2.2.4. Novel therapeutic strategies against MDSCs

Although various agents such as chemotherapeutic drugs, vitamin derivative, and mAb are widely applied against MDSCs, only few of them are known to exhibit their inhibitory effect directly on MDSCs. Moreover, restricted use of these drugs on certain conditions or cancer patients often opens the possibility to apply emerging agents that bear direct inhibitory effect on MDSCs.

Earlier investigations reveal CuNG as a strong immunomodulator that successfully induce the conversion of immunosuppressive TAMs toward proimmunogenic type, enhancing Th1 response sufficient to resolve drug-resistant cancer in animal model. Interestingly, the effect of CuNG is not exclusive to TAMs; rather it also targets MDSCs which happens to be another strong negative regulator of tumor immunity [55]. Treatment with CuNG in drug-resistant mice severely abrogates the accumulation of MDSCs in spleen as well as tumor site. Further study discloses that this reduction was associated with enhanced Th1 responses (evident from increase in IFN-γ production) and diminished Th2 responses (decrease in IL-4 production) as well as decline in Foxp3<sup>+</sup> Treg numbers [55]. Detailed investigation reveals the underlying mechanism that is associated with Fas-FasL interaction between MDSCs and CD4<sup>+</sup> T cells. Treatment of CuNG increases the FasR expression in MDSCs and FasL in CD4<sup>+</sup> T cells. Although FasL expression is enhanced, FasR expression in CD4<sup>+</sup> T cells was not observed following the treatment that determines the specificity of CuNG [55].

Another study that deals with metal chelate of Schiff's base is associated with the immunomodulation of tumor microenvironment. Metal chelate of manganese (MnNG) can have a number of valence states and possess the ability to modulate the immunosuppressive TME by reducing the number of tumor-associated MDSCs in drug-resistant tumor-bearing mice. A closer look at MnNG-mediated action reveals that it promotes MDSC differentiation into dendritic cells but not toward macrophages [56]. Additionally, it helps in the proliferation of T cells that ultimately skew Th2 immunity toward protective Th1 response. Thus MnNG may help in the differentiation of immunosuppressive MDSCs toward dendritic cells and with the upregulation of costimulatory molecules, namely CD80 and CD86, enabling them to act as better antigenpresenting cells (APCs) that ultimately generate protective antitumor immunity [56]. However, the plausible molecular mechanism underlying MnNG-mediated differentiation of MDSCs in TME is still elusive and warrants further investigations.

## 2.3. T-regulatory cells in cancer

led to MDSC-derived IL-1β secretion and angiogenesis [53]. Attempts have also been made to deplete Gr-1+ MDSCs by using anti-Gr-1 antibody that result in delayed tumor growth in mice [53]. However, this antibody also targets neutrophils, thus lacking the necessary specificity for

Fourthly, another attractive way to control MDSC function would be interrupting the underlying signaling pathways that are responsible for the production of suppressive factors by these cells. The molecules that can be effectively targeted for this purpose include cyclooxygenase 2, arginase 1, iNOS, and indoleamine 2,3-dioxygenase [52]. Since ARG1 and iNOS are the primary enzymes responsible for MDSC immunosuppression, these enzymes are the most likely targets for novel therapeutic interventions. Various different drugs including nitroaspirin, COX-2 inhibitors, and phosphodiesterase-5 (PDE5) inhibitors have been shown to profoundly inhibit both ARG1 and iNOS activity in MDSC. By removing MDSC suppressive mediators, these drugs exhibited a potent ability to restore antitumor immune responses and delayed tumor progression in several mouse models [52–54]. Interestingly, in addition to inhibiting MDSC function, COX2 inhibitors also blocked the systemic development of MDSC as well as CCL2-mediated accumulation of these cells in the tumor microenvironment in a

Although various agents such as chemotherapeutic drugs, vitamin derivative, and mAb are widely applied against MDSCs, only few of them are known to exhibit their inhibitory effect directly on MDSCs. Moreover, restricted use of these drugs on certain conditions or cancer patients often opens the possibility to apply emerging agents that bear direct inhibitory effect

Earlier investigations reveal CuNG as a strong immunomodulator that successfully induce the conversion of immunosuppressive TAMs toward proimmunogenic type, enhancing Th1 response sufficient to resolve drug-resistant cancer in animal model. Interestingly, the effect of CuNG is not exclusive to TAMs; rather it also targets MDSCs which happens to be another strong negative regulator of tumor immunity [55]. Treatment with CuNG in drug-resistant mice severely abrogates the accumulation of MDSCs in spleen as well as tumor site. Further study discloses that this reduction was associated with enhanced Th1 responses (evident from increase in IFN-γ production) and diminished Th2 responses (decrease in IL-4 production) as

mechanism that is associated with Fas-FasL interaction between MDSCs and CD4<sup>+</sup>

Treatment of CuNG increases the FasR expression in MDSCs and FasL in CD4<sup>+</sup>

following the treatment that determines the specificity of CuNG [55].

