## 3 Preclinical Evidence Evaluating the Effects of Phytochemicals on Immune Checkpoints  
Although the effects of phytochemicals on the immune system and tumor microenvironment have been extensively studied [12; 15], the impact of phytochemicals on the immune checkpoints began receiving increased interest following ICIs' entrance into clinical practice. Phytochemicals have been suggested to have the potential to modulate the therapeutic effects of immune checkpoint inhibitors through the regulation of several signaling pathways (Figure 1). Preclinical studies with variable phytochemicals were conducted on several tumor types, including NSCLC, breast cancer, colorectal cancer, hepatocellular cancer, melanoma, head and neck cancer, and glial tumors (Table 1). While most studieshave focused on phytochemicals' effects on the PD-1 and PD-L1 pathways, one study reported the blockage of both PD-1/PD-L1 and the CTLA-4/CD80 interactions by the flavonoids eriodictyloI fisetin and quercetin liquiritigenin [16]. The phytochemicals mainly caused decreases in the tumor PD-L1 expression. In contrast, Z-guggulsterone, a phytosterol, increased PD-L1 mRNA expression and transcription in a dose-dependent manner via the activation of the AKT and ERK1/2 signaling pathways [17]. While the authors suggested that increased PD-L1 expression could be an opportunity to develop combinations with synergism, the increased PD-1 expression secondary to AKT and ERK1/2 activations could reduce ICI efficacy as a result of the pivotal roles of AKT and ERK1/2 activation in resistance to immunotherapy in clinical studies [18; 19]. Interestingly, Phoenix dactylifera extract (including gallic acid, coffee acid, and ellagic acid phytochemicals) increased cardiac and kidney PD-1 expressions in a mouse model exposed to Adriamycin [20]. The Phoenix dactylifera extract led to attenuated cardiotoxicity and nephrotoxicity secondary to decreased oxidative stress and apoptotic pressure. The authors suggested that increased PD-1 levels could protect from cardiotoxicity and nephrotoxicity via reduced oxidative stress and apoptotic pressure [20]. If their observations could be confirmed in clinical trials, the phytochemicals could be used for toxicity prevention in ICI-treated patients. Additionally, increased PD-1 expression and corresponding immuno  
Figure 1: Schematic figure representing some interactions between phytochemicals, T-cells, and cancer cells. Phytochemicals have been suggested to have the potential to modulate the therapeutic effects of immune checkpoint inhibitors and PD-L1 expression through the regulating of several signaling pathways, such as EGFR/Akt. Some phytochemicals can regulate PD-L1 expression, while others inhibit PD-L1 glycosylation or PD-L1/PD-1 binding, such as quercetin, saponins, and fisetin. Abbreviations: EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; MHC: major histocompatibility complex; PD-1: programmed cell death protein 1; PD-L1: programmed death ligand 1 (PD-L1). suppression could be exercised as a strategy to prevent or mitigate immune-related adverse events by immunotherapy. The STAT pathway was the other commonly affected pathway from phytochemicals, with five studies reporting STAT pathway aberrations in addition to changes in the PD-1/PD-L1 pathway [21; 22; 23; 24; 25]. While STAT1 and STAT2 involve the constitution of the antitumor response via cross-talks with IFN, STAT3 has pro-oncogenic and immunosuppressive properties [26]. Phytochemicals created a consistent inhibitory effect on the STAT3 pathway, and STAT3 down-regulation was among the drivers of PD-L1 suppression in several studies [22; 25]. Contrary to the inhibition of immune-suppressive STAT3 by phytochemicals, [PERSON] et al. demonstrated an inhibitory effect on STAT1 by apigenin, a flavonoid, and observed a greater decrease in PD-L1 by apigenin than curcumin secondary to more potent inhibition of STAT1 by apigenin [23].