EP2 and EP4 blockade prevents tumor-induced suppressive features in human monocytic myeloid-derived suppressor cells

Tumors educate their environment to prime the occurrence of suppressive cell subsets, which enable tumor evasion and favors tumor progression. Among these, there are the myeloid-derived suppressor cells (MDSCs), their presence being associated with the poor clinical outcome of cancer patients. Tumor-derived prostaglandin E2 (PGE2) is known to mediate MDSC differentiation and the acquisition of pro-tumor features. In myeloid cells, PGE2 signaling is mediated via E-prostanoid receptor type 2 (EP2) and EP4. Although the suppressive role of PGE2 is well established in MDSCs, the role of EP2/4 on human MDSCs or whether EP2/4 modulation can prevent MDSCs suppressive features upon exposure to tumor-derived PGE2 is poorly defined. In this study, using an in vitro model of human monocytic-MDSCs (M-MDSCs) we demonstrate that EP2 and EP4 signaling contribute to the induction of a pro-tumor phenotype and function on M-MDSCs. PGE2 signaling via EP2 and EP4 boosted M-MDSC ability to suppress T and NK cell responses. Combined EP2/4 blockade on M-MDSCs during PGE2 exposure prevented the occurrence of these suppressive features. Additionally, EP2/4 blockade attenuated the suppressive phenotype of M-MDSCs in a 3D coculture with colorectal cancer patient-derived organoids. Together, these results identify the role of tumor-derived PGE2 signaling via EP2 and EP4 in this human M-MDSC model, supporting the therapeutic value of targeting PGE2-EP2/4 axis in M-MDSCs to alleviate immunosuppression and facilitate the development of anti-tumor immunity.

In this study, we examine the role of EP2 and EP4 signaling in the differentiation, phenotype, and function of moMDSCs upon tumor-derived PGE2 contact.We show that exposure of human monocytes to tumor-derived PGE2 leads to a suppressive state that resembles that of M-MDSCs and is attenuated upon EP/EP4 blockade.Using a human M-MDSC model, we show that tumorderived PGE2 steers moMDSCs towards the acquisition of a suppressive phenotype as seen by the upregulation of CD14, IL-10 and PGE2 production, or the downregulation of HLA-DR.Additionally, PGE2-EP2/4 signaling boosts the suppressive function of moMDSCs over the development of proinflammatory responses in NK cells, T cells, and moDCs.Blockade of EP2 and EP4 demonstrated that both receptors contribute to the acquisition of this suppressive state, with EP4 rather than EP2 mediating this effect.Combined EP2/4 blockade completely impairs the arising of such phenotype and function.Using a 3D co-culture with M-MDSCs and colorectal cancer patient-derived organoids, blockade of EP2 and EP4 attenuates the suppressive phenotype of moMDSCs.Altogether, these results illustrate the role of EP2 and EP4 in human M-MDSCs as well as the relevance of targeting the PGE2-EP2/4 axis in human MDSCs for the treatment of cancer.
In vitro generation of moMDSCs and moDCs moMDSCs and moDCs were differentiated in vitro by culturing monocytes at a concentration of 0.5 million cells per ml in T25 flasks containing a final volume of 4 ml X-VIVO medium (Lonza) supplemented with 2% human serum (HS) (Sanquin, the Netherlands) for 7 days.For the generation of moMDSCs, monocyte cultures were supplemented with 60 U/ml of GM-CSF (130-093-867, Miltenyi Biotec) and 10 ng/ml of IL-6 (130-093-933, Miltenyi Biotec).For moDC generation, monocyte cultures were supplemented with 450 U/ml of GM-CSF (130-093-867, Miltenyi Biotec) and 300 U/ml of IL-4 (130-093-924, Miltenyi Biotec).On day 3 of differentiation, moMDSCs and moDC cultures were supplemented with 1 ml of X-VIVO supplemented with 2% HS and 5x concentrated IL-6 and GM-CSF in the case of moMDSCs, and 5x concentrated IL-4 and GM-CSF in the case of moDCs.To generate mature moDCs, on day 6 of differentiation, moDC cultures were further stimulated with 2 µg/ml of poly (I:C) (Invivogen) for 24 hours prior to further use in functional experiments.

