Figure 3 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Establishment of a TE8-Luc orthotopic esophageal cancer model and a non-invasive semi-quantitative monitoring method using the IVIS imaging system. A) IVIS images were obtained once a week from 3 to 7 weeks after TE8-Luc orthotopic tumor inoculation into the abdominal esophagus. B) Luminescent intensity of photons emitted from each tumor and its surroundings in the images in (A) was quantified. Mouse numbering corresponds to the numbering in (A).
Source publication
Orthotopic models of various types of tumors are widely used in anti-tumor therapeutic experiments in preclinical studies. However, there are few ways to appropriately monitor therapeutic effect in orthotopic tumor models, especially for tumors invisible from the outside. In this study we aimed to establish a non-invasive semi-quantitative biolumin...
Similar publications
Background:
Currently ketogenic diets (KDs) are hyped as an anti-tumor intervention aimed at exploiting the metabolic abnormalities of cancer cells. However, while data in humans is sparse, translation of murine tumor models to the clinic is further hampered by small sample sizes, heterogeneous settings and mixed results concerning tumor growth re...
Citations
... 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.
... To monitor the tumor growth using a noninvasive, semiquantitative bioluminescent imaging method, 41 we established TE8 cells that stably express luciferase (TE8-luc). When the TE8-luc cells were subcutaneously implanted into athymic mice, a significant and strong correlation was observed between tumor volume and the luciferase emission level (r = 0.945, p < 0.001, data not shown). ...
... 39,40,45 In addition, our orthotopic tumor model enables accurate monitoring of tumor growth, without invasive procedures, through bioluminescence imaging. 41,46 Using the orthotopic tumor model, we find that an intratumoral inoculation with G47D is highly efficacious. Unlike in the mouse model, a direct intratumoral injection into esophageal cancer in patients is easily doable via endoscope, so it could become a principal means of treating EC in clinical settings. ...
... Orthotopic implantation of TE8-luc cells (2 Â 10 6 cells) was performed in 6-week-old female athymic mice as previously reported. 41 Briefly, a slanting 10-mm skin incision was made in the left upper abdomen under anesthesia. The stomach was pulled down with tweezers so that the abdominal esophagus could be seen. ...
Treatment options are limited for esophageal carcinoma (EC). G47Δ, a triple-mutated, conditionally replicating herpes simplex virus type 1 (HSV-1), exhibits enhanced killing of tumor cells with high safety features. Here, we studied the efficacy of G47Δ using preclinical models of human EC. In vitro, G47Δ showed efficient cytopathic effects and replication capabilities in all eight human esophageal cancer cell lines tested. In athymic mice harboring subcutaneous tumors of human EC (KYSE180, TE8, and OE19), two intratumoral injections with G47Δ significantly inhibited the tumor growth. To mimic the clinical treatment situations, we established an orthotopic EC model using luciferase-expressing TE8 cells (TE8-luc). An intratumoral injection with G47Δ markedly inhibited the growth of orthotopic TE8-luc tumors in athymic mice. Furthermore, we evaluated the safety of applying G47Δ to the esophagus in mice. A/J mice inoculated intraesophageally or administered orally with G47Δ (10⁷ plaque-forming units [pfu]) survived for more than 2 months without remarkable symptoms, whereas the majority with wild-type HSV-1 (10⁶ pfu) deteriorated within 10 days. PCR analyses showed that the G47Δ DNA was confined to the esophagus after intraesophageal inoculation and was not detected in major organs after oral administration. Our results provide a rationale for the clinical use of G47Δ for treating EC.
... Subsequently, all tumor changes were compared with the reference point over the course of treatment and were recorded every 10 days. Notably, the measured luminescence intensities during this period reflected the changes in tumor size [12]. Ten minutes before imaging, the experimental animals received an IP injection of 150 mg/kg D-luciferin potassium salt (Sigma Life Science, St. Louis, MO, USA). ...
