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Abstract
It is routine to genetically modify cells to express fluorescent or bioluminescent reporter proteins to enable tracking or quantification of cells in vitro and in vivo. Herein, we characterized the stability of luciferase reporter systems in C4-2B prostate cancer cells in mono-culture and in co-culture with bone marrow-derived mesenchymal stem/stromal cells (BMSC). An assumption made when employing the luciferase reporter is that the luciferase expressing cell number and bioluminescence signal are linearly proportional. We observed instances where luciferase expression was significantly upregulated in C4-2B cell populations when co-cultured with BMSC, resulting in a significant disconnect between bioluminescence signal and cell number. We subsequently characterized luciferase reporter stability in a second C4-2B reporter cell line, and six other cancer cell lines. All but the single C4-2B reporter cell population had stable luciferase reporter expression in mono-culture and BMSC co-culture. Whole-genome sequencing revealed that relative number of luciferase gene insertions per genome in the unstable C4-2B reporter cell population was lesser than stable C4-2B, PC3 and MD-MBA-231 luciferase reporter cell lines. We reasoned that the low luciferase gene copy number and genome insertion locations likely contributed to the reporter gene expression being exquisitely sensitive BMSC paracrine signals. In this study, we show that it is possible to generate a range of stable and reliable luciferase reporter prostate- and breast- cancer cell populations but advise not to assume stability across different culture conditions. Reporter stability should be validated, on a case-by-case basis, for each cell line and culture condition.
Highlights
Quantifying the number of a specific type in complex co-cultures, or in animals, requires that cells of interest are distinguishable from neighboring cells
We observed instances where the luciferase bioluminescence signal generated by C4-2B cells, an LNCaP-derived cell line isolated from metastatic prostate cancer cells from the lumbar spine of castrated mice[12,13], was significantly greater when these cells were co-cultured with bone marrow-derived mesenchymal stromal cells (BMSC), compared to mono-culture
To validate that the CMV promoter was driving luciferase gene expression in the C4-2B-CMV1, we used PCR to demonstrate that the CMV and luciferase genes were in the genome (Supplementary Fig. 2a) and adjacent to each other (Supplementary Fig. 2b)
Summary
Quantifying the number of a specific type in complex co-cultures, or in animals, requires that cells of interest are distinguishable from neighboring cells. The underlying assumption when using bioluminescence to estimate the number of cells in a co-culture, or in an animal, is that there is a linear relationship between the bioluminescence signal and the number of viable cells expressing the reporter protein (typically luciferase) This requires that expression and production of the luciferase enzyme is stable in cell populations over time, and across different culture conditions. We observed instances where the luciferase bioluminescence signal generated by C4-2B cells, an LNCaP-derived cell line isolated from metastatic prostate cancer cells from the lumbar spine of castrated mice[12,13], was significantly greater when these cells were co-cultured with BMSC, compared to mono-culture These observations suggested that a luciferase reporter may not be a viable method to estimate relative cancer cell number under co-culture conditions. We performed whole-genome sequencing on two additional stable reporter cell lines in an effort to determine if reporter stability could be predicted from sequence data
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