An Immunomodulatory Gallotanin-Rich Fraction From Caesalpinia spinosa Enhances the Therapeutic Effect of Anti-PD-L1 in Melanoma

PD-1/PD-L1 pathway plays a role in inhibiting immune response. Therapeutic antibodies aimed at blocking the PD-1/PD-L1 interaction have entered clinical development and have been approved for a variety of cancers. However, the clinical benefits are reduced to a group of patients. The research in combined therapies, which allow for a greater response, is strongly encouraging. We previously characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) with antitumor activity in both melanoma and breast carcinoma, as well as immunomodulatory activity. We hypothesize that the combined treatment with P2Et and anti-PD-L1 can improve the antitumor response through an additive antitumor effect. We investigated the antitumor and immunomodulatory activity of P2Et and anti-PD-L1 combined therapy in B16-F10 melanoma and 4T1 breast carcinoma. We analyzed tumor growth, hematologic parameters, T cell counts, cytokine expression, and T cell cytotoxicity. In the melanoma model, combined P2Et and anti-PD-L1 therapy has the following effects: decrease in tumor size; increase in the number of activated CD4+ and CD8+ T cells; decrease in the number of suppressor myeloid cells; increase in PD-L1 expression; decrease in the frequency of CD8+ T cell expressing PD-1; improvement in the cytotoxic activity of T cells; and increase in the IFNγ secretion. In the breast cancer model, P2Et and PD-L1 alone or in combination show antitumor effect with no clear additive effect. This study shows that combined therapy of P2Et and anti-PD-L1 can improve antitumor response in a melanoma model by activating the immune response and neutralizing immunosuppressive mechanisms.


INTRODUCTION
Cancer is a major public health problem and remains a main cause of mortality and morbidity worldwide. Cancer patients have traditionally been treated with chemotherapy and radiotherapy despite their significant toxicity and lack of effectiveness in all patients. In the last decade the role of the immune system in the control of tumor growth and progression has been well established, and several immunotherapies have been designed. Cancer vaccines, antibody-mediated immune modulation, adoptive T-cell transfer (1), and strategies to induce immunogenic cell death (2) have been tested in patients. However, to date immunotherapy has shown durable clinical benefit in only a small subset of patients. Consequently, the search for alternative treatments, therapies or combined strategies against cancer is highly important (3).
Antibodies against immune-checkpoint molecules, as programmed cell death protein-1 (PD-1), aim to neutralize immunosuppression of tumor infiltrating lymphocytes (TILs) have been approved for a variety of cancers (4)(5)(6)(7). Unfortunately, only a minority of patients benefit from this checkpoint blockade across many types of cancer. These checkpoint inhibitors seem to be more effective when there has been a prior immune system activation associated with a sufficient number of TILs and a higher expression of PD-1 ligand (PD-L1) on tumor cells (4,5,8,9). Thus, to improve the number of patients who benefit from PD-1 blockade, PD-1/PD-L1 antibodies are being used together and/or in combination with other anticancer agents or immunotherapies. The U.S. Food and Drug Administration (FDA) has approved the uses of atezolizumab (a monoclonal anti-PD-L1 antibody) in combination with albumin-linked paclitaxel (nab-Paclitaxel) for the treatment of patients with unresectable locally advanced or metastatic triple-negative breast cancer tumors expressing PD-L1 (10).
In our previous studies, we got a gallotannin-rich extract from Caesalpinia spinosa (P2Et) that has been previously reported to have antitumor activity in murine melanoma and breast cancer models (11)(12)(13). Caesalpinia spinose, commonly called Dividivi or Tara has been traditionally used by Colombian indigenous located on the Caribbean coast. It is a shrub with a pantropical distribution in forests, savannas, and semi-deserts. Dividivi fruits have 40 to 60% of hydrolyzable tannins with gallic acid as the main constituent. Tannic acids present in Dividivi inhibit the growth of tumors induced by chemical agents (14) and the carcinogenesis induced by UV light in mice (15). Also, gallic acid shows antioxidant, anti-allergenic, anti-mutagenic, anti-carcinogenic, and anti-inflammatory properties (16) and decreased proliferation of cervical cancer cells, leukemia, and melanoma (17,18).
In our hands, P2Et extract induces immunogenic cell death, displaying calreticulin on the cell surface, and ATP secretion in both breast and melanoma models (19). Additionally, in B16-F10 melanoma model we demonstrated that P2Et's antitumor activity is partially abolished in immunodeficient mice, indicating that the antitumor activity of the P2Et treatment is highly dependent on the immune system (11). The treatment of C57BL/6 or BALB/c healthy mice with P2Et increased the number of CD4 + and CD8 + activated T, NK, regulatory T, dendritic and MDSC cells in lymphoid organs. However, in tumor-bearing animals, P2Et decreased the number of intratumor myeloid-derived suppressor cells (MDSCs) and increased the number of CD4 + and CD8 + T cells (20), suggesting a role in the modulation of the immune response, which is different in relation to the presence or not of tumors. According to the above studies, we hypothesize that the combined treatment with P2Et extract and anti-PD-L1 can improve the antitumor response through an additive antitumor effect.
In the present study, we investigated the antitumor and immunomodulatory activity of P2Et and anti-PD-L1 combined therapy in two different murine models, B16-F10 melanoma and 4T1 breast carcinoma. To this aim, we analyzed tumor growth, hematologic parameters, T cell counts, cytokine expression, and T cell cytotoxicity. Overall, we found that combined therapy with P2Et and anti-PD-L1 improves the antitumor response in the melanoma model by activating the immune response and neutralizing immunosuppressive mechanisms. In contrast, and surprisingly, no additive effect of the combination was observed in the breast cancer model.

