Endothelial Ca2+ Signaling and the Resistance to Anticancer Treatments: Partners in Crime

Intracellular Ca2+ signaling drives angiogenesis and vasculogenesis by stimulating proliferation, migration, and tube formation in both vascular endothelial cells and endothelial colony forming cells (ECFCs), which represent the only endothelial precursor truly belonging to the endothelial phenotype. In addition, local Ca2+ signals at the endoplasmic reticulum (ER)–mitochondria interface regulate endothelial cell fate by stimulating survival or apoptosis depending on the extent of the mitochondrial Ca2+ increase. The present article aims at describing how remodeling of the endothelial Ca2+ toolkit contributes to establish intrinsic or acquired resistance to standard anti-cancer therapies. The endothelial Ca2+ toolkit undergoes a major alteration in tumor endothelial cells and tumor-associated ECFCs. These include changes in TRPV4 expression and increase in the expression of P2X7 receptors, Piezo2, Stim1, Orai1, TRPC1, TRPC5, Connexin 40 and dysregulation of the ER Ca2+ handling machinery. Additionally, remodeling of the endothelial Ca2+ toolkit could involve nicotinic acetylcholine receptors, gasotransmitters-gated channels, two-pore channels and Na+/H+ exchanger. Targeting the endothelial Ca2+ toolkit could represent an alternative adjuvant therapy to circumvent patients’ resistance to current anti-cancer treatments.


Introduction
An increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) has long been known to play a crucial role in angiogenesis and arterial remodeling [1][2][3][4][5]. Accordingly, growth factors and cytokines, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), angiopoietin and stromal derived factor-1α (SDF-1α), trigger robust Ca 2+ signals in vascular endothelial cells [6][7][8][9][10][11][12], which recruit a number of downstream Ca 2+ -dependent pro-angiogenic decoders. These include, but are not limited to, the transcription factors, Nuclear factor of activated T-cells (NFAT), Nuclear factor-kappaB (NF-κB) and cAMP responsive element binding protein (CREB) [8,13,14], myosin light chain kinase (MLCK) and myosin 2 [8,15], endothelial nitric oxide synthase (eNOS) [16,17], extracellular signal-regulated kinases 1 2 (ERK 1/2) [18,19] and Akt [19,20]. Not surprisingly, therefore, subsequent studies clearly revealed that endothelial Ca 2+ signals may also drive tumor angiogenesis, growth and metastasis [3,[21][22][23][24]. However, the process of tumor vascularization is far more complex than originally envisaged [25]. Accordingly, the angiogenic switch, which is the initial step in the multistep process that ensures cancer cells with an adequate supply of oxygen and nutrients and provides them with an escape route to enter peripheral circulation, is triggered by the recruitment of bone marrow-derived endothelial progenitor cells (EPCs), according to a process termed vasculogenesis [26][27][28]. Similar to mature endothelial cells, EPCs require an increase in [Ca 2+ ] i to proliferate, assembly into capillary-like tubular networks in vitro and form patent neovessels in vivo [29][30][31]. Of note, intracellular Ca 2+ signals finely regulate proliferation and in vitro tubulogenesis also in tumor-derived EPCs (T-EPCs) [23,32,33]. An established tenet of neoplastic transformation is the remodeling of the Ca 2+ machinery in malignant cells, which contributes to the distinct hallmarks of cancer described by Hanahan and Weinberg [34][35][36]. Tumor endothelial cells (T-ECs) and T-EPCs do not derive from the malignant clone, but they display a dramatic dysregulation of their Ca 2+ signaling toolkit [29,32,37]. The present article surveys the most recent updates on the remodeling of endothelial Ca 2+ signals during tumor vascularization. In particular, it has been outlined which Ca 2+ -permeable channels and Ca 2+ -transporting systems are up-or down-regulated in T-ECs and T-EPCs and how they impact on neovessel formation and/or apoptosis resistance in the presence of anti-cancer drugs. Finally, the hypothesis that the remodeling of endothelial Ca 2+ signals may be deeply involved in tumor resistance to standard therapeutic treatments, including chemotherapy, radiotherapy and anti-angiogenic therapy is widely discussed.
Herein, the mechanisms whereby the remodeling of the endothelial transportome, i.e., the specific arsenal of ion channels and transporters expressed by vascular endothelial cells and ECFCs, confers resistance to anti-cancer therapies have been subdivided into two main categories: (1) enhanced neovascularization, which attenuates the therapeutic outcome of anticancer treatments by nourishing cancer cells with O 2 and nutrients and removing their catabolic waste, and further provides them with a direct access to peripheral circulation, thereby favoring metastasis ( Figure 1 and Table 1); and (2) resistance to apoptosis, which hampers the cellular stress induced by chemo-and radiotherapy on tumor endothelial cells and interferes with the dismantling of cancer vasculature ( Figure 2 and Table 2). and radiotherapy on tumor endothelial cells and interferes with the dismantling of cancer vasculature ( Figure 2 and Table 2).  The endothelial Ca 2+ transportome is remodeled to promote tumor endothelial cell resistance to apoptosis. The sequence of events is illustrated by the black arrows. Downward arrows indicate the down-regulation of a specific Ca 2+ -permeable channel/transporter or of a precise cellular process. Upward arrows indicate the over-expression of a specific Ca 2+ -permeable channel or the stimulation of a precise cellular process. and radiotherapy on tumor endothelial cells and interferes with the dismantling of cancer vasculature ( Figure 2 and Table 2).    The generic term EPC, in this context, refers to circulating pro-angiogenic cells which cannot be grouped into the ECFC sub-family and are likely to belong to the myeloid lineage.
Subsequently, the role of TRPV4 was investigated in prostate adenocarcinoma-derived endothelial cells (A-TECs). Unlike B-TECs, TRPV4 was down-regulated in A-TECs, which increased their sensitivity towards extracellular matrix stiffness, boosted their migration rate and favored the development of an aberrant (i.e., non-uniform, abnormally dilated and leaky) tumor vascular network [90]. This feature gains therapeutic relevance as the resultant hostile (i.e., low extracellular pH, hypoxia, and high interstitial pressure) microenvironment fuels tumor progression and hampers the efficacy of chemotherapy, radiation therapy, anti-angiogenic therapy immunotherapy [117,118]. Accordingly, overexpression or pharmacological activation of TRPV4 with GSK restored A-TEC mechanosensitivity and normalized their abnormal tube formation in vitro by inhibiting enhanced basal Rho activity [91]. Moreover, the daily intraperitoneal injection of GSK was able to normalize tumor vasculature in a xenograft mouse model of Lewis Lung Carcinoma (LLC), thereby improving cisplatin delivery and causing significant tumor shrinkage [91]. In addition, TRPV4-mediated Ca 2+ entry reduced A-TEC proliferation in vitro by inhibiting the extracellular signal-regulated kinases 1/2 [92]. This mechanism further contributes to GSK-induced dismantling of LLC vasculature in vivo [92]. Therefore, remodeling of TRPV4-mediated Ca 2+ entry may be used to effectively target tumor vascularization, although the most effective approach may depend on the tumor type. Accordingly, TRPV4 should be inhibited to halt tumor vascularization in breast cancer, while it must be stimulated to normalize tumor vasculature in LLC [23].

