The Use of Phytochemicals to Improve the Efﬁcacy of Immune Checkpoint Inhibitors: Opportunities and Challenges

: Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy and reshaped medical oncology practice over the past decade. However, despite unprecedented and durable clinical responses, most patients eventually fail to respond to ICI therapy due to primary or acquired resistance. There is a great need for complementary alternative medicine, such as botanicals and nutritional supplements, because of their capability to modulate a myriad of molecular mechanisms to prevent immunotherapy resistance and reduce its adverse effects. Mounting evidence suggests that phytochemicals, biologically active compounds derived from plants, can favorably regulate key signaling pathways involved in tumor development and progression. In addition, phytochemicals have been found to exert anticancer effects by altering the expression of checkpoint inhibitors of the immune response. The immunomodulatory activity of phytochemicals in the tumor microenvironment has recently received immense interest. Based on these immunomodulatory activities, phytochemicals could be candidates for combination with ICIs in future clinical studies. The current review focuses on the available evidence for combining phytochemicals with a discussion on the promising opportunities to enhance the efﬁcacy of immune checkpoint inhibitors and potential challenges resulting from these combinations. on phytochemicals’ effects on the PD-1 and PD-L1 pathways, one study the blockage of both PD-1/PD-L1 and the CTLA-4/CD80 interactions by the ﬂavonoids eriodictyol ﬁsetin and quercetin liquiritigenin 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 While the 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


Introduction
Immune checkpoint inhibitors (ICIs) have dramatically changed the oncology landscape and become a vital part of cancer care [1]. ICIs have demonstrated efficacy in several scenarios, such as monotherapy, combination with chemotherapy or targeted therapy, and first-line and subsequent treatment lines in almost all cancers [2][3][4]. The monoclonal antibodies against PD-1 and PD-L1 are the foundations of modern immunotherapy, and currently, eight anti-PD-1/PD-L1 agents are licensed in several different indications [5,6]. Still, many patients do not respond to ICIs, and resistance is inevitable in previously responding patients. Baseline and on-treatment immune dysregulation and immune exhaustion are the main proposed reasons for this treatment resistance [7]. There is an urgent need for novel approaches, such as combinations, to modify tumor microenvironments and mobilize the immune system to fight against tumors more effectively.
Phytochemicals are plant-based bioactive chemicals with diverse health-promoting effects, including cancer prevention. Removal of antioxidant stress, prevention of DNA

Literature Search and Review Structure
We conducted a literature review from the PubMed, Medline, and Embase databases to perform filtering of published studies. The MeSH search terms were "immunotherapy" OR "immune checkpoint" AND "PD-1" OR "PD-L1" OR "CTLA-4" OR "phytochemical" OR "phytonutrient" OR "microbiome". Additionally, we conducted PubMed searches for the individual phytochemical names. We included original articles in the English language that evaluate the effects of phytochemicals on immune checkpoints, ICI and phytochemical combinations, and the association between the microbiome and ICI efficacy.
The review consisted of four sections. The first section was about "Preclinical Evidence Evaluating the Effects of Phytochemicals on Immune Checkpoints", for which we reviewed the available evidence on the effects of phytochemicals on immune checkpoints in cell line and animal models. In the second section of the review, "The Preclinical Studies Evaluating the Efficacy of Phytochemical and Immune Checkpoint Inhibitor Combinations", we reviewed the studies evaluating phytochemical and ICI combinations. Due to a lack of human data, we included preclinical studies only. In "The Clues from the Microbiome Studies on the Benefit of Phytochemicals in Immunotherapy Efficacy", we reviewed the association between phytochemicals and the microbiome and the therapeutic opportunities that stemmed from phytochemicals' effects on the microbiome. In the last part of the review, we discussed the further clinical perspectives and challenges related to phytochemical and ICI combinations in the clinic and areas needing improvement to develop effective combinations.