Plant Material
Caesalpinia spinosa pods were collected in Villa de Leyva, Boyaca, Colombia and identified by Luis Carlos Jimeńez from the Colombian National Herbarium (voucher specimen number COL 523714, Colombian Environmental Ministry agreement number 1470 related to the use of genetic resources and derived products). The P2Et extract was produced under GMP conditions and chemically characterized as previously described (19,21). established protocols of the Ethics Committee of the Faculty of Sciences, PUJ, and National and International Legislation for Live Animal Experimentation (Colombia Republic, Resolution 08430, 1993; National Academy of Sciences, 2010). The present study was approved by the ethics committee of the Faculty of Sciences, PUJ, on August 9, 2018. Each specific protocol was also approved by the animal experimentation committee of PUJ. Mice were maintained in polyethylene cages with food and water provided ad libitum, on a 12-h light/dark cycle at 20-22°C and 40-60% humidity.  (11). In all experimental settings, the size of the tumors was assessed three times per week with Vernier calipers, and the volume was calculated according to the formula V (mm 3 ) = L (major axis) × W 2 (minor axis)/2 (23). Mice were euthanized by CO 2 inhalation, and then spleen, tumordraining lymph nodes (TDLN), and tumor were removed and processed. Looking for sufficient statistical power adjusted to the standard deviation and to the proportion of losses in each model, six mice were included for each treatment group.

Hematology
Approximately 700 µl of blood was collected by cardiac puncture immediately after euthanasia into tubes containing EDTA. A part of the blood was used to evaluate hematological parameters on the MICROS-60 hematology analyzer (Horiba ABX-Diagnostics, Montpellier, France). The remaining blood was used to separate plasma and evaluate cytokine levels.

Cytokine Assay
Cytokine evaluation was performed using a Cytometric Bead Array (CBA) mouse Th1, Th2, Th17 cytokine kit (BD Biosciences) according to the manufacturer's instructions. Experiments were performed twice, and each experiment was performed in duplicate. Events were acquired using a FACSAria II flow cytometer (BD Immunocytometry Systems), and the results were subsequently analyzed using FCAP array software version 3.0 (BD Biosciences). Data were log-transformed and plotted as the mean ± SEM.

