Introduction

Cyclooxygenase-2 (COX-2) is an inducible enzyme that catalyzes synthesis of proinflammatory mediators such as prostaglandins, thromboxanes, and cytokines. COX-2 overexpression is frequently observed in human cancers and is associated with poor prognosis and progression [1]. We generated genetically modified human triple negative breast cancer (TNBC) cells to overexpress COX-2 to better investigate the role of COX-2 in promoting cancer aggressiveness [2]. Previously, we found that modulation of COX-2 expression alters the choline and lipid metabolism in breast cancer cells [3]. We used 1H magnetic resonance spectroscopy (MRS) to investigate the effects of tumor COX-2 overexpression on spleen metabolism. This will provide a broader understanding of how COX-2 expressing cancers can impact the metabolic profile of vital organs that form the tumor macroenvironment.

Methods

A vector expressing the COX-2 gene was cloned and constructed to establish triple negative SUM-149 cells stably overexpressing COX-2 (SUM-149-COX-2) or cells with an empty vector (SUM-149-EV) as previously reported [2]. These cells were inoculated in the mammary fat pad of SCID mice. Mouse organs were harvested once tumor volumes were approximately 500 mm3. Spleen samples were cryopulverized in liquid nitrogen followed by dual-phase extraction using methanol, chloroform and water. Water-soluble metabolites were identified through 1H MRS performed on a 750 MHz spectrometer. Data were acquired on a Bruker Avance III 750 MHz (17.6T) MR spectrometer equipped with a 5 mm broad band inverse (BBI) probe. Data processing, analysis and quantification were performed with Topspin 3.5 software.

Results

Representative spleen 1H MR spectra obtained from a SUM-149-COX-2 tumor-bearing mouse and a SUM-149-EV tumor-bearing mouse are shown in Figure 1 for (A) the glutamate region and (B) for the lactate region. Data summarized from the spleens of four mice in the SUM-149-EV group and five mice in the SUM-149-COX-2 group are shown in Figure 2 for (A) glutamate, (B) glutamine, (C) glutamine/glutamate, (D) lactate, (E) glucose and (F) glucose/lactate. These data demonstrate the significant effects of tumor COX-2 overexpression on glutamine/glutamate and glucose metabolism in the spleen.

Discussion

Our data identified a significant increase of glutamate and lactate and a significant decrease of the glutamine/glutamate and glucose/lactate ratios in the spleens of mice with COX-2 overexpressing SUM-149 tumors compared to SUM-EV tumors. These metabolic changes must have been affected by the tumor secretome and highlight the impact cancers may have on critical organs. The spleen is a secondary lymphoid organ important for immunometabolism and T cell activation. Transport of glutamine and its conversion are required for metabolic homeostasis during sepsis and inflammation [4]. The metabolic changes observed here suggest that the COX-2 tumor secretome may be affecting spleen metabolism and may systemically contribute to the poor prognosis associated with these cancers. These insights can, in part, be applied in metabolic strategies to improve quality of life and treatment outcome.

Acknowledgements

Supported by NIH R01CA193365 and R35CA209960.

References

1. Hoellen F et al., Anticancer research. 2011;31(12):4359-67. 2. Krishnamachary B et al., Oncotarget. 2017;8(11):17981-94. 3. Shah T et al., NMR in biomedicine. 2012;25(5):746-54. 21953546. 4. Nakaya M et al., Immunity vol. 40,5 (2014): 692-705.