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Development of patient-derived orthotopic xenografts generating distant metastases. (A) Schematic representation of the injection of patient-derived subcutaneous xenografts of GC (PDX) cells into the subserosa of the stomach of NSG mice. Cells were previously transduced with lentiviruses encoding the luciferase gene (MND-luciferase) and were then amplified subcutaneously before orthotopic xenograft. (B) Representative images of bioluminescence imaging on live animals at 6.5 weeks post-orthotopic xenograft. (C) Photon quantification of GC07 and GC10 tumor growth on live animals (upper panels) or on recovered organs at end points (lower panels). GC07, n = 15 mice; GC10, n = 16 mice. (D) Representative images of the tumor developed in the stomach, and of macro-metastases developed in the lung and the liver for the GC10 case (pointed out by black arrows). * p < 0.05 in one-way ANOVA test.
Source publication
Gastric cancer is the third leading cause of cancer mortality worldwide. Cancer stem cells (CSC) are at the origin of tumor initiation, chemoresistance, and the formation of metastases. However, there is a lack of mouse models enabling the study of the metastatic process in gastric adenocarcinoma (GC). The aims of this study were to develop origina...
Citations
... Gastric cancer cell lines and patient-derived xenograft cell culture MKN45 (RRID:CVCL_0434) and AGS (RRID:CVCL_0139) ATCC cells lines, authenticated using short tandem repeat (STR) profiling within the last three years and mycoplasma-free PCR tested, were cultured in RPMI 1640-Glutamax and DMEM F12-Glutamax media respectively, supplemented with 10% heat-inactivated foetal bovine serum (FBS) (all from Thermo Fisher Scientific, Villebon sur Yvette, France) and 50 µg/ml vancomycin (Invitrogen, Cergy-Pontoise, France) at 37°C in a 5% CO 2 humidified atmosphere. Patient-derived xenograft (PDX) cells GC07 cells [12,42] were cultured in DMEM F12-Glutamax medium, supplemented with 10% FBS and 50 µg/ml vancomycin. ...
Gastric cancer’s (GC) bad prognosis is usually associated with metastatic spread. Invasive cancer stem cells (CSC) are considered to be the seed of GC metastasis and not all CSCs are able to initiate metastasis. Targeting these aggressive metastasis-initiating CSC (MIC) is thus vital. Leukaemia inhibitory factor (LIF) is hereby used to target Hippo pathway oncogenic members, found to be induced in GC and associated with CSC features. LIF-treated GC cell lines, patient-derived xenograft (PDX) cells and/or CSC tumourspheres underwent transcriptomics, laser microdissection-associated proteomics, 2D and 3D invasion assays and in vivo xenograft in mice blood circulation. LIFR expression was analysed on tissue microarrays from GC patients and in silico from public databases. LIF-treated cells, especially CSC, presented decreased epithelial to mesenchymal transition (EMT) phenotype and invasion capacity in vitro, and lower metastasis initiation ability in vivo. These effects involved both the Hippo and Jak/Stat pathways. Finally, GC’s high LIFR expression was associated with better clinical outcomes in patients. LIF treatment could thus represent a targeted anti-CSC strategy to fight against metastatic GC, and LIFR detection in primary tumours could constitute a potential new prognosis marker in this disease.
... We suspect that suboptimal surgical procedures and the use of inappropriate needle sizes or sample volumes could contribute to an increased risk of cell leakage. Previous studies have reported the use of needles with various diameters, including 30 G [19,30], 29 G [18], and 26 G [31], for the orthotopic injection of tumor cells, while some studies did not specify the needle size that was used [12]. In our study, we systematically compared the effects of different injection volumes and needle sizes on cell leakage. ...
