- Open Access
Proton pump inhibitors as anti vacuolar-ATPases drugs: a novel anticancer strategy
Journal of Experimental & Clinical Cancer Researchvolume 29, Article number: 44 (2010)
The vacuolar ATPases are ATP-dependent proton pumps whose functions include the acidification of intracellular compartments and the extrusion of protons through the cell cytoplasmic membrane. These pumps play a pivotal role in the regulation of cell pH in normal cells and, to a much greater extent, in tumor cells. In fact, the glucose metabolism in hypoxic conditions by the neoplasms leads to an intercellular pH drift towards acidity. The acid microenvironment is modulated through the over-expression of H+ transporters that are also involved in tumor progression, invasiveness, distant spread and chemoresistance. Several strategies to block/downmodulate the efficiency of these transporters are currently being investigated. Among them, proton pump inhibitors have shown to successfully block the H+ transporters in vitro and in vivo, leading to apoptotic death. Furthermore, their action seems to synergize with conventional chemotherapy protocols, leading to chemosensitization and reversal of chemoresistance. Aim of this article is to critically revise the current knowledge of this cellular machinery and to summarize the therapeutic strategies developed to counter this mechanism.
Tumor cells rely on H+ exchangers to relieve themselves from the dangerous protons byproduct of cancer metabolism that could trigger a cascade of lytic enzymes that ultimately would lead to self-digestion. Among these the most investigated are the vacuolar H+-ATPases (V-ATPases). V-ATPases are ATP dependent H+ transporters that utilize the energy freed by the hydrolysis of ATP with the active transport of protons from the cytoplasm to the lumen of intracellular compartments or, if located within the cytoplasmic membrane, the extracellular compartment [1–4]. Structurally speaking, the V-ATPases are composed of a peripheral domain (V1) that carries out ATP hydrolysis and an integral domain (V0) responsible for exchanging protons. The peripheral domain is made up of eight subunits (A-H) while the integral domain contains six subunits (a, c, c', c", d and e). V-ATPases work through a rotary mechanism in which ATP hydrolysis within V1 promotes the rotation of a central rotary domain, relative to the remainder of the complex, while the rotation of a proteolipid ring belonging to V0 domain moves protons through the membrane [5–7]. Two important physiological mechanisms of regulating V-ATPase activity in vivo are reversible dissociation of the V1 and V0 domains and changes in coupling efficiency of proton transport and ATP hydrolysis [8–15]. Malignant tumor cells overexpress lysosomal proteins on the cell surface, with deranged lysosomal activities, including acidification of internal vesicles, possibly involving altered V-ATPase function [16, 17]. The acidic tumor environment is a consequence of anaerobic glucose metabolism with secondary production of lactates byproducts through the upregulation of hypoxia-inducible factor 1α  or can be due to inadequate tumor perfusion, hypoxia secondary to disordered tumor growth or enhanced transmembrane pH regulation. These pumps, coupled with other ion exchangers, play a key role in the establishment and maintenance of malignant tumor environment and promote the selection of more aggressive cell phenotypes able to survive in this highly selective ambient.
Role of V-ATPases in tumor spread
V-ATPases play a critical role in the maintenance of an appropriate relatively neutral intracellular pH, an acidic luminal pH, and an acidic extracellular pH by actively pumping protons either through ion exchange mechanisms or by segregating H+ within cytoplasmic organelles that are subsequently expelled . It is hypothesized that the low extracellular pH of tumors might trigger proteases, leading to the dissolution of extracellular matrix. This phenomenon, as is well known, significantly contributes to tumor invasion and dissemination [21, 22]. In fact, tumor invasion depends on tumor acidifying ability that ultimately leads to secretion and activation of several classes of proteases [23, 24]. It is indeed known that low extracellular pH can trigger several proteases such as MMP-2, MMP-9, cathepsin B, and cathepsin L and result in acidity-induced up-regulation of the proangiogenic factors VEGF-A and IL-8 [25, 26]. As a consequence, the neutralization of these mechanisms has been actively pursued by many investigators who have been only partially successful, since so far it has been possible to block one or more MMPases but not all them simultaneously . A recent publication points out that by inhibiting of V-ATPases through RNA interference, it was possible to prevent cancer metastases in a murine model . This approach offers a new strategy to cope with the process of tumor spread (that is mediated by a continuous process of extracellular matrix degradation and tumor angiogenesis) by raising the extracellular tumor pH, thus arresting the activation of matrix degradating proteases. Finally, besides being a potential target of anticancer drugs, it is conceivable that V-ATPases might become a predictive factor of tumor behaviour and final outcome through the immunohistochemical evaluation of their expression and cellular distribution in tumor biopsies [29–31].
