Potential utility of eGFP-expressing NOG mice (NOG-EGFP) as a high purity cancer sampling system
© Shima et al.; licensee BioMed Central Ltd. 2012
Received: 20 March 2012
Accepted: 20 May 2012
Published: 6 June 2012
It is still technically difficult to collect high purity cancer cells from tumor tissues, which contain noncancerous cells. We hypothesized that xenograft models of NOG mice expressing enhanced green fluorescent protein (eGFP), referred to as NOG-EGFP mice, may be useful for obtaining such high purity cancer cells for detailed molecular and cellular analyses.
Pancreato-biliary cancer cell lines were implanted subcutaneously to compare the tumorigenicity between NOG-EGFP mice and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. To obtain high purity cancer cells, the subcutaneous tumors were harvested from the mice and enzymatically dissociated into single-cell suspensions. Then, the cells were sorted by fluorescence-activated cell sorting (FACS) for separation of the host cells and the cancer cells. Thereafter, the contamination rate of host cells in collected cancer cells was quantified by using FACS analysis. The viability of cancer cells after FACS sorting was evaluated by cell culture and subsequent subcutaneous reimplantation in NOG-EGFP mice.
The tumorigenicity of NOG-EGFP mice was significantly better than that of NOD/SCID mice in all of the analyzed cell lines (p < 0.01). Sorting procedures enabled an almost pure collection of cancer cells with only slight contamination by host cells. Reimplantation of the sorted cancer cells formed tumors again, which demonstrated that cell viability after sorting was well maintained.
This method provides a novel cancer sampling system for molecular and cellular analysis with high accuracy and should contribute to the development of personalized medicine.
Cancer xenograft models of immunodeficient mice are widely applied in various cancer research areas. Recently, xenografted human tumors are commonly used for preclinical drug testing, including biomarker discovery. [1, 2] It has been reported that there is a close correlation between the effects in xenografts and clinical outcomes, in terms of both drug resistance and sensitivity.  An eventual goal of such preclinical studies using mouse xenograft models is the realization of personalized medicine. Molecular analyses using clinical specimens or xenografted tumors are essential in research for personalized medicine, and high purity samples of sufficient volume are necessary for precise analyses. In general, mouse xenografts are superior to clinical specimens because of the abundance and renewability of the tumor samples.
Tumors consist of two components, i.e. cancer cells and stroma. Stromal cells derived from murine cells within the xenografted tumors. Even though tumor tissue acquired from patients is transplanted, human stromal cells are ultimately replaced by murine stromal cells . Accordingly, contamination by stromal cells hinders precise analyses of cancer cells using tumor tissue. Although stromal cells need to be removed from tumor tissue as much as possible to obtain accurate results, it is still technically difficult to collect high purity cancer cells without contamination by stromal cells. As technologies of comprehensive analyses (e.g., high-resolution microarray, next-generation sequencing and proteomics) are progressing rapidly, high purity samples uncontaminated by stromal cells are necessary for such advanced technology. Therefore, it is very important to establish a method of separating cancer cells and stromal cells clearly and collecting cancer cells uncontaminated by stromal cells.
On the other hand, athymic nude mice, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice or NOD.Cg-Prkdc scid Il2rg tm1Sug /ShiJic (NOG) mice are routinely used for mouse xenograft models of cancer. Among these types of mice, NOG mice show the most severe immunodeficient state. Machida and colleagues have reported that NOG mice have higher susceptibility to xenografted tumors than other immunodeficient mice . Thus, NOG mice are very useful for the transplantation of tumor tissue.
In 2008, Niclou and colleagues reported that NOD/SCID mice with ubiquitous expression of enhanced green fluorescent protein (eGFP) were useful for the clear separation of tumor cells and mouse stromal cells in subcutaneous xenografted tumors by fluorescence activated cell sorting (FACS), and demonstrated that the contamination by stromal cells after the removal of eGFP-expressing cells was slight.  Meanwhile, Suemizu et al. generated NOG mice expressing eGFP ubiquitously (NOG-EGFP) and clarified that NOG and NOG-EGFP mice have equivalent immunodeficient states.  However, there are no reports to study cancer xenograft of NOG-EGFP mice.