Cell culture and conditioned media preparation
Human melanoma cells (A375) were tested to be mycoplasmafree and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) 1% Antibiotic-Antimycin (AA, Gibco).To generate the melanoma cell line conditioned media (CM), A375 cells were cultured in T75 flasks at a concentration of 0.3 million cells per ml in T75 flasks with a final volume of 14 ml.After three days of culture, the supernatant was harvested and centrifuged for 5 minutes at 1500 rpms to get rid of cellular debris present in the media.Resulting CM was aliquoted and stored at -20 C until use.

MoMDSC stimulation
To determine the individual role of EP2 and EP4 on moMDSCs, the specific EP2 antagonist AH6809 (Cayman chemicals) and specific EP4 antagonist L161-982 (Cayman chemicals) were used.For EP2 blockade, 40 µM of EP2 antagonist was used, whereas 3 µM was used for the blocking of EP4 receptor.The same concentrations were used when performing the combined EP2/4 blockade.On day 5 of moMDSC differentiation, cultures were treated with the indicated antagonist two hours prior to exposure to the melanoma-derived CM.After 3 hours, 2 ml of CM were added to each T25 flask.
To establish the role of CM addition, moMDSCs were instead provided with 2 ml of fresh media, depicted as untreated moMDSCs.On day 7, after 48 hours of CM treatment, moMDSCs were harvested.For harvesting, moMDSCs culture media was removed and replaced by cold PBS supplemented with 0.1% BSA and 0.2 mM of EDTA followed by incubation for 1 hour at 4°C.Thereafter, flasks were tapped and moMDSCs cell suspension was harvested and centrifuged prior to further analysis.

Generation of CM educated monocytes
The impact of CM on monocyte development was tested by culturing monocytes at a concentration of 0.5 million cells per ml in 6 well plates with 2 ml of X-VIVO media supplemented with 2% HS.To determine the role of EP2/4 during differentiation, monocyte cultures were supplemented with 40 µM and 3 µM of EP2 antagonist and EP4 antagonist respectively.After 2 hours, monocyte cultures were treated with 0.5 ml of CM.On day 3, the differentiated monocytes were harvested for flow cytometric analysis.For day 6 of differentiation, day 3 monocytes were restimulated with the indicated concentrations of aEP2/4 and exposed to a second dose of 0.5 ml CM.On day 6, the resulting differentiated monocytes were harvested and either analyzed by flow cytometry or replated for the performance of functional experiments.

Cytokine and PGE2 production measurement
For the detection of IL-6, IL-10, and PGE2 production, harvested moMDSCs were replated in 96 U bottom plates at a concentration of 0.5 million cells per ml at a final volume of 200 µL of X-VIVO medium supplemented with 2% HS and 100 ng/ml LPS.After 24 hours of culture, the moMDSCs supernatants were harvested and analyzed by ELISA for the presence of IL-6 (88-7106-77, Invitrogen), IL-10 (88-7066-77 Invitrogen), and PGE2 (EHPGE2 ThermoFisher) according to manufacturer's instructions.

MoMDSCs suppression of moDC mediated T cell polarization assay
The ability of moMDSCs to modulate the expansion of proinflammatory T cell populations by activated DCs was determined by coculturing moDCs, allogeneic naïve CD4 T cells, and moMDSCs at a ratio 1:10:5 in 96 U bottom plates.On day 6 of coculture, half of the culture medium containing the expanding T cells was removed.Equal volume of fresh X-VIVO medium supplemented with 2% HS was added and T cell cultures were further supplemented with 20 U/ml of IL-2 (130-097-773, Miltenyi Biotec).From this day onwards, cell culture media was refreshed as described above with new media containing 20 U/ml of IL-2 every 2 days until day 12 of coculture.Resulting T cell populations were harvested and characterized either for the presence of Tregs or the intracellular cytokine expression.For determining the presence of Tregs, T cells were stained with the viability dye eFluor 780 (1:2000, ThermoFisher) in cold PBS for 15 minutes.T cells were then fixed using the FOXP3 staining kit (00-5523-00, ThermoFisher) according to manufacturer's instructions.After fixation, cells were incubated in wash/permeabilization buffer supplemented with 5% HS for 15 minutes.Then, cells were washed and stained for CD127 PE (1:100 12-12780-42, eBioScience), CD25 APC (1:100 555434, BD Biosciences), and FOXP3 A488 (1:50 53-4776-42, eBioscience) for 20 minutes.Stained cells were washed twice prior to flow cytometric analysis (see Supplementary Figure 5B for gating strategy).