Role of V-ATPases in chemoresistance
The acidic microenvironment caused by changes in the pH gradient between the intracellular and the extracellular compartments as well as the pH gradient between the cytoplasm and the intracellular organelles can be significantly involved in the mechanism of drug resistance [32, 33]. There are several mechanisms involved in this phenomenon, including decreased uptake or neutralization of weakly basic drugs by the acidic tumor microenvironment or the sequestration of chemotherapy drugs within lysosomal vesicles [32–36]. An accelerated turnover of acidic vesicles may represent an additional tumor strategy of drug resistance based on counteracting current transportation . Several investigators developed new approaches to better characterize tumor pH in animal models [38, 39] mostly through imaging systems in order to identify novel targets. As a result, new approaches have been developed to modulate drug efficacy within the low pH tumor milieu including the use of RNA interference, bicarbonates or the induction of metabolic alkalosis [40–43]. Finally, two recently published articles describe the chemosensitizing action of proton pump inhibitors (omeprazole) in a murine model of orthotopic cutaneous melanoma, a well known chemo-refractory neoplasm, opening a novel field of investigation [44, 45].
Pump inhibitors as antitumor drugs
The various functions played by V-ATPases in tumors, including proliferation, tumorigenesis, drug resistance and tumor progression, make them potential targets for preclinical investigators and clinicians. The evidence that the expression of such proteins within tumor cells is increased in chemoresistant phenotypes and the fact that this expression could be induced by anticancer drugs, prompted oncologists to pursue the pharmaceutical neutralization of this tumor function [46–48]. Molecular and pharmacological therapy of these biological targets is technically extremely difficult and may carry a significant degree of toxicity. On the other hand, proton pump inhibitors are normally adopted in the treatment of gastritis, Zollinger-Ellison syndrome and, limitedly to veterinary oncology, gastric hyperacidity secondary to mast cell tumors in dogs and cats . These drugs have been shown to be highly effective at inhibiting V-ATPases in vitro and well tolerated and extremely efficacious in murine models, resulting in increased chemotherapy efficacy and improved tumor control [44, 45, 50]. Moreover, the same schedule has been able to revert chemoresistance to 5 fluorouracil, cisplatin and doxorubicin resulting in a caspase-independent cell death. Table 1 summarizes the different efflux pumps identified so far within tumor cells and their role in the maintenance of acid-base homeostasis and provides a short list of references for each pump [21, 35, 51–59].
As a rule of thumb it is reasonable to speculate that proton pump inhibitors, being pro-drugs needing acidity to be transformed in the active drug , might be more active in the most acidic tumors. Some reports have shown that metastatic tumors are more acidic then primary tumors, but also that solid tumors, either carcinoma or melanomas or sarcomas, are more acidic than systemic tumors (i.e. leukemia). It appears therefore conceivable that proton pump inhibitors might be more active against very malignant, often entirely unresponsive to current therapy, tumors. In support to this hypothesis it has also been shown that metastatic melanoma cells may be grown in acidic condition while cells deriving from primary tumors die when cultured in the same condition, needing longer periods of adaptation to select acid-resistant cells . In fact, acidic condition increases susceptibility of metastatic melanoma cells to proton pump inhibitors . Results of ongoing and future clinical trials hopefully will provide the proof of concept that inhibition of the proton pump may represent a new approach in the war against cancer, by both improving chemotherapy and inducing tumor self-digestion.
