0±6.8 vs. 41.2±7.2 days, p<0.001). The rate of hepatic metastasis was significantly higher in SIM than in SOIM (76.9% vs. 38.4%, p=0.038). MACC1 was expressed in Colo201, HCT116, HT29, LS513, SW620, and WiDr cells but not in SW480 cells. All hepatic metastases in SIM mice expressed MACC1, and metastatic HCT116 cells had significantly greater expression than did the original HCT116 cells (p<0.001). CONCLUSION: With a higher rate of hepatic metastasis with clonal dynamics in a shorter observation time than the SOIM, SIM appears to be a good animal model for identifying new targets and in drug development for colorectal cancer liver metastasis. SOIM should also be considered for the study of the full steps of metastasis.

Evaluation of a Cell-Free Collagen Type I-Based Scaffold for Articular Cartilage Regeneration in an Orthotopic Rat Model.

The management of chondral defects represents a big challenge because of the limited self-healing capacity of cartilage. Many approaches in this field obtained partial satisfactory results. Cartilage tissue engineering, combining innovative scaffolds and stem cells from different sources, emerges as a promising strategy for cartilage regeneration. The aim of this study was to evaluate the capability of a cell-free collagen I-based scaffold to promote cartilaginous repair after orthotopic implantation in vivo. Articular cartilage lesions (ACL) were created at the femoropatellar groove in rat knees and cell free collagen I-based scaffolds (S) were then implanted into right knee defect for the ACL-S group. No scaffold was implanted for the ACL group. At 4-, 8- and 16-weeks post-transplantation, degrees of cartilage repair were evaluated by morphological, histochemical and gene expression analyses. Histological analysis shows the formation of fibrous tissue, at 4-weeks replaced by a tissue resembling the calcified one at 16-weeks in the ACL group. In the ACL-S group, progressive replacement of the scaffold with the newly formed cartilage-like tissue is shown, as confirmed by Alcian Blue staining. Immunohistochemical and quantitative real-time PCR (qRT-PCR) analyses display the expression of typical cartilage markers, such as collagen type I and II (ColI and ColII), Aggrecan and Sox9. The results of this study display that the collagen I-based scaffold is highly biocompatible and able to recruit host cells from the surrounding joint tissues to promote cartilaginous repair of articular defects, suggesting its use as a potential approach for cartilage tissue regeneration.

Orthotopic foetal lung tissue direct injection into lung showed a preventive effect against paraquat-induced acute lung injury in mice.

OBJECTIVES: Lung transplantation is the only effective therapy for patients with end-stage lung disease but an organ shortage crisis necessitates the development of alternative therapies. Recent studies have highlighted the potential of foetal tissue transplantation to facilitate the regeneration of vital organs such as liver that have been damaged by lethal diseases. Herein, with the aim of restoring pulmonary function, we hypothesized that allogenic foetal lung tissue implantation would attenuate severe respiratory failure. METHODS: Lung tissue from the foetuses of pregnant green fluorescent protein-C57BL/6 mice at 13.5 days of gestation was injected into the left lungs of recipient mice. Severe lung injury was induced by paraquat, and we analysed the survival rate and pathohistological findings after 1 month. RESULTS: The survival rate of the therapy group was 39%, which was significantly higher than the vehicle group at 5.9% (P = 0.034). Immunochemical staining showed that positive cytoplasmic stained cells with anti-interleukin-10 antibody were identified in the gland-like structure of embryonic day 13.5 foetal lung. At 4 weeks after orthotopic implantation, haematoxylin and eosin staining showed reduced lung inflammatory cells, reduced lung oedema and increased active cell proliferation of foetal lung cells. Lung injury score showed that the airway septal thickening revealed statistically significant differences between vehicle and foetal lung therapy (P < 0.001). CONCLUSIONS: Immature foetal lungs improved the survival rate of mice with paraquat-induced severe lung injury, establishing the need for systematic follow-up studies. The anti-inflammatory cytokine in the tissue from embryonic day 13.5 foetal lung might suppress severe lung injury.

Detection of Mediastinal Lymph Node Metastases Using Indocyanine Green (ICG) Fluorescence Imaging in an Orthotopic Implantation Model.