Cytometry
Briefly, 1 × 10 6 cells were stained with LIVE/DEAD Fixable Aqua for 20 min in dark conditions at room temperature. After washing with PBS 2% FBS, the cells were stained for 30 min at 4°C in dark conditions with the surface antibodies at final concentration of 1 µg/ml according to the designed multicolor panels. Then, the cells were acquired by flow cytometry using a FACSAria II flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA), and the results were subsequently analyzed using FlowJo 9.3.2 software (Tree star, Ashland, OR).

Cytotoxicity Assay by Flow Cytometry, CFSE/7-Amino Actinomycin D
To expand tumor-specific cytotoxic T cells, splenocytes (3 × 10 6 ) from each group of B16-F10 or 4T1 tumor-bearing mice were plated in 24-well plates. Cells were cultured in a total volume of 3 ml of RPMI-1640 (Eurobio) supplemented with 10% FBS (Eurobio), 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.01 M HEPES buffer, 1 mM sodium pyruvate (Eurobio), IL-2 (10 UI/ml), IL-7 (1 ng/ml), and B16-F10 or 4T1 cell lysate (20 µg/ml). The cells were then incubated in a humidified environment at 37°C and 5% CO 2 for four days. Later, the cells were restimulated with the respective cell lysate (20 µg/ml) and cultured for three additional days. Then, cells were collected and resuspended in medium for the cytotoxicity a s s a y . B 1 6 -F 1 0 o r 4 T 1 c e l l s w e r e l a b e l e d w i t h carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher Scientific, MA, USA) at a final concentration of 1 µM for 20 min at 37°C following the manufacturer's recommendations. After quenching, the labeling reaction was stopped by the addition of complete culture medium, followed by a 5-min incubation at 37°C. After 2 washes, the CFSE-labeled target cells were resuspended and used for the cytotoxicity assay. The cell concentration was adjusted to 5 × 10 5 cells/ml, and 100 µl/ well, and plated into 96-well plates. Splenocytes were added at 10:1 and 20:1 effector-target (E:T) ratios. The plates were incubated in a humidified atmosphere of 5% CO 2 and 37°C. After 12 h, the wells were harvested and labeled with 7-amino actinomycin D (7-AAD) to stain dead cells. All cells in each tube were acquired on a FACSAria II instrument (BD Immunocytometry Systems) flow cytometer, and the results were analyzed using FlowJo software (Tree Star). Analysis was performed by gating on the target cells and measuring the 7-AAD-positive cells (24). Cells positive for both 7-AAD and CFSE were considered lysed. Additionally, we calculated the percentage of cell loss in each well assuming that the number of target cells read from the 0:1 effector-target ratio was 100% of events (25). This percentage was added to the percentage of dead cells, and the percentage of cytotoxic activity was calculated using the following equation:

Statistical Analysis
Statistical analysis of the significance between two groups was calculated using the Mann-Whitney U test. Differences among subject groups were evaluated using Kruskal-Wallis and Dunn's posttest for multiple comparisons. For all cases, the differences were considered statistically significant when p < 0.05. GraphPad Prism version 6.0 for Mac OS X statistics software (GraphPad Software, San Diego, CA) was used for the statistical analyses.

