Retinoic Acid Inhibits Tumor-Associated Mesenchymal Stromal Cell Transformation in Melanoma

Bone marrow mesenchymal stem/stromal cells (BMSCs) can be transformed into tumor-associated MSCs (TA-MSCs) within the tumor microenvironment to facilitate tumor progression. However, the underline mechanism and potential therapeutic strategy remain unclear. Here, we explored that interleukin 17 (IL-17) cooperating with IFNγ transforms BMSCs into TA-MSCs, which promotes tumor progression by recruiting macrophages/monocytes and myeloid-derived suppressor cells (MDSCs) in murine melanoma. IL-17 and IFNγ transformed TA-MSCs have high expression levels of myelocyte-recruiting chemokines (CCL2, CCL5, CCL7, and CCL20) mediated by activated NF-κB signaling pathway. Furthermore, retinoic acid inhibits NF-κB signaling, decreases chemokine expression, and suppresses the tumor-promoting function of transformed TA-MSCs by prohibiting the recruitment of macrophages/monocytes and MDSCs in the tumor microenvironment. Overall, our findings demonstrate that IL-17 collaborating with IFNγ to induce TA-MSC transformation, which can be targeted by RA for melanoma treatment.

TNFα, a proinflammatory cytokine highly expressed in tumor inflammatory environment, can transform BMSCs to TA-MSCs, which produce high-level CCR2 ligands to promote tumor growth by recruiting monocytes/macrophages (Ren et al., 2012). Interleukin 17 (IL-17) is an important proinflammatory cytokine secreted by CD4 + Th17 and CD8 + Tc17 cells and highly expressed in tumor microenvironment (Miossec et al., 2009). Deletion of IL-17 reduces MDSCs in tumor microenvironment and inhibits tumor growth (He et al., 2010;Wu et al., 2014). However, whether IL-17 participates in TA-MSC transformation to support tumor growth within tumor microenvironment remains unknown. Retinoic acid (RA), a metabolite of vitamin A (Cunningham and Duester, 2015), can induce differentiation of acute promyelocytic leukemia cells (de Thé, 2018). Studies suggest that RA could inhibit solid tumor growth and regulate the tumor microenvironment (Abu et al., 2005;Bolis et al., 2020;Sun et al., 2020). Here, we found that IL-17 incorporating with IFNγ transforms BMSCs into TA-MSCs to promote tumor growth, which is inhibited by RA treatment in melanoma.

IL-17 and IFNγ Transform BMSCs Into TA-MSCs to Facilitate Melanoma Progress in vivo
To explore the role of IL-17 in transforming BMSCs to TA-MSCs, we investigated the tumor growth co-engrafted with BMSCs and IL-17 transformed MSCs. We subcutaneously inoculated B16F0 melanoma cells with normal BMSCs or BMSCs pretreated with IL-17 and IFNγ, respectively, or jointly into C57BL/6 mice ( Figure 1A). The B16F0 melanoma cells with BMSCs, which were pretreated with IL-17 and IFNγ jointly, gave more aggressive tumor growth compared to control B16F0 melanoma cells (5.2-fold increase in tumor weight, and 4-fold increase in tumor size). However, B16F0 melanoma cells with normal BMSCs or BMSCs treated with IL-17 and IFNγ respectively, did not show a significant difference in tumor weight or volume compared to control B16F0 melanoma cells (Figures 1B,C). This indicated that IL-17 incorporated with IFNγ to stimulate the BMSC to TA-MSC transformation, which promoted tumor growth in melanoma. Furthermore, we investigated that whether IL-17 and IFNγ transformed TA-MSCs can recruit monocytes/macrophages and MDSCs. Our FACS assay showed that the myelocytes, including macrophages, monocytes, and neutrophils, were dramatically increased in peripheral blood when melanoma mice were co-inoculated with IL-17 and IFNγ transformed TA-MSCs (1. 5-, 2. 5-, and 1.6-fold increase, respectively) ( Figure 1D). However, no significant increase of circulating T cells was observed in mice co-engrafted with pretreated TA-MSCs compared to mice with control melanoma cells ( Figure 1E). No significant increase of either myelocytes or T cells was observed in peripheral blood when melanoma mice were co-inoculated with normal BMSCs or BMSCs pretreated with IL-17 or IFNγ individually ( Figure 1D). More importantly, the increased macrophages, monocytes, and MDSCs were observed in the tumor microenvironment (2. 5-, 3. 5-, and 1.6-fold increase, respectively) when melanoma mice were co-engrafted with IL-17 and IFNγ transformed TA-MSCs ( Figure 1F). However, co-inoculation of normal BMSCs or BMSCs pretreated with IL-17 or IFNγ individually did not increase the numbers of macrophages and monocytes in the tumor microenvironment, and the BMSC induced slight increase of MDSCs was not statistically significant ( Figure 1F). Furthermore, tumor-resident T cells were not regulated by control BMSCs or by transformed TA-MSCs ( Figure 1G).
Overall, our observations showed that IL-17 and IFNγ jointly but not individually transformed normal BMSCs into TA-MSCs, and the IL-17 and IFNγ transformed TA-MSCs can recruit myelocytes into the tumor microenvironment to promote tumor growth.
Taken together, these data illustrated that IL-17 and IFNγ synergistically increased the expression of immunosuppressive factors and myelocyte recruiting factors in BMSCs, but IL17 or IFNγ individual treatment had a limited effect on TA-MSC transformation.