In this study, we hypothesized that NOG-EGFP mice are potentially useful for the collection of cancer cells without contamination by stromal cells and would also have the advantage of easy engraftment. Here we compare the tumorigenicity between NOG-EGFP and NOD/SCID mice and show the degree of contamination by stromal cells after removal of eGFP-expressing cells in the xenografted tumors of NOG-EGFP mice by FACS. Furthermore, we demonstrate the viability of the collected cancer cells by cell culture and subsequent inoculation.
Materials & methods
All animal experiments conformed to the guidelines of the Institutional Animal Care and Use Committee of Tohoku University and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Tohoku University. The protocol was approved by the Ethics Review Committee of Tohoku University.
6 week-old female NOG-EGFP (formally, NOD.Cg-PrkdcscidIl2rgtm1SugTg (Act-eGFP) C14-Y01-FM1310sb/ShiJic) mice and NOG mice were kindly provided by Central Institute for Experimental Animals (Kawasaki, Japan). NOD/SCID mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Female heterozygous NOG-EGFP mice were mated with male NOG mice in order to breed the NOG-EGFP mice under the permission of Central Institute for Experimental Animals. Since their offspring were NOG mice or NOG-EGFP mice, the fluorescence of NOG-EGFP mice was confirmed by a hand-held UV lamp (COSMO BIO, Tokyo, Japan). Thereafter, NOG-EGFP mice were used in the experiments. The animals were housed under pathogen-free conditions on a 12-hour light cycle and with free access to food and water.
Human pancreatic cancer cell lines (MIA Paca2 and AsPC-1) and human cholangiocarcinoma cell lines (HuCCT1 and TFK-1) were obtained from the Cell Resource Center for Biomedical Research of Tohoku University. HuCCT1, TFK-1 and AsPC-1 were cultured in RPMI-1640 media (Sigma-Aldrich, MO, USA) with 10% heat-inactivated fetal bovine serum (FBS) (SAFC Biosciences, MO, USA) and 1% penicillin/streptomycin (P/S) (Gibco/Life Technologies, CA, USA) at 37°C in an atmosphere of 5% CO2 and 95% air. Dulbecco modified Eagle medium (DMEM) (Gibco/Life Technologies) was used for culture of MIA PaCa2 cells.
We confirmed that organs and cells obtained from NOG-EGFP mice could be fluorescently visualized. In detail, after euthanizing NOG-EGFP mice, internal organs were placed on a tray and imaged using an IVIS® Spectrum system (Caliper Life Sciences, MA, USA). Skin fibroblasts of NOG-eGFP mice were cultured in RPMI-1640 media with 10% FBS and 1% P/S. Subsequently, cultured fibroblasts on dishes were visualized using a Keyence BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan).
Cell transplantation in NOG-EGFP and NOD/SCID mice
Patient-derived cancer xenografts
Resected specimens of pancreatic cancer tissue were cut into 2–3mm3 pieces in antibiotic-containing RPMI-1640 media. Under anesthesia with pentobarbital (Abbott Laboratories, IL, USA), and sevoflurane (Maruishi Pharmaceutical, Osaka, Japan), the pieces of the tumors were implanted subcutaneously into each side of the lower back in 6–8–week-old female NOG-EGFP mice. Tumors were harvested upon reaching a volume of 1,500 mm3 and provided for immunohistochemistry.
Subcutaneous tumors of NOG-EGFP xenografts were fixed in 10% formalin before embedded in paraffin. After blocking, immunohistochemistry for eGFP was performed using a rabbit anti-GFP (ab290, Abcam, MA USA) at a dilution of 1:1000 incubated for 1hour at 25°C. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Nichirei Biosciences, Tokyo, Japan) was used as the secondary antibody. Peroxidase visualization was done using 3,3'-Diaminobenzidine (DAB). All techniques including H&E staining were performed by Animal Pathology Platform, Biomedical Research Core of Tohoku University Graduate School of Medicine.