In conclusion, proton pump inhibitors might become a crucial addition to the pharmaceutical "armoury" of oncologists in consideration of their low cost, minimal toxicity and high efficacy. Further preclinical and clinical trials are ongoing to provide the clinical proof of concept for the use of proton pump inhibitors in the treatment of malignant cancers.
Finbow ME, Harrison MA: The vacuolar H+-ATPase: a universal proton pump of eukaryotes. Biochem J. 1997, 324: 697-712.
Forgac M: Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat Rev Mol Cell Biol. 2007, 8: 917-929. 10.1038/nrm2272.
Cipriano DJ, Wang Y, Bond S, Hinton A, Jefferies KC, Qi J, Forgac M: Structure and regulation of the vacuolar ATPases. Biochim Biophys Acta. 2008, 1777: 599-604. 10.1016/j.bbabio.2008.03.013.
Jefferies KC, Cipriano DJ, Forgac M: Function, structure and regulation of the vacuolar (H+)-ATPases. Arch Biochem Biophys. 2008, 476: 33-42. 10.1016/j.abb.2008.03.025.
Arai H, Terres G, Pink S, Forgac M: Topography and subunit stoichiometry of the coated vesicle proton pump. J Biol Chem. 1988, 263: 8796-8802.
Xu T, Vasilyeva E, Forgac M: Subunit interactions in the clathrin-coated vesicle vacuolar (H(+))-ATPase complex. J Biol Chem. 1999, 274: 28909-28915. 10.1074/jbc.274.41.28909.
Ohira M, Smardon AM, Charsky CM, Liu J, Tarsio M, Kane PM: The E and G subunits of the yeastV-ATPase interact tightly and are both present at more than one copy per V1 complex. J Biol Chem. 2006, 281: 22752-22760. 10.1074/jbc.M601441200.
Sautin YY, Lu M, Gaugler A, Zhang L, Gluck SL: Phosphatidylinositol 3-kinase-mediated effects of glucose on vacuolar H+-ATPase assembly, translocation, and acidification of intracellular compartments in renal epithelial cells. Mol Cell Biol. 2005, 25: 575-589. 10.1128/MCB.25.2.575-589.2005.
Trombetta ES, Ebersold M, Garrett W, Pypaert M, Mellman I: Activation of lysosomal function during dendritic cell maturation. Science. 2003, 299: 1400-1403. 10.1126/science.1080106.
Feng Y, Forgac M: A novel mechanism for regulation of vacuolar acidification. J Biol Chem. 1992, 267: 19769-19772.
Feng Y, Forgac M: Cysteine 254 of the 73-kDa A subunit is responsible for inhibition of the coated vesicle (H+)-ATPase upon modification by sulfhydryl reagents. J Biol Chem. 1992, 267: 5817-5822.
Feng Y, Forgac M: Inhibition of vacuolar H(+)-ATPase by disulfide bond formation between cysteine 254 and cysteine 532 in subunit A. J Biol Chem. 1994, 269: 13224-13230.
Forgac M: The vacuolar H+-ATPase of clathrin-coated vesicles is reversibly inhibited by Snitrosoglutathione. J Biol Chem. 1999, 274: 1301-1305. 10.1074/jbc.274.3.1301.
Xu T, Forgac M: Subunit D (Vma8p) of the yeast vacuolar H+-ATPase plays a role in coupling of proton transport and ATP hydrolysis. J Biol Chem. 2000, 275: 22075-22081. 10.1074/jbc.M002983200.
Kawasaki-Nishi S, Bowers K, Nishi T, Forgac M, Stevens TH: The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis. J Biol. 2001, 276: 47411-47420.
Saitoh O, Wang WC, Lotan R, Fukuda M: Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials. J Biol Chem. 1992, 267: 5700-5711.
Glunde K, Guggino SE, Solaiyappan M, Pathak AP, Ichikawa Y, Bhujwalla ZM: Extracellular acidification alters lysosomal trafficking in human breast cancer cells. Neoplasia. 2003, 5: 533-545.
Gatenby RA, Gillies RJ: Why do cancers have high aerobic glycolysis?. Nat Rev Cancer. 2004, 4: 891-899. 10.1038/nrc1478.