Statistical analysis
Statistical analysis was performed with Graphpad Prism 8 (Version 8.0.2.GraphPad Software San Diego, CA, USA).Unless indicated otherwise, data are depicted as mean ± SEM in bar scattered dot plots.Significance was determined as indicated in the legend of each figure.MoMDSCs phenotype data were analyzed using either a one-way analysis of variance (ANOVA) followed by multiple comparison Tukey correction, or a Friedman test followed by Dunn's multiple comparison test.Significance across the functional experiments was determined using either a one-way ANOVA followed by a Dunnett's test, or a Friedman test followed by a Dunn's multiple comparison test.Differences among the CM differentiated monocytes or moMDSCs cocultured with CRC-PDTOs were performed using either a paired t-test or a Wilcoxon test.The statistical significance is depicted as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Results
Melanoma-derived PGE2 steers human monocytes towards an MDSC-like state Monocytes exposed to tumor signals acquire a suppressive phenotype and function that recapitulate that of MDSCs (24,25,55).As model to determine the role of tumor-derived PGE2 signaling during monocyte development into MDSC-like cells, monocytes were cultured for 6 days with conditioned media (CM) derived from the melanoma cell line A375 (Figure 1A), detected to produce PGE2 (see Supplementary Figure 1A).To establish the effect of PGE2-EP2/4 signaling during MDSC-like differentiation, monocytes were incubated with specific antagonists for EP2 and EP4 (aEP2/4).Phenotype analysis after 3 and 6 days showed that EP2/4 blockade limited the expression of CD14, MerTK, CCR5, and CD11b (Figures 1B, C).No significant changes were detected for the markers HLA-DR and CD86.To assess the functional relevance of targeting PGE2-EP2/4 axis, monocytes cultured for 6 days in the presence of CM were harvested and cocultured with autologous NK and T cells.aEP2/4 attenuated the suppressive function of CM-educated monocytes as seen by the increased T cell proliferation and increased expression of the pro-inflammatory cytokines TNFa and IFNg in NK cells (Figure 1D).Overall, these data illustrate the role PGE2-EP2/4 axis in the priming of a suppressive MDSC-like state of monocytes.

PGE2-EP2/4 signaling mediates the occurrence of a suppressive moMDSC phenotype
PGE2 is well known as a modulator of the phenotype of myeloid cells, including different cell models of mouse and human MDSCs (23,24,53,54).As a human M-MDSC model, we used moMDSCs, generated from monocytes by culturing them for 5 days in the presence of IL-6 and GM-CSF, previously reported to express typical MDSC markers such as CD11b or CD33 (21,31).To distinguish the effects of tumor-derived PGE2 signaling trough the different PGE2 receptors, A375-derived CM was added to moMDSCs that were preincubated for 2 hours with aEP2, aEP4, or the combination designated as aEP2/4 (Figure 2A).After 48 hours, CM treatment led to the upregulation of CD14, MerTK, and CCR5, and the downregulation of HLA-DR and CD86 (Figures 2B,  C).Although not significant, we noted a tendency towards the upregulation of CD11b.EP blockade demonstrated both EP2 and EP4 to contribute to the induction of this phenotype.Of note, aEP4 more robustly than aEP2 prevented the acquisition of this phenotype.Combined aEP2/4 blockade prevented the acquisition of the phenotype yielded by the tumor-derived CM on moMDSCs.

PGE2-EP2/4 signaling induces the production of suppressive mediators by moMDSCs
Suppressive M-MDSCs exploit a variety of mechanisms to establish a suppressive environment, such as the secretion of suppressive mediators (22).To determine the effect of PGE2-EP2/ 4 axis on the secretome of moMDSCs, we measured the levels of IL-6, IL-10, and PGE2.MoMDSCs exposed to CM displayed a robust upregulation of these three soluble mediators.aEP2 modestly inhibited the production of these mediators, whereas aEP4 significantly impaired their production (Figure 3).Similar to aEP4 treatment, the combined aEP2/4 inhibited the secretion of these three mediators, recapitulating the production levels of moMDSCs unexposed to CM.Overall, these results identify the PGE2-EP2/4 axis as a major modulator of IL-10, IL-6, and PGE2 production by moMDSCs, with EP4 more robustly than EP2 mediating this feature.

