Endocannabinoid System and Tumour Microenvironment: New Intertwined Connections for Anticancer Approaches

The tumour microenvironment (TME) is now recognised as a hallmark of cancer, since tumour:stroma crosstalk supports the key steps of tumour growth and progression. The dynamic co-evolution of the tumour and stromal compartments may alter the surrounding microenvironment, including the composition in metabolites and signalling mediators. A growing number of evidence reports the involvement of the endocannabinoid system (ECS) in cancer. ECS is composed by a complex network of ligands, receptors, and enzymes, which act in synergy and contribute to several physiological but also pathological processes. Several in vitro and in vivo evidence show that ECS deregulation in cancer cells affects proliferation, migration, invasion, apoptosis, and metastatic potential. Although it is still an evolving research, recent experimental evidence also suggests that ECS can modulate the functional behaviour of several components of the TME, above all the immune cells, endothelial cells and stromal components. However, the role of ECS in the tumour:stroma interplay remains unclear and research in this area is particularly intriguing. This review aims to shed light on the latest relevant findings of the tumour response to ECS modulation, encouraging a more in-depth analysis in this field. Novel discoveries could be promising for novel anti-tumour approaches, targeting the microenvironmental components and the supportive tumour:stroma crosstalk, thereby hindering tumour development.


The Endocannabinoid System
Thirty years after the isolation of Delta-9-tetrahydrocannabinol (∆9-THC) and cannabinol (CBD), the two phytocannabinoids mostly represented in the inflorescences of Cannabis Sativa, the endocannabinoid system (ECS), a complex intercellular communication system, was identified and characterised. It is composed by bioactive lipid derivatives with binding affinity similar to THC and CBD, known as endocannabinoids (eCBs), a heterogeneous class of receptors and a complex of enzymes responsible for the synthesis, transport, and hydrolysis of eCBs.
The production of eCBs begins with a synthesis "on demand" from membrane phospholipid precursors. N-acyl transferase (NAT) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) are the canonical enzymes which synthesise AEA [1][2][3]. On the other hand, a first hydrolysis by phospholipase C-β (PLC-β) and a second reaction by diacylglycerol lipase (DAGL) are required for 2-AG formation [4][5][6]. Once synthetised, The ECS controls many biological events, such as synaptic plasticity, neuroprotection, immune response modulation, energy homeostasis [11][12][13], and over the past twenty years, a growing number of evidence has shown that its alteration correlates with the onset of various diseases including cancer [14,15].

Receptors
Cannabinoid receptors (CBRs) are members of the large family of seven α-helical transmembrane G protein coupled receptors (GPCRs) activating Gi/0 proteins [16,17]. CB1R (chromosome 6q15) is predominantly located in central nervous system where it modulates the release of neurotransmitters, playing a pivotal role in memory, motor coordination and emotional processes [18][19][20]. Some peripheral tissues, such as liver, heart, skeletal muscle, adipose tissue, and gastro-intestinal tract, also express CB1R [21,22]. On the other hand, CB2R (chromosome 1p36.11) is predominantly expressed on lymphoid organs and immune cells eliciting anti-inflammatory and immunosuppressive functions. However, it was also identified in certain regions of nervous system [23].

Receptors
Cannabinoid receptors (CBRs) are members of the large family of seven α-helical transmembrane G protein coupled receptors (GPCRs) activating G i/0 proteins [16,17]. CB1R (chromosome 6q15) is predominantly located in central nervous system where it modulates the release of neurotransmitters, playing a pivotal role in memory, motor coordination and emotional processes [18][19][20]. Some peripheral tissues, such as liver, heart, skeletal muscle, adipose tissue, and gastro-intestinal tract, also express CB1R [21,22]. On the other hand, CB2R (chromosome 1p36.11) is predominantly expressed on lymphoid organs and immune cells eliciting anti-inflammatory and immunosuppressive functions. However, it was also identified in certain regions of nervous system [23].
PPARα and PPARγ represent novel discovered CBRs. PPARα is widely distributed in metabolically active tissues (e.g., liver, heart, and muscle), controlling fatty acid catabolism and inflammatory processes, while PPARγ (isoform -γ1, -γ2, and -γ3) is differentially expressed in tissues (-γ1 ubiquitous; -γ2 in adipose tissue; -γ3 in macrophages) and involved in adipocyte formation, insulin sensitivity, inflammation [53,86]. Fibrates and thiazolidinediones are the main ligands of PPARα and PPARγ, respectively, and they are used clinically in the treatment of hyperlipidemia (fibrates) and in the treatment of type 2 diabetes (thiazolidinediones). To date, it is known that several cannabinoids can activate these receptors (e.g., PEA, OEA activate PPARα; e.g., THC, AEA and 2-AG activate PPARγ) Cells 2021, 10, 3396 7 of 34 and that PPARs have an involvement in cancer (e.g., colon, ovarian, breast and prostate cancer) [55,56]. However, many aspects have to be clarified and, therefore, there is a clear need for additional studies in this context.
Interestingly, recent discoveries pointed out that CBRs can create heterodimers with other receptors. In particular, it is known that GPR55 dimerises with CB2R in several tumours such as breast cancer, with implications in cancer malignancy [87]. Moreover, these heteromers are over-expressed in bones and hematopoietic cells, probably regulating cancer-related processes. CB2R can also be coupled to CXCR4 in breast and prostate cancers where the heteromers impact on cell proliferation, adhesion, invasion, and metastatic processes. Therefore, it was found that CB2R agonists can reduce CXCR4 activity and hinder the effects resulting from CXCR4-agonists [88,89]. A recent known hallmark of cancer is the heteromers CB2R-HER2, typical of breast cancers. In this context, it was demonstrated that cannabinoid agonists at CB2R can lead to disruption of these heteromers, hampering HER2 activity (discussed below) [66,90].

