Tumor-Associated Mast Cells in Urothelial Bladder Cancer: Optimizing Immuno-Oncology

Urothelial bladder cancer (UBC) is one of the most prevalent and aggressive malignancies. Recent evidence indicates that the tumor microenvironment (TME), including a variety of immune cells, is a critical modulator of tumor initiation, progression, evolution, and treatment resistance. Mast cells (MCs) in UBC are possibly involved in tumor angiogenesis, tissue remodeling, and immunomodulation. Moreover, tumor-infiltration by MCs has been reported in early-stage UBC patients. This infiltration is linked with a favorable or unfavorable prognosis depending on the tumor type and location. Despite the discrepancy of MC function in tumor progression, MCs can modify the TME to regulate the immunity and infiltration of tumors by producing an array of mediators. Nonetheless, the precise role of MCs in UBC tumor progression and evolution remains unknown. Thus, this review discusses some critical roles of MCs in UBC. Patients with UBC are treated at both early and late stages by immunotherapeutic methods, including intravenous bacillus Calmette–Guérin instillation and immune checkpoint blockade. An understanding of the patient response and resistance mechanisms in UBC is required to unlock the complete potential of immunotherapy. Since MCs are pivotal to understand the underlying processes and predictors of therapeutic responses in UBC, our review also focuses on possible immunotherapeutic treatments that involve MCs.


Introduction
Urothelial bladder cancer (UBC) is a common disease with high morbidity and mortality rates, accounting for around 2.1% of all deaths due to cancer per year [1][2][3][4][5][6][7]. As the high rate of recurrence and the need for long-term surveillance greatly increased the economic burden of UBC patients, exploring optimized and personalized therapeutic modalities against UBCs is a rapidly evolving and expanding field in adjuvant and definitive settings [4,8,9]. Tumoral depth of invasion and detrusor invasiveness are the most significant variable for progression, recurrence, and survival in UBC [6].

Mast Cells in UBC: A Double-Edged Sword
The function of MCs in many tumor types is complex and remains controversial [19], as they either promote or inhibit tumor growth, depending on the type and stage of cancer and their localization in the TME (Figure 1) [15][16][17]19]. Activated MCs may selectively produce pro-and anti-inflammatory chemicals, and their phenotypes may alter in response to the TME, thereby making them analogous to a "double-edged sword" [15,19]. For example, MCs that secrete IL-1, IL-4, IL-6, and tumor necrosis factor (TNF)-α can actively eliminate tumor cells and halt tumorigenesis [25,39]. On the contrary, it is well established that various subsets of MCs infiltrate tumors at various phases of progression; the MC count is related to the stage, prognosis, and invasiveness of the tumor [16,17,19]. The mechanism by which MCs promote tumor growth is very complex and involves tissue remodeling, angiogenesis, and immune modulation [40,41]. The existence of a potential connection between MCs, chronic inflammation, and cancer has been proposed for an extended period [29]. In chronic inflammation, the tissue interstitium is associated with oxidative stress, edema, enzymatic stress, persistent leukocyte stimulation, lymphangiogenesis, angiogenesis, and fibrosis [19,[42][43][44][45]. Chronic inflammation induces angiogenesis directly and promotes tumor growth and immune suppression [29]. Additionally, the involvement of a noxious factor can promote the participation of MCs in the chronicity of an inflammatory response [19,24,46,47].