Fais S, De Milito A, You H, Qin W: Targeting vacuolar H+-ATPases as a new strategy against cancer. Cancer Res. 2007, 67: 10627-10630. 10.1158/0008-5472.CAN-07-1805.
Nishi T, Forgac M: The vacuolar (H+)-ATPases nature's most versatile proton pumps. Nat Rev Mol Cell Biol. 2002, 3: 94-103. 10.1038/nrm729.
Martinez-Zaguilan R, Lynch RM, Martinez GM, Gillies RJ: Vacuolar-type H(+)-ATPases are functionally expressed in plasma membranes of human tumor cells. Am J Physiol. 1993, 265: 1015-29.
Martínez-Zaguilán R, Seftor EA, Seftor RE, Chu YW, Gillies RJ, Hendrix MJ: Acidic pH enhances the invasive behavior of human melanoma cells. Clin Exp Metastasis. 1996, 14: 176-186. 10.1007/BF00121214.
Razaq S, Wilkins RJ, Urban JP: The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus. Eur Spine J. 2003, 12: 341-319. 10.1007/s00586-003-0582-3.
Webb SD, Sherratt JA, Fish RG: Modelling tumour acidity and invasion. Novartis Found Symp. 2001, 240: 169-181. full_text. discussion 181-185.
Koukourakis MI, Giatromanolaki A, Sivridis E, Bougioukas G, Didilis V, Gatter KC, Harris AL, Tumour and Angiogenesis Research Group: Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer tissues is linked to tumour hypoxia, angiogenic factor production and poor prognosis. Br J Cancer. 2003, 89: 877-885. 10.1038/sj.bjc.6601205.
Rofstad EK, Mathiesen B, Kindem K, Galappathi K: Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice. Cancer Res. 2006, 66: 6699-6707. 10.1158/0008-5472.CAN-06-0983.
Coussens LM, Fingleton B, Matrisian LM: Matrix metalloproteinase inhibitor and cancer: trials and tribulations. Science. 2002, 295: 2387-2392. 10.1126/science.1067100.
Lu X, Qin W, Li J, Tan N, Pan D, Zhang H, Xie L, Yao G, Shu H, Yao M, Wan D, Gu J, Yang S: The growth and metastasis of human hepatocellular carcinoma xenografts are inhibited by small interfering RNA targeting to the subunit ATP6L of proton pump. Cancer Res. 2005, 65: 6843-6849. 10.1158/0008-5472.CAN-04-3822.
Sennoune SR, Bakunts K, Martínez GM, Chua-Tuan JL, Kebir Y, Attaya MN, Martínez-Zaguilán R: Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity. Am J PhysiolCell Physiol. 2004, 286: 1443-1452. 10.1152/ajpcell.00407.2003.
Rojas JD, Sennoune SR, Maiti D, Bakunts K, Reuveni M, Sanka SC, Martinez GM, Seftor EA, Meininger CJ, Wu G, Wesson DE, Hendrix MJ, Martínez-Zaguilán R: Vacuolar-type H+-ATPases at the plasma membrane regulate pH and cell migration in microvascular endothelial cells. Am J Physiol Heart Circ Physiol. 2006, 291: 1147-1157. 10.1152/ajpheart.00166.2006.
Hinton A, Sennoune SR, Bond S, Fang M, Reuveni M, Sahagian GG, Jay D, Martinez-Zaguilan R, Forgac M: Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells. J Biol Chem. 2009, 284: 16400-16408. 10.1074/jbc.M901201200.
Mahoney BP, Raghunand N, Bagget B, Gillies RJ: Tumor acidity, ione trapping and chemotherapeutics I. Acid pH effects the distribution of chemotherapeutic agents in vitro. Biochem Pharmacol. 2003, 66: 1207-1218. 10.1016/S0006-2952(03)00467-2.
Simon S, Roy D, Schindler M: Intracellular pH and the control of multidrug resistance. Proc Nat Acad Sci USA. 1993, 91: 1128-1132. 10.1073/pnas.91.3.1128.
