Impact of the acid microenvironment on bone cancers

Altered tumor metabolism and intratumoral acidosis in bone cancers

High glycolytic activity is a common feature of many types of cancers, including sarcomas and BM [ , ]. The shift to a primarily glycolytic phenotype results from the energetic need of finding alternative energy sources, other than oxygen, that is exacerbated by anarchic and dysfunctional perfusion of cancer tissue, ultimately leading to hypoxia. However, as elegantly described by Otto Warburg in 1956 [ ], glycolysis in cancer cells may also occur at normal oxygen tension.

In bone cancers, hypoxia is the consequence of both increased growth of bone-invading cancer cells, that is associated with a high oxygen consumption rate, and the intrinsic hypoxia of bone microenvironment. Indeed, hypoxia is a relevant contributor to bone biology and physiology [ ]. As a demonstration, in the medullary cavity of animal models, pO ₂ values range 11.7–31.7 mm Hg (1.5%–4.2%), with a mean of 20.4 mm Hg (2.7%) [ ].

The switch to glycolysis, both under normoxia and hypoxia, follows the activation of the hypoxia-inducible factor-1 (HIF-1) that drives the transcription of crucial enzymes of the glycolytic pathway [ ]. As a final result, increased glycolytic rate conveys to the intracellular accumulation of protons in the cytoplasm, but also in the extracellular space. Indeed, in order to tightly control the intracellular pH range within a narrow range, cancer cells secrete substantial amounts of protons outside the cells through different mechanisms [ ]. In cancer research, the coupling of tumor acidification and upregulation of glucose metabolism has increasingly gained attention, and new in vivo imaging tools, through the combination of PET and MRI-CEST, have been developed [ ]. On this regard, it is noteworthy that ⁸ F-FDG PET has been used for the evaluation, staging, and surveillance of patients with bone sarcomas and that the maximum SUV is related with sarcoma grade and overall survival [ , ].

Finally, in both BM and bone sarcomas, glycolysis may not be the only metabolic trigger of tumor interstitial acidosis, since it may also derive from the hydration of excessive production of CO ₂ in the more oxidative areas of the tumor [ ]. However, this mechanism has not been completely explored.

Mechanisms of proton extrusion in bone cancers

Cytosolic acidification is extremely toxic in both normal and cancer cells: it is one of the first steps to apoptosis, with no return [ ]. Bone cancer cells get rid of excessive intracellular accumulation of protons through an armamentarium of pumps and transporters that are located at the plasma membrane or at the lysosomal membrane and that strongly acidifies the extracellular space through direct pumping/transport or through exocytosis, respectively [ ]. This armamentarium includes the vacuolar H+-ATPase (V-ATPase), the Na ⁺ /H ⁺ exchanger isoform 1 (NHEs, mainly the NHE1), the monocarboxylate transporters (MCTs, mainly MCT1, also known as the lactate-proton symporter), the Na ⁺ -dependent Cl ⁻ /HCO ³⁻ exchanger, and the carbonic anhydrases (CAs) isozymes, mainly CAII, CAIX, and CAXII [ ]. In Tables 24.1–24.4 , the studies describing the expression and the role of these molecules in the extracellular acidification and in bone cancer behavior are reported. Several drugs have been tested to target these ion pumps/transporters as anticancer therapy. For a more extensive discussion, see section “ Hampering proton pump/ion transporters .”

The review of the current literature reveals that the most studied ion/proton pump/transporter is the V-ATPase in bone sarcomas and BM, followed by CAIX only for bone sarcoma. V-ATPase is a family of ATP-powered proton pumps that are mainly located at the lysosomal membrane and that acidify the intralysosomal space. In highly acidifying cells, V-ATPase can be also found at the cytoplasmic membrane to pump protons outside the cells. V-ATPase is formed by an ATP-hydrolytic domain (V1) and a proton-translocation domain (V0) [ ]. V-ATPase activity is an energy-consuming process and requires the tight association of all the components of the complex, which is ensured by the C ring [ ]. Studies on the expression and the activity of V-ATPase in bone cancers have been based on the analysis of preclinical models and, only in a few cases of tumor tissues, also in clinical series.