Mast Cells in UBC: A Double-Edged Sword
The function of MCs in many tumor types is complex and remains controversial [19], as they either promote or inhibit tumor growth, depending on the type and stage of cancer and their localization in the TME (Figure 1) [15][16][17]19]. Activated MCs may selectively produce pro-and anti-inflammatory chemicals, and their phenotypes may alter in response to the TME, thereby making them analogous to a "double-edged sword" [15,19]. For example, MCs that secrete IL-1, IL-4, IL-6, and tumor necrosis factor (TNF)-α can actively eliminate tumor cells and halt tumorigenesis [25,39]. On the contrary, it is well established that various subsets of MCs infiltrate tumors at various phases of progression; the MC count is related to the stage, prognosis, and invasiveness of the tumor [16,17,19]. The mechanism by which MCs promote tumor growth is very complex and involves tissue remodeling, angiogenesis, and immune modulation [40,41]. The existence of a potential connection between MCs, chronic inflammation, and cancer has been proposed for an extended period [29]. In chronic inflammation, the tissue interstitium is associated with oxidative stress, edema, enzymatic stress, persistent leukocyte stimulation, lymphangiogenesis, angiogenesis, and fibrosis [19,[42][43][44][45]. Chronic inflammation induces angiogenesis directly and promotes tumor growth and immune suppression [29]. Additionally, the involvement of a noxious factor can promote the participation of MCs in the chronicity of an inflammatory response [19,24,46,47].

Evidence for Pro-Tumorigenic Roles of Mast Cells in Urothelial Bladder Cancer
Interactions between MCs and bladder tumor cells may result in the activation of MCs and release of mediators ( Figure 2) [15,25,48]. Upon activation, MCs produce multiple growth factors, angiogenic factors, and pro-inflammatory chemicals that contribute to the aggressive phenotypes of tumor cells [15,25,48]. Moreover, MCs infiltrate tumors and promote their proliferation and invasion [49]. Their recruitment to the tumors increases

Evidence for Pro-Tumorigenic Roles of Mast Cells in Urothelial Bladder Cancer
Interactions between MCs and bladder tumor cells may result in the activation of MCs and release of mediators ( Figure 2) [15,25,48]. Upon activation, MCs produce multiple growth factors, angiogenic factors, and pro-inflammatory chemicals that contribute to the aggressive phenotypes of tumor cells [15,25,48]. Moreover, MCs infiltrate tumors and promote their proliferation and invasion [49]. Their recruitment to the tumors increases the interaction between estrogen receptors (ERs) and C-C Motif Chemokine Ligand 2 (CCL2), wherein CCL2 promotes epithelial-to-mesenchymal transition (EMT) and the production of matrix metalloproteinases (MMP) in the tumor location. Therefore, this indicates that the activation of the ERβ/CCL2/EMT/MMP axis by the MCs increases UBC invasion [49]. MCs operate by increasing the motility, proliferation, and differentiation of endothelial cells and promote tumor-endothelial cell adhesion [25]. Analysis of the UBC tumor tissues revealed a strong association between the microvessel density and the number of MCs present in the tumoral zone [50]. Numerous angiogenic mediators are produced by the MCs in the TME, including OX40L, VEGF, IL-8, nerve growth factor, TNF-α, TGF-β, tryptase, histamine, CXCL12, CXCL8, urokinase-type plasminogen activator, prostaglandin E2 (PGE2), platelet-derived growth factor-β, fibroblast growth factor-2 (FGF-2), IL-6, IL-8, and thymidine phosphorylase [15][16][17]19,25,41,[51][52][53]. Notably, the overall function of MCs in tumor angiogenesis is dependent on the stimuli that activates them and the subsequent mediator produced by them [25,54]. the interaction between estrogen receptors (ERs) and C-C Motif Chemokine Ligand 2 (CCL2), wherein CCL2 promotes epithelial-to-mesenchymal transition (EMT) and the production of matrix metalloproteinases (MMP) in the tumor location. Therefore, this indicates that the activation of the ERβ/CCL2/EMT/MMP axis by the MCs increases UBC invasion [49]. MCs operate by increasing the motility, proliferation, and differentiation of endothelial cells and promote tumor-endothelial cell adhesion [25]. Analysis of the UBC tumor tissues revealed a strong association between the microvessel density and the number of MCs present in the tumoral zone [50]. Numerous angiogenic mediators are produced by the MCs in the TME, including OX40L, VEGF, IL-8, nerve growth factor, TNFα, TGF-β, tryptase, histamine, CXCL12, CXCL8, urokinase-type plasminogen activator, prostaglandin E2 (PGE2), platelet-derived growth factor-β, fibroblast growth factor-2 (FGF-2), IL-6, IL-8, and thymidine phosphorylase [15][16][17]19,25,41,[51][52][53]. Notably, the overall function of MCs in tumor angiogenesis is dependent on the stimuli that activates them and the subsequent mediator produced by them [25,54]. Tumor-infiltrating MCs directly affect the aggressiveness of a tumor cell. However, they indirectly function inside the TME by interacting with other immune cells and promoting or suppressing immunological responses [15,25,40]. Since the MCs interact with immune suppressor cells, such as MDSCs and Tregs, they can substantially influence the development and functional capability of tumor immunity [15,19,[55][56][57][58][59][60][61]. For example, MDSCs are recruited to tumor locations by CCL2 and potentially CXCL1 and CXCL2, where direct contact with MCs or with histamine increases their inhibitory function [19,55]. The MC stimulates the production of IL-17 via MDSC, mobilizing Tregs and enhancing their suppressive activity on cytotoxic T cell [19,57]. Subsequently, IL-17 indirectly recruits Tregs and increases their suppressor function and stimulates IL-9 production, thus enhancing the pro-tumorigenic function of the MCs in the TME [19,57]. Remarkably, TGF-β and IL-10 produced by the MCs promote the development of Tregs, downregulate expression of costimulatory molecules in DCs, and reduce the production of proinflammatory cytokines by TAMs. They also suppress antigen-specific T cell responses Tumor-infiltrating MCs directly affect the aggressiveness of a tumor cell. However, they indirectly function inside the TME by interacting with other immune cells and promoting or suppressing immunological responses [15,25,40]. Since the MCs interact with immune suppressor cells, such as MDSCs and Tregs, they can substantially influence the development and functional capability of tumor immunity [15,19,[55][56][57][58][59][60][61]. For example, MDSCs are recruited to tumor locations by CCL2 and potentially CXCL1 and CXCL2, where direct contact with MCs or with histamine increases their inhibitory function [19,55]. The MC stimulates the production of IL-17 via MDSC, mobilizing Tregs and enhancing their suppressive activity on cytotoxic T cell [19,57]. Subsequently, IL-17 indirectly recruits Tregs and increases their suppressor function and stimulates IL-9 production, thus enhancing the pro-tumorigenic function of the MCs in the TME [19,57]. Remarkably, TGF-β and IL-10 produced by the MCs promote the development of Tregs, downregulate expression of costimulatory molecules in DCs, and reduce the production of pro-inflammatory cytokines by TAMs. They also suppress antigen-specific T cell responses and enhance fibrosis [55,[58][59][60]. The presence of more tumor-specific IFN-γ + CD8 + T cells in the tumor-draining lymph nodes of MC-deficient mice than that in wild-type mice supports the notion that MCs suppress tumor-specific T cell response in UBC [55,56].

Cytokine Milieu in the Tumor Microenvironment Generated by Mast Cells
Histamine, a primary mediator secreted by MCs, exhibits either tumorigenic or antitumor downstream effects depending on the TME and its receptors H1R, H2R, H3R, or H4R [25,55,[62][63][64][65]. Depending on which receptor is activated, histamine may promote the function of particular Th subtypes or Treg responses and alter immunophenotype of monocytes; thus, immunosuppressive signals to NK cells are downregulated [55,[63][64][65]. Tryptase, a serine protease produced during MC activation, stimulates angiogenesis and ECM breakdown, resulting in tumor progression and metastasis [25,38,55,62]. Based on these findings, three MC tryptase inhibitors, nafamostat mesylate, tranilast, and gabexate mesylate, have been demonstrated as anti-cancer agents in multiple solid tumor types either in conjunction with other cancer treatments or as an individual treatment [55,[66][67][68]. Although tryptase inhibitors reduce angiogenesis and activation of MMPs, they also exhibit other activities, such as suppression of TGF-β, inhibition of other proteases, downregulation of NF-κB, and inhibition of the MAPK signaling pathway [55,69].