PD-L1 Expression Is Modulated by P2Et
Extract in B16-F10 and 4T1 Cells P2Et Treatment Enhances the Response to aPD-L1 immunotherapy in B16-F10 Tumor-Bearing Mice In addition to the properties of P2Et extract to induce immunogenic cell death and to modulate the immune response (11)(12)(13)20), it also upregulates the PD-L1 expression in certain cell lines, which could sensitize cancer cells to the anti-PD-L1 antibody. To check this hypothesis, we tested a combined therapy of P2Et and anti-PD-L1 (aPD-L1) in B16-F10 tumor-bearing mice (Figure 2A). 1 × 10 5 B16-F10 cells were injected s.c to C57BL/6 mice, and 5 days after, mice were treated with P2Et, aPD-L1, P2Et plus aPD-L1 or PBS. Mice treated with P2Et plus aPD-L1 showed the smallest tumor size compared to the other groups, even when compared to the P2Et only treated mice, while therapy with only aPD-L1 did not impair tumor growth compared to the PBS control group ( Figure 2B). In addition, mice treated with P2Et plus aPD-L1 showed a higher percentage or survival (Supplementary Figure 3).
In order to evaluate whether the modulation of PD-L1 on the tumor cell could be related to a possible adjuvant activity of PD-L1 in vivo, we evaluated the effect of combined therapy of P2Et and aPD-L1 in 4T1 tumor-bearing mice (Figure 2A). Mice treatment with P2Et delayed tumor growth as previously observed (12,20), but although aPD-L1 and P2Et plus aPD-L1 treatments, as P2Et alone, had a positive effect on tumor growth, no synergistic activity was observed on 4T1 tumorbearing mice ( Figure 2C). P2Et extract has an important antioxidant activity (19) that could protect host cells from oxidative stress during cancer expansion and progression. Thus, we evaluated hematological parameters in normal and treated tumor-bearing mice. In the B16-F10 model, there was no change in leukocytes, platelets, lymphocytes, monocytes, and granulocytes' counts in mice treated with P2Et alone or in combination with aPD-L1 compared with healthy mice ( Figure 3A). Mice treated with P2Et or P2Et plus aPD-L1 had a higher number of leukocytes, lymphocytes, monocytes, and granulocytes compared to the untreated mice. However, in the 4T1 model, the only populations that remained in the normal range were platelets in mice treated with P2Et plus aPD-L1, and monocytes and granulocytes in mice treated with aPD-L1 ( Figure 3B). In conclusion, a significant recovery of hematological parameters was evidenced in melanoma model when P2Et alone or in combination with aPD-L1 was used.

P2Et Treatment Increases PD-L1 Expression in Tumor Cells In Vivo
Given that the expression of PD-L1 in tumor cells may affect the response to immune blockade therapy, we evaluated PD-L1 expression in tumor CD45 negative cells recuperated from mice. In the melanoma model, P2Et extract alone or in combination with aPD-L1 significantly increased surface PD-L1, (Figures 4A, B) and PD-L1 mRNA expression in tumors ( Figure 4C). In contrast, it significantly decreased the frequency of CD8 + T cells expressing PD-1 receptor ( Figure 4D). This suggests that P2Et can improve the effector response of CD8 + T cells. P2Et treatment did not modulate the PD-L1 expression in the breast 4T1 model in vivo (Figures 4E-G) as previously shown ( Figure 1) and neither did it modulate the frequency of T cells expressing PD-1 ( Figure 4H). In both models, a significant decrease in PD-L1 surface expression after aPD-L1 treatment compared with other groups (Figures 4A,  B, E, F) was found. However, there has been a diminution due to a masking effect of therapeutic aPD-L1 antibody (Supplementary Figure 2). This would explain the discrepancy between protein level and mRNA expression in the P2Et plus aPD-L1 group of B16-F10 model ( Figures 4A, C).
Recent studies have shown that adjacent cells around the tumor (dendritic or macrophages), express higher levels of PD-L1 and play an important role in the response to immunotherapy (8,27,28). Therefore, we assessed the PD-L1 expression also in tumor infiltrating CD45 + cells in both models, but no significant differences were found among the different treatments (data not shown). The fine analysis of the type of cells present in the tumor allowed showing that the relationship between tumor cells and immune cells varies depending on the type of treatment. Therefore, in melanoma model the therapy with P2Et or P2Et plus aPD-L1 induces a significant increase in the number of tumor-infiltrating CD45 + cells per mg of tissue, compared to PBS or aPD-L1 groups ( Figure 5A). Within the CD45 + cells, the population of CD4 + and CD8 + T cells was analyzed ( Figure 5B) and an increase of cell numbers was found in mice treated with P2Et or with the combination ( Figure 5C). In addition, P2Et or P2Et plus aPD-L1 treatment, also induced a high frequency of activated (CD44 + ) CD4 + and CD8 + T cells ( Figure 5D). The assessment of other immune cell populations in the tumor ( Figure 5E) showed that the three treatment strategies decreased the number of MDSC, which due to lack of functional evaluation are called myeloid-derived suppressor like cells (MDSC-LCs) ( Figure 5F) (29). However, the number of tumor-infiltrating monocytic MDSC (M-MDSC) was lower with P2Et plus aPD-L1 treatment compared to P2Et alone ( Figure 5F). Recently it was described that PD-L1 expression on dendritic cells (DCs) and macrophages correlated with the efficacy of immunotherapy in ovarian cancer and melanoma (30). We found that P2Et treatment increased the frequency of conventional DCs (cDCs) in TDLN and also increased PD-L1 expression in these cells and in macrophages (Supplementary Figures 4A, B), suggesting that the P2Et-based therapy alone may trigger counter regulatory immune loops, thus providing a rationale for combination with immune check point blockade. However, no differences were found in the spleen (Supplementary Figure 5). Similar results were found in the 4T1 model. The treatment with P2Et, aPD-L1 or P2Et plus aPD-L1 significantly increased the number of CD45 + cells per mg of 4T1 tumor (Supplementary Figure 6A), with an increase in CD4 + and CD8 + T cells (Supplementary Figure 6B). Likewise, we found an increase in the frequency of activated CD44 + CD4 + and CD44 + CD8 + T cells when treated with P2Et plus aPD-L1, and an increase of CD44 + CD8 + T cells when treated with P2Et (Supplementary Figure 6C). Moreover, we found a significant decrease in the total number of MDSC-LC after all the treatments compared with non-treated mice, mainly due to the decrease in polymorphonuclear MDSC (PMN-MDSC) cells (Supplementary Figure 6D). Furthermore, we showed that P2Et plus aPD-L1 treatment increased the frequency of cDC, and P2Et alone increased their PD-L1 expression. However, in the macrophage population no differences were observed among groups (Supplementary Figures 4C, D).