Simple Summary
Despite remarkable progress in treating early-stage gastric cancer (GC), the clinical outcomes for patients with advanced disease remain very poor. Tissue invasion and metastasis constitute the major causes of cancer-related deaths, including GC. This highlights the urgent need to develop animal models that can recapitulate these processes to develop novel therapeutic strategies. We developed a highly reproducible and cost-effective procedure to establish orthotopic GC xenografts showing high engraftment and metastatic rates via the direct implantation of tumor cell suspensions. Compared with the routine method to establish orthotopic xenograft models by engrafting intact tumor fragments, our approach significantly shortens the experimental timeline and allows for the flexible adjustment of the number of tumor cells implanted to control the rate of tumor progression. Both dose- and time-dependent progressions of tumor invasion and metastasis were nicely recapitulated in our model. Our work provides valuable tools for studying GC progression and metastasis and developing effective therapies.
Abstract
Although the implantation of intact tumor fragments is a common practice to generate orthotopic xenografts to study tumor invasion and metastasis, the direct implantation of tumor cell suspensions is necessary when prior manipulations of tumor cells are required. However, the establishment of orthotopic xenografts using tumor cell suspensions is not mature, and a comparative study directly comparing their engraftment and metastatic capabilities is lacking. It is unclear whether tumor fragments are superior to cell suspensions for successful engraftment and metastasis. In this study, we employed three GC cell lines with varying metastatic capacities to stably express firefly luciferase for monitoring tumor progression in real time. We successfully minimized the risk of cell leakage during the orthotopic injection of tumor cell suspensions without Corning Matrigel by systematically optimizing the surgical procedure, injection volume, and needle size options. Comparable high engraftment and metastatic rates between these two methods were demonstrated using MKN-45 cells with a strong metastatic ability. Importantly, our approach can adjust the rate of tumor progression flexibly and cuts the experimental timeline from 10–12 weeks (for tumor fragments) to 4–5 weeks. Collectively, we provided a highly reproducible procedure with a shortened experimental timeline and low cost for establishing orthotopic GC xenografts via the direct implantation of tumor cell suspensions.
... The PI3K/AKT/mTOR pathway regulates GC cells' function through various mechanisms, including promoting cell invasion, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, and activating tumor chemotherapy resistance. Pan-PI3K inhibitors such as BKM120, BAY80-6946, and PI3K subunit selective inhibitors such as BYL719 and TAK117 showed sound anti-tumor effects in in vitro experiments of GC [78][79][80][81][82][83]. Still, they did not produce promising results in clinical studies. ...
Gastric cancer (GC) has emerged as a significant issue in public health all worldwide as a result of its high mortality rate and dismal prognosis. AT-rich interactive domain 1 A (ARID1A) is a vital component of the switch/sucrose-non-fermentable (SWI/SNF) chromatin remodeling complex, and ARID1A mutations occur in various tumors, leading to protein loss and decreased expression; it then affects the tumor biological behavior or prognosis. More significantly, ARID1A mutations will likely be biological markers for immune checkpoint blockade (ICB) treatment and selective targeted therapy. To provide theoretical support for future research on the stratification of individuals with gastric cancer with ARID1A as a biomarker to achieve precision therapy, we have focused on the clinical significance, predictive value, underlying mechanisms, and possible treatment strategies for ARID1A mutations in gastric cancer in this review.
... Meanwhile, organ metastasis was observed in the lung and liver with the formation of peritoneal metastasis. 49 It seems that the aggressiveness of primary tumor is important to the success rates of PDX. However, several studies have shown that no significant association between PDX growth and the tumor characteristics. ...
Peritoneal metastasis is a challenging aspect of clinical practice for gastric cancer. Animal models are crucial in understanding molecular mechanisms, assessing drug efficacy, and conducting clinical intervention studies, including those related to gastric cancer peritoneal metastasis. Unlike other xenograft models, peritoneal metastasis models should not only present tumor growth at the transplant site, but also recapitulate tumor cell metastasis in the abdominal cavity. Developing a reliable model of gastric cancer peritoneal metastasis involves several technical aspects, such as the selection of model animals, source of xenograft tumors, technology of transplantation, and dynamic monitoring of the tumor progression. To date, challenges remain in developing a reliable model that can completely recapitulate peritoneal metastasis. Thus, this review aims to summarize the techniques and strategies used to establish animal models of gastric cancer peritoneal metastasis, providing a reference for future model establishment.