Cannabinoid Receptor Agonists
Cannabinoid receptor agonists represent a group of phyto-, endo-, and synthetic cannabinoids able to bind cannabinoid receptors with different affinities and efficacies. Based on their chemical structure, they are distinguished into classical, non-classical compounds, aminoalkylindoles, and eicosanoids [91].
Non-classical ligands share the similar bicyclic and tricyclic structure of ∆9-THC, lacking the pyran ring. Among these, CP 55,940 works as a full agonist at both CBRs, acting in a nanomolar range of affinity. Although this compounds directly target both CB1R and CB2R, the higher specificity for CB2R represent a molecular approach to overcome the psychotropic effects given by CB1R. Indeed, CB2R selective agonists, such as JWH-133 (classical), JWH-015, and AM1241 (aminoalkylindoles), are becoming optimal candidates for anticancer therapy.
The cannabimimetic aminoalkylindole WIN 55,212-2 shares an intrinsic activity for both CBRs similar to CP 55,940 and HU-210, however it showed to be more specific for CB2R [92,93].
For what concerns eicosanoids, the class principally includes AEA and 2-AG, endogenous ligands for CB1R and CB2R, although AEA is a partial agonist for CB2R with respect to 2-AG [91]. Together with L-α-lysophosphatidylinositol (LPI) and virodhamine, AEA and 2-AG modulate the activity of GPR55 [94]. Moreover, AEA is considered a full agonist (endovanilloid) at TRPV1 in different tumour cell lines, while 2-AG is weakly active on TRPV1 [95]. Finally, it has been also reported that AEA and 2-AG activate PPARα and -γ [96].
Although still under study, the anti-cancer activity of several CBRs' agonists has now been assessed in many types of tumours ( Table 2). Most of them exert anti-proliferative and apoptotic outcomes by activating different receptors and signalling pathways dependent on the specific cancer types (see the table for detail). A reduction in the migratory/invasive potential of cancer cells was also reported for some CBR agonists, mainly driven by the inhibition of metalloproteinases.

Other Agonists
R(+)-Methanandamide and Metfluoroanandamide (Met-F-AEA) are two non-hydrolysable and metabolically stable AEA analogues, with main affinity for CB1R. In prostate and human cervical cancer cells, R(+)-Methanandamide exerted anti-proliferative and proapoptotic functions, respectively [97,98]. The compound induced an arrest of cell cycle in G0/G1 phase and induced necrosis in gastro-intestinal cancer in vitro [99]. In breast cancer Met-F-AEA inhibited cell cycle, tumour cell adhesion and migration, interfering with the RhoA/ROCK signalling pathway and FAK phosphorylation [100][101][102]. In lung, gastrointestinal and thyroid tumours apoptosis, and cell growth were modulated [103][104][105]. The inhibition of cell growth was also reported in melanoma in vitro [106].
PEA is an endogenous fatty acid amide related to AEA. It principally agonises PPARα and also activates GPR55 and TRPV1, with modalities yet to be clarified. Moreover, it is known that PEA is an indirect activator of CBRs, as inhibitor of FAAH which increases AEA and 2-AG levels [107]. It induced cell death in high-grade astrocytoma/neuroblastoma cells, it slowed down melanoma cell survival; moreover, it potentiated the cytotoxic effect of AEA on human breast cancer in vitro [52,108,109].
As aforementioned, JWH-015 and JWH-133 are selective for CB2R. In PC-3 prostate cancer, metastatic breast cancer MCF-7 cells and lung cancer cell lines, JWH-015 had antiproliferative action; moreover, it inhibited the activation of EGFR in ERα breast cancer cells [98,115,116]. In lung cancer the compound attenuated growth factor-direct in vitro chemotaxis and chemo-invasion by reducing focal adhesion complexes, inhibiting Akt phosphorylation and MMP-9 expression/activity [25]. JWH-133 counteracted proliferation and migration of glioma, breast cancer cells and decreased trans-endothelial migration of melanoma cells [117][118][119].