PGE2-EP2/4 educated moMDSCs suppress autologous T cell responses
M-MDSCs are potent suppressors of T cell immune responses as they inhibit T cell proliferation and the production of proinflammatory cytokines such as IFNg (20,21,(24)(25)(26)28).To assess the ability of moMDSCs to modulate T cell proliferation, moMDSCs were cocultured with autologous T cells labelled with CFSE dye for three days (Figure 4A).Additionally, moMDSCs were cocultured with T cells not labelled with CFSE for the intracellular detection of cytokines.Prior to coculture, T cells were primed to proliferate by using activating anti-CD3/CD28 dynabeads.Exposure to CM led to moMDSCs with a superior ability to suppress the proliferation of autologous T cells (Figure 4B).Cytokine analysis demonstrated that CM treated moMDSCs yielded T cells with a reduced production of IFNg, TNFa, and IL-2, cytokines associated with active pro-inflammatory T cell responses (Figure 4C).Tumor-derived PGE2 signaling induces the production of soluble factors on moMDSCs via EP2 and EP4.In vitro generated moMDSCs were treated with either aEP2, aEP4, or aEP2/4 prior to exposure to melanoma-derived CM.After 48 hours, moMDSCs were harvested, replated, and stimulated with 100 ng/ml of LPS.After overnight restimulation, moMDSCs supernatants were harvested and analyzed for the presence of the cytokines IL-6 and IL-10, and the lipid mediator PGE2 by ELISA.Data shows the pg/ml of each of these soluble factors.Bar graphs show the mean ± SEM and each data point represents an individual moMDSC donor (n>8) tested in three independent experiments.P values were calculated on raw concentration values with a Friedman test followed by a Dunn's multiple comparison test (*p 0.05; **p 0.01; ***p 0.001).ns, non significant.

A B C
Tumor-derived PGE2 signaling mediates the occurrence of a suppressive moMDSC phenotype via EP2 and EP4.Given the strong phenotypical modulation of moMDSCs achieved by blocking EP2 and EP4, we further assessed whether this inhibition could recover T cell function upon moMDSC coculture.aEP2 tended to partly recover T cell proliferation, whereas aEP4 significantly restored T cell proliferation to the levels of moMDSCs unexposed to the CM.As expected, combined aEP2/4 restored T cell proliferation to a level similar to that induced by aEP4 (Figure 4B).Although not significantly, cytokine analysis showed both EP receptors similarly contribute to the downregulation of IFNg, IL-2, and TNFa production by T cells after moMDSC coculture.The combined aEP2/4 further upregulated the expression of these cytokines compared to the individual blockade of EP2 and EP4 (Figure 4C).Further characterization of CD4 and CD8 T cells within the cocultures demonstrated these suppressive features to be similarly present across CD4 and CD8 T cells (see supplementary Figures 3C, D).

PGE2-EP2/4 signaling potentiates the suppressive ability of moMDSCs over NK cells
Tumor cell clearance requires active immune responses by cytotoxic lymphocytes, including NK cells.PGE2 signaling on M-MDSCs has demonstrated to mediate NK cell dysfunction in the tumor tissue, enabling tumor progression (28,29).To investigate the relevance of M-MDSCs on NK cells, moMDSCs were cocultured with autologous NK cells pre-stimulated with IL-2 (Figure 5A).After three days, NK cells were characterized for the expression of different activation surface markers and the production of pro-inflammatory cytokines.CM treated moMDSCs suppressed the production of IFNg and TNFa, whereas no changes were detected for granzyme B (Figure 5B).NK activation marker analysis showed CM-treated moMDSCs to suppress the expression of CD25, CD69, and NKG2D, whereas NKp46 levels remained unchanged (Figure 5C).Of note, a significant downregulation of the lineage marker CD56 on NK cells was observed.EP2 and EP4 modulation demonstrated aEP4 rather than aEP2 to primarily recover the expression of CD25, CD69, NKG2D, IFNg, and TNFa.Combined aEP2/4 further recovered the expression of these markers compared to aEP4 only, resembling the NK suppression level induced by untreated moMDSCs.Overall, these results indicate the synergistic contribution of EP2 and EP4 on moMDSCs during NK cell suppression, with EP4 primarily mediating such feature.