Piezo Proteins
Piezo1 and Piezo2 proteins are two recently identified non-selective cation channels that mediate mechanosensory transduction in mammalian cells [119,120]. Piezo proteins are gigantic homotetrameric complexes endowed with one or four ion-conducting pores: each subunit comprises over 2500 amino acids and presents 24-40 predicted transmembrane domains [119]. Piezo channels are Ca 2+ -permeable and, therefore, lead to robust Ca 2+ entry in response to mechanical deformation of the plasma membrane; unlike TRPV4 channels [105], Piezo-mediated Ca 2+ entry is directly activated by tension within the lipid bilayer of the plasma membrane rather than by physical coupling to the sub-membranal cytoskeleton or intracellular second messengers [120]. A recent study demonstrated that the endothelial Piezo1 was activated by laminal shear stress to drive embryonic vascular development [81]. Piezo1 promoted vascular endothelial cell migration, alignment and re-alignment along the direction of blood flow by engaging the Ca 2+ -dependent decoders, eNOS and calpain [81,121]. More recently, Piezo2 was found to be up-regulated in T-ECs from mouse xenografted with GL261 glioma cells [93]. Knocking down Piezo2 with a selective small interfering RNA (siRNA) reduced glioma angiogenesis and normalized tumor neovessels [93]. Moreover, suppressing Piezo2 expression decreased VEGF-and interleukin-1β-induced angiogenesis in the mouse corneal neovascularization model [93]. Finally, Piezo2-mediated Ca 2+ entry elicited the Ca 2+ -dependent transcription of Wnt11 and, consequently, the nuclear translocation of β-catenin in HUVECs, thereby promoting their angiogenic activity in vitro [93]. Although this mechanism remains to be confirmed in T-ECs, Piezo2 stands out as a crucial regulator of tumor angiogenesis and should be probed as a novel target for more effective anti-cancer treatments.

P2X7 Receptors
ATP and its metabolite, adenosine, are major constituents of tumor microenvironment and may differently affect tumor growth, immune cells and tumor-host interaction by activating a wealth of metabotropic (i.e., P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) and ionotropic (P2X1-2X8,) receptors [122]. Of note, ATP has long been known to stimulate angiogenesis though metabotropic P2y receptors [123]. Nevertheless, a recent investigation demonstrated that P2X7 stimulates tumor angiogenesis in vivo. Two different tumor cell lines, i.e., HEK293 and CT26 colon carcinoma cells, were transfected with P2X7 receptors and subsequently xenografted into immunodeficient or immunocompetent BALB/c mice, respectively. Tumor growth and angiogenesis were significantly enhanced by P 2X7 expression; consequently, pharmacological inhibition (with AZ10606120) or genetic silencing of P 2X7 decreased tumor growth and dramatically reduced vascular density [124]. This study further confirmed that P2X7 receptors were significantly up-regulated in several types of cancer cells, including those from breast cancer, and stimulated angiogenesis by promoting VEGF release [124]. More recently, it was found that P2X7 receptors were over-expressed also in B-TECs [94]. This study revealed that the activation of these purinergic receptors with high doses of ATP (>20 µM) and BzATP, a selective P 2X7 agonist, inhibited B-TEC, but not HDMEC, migration in vitro. The anti-angiogenic effect of P 2X7 was mediated by the Ca 2+ -sensitive adenylate cyclase 10 (AC10), which increased cyclic adenosine monophosphate (cAMP) and recruited EPAC-1 to dampen cell migration by inducing cytoskeletal remodeling [94]. Moreover, P2X7 receptors-induced cAMP production stabilized bidimensional tumor vessels by favoring pericyte attraction towards B-TECs and reducing endothelial permeability [94]. Intriguingly, hypoxia prevented the anti-angiogenic ability of P 2X7 receptors by likely reducing their expression [94,125]. These data, therefore, strongly suggest that stimulating P2X7 receptors could provide an efficient strategy to normalize tumor vasculature, thereby enhancing the delivery of cytotoxic drugs and of O 2 for radiotherapy. In this context, it should be pointed out that P2X7 receptors target hematopoietic EPCs to glioblastoma [126]. Although this investigation was conducted on healthy cells, and remains therefore to be validated in T-EPCs, it suggests that ATP may differentially affect tumor endothelial cells and T-EPCs. Alternatively, the effect exerted by P2X7 con T-EPC fate could be cancer-dependent and needs to be further investigated.

