Altered pH gradient at the plasma membrane of osteosarcoma cells is a key mechanism of drug resistance

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, shortterm acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance

Primers and probes.

<p>Gene symbol, name, function, accession number, primer sequences and selected probe are shown.</p

Gene expression of stem-cell markers confirmed the enhancement of stem-like features of CSC when are co-cultured with MSC in transwell.

<p><b>(A)</b> Scheme of the co-culture system of HOS-CSCwith MSC used in this study. The co-culturing was prolonged for 3 days; <b>(B)</b> Sox2, Oct4, Nanog, and CXCR4 expression evaluated by Real Time PCR in HOS-CSC spheres that were cultured alone or with MSC (in transwell). Gene expression of CSC was also compared to parental HOS adherent cells (T0) (*p<0.05); <b>(C)</b> CXCR4 was also evaluated by ELISA. (*p<0.05). Note that the levels of Oct4, Nanog, and CXCR4 markers in the spheres were significantly higher than the parental cell line. For Sox2, we saw a similar trend of increase in CSC spheres respect to parental cells, although this increase was more evident when CSC where co-cultured with MSC.</p

The co-culturing of OS cells with MSC enhances the spherogenic potential and protein expression of stem-related markers in OS-CSC spehers.

<p><b>(A)</b> Scheme of the assays showed in this figure. <b>(B)</b> Representative pictures of the formed sphere of CSC in different conditions: with or w/o MSC in the upper chamber, see panel A (scale bar 500 μm, black arrows show the spheres with higher size); <b>(C)</b> spheres of CSC shown in panel A were counted and expressed as sphere forming efficiency (number of spheres formed / number of cells seeded × 100) (*p<0.05); <b>(D)</b> Average diameter of the counted spheres of CSC in panel C; <b>(E)</b> Stem cell markers of CSC spheres were evaluated by proteome expression profiler (left panel, image of the blotted membranes; right panel, densitometric evaluation of the same membranes and graphical representation of the obtained data); Oct4, Sox2 and Nanog have been highlighted on the membrane. Map of the blotted membrane and human pluripotent stem cell array coordinates are also shown.</p

Model for the circuit between MSC and HOS-CSC.

<p>Recruitment of MSC to the tumor environment leads to enhanced proliferation of OS stem cells. The presence of CSC, in turn, leads to a consistent secretion of TGFβ1 that activates a stromal autocrine loop that might be responsible for the activation of NF-kB genes and IL-6 secretion by MSC. Indeed, neutralization of TGFβ1 reduces the amount of secreted IL-6. Pro-tumorigenic effects of MSC, via IL-6, including induction of HOS-CSC migration and sphere growth, can be counteracted also by IL-6 neutralizing antibody. The presence of MSC is also responsible for increased expression of adhesion molecules involved in intra- or extra-vasation and the expression of MET can be counteracted by IL-6 neutralization.</p