MCs release chymase and leukocyte elastase during inflammatory processes and degranulation. These molecules act on matrix-associated latent TGF-β complexes, releasing the latent TGF-β from the subendothelial ECM [15,41]. TGF-β acts a tumor suppressor during the premalignant phases of tumorigenesis, but it promotes tumor growth in the later stages, resulting in metastasis [19,70]. Additionally, TGF-β can directly suppress the immunological functions of effector T cells, NK cells, and B cells [41,71].
A clinical study demonstrated that the number of IL-17-positive MCs increased in few patients with carcinoma in situ while they underwent BCG therapy [88]. It was speculated that this occurrence was because of the release of IL-17 by the MCs in response to the BCG treatment. Subsequently, the MCs stimulate IL-8 production in the urothelium [88]. Further, it was hypothesized that neutrophils and various other immune cells were thereafter chemotactically attracted to the tumor site, thus amplifying the overall immune response [88]. This study can help stratify the cases that can have better survival and respond much better to the BCG treatment [72,89].

Manipulating Mast Cells to Boost Treatment-Induced Immunogenic Cell Death
Increased tumor-infiltration by MCs prior to surgery is significantly associated with poor response to pre-surgical chemotherapy in the aggressive form of locally advanced solid tumors [16,55]. Moreover, recent data indicate that increased tumor-infiltration by MCs predicts a poor response to ICIs that target PD-1 in melanoma [55,90]. Notably, increased infiltration of tumors by stromal MCs is an independent prognostic marker that indicates an unfavorable prognoses for patients with MIBC [91]. A retrospective investigation evaluating the effectiveness of adjuvant chemotherapy in patients with MIBC discovered that individuals who had minimal tumor-infiltrating MCs have a low risk of mortality and cancer recurrence [91]. Furthermore, differential gene expression profiles in MIBC specimens revealed that bladder tumors with a low number of invading MCs expressed more genes associated with immune activation [91]. Spatial analysis revealed close proximity between CTLs and the MCs, highlighting MCs as promising therapeutic targets that can improve current therapeutic strategies against UBC [16].
Most importantly, existing literature indicates that MCs play a critical role in orchestrating an initial antitumor immune response but may also be responsible for inducing resistance against ICIs [55]. Tumor RNA-seq, multiplexed imaging, and immunohistochemistry labeling have shown elevated chemokine expression, particularly with the recruitment of MCs and FOXP3 + Tregs at selected tumor locations [90]. These tumor-invading cells are linked with decreased expression of HLA-class I molecules on the tumor cells, a deficiency of CD8 + T cells in tumor locations, and efficient killing of tumor cells and eventual immunological escape following anti-PD-1 treatment [90]. When anti-PD-1 is combined with sunitinib or imatinib, MC numbers are depleted, and tumors completely regress, suggesting that MC depletion may improve the effectiveness of anti-PD-1 treatment [90]. Moreover, inhibition of MC-associated PD-L1 improved tumor control and boosted tumor-infiltration by CD3 + T-cells and elevated IFN-γ and granzyme B production [55].
Resistance to anti-PD-1 therapy mediated by the MCs requires greater research to discover new treatment avenues [90]. As the MCs are associated with resistance to anti-PD-1 treatment, their depletion enhances patient response for anti-PD-1 therapy [55,90]. Generally, therapeutics directed against the MCs in cancer have one of the three mechanisms of action: decreasing the number of MCs, modifying MC activity and phenotype, and altering the mediators produced by the MCs and their downstream functions [55]. c-KIT inhibitors, MC stabilizers, FcεR1 signaling pathway activators/inhibitors, antibodies against inhibitory receptors and ligands, and TLR agonists and modulators of MC mediators are all possible treatment approaches ( Figure 3) [55].
Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 14 [88]. Further, it was hypothesized that neutrophils and various other immune cells were thereafter chemotactically attracted to the tumor site, thus amplifying the overall immune response [88]. This study can help stratify the cases that can have better survival and respond much better to the BCG treatment [72,89].

Manipulating Mast Cells to Boost Treatment-Induced Immunogenic Cell Death
Increased tumor-infiltration by MCs prior to surgery is significantly associated with poor response to pre-surgical chemotherapy in the aggressive form of locally advanced solid tumors [16,55]. Moreover, recent data indicate that increased tumor-infiltration by MCs predicts a poor response to ICIs that target PD-1 in melanoma [55,90]. Notably, increased infiltration of tumors by stromal MCs is an independent prognostic marker that indicates an unfavorable prognoses for patients with MIBC [91]. A retrospective investigation evaluating the effectiveness of adjuvant chemotherapy in patients with MIBC discovered that individuals who had minimal tumor-infiltrating MCs have a low risk of mortality and cancer recurrence [91]. Furthermore, differential gene expression profiles in MIBC specimens revealed that bladder tumors with a low number of invading MCs expressed more genes associated with immune activation [91]. Spatial analysis revealed close proximity between CTLs and the MCs, highlighting MCs as promising therapeutic targets that can improve current therapeutic strategies against UBC [16].
Most importantly, existing literature indicates that MCs play a critical role in orchestrating an initial antitumor immune response but may also be responsible for inducing resistance against ICIs [55]. Tumor RNA-seq, multiplexed imaging, and immunohistochemistry labeling have shown elevated chemokine expression, particularly with the recruitment of MCs and FOXP3 + Tregs at selected tumor locations [90]. These tumor-invading cells are linked with decreased expression of HLA-class I molecules on the tumor cells, a deficiency of CD8 + T cells in tumor locations, and efficient killing of tumor cells and eventual immunological escape following anti-PD-1 treatment [90]. When anti-PD-1 is combined with sunitinib or imatinib, MC numbers are depleted, and tumors completely regress, suggesting that MC depletion may improve the effectiveness of anti-PD-1 treatment [90]. Moreover, inhibition of MC-associated PD-L1 improved tumor control and boosted tumor-infiltration by CD3 + T-cells and elevated IFN-γ and granzyme B production [55].
Resistance to anti-PD-1 therapy mediated by the MCs requires greater research to discover new treatment avenues [90]. As the MCs are associated with resistance to anti-PD-1 treatment, their depletion enhances patient response for anti-PD-1 therapy [55,90]. Generally, therapeutics directed against the MCs in cancer have one of the three mechanisms of action: decreasing the number of MCs, modifying MC activity and phenotype, and altering the mediators produced by the MCs and their downstream functions [55]. c-KIT inhibitors, MC stabilizers, FcεR1 signaling pathway activators/inhibitors, antibodies against inhibitory receptors and ligands, and TLR agonists and modulators of MC mediators are all possible treatment approaches ( Figure 3) [55]. Reduced MC numbers can be achieved by inhibiting the final step in the development of MCs from myeloid precursor cells. Other alternatives include lowering the growth factors required for cell survival or limiting the recruitment of MCs to tumors [55]. The SCF is a cytokine that binds to the c-KIT receptor and is critical for MC differentiation, survival, proliferation. Thus, MCs can be targeted pharmacologically by small molecule inhibitors targeting c-KIT used in clinical practice, including nilotinib, dasatinib, sunitinib, midostaurin, ibrutinib, and masitinib [55,92]. Monoclonal antibodies targeting c-KIT, such as CDX-1058 and CDX-0159, are in clinical development for inflammatory disease and c-KIT-positive solid tumors (NCT02642016) and have a greater specificity for the intended target than the TKIs [55]. However, clinical translation is hampered by the plasticity and context-based functions of the MCs [55]. Therefore, it is critical to include patient samplebased translational research into an investigation on the biologic relevance and therapeutic efficacy of MC-directed treatments [55].