Stim1, Orai1 and Canonical Transient Receptor Potential 1 (TRPC1)
SOCE represents the most important Ca 2+ entry pathway supporting the pro-angiogenic activity of human ECFCs [29,33,56]. Accordingly, TRPV4 boosted ECFC proliferation rate only when accompanied by the administration of a robust dose of growth factors [86,127], whereas TRPV1 stimulated ECFC proliferation and tubulogenesis by mediating the intracellular intake of anandamide in a Ca 2+ -independent manner [128]. SOCE is activated by the pharmacological (by blocking SERCA-mediated Ca 2+ sequestration) or physiological (by stimulating InsP 3 Rs) depletion of the ER Ca 2+ stores and is mediated by the dynamic interplay between Stim1, Orai1 and TRPC1 [29, 56,58,84]. It is, however, still unknown whether Orai1 and TRPC1 form two independent Stim1-gated Ca 2+ -permeable routes [129] or assemble into a unique heteromeric supermolecular complex in ECFCs [130]. A recent series of studies revealed that SOCE maintained VEGF-induced intracellular Ca 2+ oscillations and promoted ECFC proliferation and in vitro tubulogenesis by recruiting the Ca 2+ -dependent transcription factor, NF-κB [58,83]. Of note, SOCE was significantly enhanced in metastatic renal cellular carcinoma (RCC)-derived ECFCs (RCC-ECFCs) due to the up-regulation of Stim1, Orai1 and TRPC1 [95]. Similar to normal cells, the pharmacological blockade of SOCE with YM-58483/BTP2 or with low micromolar doses of lanthanides prevented proliferation and tube formation in RCC-ECFCs [95]. This finding strongly suggests that SOCE could provide an alternative target for the treatment of metastatic RCC [32,131], which develops either intrinsic or acquired refractoriness towards conventional treatments, such as anti-VEGF inhibitors and anti-mammalian target of rapamycin (mTOR) blockers [132]. As more extensively discussed below, the overall remodeling of the intracellular Ca 2+ toolkit in T-ECFCs could indeed be responsible for the relative or complete failure of standard therapies in RCC patients. Conversely, SOCE was not significantly up-regulated in breast cancer-derived ECFCs (BC-ECFCs) [96]. Accordingly, Orai1 and TRPC1 expression were not significantly altered, while Stim1 was significantly more abundant as compared to control cells. Nevertheless, a tight stoichiometric ratio between Stim1, Orai1 and TRPC1 is required for SOCE to be activated [133]. If all Orai1 and TRPC1 channel proteins are gated by the physiological levels of Stim1, any increase in Stim1 expression will not be sufficient to enhance SOCE as there will be no further channels available on the plasma membrane. Similar to RCC-ECFCs, however, the pharmacological inhibition of SOCE abrogated BC-ECFC proliferation and tube formation, thereby confirming that Orai1 and TRPC1 could serve as reliable targets to interfere with tumor vascularization, although this hypothesis remains to be validated in vivo [134,135]. The strict requirement of Stim1 for tumor vascularization is further suggested by the recent finding that Stim1 transcription in hypoxic tumors is finely regulated by HIF-1 [136]. SOCE, in turn, was found to stimulate HIF-1 accumulation in hypoxic cancer cells by engaging Ca 2+ /calmodulin-dependent protein kinase II and p300 [136]. Therefore, targeting SOCE could also affect the expression of the primary transcription factor responsible for RCC and breast cancer growth and metastasis [137,138]. Of note, HIF-1 has been shown to control also TRPC1 expression [139], although it is still unclear whether this regulation also occurs in tumor microenvironment and, if so, why TRPC1 is up-regulated in RCC-ECFCs, but not in RCC-ECFCs.
The role played by SOCE in tumor vascularization has, finally, been uncovered also in infantile hemangioma (IH), the most common childhood malignancy which may cause disfigurement, ulceration and obstruction and, if not treated, ultimately leads to patients' death [140]. IH is a vascular tumor that arises as a consequence of dysregulation of angiogenesis and vasculogenesis [140]. The clonal expansion of an endothelial progenitor/stem cell population, which is closely reminiscent of ECFCs, is deeply involved in IH vascularization [141,142]. A recent investigation provided the evidence that Stim1, Orai1 and TRPC1 drive the higher rate of IH-derived ECFC (IH-ECFC) growth as compared to control cells [97]. Stim1, Orai1 and TRPC1 were not up-regulated in IH-ECFCs; however, the ER Ca 2+ store was depleted to such an extent that Stim1 was basally activated and gated the constitutive activation of Orai1 and TRPC1 [97]. Stim2 displays a lower Ca 2+ affinity as respect to Stim1 and supports basal Ca 2+ entry in HUVECs [143]. Nevertheless, the pharmacological abrogation of Stim2 silencing did not affect constitutive SOCE in IH-ECFCs [97]. Constitutive SOCE boosted IH-ECFC proliferation by enhancing NO release [97], thereby emerging as an alternative target to treat IH in propranolol-resistant patients [144].

Neuronal Nicotinic Receptors (nAchRs)
nAchRs belong to a super-family of Cys-loop ligand-gated non-selective cation channels that are physiologically activated by acetylcholine, mediate fast synaptic transmission in neurons and, by virtue of their resolvable Ca 2+ -permeability, control a number of Ca 2+ -dependent processes, including neurotransmitter release and synaptic plasticity [145,146]. However, nAchRs are also largely expressed in non-neuronal brain cells, such as astrocytes, in epithelial cells and in several types of vascular cells, including smooth muscle cells and endothelial cells [75,[147][148][149]. It has been established that α7 homomeric nAchRs (α7-nAchRs) promote endothelial cell proliferation, migration and tube formation both in vitro and in vivo by recruiting an array of Ca 2+ -dependent effectors [98,99]. These include eNOS, mitogen-activated protein kinase, phosphoinositide 3-kinase (PI3K), NF-κB, matrix metalloproteinase-2 and -9 [98,99,150]. In addition, α7-nAChRs were shown to induce the JAK2/STAT3 signaling cascade to promote endothelial cell survival [151]. Intriguingly, α7-nAchRs possess the highest Ca 2+ -permeability among the known nAchR subtypes [152]. These pieces of evidence ignited the hypothesis that nicotine accelerated tumor growth by stimulating endothelial α7-nAchRs, thereby promoting angiogenesis and tumor vascularization [147,149]. In support of this model, nicotine induced tumor growth in a mouse model of LLC by stimulating endothelial cell proliferation and tube formation. Nicotine-induced tumor vascularization was significantly reduced by pharmacological blockade (with mecamylamine or hexamethonium) as well as by genetic silencing of α7-nAChRs. The signaling pathways recruited by α7-nAChRs to sustain tumor angiogenesis were not deeply investigated, but nicotine stimulated endothelial cells to release NO, prostacyclin and VEGF [98,99]. It should, however, be pointed out that the expression and role of α7-nAChRs in T-ECs has not been investigated, yet. Nevertheless, hypoxia has been shown to increase α7-nAChRs expression in a mouse model of hindlimb ischemia [98], whereas α7-nAChRs may stimulate HIF-1αtranscription [153]. These observations support the hypothesis that α7-nAChRs are actually over-expressed in T-ECs.
In addition to promoting angiogenesis, nicotine could recruit α7-nAChRs to boost vasculogenesis. A recent study revealed that nicotine induced proliferation, migration and tube formation also in ECFCs and that this effect was inhibited by mecamylamine or α-bungarotoxin [100]. Moreover, nicotine triggered EPC mobilization from bone marrow in a cohort of mice xenografted with colorectal cancer cells, thereby fostering tumor growth and vascularization [101]. Lastly, exposure to second hand smoke stimulated tumor angiogenesis and increased the number of circulating EPCs in a mouse model of LLC by enhancing VEGF release: mecamylamine, however, halted VEGF release, thereby reducing tumor size and capillary density. The pro-angiogenic effect of nicotine was, therefore, likely to be mediated by nAchRs [154]. We are yet to know whether and how α7-nAChRs are altered in T-ECs and T-EPCs. Nevertheless, these ionotropic receptors could be regarded as a promising target for alternative anti-angiogenic therapies.

