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Orthotopic ESCC cell line model. (a) Different cell numbers were evaluated for orthotopic tumor formation for four ESCC cell lines. (b) Xenogen images of 10⁵ cells for four ESCC cell lines expressing luciferase illustrate the growth of the tumor with (A) KYSE150 luc, (B) KYSE30 luc, (C) 81-T luc, and (D) SLMT-1 luc. (c) The bioluminescence live animal images displaying coronal, sagittal, and transaxial cross-section views of the animal and providing tumor depth and location information of a KYSE150 orthotopic tumor in 3D taken by the Xenogen IVIS Spectrum.
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Increasing evidence indicates tumor-stromal interactions play a crucial role in cancer. An in vivo esophageal squamous cell carcinoma (ESCC) orthotopic animal model was developed with bioluminescence imaging established with a real-time monitoring platform for functional and signaling investigation of tumor-stromal interactions. The model was produ...
Citations
... Кроме этого, описанная экспериментальная модель позволяет исследовать взаимодействие опухоли и стромы [14]. Другим серьезным недостатком этой модели является необходимость использования специализированных методов визуализации, например, система визуализации in vivo в сочетании с использованием биолюминесцентной технологии для мониторинга развития опухоли [20,21]. Таким образом, эту экспериментальную модель в основном используют в исследованиях, посвященных биологии опухоли, на ее поздних стадиях развития, а также специфической активности новых антибластомных препаратов. ...
Важную роль в экспериментальных исследованиях противоопухолевых соединений играют модели ксеногенных опухолей in vivo. Несмотря на достигнутые в этой области успехи, вероятность того, что новое лекарственное противоопухолевое средство будет одобрено для использования в онкологической клинике, остается невысокой. Такая низкая воспроизводимость экспериментальных результатов в клинике связана с тем, что существующие стандартные модели злокачественных новообразований in vivo, имеют невысокую прогностическую значимость. Тем не менее эксперименты на лабораторных моделях ксеногенных опухолей играли и продолжают играть ключевую роль в апробации и продвижении потенциальных противоопухолевых препаратов в клинику. В данном обзоре представлен анализ отечественной и зарубежной литературы, посвященной созданию и применению ксеногенных моделей in vivo в области экспериментальной онкологии.
... To better understand lymphatic metastasis of human ESCC, there is a need of developing proper animal models. In the literature, two previous studies reported lymphatic permeation and lymph node metastasis of human ESCC cells (i.e., TE8, KYSE150) when inoculated orthotopically into the esophagus of immunodeficient mice [13,14]. However, the lymphatic system in the mouse esophagus has not been characterized to our best knowledge. ...
... Approximately, 25% of cancers showed enlarged lymph nodes and pan-cytokeratin + cancer cells inside the lymph nodes in the para-tracheo-pharyngeal-esophageal regions [19]. Orthotopic xenograft models using human ESCC cells (i.e., TE8, KYSE150) in immunodeficient mice generated lymphatic permeation and lymph node metastasis in two previous reports [13,14]. In this study, mouse MOC2 cells and LLC-eGFP cells were drained from the submucosa to esophageal lymphatic vessels (lymphatic permeation), peri-esophageal lymph nodes, and distant organs (lung and liver) in immunocompetent mice (Suppl. ...
Background
Lymphatic metastasis is commonly seen in patients with esophageal squamous cell carcinoma (ESCC). Both lymphatic metastasis and the number of involved nodes are prognostic for post-operative survival. To better understand lymphatic metastasis of ESCC, there is a need to develop proper animal models.
Aims
This study is aimed to characterize the morphology and function of the lymphatic drainage system in the mouse esophagus.
Methods
Immunostaining and fluorescence imaging were used to visualize the lymphatic drainage system in the mouse esophagus. Tracers and cancer cells were orthotopically inoculated into the submucosa of the mouse esophagus to mimic lymphatic metastasis of T1 ESCC.