Cannabinoid Receptor Antagonists/Inverse Agonists
Due to the "double face" of cannabinoid receptors, that are not only involved in tumour suppression but also in tumour development and progression, research in the field of cannabinoids has progressed further towards the synthesis of receptor antagonist compounds (Table 3). In this regard, SR141716 (Rimonabant) and CBD are well-known CB1R antagonists. Specifically, CBD can also act as inverse agonist/negative allosteric modulator at CB1R and partial agonist at CB2R [120,121]. In addition, several reports demonstrated that it can activate TRPV1, PPAR-γ and antagonise GPR55 [122][123][124]. AM251 and 6-iodopravadoline (AM-630) are two synthetic cannabinoid CB2R inverse agonists. However, AM251 is also reported to activate GPR55 [125]. CID16020046 is a selective GPR55 antagonist recently used in the anti-cancer research.
In breast cancers SR141716 blocked cell proliferation, effect also found in gastrointestinal tumours where the compound, after arresting tumour cell population in G2/M phase, induced mitotic catastrophe [126,127]. In glioma cells SR141716 activated caspasedependent apoptosis via G1 phase stasis and it up-regulated the cell membrane MICA/B, a potent a stress-induced ligand for the natural-killer group 2, member D (NKG2D) receptor, expressed in NKs [128]. Moreover, Fiore et al. reported that SR141716 impacted in chemoresistance and cancer stemness through inhibition of Wnt/β-catenin pathway in primary colon cancer stem cells (CSCs) [129].
CBD induced apoptosis pathways in gastro-intestinal cancers through excessive ROS production, ER stress and Noxa activation [130,131]. Apoptosis linked to ER-stress was also reported in breast, prostate and in GBM [132][133][134]. Interestingly, in lung and breast cancers CBD stimulated apoptosis by direct and indirect regulation of PPAR-γ [135,136]. In breast cancer, CDB induced a crosstalk between apoptosis and autophagy modulating cancer cell death [137]. CDB induced cell cycle arrest and reduction in cell proliferation in gastro-intestinal, breast, prostate and in brain cancers [130,133,138]. The reduction in cell migration and invasion was reported in gastro-intestinal cancer, lung cancer, and breast Cells 2021, 10, 3396 9 of 34 cancer [138][139][140][141][142]. In GMB and glioma, CBD reduced cell invasion through inhibition of Id-1 expression and downregulation of proteins specifically involved in growth, invasion and angiogenesis (e.g., MMP-9, TIMP-4, VEGF, TGF-β) [143,144]. Moreover, CBD increased the uptake of doxorubicin (DOXO) in breast cancer cells, it detained synergistic anti-proliferative effects with docetaxel and/or bicalutamide in prostate cancer cells, it increased chemosensitivity to Temozolomide (TMZ), Carmustine (BCNU), and DOXO in glioma [134,145,146]. However, in glioma, CBD failed to exert good cytotoxicity if compared to its activity in combination with HSP inhibitors [147].
AM251 decreased proliferation and migration in renal cell carcinoma. Moreover, it induced apoptosis by upregulating Bax and decreasing Bcl-2 expression [148]. Induction of apoptosis was also addressed in pancreatic cancer via receptor independent mechanisms. Despite these effects, it was fond that the compound reverts the anti-tumour activities of Met-F-AEA and ACEA, in gastro-intestinal and breast cancer, respectively, impacting on proliferation and cancer cell potential [104,112]. Few evidences are reported for AM-630 and CID16020046 in tumours, however it is known that AM-630 induced cell cycle arrest in G2/M phase and it inhibited migration in renal cell carcinoma [149]. In gastro-intestinal cancer CID16020046 inhibited cancer cell growth through downregulation of ERK1/2 phosphorylation and it decreased migration and the ability of adhesion to endothelial cells [150,151]. In breast cancer the compound reduced migration and chemoresistance through downregulation of multidrug resistance exporters, such as the breast cancer resistance protein (BCRP) [152].

Cannabinoid Enzymatic Dysregulations in Cancer
Synthesis and degradation of endocannabinoids were found deregulated in several malignant tissues. Downregulation and altered activity of NAPE-PLD and FAAH, followed by a reduction in AEA, was found in glioma tissues where, on the contrary, increased 2-AG levels correlated with a decrease in MAGL and an unchanged DAGL-α expression [153]. In endometrial carcinoma, AEA amounts were higher with respect to healthy tissues, as a consequence of FAAH reduction and NAPE-PLD increase [154]. On the contrary, in prostate adenocarcinomas biopsies FAAH protein expression increased in comparison to non-tumour biopsies [155]. In HCC MAGL expression was higher in patient with poor prognosis [156]. To date, MAGL and FAAH inhibitors are the most studied cannabinoid enzymatic targeting strategies.

MAGL Inhibitors
JZL184 and URB-602 are two examples of MAGL inhibitors. Nomura et al. attested that JZL184 impacted on PC3 cell migration, invasion, and survival [157]. In CRC cell lines, the compound decreased proliferation, increased apoptosis (by regulating the expression of Bcl-2 and Bax), and it improved tumour cell sensitivity to 5-fluorouracil. Moreover, in these tumour cells, it suppressed migration and altered the expression of EMT markers for example increasing E-cadherin, decreasing Vimentin and the Snail family transcriptional repressor 1 (SNAI1) [158]. Zhang et al. demonstrated that JZL-184 reduced HCC proliferation, apoptosis and invasion in vitro, suggesting that MAGL activated both proliferation and invasion of HCC cells through unknown mechanisms involving the prostaglandin E2 (PGE2) and lysophosphatidic acid (LPA) [159]. In osteotropic breast and prostate cancer cells, JZL184 reduced migration and invasion. Moreover, the compound blocked the ability of these cells to stimulate osteoclastogenesis in co-cultured models. JZL184 also inhibited the differentiation of primary osteoblast and early differentiated osteosarcoma cells. In vivo it prevented the formation of bone nodule in the presence and absence of the highly metastatic osteosarcoma cells. Given the evidence, JZL184 could therefore be considered an optimal candidate for the treatment of cancer-associated bone diseases. However, it exerted paradoxical reduction in bone volume via an effect that involved the activation of the skeletal endocannabinoid system. Further investigations are therefore needed in this context [160]. For what concerns URB-602, it inhibited tumour growth and angiogenesis in xenograft models of colon carcinogenesis. Moreover, it attenuated azoxymethane-induced pre-neoplastic lesions, polyps and tumours in vivo [161]. Accordingly, González et al. reported that the compound, given with Cannabigerol (CBG), weakly antagonist at CB1R, and O-1602 (synthetic GPR55 agonist) induced apoptosis, reduced angiogenesis, tumour volume, and aberrant crypt foci (ACF) on colorectal cancer (CRC) models in vivo [162].

FAAH Inhibitors
In lung cancer, the two FAAH inhibitors Arachidonoyl serotonin (AA-5HT) and URB597 reduced invasion of A549 cells, upregulating TIMP-1. Moreover, the compounds inhibited metastatic processes in vivo [163]. Always in lung cancer, Ravi et al. reported that URB597 downregulated cyclin D1 and CDK4, it activated apoptosis (via caspase-9 and PARP) and it inhibited MMP-2 and stress fibre formation [103]. In non-small lung cancer (NSCLC) URB597 in combination with Met-F-AEA significantly reduced in vitro EGF-induced proliferative and chemotactic activities with respect to Met-F-AEA given alone. Moreover, in combination with PEA, the compound decreased B16 melanoma cancer cell viability [108].