Gasotransmitters-Activated Ca 2+ -Permeable Channels
Gaseous mediators or gasotransmitters are endogenous signaling messengers that, although being toxic at high concentrations, regulate a multitude of physiological processes, ranging from the regulation of vascular tone to synaptic plasticity and mitochondrial bioenergetics [155][156][157][158]. The gasotransmitters NO and hydrogen sulphide (H 2 S) have recently been shown to stimulate endothelial cells through an increase in [Ca 2+ ] i [6,156,159], while the role of CO in angiogenesis is less clear [160]. NO promotes angiogenesis and disease progression in several types of malignancies [161,162], including breast cancer [163]. The administration of two structurally unrelated NO donors, i.e., S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP), was recently found to trigger Ca 2+ influx and migration in B-TECs [164]. These effects were mimicked by elevating endogenous NO release with L-arginine [164], which is the physiological substrate for eNOS [156]. Of note, AA-induced TRPV4 activation in B-TECs was inhibited by preventing NO production with N G -nitro-L-arginine methyl ester (L-NAME) [88,164]; moreover, AA-and NO-induced Ca 2+ entry were both sensitive to protein kinase A (PKA) inhibition [164]. It is, therefore, likely that NO elicits Ca 2+ entry in B-TECs by gating TRPV4. In agreement with this hypothesis, TRPV4 may be activated by NO through direct S-nitrosylation [71] and is phosphorylated by PKA upon AA stimulation in vascular endothelial cells [70]. Finally, NO-induced Ca 2+ entry and migration were dramatically reduced in HDMECs [164], in which TRPV4 expression was significantly down-regulated [88]. Besides TRPV4, however, NO is able to recruit multiple TRP channels, such as TRPC1, TRPC4, TRPC5, TRPV1, and TRPV3 [71], some of which are up-regulated in T-ECFCs [95,165]. Unfortunately, it is still unclear whether NO elicits intracellular Ca 2+ entry in these cells. Although future work is mandatory to understand whether NO stimulates TRP channels, as well as other Ca 2+ -permeable channels, to promote tumor vascularization, endothelial Ca 2+ signaling is emerging as an attractive target to prevent its pro-tumorigenic effect.
H 2 S has also been shown to promote angiogenesis in a Ca 2+ -dependent manner. For instance, H 2 S mediated VEGF-induced Ea.hy926 cell proliferation and migration by inducing InsP 3 -dependent ER Ca 2+ release without the contribution of extracellular Ca 2+ entry [6]. The components of the endothelial Ca 2+ toolkit recruited by H 2 S may, however, vary depending on the vascular bed [166]. H 2 S induced ER-dependent Ca 2+ release through InsP 3 Rs and RyRs followed by a sustained SOCE in primary cultures of human saphenous vein endothelial cells [167], whereas it recruited the reverse mode of NCX by gating a Na + -and Ca 2+ -permeable pathway in rat aortic endothelial cells [168] and HDMECs [21]. Conversely, NaHS did not elicit any resolvable elevation in [Ca 2+ ] i in ECFCs [6] and its role in neovasculogenesis in vivo operated by truly endothelial precursors remains to be elucidated [169]. H 2 S-induced Ca 2+ signals may be translated into a pro-angiogenic signal by multiple Ca 2+ -dependent decoders, including the PI3K/Akt and the ERK/p38 signaling pathways [155,156]. Growing evidence demonstrated that H 2 S drove disease progression and angiogenesis in several types of tumor, such as RCC and colorectal cancer [169]. Intriguingly, sodium hydrosulfide (NaHS), a widely employed H 2 S donor, induced intracellular Ca 2+ signals in both B-TECs and HDMECs; however, NaHS-elicited Ca 2+ signals were enhanced and arose within a significantly lower range (nanomolar vs. micromolar) in B-TECs [21]. Consequently, NaHS promoted proliferation and migration in B-TECs, but not in control endothelial cells [21]. The Ca 2+ response to H 2 S was mediated by a Ca 2+ -permeable non-selective cation channel and was sustained by membrane hyperpolarization through the activation of a K + conductance [21], likely an ATP-sensitive K + channel [157]. The molecular nature of this Ca 2+ -permeable route is yet to be identified [166]. Nevertheless, H 2 S is able to stimulate TRPV3 and TRPV6 in bone marrow-derived mesenchymal cells by direct sulfhydration of some of Cys residues within their protein structure [170]. Moreover, H 2 S activated TRPA1 in RIN14B cells [171]. Deciphering the molecular target of H 2 S in tumor endothelium is, therefore, mandatory to devise alternative anti-cancer treatments. In addition, both eNOS and cys-tathionine gamma lyase (CSE), the enzyme which catalyzes H 2 S production in vascular endothelial cells, are Ca 2+ -sensitive [16,42,172]. Therefore, targeting a Ca 2+ entry/release pathway tightly coupled to either eNOS (i.e., Orai1, [173]) or CSE (yet to be identified) has the potential to interfere with multiple pro-angiogenic pathways and, therefore, exert a more profound anti-tumor effect.

Connexin 40 (Cx40)
Connexin (Cx) hemichannels, also termed connexons, have long been known to provide the building blocks of gap junctions, thereby enabling the transfer of small solutes, ions and signaling molecules, such as Ca 2+ and InsP 3 , between adjacent cells [174]. Three diverse Cx isoform exist in vascular endothelial cells, i.e., Cx37, Cx40, and Cx43, and synchronize robust NO release induced by extracellular autacoids by mediating intercellular Ca 2+ communication [175,176]. In addition, unopposed Cx hemichannels were found to mediate extracellular Ca 2+ entry and NO release in endothelial cells from different vascular beds [16,74,[177][178][179]. Earlier work suggested that Cxs served as tumor suppressors and were down-regulated in cancer, thereby affecting vascular integrity and reducing vascular leakage [180][181][182]. However, a recent study challenged this dogma by showing that Cx40 was over-expressed in T-ECs and promoted disease progression and angiogenesis in melanoma and urogenital cancers [102]. Cx40 stimulated tumor growth by inducing eNOS recruitment, which strongly suggest that intracellular Ca 2+ levels increased during the angiogenic process [102]. Intriguingly, targeting Cx40 function with 40 Gap27, a peptide that has long been use to inhibit Cx40-mediated intercellular communication and extracellular Ca 2+ entry [1,16,178], normalized tumor vasculature and enhanced the efficacy of the chemotherapeutic drug, cyclophosphamide [102]. Therefore, although these findings remain to be confirmed in other tumor types, and the role served by Ca 2+ is still unclear, Cx40 deserves careful consideration for the design of new anticancer drugs.