Results
Using immunostaining of a lymphatic vessel marker (LYVE1), we found that lymphatic vessels were located in the submucosa and muscularis propria of the mouse esophagus, similar to the human esophagus. In the esophagus of ProxTom mice expressing tdTomato in the lymphatic vessels, we discovered a microscopic meshwork of lymphatic vessels. Functionally, orthotopically inoculated tracers (Indian ink and FITC-dextran) were drained from the submucosa into peri-esophageal lymph nodes via lymphatic vessels. Orthotopically inoculated mouse cancer cells (LLC-eGFP, MOC2) metastasized from the submucosa to lymphatic vessels, peri-esophageal lymph nodes, and distant organs (liver and lung) in immunocompetent mice. Similarly, in immunodeficient mice, orthotopically inoculated human ESCC cells (KYSE450-eGFP-Luc) metastasized via the same route.
Conclusion
We have characterized the morphology and function of the lymphatic drainage system of the mouse esophagus. These observations lay a foundation for mechanistic and therapeutic studies on lymphatic metastasis of T1 ESCC.
... The mice had free access to food (sterilized special mouse pellet feed) and water, and were regularly subjected to ultraviolet irradiation for disinfection. The KYSE-150 cells [5×10 5 cells in 100 μL of phosphate-buffered saline (PBS)] (27) were injected subcutaneously into the back of each 4-week-old mouse. The mice were observed every 2 days for at least 8 weeks, and cervical dislocation was then performed to euthanize the mice. ...
Background:
miR-488-3p has been reported to play an important role in cancer progression and metastasis. The protein 53 (P53) gene serves as a mediator and biomarker of esophageal squamous cell carcinoma (ESCC). However, the molecular mechanism underlying miR-488-5p in the pathology of ESCC through the P53 pathway has not been examined.
Methods:
The expression levels of miR-488-5p were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cytological experiments were performed to evaluate the biological functions of miR-488-5p. A bioinformatics analysis was performed to determine the pathways and key miR-488-5p targets associated with ESCC. Correlations between miR-488-5p and P53 signaling pathways were validated by western blotting and the dual luciferase reporter gene system. Finally, the expression level of miR-488-5p was regulated and tumor formation experiments were performed in nude mice.
Results:
The qRT-PCR analysis showed that MiR-488-5p expression was more upregulated in the KYSE-150 group than the HEEC group. In the KYSE-150 cells, the colony formation assay and flow cytometry analysis indicated that the miR-488-5p inhibitor inhibited cell viability and increased cell apoptosis; however, these effects were recovered by P53 knockdown (KD). In addition, cell invasion and cell migration were inhibited by the miR-488-5p inhibitor, but were also improved by P53 KD. Similarly, the miR-488-5p inhibitor induced the expression of P53 and P21 than normal control (NC) group in which miR-488-5p expression was normal, while P53 KD prevented the effects of the miR-488-5p inhibitor in KYSE-150 cells. Additionally, we found that tumor size was obviously smaller in miR-488-5p overexpression (OE)+ P53 OE mice than miR-488-5p OE mice. Hematoxylin and eosin and immunohistochemistry staining also revealed similar results.
Conclusions:
Our results suggest that miR-488-5p promotes ESCC progression by suppressing the P53 pathway. These findings should provide novel ideas for ESCC therapies.
... Next, EC109-luc cells suspended in PBS with an equal volume of Matrigel (Corning, Tewksbury, MA, USA) at a concentration of 2 × 10 6 cells/25 μL were injected into the anterior wall of the abdominal esophagus using a 29-gauge insulin syringe. The small bulge at the injection site indicates a successful injection[31]. Finally, the abdomen was closed using absorbable sutures (Jinhuan Medical Products Co., LTD, Shanghai, China). ...