CAIX is one of the 14 carbonic anhydrase isoforms found in humans. Carbonic anhydrases are a large family of transmembrane dimeric zinc metalloenzymes with an extracellular active site that catalyzes the reversible hydration of carbon dioxide and that facilitates acid secretion in different cell types [ ]. Evidence on the expression of CAIX in bone cancers has been largely based on the analysis of human cancer tissues.

Previous reports on the characterization of extracellular acidification in bone cancers, by using in vitro preclinical models and different techniques to measure pHe, like using macro- or microelectrodes or measuring the extracellular acidification rate (ECAR) values by the Seahorse technologies, have shown that the activity of these pumps/transporters is responsible for a strong acidification of the medium or in close proximity to the cells, very close to the cell membrane, respectively [ ]. Often, in the same cancer cells, the enhanced acidification ability has also been demonstrated by using acridine orange or lysosensor staining, as it has been demonstrated in soft-tissue sarcoma–derived stem cells [ , , ].

In summary, there is consistent evidence that a high glycolytic rate and the subsequent activation of different ion pumps and transporters in cancer cells are a major cause of tumor interstitial acidosis, both in BM and in bone sarcomas.

Bone resorption as a source of extracellular acidification

In the bone soil, to expand and invade the surrounding normal tissue, bone cancer cells degrade the hard extracellular matrix by directly or indirectly stimulating the activity of osteoclasts, the highly specialized bone-resorbing cells. For an extensive review of the molecular mechanism of BM and the paracrine circuit between cancer cells and osteoclasts, see Ref. [ ]. In summary, the induction of osteoclast activity can be triggered by a plethora of growth factors that are also commonly active under physiological conditions in bone remodeling and that can be secreted by cancer cells, or by tumor-stimulated osteoblasts, the bone-forming cells. Among these, the most important is the receptor activator of nuclear factor-kappa B ligand (RANKL). Once stimulated, mature osteoclasts can resorb bone through the execution of a dynamic multistep process. First, osteoclasts migrate and attach to the bone surface targeted for degradation and removal, thereby forming a tight “sealing zone.” Then, the plasma membrane polarizes to form the resorption organelle, the ruffled border, a unique folded highly permeable membrane facing to the resorbing bone surface [ ]. Next, to dissolve the mineralized component of bone, osteoclasts secrete hydrochloric acid into the resorption lacunae (Howship lacunae) mainly via the plasma membrane (a3 isoform) V-ATPase [ ]. Proton pumping performed by osteoclasts during bone resorption activity is a highly energy-consuming process that is mainly based on osteoclast glycolytic metabolism [ ]. Remarkably, a3 expression is 100-fold higher in osteoclasts compared to other cell types [ ]. V-ATPase activity is also coupled with the activity of the chloride channel ion-proton antiporter ClC-7 [ ], and both proteins are clustered at the ruffled border domain. As a consequence, in Howship lacunae, the pH reaches very low values, around 4.5 [ ]. At the end of the resorption process, protons pumped into Howship lacunae diffuse in the extracellular space, thereby causing further acidification of the tumor microenvironment. Adversely, proteinaceous component of the matrix, mainly type I collagen, is degraded through the activity of the osteoclast-derived cysteine proteinase cathepsin K.

In addition to osteoclasts, acid-mediated resorption of the bone mineralized matrix can also be performed by osteocytes. Osteocytes are the final fully differentiated form of osteoblasts that are entrapped in the hard matrix and directly remodel the bone walls of their lacuna-canalicular systems in a process known as osteocytic perilacunar/canalicular remodeling. As for osteoclasts, this process is based on the combined activity of matrix metalloproteinases (MMPs), vacuolar type H ⁺ -ATPases that secrete acid [ , ], and other enzymes, such as cathepsin K and carbonic anhydrases [ ]. However, the interplay between tumor cells and osteocytes is almost completely unexplored and it is unknown if perilacunar remodeling can be induced by invading cancer cells.

In conclusion, these findings show that in addition to tumor cells, also tumor-induced bone-resorbing cells contribute to the acidification of intratumoral microenvironment, both in bone sarcomas and in BM.