RA Inhibits TA-MSC Transformation and Further Suppresses Melanoma Progress in vivo
RA inhibits Th17 differentiation (Mucida et al., 2007;Elias et al., 2008), which suggested that RA might inhibit IL-17 signaling. Therefore, we investigated whether RA regulates IL-17 mediated TA-MSC transformation. We simultaneously supplied RA during IL-17 and IFNγ mediated TA-MSC transformation, and further performed co-engrafted cell-derived xenograft experiments with B16F0 melanoma cells and transformed TA-MSCs or RA treated TA-MSCs ( Figure 3A). Intriguingly, RA supplement dramatically inhibited the tumor-promoting capacity of IL-17 and IFNγ transformed TA-MSCs, which had 68% reduction in tumor weight ( Figure 3B) and 53% reduction in tumor volume compared to TA-MSCs without RA treatment ( Figure 3C). To explore the underline mechanism, we further analyzed the myelocytes in peripheral blood and tumor microenvironment. We surprisingly found that RA remarkably inhibited the myelocyte recruiting function of TA-MSCs, with 64% decrease of macrophages, 53% decrease of monocytes, and 89% decrease of neutrophils in peripheral blood ( Figure 3D). More importantly, RA-treated TA-MSCs completely failed to recruit macrophages, monocytes, and MDSCs into the tumor microenvironment (85, 83, and 108% decrease, respectively) ( Figure 3F). Consistent with our previous observation, no significant change of circulating T cells ( Figure 3E) or tumor-resident T cells ( Figure 3G) was observed in mice co-engrafted with TA-MSCs or RA treated TA-MSCs compared to mice with control BMSCs.
Overall, our data demonstrated RA treatment significantly blocked IL-17 and IFNγ mediated TA-MSC transformation in promoting tumor growth in melanoma.

