Abstract
BackgroudA key strategy for the study of the tumor microenvironment is to implant human tumor cells in an immunodeficient rodent strain ubiquitously expressing a fluorescent marker. Here, a novel nude rat expressing a green fluorescent protein (GFP) transgene was established and engrafted with primary human tumor tissue in order to separate tumor from stromal cell populations for subsequent molecular analysis.MethodsSD-TG (GFP) 2BalRrrc transgenic rats were crossed with HsdHan™: rnu/rnu Rowett nude rats to develop a GFP expressing immunocompromised rat. PCR and flow cytometry were used to follow the GFP genotype and phenotype in newborns. After three to four generations, animals were implanted with 4 T1 dsRed murine breast cancer cells or primary human glioblastoma (GBM) biopsies to generate xenografts for subsequent separation by fluorescence-activated cell sorting (FACS).ResultsFluorecence microscopy and reverse transcription-PCR demonstrated that GFP, under the control of the human ubiquitin C promoter, was stably maintained and expressed in diverse organs over several generations. Immunophenotyping of blood samples by flow cytometry confirmed that the immunodeficient features of the parental rat strain, HsdHan™: rnu/rnu, were retained in the GFP nude rat. Both the murine cell line and human GBM biopsies engrafted, and stromal cell populations were isolated from dissociated xenografts by FACS to > 95% purity.ConclusionsA GFP transgene was stably introduced into a nude rat background in which human and murine cancer cells successfully engrafted. This animal strain provides a novel in vivo system for detailed cellular and molecular characterization of tumor-stroma interactions.
Highlights
The development of a human tumor is a collaboration between a mutated cell and its microenvironment
Generation of a green fluorescent protein (GFP)-expressing nude rat In order to obtain a nude rat expressing GFP, the SD-TG (GFP) 2BalRrrc transgenic rats were crossbred with HsdHanTM: rnu/rnu Rowett nude rats
Newborn rats were genotyped at each generation for the presence of the GFP transgene by PCR of genomic DNA isolated from rat tails (Figure 1B)
Summary
The development of a human tumor is a collaboration between a mutated cell and its microenvironment. The idea that tumor growth was in part dependent on normal cell types originated over 100 years ago when Stephen Paget proposed the “seed and soil” hypothesis in 1889 [1]. His original studies addressed the pattern of development of metastasis in human patients, modern studies have revealed that a complex crosstalk between stromal compartments and tumor cells exists at any point during neoplastic progression [2,3,4,5]. It is composed of many different cell types, including cancerassociated fibroblasts (CAFs), endothelial cells, and diverse immune cells, as well as extracellular matrix [5]. The ability to separate stromal from tumor cells to high purity is essential in order to study tumor-stroma interactions
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