Targeting mast cells in gastric cancer with special reference to bone metastases

Abstract

Bone metastases from gastric cancer (GC) are considered a relatively uncommon finding; however, they are related to poorer prognosis. Both primary GC and its metastatic progression rely on angiogenesis. Several lines of evidence from GC patients strongly support the involvement of mast cells (MCs) positive to tryptase (MCPT) in primary gastric tumor angiogenesis. Recently, we analyzed infiltrating MCs and neovascularization in bone tissue metastases from primary GC patients, and observed a significant correlation between infiltrating MCPT and angiogenesis. Such a finding suggested the involvement of peritumoral MCPT by infiltrating surrounding tumor cells, and in bone metastasis angiogenesis from primary GC. Thus, an MCPT-stimulated angiogenic process could support the development of metastases in bone tissue. From this perspective, we aim to review the hypothetical involvement of tumor-infiltrating, peritumoral MCPT in angiogenesis-mediated GC cell growth in the bone microenvironment and in tumor-induced osteoclastic bone resorption. We also focus on the potential use of MCPT targeting agents, such as MCs tryptase inhibitors (gabexate mesylate, nafamostat mesylate) or c-KitR tyrosine kinase inhibitors (imatinib, masitinib), as possible new anti-angiogenic and anti-resorptive strategies for the treatment of GC patients affected by bone metastases.

Key Words: Bone metastases, Gastric cancer, Receptor activator of nuclear factor-κB, Angiogenesis, Osteoclastic bone resorption, Tryptase inhibitors, c-Kit receptor tyrosine kinase inhibitors, Anti-angiogenic therapy

Core tip: The activation of the stem cell factor/c-Kit receptor (c-KitR) pathway in mast cells (MCs), and tryptase release upon MCs degranulation have a pivotal role in tumor angiogenesis in several human malignancies. MCs positive to tryptase (MCPT) have been implicated in primary gastric cancer (GC) angiogenesis. Our preliminary findings indicated that bone metastasis angiogenesis from GC is also supported by infiltrating MCPTs. Overall, the evidence provides a rationale to evaluate c-KitR inhibitors that block MCs degranulation, or tryptase inhibitors that inhibit tryptase and/or the Proteinase-Activated Receptor-2 pathway, in clinical trials for bone metastasis GC patients.

INTRODUCTION

Gastric cancer (GC) is a major cause of cancer-related mortality worldwide[1]. According to GLOBOCAN estimates, there were 260000 cases of cardia GC and 691000 cases of non-cardia GC in 2012[1]. Generally, at the time of diagnosis, most patients have unresectable or metastatic disease[2]. The most common site of distant metastases is the peritoneum, followed by the liver, lung and bones[2,3]. Bone metastases are a relatively uncommon finding[4], occurring in between 1% and 20% of GCs. Notably, they represent a major discomfort because of the related pain, neurological involvement and hypercalcemia syndrome[5,6]. Recently, it was reported that bone metastases diagnosed by Fluorine-18- fluorodeoxyglucose positron emission tomography/computed tomography examinations represented 10% of an evaluated series[7]. In fact, many GC patients die from metastases in intraperitoneal organs before their bone metastatic sites are revealed. Both primary GC and its metastatic progression rely on angiogenesis[8,9], which plays a crucial role in cancer development, inducing tumor growth, invasion and metastasis[10,11]. Moreover, cancer stem cells also promote GC metastasis via close physical cellular contact and paracrine signals released from the tumor niche, both in vivo and in vitro. In fact, stroma-associated cancer stem cells promote the GC cell epithelial to mesenchymal transition (EMT), attracting circulating cancer cells to self-seed the primary tumor, again though EMT[12].

Interestingly, a large body of evidence supports the substantial involvement of mast cells (MCs) in tumor angiogenesis[9,13-17]. A preclinical pivotal study documented that the angiogenic response to subcutaneously growing B16-BL6 tumors was lower in genetically MC-deficient W/W_v mice compared with MC-sufficient^+/+ littermate mice. Moreover, the latters showed a greater propensity to develop lung metastases[18]. Accordingly, a significant increase in MCs density (MCD) was observed in several animal and human malignant tumors[19-22].

MCs secrete several classical and non-classical pro-angiogenic factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor-β (PDGF-β), interleukin-6 (IL-6), IL-8, thymidine phosphorylase (TP), chymase and tryptase[23-27].