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 studies have 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 eriodictyol 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].
practice. Phytochemicals have been suggested to have the potential to modulate the apeutic effects of immune checkpoint inhibitors through the regulation of several si ing pathways (Figure 1). Preclinical studies with variable phytochemicals were condu on several tumor types, including NSCLC, breast cancer, colorectal cancer, hepatocel cancer, melanoma, head and neck cancer, and glial tumors (Table 1). While most stu have focused on phytochemicals' effects on the PD-1 and PD-L1 pathways, one stud ported the blockage of both PD-1/PD-L1 and the CTLA-4/CD80 interactions by the f noids eriodictyol fisetin and quercetin liquiritigenin [16]. The phytochemicals m caused decreases in the tumor PD-L1 expression. In contrast, Z-guggulsterone, a tosterol, increased PD-L1 mRNA expression and transcription in a dose-dependent m ner via the activation of the AKT and ERK1/2 signaling pathways [17]. While the aut suggested that increased PD-L1 expression could be an opportunity to develop com tions with synergism, the increased PD-1 expression secondary to AKT and ERK1/2 vations could reduce ICI efficacy as a result of the pivotal roles of AKT and ERK1/2 vation in resistance to immunotherapy in clinical studies [18,19]. Interestingly, Phoenix dactylifera extract (including gallic acid, coffeic acid, an lagic acid phytochemicals) increased cardiac and kidney PD-1 expressions in a m model exposed to Adriamycin [20]. The Phoenix dactylifera extract led to attenuated diotoxicity and nephrotoxicity secondary to decreased oxidative stress and apop pressure. The authors suggested that increased PD-1 levels could protect from cardi icity and nephrotoxicity via reduced oxidative stress and apoptotic pressure [20]. If observations could be confirmed in clinical trials, the phytochemicals could be use toxicity prevention in ICI-treated patients. Additionally, increased PD-1 expression Interestingly, Phoenix dactylifera extract (including gallic acid, coffeic 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- 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, Xu 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]. These observations show that different phytochemicals could act differently on STAT proteins (immune-activating vs. immune-suppressive), and various classes of phytochemicals (i.e., flavonoid and non-flavonoid polyphenols) could have different potency on the tumor immune milieu ( Figure 2). Further delineation of these interactions for individual phytochemicals is paramount to designing successful phytochemical and immunotherapy combinations. corresponding immunosuppression 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, Xu 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]. These observations show that different phytochemicals could act differently on STAT proteins (immune-activating vs. immunesuppressive), and various classes of phytochemicals (i.e., flavonoid and non-flavonoid polyphenols) could have different potency on the tumor immune milieu ( Figure 2). Further delineation of these interactions for individual phytochemicals is paramount to designing successful phytochemical and immunotherapy combinations.

Preclinical Studies Evaluating the Efficacy of Phytochemical and Immune Checkpoint Inhibitor Combinations
Several studies have evaluated the efficacy of ICI plus phytochemical combinations using variable phytochemicals in different cancer models ( Table 2). The first report of a phytochemical and ICI combination strategy (bisdemethoxycurcumin with an anti-PD-L1 antibody) was published by Shao et al. in 2017 in a mouse bladder cancer model [27]. The researchers observed T-lymphocyte-based immune activation and removal of immune exhaustion via a decrease in the intratumoral myeloid-suppressor cells, leading to increased survival [27]. Later studies most frequently used curcumin, a non-flavonoid polyphenol, as the experimental phytochemical [28][29][30][31] and primarily focused on the colorectal cancer models [28,30,31,36], followed by NSCLC [33,38,39]. The intratumoral expansion of CD8+ or CD4+ lymphocytes and the increased levels of IFN-γ were consistent findings throughout the studies [34,42].
The decrease in PD-L1 levels was observed in five studies conducted with curcumin [28,29], lycopene [39], gallic acid [33], and melafolone [38] and could contribute to the synergism between ICI and phytochemicals by releasing the inhibitory breaks of immune checkpoints. In contrast, an increase in PD-L1 expression level was reported by Lasso et al. with gallatonin-rich Caesalpinia spinosa and anti-PD-L1 antibody combination [34]. The previous observations of PD-L1 expression increase with resveratrol and piceatannol in breast and colorectal cancer cell lines support the variable effects of different phytochemicals on immune checkpoint expressions [43], although the reason for contrasting effects on PD-L1 expression with the phytochemicals from the same class (non-flavonoid polyphenols) is yet to be defined.
While it could be problematic to expect a synergism between ICIs and phytochemicals, we think the differential effects of phytochemicals on PD-L1 expression levels could be beneficial to aid individualized treatment planning according to tumor microenvironments in different tumors. While decreasing immunosuppression secondary to decreased PD-L1 expression would be beneficial in a tumor-agnostic manner, increased ICI efficacy with increased tumoral PD-L1 expression levels was observed in NSCLC [33], melanoma [34], and bladder cancer [27] patients. Both the pretreatment with phytochemicals and the addition of phytochemicals at times of progression to increase PD-L1 expression should be evaluated as a treatment strategy, especially in tumors with an increased benefit with increased PD-L1 expressions.