... The LN metastasis model was established by injecting EC109-luc cells at a concentration of 0.8 × 10 6 cells/25 μL into the hock region of mice. Growth of orthotopic esophageal cancer and sentinel LN metastasis were monitored by bioluminescence imaging using an IVIS apparatus after intraperitoneal infection of D-luciferin (PerkinElmer, MA, USA) at a dose of 150 mg/kg body weight[31]. ...
The postoperative survival of esophageal squamous cell carcinoma (eSCC) is notably hindered by cancer recurrence due to difficulty in identifying occult metastases. Cellular mesenchymal-epithelial transition factor (c-Met), which is highly expressed in different cancers, including eSCC, has become a target for the development of imaging probes and therapeutic antibodies. In this study, we synthesized an optical probe (SHRmAb-IR800) containing a near-infrared fluorescence (NIRF) dye and c-Met antibody, which may help in NIRF-guided resection of micrometastases derived from eSCC. Cellular uptake of SHRmAb-IR800 was assessed by flow cytometry and confocal microscopy. In vivo accumulation of SHRmAb-IR800 and the potential application of NIRF-guided surgery were evaluated in eSCC xenograft tumor models. c-Met expression in human eSCC samples and lymph node metastases (LNMs) was analyzed via immunohistochemistry (IHC). Cellular accumulation of SHRmAb-IR800 was higher in c-Met-positive EC109 eSCC cells than in c-Met-negative A2780 cells. Infusion of SHRmAb-IR800 produced higher fluorescence intensity and a higher tumor-to-background ratio (TBR) than the control probe in EC109 subcutaneous tumors (P < 0.05). The TBRs of orthotopic EC109 tumors and LNMs were 3.01 ± 0.17 and 2.77 ± 0.56, respectively. The sensitivity and specificity of NIRF-guided resection of metastases derived from orthotopic cancers were 92.00% and 89.74%, respectively. IHC results demonstrated positive staining in 97.64% (124/127) of eSCC samples and 91.67% (55/60) of LNMs. Notably, increased c-Met expression was observed in LNMs compared to normal lymph nodes (P < 0.0001). Taken together, the results of this study indicated that SHRmAb-IR800 facilitated the resection of micrometastases of eSCC in the xenograft tumor model. This c-Met-targeted probe possesses translational potential in NIRF-guided surgery due to the high positive rate of c-Met protein in human eSCCs.
... Tumor size was measured twice a week, and the volume of the tumor was calculated with the formula: V = 1/2× length × width 2 . For orthotopic injection, 2 × 10 5 cells expressing Luciferase were injected into the muscularis externa of the esophagus of 6-week-old NOD/SCID mice, as described in [12]. Three days after implantation, the mice were divided into two groups (NS or 50 mg/kg CGA) according to in vivo bioluminescence imaging using an IVIS Spectrum Imaging System (Perki-nElmer, MA, USA). ...
Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China, accompanied by an extremely high mortality rate. Chlorogenic acid (CGA) is a small-molecule compound, that has been shown to have a wide range of biological activities, including antitumor. However, the efficacy and molecular mechanism of CGA on ESCC remains unknown. In this study, we confirmed the inhibition of proliferation by CGA in ESCC cells, as well as the reduction of ESCC xenograft volume by CGA in vivo. In addition, CGA also suppressed both the migration and invasion of ESCC cells in vitro. In a carcinogen-induced murine model of ESCC, hyperplasia of the esophagus was slowed by CGA, while mice suffering from ESCC that were treated with CGA had longer survival times than mice in the control group. The measurement of pluripotency factors (BMI1, SOX2, OCT4 and Nanog) that are related to poor prognosis revealed reduced expression of both BMI1 and SOX2, but not of OCT4 or Nanog, in ESCC cells, in both a dose- and time-dependent manner. Together, our initial findings demonstrate that CGA suppresses ESCC progression, downregulates the expression of BMI1 and SOX2, and provide an anti-tumor candidate for ESCC therapy.