Notably, tryptase is the most powerful non-classical pro-angiogenic factor released by MCs upon c-Kit receptor (c-KitR) TK activation[28]. Tryptase induced endothelial cell (EC) proliferation in a matrigel assay[29] and in vivo in a chick embryo chorioallantoic membrane (CAM) assay[27]: tryptase inhibitors suppressed both effects consistently. Tryptase activates the proteinase-activated receptor-2 (PAR-2) expressed on ECs, directly stimulating their proliferation[17,30]. Interestingly, PAR-2 expression is greater in tumor tissues than in normal tissues[31]. Moreover, it also promotes the degradation of extracellular matrix (ECM) components via metalloproteinases activation, resulting in the release of ECM-bound latent pro-angiogenic factors (e.g., VEGF and FGF-2)[32,33]. Most notably, many human studies have established a positive correlation between MCD positive to tryptase (MCDPT) and microvascular density (MVD) in tumor tissue[9,13,14,34-39].

Several findings from patients strongly support the crucial involvement of MCDPT in primary gastric tumor angiogenesis[28,39-41]. There is also a significant correlation between MCDPT and angiogenesis in bone metastases from GC patients[42].

Starting from these preliminary results, we aim to review: (1) the involvement of MCDPT in GC angiogenesis; (2) the specific role that MCDPT might play in bone metastases angiogenesis; and (3) the potential targeting of MCDPT by tryptase or c-KitR tyrosine kinase (TK) inhibitors in metastatic gastric patients with special reference to bone metastases.

MCDPT AND TUMOR ANGIOGENESIS

MCs are cells that originate in the bone marrow from pluripotent CD34⁺ hematopoietic stem cells[43]. Precursors of MCs migrate through the circulation to their target tissues, completing their maturation process into granulated cells, under the influence of several microenvironment growth factors[44]. The most important of the above factors is the ligand for the c-KitR TK, stem cell factor (SCF), secreted mainly by fibroblasts and ECs. SCF also regulates development, survival and de novo proliferation of MCs[33,45]. MCs express the high affinity IgE receptor, which, following IgE activation, triggers release of their stored pro-angiogenic factors. In this way a link between immunity and angiogenesis is established by MCs. Among the other IgE-independent MCs activation mechanisms, a wide variety of other surface receptors for cytokines, chemokines, immunoglobulins, complement and bacterial products, are also described[46].

MCs granules are key functional elements, characterized by two distinct secretory patterns: exocytosis or piecemeal degranulation[33]. Interestingly, this latter mechanism, representing a slow and selective pathway of cell secretion, has been more frequently observed in MCs infiltrating areas of chronic inflammation, such as tumor tissues[47]. Correspondingly, a link between MCs, chronic inflammation and cancer has been long suggested[23,47]. Thus, MCs are one of the earliest and major inflammatory cell types recruited into the tumor microenvironment[9,13,14,39,48].

In particular, several data from human studies have highlighted a strong linear correlation between MCDPT and pathological angiogenesis sustaining several solid tumors, such as human malignant melanoma, endometrial carcinoma, breast cancer, GC, colorectal cancer (CRC) and pancreatic ductal adenocarcinoma[9,14,20,21,28,34-39,49-51]. Remarkably, MCDPT correlated with angiogenesis, tumor aggressiveness and poor tumor outcome in most clinical investigations[9,16,23,33], suggesting a prognostic significance for MCDPT in several tumors[14,21,35,39,50-52]. Moreover, Ammendola et al[28] demonstrated a positive correlation among MCDPT, c-KitR expressing cells and MVD in tumor tissue specimens from surgical GC patients, confirming that c-KitR activation on MCs surface, resulting in tryptase degranulation from activated MCs, has a pivotal function in tumor angiogenesis[24,53].

Thus, MCDPT may represent a novel and attractive therapeutic target of anti-angiogenic cancer therapy[33,45]. Indeed, MCDPT-targeted therapeutic approaches could limit angiogenesis in growing tumors, potentially decreasing tumor growth and metastasis. Therefore, tryptase inhibitors, such as gabexate mesylate and nafamostat mesylate[54], or c-KitR inhibitors, such as imatinib and masitinib[10,55,56], should be evaluated in clinical trials as new anti-angiogenic agents in combination with chemotherapy.