∆9-THC (classical)
Gastro-intestinal cancer Induction of apoptosis through CB1R-mediated inhibition of RAS-MAPK/ERK and PI3K-Akt survival signalling cascades [164] HCC Anti-proliferative action associated with accumulation of ceramide, ER-stress and PPARγ activity Autophagy-mediated cell death in combination with JWH-015 [69,165] Lung cancer Inhibition of tumour cell growth (reduction in 3H thymidine and 14C-uridine uptake) Inhibition of EGF-induced proliferation/migration and invasion, reduction in EGF-induced phosphorylation of ERK1/2, ERK1/2 and Akt [166,167] Breast cancer Disruption of HER2-CB2R heteromers leading to HER2-proteasome degradation Induction of cell cycle arrest through Cdc2 downregulation, leading to apoptosis Reduction in 17β-oestradiol-induced proliferation [90,168,169] Prostate cancer Induction of apoptosis independent from CBRs [170] Pancreatic tumour Induction of apoptosis through de novo synthesis of ceramide and consequent upregulation of ER stress related genes p8, ATF-4 and TRB3 [171] Brain cancer Inhibition of cell proliferation, induction of cycle arrest, ROS production and apoptosis, given alone or in combination with CBD Autophagy-mediated cancer cell death Inhibition of MMP-2 expression and cell invasion in cultured glioma cells via ceramide accumulation and activation of p8 stress protein Increase in radiosensivity in combination with CBD [132,[172][173][174][175] Endometrial cancer Inhibition of migration through down regulation of MMP-9 [176] Leukaemia Induction of apoptosis via MAPK pathway Reversion of multidrug resistance together with CBD Sensitisation to cytotoxic effects of chemotherapy [177][178][179] Melanoma Induction of cell cycle arrest through Akt inhibition, activation of autophagy-mediated apoptosis [180,181] WIN 55,212-2 (aminoalkyndole)

Gastro-intestinal cancer
Inhibition of cell proliferation and induction of apoptosis. Inhibition of Akt, downregulation of MMP-2 and VEGF-A Inhibition of cell migration/invasion and EMT markers through COX2 downregulation [182][183][184] Prostate cancer Inhibition of cell growth, induction of apoptosis, decrease in AR, PSA, PCNA and VEGF in LNCaP Prevention of neuroendocrine differentiation of LNCaP by inhibition of PI3K/Akt/mTOR axis and stimulation of AMPK [185,186] Renal carcinoma Inhibition of proliferation and cell viability. Induction of G0/G1 cell cycle arrest, apoptosis and reduced proliferation into 3D spheres [187] Osteosarcoma Inhibition on cell migration with reduction in MMP-2 and MMP-9 [188] Lung and testicular cancer Induction of apoptosis [189]

Gastro-intestinal cancer
Induction of G0/G1 cell cycle arrest and apoptosis Reduction in cell proliferation through activation of Wnt5a non-canonical pathway Inhibition of cell proliferation induced by FAS-death receptor translocation in lipid rafts, mediated by GPR55 activation [99,190,191] Lung cancer Reduction in tumour cell spreading, mimicking the anti-invasive action of FAAH inhibitors (same effect given by 2-AG, OEA, PEA) [163] Breast cancer Inhibition of cell proliferation through downregulation of adenylate cyclase and activation of MAPK, exerting downregulation on prolactin and tyrosine kinase levels [192][193][194] Prostate cancer Reduction in EGF-induced cell proliferation, induction of apoptosis and necrosis through EGFR downregulation Induction of apoptosis mediated by activation of ERK and inhibition of AKT signalling pathways (same effect given by 2-AG and Met-F-AEA) [195,196] Non-melanoma skin cancer Induction of apoptosis mediated by oxidative stress and CBR-independent signalling [197] Lymphoma Reduction of tumour cell viability [198] R(+)-Methanandamide (stable AEA analogue)

Breast cancer
Induction of cell cycle arrest correlated with Chk1 activation, Cdc25A degradation and downregulation of Cdk2 activity Inhibition of adhesion and migration, interfering with the RhoA/ROCK signalling pathway and FAK phosphorylation [100][101][102] Melanoma Inhibition of cell growth [106] Lung cancer Induction of G0/G1 cell cycle arrest leading to apoptosis (in combination with UR597) [103] Gastro-intestinal cancer Increase in AEA availability, induction of oestrogen receptor β expression, decrease in proliferation rate due to CB1 up-regulation through the transcriptional activation of CNR1 promoter (CRC) [104] Thyroid cancer Induction of apoptosis via p53 and p21 [105] PEA Brain cancer Induction of cell death [52] Melanoma Reduction of melanoma cell survival in combination with URB597 [108] Breast cancer Increase in cytotoxic effect of AEA [109] ACEA Gastro-intestinal cancer Activation of apoptosis through TNF-α-mediated ceramide de novo synthesis [110] HCC Reduction of cell viability, invasion and MMP-2/MMP-9 expression [111] Breast cancer Inhibition of invasion in breast cancer stem cells [112] Pancreatic cancer Induction of ROS-mediated autophagy via activation of AMPK, inhibition of energetic metabolism. Decrease in GAPDH and PMK2 expression. Increase the anticancer potential of gemcitabine [113]

JWH-015
Prostate cancer Inhibition of cell growth and apoptosis induction via de novo synthesis of ceramide.
Signalling pathways include JNK activation and Akt inhibition. [98] Breast cancer Reduction of tumour growth, chemotaxis and wound healing. (block of the chemokine receptor CXCR4 signalling) Inhibition of EGFR activation in ERα breast cancer cells [115,199] Lung cancer Attenuation of growth factor-directed in vitro chemotaxis and chemo-invasion. Reduction in focal adhesion complex. Inhibition of Akt phosphorylation and reduction in MMP-9 expression and activity [25] JWH-133 Brain cancer Inhibition of glioma cell viability [118] Breast cancer Decrease in cell proliferation, induction of apoptosis, inhibition of cell migration [119] Melanoma Decrease in trans-endothelial migration in vitro [117]