Another therapeutic strategy for targeting the MCs is the prevention or abrogation of MC activation [25,55,93,94]. Targeting MC activation by inhibiting the secretion of mediators using tyrosine kinase inhibitors (TKIs; imatinib, sunitinib, and masitinib) or using tryptase inhibitors (gabexate mesylate and nafamostat mesylate) can be beneficial as an anti-cancer therapy [25,55,90,95,96]. Furthermore, stabilizing medications, such as cromolyn sodium, are often used in allergy disorders to inhibit degranulation in MCs and have been studied in various preclinical solid tumor models [25,55,93,94]. For instance, in a study on MYC-induced pancreatic neuroendocrine tumors, association of MYC activation with MC recruitment was observed to be necessary for tumor growth, and treatment with cromolyn sodium inhibited MC degranulation and reduced tumor growth [55,93].
Since MCs and MC-induced inflammation induce anti-tumor responses, studies have proposed the use of anti-tumor IgE antibodies for cancer immunotherapy [55,[100][101][102]. The high density of FcεR1 and the prolonged half-life of antibodies makes this an appealing treatment approach, particularly for malignancies with high MC infiltration. Omalizumab, a humanized monoclonal antibody directed against IgE has been demonstrated to be effective in severe allergic asthma and is often administered to patients today. In vitro experiments using humanized monoclonal anti-HER-2/neu IgE and humanized anti-CD20 IgE targeted MC degranulation and reduced tumor cell proliferation [55,101]. Combining anti-MUC-1 IgE with chemokines that target the MCs in an MUC-1-expressing 4T1 murine breast cancer model resulted in tumor rejection. Importantly, the 4T1 cells were also subsequently rejected on the contralateral flank in the absence of either the IgE antibody or chemokines, suggesting the activation of a memory immune response [55,100]. Of note, anti-tumor IgE antibodies are limited to targetable tumor antigens, such as HER2, CD20, and MUC-1; thus, FGFR 2/3 and necti-4 in UBC are potential tumor-specific targets [55].
Given the abundance and plasticity of the MCs, there have been considerable attempts to favorably modify the function of pre-existing intra-tumoral MCs to induce an antitumor response rather than to deplete MC levels [55,103,104]. Targeting TLRs is a feasible therapeutic approach for doing this, either by direct inhibition with synthetic TLR agonists or indirect inhibition with natural TLR agonists produced as intermediates in response to other immunotherapies [58,103,104]. Conventionally, cancer immunotherapy targeting TLRs has focused on increasing TLR activity in macrophages, DCs, and B cells. Presently, the critical role of TLRs on monocytes and macrophages in defining cancer immunity has been gaining momentum [58,103,104]. Furthermore, antibodies directed against inhibitory cell surface receptors to suppress MC activation are another avenue of active research [58]. Recently, SIGLEC-8 was discovered as an inhibitory receptor that is mostly expressed on the surface of the MCs, eosinophils, and, to a lesser degree, basophils [103,104]. When bound to its ligand, it directly induces antibody-dependent cell-mediated cytotoxicity and decreases degranulation in the MCs [58]. Remarkably, antolimab is a humanized IgG1 monoclonal antibody that inhibits SIGLEC-8 and, therefore, decreases MC activation and inflammation in anaphylaxis mice models [58].

Take-Home Messages and Challenges
Therapeutic strategies that disrupt the function of the MCs or that of their mediators are common [29]. Notably, the MCs can serve as a novel target for adjuvant therapy for cancers, as they can selectively inhibit angiogenesis, tissue remodeling, and tumorpromoting molecules, thus secreting cytotoxic cytokines and preventing MC-mediated immune suppression [29]. Pre-clinical studies in experimental models, using anti-c-KIT antibodies [105], anti-TNFα antibodies [106], or the MC stabilizer cromolyn [93], have shown promising results [29]. The c-KIT pathway is often activated in tumors, identifying c-Kit as a proto-oncogene. As a result, targeting of the c-KIT pathway was considered to be an optimal approach for a tumor-specific treatment [29]. Consequently, novel TKIs may be effective against wild type c-KIT, which is expressed by the tumor-infiltrating MCs, and may be beneficial in eliminating the MCs. Additionally, c-KIT-targeted therapy with TKIs may ideally work against both tumor and stromal cells [29]. Despite seeking to positively regulate the anti-tumor capabilities of the MCs, clinical evaluation of toxicities is necessary to ensure that critical biological responses necessary for the health of the host are not negatively affected [55].