PGE2-EP2/4 signaling on moMDSCs steers moDC mediated T cell responses towards suppressive T cell populations
DCs are key initiators of anti-tumor immunity via priming tumor antigen specific T cell responses (58).However, this antitumoricidal DC feature is often impaired in cancer patients due to the suppressive TME.Although this outcome likely arises from tumor-mediated DC dysfunction, we hypothesize that environmental M-MDSCs can further steer or compromise the development of pro-inflammatory T cell responses regardless of the activation status of DCs.To address this question, moMDSCs were cocultured with moDCs previously treated with poly I:C and allogeneic naïve CD4 T cells (Figure 6A).After coculture, the resulting T cell populations were analyzed for the expression of the pro-inflammatory cytokines IFNg, TNFa, and IL-2 (Figures 6B,  6C).Additionally, Treg occurrence was assessed by determining the expression of CD127, CD25, and FOXP3 (Figure 6C).T cell analysis showed CM-treated MDSCs in coculture with moDCs to robustly suppress IFNg responses.Although not significant we observed a tendency towards the downregulation of TNFa and IL-2, and a modest increase in Tregs.EP2 and EP4 blockade demonstrated both receptors contribute to this phenotype, although we noted a higher EP4 contribution compared to that of EP2.Combined aEP2/4 on moMDSCs recovered IFNg and TNFa production to the level of untreated moMDSCs.Additionally, aEP2/4 significantly diminished the enrichment of Tregs compared to CM treated moMDSCs.Collectively, these results show that PGE2-EP2/4 signaling in moMDSCs skew moDC-mediated T cell responses towards the expansion of T cells with a suppressive rather than a pro-inflammatory profile.
EP2/4 blockade attenuates the suppressive phenotype of moMDSCs in 3D cocultures with colorectal cancer patient-derived tumor organoids To assess the impact of targeting EP2/4 signaling in a model that better recapitulates the TME, we performed a 3D coculture of moMDSCs with a CRC-PDTO derived from liver metastasis.
MoMDSCs were co-cultured in a collagen matrix together with the CRC-PDTOs to allow PDTOs and moMDSCs interaction (Figure 7A).After 24 hours, cocultures were treated with aEP2/ 4. Next day, collagen cultures were dissociated, and the resulting cell suspension was stained and analyzed for flow cytometry.MoMDSCs were distinguished based on positivity for CD11b (Figure 7B).aEP2/4 treatment of the cocultured cells led to M-MDSCs with lower expression levels of CD11b, MerTK, and CCR5, and higher levels of CD86 (Figure 7C).Although not significant, we noted aEP2/4 tended to downregulate the expression of CD14.Overall, aEP2/4 can significantly attenuate the suppressive phenotype of moMDSCs cultured in a 3D TME model.