P2Et in Combination With aPD-L1 Enhances the Effector Response of Cytotoxic Cells in Melanoma and Breast 4T1 Model
To modulate the immune response in the B16-F10 melanoma model based on the known capacity of P2Et, we evaluated the production of cytokines in each group. Interestingly, a significant increase in the plasma concentrations of IFNg was found in the aPD-L1, P2Et and P2Et plus aPD-L1 groups compared to the control group, while a significant decrease in the plasma concentrations of IL-10 was found when P2Et was used ( Figure 6A). In contrast, a significant increase in the plasma concentrations of IL-10 and IL-17 was found in aPD-L1 or P2Et plus aPD-L1 treated 4T1-BALB/c mice as compared to B16-C57BL/c mice ( Figure 6B) with a downward trend in IL-6 (data not shown), confirming our previous results (12). Taking into account the potential role of IL-10 and IL-17 in tumor promotion (31)(32)(33), these results may explain the differences in the treatment outcome in both models. No differences were found in the plasma concentrations of IL-4 and IL-2 (data not shown) between groups. Finally, we also assessed the cytotoxic capacity of spleen cells after expanding tumor-specific cytotoxic T cells, as explained in the Materials and Methods section. We found that P2Et plus aPD-L1 treatment induced a higher cytotoxic potential of cytotoxic cells both in melanoma and in the breast cancer model (Figures 6C, D), suggesting that combined therapy improves CD8 + T cell effector functions, which would entail a better response to immunotherapy.