Two-Pore Channels (TPCs)
The ER is the largest endogenous Ca 2+ store in vascular endothelial cells by accounting for ≈75% of the total Ca 2+ storage capacity [2]. The remainder 25% of the total stored Ca 2+ is located within the mitochondria and the acidic Ca 2+ stores of the endolysosomal (EL) system [2]. As more widely illustrated in [196], the EL Ca 2+ store releases Ca 2+ through many Ca 2+ -permeable channels, including Mucolipin TRP 1 (TRPML1), Melastatin TRPM 2 (TRPM2) and two-pore channels 1 and 2 (TPC1-2) [197,198]. The newly discovered second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), is the physiological stimulus that gates TPC1-2 in response to extracellular stimulation [196,199,200]. NAADP-induced EL Ca 2+ release is, in turn, amplified by juxtaposed ER-embedded InsP 3 Rs and RyRs through the CICR process, thereby initiating a regenerative Ca 2+ wave [196,200,201]. NAADP-gated TPC2 channels are also expressed in vascular ECs [202,203], whereas N-ECFCs display larger amounts of TPC1 [86,204]. A recent study demonstrated that NAADP-induced Ca 2+ signals promoted tumor vascularization and metastasis in murine models xenografted with B16 melanoma cells [205]. Of note, the pharmacological blockade of NAADP-induced Ca 2+ release with Ned-19 dampened melanoma growth, vascularization and lung metastasis [205]. Future work will have to assess whether TPC2 channels are up-regulated in T-ECs and whether NAADP-induced Ca 2+ signaling also drive T-ECFC incorporation into tumor neovessels. However, TPCs stand out as promising targets to develop alternative anti-angiogenic treatments.

Canonical Transient Receptor Potential 5 (TRPC)
TRPC5 forms a homotetrameric Ca 2+ -permeable channel that is gated upon PLCβ activation by Gq/11-coupled membrane receptors through a yet to be identified signaling cascade [2,206,207]. Accordingly, although some studies reported that TRPC5 is recruited in a store-dependent manner by Stim1 [208], it has been proposed that TRPC5 activation by PLCβ does not involve ER store depletion [209]. In addition, TRPC5-mediated Ca 2+ entry is elicited by several physiological messengers, including reduced thioredoxin, protons, sphingosine-1-phosphate, lysophospholipids, NO and Ca 2+ itself [207]. Finally, TRPC5 presents a spontaneous activity that is increased by lanthanides, cold temperatures (47 • C to 25 • C) and membrane stretch; consequently, TRPC5 serves as a cold sensor in the peripheral nervous system [210]. Of note, TRPC5 may establish physical associations with a multitude of molecular partners, including TRPC1, TRPC4 and TRPC6, which regulate its membrane localization and biophysical properties [207]. TRPC5 differently tunes angiogenesis depending on the vascular bed. For instance, TRPC5 promoted proliferation and tube formation by inducing intracellular Ca 2+ oscillations in EA.hy926 cells [211]. Conversely, a TRPC6-TRPC5 channel interaction inhibited angiogenesis by decreasing the rate of migration in bovine aortic ECs (BOECs). In this context, TRPC6-mediated Ca 2+ entry triggered an ERK-mediated phosphorylation cascade that leads to MLCK activation and TRPC5 externalization on the plasma membrane [212,213]. It has recently been shown that endothelial TRPC5 could underlie the development of chemoresistance to anticancer drugs in both breast cancer [104,214,215] and colorectal carcinoma [216]. P-glycoprotein (P-gp), also termed multidrug resistance protein 1 (MDR1), is a multidrug efflux transporter that expels xenobiotics out from the cytoplasm into the extracellular milieu [217]. P-gp overexpression, therefore, confers resistance to malignant cells, which become insensitive to a wide range of cancer chemotherapeutics, including adriamycin, vincristine, taxol, and anthracyclines [217]. Earlier evidence demonstrated that TRPC5 was up-regulated and induced P-gp overexpression by hyper-stimulating the Ca 2+ -dependent transcription factor, NFATc3 in chemoresistant MCF-7 breast cancer cells [214]. In agreement with this observation, microRNA 320a (miR-320a), which is able to associate with and degrade TRPC5 and NFATc3 transcripts in normal cells, was down-regulated in chemoresistant breast cancer cells due to the hypermethylation of its promoter sequence [218]. TRPC5 up-regulation induced resistance to adriamycin, paclixatel, epirubicin, mitoxantrone and vincristine [214]. Additionally, TRPC5-mediated Ca 2+ entry promoted transcription of HIF-1α gene, thereby boosting VEGF release and enhancing tumor angiogenesis [219]. Remarkably, TRPC5-based chemoresistance could be shuttled to tumor endothelial via intercellular communication. Adriamycin-resistant MCF-7 cells could pack the up-regulated TRPC5 channels into mobile extracellular vesicles (EVs), which are released in tumor microenvironment and transferred their signaling content to surrounding endothelial cells. This scenario is supported by the observations that HDMECs exposed to TRPC5-contanining EVs, which were collected from adriamycin MCF-7 breast cancer cells, over-expressed the TRPC3-NFATc3-P-gp signaling pathway and developed resistance to adriamycin-induced apoptosis [104]. Moreover, TRPC5-containing vesicles were identified in peripheral blood of breast cancer patients receiving chemotherapy and of nude mice bearing adriamycin-resistant MCF-7 tumor xenografts [215]. Furthermore, P-gp production was enriched in tumor endothelium of adriamycin-resistant MCF-7 xenografts than in other sites and was sensitive to TRPC5 inhibition with a specific siRNA (siTRPC5) [104]. These data, therefore, suggest that TRPC5 provide a promising target to design alternative adjuvant anticancer treatments [220]. Accordingly, a blocking TRPC5 antibody reduced P-gp expression, retarded cancer growth and boosted paclitaxel-induced tumor regression in chemoresistant breast cancer in vivo [104,214,215]. The endothelial effects of TRPC5 in breast cancer are seemingly limited to T-ECs, as BC-ECFCs do not express this channel [96]. Future work will have to assess whether, besides conferring B-TECs with the resistance to chemotherapeutic drugs, TRPC5 up-regulation accelerates breast cancer angiogenesis.