Discussion
Tumor-derived PGE2 is well established as major factor mediating the development of MDSCs as well as boosting a protumor phenotype and function (20,24,28,34,35).Yet, the phenotypical and functional implications of tumor-derived PGE2 signaling via either EP2 and EP4, or the relevance of targeting these receptors in human M-MDSCs exposed to tumor-derived PGE2 is poorly defined.In this study, we show that EP2 and EP4 contribute to the induction of a suppressive phenotype and function using an in vitro model for M-MDSCs.We show that the combined blockade of EP2 and EP4 attenuates a pro-tumor phenotype and function of moMDSCs exposed to tumor-derived PGE2 in 2D using melanoma CM and in a 3D coculture with CRC-PDTOs.M-MDSCs in cancer patients are primarily derived from bone marrow precursors.However, cumulative evidence suggests that blood monocytes can also serve as precursors for M-MDSCs upon encountering tumor-derived signals (20,24,25,33,55).Exposure of freshly isolated monocytes to CM derived from the melanoma cell line A375 led to the acquisition of a pro-tumor state.In line with a prior study using a COX inhibitor (24), targeting EP2/4 signaling during monocyte differentiation limited the expression of suppressive markers, including CD14 or CD11b.We also noted an attenuation of the suppressive capacity of these cells, as seen by the increased T cell proliferation and superior expression of IFNg and TNFa on NK cells.Noteworthy, CCR5 expression on MDSCs has been demonstrated to correlate with a superior suppressive capacity (59).In line with this publication, we noted that EP2/4 blockade attenuated the expression of CCR5 indeed correlating with a reduced suppressive capacity.Besides being indicative for the MDSC suppressive ability, CCR5 expression enables MDSC recruitment into tumor tissue.Knowing that PGE2 can be found at high levels in the blood of melanoma patients (data not shown) we hypothesize that circulating PGE2 might fine-tune blood monocytes to acquire a suppressive phenotype, mediate CCR5 expression, and facilitate their recruitment into the tumor tissue.
PGE2-EP2/4 signaling is involved not only in the differentiation of MDSCs, but also in the acquisition of a pro-tumoral suppressive phenotype (23,24,53,54).In the here used human M-MDSC model, we observed that upon exposure to melanoma CM, moMDSCs further acquired a suppressive phenotype, characterized by the upregulation of CD14, CCR5, MerTK, and the downregulation of HLA-DR and CD86.Additionally, CM treatment boosted moMDSCs production of IL-6, IL-10, and PGE2, soluble mediators associated with tumor-promoting inflammation.EP2 and EP4 blockade demonstrated both receptors to contribute to the acquisition of this phenotype.However, we noted a superior EP4 contribution rather than that of EP2 for the acquisition of such features.These data are in line with previous mice studies where EP4 blockade prevented the downregulation of activating myeloid markers such as HLA-DR and CD86 on M-MDSCs (52,53,60), or hampered the expression of PGE2, IL-6, and IL-10 (53).Additionally, although there are limited studies conducted on human M-MDSCs, studies using moDCs have demonstrated that PGE2 via EP2 and EP4 boosts CD14, COX2, and IL-10 expression together with the downregulation of HLA-DR (20, 61), resembling the effect here reported for our in vitro M-MDSC model.
M-MDSCs display a variety of suppressive functional features, which hamper the induction of pro-inflammatory immune responses and skews the host immune system towards the acquisition of a tolerogenic and regulatory state, enabling tumor progression.These pleiotropic mechanisms of suppression are dependent on the abundance of different environmental tumor factors.In the here used M-MDSC model, we noted that providing melanoma CM to moMDSCs led to the suppression of autologous T cells.In line with prior studies (20,21,24,25), coculture with autologous T cells demonstrated to limit T cell proliferation and to reduce the expression of pro-inflammatory cytokines such as IFNg and TNFa.Additionally, we noted that CM-treated moMDSCs suppressed autologous NK cell responses.CM-treated moMDSCs led to the downregulation of NK cell activation markers including NKG2D, CD25, CD69 and the expression of the cytokines IFNg and TNFa after co-culture with M-MDSCs.Addition of M-MDSC to moDCs and naïve CD4 T cell cocultures yielded T cells with reduced production of IFNg and TNFa.Targeting PGE2-EP2/4 axis on moMDSCs successfully recovered the moDC-induced T cell and NK cell responses.In these cultures, a modest reduction of the Treg was observed upon EP2/4 blockade, aligning with a previous mice study (53).PGE2 involvement in the suppression of T and NK cell responses has been previously reported for mice MDSCs and human moDCs, where PGE2 primed MDSC-like features (20,28,50,53,61).Although we noted that T-and NK cell responses were reversed either partly (when individually targeting EP2 and EP4) or completely (when simultaneously targeting EP2 and EP4), we noted a different EP implication, with EP4 rather than EP2 primarily mediating these suppressive features of moMDSCs.The predominant involvement of EP4 for the prevention of PGE2-mediated M-MDSC suppression of T cells and NK cells has already been demonstrated for mice MDSCs (28,52,54).Although EP4 targeting in mice displays a therapeutic benefit in different tumor models, few studies have explored the potential benefit of targeting EP2, EP4, or both.For example, two studies in mice demonstrated EP2 next to EP4 targeting to impair the development of MDSCs and limit tumor growth (49,50).A recent publication exploring a dual EP2/4 antagonist has demonstrated to outperform the individual targeting of EP4 or EP2 for immunomodulation, hence preventing tumor growth (62,63).The limited available studies with human cells use monocytes and moDCs demonstrate a similar contribution of EP2 and EP4 during the acquisition of MDSC-like features (20,28,53).Noteworthy, a recent study demonstrated that EP2 is expressed on blood MDSCs from colorectal cancer patients (50), underscoring the potential functional implication of EP2.We thus hypothesize that human MDSCs rely on both EP2 and EP4 signaling for acquiring a fully suppressive state.
To further evaluate the relevance of targeting EP2/4 signaling in a tumor model that better recapitulates the TME, we cocultured moMDSCs with CRC-PDTO.Notably, the combined application of EP2/4 antagonists within the coculture attenuated the development of a suppressive phenotype, as seen by the reduced CD11b, MerTK or CCR5 expression.Based on our observations and current literature, we propose that both EP2 and EP4 fosters human M-MDSC transition towards a suppressive pro-tumor state able to dampen T cell and NK cell responses, arguing for the combined usage of EP2 and EP4 antagonists to prevent a tumor-derived PGE2 imprinted phenotype of M-MDSCs.Although we show here the key role of EP2 and EP4 in in vitro generated human M-MDSCs, future research is required to validate the EP2/4 relevance on other blood MDSCs subsets.In different types of tumors, PMN-MDSCs, instead of the here investigated M-MDSCs model, have been reported to play a major role in promoting a suppressive TME.Unlike M-MDSCs, there are currently no cell models to study human blood PMN-MDSCs.Altogether, further exploration of PGE2-EP2/4 axis on blood MDSC subsets could serve to further establish the potential of targeting EP2/4 for therapeutic intervention in cancer patients.
Prior studies have demonstrated that PGE2-EP2/4 targeting can modulate immune cell function in different tumor models and enhance anti-tumor immunity (62)(63)(64).This illustrates the potential of targeting EP2/4 for the treatment of cancer.In fact, there are several ongoing phase I clinical trials for the treatment of cancer, either targeting EP4 or the combined EP2/4 (NCT04344795, NCT03155061) (65,66).Using an in vitro cell model for M-MDSCs, our results further support the evidence that targeting EP2 and EP4 possess a therapeutic value for the immune modulation of the host immune system, alleviate tumor-derived suppression and thus, promote the development of anti-tumor immunity.