BACKGROUND: The method of quickly identifying metastatic mediastinal lymph nodes has become an urgent problem for lung cancer surgery. Indocyanine green (ICG) has the characteristic of being retained in or around the lymph nodes; its pharmacokinetic characteristics and optimal imaging time have not yet been elucidated. MATERIALS AND METHODS: The IVIS Lumina Imaging System was used to detect near infrared (NIR) fluorescence signals at different ICG doses, times and excitation/emission wavelengths in vitro. An artificial lymphogenous metastatic model of squamous lung carcinoma was established in 32 SCID-CB17 mice using Ma44.3 cells. An intratracheal injection of 1.25 ml/kg ICG (1.25×10-2 mg/ml) was performed, then 780 nm Ex and 845 nm Em were used to visualize ICG at four different times. The metastatic mediastinal lymph nodes and the implanted local tumor site in the left lung were confirmed with bioluminescence and hematoxylin and eosin (H&E) staining of pathological specimens. RESULTS: ICG had the strongest NIR fluorescence signal when using 780 nm Ex and 845 nm Em at 2 to 4 h after administrating 1.25×10-2 mg/ml ICG in vitro. Combined with pathological H&E examination, fluorescence imaging of ICG reflected true-positive mediastinal metastasis of the mediastinum at 0.5 h and 2 h after the injection of ICG in vivo. While true-positive local tumor growth at the site of implantation in the left lung was reflected within 4 h after the injection of ICG. CONCLUSION: ICG was able to display the metastatic mediastinal lymph nodes within 2 h after endotracheal injection in an orthotopic squamous lung carcinoma implantation model.

Novel Breast Cancer Brain Metastasis Patient-Derived Orthotopic Xenograft Model for Preclinical Studies.

The vast majority of mortality in breast cancer results from distant metastasis. Brain metastases occur in as many as 30% of patients with advanced breast cancer, and the 1-year survival rate of these patients is around 20%. Pre-clinical animal models that reliably reflect the biology of breast cancer brain metastasis are needed to develop and test new treatments for this deadly condition. The patient-derived xenograft (PDX) model maintains many features of a donor tumor, such as intra-tumor heterogeneity, and permits the testing of individualized treatments. However, the establishment of orthotopic PDXs of brain metastasis is procedurally difficult. We have developed a method for generating such PDXs with high tumor engraftment and growth rates. Here, we describe this method and identify variables that affect its outcomes. We also compare the brain-orthotopic PDXs with ectopic PDXs grown in mammary pads of mice, and show that the responsiveness of PDXs to chemotherapeutic reagents can be dramatically affected by the site that they are in.

A new molecular probe: An NRP-1 targeting probe for the grading diagnosis of glioma in nude mice.

Magnetic resonance molecular imaging, as a safe imaging technology, provides a new idea for the early qualitative and hierarchical diagnosis of gliomas. The purpose of this study was to design and evaluate the value of neuropilin-1 (NRP-1) targeting molecular probes in the hierarchical diagnosis of gliomas. First, we created an NRP-1 targeted magnetic resonance molecular probe (USPIO-PEG-tLyP-1) by combining the polypeptide tLyP-1 with ultra-small superparamagnetic iron oxide nanoparticles (USPIONs), detecting the physical properties by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Second, in vivo experiments, we established two different degrees of malignant gliomas in-situ in nude mice by injecting U87 and CHG-5 cells. Then, to detect the binding ability of the probe with different grades of tumour tissues, we injected the probe into the tumour-bearing mice through the tail vein. Next, MRI was performed before injection, and 6 h, 12 h, 24 h after injection, and we found significantly more iron particles in the tumour tissues of U87 tumour-bearing mice than in tumour tissues of CHG-5 tumour-bearing mice. The signal intensities of the T2-weighted images of the tumour tissues of each group as well as microscopic observations by Prussian blue staining indicated that the binding ability of this molecular probe to U87 glioma (HGG) with high NRP-1 expression was significantly greater than that of CHG-5 glioma (LGG) with low NRP-1 expression (P < 0.01). Therefore, this study confirms that the novel molecular probe USPIO-PEG-tLyP-1 can be used for the grading diagnosis by MRI for gliomas of high and low grade with different NRP-1 expression levels.

Establishment of a stable hepatic metastasis mouse model of murine colorectal cancer by microsurgical orthotopic implantation.

BACKGROUND: Liver metastasis is a common cause of death from colorectal cancer (CRC). In this paper we developed a liver metastasis mouse model by microsurgical orthotopic implantation (MSOI) to illuminate the CRC progression with an eye toward developing effective drug treatment. METHODS: Murine colon carcinoma CT-26 cells were cultured and then injected to male BALB/c athymic nude mice right flank to generate subcutaneous implantation tumor with 2×107 CT-26 cell suspension in DMEM. Tumor tissue at an average size of 1 cm3 was injected into another nude mice right flank with 20-gauge inoculating needle. Between fourth and sixth generations, tumor tissue sewn into the cecal surface establishes orthotopic transplanted CRC model by MSOI. Then on the 7th, 14th, 21st and 35th day, body weight, abdomen circumference, volume of ascites and local tumor weight were observed and weighed. On the 21st day and 35th day, local tumor rate was calculated, and metastatic tumors of other organs were observed. Tumor tissue was stained by HE for pathologic analysis. RESULTS: On the 35th day, body weight and abdomen circumference of the model group were significantly higher than the control group (P<0.01). Local tumor weight increased rapidly from the 21st d to the 35th d (P<0.01), and take rate was high (100%). Metastatic tumor appeared only in liver on the 21st day and then invaded to liver, stomach, retroperitoneal lymph node and abdominal wall on the 35th day. The metastatic rate of liver tumor respectively was 83.3% and 100% on the 21st day and 35th day, but liver function remained normal. Pathologic analysis showed that colorectal tumor invaded the normal tissue of liver, abdominal wall and stomach. CONCLUSIONS: A stable hepatic metastasis mouse model of murine CRC was established by MSOI.