Gastro-intestinal cancer
Induction of G2/M cell cycle arrest and mitotic catastrophe Synergic effect in combination with oxaliplatin, blocking cell proliferation Impact in chemoresistance and cancer stemness, retain of architecture and heterogeneity of human healthy organoids in ex vivo cultures through inhibition of Wnt/β-catenin canonical pathway [126,129,200,201] Brain cancer Induction of cell proliferation arrest, caspase-dependent apoptosis and upregulation of the NKG2D ligand MICA/B [128] Breast cancer Inhibition of cell proliferation via CB1R-interaction with lipid rafts [127] CBD (antagonist, inverse agonist and negative allosteric modulator at CB1R/partial agonist at CB2R)

Gastro-intestinal cancer
Induction of G0/G1 cell cycle arrest through downregulation of CDK2-cyclin E. Activation of mitochondrial-dependent apoptosis pathway by increasing ROS production Reduction of cell migration Protection of DNA from oxidative damage, increase in endocannabinoid levels, reduction in proliferation through CB1R, TRPV1 and PPARγ involvement.
Reduction of invasion and cell migration Induction of apoptosis through excessive ROS production, ER stress and Noxa activation

Pancreatic cancer
Induction of apoptosis via receptor-independent mechanisms [204] Gastro-intestinal cancer Reversion of the Met-F-AEA anti-proliferative effect [104] Breast cancer Reversion of the effect of ACEA on the decrease in the invasive potential of breast cancer stem cells [112] Renal cell carcinoma Decrease in proliferation, induction of apoptosis by upregulating Bax and decreasing Bcl-2. Inhibition of cell migration [148]
The TME participates through dynamic interactions to neoplastic development, actively contributing to a plethora of phenomena, such as proliferation, angiogenesis, invasion, immune escape, and metastatic spread [210][211][212].
ECM is one of the major components of the TME. The composition of the ECM can undergo substantial variation among different tumours/tissues, as well as during tumour evolution, being susceptible to variation in protein abundance, glycoproteins, and proteoglycans composition and/or ECM stiffness [213]. In addition to its supporting role, ECM is also directly and indirectly involved in different aspects of cancer cell progression, i.e., cell survival, proliferation, adhesion, and migration [214]. Moreover, ECM contributes to the development of blood vessels to supply the tumour mass and to support the metabolic processes [215]. The establishment of an hypoxic environment also concur to promote the HIF-dependent VEGF expression, resulting in an abnormal development of neo-angiogenesis and in the suppression of the anti-tumour immune response [216,217]. ECs also release angiocrine factors and promote a rearrangement of vessel architecture characterised by altered permeability which is crucial for tumour cells metastatic spreading [218,219].
Among the cellular components of the TME, CAFs exert a prominent role in several tumours. CAFs establish a biunivocal crosstalk with cancer cells and other accessory cells, mainly acting through the release of an altered secretome and producing a large amount of growth factors, cytokines, chemokines, and metabolites, that influence tumour evolution [220][221][222]. Through the release of soluble factors, CAFs are able to promote EMT in cancer cells, endowing them with pro-invasive features and stem-like properties, ultimately favouring metastatic dissemination [223][224][225]. CAFs are also responsible for the arising of a pro-inflammatory milieu, for new vessels formation and for the recruitment of immune cells. In particular, CAFs exert an immunomodulating role, mainly enhancing the M2/M1 macrophage and the Treg/Th1 ratio [226][227][228]. Tumour-associated pericytes (TAPs), in addition to their supportive role to the angiogenic process, also actively contribute to TME immunomodulation through disruption of anti-tumour T cells responses [229,230]. In some contexts, the presence of cancer-associated adipocytes (CAAs) is an additional support to tumour development. They represent a lipid reservoir for cancer cells, sustaining their energetic demands [231]. In addition, CAAs exhibit an impairment in adipocyte differentiation markers and over-expression of cytokines, proteases and growth factor termed adipokines, altogether emerging as key actors in neoplastic progression [232][233][234].