DISCUSSION
In the current study, we evaluated the combined treatment of P2Et, a polyphenol-rich extract obtained from Caesalpinia spinosa, and aPD-L1 antibody for melanoma and breast cancer in mouse models. This in accordance with the fact that, in breast cancer, there is no high response to immunotherapy compared to that observed in other types of cancer such as melanoma, lung, kidney cancer, among others (34). We confirm the antitumor activity of P2Et both on melanoma B16-F10 and breast cancer 4T1 tumor models (11,13). We then demonstrate that P2Et extract improves the response to treatment with aPD-L1 antibody in the murine melanoma model, finding that the combined therapy increases PD-L1 expression in tumor cells, maintains most hematological parameters in the normal range, modulates the immune response and enhances the effector response of cytotoxic cells. By contrast although experiments on the number of TILs and the effector response of cytotoxic cells revealed favorable results in the breast cancer model, combined therapy had no effects in tumor growth compared to individual therapy with P2Et or aPD-L1. These results reflect the necessity to evaluate individual heterogenicity of the response, and its relationship with the tumor type and genetic background of the individual. We observed heterogenous responses of PD-L1 expression on different tumor cells treated with P2Et extract. The mechanisms governing the PD-L1 expression are not well understood (35). PD-L1 is not only expressed in tumors, but also on the surface of B lymphocytes, monocytes, natural killer cells, macrophages, and vascular endothelial cells (36). However, the mechanisms governing the expression on tumor cells and immune cells seem to be different. Different polyphenols have been evaluated in their ability to modulate the expression of PD-L1 or to synergize with other therapies (37,38). This seems to depend on the microenvironment context, the dose of the compounds, and the type of cells to which they are directed (39). A recent study showed that two polyphenolic compounds, curcumin and apigenin, decrease the PD-L1 expression on melanoma cells in vitro and in vivo in a murine model, diminishing the tumor growth and increasing immune cell infiltration (40). Nonetheless, other studies have shown that an increase in PD-L1 expression may enhance therapeutic effect of aPD-L1 antibodies. In fact, treatments with resveratrol and piceatannol alone or in combination elicited an upregulation of PD-L1 in some breast and colorectal cancer cell lines (37).
In tumors, PD-L1 expression can be regulated genetically and epigenetically. Amplifications and deletions of the PD-L1 gene have been identified on primary tumor cells, and both have been related with worse prognosis of the disease (41). Complexity of anti-PD-L1 treatment effectivity is evidenced by the observation that in some melanoma patients with detection of PD-L1 by immunohistochemistry, a positive clinical response to anti-PD-L1 treatment has been observed, while a bad response have been also observed in spite of a strong PD-L1 expression (42,43). PD-L1 levels may be upregulated by different intracellular signals as Akt activation or PTEN dysfunction (44) or, by regulation of micro-RNAs (45). Natural products act on these intracellular signals modulating several tumorigenic signals (46), having also effect on microRNAs. Therefore, the identification of mechanisms involved in PD-L1 modulation by P2Et or other natural products requires a holistic approach including integrate omics science and systems biology.
As we mentioned before, mechanisms implied in PD-L1 expression on tumor and immune cells seems to be different. The study of PD-L1 expression on non-small cell lung carcinoma (NSCLC), shows that reduced methylation of the PD-L1 promoter on tumor but not immune cells, increases the PD-L1 expression in response to external stimuli (47). Immune response by itself, regulates PD-L1 expression though interferon signaling (48) and even signals inducing epithelialto-mesenchymal transition of tumor cells, improves tumor responses to anti-PD-L1 antibodies (49).
In this study, we show that P2Et modulates the gene expression of PD-L1, as well as their protein expression, and this differs among the different cell lines studied. Although the factors involved were not studied, it is important to highlight that while the B16-F10 cell line increased gene expression and the protein response to P2Et, the 4T1 line showed a significant increase only in the presence of CoCl 2 . However, our results suggest that while P2Et exerts an activating effect of PD-L1 expression on the B16-F10 cells, it has little to no influence in 4T1 cells. These differences appear to be related to the intrinsic differences of tumor cells, but also to the in vivo tumor response in different mice strains. Different mice strains may have different immune status and thus may display different immune infiltrates in tumors which in turn can influence the outcome of immunotherapy. Thus, Sellers and al., have been shown immune variations specific to the strain (50). The BALB/c mice are considered a Th2 prone strain, whereas a Th1 response characterizes the C57BL/6 mice. Nevertheless, this also depends on the type of stimulus (51). In this sense, the production of