... Two main approaches are employed to implant ESCC cells/xenografts in the animal esophagus, for which they differ at the site of tumor implantation. One method implants ESCC cells/xenografts in the upper region of the esophagus [17,18], while the other implants ESCC cells/xenografts in the lower end of the esophagus at the abdominal region near the gastroesophageal junction [19][20][21][22]. Recently, we have also reported the survival comparison between animals with orthotopic tumor developed at the upper esophageal region and the lower region, and found that those with tumor at the abdominal region have better survival [23]. ...
... However, it also suffers Subcutaneous YQ23 alone or combined with cisplatin or 5-fluorouracil [16] Subcutaneous Ginsenoside Rg3 alone or combined with paclitaxel and cisplatin [15] Subcutaneous Afatinib [39] Orthotopic Temsirolimus [14] Patient-derived Lapatinib alone and in combination with 5-fluorouracil or oxaliplatin [25] Patient-derived Cisplatin and 5-fluorouracil [26] Patient-derived Trastuzumab [27] from the same limitation as the subcutaneous model by using ESCC cell lines as the source materials for tumor establishment. Another major disadvantage of this model is the need to use specialized imaging methods, e.g. the in vivo imaging system coupled with the use of bioluminescent technology [19,20,22], to monitor tumor growth. Despite these limitations, the strengths of this model make it a valuable animal model for use in mid to late stage research. ...
... When this model was used to reveal the tumor suppressing effect of a transmembrane protease DESC1, slower tumor growth rate was observed with the use of DESC1-expressing KYSE-150 ESCC cells when compared to those using control cells [22]. In an independent study using this model to examine the tumor phenotype of a tumor-related protein kinase AKT, an obvious tumor-suppressing effect was found associated with the knockdown of AKT, such that a reduced growth rate of orthotopic tumors was readily detected [19]. Aside its use to study tumor growth, this model can be applied to investigate the mechanism of tumor invasion. ...
Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer worldwide and highly prevalent in less developed regions. Management of ESCC is challenging and involves multimodal treatments. Patient prognosis is generally poor especially for those diagnosed in advanced disease stage. One factor contributing to this clinical dismal is the incomplete understanding of disease mechanism, for which this situation is further compounded by the presence of other limiting factors for disease diagnosis, patient prognosis and treatments. Tumor xenograft animal models including subcutaneous tumor xenograft model, orthotopic tumor xenograft model and patient-derived tumor xenograft model are vital tools for ESCC research. Establishment of tumor xenograft models involves the implantation of human ESCC cells/xenografts/tissues into immunodeficient animals, in which mice are most commonly used. Different tumor xenograft models have their own advantages and limitations, and these features serve as key factors to determine the use of these models at different stages of research. Apart from their routine use on basic research to understand disease mechanism of ESCC, tumor xenograft models are actively employed for undertaking preclinical drug screening project and biomedical imaging research.
... The assay was performed as previously described [16]. Briefly, 8×10 5 cells of luciferase-labeled KYSE150luc cells were injected into the esophagus of BALB/cAnN-nu (nude) mice. ...
... Compared to conventional subcutaneous nude mouse tumorigenicity model, the orthotopic ESCC model recapitulates more closely the microenvironment of the tumor in its organ of origin and is more reflective of the actual response observed in the esophageal tumor. Thus, the ESCC orthotopic model was used as it is a better choice for drug response study due to the unique tumor microenvironment [16,36]. Consistent with the in vitro study, the in vivo study also shows that the treatment of carboplatin plus paclitaxel and 5-FU plus cisplatin caused an induction of PD-L1 level in the ESCC orthotopic tumors, as demonstrated by the Western blotting and IHC staining results. ...