Prostate cancer is one of the most common cancers in men. The heterogeneity and mutations exhibited by prostate cancer cells often results in the progression to incurable metastatic castration-resistant prostate cancer (mCRPC). Our previous investigations demonstrated that the virus-like particles (VLPs) of JC polyomavirus (JCPyV) can deliver exogenous genes to prostate cancer cells for expression. JCPyV VLPs packaging pPSAtk (PSAtk-VLPs) possess the ability to transcriptionally target and selectively induce cytotoxicity in prostate cancer cells in vitro and in vivo, as pPSAtk can only express the thymidine kinase gene, a suicide gene, in androgen receptor-positive cells. To further investigate whether PSAtk-VLPs inhibit the growth of metastasized prostate cancer cells, we established an animal model of bone-metastatic prostate cancer to compare PSAtk-VLPs with leuprorelin acetate and enzalutamide, hormonal agents commonly used in clinical settings, and investigated the effectiveness of PSAtk-VLPs. In the present study, we observed that PSAtk-VLPs effectively inhibited the growth of prostate cancer cells that had metastasized to the bone in the metastatic animal model. In addition, PSAtk-VLPs showed a higher effectiveness than hormone therapy in this animal model study. These results suggest that PSAtk-VLPs may serve as a treatment option for mCRPC therapy in the future.
... The surveillance of tumor progression in all humanderived orthotopic bladder xenografts still highly depends on either end-point pathological and immunohistochemical analyses, or in vivo imaging technologies such as PET-CT (positron emission tomography-computed tomography), 8,9 MRI (magnetic resonance imaging), 10 ultrasound imaging, 11 and bioluminescence imaging. 12,13 However, end-point histological analyses cannot provide real-time information regarding tumor progression. The real-time in vivo imaging approaches, while being able to estimate the tumor size during growth, require expensive equipment, highly skilled personnel, time-consuming steps, imaging agents, hair removal, and/or anesthetics, and are limited by the device capacity to process a large number of mice. ...
The human-derived orthotopic xenograft mouse model is an effective platform for performing in vivo bladder cancer studies to examine tumor development, metastasis, and therapeutic effects of drugs. To date, the surveillance of tumor progression in real time for orthotopic bladder xenografts is highly dependent on semi-quantitative in vivo imaging technologies such as bioluminescence. While these imaging technologies can estimate tumor progression, they are burdened with requirements such as anesthetics, specialized equipment, and genetic modification of the injected cell line. Thus, a convenient and non-invasive technology to quantitatively monitor the growth of bladder cancer in orthotopic xenografts is highly desired. In this work, using a microfluidic chemiluminescent ELISA platform, we have successfully developed a rapid, multiparameter urine-based and non-invasive biomolecular prognostic technology for orthotopic bladder cancer xenografts. This method consists of two steps. First, the concentrations of a panel of four urinary biomarkers are quantified from the urine of mice bearing orthotopic bladder xenografts. Second, machine learning and principal component analysis (PCA) algorithms are applied to analyze the urinary biomarkers, and subsequently, a score is assigned to indicate the tumor growth. With this methodology, we have quantitatively monitored the orthotopic growth of human bladder cancer that was inoculated with low, medium, and high cancer cell numbers. We also employed this method and performed a proof of principle experiment to examine the in vivo therapeutic efficacy of the EGFR inhibitor, dacomitinib.
... Bioluminescence imaging (BLI), as a noninvasive and sensitive optical approach, can evaluate the magnitude and distribution of the inoculated cells in intact small animals, and provides the longitudinal assessment and real time monitoring of disease progression. So far, BLI has been widely used for monitoring tumor [4][5][6] and tracking stem cells in vivo [7,8], and monitoring genes [9,10] and evaluating protein stability and interaction in vitro [9]. ...
Uterine fibroid is one of the most common solid tumors occurring in reproductive age women. Lack of accurate methods for In vivo quantitative assessment of uterine fibroid progression severely impedes the basic research and drug screen of this disease. To solve this problem, the correlation between bioluminescence imaging (BLI) and initial cell number used to form xenograft was investigated in this study. The results showed that both subcutaneous (SC) and intraperitoneal (IP) D-luciferin administration led to fast increase of bioluminescence signal (BLS) intensity and caused large variation of peak signal intensity of xenografts through the analysis of BLI kinetic curves. We found that a distinct linear stage appeared in xenograft BLI curve for each mouse subjected to IP-injection of D-luciferin. Moreover, a high positive correlation was found between linear slope and the initial number of human uterine fibroid smooth muscle cells (fSMCs) used for xenograft formation. Our research indicates that the slope of linear stage in BLI curve is more appropriate for in vivo quantitative assessment of human uterine fibroid xenograft.