IL-17 could explain some differences between the strains. Although it is true that IL-17 has been correlated with tumor progression in the 4T1 and B16 models (52, 53), we did not observe important differences in the plasma levels of this cytokine in the B16 model, but in the 4T1 model. Interestingly, treatment with PD-L1 alone or accompanied by P2Et significantly increased IL-17 in 4T1 mice, but not in B16. The mechanisms that regulate the activation of LTh17 depend on complex orchestrations of cytokines in the tumor microenvironment and their function can be dual (54). For example, in breast cancer it has been clearly observed that IL-23 induced by PGE2 (55) plays a very important role in the recruitment and activation of LTh17 related to tumor evolution. However, it is not clear what the role of PGE2 is in melanoma, and if this complex cytokine interaction is going to occur in the same way. In fact, it has recently been shown that IL-23 plays a dual role in the antitumor immune response in melanoma, depending on tumor immunogenicity (56).
Another important parameter that can influence the type of response generated is the time and kinetics to which the treatments are added. In this work, we chose to use both treatments simultaneously; however, some experimental data show that these factors can have an effect on the type of response generated (57). Moreover, our results showed heterogeneity in the modulation of PD-L1 expression in the tumor microenvironment related with the tumor model. Similarly, Grasselly et al. reported that the antitumor activity of cytotoxic chemotherapy combined with immune checkpoint inhibitors was model-dependent (57).
The protective activity of some polyphenols has been previously reported (39,58). However, there are few studies where its protective potential in co-treatment with immune checkpoints blockade has been evaluated (38). In previous studies of our group, we have observed the potential of P2Et in the induction of a specific antitumor immune response in both B16-F10 (11,20) and 4T1 (12,13,20) models. We have also reported a preferential migration of CD45 + cells to the tumor and peripheral lymphoid organs, allowing a better antigenic presentation through the activation of DC and the decrease of MDSCs and regulatory T cell suppressors (11,20). These findings suggest that P2Et can regulate PD-L1 expression both directly acting on tumor cells, as previously exposed, but also by the way of immune system modulation. It could not be ruled out that other immune checkpoint inhibitors, similarly regulated, might be modulated by this extract, favoring immune response activation which might be evaluated. In this way, effectiveness of the clinical response to the blockade of PD-L1 and PD-1 has been associated with an increase in TILs (5-7, 12, 23), and in this work we have shown that the use of P2Et favored the intra-tumoral and peripheral activation of the immune response.
Moreover, in both models we observed that treatment with P2Et alone or in combination with anti PD-L1 decreases the frequency of intratumoral MDSC-LC (20). The MDSC are currently presented as one of the main immunosuppressive elements of the antitumor response (59). Indeed, the antitumor activity of the polyphenols through the inhibition of the suppressive function of the MDSC and other suppressor mechanisms has been evidenced for the epigallocatechin gallate and the curcuma, among others (60,61). Other important effect of P2Et in B16-F10 tumor-bearing mice was the increase of CD8 + T cells frequency but the decrease of the frequency of CD8 + T cells expressing PD-1, suggesting that PD-1 low/neg cells are expanding upon the treatment.
We demonstrate that mice receiving combined therapy had strengthened T cell response. In tumor tissues, most of the infiltrated T cells displayed an activated phenotype, which may be correlated with a better effector response. In the spleen, cytotoxic T cells showed greater cytotoxic activity and, consequently, may be involved with better control of tumor progression in both models. Comparable results have been reported using a curcumin analogue in combination with anti-PD-L1 in murine bladder cancer (38), perhaps because curcumin has been shown to stimulate the immune response (62), as well as P2Et extract (11,12,20).
In summary, these findings suggest that P2Et extract sensitizes B16-F10 cells by increasing PD-L1 expression for an enhanced response to PD-L1 blockade, effect not found in murine breast cancer model, achieving that combination treatment improve antitumor response in the melanoma model by enhancing CD8 + T cells activity and suppressing MDSCs.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the ethics committee of the Faculty of Sciences, Pontifical Javeriana University and Animal experimentation committee of Pontifical Javeriana University.

AUTHOR CONTRIBUTIONS
PL and AG-C designed and executed the experiments, and acquired and interpreted the data. PL and SF drafted the manuscript. CU developed the in vivo animal experiments, acquired and interpreted the data. AB, AD, AM-U, and PR interpreted and analyzed the data. SF leader of the project and designed the experiments, interpreted the results, and revised the manuscript. All authors contributed to the article and approved the submitted version.