Inositol-1,4,5-Trisphosphate (InsP 3 ) Receptors (InsP 3 Rs)
InsP 3 Rs are non-selective cation channels which mediate ER-dependent Ca 2+ release, thereby controlling multiple endothelial cell functions, including bioenergetics, apoptosis, angiogenesis and vasculogenesis (see Paragraph 2. Ca 2+ signaling in normal endothelial cells: a brief introduction). Three distinct InsP 3 R isoforms exist in both vascular endothelial cells and ECFCs [2,29], i.e., InsP 3 R1, InsP 3 R2 and InsP 3 R3, which may associate into homo-or hetero-tetrameric ER-embedded channels [2]. It has recently been shown that InsP 3 Rs were dramatically down-regulated in RCC-ECFCs, thereby preventing the onset of VEGF-induced intracellular Ca 2+ oscillations, proliferation and in vitro tubulogenesis [95] (Figure 3). More specifically, RCC-ECFCs only expressed InsP 3 R1, while InsP 3 R2 and InsP 3 R3 were absent [95]. This result was surprising as InsP 3 R1 was transcriptionally regulated by HIF-2 in human RCC cancer cell lines [221]. The failure of the pro-angiogenic Ca 2+ response to VEGF also involves the chronic reduction in the ER Ca 2+ concentration ([Ca 2+ ] ER ) in RCC-ECFCs, as monitored by using an ER-targeted aequorin Ca 2+ indicator [87,222]. Therefore, in contrast with the widely accepted belief that VEGF sustains the angiogenic switch [223], VEGF does not stimulate ECFC-dependent neovessel formation in RCC patients [27,32]. This observation shed novel light on the refractoriness to anti-VEGF therapies in individuals suffering from RCC [224][225][226]. It has long been known that humanized monoclonal anti-VEGF antibodies, such as bevacizumab, or small molecule tyrosine kinase inhibitors, such as sorafenib and sunitinib, did not increase the overall survival of RCC patients, who ultimately developed secondary (acquired) resistance and succumbed because of tumor relapse and metastasis. In addition, targeting VEGF-dependent pathway proved to be ineffective in a large cohort of subjects, who displayed intrinsic refractoriness to these anti-VEGF drugs and did not show any improvement in their progression free survival [227,228]. ECFCs play a key role during the early phases of the angiogenic switch that supports tumor vascularization and metastasis [27,28,229]. If tumor vasculature is dismantled by anti-VEGF drugs, the following drop in P O2 will release in circulation a cytokine storm that attracts ECFCs from their bone marrow and/or vascular niches. ECFCs will home to the shrunk tumor, but, being insensitive to VEGF, will not be affected by the presence of anti-VEGF drugs. Consequently, they will proliferate in response to the mixture of growth factors liberated in tumor microenvironment and will restore blood supply to cancer cells [32]. Remodeling of the Ca 2+ toolkit in RCC-ECFCs could, therefore, underlie the resistance to anti-angiogenic therapies in RCC patients. Similar data were obtained in BC-ECFCs, in which the significant reduction in [Ca 2+ ] ER prevented VEGF from triggering robust intracellular Ca 2+ oscillations, proliferation and tube formation, although the pattern of InsP 3 R expression remained unchanged [37,96]. Again, this result is consistent with notion that also breast cancer patients present intrinsic or secondary refractoriness to anti-VEGF therapies [54,230]. The reduction in [Ca 2+ ] ER observed in several types of tumor-associated ECFCs, including IH-ECFCs [95][96][97], was likely to reflect the down-regulation of SERCA2B activity [87,95]. Accordingly, ATP-induced InsP 3 -dependent ER Ca 2+ release in RCC-ECFCs decayed to resting Ca 2+ levels with slower kinetics as compared to normal ECFCs [95]. Intriguingly, the gene expression profile of RCC-and BC-ECFCs resulted to be dramatically different with respect to normal cells [37]: BC-ECFCs and RCC-ECFCs presented, respectively, 382 and 71 differently expressed genes (DEGs) as compared to healthy cells, including TMTC1 [37]. TMTC1 is a tetratricopeptide repeat-containing adapter protein, which binds to and inhibits SERCA2B, thereby reducing ER Ca 2+ levels and dampening agonist-induced intracellular Ca 2+ release [231]. It is conceivable that TMTC1 up-regulation in T-ECFCs contributes to the chronic underfilling of their ER Ca 2+ reservoir. In further agreement with this observation, electron microscopy revealed that both RCC-and BC-ECFCs presented dramatic ultrastructural differences as compared to control cells [87,96]. In particular, T-ECFCs presented a remarkable expansion of ER volume, whereas mitochondria were more abundant and very often elongated as compared to N-ECFCs [87,96]. This ultrastructural remodeling is consistent with the ER stress caused by the chronic reduction in [Ca 2+ ] ER [232,233].

Targeting the Endothelial Ca 2+ Toolkit to Circumvent the Resistance to Anticancer Treatments
Remodeling of the Ca 2+ toolkit in tumor cells led many authors to search for alternative strategies to treat cancer. Intracellular Ca 2+ signaling controls most, if not all, the so-called cancer hallmarks and could, therefore, be targeted to inhibit or, at least, retard tumor growth and metastasis [35,36,[234][235][236][237][238][239][240]. As shown above, the Ca 2+ transportome is also altered in stromal cancer cells, [32,241,242], including endothelial cells and ECFCs. Remodeling of the endothelial Ca 2+ toolkit could play a crucial role in the refractoriness to anticancer treatments, by supporting tumor vascularization and decreasing the susceptibility to pro-apoptotic stimuli. Therefore, the endothelial Ca 2+ transportome might provide an efficient target for adjuvant therapies to conventional anti-cancer treatments. Three strategies could be pursued to improve the therapeutic outcome of standard therapies by interfering with the endothelial Ca 2+ machinery: (1) blocking Ca 2+ signaling to dampen angiogenesis and/or vasculogenesis; (2) stimulating Ca 2+ entry to normalize tumor vessels, thereby improving the delivery and efficacy of chemo-, radio-and immunotherapy; and (3) manipulating Ca 2+ signaling to endothelial cell apoptosis and dismantle tumor vasculature.