The Involvement of the Endocannabinoid System in the Tumour Microenvironment
Alongside the widely investigated effect of cannabinoid on cancer cells, as deeply aforementioned, evidence are now emerging about the direct effect of ECS on accessory cells of the TME.
The different cellular components of the TME surrounded the tumour as a dense neuralgic network, generating intense crosstalk that supports and promotes neoplastic growth, and they act as allies in all phases of cancer progression, up to the metastatic process. Given the wide effects of ECS, as discussed above, the deregulation of different components of the ECS machinery in the accessory cells may cover an important role in shaping TME.
The microenvironmental cells that are most affected by ECS are the components of the immune system. Immune cells (innate and adaptative immunity) are known to release eCBs and they are also able to respond to these ligands [235,236]. Indeed, CBR expression has been found in several immune components, among which dendritic cells, T-and B-cells, macrophages, and NK cells [237][238][239][240][241]. CB1R and CB2R are mainly expressed in the immune system and their levels are increased upon exposure to inflammatory cytokines (e.g., IL-6, TNF-α) [242]. These cytokines are common in cancer diseases and associated with improvement of cell growth [243][244][245]. The inflammatory milieu also mediates the modulation of eCBs [242]. It is known that cytokines stimulation is able to increase the CBRs expression in peripheral blood mononuclear cells (PBMCs) [246]. However, Staiano et al. reported that the high expression of CB1R and CB2R in lung-resident macrophages inhibited the release of several factors (VEGF-A) in vitro inflammatory environments [247].
Notably, the effects of CBs in tumour immune regulation can be different and dependent on tumour types. In murine TNBC cells, the treatment with CBD reduced the recruitment of the M2 sub-population of macrophages in the primary tumour and in the metastatic site. Indeed, CBD-treated cancer cells exhibited a reduction in secreted granulocyte macrophage colony-stimulation factor (GM-CSF) and C-C motif chemokine ligand 3 (CCL3) cytokines, which are important for macrophage recruitment and activation [142]. Similarly, in melanoma cells THC treatment reduced infiltration of macrophages and neutrophils and interfered with cytokines production [248].
Recent evidence highlighted a central role of CB2R in regulating tumour immunity in melanoma. High intra-tumoural CB2R gene expression correlated to the improvement of overall survival. In particular, CB2R is predominantly expressed in B cells and responsible for their differentiation. The impairment of CB2R expression led to less differentiated B cells, favouring the induction of regulatory T cells (Treg) and the generation of an immunosuppressive microenvironment in Cnr2 −/− mice [249].
In NSCLC, the administration of the CB2R agonist JWH-015 strongly reduced recruitment of macrophages to the tumour site, thereby inhibiting macrophage induced EMT and the acquisition of pro-invasive skills and contributing to the blockade of tumour progression given by the interplay between cancer and microenvironment host cells, thus indicating tumour regressive property in A549, CALU-1 cells and in vivo mouse model [250]. Recently, Haustein and co-workers showed that Met-F-AEA treatment of lung cancer cells led to ICAM-1 upregulation and increased their susceptibility to the cytolytic action of LAK cells, suggesting a novel anti-cancer action mode of cannabinoids [202].
Several studies demonstrated that human DCs expressed CBRs and produced eCBs [12,251]. In the pancreatic ductal adenocarcinoma murine model, 2-AG administration exhibited a direct anti-tumour effect by inducing DC phenotypic maturation and the production of pro-inflammatory cytokines, but also significantly promoted an immunosuppressive microenvironment via increasing the suppressive immune cell population of myeloid-derived suppressor cells (MDSCs) [252].
In HCC, inactivation of CB2R altered immune infiltrates, for instance, leading to inhibition of CD4+ T-cell-recruitment. Although the authors did not investigate the specific lymphocyte population, it seems that the increase in tumour growth in CB2R-deficient HCC model was related to a malfunctioning response in immunosurveillance [253].
In CRC, the GPR55 knockout in mouse models resulted in the alteration of immune composition, with an increase in the amount of CD4+/CD8+ T cells, indicating a relation between GPR55 impairment and a more favourable prognosis [150].
In a model of colon cancer, MAGL deficiency drives CB2R-TLR4 axis-dependent macrophages polarisation towards an M2-phenotype, through 2-AG-CB2R signalling, which contributed to suppressing cancer-related CD8+ T-cells. In mice bearing MAGLdeficient macrophages, treatment with CB2R antagonists created a hurdle to cancer progression, providing potential therapeutic targets [254].
On the contrary, in glioblastoma stem cells (GSCs) ARS2/MAGL axis promoted cancer progression and M2-like TAM polarisation. Furthermore, pharmacological targeting of MAGL impacted on survival rate in vivo xenograft model and offered benefits in patients with glioblastoma multiforme (GBM) [255]. Moreover, cannabinoids have been shown to modulate chemotaxis in various immune cell types [256][257][258]. In line with this concept, THC leads to a CB2R-dependent inhibition of peritoneal murine macrophages through activation of RANTES/CCL5 signalling [259]. A parallel report, by other authors, demonstrated that THC treatment of murine macrophages inhibited their chemotaxis towards CCL2 [260].
In solid tumours, CAFs are one of the most abundant cellular components of the TME. A recent publication shed new light on an intriguing role of the ECS in regulating stromal reactivity in a prostate cancer cell model. Indeed, it was shown that patient-derived CAFs, in response to tumour-secreted inflammatory cytokines, upregulated both CB1R and CB2R compared to the healthy counterpart. Interestingly, treatment with WIN 55,212.2 was able to revert CAF activated phenotype or to prevent tumour-induced healthy prostate fibroblast activation, thereby interfering with the supportive role of the stromal component in prostate cancer [261]. These observations reinforce the therapeutic potential of WIN 55,212-2 in prostate cancer treatment and stress the importance of targeting the endocannabinoid system to simultaneously hinder both cancer cells and stromal compartments and to disrupt their pro-aggressive interplay.
Although in a non-cancer model, it has been reported that UVA-UVB exposition of human skin fibroblast increased CB1R/2 and GPR55 expression, resulting in a proinflammatory and pro-oxidant response [262]. This could suggest new approaches to interfere with the stromal ECS in order to prevent UV-induced inflammation and/or redox imbalance. The alteration of CB1R expression was also reported during differentiation of human fibroblast into neurons. Although CB1R was not detected in the early stages of neurogenesis, it was markedly increased during transformation of pluripotent stem cells into differentiated neurons [263].
A few evidence are also available about the impact of cannabinoids on ECs and the angiogenic process in cancer. It was reported that CBD is able to inhibit human umbilical vein endothelial cells (HUVEC) migration, invasion, and sprouting, and that these effects are related to a negative modulation of several prominent factors including MMP-9, TIMP1, PAI1, urokinase-type plasminogen activator (uPA), CXCL16, IL-8, Endothelin-1 (ET-1), and platelet derived growth factor-AA (PDGF-AA), which are involved in the primary vascular endothelial cell functions [264]. In addition, AEA-treated breast cancer cells were found to impair endothelial cell proliferation, which correlated with a decreased synthesis of VEFG, leptin, interferon-γ and thrombopoietin [265]. The anti-angiogenic activity was also demonstrated in thyroid cancer cells, using Met-F-AEA. In particular, this compound reduced endothelial cells proliferation and led to apoptotic process via CB1R. [266].
Recently, Luo and colleagues showed that THC is implicated in the crosstalk between cancer cells and vascular endothelial cells in a Stat1-dependent manner. In particular, the authors demonstrated that THC-treated HCCT116 cells were able to promote tube formation and human-induced pluripotent stem cell-derived vascular endothelial cells (hiPSC-VECs) migration. The genetic and pharmacological Stat1 inhibition interfered with these pro-angiogenic effects, dampening the crosstalk between cancer and endothelial cells [267].
Experimental evidence also demonstrated a role for CBRs activation in mediating cancer cells extravasation and, in turn, metastatic dissemination. In particular, it was reported that CB2R activation with JWH-133 significantly interferes with human melanoma cells adhesion to brain endothelial cells, thereby decreasing the trans-endothelial migration ability and reducing brain melanoma metastases [117].
Additionally, recent studies reported that, although in a non-neoplastic condition, the endogenous 2-AG and AEA, as well as the exogenous cannabinoid WIN 55,212-2, promoted the vasorelaxation of rat retinal capillaries in a CB1R-dependent manner, by modulating the nitric oxide-cyclic guanosine monophosphate pathway, thus resulting in an increase in capillary diameter and pericyte width [268]. The ability of cannabinoids to dilate the retinal capillaries may have therapeutic implications for retinal vascular diseases, and the CB1R may provide a new target for the regulation of the retinal blood flow.
ECS was also involved as a mandatory role in fat storage through different processes, including direct effects on adipocytes (e.g., proliferation and differentiation), contributing to the development of obesity and metabolic diseases [269][270][271]. The presence of CAAs in the tumour milieu, of several solid cancers (e.g., in breast cancer) is associated with cancer malignancy and it may be supported by the catabolic effects of ECS. Two different studies reported that the alteration of the catabolic activity in adipocytes led to tumour progression through PPARγ downregulation in co-cultivated adipocytes [272,273]. Moreover, the authors discovered that breast cancer cells co-cultivated with mature adipocytes exhibited an aggressive phenotype, leading to induction of EMT in co-culture condition [273].
Although further investigation and clarification are needed, these studies suggest a role for the ECS in regulating the TME composition and reactivity, paving the way to novel pharmacological approaches to fight cancer progression, that not only directly target cancer cells, but also counteract the supportive role of accessory cells (Figure 4).
Additionally, recent studies reported that, although in a non-neoplastic condition, the endogenous 2-AG and AEA, as well as the exogenous cannabinoid WIN 55,212-2, promoted the vasorelaxation of rat retinal capillaries in a CB1R-dependent manner, by modulating the nitric oxide-cyclic guanosine monophosphate pathway, thus resulting in an increase in capillary diameter and pericyte width [268]. The ability of cannabinoids to dilate the retinal capillaries may have therapeutic implications for retinal vascular diseases, and the CB1R may provide a new target for the regulation of the retinal blood flow.
ECS was also involved as a mandatory role in fat storage through different processes, including direct effects on adipocytes (e.g., proliferation and differentiation), contributing to the development of obesity and metabolic diseases [269][270][271]. The presence of CAAs in the tumour milieu, of several solid cancers (e.g., in breast cancer) is associated with cancer malignancy and it may be supported by the catabolic effects of ECS. Two different studies reported that the alteration of the catabolic activity in adipocytes led to tumour progression through PPARγ downregulation in co-cultivated adipocytes [272,273]. Moreover, the authors discovered that breast cancer cells co-cultivated with mature adipocytes exhibited an aggressive phenotype, leading to induction of EMT in co-culture condition [273].
Although further investigation and clarification are needed, these studies suggest a role for the ECS in regulating the TME composition and reactivity, paving the way to novel pharmacological approaches to fight cancer progression, that not only directly target cancer cells, but also counteract the supportive role of accessory cells (Figure 4).