1
FIGURE 1 Tumor-derived PGE2 skews monocytes via EP2/EP4 receptors towards a suppressive state.(A) To examine the effect of PGE2-EP2/4 signaling during M-MDSC like cell development, monocytes were treated with aEP2/4 prior to exposure to melanoma cell line-derived CM.After day 3 and day 6 of treatment, the resulting cells were analyzed for the expression of MerTK, CD11b, CD14, CCR5, HLA-DR, and CD86.(B) Histograms showing the expression levels of CD14 of a representative donor with and without aEP2/4 treatment on day 3 and day 6.(C) Graphs show either the percentage of positive cells for MerTK, CCR5 and CD86, or the geometric mean fluorescence intensity (gMFI) of CD11b, CD14 and CCR5.Each dot represents the mean ± SD of monocytes from different donors (n≥3) combined from two independent experiments.(D) The functional relevance of targeting aEP2/4 was determined by coculturing the resulting M-MDSC-like cells with autologous T cells and NK cells.After three days, T cell proliferation was determined as loss of CFSE dye.For NK cell suppression, the intracellular levels of IFNg and TNFa were determined.Violin plots show the mean, and each data point represents an individual monocyte donor (n=5) from a single independent experiment.P values were calculated with either a paired t-test or a Wilcoxon test (*p 0.05; **p 0.01; ***p 0.001).ns, non significant.

FIGURE 3
FIGURE 3 (A) In vitro generated moMDSCs were treated with aEP2, aEP4 or aEP2/4 prior to exposure to melanoma-derived CM.After 48 hours, moMDSCs were analyzed for the expression of CD14, MerTK, CCR5, CD11b, CD86, and HLA-DR by flow cytometry.(B) Representative histograms showing the expression levels of CD14 on untreated moMDSCs and moMDSCs treated with CM with or without aEP2/4 treatment.Graph represents the relative CD14 gMFI expression.Bar graph shows the mean ± SEM and each data point represents an individual M-MDSCs donor (n=7) tested in two independent experiments.(C) Bar graphs showing relative gMFI levels of MerTK, CCR5, CD11b, CD86, and HLA-DR.Relative gMFIs were determined by normalizing to gMFI levels of the untreated moMDSCs for every donor.Bar graphs show the mean ± SEM and each data point represents an individual moMDSCs donor (n>6) tested in two independent experiments.P values were calculated on raw gMFI values with either a Friedman test followed by a Dunn's test or a oneway ANOVA with Tukey test for multiple comparison correction (*p 0.05; **p 0.01; ***p 0.001).ns, non significant.