Our understanding of the MC immunobiology in cancer is limited because majority of the research has been conducted in vitro using human and murine samples, as the MC biology differs inherently in both these samples. Moreover, it is difficult to decipher the specificity of the MC mediators, such as cytokines and chemokines, as they are secreted by multiple cell types [55]. The function of an MC depends on factors, such as the cancer type and stage, likely treatment history, concurrent anti-cancer therapies, and on the activation and location of MCs within a tumor and how they are altered by the therapeutics being investigated [55]. Advances in immune monitoring provide in-depth profiling data on immune cells and include techniques, such as single cell sequencing technologies, functional assays to assess polyfunctional responses, and multiplexed immunohistochemistry to determine spatial organization and interaction of the cells [55]. Studying the pharmacodynamic changes in intratumoral MCs and mediators in patient samples subjected to these therapies and studying the role of MCs specifically in these therapies in pre-clinical studies can provide an added insight into cancer therapy targeting the MCs [55]. Thus, discovering therapeutically relevant characteristics and mechanisms in the MCs and understanding the longitudinal changes in these features and pathways in response to systemic therapies is critical to determine an optimal therapeutic approach [55]. Nevertheless, preclinical studies, such as combination trials involving PD-1 immune checkpoint inhibition in multiple malignancies, indicate that discovery of optimum treatment combinations may be more necessary than monotherapy involving MCs in UBC [55]. Notably, it is critical to combine rational trial design with pharmacodynamic evaluations to discover the most effective treatment responses and possible resistance mechanisms when designing clinical trials to evaluate novel combinations [55]. The timing and agent selection are critical factors in a combination therapy [107].
As one of the earliest cells to infiltrate tumors, MCs are crucial components of the immune system that can induce angiogenesis and aid tumor progression. Opposingly, the MCs are beneficial to patients as they selectively recruit various immune cells to the tumor region. Their role in the development and progression of UBC needs a detailed investigation. Moreover, their role may vary depending on the stage and invasiveness of the cancer. Thus, further investigation into the recruitment of MCs to tumors and the functional role of MC mediators can delineate the potential of MCs as novel immunotherapeutic targets against UBC. Furthermore, interaction of the MCs with other cell types in the TME should be studied further to elucidate all possible interactions and prognostic implications. Focusing on the activation of MCs and the release of inflammatory cytokines in UBC may help develop innovative UBC-control strategies. Moreover, study on the architecture and geographic distribution of MCs can provide further insights into their involvement in UBC biology. Profiling the heterogeneity of MCs in benign and malignant solid tumors can target and avoid MC-mediated tumor angiogenesis. Additionally, particular attention must be paid to determine the composition of MCs in the TME in UBC, since contact with the complex tumor environment has been demonstrated to affect the functional expression of various membrane receptors. To monitor the onset of ICD and its subsequent consequences on MC immunobiology in UBCs, clinical studies must regularly integrate a strong biomarker strategy [108]. These biomarkers must assess the type of cell death and quantify the release of DAMPs, as well as determine the number, identity, and location of immune cells involved in a functioning adaptive immune response [108]. Biomarkers should distinguish between natural and therapeutically-mediated ICD by focusing on cell types that are most sensitive to DAMPs [108]. Since the end stage of an ICD is a protective T cell response, T cell numbers should also be quantified to determine the effectiveness of treatment strategies [108].