Glycerophosphocholine (GPC) is an important component of the elevated total choline signal in cancer, which is detected at 3.2 ppm by ¹H magnetic resonance spectroscopy (MRS) in vivo. A relative decrease of GPC with respect to phosphocholine (PC) was observed with malignant progression of breast cancer ¹. The two genes glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5 ²) and 6 (GDPD6 ³) are overexpressed in breast cancer cells as compared to normal epithelium, and cleave GPC to glycerol-3-phosphate and free choline. In this study, we investigated the functional roles of GDPD5 and GDPD6 in breast cancer cells in a series of gene silencing experiments.Lentiviral vectors based on pRRL-green fluorescent protein (GFP) containing shRNA against GDPD5 (shGDPD5) or shRNA against GDPD6 (shGDPD6) were constructed to constitutively knock down these two genes. Lentiviral particles of empty vector, vector with shGDPD5 or shGDPD6 were produced and utilized to transduce the breast cancer cell lines MCF-7 and MDA-MB-231, and the immortal nonmalignant breast epithelial cell line MCF-12A. Cell numbers were quantified by manual counting of viable cells using trypan blue exclusion. Photomicrographs were taken using a Nikon TS100 inverted fluorescence microscope. RNA was isolated using the Qiagen RNeasy kit, and reverse transcription to cDNA was performed for viable MCF-12A knockdown cell lines. SYBR Green quantitative PCR (qPCR) was used to quantify the knockdown efficiency of these vectors. High-resolution (HR) ¹H MR spectra were obtained from dual-phase extracts of cells with GDPD5 or GDPD6 siRNA knockdown as well as non-target controls. Triple-negative MDA-MB-231 breast cancer cells did not survive constitutive silencing of either GDPD5 or GDPD6, while cells transduced with empty-vector containing lentiviral particles proliferated well (Fig. 1A-B). Comparable results were obtained in estrogen-receptor positive MCF-7 cells (data not shown). HR ¹H MRS demonstrated that the GPC signal significantly increased following transient silencing with siRNA against GDPD6, while silencing of GDPD5 resulted in a marginal increase of GPC compared to non-target controls (Fig. 1C). However, nonmalignant MCF-12A cells survived constitutive GDPD5 and GDPD6 knockdown, as evident by GFP expression in transduced cells as well as qRT-PCR demonstrating successful GDPD5 or GDPD6 knockdown (Fig. 2).Previous work showed that GDPD5 is associated with malignant progression of breast cancer ² and GDPD6 ³ with cancer cell migration. Our study showed for the first time that the expression of these two genes is essential for breast cancer cell survival, but not for the survival of the nonmalignant breast epithelial cell line MCF-12A, which has a low expression level of GDPD5 and a high cellular concentration of GPC. This observation suggests that both GDPD5 and GDPD6 are potential molecular targets for cancer therapy. GDPD5 and GDPD6 are interesting theranostic targets whose knockdown or inhibition can kill cancer cells, which can be monitored by non-invasive MRS.