... There are a number of xenograft models; however, the most prevailing in CSC research are the patient-derived xenografts (PDX) and the cell line-derived xenograft (CDX) models. In these models, mice are engrafted with either patient or cell line-derived cells, respectively [80][81][82][83][84]. It has been shown that CSCs, even when injected at low numbers, have the capacity to efficiently generate tumors that are heterologous and which exhibit similar histological properties as the tumor of origin. ...
Cancer is a multifactorial, complex disease exhibiting extraordinary phenotypic plasticity and diversity. One of the greatest challenges in cancer treatment is intratumoral heterogeneity, which obstructs the efficient eradication of the tumor. Tumor heterogeneity is often associated with the presence of cancer stem cells (CSCs), a cancer cell sub-population possessing a panel of stem-like properties, such as a self-renewal ability and multipotency potential. CSCs are associated with enhanced chemoresistance due to the enhanced efflux of chemotherapeutic agents and the existence of powerful antioxidant and DNA damage repair mechanisms. The distinctive characteristics of CSCs make them ideal targets for clinical therapeutic approaches, and the identification of efficient and specific CSCs biomarkers is of utmost importance. Aldehyde dehydrogenases (ALDHs) comprise a wide superfamily of metabolic enzymes that, over the last years, have gained increasing attention due to their association with stem-related features in a wide panel of hematopoietic malignancies and solid cancers. Aldehyde dehydrogenase 1B1 (ALDH1B1) is an isoform that has been characterized as a marker of colon cancer progression, while various studies suggest its importance in additional malignancies. Here, we review the basic concepts related to CSCs and discuss the potential role of ALDH1B1 in cancer development and its contribution to the CSC phenotype.
... Tumours were monitored twice a week up to 12 weeks using a calliper. Tumours were collected, fixed in a 3.7% buffered-formaldehyde solution and embedded in paraffin following standard procedures as described [4,23,25]. ...
... GC10 PDX cells and MKN45 cells, previously transduced with a lentivirus encoding luciferase gene [25], were grown subcutaneously in NSG mice and tumours were dissociated and immuno-stained for FACS cell sorting as described in upper sections. 10000 GC10 cells and 2500 CD44v3+ and CD44v3-MKN45 cells were injected into the sub-serosa of the stomach of 6 weeks old male NSG mice [25]. ...
... GC10 PDX cells and MKN45 cells, previously transduced with a lentivirus encoding luciferase gene [25], were grown subcutaneously in NSG mice and tumours were dissociated and immuno-stained for FACS cell sorting as described in upper sections. 10000 GC10 cells and 2500 CD44v3+ and CD44v3-MKN45 cells were injected into the sub-serosa of the stomach of 6 weeks old male NSG mice [25]. Surgery procedures are detailed in Supplemental Methods. ...
Background
Cancer stem cells (CSCs) are at the origin of tumour initiation and progression in gastric adenocarcinoma (GC). However, markers of metastasis-initiating cells remain unidentified in GC. In this study, we characterized CD44 variants expressed in GC and evaluated the tumorigenic and metastatic properties of CD44v3+ cells and their clinical significance in GC patients.
Methods
Using GC cell lines and patient-derived xenografts, we evaluated CD44+ and CD44v3+ GC cells molecular signature and their tumorigenic, chemoresistance, invasive and metastatic properties, and expression in patients-derived tissues.
Results
CD44v3+ cells, which represented a subpopulation of CD44+ cells, were detected in advanced preneoplastic lesions and presented CSCs chemoresistance and tumorigenic properties in vitro and in vivo. Molecular and functional analyses revealed two subpopulations of gastric CSCs: CD44v3+ CSCs with an epithelial-mesenchymal transition (EMT)-like signature, and CD44+/v3– CSCs with an epithelial-like signature; both were tumorigenic but CD44v3+ cells showed higher invasive and metastatic properties in vivo. CD44v3+ cells detected in the primary tumours of GC patients were associated with a worse prognosis.
Conclusion
CD44v3 is a marker of a subpopulation of CSCs with metastatic properties in GC. The identification of metastasis-initiating cells in GC represents a major advance for further development of anti-metastatic therapeutic strategies.