SOCE is, perhaps, the most suitable target to affect tumor vasculature by inhibiting both angiogenesis and vasculogenesis. Although there is no report of SOCE expression in T-ECs, Stim1 and Orai1 control proliferation and tube formation in normal endothelial cells, such as rat CMECs [7], bovine brain capillary endothelial cells [243], mouse lymphatic endothelial cells [244], and HUVEC [57,58]. Moreover, the pharmacological blockade of SOCE attenuates the rate of cell growth and abrogates in vitro tubulogenesis in RCC-, IH-and BC-derived ECFCs [95][96][97]. In addition, SOCE controls proliferation, migration and metastasis in a multitude of different cancer cell lines [134,135], which expands the cellular targets of SOCE inhibitors to the whole tumor microenvironment. We [131,135] and others [133,245] have recently described the Orai1 and TRPC1 inhibitors, some of which have been listed in Table 1, that could serve as a molecular template to design novel anticancer drugs. Unfortunately, none of these drugs have reached the milestone of being approved by US Food and Drug Administration (FDA) due to their scarce selectivity and high toxicity. For instance, carboxyamidotriazole (CAI), a non-selective blocker of Ca 2+ signaling, was originally used to inhibit angiogenesis in vitro and tumor vascularization in vivo [131,[246][247][248]. Depending on the cell type, CAI was able to block SOCE by occluding the mitochondrial Ca 2+ uniporter [249,250] or reducing InsP3 synthesis, which in turn prevents InsP3-dependent Ca 2+ release and Stim activation [56,251,252]. Intriguingly, CAI inhibited proliferation and tube formation also in RCC-and BC-ECFCs by preventing InsP3-dependent ER Ca 2+ release [95,96]. Additionally, CAI was found to block growth and motility in several types of cancer cell lines [252,253]. Therefore, phase I-III clinical trials were launched to assess CAI toxicity and tolerability in patients suffering from several types of malignancies, including RCC, breast cancer, ovarian cancer, melanoma, non-small cell lung

Targeting the Endothelial Ca 2+ Toolkit to Circumvent the Resistance to Anticancer Treatments
Remodeling of the Ca 2+ toolkit in tumor cells led many authors to search for alternative strategies to treat cancer. Intracellular Ca 2+ signaling controls most, if not all, the so-called cancer hallmarks and could, therefore, be targeted to inhibit or, at least, retard tumor growth and metastasis [35,36,[234][235][236][237][238][239][240]. As shown above, the Ca 2+ transportome is also altered in stromal cancer cells [32, 241,242], including endothelial cells and ECFCs. Remodeling of the endothelial Ca 2+ toolkit could play a crucial role in the refractoriness to anticancer treatments, by supporting tumor vascularization and decreasing the susceptibility to pro-apoptotic stimuli. Therefore, the endothelial Ca 2+ transportome might provide an efficient target for adjuvant therapies to conventional anti-cancer treatments. Three strategies could be pursued to improve the therapeutic outcome of standard therapies by interfering with the endothelial Ca 2+ machinery: (1) blocking Ca 2+ signaling to dampen angiogenesis and/or vasculogenesis; (2) stimulating Ca 2+ entry to normalize tumor vessels, thereby improving the delivery and efficacy of chemo-, radio-and immunotherapy; and (3) manipulating Ca 2+ signaling to endothelial cell apoptosis and dismantle tumor vasculature. SOCE is, perhaps, the most suitable target to affect tumor vasculature by inhibiting both angiogenesis and vasculogenesis. Although there is no report of SOCE expression in T-ECs, Stim1 and Orai1 control proliferation and tube formation in normal endothelial cells, such as rat CMECs [7], bovine brain capillary endothelial cells [243], mouse lymphatic endothelial cells [244], and HUVEC [57,58]. Moreover, the pharmacological blockade of SOCE attenuates the rate of cell growth and abrogates in vitro tubulogenesis in RCC-, IH-and BC-derived ECFCs [95][96][97]. In addition, SOCE controls proliferation, migration and metastasis in a multitude of different cancer cell lines [134,135], which expands the cellular targets of SOCE inhibitors to the whole tumor microenvironment. We [131,135] and others [133,245] have recently described the Orai1 and TRPC1 inhibitors, some of which have been listed in Table 1, that could serve as a molecular template to design novel anticancer drugs. Unfortunately, none of these drugs have reached the milestone of being approved by US Food and Drug Administration (FDA) due to their scarce selectivity and high toxicity. For instance, carboxyamidotriazole (CAI), a non-selective blocker of Ca 2+ signaling, was originally used to inhibit angiogenesis in vitro and tumor vascularization in vivo [131,[246][247][248]. Depending on the cell type, CAI was able to block SOCE by occluding the mitochondrial Ca 2+ uniporter [249,250] or reducing InsP 3 synthesis, which in turn prevents InsP 3 -dependent Ca 2+ release and Stim activation [56,251,252]. Intriguingly, CAI inhibited proliferation and tube formation also in RCC-and BC-ECFCs by preventing InsP 3 -dependent ER Ca 2+ release [95,96]. Additionally, CAI was found to block growth and motility in several types of cancer cell lines [252,253]. Therefore, phase I-III clinical trials were launched to assess CAI toxicity and tolerability in patients suffering from several types of malignancies, including RCC, breast cancer, ovarian cancer, melanoma, non-small cell lung carcinoma, and gastrointestinal (stomach and pancreas) adenocarcinomas [56,248,254,255]. As discussed elsewhere [131], this drug caused disease stabilization when administrated alone or as adjuvant of chemo-or radio-therapy, and induced well tolerable side effects in most patients, such diarrhea, nausea and/or vomiting, fatigue and constipation. The therapy was discontinued only in RCC patients, who underwent disease progression and suffered from unacceptable toxicities, such as neuropsychiatric difficulties and asthenia [256]. As mentioned earlier, however, the effect of CAI is not directed towards the SOCE machinery, but is indirect. In addition to SOCE, CAI may also block TRPV4 and ER leakage channels [89,95,96,131]. A recent investigation, however, screened a library of >1800 FDA-approved drugs to search for specific SOCE blockers and identified five novel compounds, i.e., leflunomide, teriflunomide, lansoprazole, tolvaptan and roflumilast, that could be successfully used in therapy (leflunomide and terifluonomide) or provide the template to design more selective Orai1 inhibitors (i.e., lansoprazole, tolvaptan and roflumilast) [257].
An alternative strategy consists in stimulating endothelial Ca 2+ signaling to induce tumor normalization by activating distinct Ca 2+ entry routes depending on the tumor type. For instance, TRPV4-mediated Ca 2+ entry drives tumor normalization in LLC [90,91], whereas P 2X7 receptors could be targeted to normalize tumor vasculature in breast cancer [94]. Tumor normalization, in turn, represents a promising adjuvant approach to facilitate cancer therapy by increasing the diffusion of chemotherapeutic drugs, improving radiotherapy efficiency and favoring the recruitment of tumor-killing immune cells [118,258]. Several synthetic agonists may selectively induce TRPV4 opening, such as 4αPDD derivatives, RN-1747, and JNc-440. Moreover, GlaxoSmithKline commercialized several patent applications of small molecule TRPV4 activators, the most famous of which is GSK [105,259]. Likewise, BzATP is regarded as the most potent P 2X7 receptor agonist, while 2-meSATP and ATPγS are only partial agonists and αβ-meATP and βγ-meATP exert a rather weaker on activation [260]. Clearly, further studies are required to uncover additional components of the endothelial Ca 2+ toolkit potentially implicated in tumor normalization. Nevertheless, a recent investigation reported that angiopoietins, which induce vessel maturation by regulating the interaction between luminal endothelial cells and mural cells, such as vascular smooth muscle cells and pericytes, stimulate HUVEC migration by promoting ER-dependent Ca 2+ release through InsP 3 Rs and RyRs [261]. These findings lend further support to the hypothesis that targeting the endothelial Ca 2+ signaling provides a suitable means to accelerate the dismantling of tumor vasculature by standard anticancer therapies.
