Cancer associated bone pain (CIBP) is a common event in patients with advanced disease with bone metastases (BM), significantly impairing their quality of life. Treatment options are limited and mainly based on the use of opioids with unacceptable side effects. Local acidosis is a well-known cause of pain since it directly stimulates nociceptors that express acid-sensing ion channels and densely innervate bone. In BM, local acidosis derives from osteoclast bone resorption activity and from the acidification by glycolytic tumor cells. Here we speculated that the pH lowering of intratumoral interstitial fluid also promotes nociceptors sensitization and hyperalgesia through the activation of cells of mesenchymal origin in BM microenvironment that might release inflammatory and nociceptive mediators.

As a model of breast cancer that can metastatise to the bone we used MDA-MB-231 (MDA), and a subclone with a higher tendency to form osteolytic BM (bmMDA). We evaluated the basal expression of proton pumps/ion transporters by Real-Time PCR (Q-RT-PCR). To evaluate the effect of extracellular acidosis on mesenchymal tumor-associated stroma, we used human osteoblast primary cultures from healthy donors and cancer-associated fibroblasts isolated with specific immunobeads from the tumor biopsies of patient with BM. We exposed the cells to pH 6.8 medium at different time points (between 3 to 24 hours). After the short-term incubation with acidosis, for the expression of and acid-sensing ion channels, inflammatory cytokines and nociceptive mediators that can produce hyperalgesia, we used both a wide screening through a deep-sequencing approach and Q-RT-PCR, and ELISA. Xenograft for osteolytic BM induced by intratibial injection of bmMDA were treated with Omeprazole and monitored for CIBP through several cognitive tests.

We found a significantly higher extracellular proton efflux and expression of proton pumps/ion transporters associated with the acid-base balance, the monocarboxylate transporter 4 (MCT4), the carbonic anhydrase (CA9), and the vacuolar ATPase (V-ATPase) V₁G₁ subunit, and V₀c subunitin bmMDA, a subclone that is prone to form BM in respect to the parental cell line MDA-MB-231. In mesenchymal stromal cells, osteoblasts and cancer-associated fibroblasts, the incubation with pH 6.8 induced the expression of the achid-sensing ion channels AISC3/ACCN3 and AICS4/ACCN4, as well as of the nociceptive modulators nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), and of the inflammatory cytokines interleukin 6 (IL6) and 8 (IL8), and Chemokine (C-C motif) ligand 5 (CCL5). Furthermore, the targeting of V0c subunit to inhibit intratumoral acidification significantly reduced CIBP in mice model of BM.

In this study we demonstrated for the first time that, in addition to the direct acid-sensing neuronal stimulation, the acidic microenvironment of BM causes hyperalgesia through the activation of an inflammatory reaction in the tumor-associated mesenchymal stroma at the tumor site, thereby offering as a new target for palliative treatment in advanced cancer.

The glycolytic-based metabolism of cancers promotes an acidic microenvironment that is responsible for increased aggressiveness. However, the effects of acidosis on tumour metabolism have been almost unexplored, and the metabolic adaptation of cancer cells to acidosis has never been compared with the metabolic response of normal cells.

In this study, to pinpoint for the first time the different metabolic profiles between osteosarcoma (OS) cells and normal human fibroblasts (Fb) under short-term acidosis, we used capillary electrophoresis with time-of-flight mass spectrometry (CE-TOFMS). We also screened alterations of the epigenetic profiles – DNA methylation and histone acetylation – of OS cells and compared it with those of normal Fb.

Using CE-TOFMS, we observed a significant metabolic difference associated with glycolysis repression (dihydroxyacetone phosphate), increase of amino acid catabolism (phosphocreatine and glutamate) and urea cycle enhancement (arginino succinic acid) in OS cells compared with normal Fb. Noteworthy, metabolites associated with chromatin modification, like UDP-glucose and N⁸-acetylspermidine, decreased more in OS cells than in normal Fb. Further, combined bisulfite restriction analysis (COBRA) and acetyl-H3 immunoblotting indicated an epigenetic stability in OS cells than in normal Fb, and OS cells were more sensitive to an HDAC inhibitor under acidosis than under neutral condition.

Our data suggest that acidosis promotes a metabolic reprogramming that can contribute to the epigenetic maintenance under acidosis only in OS cells, and then the acidic microenvironment should be considered for future therapeutic approaches. The application of epigenetic modulators will be able to become an effectively therapeutic option to selectively target malignancies under the acidic microenvironment.