... Gastric cancer is one of the commonest types of gastrointestinal malignancies and contributes to the second-highest cancer mortality rate worldwide [59][60][61]. Despite surgical excision is the primary remedies for early-stage and locally advanced gastric cancer, patients are usually diagnosed at a terminal period that is short of effectual therapies [62][63][64][65]. ...
Long noncoding RNAs (lncRNAs) are defined as a class of non-protein-coding RNAs that are longer than 200 nucleotides. Previous studies have shown that lncRNAs play a vital role in the progression of multiple diseases, which highlights their potential for medical applications. The lncRNA hepatocyte nuclear factor 1 homeobox A (HNF1A) antisense RNA 1 (HNF1A-AS1) is known to be abnormally expressed in multiple cancers. HNF1A-AS1 exerts its oncogenic roles through a variety of molecular mechanisms. Moreover, aberrant HNF1A-AS1 expression is associated with diverse clinical features in cancer patients. Therefore, HNF1A-AS1 is a promising biomarker for tumor diagnosis and prognosis and thus a potential candidate for tumor therapy. This review summarizes current studies on the role and the underlying mechanisms of HNF1A-AS1 various cancer types, including gastric cancer, liver cancer, glioma, lung cancer, colorectal cancer, breast cancer, bladder cancer, osteosarcoma, esophageal adenocarcinoma, hemangioma, oral squamous cell carcinoma, laryngeal squamous cell carcinoma, cervical cancer, as well as gastroenteropancreatic neuroendocrine neoplasms. We also describe the diagnostic, prognostic, and therapeutic value of HNF1A-AS1 for multiple cancer patients.
... Several human GAC cell lines have been used to create orthotopic mouse models of PC [15][16][17][18][19][20]. However, most of these cell lines (e.g., NCI-N87, MKN-45, MKN-28, AGS, Snu-16) or patient-derived cells (e.g., GC04, GC07, GC10, GTX-085) have not formed PC after orthotopic implantation unless manipulated [15][16][17][18][19]. Yanagihara K et al. established several subclones from two PC cell lines by repeated selection (10-cycles) and formed PC only 30% of the time and took several months before PC could be identified [20]. ...
... Several human GAC cell lines have been used to create orthotopic mouse models of PC [15][16][17][18][19][20]. However, most of these cell lines (e.g., NCI-N87, MKN-45, MKN-28, AGS, Snu-16) or patient-derived cells (e.g., GC04, GC07, GC10, GTX-085) have not formed PC after orthotopic implantation unless manipulated [15][16][17][18][19]. Yanagihara K et al. established several subclones from two PC cell lines by repeated selection (10-cycles) and formed PC only 30% of the time and took several months before PC could be identified [20]. ...
... To monitor tumor growth and metastases in vivo, we transduced all three cell lines with lentivirus expressing mCherry-Luciferase (Fig. 1B). We also used mCherry-Luciferase to label the two commercially available cell lines, Snu-1 and MKN45, which have been used for orthotopic models, for comparison [15][16][17]. After injection of cells, the tumor growth was monitored by BLI imaging weekly (Fig. 6B & C). ...
Background
Gastric adenocarcinoma with peritoneal carcinomatosis (PC) is therapy resistant and leads to poor survival. To study PC in depth, there is an urgent need to develop representative PC-derived cell lines and metastatic models to study molecular mechanisms of PC and for preclinical screening of new therapies.
Methods
PC cell lines were developed from patient-derived PC cells. The tumorigenicity and metastatic potential were investigated by subcutaneously (PDXs) and orthotopically. Karyotyping, whole-exome sequencing, RNA-sequencing, and functional studies were performed to molecularly define the cell lines and compare genomic and phenotypic features of PDX and donor PC cells.
Results
We established three PC cell lines (GA0518, GA0804, and GA0825) and characterized them in vitro. The doubling times were 22, 39, and 37 h for GA0518, GA0804, and GA0825, respectively. Expression of cancer stem cell markers (CD44, ALDH1, CD133 and YAP1) and activation of oncogenes varied among the cell lines. All three PC cell lines formed PDXs. Interestingly, all three PC cell lines formed tumors in the patient derived orthotopic (PDO) model and GA0518 cell line consistently produced PC in mice. Moreover, PDXs recapitulated transcriptomic and phenotypic features of the donor PC cells. Finally, these cell lines were suitable for preclinical testing of chemotherapy and target agents in vitro and in vivo .