... [48][49][50] We have established and reported this esophageal orthotopic xenograft model, in which tumor volume and metastatic activity were evaluated by luciferase emission. 51 In our study, we successfully transfected TE4 cells with luciferase and applied TE4-luc cells to evaluate metastatic activity. Although our model revealed that cocultured cells metastasize to the liver, lymph nodes and peritoneum more than cancer cells alone, all cocultured tumors intriguingly showed locoregional lymph node metastasis and a significant increase in the number of lymph node metastases. ...
Lymph node metastasis is a pathognomonic feature of spreading tumors, and overcoming metastasis is a challenge in attaining more favorable clinical outcomes. Esophageal cancer is an aggressive tumor for which lymph node metastasis is a strong poor prognostic factor, and the tumor microenvironment (TME), and cancer‐associated fibroblasts (CAFs) in particular, has been implicated in esophageal cancer progression. CAFs play a central role in the TME and have been reported to provide suitable conditions for the progression of esophageal cancer, similar to their role in other malignancies. However, little is known concerning the relevance of CAFs to the lymph node metastasis of esophageal cancer. Here, we used clinical samples of esophageal cancer to reveal that CAFs promote lymph node metastasis and subsequently verified the intercellular relationships in vitro and in vivo using an orthotopic metastatic mouse model. In the analysis of clinical samples, FAP⁺ CAFs were strongly associated with lymph node metastasis rather than with other prognostic factors. Furthermore, CAFs affected the ability of esophageal cancer cells to acquire metastatic phenotypes in vitro; this finding was confirmed by data from an in vivo orthotopic metastatic mouse model showing that the number of lymph node metastases increased upon injection of cocultured cancer cells and CAFs. In summary, we verified in vitro and in vivo that the accumulation of CAFs enhances the lymph node metastasis of ESCC. Our data suggest that CAF targeted therapy can reduce lymph node metastasis and improve the prognosis of patients with esophageal cancer in the future.
... 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. ...
... Indeed, Matrigel has been widely used in other studies that develop tumor xenografts at the esophagus or at other body sites, e.g. pancreas[6,30,31]. ...
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.
... 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.
... 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 [90][91][92][93][94][95], ...
... The mice were housed 2~3 animals per cage in pathogen free conditions, under 12 h light/dark cycle, 22 ± 2˚C room temperature, and 40-60% relative humidity with ad libitum food and drinking water. Orthotopic esophageal cancer model was performed according to a previous study [21]. A skin incision was made in the middle of the upper abdomen from the xiphoid process. ...
The β-nitrostyrene family have been implicated for anti-cancer property. However, the pharmacological role of β-nitrostyrene in esophageal cancer remain unclear. Here, a β-nitrostyrene derivative, CYT-Rx20, was synthesized and assessed for its anti-cancer activities and underlying mechanism in esophageal cancer. CYT-Rx20 induced cytotoxicity in esophageal cancer cells by promoting apoptosis through activation of caspase cascade and poly(ADP-ribose) polymerase (PARP) cleavage. Besides, CYT-Rx20 inhibited esophageal cancer cell migration and invasion by regulating the expression of epithelial to mesenchymal transition (EMT) markers. CYT-Rx20 decreased cell viability and migration through suppression of the PI3K/AKT and STAT3 pathways. Of note, the cytotoxicity and anti-migratory effect of CYT-Rx20 were enhanced by co-treatment with SC79 (AKT activator) or colivelin (STAT3 activator), suggesting the dependency of esophageal cancer cells on AKT and STAT3 for survival and migration, an oncogene addiction phenomenon. In xenograft tumor-bearing mice, CYT-Rx20 significantly reduced tumor growth of the implanted esophageal cancer cells accompanied by decreased Ki-67, phospho-AKT, and phospho-STAT3 expression. In orthotopic esophageal cancer mouse model, decreased tumor growth and lung metastasis with reduced Ki-67 and phospho-STAT3 expression were observed in mice treated with CYT-Rx20. Together, our results suggest that CYT-Rx20 is a potential β-nitrostyrene-based anticancer compound against the tumor growth and metastasis of esophageal cancer.