4 PGE2
FIGURE 4 PGE2-educated moMDSCs suppress autologous T cell responses via EP2 and EP4.In vitro generated moMDSCs were treated with either aEP2, aEP4, or aEP2/4 prior to exposure to melanoma-derived CM.After 48 hours, moMDSCs were harvested and replated for posterior coculturing with T cells.(A) moMDSCs were cocultured with autologous T cells previously primed to proliferate using activating dynabeads (anti-CD3/CD28).(B) Bar graphs showing the relative T cell proliferation.Relative proliferation was determined by normalizing to the T cell proliferation given by untreated moMDSCs.Each data point represents an individual moMDSC donor (n=5) tested in two independent experiments.(C) Production of IFNg, IL-2, and TNFa by T cells was determined by intracellular staining.Bar graphs show the percentage of T cells positive for the indicated cytokine.Each data point represents an individual moMDSC donor (n=5) from a single independent experiment.P values were calculated on raw percentage values with one-way ANOVA followed by a Dunnett's comparison test (*p 0.05; **p 0.01; ***p 0.001).ns, non significant.
FIGURE 5 PGE2-EP2/4 signaling potentiates the NK cell-suppressive ability of moMDSCs.In vitro generated moMDSCs were treated with either aEP2, aEP4, or aEP2/4 prior to exposure to melanoma-derived CM.After 48 hours, moMDSCs were harvested and replated for coculturing with NK cells.(A) moMDSCs were cocultured with autologous NK cells preactivated with IL-2.(B) Production of IFNg, TNFa, and GrzB by NK cells was determined by intracellular staining.Bar graphs are showing the percentage of positive NK cells for the indicated cytokine.Each data point represents and individual moMDSC donor (n=5) from a single independent experiment.(C) Bar graphs show the expression levels of different NK cell markers, either in percentage (NKG2D, NKp46, CD25, CD69) or relative gMFI (CD56).Relative CD56 gMFI expression was determined by normalizing to the expression levels of the untreated condition for every donor.Each data point represents an independent moMDSC donor (n>6) tested in two independent experiments.P values were calculated on the percentage or raw gMFI values with either a Friedman test followed by a Dunn's test or a one-way ANOVA followed by a Dunnett's comparison test (*p 0.05; **p 0.01; ***p 0.001).ns, non significant.
FIGURE 6 PGE2-EP2/4 signaling in moMDSCs steers moDC-mediated T cell responses towards the induction of suppressive T cell populations.(A) In vitro generated moMDSCs were treated with aEP2, aEP4, or aEP2/4 prior to exposure to melanoma-derived CM.After 48 hours, moMDSCs were harvested and replated for coculturing with moDCs and naïve CD4 T cells.To assess the impact of moMDSCs on the ability of moDCs to induce pro-inflammatory T cell responses, moMDSCs were cocultured with moDCs and allogeneic naïve CD4 T cells for 12 days.(B) Histograms show the intracellular IFNg levels in T cells after coculture from a representative donor.(C) Percentage of T cells positive for intracellular IFNg, IL-2, and TNFa expression.Bar graphs show the mean ± SEM and each data point represents an individual M-MDSCs donor (n=7) tested in two independent experiments.(C) T cells were analyzed for the expression of CD125, CD25, and FOXP3 to identify Tregs.Bar graphs show the percentage of CD127-CD25+FOXP3+ cells.Each data point represents expanded T cells from an individual moMDSC donor (n=8), derived from two independent experiments, and the bar graphs show the mean ± SEM.P values were calculated with either a Friedman test followed by a Dunn's test or a one-way ANOVA with a Dunnett's test for multiple comparison (*p 0.05; **p 0.01; ***p 0.001).ns, non significant.