Raghunand N, Mahoney BP, Gillies RJ: Tumor acidity, ion trapping and chemotherapeutics. II. pH-dependent partition coefficients predict importance of ion trapping on pharmacokinetics of weakly basic chemotherapeutic agents. BiochemPharmacol. 2003, 66: 1219-1229.
Martínez-Zaguilán R, Raghunand N, Lynch RM, Bellamy W, Martinez GM, Rojas B, Smith D, Dalton WS, Gillies RJ: pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines. Biochem Pharmacol. 1999, 57: 1037-1046. 10.1016/S0006-2952(99)00022-2.
Raghunand N, Martínez-Zaguilán R, Wright SH, Gillies RJ: pH and drug resistance. II. Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs. Biochem Pharmacol. 1999, 57: 1047-1058. 10.1016/S0006-2952(99)00021-0.
Bobichon H, Colin M, Depierreux C, Liautaud-Roger F, Jardillier JC: Ultrastructural changes related to multidrug resistance in CEM cells: role of cytoplasmic vesicles in drug exclusion. J Exp Ther Oncol. 1996, 1: 49-61.
Raghunand N, Altbach MI, van Sluis R, Baggett B, Taylor CW, Bhujwalla ZM, Gillies RJ: Plasmalemmal pH-gradients in drug-sensitive and drug-resistant MCF-7 human breast carcinoma xenografts measured by 31P magnetic resonance spectroscopy. Biochem Pharmacol. 1999, 57: 309-312. 10.1016/S0006-2952(98)00306-2.
Raghunand N: Tissue pH measurement by magnetic resonance spectroscopy and imaging. Methods Mol Med. 2006, 124: 347-364.
Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Taylor CW, Paine-Murrieta G, Roe D, Bhujwalla ZM, Gillies RJ: Enhancement of chemotherapy by manipulation of tumour pH. Br J Cancer. 1999, 80: 1005-1011. 10.1038/sj.bjc.6690455.
You H, Jin J, Shu H, Yu B, De Milito A, Lozupone F, Deng Y, Tang N, Yao G, Fais S, Gu J, Qin W: Small interfering RNA targeting the subunit ATP6L of proton pump V-ATPase overcomes chemoresistance of breast cancer cells. Cancer Lett. 2009, 280: 110-119. 10.1016/j.canlet.2009.02.023.
Robey IF, Baggett BK, Kirkpatrick ND, Roe DJ, Dosescu J, Sloane BF, Hashim AI, Morse DL, Raghunand N, Gatenby RA, Gillies RJ: Bicarbonate increases tumor pH and inhibits spontaneous metastases. Cancer Res. 2009, 69: 2260-2268. 10.1158/0008-5472.CAN-07-5575.
Raghunand N, Mahoney B, van Sluis R, Baggett B, Gillies RJ: Acute metabolic alkalosis enhances response of C3H mouse mammary tumors to the weak base mitoxantrone. Neoplasia. 2001, 3: 227-235. 10.1038/sj.neo.7900151.
Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, Montinaro A, Marra M, Lugini L, Logozzi M, Lozupone F, Federici C, Iessi E, Parmiani G, Arancia G, Belardelli F, Fais S: Effect of proton pump inhibitor pretreatment on resistance of solid tumors to cytotoxic drugs. J Natl Cancer Inst. 2004, 96: 1702-1713. 10.1093/jnci/djh305.
De Milito A, Canese R, Marino ML, Borghi M, Iero M, Villa A, Venturi G, Lozupone F, Iessi E, Logozzi M, Mina PD, Santinami M, Rodolfo M, Podo F, Rivoltini L, Fais S: pH-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity. Int J Cancer. 2009,
Murakami T, Shibuya I, Ise T, Chen ZS, Akiyama S, Nakagawa M, Izumi H, Nakamura T, Matsuo K, Yamada Y, Kohno K: Elevated expression of vacuolar proton pump genes and cellular pH in cisplatin resistance. Int J Cancer. 2001, 93: 869-874. 10.1002/ijc.1418.