Tumor-sealing Surgical Orthotopic Implantation of Human Colon Cancer in Nude Mice Induces Clinically-relevant Metastases Without Early Peritoneal Carcinomatosis.

BACKGROUND: Surgical orthotopic implantation of human colon cancer tissue to the ceca of mice has been used to mimic behavior of cancer in human patients for the development of precision cancer medicine. However, with the current method of serosal surface implantation (SSI) of pieces of human colon cancer tissue, cancer cells are exposed to the peritoneum, which can artificially increase the rate of peritoneal carcinomatosis (PC) during the disease course. The objective of the present study was to introduce a tumor-sealing method (TSM) and compare it with SSI for the ability to produce clinically-relevant metastases without artificial PC. MATERIALS AND METHODS: HCT116 colon cancer cells transfected with green fluorescence protein (GFP) were cultured and then injected into the subcutaneous layer of athymic nude mice. Subcutaneous tumors were allowed to grow sufficiently to supply adequate tumor for orthotopic implantation. For SSI, a 1 mm3-sized tumor fragment was sutured to partially torn serosa of the cecum. For TSM, the blind end of the cecum was folded over the tumor fragment and sealed with sutures. At 20 days after implantation, all mice were opened to visualize PC by intravital fluorescence imaging. At necropsy, distant metastasis was investigated using frozen section of whole blocks of organs. RESULTS: At 20 days after implantation, PC rates in the SSI group and the TSM group were 80% (12/15) and 20% (3/15), respectively (p<0.001). The liver metastasis rate was 41.7% (5/12) in the SSI group and 50% (5/10) in the TSM group (p=0.696). The lung metastasis rate was 0% (0/12) in the SSI group and 10% (1/10) in the TSM group (p=0.201). The mean survival of mice without PC on the 20th day was significantly longer than that of mice with PC on the 20th day (69.1±14.7 vs. 44.5±12.4 days, p=0.001). CONCLUSION: These results suggest that TSM might be a more patient-like and useful method as a model of metastatic colon cancer than SSI.

Efficacy of Recombinant Methioninase (rMETase) on Recalcitrant Cancer Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models: A Review.

An excessive requirement for methionine (MET), termed MET dependence, appears to be a general metabolic defect in cancer and has been shown to be a very effective therapeutic target. MET restriction (MR) has inhibited the growth of all major cancer types by selectively arresting cancer cells in the late-S/G2 phase, when they also become highly sensitive to cytotoxic agents. Recombinant methioninase (rMETase) has been developed to effect MR. The present review describes the efficacy of rMETase on patient-derived orthotopic xenograft (PDOX) models of recalcitrant cancer, including the surprising result that rMETase administrated orally can be highly effective.

Molecular and Physiological Evaluation of Pancreatic Cancer-Induced Cachexia.

Cachexia, a complex metabolic syndrome, is characterized by involuntary weight loss along with muscle wasting and fat depletion leading to poor quality of life of patients. About 80% of pancreatic cancer patients exhibit cachectic phenotype at the time of diagnosis. Here, we present the several molecular and physiological parameters, which we utilize to study the pancreatic cancer-induced cachexia in in vitro models and preclinical mice models of pancreatic cancer. We have described myotube and adipocyte-based in vitro models of muscle and fat wasting, including methods of cell culture, differentiation, and treatment with cancer cell-conditioned medium. Furthermore, we have explained the methods of evaluation of key cachectic markers for muscles. Next, we have detailed the orthotopic implantation mouse models of pancreatic cancer and evaluation of different physiological parameters, including body weight, food intake, body composition analysis, glucose tolerance test, insulin resistance test, grip strength measurement, and rotarod performance test. We have also explained morphological parameters and molecular markers to evaluate the muscle wasting in pancreatic cancer-induced cachexia.

A severe combined immunodeficiency disease mouse model of human adenocarcinoma with lepidic-predominant growth.

Severe combined immunodeficiency disease (SCID) mice with human lepidic adenocarcinoma were established by the intrabronchial implantation of fresh surgically resected specimens. Human pulmonary adenocarcinoma tissue from 16 different cases was transplanted into SCID mice, and SCID mouse tumors were established from four of these cases (25%). Among the four tumors, the tumor cells of two SCID mice showed replacement lepidic growth of mouse alveolar structures accompanied by multiple intrapulmonary lesions. Human lung carcinoma cell lines showing lepidic growth are rare and the xenograft models using the SCID mouse model developed in the current study will be useful for analyzing the growth and/or progression patterns and clinical behavior of lepidic adenocarcinoma, the major histological subtype of human carcinoma of the lung.