MCDPT, ANGIOGENESIS, AND IMMUNE SUPPRESSION IN HUMAN CANCERS: THE DUAL VALUE OF TARGETING MCDPT

Different infiltrating cell populations form the tumor microenvironment, enabling a pathological condition that directly promotes angiogenesis, tumor spread and invasion[57] and, concurrently, hamper the immune response against tumors[58]. However, a relationship between high MCD and improved overall survival has also been reported in some human studies[59], signifying a diversified and not yet well-understood role for MCs in cancer. Note that, with reference to this relationship between high MCD and favorable prognosis, MCs are able to modulate both innate and adaptive immune responses[60]. In this context, MCs are involved in innate immunity by releasing tumor necrosis factor-α (TNF-α) and interleukins (IL-1, 4, 6) that, in turn, help to kill tumors[24,46]. In addition, MCs express both the major histocompatibility class (MCH) II antigen and its co-stimulatory molecule, which activate adaptive T and B cell responses against tumors[61]. Finally, MCs cooperate with natural killer cells to reject cancer cells[61]. On the other hand, Shin et al[62] reported the critical involvement of MCs’ tryptase in eosinophils recruitment, suggesting a role in the induction of a pro-inflammatory microenvironment. In this way, MCDPT may also induce angiogenesis via several pro-inflammatory cytokines that act as pro-angiogenic factors[23,33]. Taken together, the data confirm the potential dual value of MCPT as a therapeutic target for tryptase inhibitors or c-KitR tyrosine kinase inhibitors, which could both prevent MCDPT-mediated immune suppression and promote/unleash protective anti-tumor immune responses through the selective inhibition of MCDPT-mediated angiogenesis, resulting in tumor regression.

MCDPT AND ANGIOGENESIS IN HUMAN GC

Several studies support the view that inflammation is a critical component of GC development and progression[24,53,63]. In turn, increasing evidence suggests an intimate relationship between angiogenesis, inflammation and GC progression[16]. In the GC microenvironment, many types of inflammatory cells exist, and among them, MCs promote GC angiogenesis by secreting pro-angiogenic factors and cooperating with stromal and malignant cells. In turn, neovascularization helps to maintain inflammation by promoting the migration of inflammatory cells to the site of inflammation[16,63,64].

From a translational point of view, a significant positive correlation between MCDPT and MVD has been confirmed in cancer tissue specimens. Interestingly, specimens from patients with advanced histological stages of the disease showed a higher MCD than those from patients with early stage disease[65,66].

Similarly, Ribatti et al[9] observed that stage IV GC shows a higher degree of vascularization than the earlier stages, and that MCDPT increases in parallel with malignancy grade and is highly correlated with the extent of angiogenesis in primary tumor tissue. Zhao et al[67] reported a significant positive correlation between the increased infiltration of MCDPT and the progression of disease in GC patients, documenting an association between a high level of tryptase expression and advanced tumor stage, which is a marker of poor prognosis. In agreement with the above findings, a more recent investigation evaluating biopsy specimens from 25 GC patients highlighted a positive correlation between MCDPT, c-KitR expressing cells and MVD, using immunohistochemistry and image analysis methods[28]. Furthermore, in a series of 41 gastrointestinal cancer patients, a positive correlation between MCDPT and the number of metastatic lymph nodes harvested, and between MCDPT in primary tumor tissue and in metastatic lymph node tissue, were observed[39]. These results suggested that MCs’ tryptase, like VEGF, could be involved in tumor lymphangiogenesis, which, in turn, is correlated with lymph node metastases, both in experimental cancer models and several human cancers[68]. Table 1 summarizes the above studies correlating MCD with angiogenesis or negative prognostic factors.

Table 1 Principal studies that correlate mast cell density and mast cells density positive status to tryptase with angiogenesis or negative prognostic factors in gastric cancer patients.

Ref.	Disease stage/main stages	Chemotherapy	Patients (n)/site	Methods of MCs identification	Correlation	P value
Ribatti et al[9], 2010	All TNM stages (mainly II-IV)	No	30	Immunohistochemistry primary anti-tryptase and anti-chymase Abs	MCD and Angiogenesis and advanced tumor stage	< 0.05
Zhao et al[67], 2012	All TNM stages (mainly II-IV)	No	60	Immunohistochemistry primary anti-tryptase Ab	MCD and advanced tumor stage	NS
Ammendola et al[28], 2013	TNM stage III	Nr	25	Immunohistochemistry primary anti-tryptase Ab	MCDPT and MVD and c-KitR-EC	0.001
Ammendola et al[32], 2013	TNM stage III	Nr	19	Immunohistochemistry primary anti-tryptase Ab	MCDPT and no. of metastatic lymph nodes	0.01

Ab/s: Antibody/antibodies; MCD: Mast cell density; NR: Not reported; MCDPT: Mast cells density positive to tryptase; c-KitR EC: c-Kit receptor expressing cell.