Cannabinoid-Based Antineoplastic Treatment-Preclinical Studies
Throughout history, cannabinoids have been considered for their therapeutic features and, in recent decades, they go into the spotlight due to their important role in cancer. However, despite the growing studies to understand the mechanisms by which ECS impacts on cancer progression, there are still few developments in this field that consent to overcome the preclinical phase.
Only four pilot clinical studies with cannabinoids in the anti-tumoural therapy were carried out [274][275][276][277]. However, due to the small number of enrolled patients, these studies could not define reliable data on large cohort. Nevertheless, in a future vision, considering the results obtained in vitro and in preclinical models as a firm basis, cannabinoids may potentially be proposed as innovative therapeutic approaches in various cancer types.
In the last decade, several murine preclinical models were set up to test the anti-tumour effect of cannabinoids, as reported in Table 4.  [101,255,[287][288][289][290][291] Many authors have explored ECS modulation in preclinical glioma models [26,118,175]. Different studies highlighted that synthetic cannabinoids, such as JWH-133, WIN 55,212-2, HU-210, and KM-233, are promising as anticancer agents for glioma treatment [286]. In particular, the treatment with JWH-133 significantly decreased tumour size and tumour angiogenesis, impairing VEGFR activation and reducing blood vessel size and functionality [282,283]. A deregulated expression of Ang2, TIMP and MMPs given by the synthetic cannabinoid was also observed, resulting in the inhibition of cell invasiveness [286]. Since the upregulation of MMP-2 is associated with poor prognosis in gliomas, the effect on MMP-2 is promising for cannabinoid anti-tumour efficacy [174].
Combined treatment of THC with TMZ, the principal pharmacologic approach for GBM, increased the effect of tumour growth reduction in glioma xenografts. This effect was also observed in tumour resistant to TMZ [278]. CBD also elicited anti-tumour effects, as observed by the decrease in ld-1 and Ki67 expression in tumour both in orthotopic (U251) and xenograft glioma models, significantly prolonging mouse survival [289][290][291]. Moreover, a combination of CBD+THC increased ionising radiation anticancer therapy [175].
Both phytocannabinoids, CBG, and the synthetic HU-331 (quinone, synthesised from CBD) reduced tumour growth in colon cancer xenograft models [279,280]. These treatments also impaired angiogenesis, ACF and gave less toxicity with respect to DOXO [284,292]. CBD was also shown to mainly reduce the number of ACF, polyps and area of tumours in azoxymethane-induced colon cancer, not only impacting on angiogenesis and metastasis, but also promoting apoptotic process mediated by Noxa activation [131], [285]. In addition, the atypical cannabinoid O-1602 exerts an anti-inflammatory effect by inhibiting TNF-α expression and Stat3 and NFkB activation and promoted the decrease in tumour growth in colon cancer murine models [281].
The cannabinoid therapeutic potential was also studied in orthotopic, genetically engineered mouse models (GEMMs) and xenograft breast cancer in vivo models [293]. CBD and THC decreased tumour size and metastatic lung incidence, and in some cases, they additionally increased survival [287]. CBD also acts by decreasing EGFR signal and Akt expression [142]. In HER2+ breast cancer subtypes, THC also impacted the heteromers CB2R-HER2, selectively binding to CBR and consequently leading to HER2 inactivation and decreasing HCC1944-orthotopic xenograft mice tumour size. These findings defined a new targeted approach in HER2-positive breast cancer in in vivo models [90].
In prostate xenograft-LNCaP murine model, CBD was able to induce pro-apoptotic effects and to inhibit tumour growth [134]. On the model, synthetic compounds, such as JWH-015 and WIN 55,212-2, reduced tumour size [98]. In particular, WIN 55,212-2 caused a decrease in serum PSA level that directly correlated with cancer growth inhibition [185]. WIN 55,212-2 was effective also in skin cancers, especially in melanoma, where it reduced tumour growth, angiogenesis and metastatic potential in xenograft-B16 [180]. CBD and THC were also effective in murine models of cutaneous melanoma, leading to a reduction in tumour growth, angiogenesis, and metastatic dissemination, by inducing an increase in autophagy and apoptosis processes [181,248]. In addition, a recent discovery evidenced how the double administration of THC+Trametinib reduces cancer survival, invasion and metastatic potential of MEK inhibitor (MEKi)-resistant melanoma cells [288]. In addition, ACEA was able to inhibit, liver colonisation of human melanoma cells into severe immunodeficiency (SCID) mice [106].
There are also anti-cancer in vivo evidence regarding ECS degradative enzymes inhibitors. It has been reported that URB597, an acknowledged FAAH inhibitor, when used in combination with PEA or AEA, reduces skin cancer and CRC progression, respectively [104,108]. In lung cancer, URB597 was effective on the metastatic potential [163].
URB-602 is a MAGL inhibitor, which showed an anticancer effect on CRC growth and angiogenesis [161]. Another MAGL inhibitor, JZL184, exerted its effects mainly attenuating tumour growth and metastatic process in different murine models, among which, prostate cancer, HCC, and breast cancer, where the compound acts mainly by reducing the progression of bone metastasis [157,159,160]. Lastly, JZL184 increased the survival rate in GBM murine model [255].
These scientific findings concerning in vivo model cannabinoids' efficacy are positive and encourage more in-depth research in this area. Although considering the obstacles to overcome and the aspects to be better explored, it is extremely interesting to deepen the mechanisms through which ECS deregulation, leads to changes in the tumour milieu which then impact on cancer evolution.