The current study reveals the clinicopathological association of PD-L1 in Hong Kong esophageal squamous cell carcinoma (ESCC) patients and the differential regulation of PD-L1 by standard first-line chemotherapy in ESCC. Immunohistochemical analysis of tissue microarray data from 84 Hong Kong ESCC patients shows that PD-L1 was expressed in 21% of the tumors. Positive PD-L1 staining was significantly associated with later disease stage (stages III and IV) (P value = .0379) and lymph node metastasis (P value = .0466) in the Hong Kong cohort. Furthermore, PD-L1 expression was significantly induced in ESCC cell lines after standard chemotherapy treatments, along with EGFR and ERK activation in both in vitro studies and the in vivo esophageal orthotopic model. The endogenous expression of PD-L1 was reduced by treatment with an EGFR inhibitor (erlotinib) or by the knockdown of EGFR. Moreover, the upregulation of PD-L1 by chemotherapy was also attenuated by the treatment with erlotinib and a MAPK/MEK inhibitor (AZD6244), suggesting that PD-L1 is regulated by the EGFR/ERK pathway in ESCC. The regulation of PD-L1 by the EGFR pathway was further supported by the correlation of PD-L1 and EGFR expression observed in the commercially available tissue microarray set (P value = .028). Taken together, the current study was the first to demonstrate the upregulation of PD-L1 by chemotherapy in ESCC and its regulation through the EGFR/ERK pathway. The results suggest the potential usefulness of combined conventional chemotherapy together with anti-PD-L1 immunotherapy to achieve better treatment outcome.
... Alternatively, orthotopic tumor xenograft model can be established by developing tumor xenograft at the esophagus, thus allowing the study of tumor-stromal interaction. As this interaction is important for tumorigenesis and responses to treatments [1][2][3], this latter model is more xenograft in the animal esophagus either at the cervical or at the abdominal part [4][5][6][7][8][9]. ...
... All rights reserved. [6][7][8][9]. Although both methods have been widely used, the method on cervical esophagus seems to be more clinically relevant since most ESCC originates at this site in the patients. ...
Purpose:
Tumor xenograft model is an indispensable animal cancer model. In esophageal squamous cell carcinoma (ESCC) research, orthotopic tumor xenograft model establishes tumor xenograft in the animal esophagus, which allows the study of tumorigenesis in its native microenvironment.
Materials and methods:
In this study, we described two simple and reproducible methods to develop tumor xenograft at the cervical or the abdominal esophagus in nude mice by direct injection of ESCC cells in the esophageal wall.
Results:
In comparing these two methods, the cervical one presented with more clinically relevant features, i.e., esophageal stricture, body weight loss and poor survival. In addition, the derived tumor xenografts accompanied a rapid growth rate and a high tendency to invade into the surrounding structures. This model was subsequently used to study the anti-tumor effect of curcumin, which is known for its potential therapeutic effects in various diseases including cancers, and its analogue SSC-5. SSC-5 was selected among the eight newly synthesized curcumin analogues based on its superior anti-tumor effect demonstrated in an MTT cell proliferation assay and its effects on apoptosis induction and cell cycle arrest in cultured ESCC cells. Treatment of orthotopic tumor-bearing mice with SSC-5 resulted in an inhibition in tumor growth and invasion.
Conclusion:
Taken together, we have established a clinically relevant orthotopic tumor xenograft model that can serve as a preclinical tool for screening new anti-tumor compounds, e.g. SSC-5, in ESCC.
... Chitosan (CHT), as opposed to other naturally occurring polymers and CHT-based nanoparticles, has recently attracted much consideration in both pharmaceutical and biomedical applications due to its exceptional biological properties including biocompatibility, biodegradability, and nontoxicity [12] but with low transfection efficiency. The high buffering potential and transfection efficiency of polyethylenimine (PEI) have been explored for the delivery of DNA and other anticancer therapeutics in the management of cancer diseases [13][14][15]. Meanwhile, covalent attachment of hydrophilic polyethylene glycol (PEG) onto the surfaces of nanoparticles prolongs the circulation halflife in vivo of encapsulated chemotherapeutics, shields the surface of nanoparticles from uptake by the RES, and reduces carrier's cytotoxicity with improved colloidal stability [16]. ...