Clues from Microbiome Studies on the Benefit of Phytochemicals in Immunotherapy Efficacy
The commensal bacteria residing in the gastrointestinal system and their genome is called the gastrointestinal (GI) microbiome. The GI microbiome plays significant roles in self-defense and inflammation [68]. Recently, the GI microbiome has emerged as a predictor of ICI efficacy, and several studies have demonstrated better survival in ICI-treated patients enriched with beneficial commensal bacteria, including Akkermansia muciniphila, Bacteroides spp., and Faecalibacterium prausnitzii. Akkermansia muciniphila in particular was associated with a response to ICIs in four studies conducted on patients with three different tumors (RCC, NSCLC, and HCC) [69][70][71][72]. Whether the increases in these bacteria are secondary to a bystander effect or these bacteria play roles in ICI efficacy is highly debated. However, the restoration of ICI efficacy with Akkermansia muciniphila transplantation in ICI nonresponders mice [70] and the recent report of improved ICI efficacy with microbiome and ICI combination in metastatic renal cell carcinoma patients [73] point to an anti-tumor role of the microbiome rather than it only being a biomarker.
Several phytochemicals such as curcumin and phenols could increase levels of beneficial commensal bacteria (Table 3) and correct the dysbiosis created by oxidative stress, as shown in alcoholic liver disease and fatty liver disease models. Similarly, phytochemicals could correct dysbiosis in patients treated with ICIs. These phytochemicals are most commonly found in fiber-rich diets (Table 3). Frankel et al. reported indirect evidence of this strategy's possible benefit in melanoma patients [14]. Metabolomic analyses in 39 ICI-treated melanoma patients revealed that the anacardic acid levels were increased in responders. Most of these patients (five out of six) had dietary habits that explained the high anacardic acid levels. Based on these points, we think using phytochemicals to correct dysbiosis and increase levels of bacteria associated with ICI efficacy should be exploited in clinical trials. Additionally, further research is needed to delineate the optimal fiber type to consume for ICI efficacy. Recently, Nakajima et al. reported that a soluble fiber diet increased gut Bacteroides fragilis, previously associated with ICI response, while the insoluble fiber-rich diet reduced the Bacteroides fragilis levels in a mice model [74]. In addition, Li et al. previously demonstrated the enrichment of beneficial commensal Actinobacteria and Akkermansia in obese mice fed with nondigestible fructans, which are found in higher concentrations in bananas, compared to mice fed with cellulose [75,76]. Until the results of these trials become available, the recommendation of eating soluble fiber-rich diets and diets rich in phytochemicals that increase beneficial commensal bacteria could benefit ICI-treated patients.

Future Perspectives
Phytochemicals have several effects on promoting anti-tumor immunity and modulating the tumor microenvironment, including immune checkpoints. Based on the available preclinical evidence, phytochemicals have the potential to transform immunologically cold tumors into hot tumors and improve immunotherapy efficacy. This strategy could be especially promising for tumors that have consistently garnered less benefit from ICIs, including sarcomas and brain tumors [104,105]. The available preclinical evidence and the demonstration of higher ICI response rates in melanoma patients with increased anacardic acid levels in metabolomic studies support the progression of phytochemical and ICI combinations to clinical studies [14]. Patient-derived xenograft models would be suitable venues to assess the clinical use of phytochemicals in transforming tumor immune profiles and would be beneficial for further clinical studies.
Combining chemotherapy and ICIs and combining two ICIs (CTLA-4 and PD-1/PD-L1) has become the standard of care in the first-line treatment of NSCLC, gastric cancer, RCC, and melanoma, with improved response rates and survival [106][107][108][109][110]. The increased immune activation in the tumor microenvironment was among the main drivers of the increased efficacy of these combinations [111]. However, the efficacy of adding phytochemicals to these combinations has not been investigated yet. We think that phytochemicals could add benefits to these combinations, and further research focusing on the efficacy of adding phytochemicals to chemotherapy-ICI and ICI-ICI combinations could have implications for current clinical practice.
Another knowledge gap concerns the efficacy of using phytochemical and ICI combinations in adjuvant settings. Although it would be harder to measure the efficacy of phytochemical and ICI combinations in adjuvant settings, animal models with radiated tumors or chemoprevention models using ICI and phytochemical combinations should be designed considering the well-known efficacy of phytochemicals in chemoprevention.
Lastly, the phytochemical structure of the traditional herbs with well-known immunomodulatory effects should be thoroughly delineated to identify more candidates to combine with ICIs. For example, Artemisia products are used in China to fight malaria, allergies, and auto-immune diseases [112]. The recently conducted phytochemical analysis of Artemisia annua L. demonstrated the presence of several flavonoids, hydroxycoumarins, and phytosterols, phytochemicals with possible anticancer effects on immune checkpoints [113]. Furthermore, recent gene expression analyses in HCC about Artemisia scoparia demonstrated the increased expression of BIRC5 and secondary expression of CTLA-4 and LAG-3 immune checkpoints and a possible immune activation with this herbal medicine [114]. Further studies evaluating the phytochemical constituent of herbal medications and corresponding effects on the immune checkpoints are needed.

Conclusions
In conclusion, plant-based phytochemicals could be beneficial adjuncts to ICIs with improved immune activation and tumor microenvironment modulation. Further research is needed considering the difficult scenarios in the clinical practice and the areas needing improvement to answer more clinically oriented questions.