Overall, the majority of studies in GC support the view that MCDPT is significantly correlated with the depth of invasion, lymph node metastases, lymphatic or blood invasion, the degree of histological differentiation, and the number of blood vessels surrounding GC cells. Most of the MCs located near the neovascularization areas were degranulated, suggesting the critical role of tryptase in angiogenesis and tumor progression[8]. Correspondingly, tumors injected into mice treated with inhibitors of MCs-degranulation presented decreased vascularization, growth and metastasis[65,69].

Data from an ultra-structural study in advanced GC patients highlighted that perivascular MCs containing only tryptase in their granules exhibited piecemeal degranulation that was significantly associated with microvascular basal lamina changes (irregular thickness, multiple layers and loose association with ECs and pericytes), which fitted with a remodeling of existing microvasculature. These changes are involved in the increased vascular permeability that characterizes the tumor microvasculature[70]. Interestingly, this suggested that MCs’ tryptase, through its direct and indirect proteolytic activity on the ECM, could induce fenestrations in the endothelium of tumor microvasculature, like VEGF[71]; thus, specifically contributing to metastasis in GC.

In light of these evidence-based considerations, serum tryptase released by MCs could be an indicator for future surgery, a valid predictive factor for hematogenous invasion or metastasis, and a promising prognosis marker both in early and advanced GC. Taken together, the evidence suggests that the accumulation of MCDPT at the periphery of GC tissue might lead to increased rates of tumor vascularization, thus promoting tumor growth and metastases to distant organs. Therefore, MCDPT may represent a valuable target of anti-angiogenic therapy, either by tryptase inhibitors or c-KitR inhibitors, which may prove useful therapeutic tools to control angiogenesis-mediated tumor growth, progression and metastasis in GC.

ROLE OF MCDPT IN BONE METASTASIS ANGIOGENESIS FROM PRIMARY GC

Bone metastases from GC arise from a scattered metastatic spread of tumor cells in the bone marrow (most frequently located in the thoracic and lumbar vertebrae)[72]. These metastases are mainly osteolytic, while osteoblastic implants are rarely reported[5,6,73-75]. Notably, bone metastases in GC usually occur in the advanced stages of the disease, generally correlating with poor prognosis[2,5,6,73,74]. However, the dissemination of micrometastatic cells in the bone tissue may be evident in early GC, in which a poorly-differentiated carcinoma, the presence of signet-ring cell carcinoma, and/or the involvement of lymph node metastasis, seem to be risk factors associated with bone metastases[76]. In particular, in bone marrow micrometastases in early GC, subclinical seeding of tumor cells in the bone marrow (at the time of primary tumor resection) was detected by immunocytochemical evaluation of epithelial cytokeratin protein, a distinctive trait of epithelial cells, both normal and malignant, that would not normally be present in the hematogenous marrow[77]. According to Jauch et al[78], cytokeratin-positive cells in the bone marrow represent a surrogate marker for general disseminative metastasis, rather than the beginning of metastatic growth in patients with GC. Interestingly, patients with cytokeratin positivity in their bone marrow had a higher MVD compared with cytokeratin-negative patients[40,77]. Correspondingly, VEGF-positive tumors associated with increased MVD show cytokeratin-positive cells in the bone marrow[8]. As a result, the above reported occurrence of such micrometastases (cytokeratin-positive cells) in the bone marrow is potentially closely related to angiogenesis in the primary tumor[8,40].

These observations suggested that angiogenesis plays an essential role in micrometastatic (subclinical) seeding of GC cells in the bone tissue, in the development of bone marrow micrometastases into clinically manifest bone metastases (macroscopic disease) and in their potential to invade circulatory system for further distant dissemination from the bone microenvironment. Recently, we investigated infiltrating MCDPT and neovascularization in 15 bone tissue metastases selected from a series of 190 GC patients. We evaluated bone biopsies samples from these patients using immunohistochemistry and image analysis methods. The results showed a statistically significant correlation among MCDPT, MC-area-PT, MVD and endothelial area[42].