Conclusion
We successfully established three patient-derived PC cell lines and an improved PDO model with high incidence of PC associated with malignant ascites. Thus, these cell lines and metastatic PDO model represent excellent resources for exploring metastatic mechanisms of PC in depth and for target drug screening and validation by interrogating GAC for translational studies.
... Buparlisib (BKM120, a pan-class I PI3K inhibitor), first line treatment of metastatic head and neck epidermoid carcinomas, was found to decrease GCSC tumorigenic properties in vitro and to decrease GC metastasis in vivo [109]. Verteporfin, an FDA-approved drug for age-related macular degeneration, was repositioned in the GCSC context for its capacity to decrease YAP/TEAD transcriptional activity, cell proliferation, CD44 expression and number of tumorsphere-forming CD44+ALDHhigh GCSCs in vitro. ...
... Likewise, subcutaneous xenograft of PDX cells reflect the heterogeneity of patient tumours but still do not metastasize to distant organs [55]. In a recent study, Giraud et al. developed orthotopic PDX models in which patient-derived GC cells were xenografted directly into the stomach wall of immunodeficient mice and led after 8 weeks to distant metastases [109]. In these pre-clinical models, luciferase-encoding GC cells were traced all through the in vivo experiment allowing the monitoring of primary tumour establishment and kinetic of GC cells spread and metastasis development. ...
... In these pre-clinical models, luciferase-encoding GC cells were traced all through the in vivo experiment allowing the monitoring of primary tumour establishment and kinetic of GC cells spread and metastasis development. Using these models, the authors showed that Buparlisib treatment significantly inhibited GCSC properties in vitro and reduced the number of distant metastases in vivo when the treatment was done in the metastases starting time-lapse determined by the model [109]. This preclinical mouse model of metastatic GC represents a major advance to study anti-metastatic efficiency of new GCSC-based therapies. ...
Gastric cancer's bad incidence, prognosis, cellular and molecular heterogeneity amongst others make this disease a major health issue worldwide. Understanding this affliction is a priority for proper patients' management and for the development of efficient therapeutical strategies. This review gives an overview of major scientific advances, made during the past 5-years, to improve the comprehension of gastric adenocarcinoma. A focus was made on the different actors of gastric carcinogenesis, including, Helicobacter pylori cancer stem cells, tumour microenvironment and microbiota. New and recent potential biomarkers were assessed as well as emerging therapeutical strategies involving cancer stem cells targeting as well as immunotherapy. Finally, recent experimental models to study this highly complex disease were discussed, highlighting the importance of gastric cancer understanding in the hard-fought struggle against cancer relapse, metastasis and bad prognosis.
... 79 The success rate of PDX modeling was also different among different tumor types, and the success rate of colorectal cancer and lung cancer was significantly higher than that of prostate cancer. 80 These shortcomings promote the emergence of some improved PDX models. ...
Animal models refers to the animal experimental objects and related materials that can simulate human body established in medical research. As the second-largest disease in terms of morbidity and mortality after cardiovascular disease, cancer has always been the focus of human attention all over the world, which makes it a research hotspot in the medical field. At the same time, more and more animal models have been constructed and used in cancer research. With the deepening of research, the construction methods of cancer animal models are becoming more and more diverse, including chemical induction, xenotransplantation, gene programming, and so on. In recent years, patient-derived xenotransplantation (PDX) model has become a research hotspot because it can retain the microenvironment of the primary tumor and the basic characteristics of cells. Animal models can be used not only to study the biochemical and physiological processes of the occurrence and development of cancer in objects but also for the screening of cancer drugs and the exploration of gene therapy. In this paper, several main tumor animal models and the application progress of animal models in tumor research are systematically reviewed. Finally, combined with the latest progress and development trend in this field, the future research of tumor animal model was prospected.