Cell sorting and phenotyping of murine stromal cells
TFK-1 xenografts were used in this experiment. Freshly isolated subcutaneous tumors of NOG-EGFP mice were dissociated by mincing the tissue with scalpels, followed by incubation in RPMI-1640 media containing collagenase (Worthington Biochemical, NJ, USA) for 30 min at 37°C. After incubation, the cell suspension was filtered through a 100-μm cell strainer. The cells were resuspended in phosphate buffered saline (PBS) and sorted on a fluorescence-activated cell sorter (FACS Aria TM II Cell Sorter, BD Biosciences, Erembodegem, Belgium) on the basis of single-cell viability and the presence of GFP. For immunophenotyping, cells were incubated for 30 min at room temperature with conjugated antibodies against mouse CD31, CD90, CD49b, CD14, CD11c (CD31: 561410, CD90: 553007, CD49b: 553858, CD14: 560636 and CD11c: 560583, BD Biosciences) or conjugated isotype controls (APC-CyTM7 (Rat IgG1, κ)-560534, Alexa-Flour700 (Hamster IgG, λ1): 560555, APC (Rat IgG2a, κ): 53932, PE (Rat IgM, κ): 553943, PE-CyTM7 (Rat IgG2a, κ): 552867, BD Biosciences), as previously reported  . Analyses were performed on a FACS Aria TM II Cell Sorter (BD Biosciences).
Viability of sorted cancer cells
Xenografted tumors of TFK-1 cells in NOG-EGFP mice were harvested and separated into cancer cells and stromal cells by FACS as described above. Collected TFK-1 cells were cultured on dishes and subsequently reimplanted in NOG-EGFP mice. In order to confirm the effect of removal of eGFP-expressing cells, the subcutaneous tumors of TFK-1 cells were provided for primary cell culture without FACS sorting as a control.
Data were presented as the mean ± S.E. Statistical significance was determined by Mann–Whitney U test performing using GraphPad Prism for Windows version 5.02. Differences between experimental groups were considered significant when the p-value was <0.05.
Confirmation of eGFP expression in NOG-EGFP mice
Comparison of tumorigenic potential between NOG-EGFP and NOD/SCID mice
Separation of cancer cells and stromal cells
Cell viability after FACS sorting
The aim of the present study was to develop methods for separating mice-xenografted human cancer cells from host cells by FACS with minimal amount of contamination and also to maintain the cell viability for subsequent analyses. For this purpose, we have developed techniques that employ NOG-EGFP mice.
To date, fluorescent immunodeficient mice, i.e. GFP nude mice , NOD/SCID EGFP mice  and NOG-EGFP mice , have been established. The previous reports showed that fluorescent mice were very useful to study the details of tumor-stroma interaction [10–12]. Recently, Niclou and colleagues reported the almost complete separation of cancer cells and host cells using xenografted tumors of a glioma cell line in NOD/SCID EGFP mice. Based on this report, we evaluated the contamination rate of murine stromal cells among each cell type collected cancer cells. Our results showed similar contamination rates to those of the previous report and suggest that fluorescent mice would be very useful for the separation of cancer cells from host cells. However, the purity of the separation might be different in tumor type and implantation site since content rate of stromal cells varies in them. Further studies including orthotopic models of several organs and use of other tumor types are needed to evaluate the purity of separation. We also demonstrated that that sorted cancer cells were able to grow in vitro and in vivo. One of the advantages is that the tumor cells start to grow significantly earlier in NOG-EGFP mice than in NOD/SCID mice. Our present results provide a novel way of employing of collected cancer cells for to various subsequent analyses. In the report of the NOD/SCID EGFP xenografts, cancer cells labeled with another type of fluorescence were used for the separation study . The present study suggests that fluorescent labeling of cancer cells is not necessary for the separation of cancer cells and host cells.
On the other hand, this method is applicable for the collection of not only cancer cells but also stromal cells. The methodology using fluorescent mouse xenografts might usefully contribute to studies of cancer stromal cells.
In conclusion, NOG-EGFP has high potential utility for complete separation of cancer cells and stromal cells with minimal contamination, if any, from xenografted tumors. Further studies are needed to establish a solid methodology for the separation and collection of stromal/cancer cells, and the use of NOG-EGFP mice for this is very promising.
This work was supported by Japan Society for the Promotion of Science Grant-in-Aids for Young Scientists (B: 23791512) (HH), (B: 23791515) (TO), (B: 23791514) (MM).
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