Finally, the endothelial Ca 2+ machinery could be properly manipulated to enhance the pro-apoptotic outcome of chemo-and radiation-therapy. For instance, TRPC5-mediated Ca 2+ entry could be inhibited in B-TECs by taking advantage of a battery of novel small molecule inhibitors, such as Pico145 [262], 3,5,7-trihydroxy-2-(2-bromophenyl)-4H-chromen-4-one (AM12) [263], 2-aminobenzimidazole derivatives [264], ML204 [265], and neuroactive steroids [266]. Alternatively, the [Ca 2+ ] ER could be augmented to such an extent to induce the pro-apoptotic InsP 3 -driven ER-to-mitochondria Ca 2+ transfer. Pinton's group demonstrated that phototherapy induces a p53-dependent increase in [Ca 2+ ] mit , which leads to tumor disruption in vivo [239,267]. Moreover, cytotoxic ER-dependent Ca 2+ mobilization could be promoted by conjugating thapsigargin, a selective SERCA inhibitor, with a protease-specific peptide carrier, which is cleaved by the prostate-specific membrane antigen (PMSA) [268]. PMSA is widely expressed in the endothelium of many solid tumors [269,270], including RCC, thereby selectively favoring thapsigargin release in TME and inducing cancer and stromal cell apoptosis [268,271]. This prodrug has been termed mipsagargin or prodrug G202 and has recently been probed in a phase I clinical trials in patients suffering from refractory, advanced or metastatic solid tumors [272]. We do not know yet whether [Ca 2+ ] ER is also decreased in the endothelium of tumor neovessels, as ECFCs are likely to be replaced/diluted by local endothelial cells after the angiogenic switch [27]. Nevertheless, mipsagargin is likely to cause pro-apoptotic Ca 2+ release in all stromal cells, including T-ECs.
As outlined elsewhere [23,32,135], caution is warranted when targeting a ubiquitous intracellular second messenger, such as Ca 2+ . It should, however, be pointed out that several inhibitors of voltage-gated Ca 2+ channels, such as verapamil, nifedipine and nitrendipin, are routinely employed in clinical practice to treat severe cardiovascular disorders, including hypertension, arrhythmia, acute myocardial infarction-induced heart failure and chronic stable angina [135,273]. In agreement with this observation, a phase I clinical trial is currently assessing the therapeutic outcome of Ca 2+ electroporation on cutaneous metastases of solid tumors as compared to standard electrochemotherapy with bleomycin (https://clinicaltrials.gov/ct2/show/NCT01941901). Ca 2+ electroporation is predicted to enhance the rate of cancer cell death by resulting in cytotoxic Ca 2+ accumulation in the cytosol and in mitochondria [36]. Finally, the pharmacological inhibition of intracellular Ca 2+ signaling did not elicit any intolerable side effects, such as immune depression, bleeding or neuropathic disorders, in least three distinct models of human cancer xenografts [24, 205,274].

Conclusions
The present article discussed how remodeling of the endothelial Ca 2+ toolkit (or transportome) could contribute to the resistance to anti-cancer treatments, which hampers from the very beginning their therapeutic outcome (intrinsic resistance) or leads to tumor relapse (acquired resistance) and patients' death. The intimate relationship between endothelial Ca 2+ signaling and refractoriness to anti-cancer treatments cannot be fully appreciated by studying normal/healthy endothelial cells [32]. For instance, the role of VEGF in promoting tumor neovascularization has been extensively acknowledged based upon the observation that VEGF triggers pro-angiogenic Ca 2+ signals in normal endothelial cells [6,58,251] and ECFCs. Nevertheless, VEGF does not stimulate proliferation and tube formation in T-ECFCs, which play a crucial role in sustaining the angiogenic switch and are likely to restore tumor vasculature prior to recurrence of disease progression. Therefore, to be effective in the patients, a strategy aiming at targeting the Ca 2+ toolkit must be first probed on tumor-associated endothelial cells and ECFCs in vitro, as the their Ca 2+ machinery could be different from that of naïve cells. The protocol to isolate ECFCs from peripheral blood does not require an unreasonable volume of blood (≈40 mL), but it takes no less than three weeks [31] due to lack of ECFCs-specific membrane antigens. It will be imperative to speed up this procedure to accelerate the therapeutic translation of the findings generated by basic research. Isolating T-ECs represents a more technically demanding challenge, but several strategies were designed to collect and expand T-ECs from several types of solid cancers [275]. Further work on patients-derived T-ECs or T-ECFCs is mandatory to identify novel components of the endothelial Ca 2+ toolkit involved in the refractoriness to anti-cancer therapies. Most of the attention is, of course, currently paid to Stim1 and Orai1 [33,131] and to the multiple TRP channel subfamilies that drive physiological angiogenesis [2,131,276,277]. Additional components of the endothelial Ca 2+ transportome deserve careful investigation. For instance, Orai3 was found to up-regulated in several types of T-ECFCs [95,165], but its role in tumor vascularization is currently unknown. Of note, Orai3 may replace Orai1 as the pore-forming subunit of store-operated channels in cancer cells and could, therefore, emerge as a promising target for anti-cancer therapies [278]. NCX provides another unconventional Ca 2+ entry that regulates proliferation and tube formation in healthy endothelial cells [18,279], but has been scarcely investigated in tumor neovessels. Finally, nAchRs are not the only ionotropic receptors expressed in vascular endothelial cells. N-methyl-D-aspartate receptors (NMDARs) are widely expressed in brain microvascular endothelial cells [280], in which they recruit eNOS and stimulate NO release in response to synaptic activity [281]. Aberrant glutamate signaling has been associated to glioma growth [282] and NMDARs-mediated Ca 2+ entry could engage Ca 2+ -dependent decoders other than eNOS in brain endothelium. Therefore, the expression and role of endothelial NMDARs in glioblastoma should be carefully evaluated. [CrossRef] [PubMed]