Torigoe T, Izumi H, Ishiguchi H, Uramoto H, Murakami T, Ise T, Yoshida Y, Tanabe M, Nomoto M, Itoh H, Kohno K: Enhanced expression of the human vacuolar H+-ATPase c subunit gene (ATP6L) in response to anticancer agents. J Biol Chem. 2002, 277: 36534-36543. 10.1074/jbc.M202605200.
Torigoe T, Izumi H, Yoshida Y, Ishiguchi H, Okamoto T, Itoh H, Kohno K: Low pH enhances Sp1 DNA binding activity and interaction with TBP. Nucleic Acids Res. 2003, 31: 4523-4530. 10.1093/nar/gkg487.
Thamm DH, Vail DM: Mast cell tumors. Small Animal Clinical Oncology. Edited by: Withrow SJ, MacEwen EG. 2007, Philadelphia, PA: WB Saunders Co, 402-424. full_text. 4 edn
De Milito A, Iessi E, Logozzi M, Lozupone F, Spada M, Marino ML, Federici C, Perdicchio M, Matarrese P, Lugini L, Nilsson A, Fais S: Proton pump inhibitors induce apoptosis of human B-cell tumors through a caspase-independent mechanism involving reactive oxygen species. ncer Res. 2007, 67: 5408-5417.
Cardone RA, Casavola V, Reshkin SJ: The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer. 2005, 5: 786-795. 10.1038/nrc1713.
Semenza GL: Tumor metabolism: cancer cells give and take lactate. J Clin Invest. 2008, 118: 3835-3837.
Robertson N, Potter C, Harris AL: Role of carbonic anhydrase IX in human tumor cell growth, survival, and invasion. Cancer Res. 2004, 64: 6160-6165. 10.1158/0008-5472.CAN-03-2224.
Shimokawa O, Matsui H, Nagano Y, Kaneko T, Shibahara T, Nakahara A, Hyodo I, Yanaka A, Majima HJ, Nakamura Y, Matsuzaki Y: Neoplastic transformation and induction of H+, K+ -adenosine triphosphatase by N-methyl-N'-nitro-N-nitrosoguanidine in the gastric epithelial RGM-1 cell line. In Vitro Cell Dev Biol Anim. 2008, 44: 26-30. 10.1007/s11626-007-9067-8.
Gervasoni JE, Fields SZ, Krishna S, Baker MA, Rosado M, Thuraisamy K, Hindenburg AA, Taub RN: Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy. Cancer Res. 1991, 51: 4955-4963.
Klohs WD, Steinkampf RW: The effect of lysosomotropic agents and secretory inhibitors on anthracycline retention and activity in multiple drug-resistant cells. Mol Pharmacol. 1988, 34: 180-185.
Simon SM, Schindler M: Cell biological mechanisms of multidrug resistance in tumors. Proc Natl Acad Sci USA. 1994, 91: 3497-3504. 10.1073/pnas.91.9.3497.
Fletcher JI, Haber M, Henderson MJ, Norris MD: ABC transporters in cancer: more than just drug efflux pumps. Nat Rev Cancer. 2010, 10: 147-156. 10.1038/nrc2789.
Mullin JM, Gabello M, Murray LJ, Farrell CP, Bellows J, Wolov KR, Kearney KR, Rudolph D, Thornton JJ: Proton pump inhibitors: actions and reactions. Drug Discov Today. 2009, 14: 647-60. 10.1016/j.drudis.2009.03.014.
Lugini L, Matarrese P, Tinari A, Lozupone F, Federici C, Iessi E, Gentile M, Luciani F, Parmiani G, Rivoltini L, Malorni W, Fais S: Cannibalism of live lymphocytes by human metastatic but not primary melanoma cells. Cancer Res. 2006, 66: 3629-38. 10.1158/0008-5472.CAN-05-3204.
This work has been supported by "Grant 2009" "Malattie Rare"of the Italian Ministry of Health, bya MIUR grant and by a "ACC" Grant to E.P.S and G.C., and by the Italian Ministry of Health to S.F.
The authors declare that they have no competing interests.
All the authors read and approved the final manuscript. EPS and SF equally contributed to this work, GC supervised the other contributors and critically revised the manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.