These correlations suggested the involvement of infiltrating MCDPT in bone metastasis angiogenesis from primary GC, as well as in primary tumor neovascularization. Therefore, an MCDPT-stimulated angiogenic process could support the development of metastases in the bone tissue. From a biological point of view, MCs may be both recruited and activated by SCF[58,79], the ligand for c-KitR, and by other growth factors, such as VEGF, FGF-2 and TP, secreted by metastasized bone marrow GC cells[8,27,29,80,81]. Pro-angiogenic factors, released from activated MCs, may act in autocrine and paracrine manners, and then stimulate MCs, ECs[51,82] and GC cells, as reported by Marech et al[51]. Interestingly, the bone tumor microenvironment plays a crucial role in osteoclastogenesis by inducing the production of the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) by marrow stromal cells and osteoblasts. Tumor cells secrete parathyroid hormone-related peptide (PTH-rP) and several other osteotropic factors, including IL-6, TNF, M-CSF and prostaglandin E2 (PGE2), which upregulate the expression of RANKL on the surface of marrow stromal cells and immature osteoblasts. RANKL then binds RANK on the surface of osteoclast precursors, ultimately resulting in osteoclast formation and bone resorption. Thus, tumor-induced osteoclastic bone resorption leads to the release of growth factors, such as TGF-β, FGFs, PDGFs, insulin-like growth factors, and bone morphogenetic proteins by the bone matrix that promote tumor growth and further bone destruction by increasing the production of PTH-rP, as well as the above growth factors, resulting in further RANKL upregulation[83].

This reciprocal interplay between tumor cells and the bone tumor microenvironment results in a vicious circle that further increases tumor growth and bone destruction[83]. Crucially, MCDPT could foster this “symbiotic relationship” between bone destruction and tumor growth by releasing pivotal components of the above-described vicious circle[84,85].

The involvement of MCDPT both in metastatic bone resorption and in angiogenesis-mediated GC cell growth is also suggested by MCs ability to produce and release TGF-β[23], which stimulates osteoclast activation via the RANKL pathway[54,86].

Furthermore, TGF-β, in the presence of M-CSF and in the absence of RANKL, could induce in vitro osteoclast formation directly[87], suggesting that MCDPT, by releasing TGF-β, could also stimulate the RANKL-independent metastatic bone resorption in the bone tissue metastases from GC patients[81,88]. Based on the discussed data, MCDPT may potentially offer a novel and promising target of anti-angiogenic therapy to decrease both angiogenesis-mediated GC cell growth in the bone tissue and tumor-induced osteoclastic bone resorption[2,8,42].

TARGETING MCDPT: A POSSIBLE ANTI-ANGIOGENIC AND ANTI-RESORPTIVE STRATEGY IN BONE METASTASIS GC PATIENTS

The use of MCs’ tryptase inhibitors (gabexate mesylate, nafamostat mesylate and tranilast)[17] or c-KitR tyrosine kinase inhibitors (imatinib and masitinib)[10,28,55] could represent a potential anti-tumor strategy to inhibit both angiogenesis and RANKL-/non-RANKL-mediated osteoclast activation[42].

Unlike conventional chemotherapy, anti-angiogenic therapy does not induce a direct cytotoxic effect, but reduces neovascularization mainly by targeting small foci of proliferating cells of tumor-associated capillary endothelium or from metastatic sites. Thus, angiogenesis inhibitors generally do not cause suppression of the hematogenous bone marrow, hair loss or gastrointestinal symptoms like cytotoxic chemotherapeutics, which require pause periods to allow regeneration of normal cells. By virtue of these features, angiogenesis inhibitors need to be administered for a longer period compared with conventional cytotoxic chemotherapeutics and, consequently, anti-angiogenic therapy is administered continuously[89]. Therefore, anti-angiogenic agents may be effective for long-term administration to achieve prolonged dormancy of primary GC and GC micrometastases[40].