RA Inhibits IL-17-Stimulated Myelocyte-Recruiting Chemokine Expression in BMSCs Through Inhibiting NF-κB Signaling Pathway
To explore the molecular mechanism that RA suppressed IL-17 and IFNγ mediated TA-MSC transformation, we first analyzed the expression of immunoregulatory molecules in IL-17 and IFNγ transformed TA-MSCs. Intriguingly, RA completely blocked the increase of myelocyte recruiting chemokines expression, including CCL2, CCL5, CCL7, and CCL20, in IL-17 and IFNγ transformed TA-MSCs ( Figure 4A). We also noticed that IL-17 treatment alone also slightly increased myelocyte recruiting chemokines expression, which was also completely blocked by RA treatment (87, 89, 90, and 83% reduction) ( Figure 4B). However, RA did not inhibit the immunosuppressive molecule expression, including NOS2, PD-L1, CXCL9, CXCL10 ( Figure 4C). This indicated that RA inhibited TA-MSC transformation mainly by blocking their ability to recruit myelocytes for tumor-promoting.
To understand the molecular mechanism that RA inhibits IL-17 signaling to suppress myelocyte recruiting chemokine expression, we performed transcription analysis for BMSCs  Immunoblotting analysis of key NF-κB pathway elements in the BMSCs exposed to IL-17 with or without the RA treatment as indicated. (I) The relative mRNA expression of myelocyte-recruiting chemokines in BMSCs treated by IL-17, RA, and BetA as indicated. (J) The relative mRNA expression of myelocyte-recruiting chemokines in BMSCs treated by IFNγ, IL-17, RA, and BetA as indicated. Data represent mean ± SD of 3 independent experiments. * or ‡ p < 0.05, ** or ‡ ‡ p < 0.01, ‡ ‡ ‡ p < 0.001, ‡ ‡ ‡ ‡ p < 0.0001. ns, not significant.
Frontiers in Cell and Developmental Biology | www.frontiersin.org under RA treatment. Our RNA sequencing (RNA-seq) analysis successfully detected 14,423 genes, in which 1,474 genes were upregulated and 1,393 genes were downregulated in BMSCs after RA treatment ( Figure 4D). We noticed that NF-κB pathway, which stimulates CCL2 release in TA-MSCs (Katanov et al., 2015), was inhibited in BMSCs upon RA treatment ( Figure 4E). Strikingly, major NF-κB pathway elements were downregulated in BMSCs after RA treatment ( Figure 4F). Furthermore, RA treatment significantly inhibited IL-17 stimulated phosphorylation of RalA-p65 and IκBα, two key molecules in NF-κB pathway, in BMSCs (Figures 4G,H). This indicated that RA might inhibit NF-κB pathway to suppress the expression of myelocyte recruiting chemokines in IL-17 transformed TA-MSCs. To confirm this, we employed NF-κB specific activator, betulinic acid (BetA) (Kasperczyk et al., 2005), to rescue the suppressed NF-κB signaling in IL-17 transformed TA-MSCs upon RA treatment. Notably, BetA treatment completely rescued the expression of CCL2, CCL5, CCL7, and CCL20 in IL-17 transformed TA-MSCs under RA treatment ( Figure 4I). The rescue effect was also observed in IL-17 and IFNγ transformed TA-MSCs ( Figure 4J).
Taken together, our data showed that IL-17 activates NF-κB pathway to upregulate myelocyte recruiting chemokines in TA-MSCs, and the TA-MSC transformation was significantly blocked by RA treatment due to inhibition of NF-κB signaling pathway.
As NF-κB pathway is critical to CCL family regulation in IL-17 transformed TA-MSCs, we next investigated the role of NF-κB pathway in TNFα mediated TA-MSC transformation. Consistently, we observed that TNFα treatment activated NF-κB pathway in BMSCs, which was evidenced by the activation of NK-κB pP65 and pIκBα, which was coupled with the reduction of IκBα, at 7.5-15 min after TNFα treatment. Notably, RA treatment remarkably attenuated the activation of NFκB pathway in BMSCs under TNFα treatment (Figures 5D,E). More importantly, NF-κB activator, BetA, completely rescued the decrease of CCL2, CCL5, CCL7, and CCL20 under RA treatment in TNFα educated BMSCs ( Figure 5F) and BMSCs treated with TNFα and IFNγ simultaneously ( Figure 5G).
Taken together, these data demonstrated that RA inhibited NF-κB pathway to suppress the expression of myelocyte chemokines in TNFα educated TA-MSCs.