Concluding Remarks
Here, we provide an in-depth overview of the complex endocannabinoid system and the numerous and compelling evidence in the tumour field. The effects of ECS deregulation have been studied for decades. Several evidence explain how ECS can impact on cancer development and what are the signalling pathways that are involved in the observed effects. Most of the evidence is related to cancer cells, where ECS affects tumour initiation and progression through different mechanisms. Cannabinoids induce cell death, cell cycle arrest, inhibition of tumour angiogenesis, but they have a substantial impact also on migration and tumour invasion. However, still today many evidence are contradictory, so they need more in-depth analysis. The surrounding microenvironment is another field that has recently been explored in relation to this complex system. Although the studies are still limited and more deep investigations are needed, there are promising evidence on the deregulation of ECS in the immune system and the role of cannabinoids in cancer immune modulation. A direct effect of the ECS in regulating stromal reactivity, tumour:stroma crosstalk and endothelial cell function is also emerging. In detail, the action of phytocannabinoids (CBD, ∆9-THC), but also the treatment with an aminoalkylindoles agonist (JWH-015) on the immune component, decreases the secretion of cytokines/chemokines, recruitment, and proliferation. Furthermore, for the macrophage component, there is a decrease in the M2 population rate. The use of Met-F-AEA is able to reduce the release of angiogenic factors, proliferation, migration, and sprouting in endothelial cells. A similar result was also obtained with CBD. Finally, it is known, from a recent publication, that the cannabimimetic aminoalkylindole WIN 55,212-2 acts on stromal reactivity and reversion of activatingrelated phenotype.
This shed new light on the potential clinical application of cannabinoids to target not only cancer cells but also the supportive accessory cells and the tumour:stroma interplay. On the basis of the in vitro available studies, numerous preclinical studies in mouse models have been developed to proceed towards the design of novel pharmacological approaches, based on the use of cannabinoids both alone or in combination with already approved drugs.
Author Contributions: M.I. and G.S. wrote the manuscript and elaborated the figure. E.G., G.C. and P.C. critically reviewed and editing the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.