Furthermore, the characteristics of anti-angiogenic therapy have recently prompted the medical-scientific community to consider using anti-angiogenic agents in combination with traditional cytotoxic anti-cancer drugs to potentiate the clinical effectiveness of the latter by preventing their limitations. Moreover, MCs’ tryptase or c-KitR inhibitors could be added to available conventional cytotoxic drugs that target normal marrow and GC cells indiscriminately in the bone tissue from bone metastasis GC patients. In particular, these anti-angiogenic agents might temporarily inhibit the remodeling of tumor microvasculature and, consequently, the increase of tumor microvascular permeability in the bone metastases from primary GC patients. Starting from the consideration that tumor-associated vasculature represents the first barrier to the penetration of drugs into tumors, agents such as gabexate mesylate and nafamostat mesylate, or imatinib and masitinib, could act as anti-angiogenic modulators by impairing tumor vasculature functions in the bone tumor microenvironment, possibly resulting in the potentiation of the therapeutic response when administered in combination with cytotoxic chemotherapy, and potentially prolonging the survival time of GC patients with bone metastases.

Notably, synergism between MCs’ tryptase inhibitors and classical chemotherapy drugs in cancer treatment is exemplified by the ability of nafamostat mesylate to inhibit both in vitro and in vivo chemotherapy-induced nuclear factor kappa-B (NF-κB) activation[90,91], which is well-known to contribute to the angiogenic phenotype of several tumors[92], and chemoresistance and suboptimal therapeutic efficacy of cancer chemotherapy drugs[93]. Correspondingly, it is interesting that nafamostat mesylate has been shown to enhance the anti-tumor effect of paclitaxel against GC with peritoneal dissemination by inhibiting paclitaxel-induced NF-κB activation in mice[94]. Thus, we speculate that a combination chemotherapy of nafamostat mesylate and classical cytotoxic drugs could potentially exert a synergistic anti-tumor effect in bone metastases from GC.

Finally, MCDPT targeting agents, such as tryptase inhibitors or c-KitR receptor inhibitors, could represent a novel potential anti-angiogenic approach to inhibit tumor-induced osteoclastic bone resorption and bone destruction in bone metastasis GC patients. Intriguingly, the potential ability of MCDPT targeting to inhibit the RANKL-dependent vicious circle of cancer-induced bone destruction, supports further investigation of tryptase inhibitors or c-KitR inhibitors in clinical trials comparing them with denosumab[95] (a well-known inhibitor of the RANKL–RANK interaction), in terms of efficacy and safety, in the prevention of skeletal-related events in adult patients with bone metastases from GC. Importantly, unlike denosumab, MCDPT-targeting agents could decrease angiogenesis-mediated GC cell growth in the bone tissue and metastatic bone resorption, independently of their inhibitory activity on the cycle of bone destruction and tumor growth mediated by RANKL. Therefore, it would be interesting to investigate whether combination therapy with these agents, which are potentially anti-angiogenic and anti-resorptive, and denosumab may exert a clinically useful, synergistic effect in decreasing tumor-induced osteoclastic bone resorption and angiogenesis-mediated bone metastasis progression in primary GC patients affected by bone metastases. Accordingly, clinical studies comparing the efficacy and safety profile of MCDPT targeting agents versus other anti-resorptive drugs, such as bisphosphonates[96], in bone metastasis GC patients might be also worth performing.

CONCLUSION

A survey of the literature data indicated that MCs are important players in tumor angiogenesis and development[15,33,61], by releasing a panoply of angiogenic factors (e.g., tryptase)[23]. This is supported by human studies that highlighted a strong correlation between MCDPT and pathological angiogenesis in various solid tumors[9,14,20,21,28,34-39,49-51].

With special reference to GC, much evidence supports the involvement of MCDPT in primary gastric tumor angiogenesis, thus promoting metastases[8,9,28,39-41]. With regards to the subgroup of bone metastases, pilot published data demonstrated a significant correlation between infiltrating MCDPT and angiogenesis[42].

From a therapeutic point of view, tumors injected in mice treated with inhibitors of MCs-degranulation presented decreased vascularization and metastasis[65,69]. According to these data, targeting MCs is currently under investigation, especially in GC patients[56,97]. In particular, for the subgroup of patients with bone metastases from GC, the biological background suggests that MCDPT may stimulate the RANKL-dependent vicious circle of tumor-induced bone destruction and tumor growth, and the RANKL-independent metastatic bone resorption. This biological background further supports targeting of MCDPT as a therapeutic strategy in bone metastases.

Finally, ad hoc clinical trials might be performed to compare, in terms of efficacy and safety, these potential anti-angiogenic and anti-resorptive agents with anti-resorptive drugs currently used clinically (i.e., denosumab and bisphosphonates) for the prevention of skeletal-related events in GC patients affected by bone metastases.