Although FasL expression is enhanced, FasR expression in CD4<sup>+</sup> T cells was not observed

Another study that deals with metal chelate of Schiff's base is associated with the immunomodulation of tumor microenvironment. Metal chelate of manganese (MnNG) can have a number of valence states and possess the ability to modulate the immunosuppressive TME by reducing the number of tumor-associated MDSCs in drug-resistant tumor-bearing mice. A closer look at MnNG-mediated action reveals that it promotes MDSC differentiation into dendritic cells but not toward macrophages [56]. Additionally, it helps in the proliferation of T cells that ultimately

Treg numbers [55]. Detailed investigation reveals the underlying

T cells.

T cells.

clinical use.

on MDSCs.

mouse model of glioma [52, 54].

178 Anti-cancer Drugs - Nature, Synthesis and Cell

well as decline in Foxp3<sup>+</sup>

2.2.4. Novel therapeutic strategies against MDSCs

The tumor escape of immune surveillance is also achieved by the recruitment of immunosuppressive CD4<sup>+</sup> CD25<sup>+</sup> FoxP3+ Tregs into the tumor microenvironment. The phenotype and functional properties of Tregs are similar in mice and human. They include both natural Treg (nTreg) and locally induced Treg (iTreg) cells [57, 58]. nTreg cells are derived from thymus without specific antigenic stimulation and represents a small fraction (5–6%) of total CD4<sup>+</sup> T cells. They are characterized by the expression of CD25, FoxP3, and GITR [3]. CD4<sup>+</sup> or IL-10 expressing iTreg cells are also known to express high levels of Foxp3 and GITR [58]. Induced Tregs are emerged from naive T cells by specific modes of antigenic stimulation, especially in a particular cytokine milieu [57, 58]. Both subsets possess a strong capacity to suppress the immune system, although they differ in a distinct suppressive mechanism. A large number of CD4<sup>+</sup> CD25<sup>+</sup> FoxP3+ Treg cells are found in both the circulation and tumor site of different cancer-bearing patients and their number inversely proportional to the survival of the patients. They are either naturally occurring or locally induced Treg population [58]. Experimental observation reveals that chemokine CCL2 produced by tumor and its associated macrophages facilitate the recruitment of thymus-derived Treg cells in tumor microenvironment [3]. Defective myeloid DCs also induced IL-10+ regulatory T cells in vitro and in vivo in patients with cancer. Conversion of Treg from CD4<sup>+</sup> CD25<sup>−</sup> T cells depends on local cytokine milieu. Immunosuppressive cytokines such as TGF-β and IL-10, which are present at high levels in the tumor microenvironment, might mediate induction and differentiation of iTreg cells [58]. Recent studies also highlighted the role of MDSCs to facilitate the de novo development of Tregs through TGF-β dependent and independent pathways [34].

#### 2.3.1. Tregs and immunosuppression

Tumor-associated Tregs suppress both the innate and the adaptive immune responses. The suppressive function of tumor-induced Tregs depends on the antigen-specific stimulation of these cells. Once activated, they efficiently suppress CD4+ and CD8+ T cells in an antigen nonspecific manner [58]. This suppressive potential of Tregs depends on the cell-cell contact and soluble mediators (e.g., TGF-β and IL-10) released by Tregs. Recent evidence suggests that Treg exerts immunosuppressive function by lowering the endogenous TAA-specific T-cell immunity, thereby contributing to tumor progression [3]. Furthermore, the progressive infiltration and induction of Treg tilt the balance between Treg cells and effector T cells toward immunosuppression. This is, in part, mediated by competition for IL-2 between Tregs and conventional T cells. IL-2 promotes the differentiation of T cells into effector T cells. Interestingly, it also promotes the differentiation of immature T cells into regulatory T cells. Therefore, competitive consumption of IL-2 is also indirectly related to suppressive mechanism for Tregs in established tumors [59]. Another effective way to suppress T-cell activation and promote tolerance is to reduce the availability of tryptophan, which is performed by the IDO+ APC in the tumor microenvironment. CTLA4+ Tregs help inducing the expression of IDO in APCs through CTLA4 to mediate their suppressive activity [60]. Furthermore, Tregs also downregulate the expression of CD80 and CD86 through CTLA-4 and induce immunosuppressive costimulatory molecule B7-H4 on DCs by IL-10 and thus hamper activation of other T cells by DCs [61]. Additionally, Tregmediated suppression of natural killer (NK)-cell function is associated with TGF-β in tumorbearing mice [62].