The aim of this study is to effectively enhance antitumor activities of endostatin by preparing polymeric nanocarriers. NMR and FT-IR spectra confirmed the successful grafting of the CHT-g-PEI and CHT-g-PEI-PEG-NH 2 conjugates. SEM micrographs confirmed the shape of endostatin-loaded nanoparticles to be spherical while both TEM and zeta size results showed nanoparticle’s average size to be 100.6 nm having a positively charged surface with zeta potential of 7.95 mV. The concentrations of CHT and TPP as well as the changing pH conditions account for the increased swelling pattern of endostatin-loaded nanoparticles and influenced endostatin release in vitro. PEI increased the overall amine protonation while PEG aggravated endostatin encapsulation and release. Nanoparticles swell and release endostatin at acidic tumor pH of 6.8 compared to physiological pH of 7.4. The native CHT-g-PEI-PEG-NH 2 conjugate showed high cytocompatibility above 80% cell viability across tested formulations. Endostatin-loaded nanoparticles showed a significant reduction in cell viability across tested formulations, with 5.32% cell death at 125 μ g/mL and 13.36% at 250 μ g/mL following 24 hours’ incubation period. Interestingly, more than a fourfold (61.68%) increment in cytotoxicity was observed at nanoparticle concentration of 1000 μ g/mL . It was concluded that CHT-g-PEI-PEG-NH 2 is an effective cargo for endostatin delivery with antiangiogenic effect in squamous cell carcinoma.
... The surgical procedures to induce orthotopic esophageal tumors are technically challenging due to the location and size of the esophagus in laboratory animals (mostly mice). Five surgical approaches to the esophagus have been described: (i) median laparotomy (7)(8)(9)(10)(11)(12), (ii) median laparotomy combined with transgastric approach (13), (iii) subcostal laparotomy (14), (iv) transoral (15) and (v) cervical approach (16). Tumor take varies between 0 and 100% (mean, 80.06%), and seems to depend more on the aggressiveness of the tumor cell line, than on the surgical technique. ...
The present study aimed to investigate the orthotopic growth potential of two generally available esophageal adenocarcinoma cell lines, OE33 and OACM5 1.C, and a third in vivo selected subpopulation, OACM5 1.C SC1. One group of mice was subcutaneously injected in the hind legs. Tumor growth was measured with calipers. Another group was injected orthotopically in the distal esophageal wall through median laparotomy. Tumor development was evaluated macroscopically and confirmed microscopically. A subset of mice was evaluated with magnetic resonance imaging (MRI) to follow tumor progression. Additionally, functional cell line characteristics were evaluated in vitro (clonogenic, collagen invasion and sphere formation assays, and protein analysis of cell-cell adhesion and cytoskeletal proteins) to better understand xenograft behavior. OE33 cells were shown to be epithelial‑like, whereas OACM5 1.C and OACM5 1.C SC1 were more mesenchymal-like. The three cell lines were non‑invasive into native type I collagen gels. In vivo, OE33 cells led to 63.6 and 100% tumor nodules after orthotopic (n=12) and subcutaneous (n=8) injection, respectively. Adversely, OACM5 1.C cells did not lead to tumor formation after orthotopic injection (n=6) and only 50% of subcutaneous injections led to tumor nodules (n=8). However, the newly established cell line OACM5 1.C SC1 resulted in 33% tumor formation when orthotopically injected (n=6) and in 100% tumors when injected subcutaneously (n=8). The higher xenograft rate of OACM5 1.C SC1 (P<0.05) corresponded with a higher clonogenic potential compared to its parental cell line (P<0.0001). All models showed local tumor growth without metastasis formation. In conclusion, OACM5 1.C has a poor tumor take rate at an orthotopic and ectopic site. A subpopulation obtained through in vivo selection, OACM5 1.C SC1, gives a significant higher take rate, ectopically. Furthermore, OE33 establishes orthotopic (and subcutaneous) xenografts in mice. These models can be of interest for future studies, and their slow growth rates are a challenge for therapeutic intervention.
