Abstract
Similar to other types of cancer, acidification of tumor microenvironment is an important feature of osteosarcoma, and a major source of cellular stress that triggers cancer aggressiveness, drug resistance, and progression. Among the different effects of low extracellular pH on tumor cells, we have recently found that short-term exposure to acidosis strongly affects gene expression. This alteration might also occur for the most commonly used housekeeping genes (HKG), thereby causing erroneous interpretation of RT-qPCR data. On this basis, by using osteosarcoma cells cultured at different pH values, we aimed to identify the ideal HKG to be considered in studies on tumor-associated acidosis. We verified the stability of 15 commonly used HKG through five algorithms (NormFinder, geNorm, BestKeeper, ΔCT, coefficient of variation) and found that no universal HKG is suitable, since at least four HKG are necessary for proper normalization. Furthermore, according to the acceptable range of values, YWHAZ, GAPDH, GUSB, and 18S rRNA were the most stable reference genes at different pH. Our results will be helpful for future investigations focusing on the effect of altered microenvironment on cancer behavior, particularly on the effectiveness of anticancer therapies in acid conditions.
Highlights
Human tumors survive adverse microenvironments that derive from uncontrolled cell proliferation and anarchic tissue organization [1]
By using osteosarcoma cells, we investigated the influence of extracellular acidosis on the stability of the most widely adopted housekeeping genes (HKG) in order to identify HKG that can be reliably used for gene expression analysis
18S rRNA was in general the most expressed HKG, in acidic pH the least expressed gene was G6PD, whereas under physiological pH the least expressed gene was GUSB (Figure 1, Table 2)
Summary
Human tumors survive adverse microenvironments that derive from uncontrolled cell proliferation and anarchic tissue organization [1]. Among the different features of the adverse and altered tumor microenvironment, extracellular acidosis has been a major field of investigation as it is a direct cause of cancer progression and therapeutic resistance [2]. As for other solid malignancies, we have recently shown that acidosis modulates osteosarcoma behavior, fosters cellular metabolic switch, epigenetic modifications, and other cellular alterations that result in a more aggressive tumor phenotype characterized by increased cancer stemness, drug resistance, and invasiveness [10,11,12,13,14,15,16]. There is still an urgent need to improve the knowledge about the underlying mechanisms regulating cancer aggressiveness under low extracellular pH conditions, and gene expression analysis can provide critical information in this field. Low extracellular pH might be included among the different stimuli that can cause HKG instability
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