DISCUSSION
BMSCs can be transformed into TA-MSCs, which is featured by producing high-level CCR2 ligands to recruit monocytes/macrophages and MDSCs in promoting tumor growth (Ren et al., 2012;Wang Y. et al., 2014;Lin et al., 2016). Tumor proinflammatory cytokine, TNFα, efficiently transforms BMSCs into TA-MSCs and promotes tumor growth in lymphoma, melanoma, and breast carcinoma (Ren et al., 2012;Katanov et al., 2015). IL-17 is involved in inflammatory process and enhances the expression of an immunosuppressive molecule, NOS2, in murine hepatitis (Oukka, 2008;Han et al., 2014). However, unlike innate immune cell generated TNFα, IL-17 is derived from T cells (Oukka, 2008). IL-17 has both pro-tumor and anti-tumor effects. IL-17 inhibits tumor progression and metastasis in melanoma and colon cancer by promoting the function of T cells and NK cells (Kryczek et al., 2009;Martin-Orozco et al., 2009). However, growing evidences show that IL-17 promotes tumor growth in various solid tumors, including melanoma, breast cancer, colon cancer, and hepatocellular carcinoma (Wang et al., 2009;Grivennikov et al., 2012;Coffelt et al., 2015;Gomes et al., 2016). Genetic evidence shows that IL-17 can directly promote proliferation of transformed colonic epithelial cells tumor through its type A receptor (IL-17RA) (Wang K. et al., 2014). Here, we found that IL-17 cooperating with IFNγ to transform TA-MSCs in supporting tumor growth in melanoma. These suggested that blocking IL-17 signaling may inhibit melanoma cells through multiple mechanisms. Out work also suggested that adaptive immune cells can modulate the protumorigenic function of TA-MSCs, which recruits macrophages to support tumor growth (Ren et al., 2012). Intriguingly, we observed that IL-17 treatment alone cannot efficiently transform BMSCs to TA-MSCs, due to the less myelocyte recruiting chemokine expression, and limited ability to recruit macrophages and MDSCs. However, IFNγ remarkably strengthened the myelocyte recruiting chemokines upregulation ability of IL-17, therefore IFNγ and IL-17 synergistically promoted BMSC to TA-MSC transformation. Moreover, TNFα induces cell necroptosis and apoptosis (Locksley et al., 2001;Kalliolias and Ivashkiv, 2016), therefore TNFα transformed TA-MSCs may have limited ability to promote tumor growth. However, IL-17 promotes cell proliferation (Wu et al., 2014), presumably, IL-17 transformed TA-MSCs may have greater efficiency to promote tumor growth. Accordingly, high IL-17 level is observed in colon cancer, skin cancer, and lung cancer patients with poor clinical outcome (Marshall et al., 2016;Razi et al., 2019;Bellone et al., 2020). Recent work showed that IL-17 also regulates the protumorigenic function of cancer-associated fibroblasts (CAFs) (Mucciolo et al., 2021), which are also transformed from normal * p < 0.05, * * or ‡ ‡ p < 0.01, * * * or ‡ ‡ ‡ p < 0.001, ‡ ‡ ‡ ‡ p < 0.0001. ns, not significant.
BMSCs (Quante et al., 2011). Therefore, further studies are warranted to determine the discrepancy between TA-MSCs and CAF in regulating melanoma. ATRA has revolutionized the treatment of acute promyelocytic leukemia (de Thé, 2018). However, the application of ATRA in solid tumors remains to be explored. RA can inhibit tumor cell proliferation in melanoma (Edward and MacKie, 1989;Zhang and Rosdahl, 2005;Li and Han, 2020) and promote immune surveillance in breast cancer, colorectal cancer, and melanoma by influencing the metabolism of MDSCs, upregulating genes related to immune response, and supporting the survival of tumor-specific CD8 + T cells (Guo et al., 2012;Paroni et al., 2020;Sun et al., 2020). Conversely, RA treatment was also reported to benefits tumor progression in sarcoma and chronic lymphocytic leukemia (CLL) by promoting the protumoral differentiation of intertumoral monocytes in sarcoma and increasing CD38 expression in CLL cells Devalaraja et al., 2020). Our study showed that RA treatment almost completely inhibited the increase of myelocyte recruiting ability of IL-17 and IFNγ transformed TA-MSCs, although it barely influenced the expression of the immunosuppressive molecules induced by IFNγ. RA treatment successfully inhibited the BMSC to TA-MSC transformation and significantly inhibited tumor growth in melanoma, which opens an avenue for tumor microenvironment targeting therapy.
Collectively, our study identified IL-17 can educate healthy BMSCs into TA-MSCs, and uncovered a new therapeutic approach to target TA-MSCs by RA. This finding may extend the mechanism and application of RA in tumor therapy.
C57BL/6 mice were bred under specific pathogen-free conditions in the animal facility of Sun Yat-sen university. All animal protocols were approved by our Institutional Animal Care and Use Committee.

Cell Culture
BMSCs were isolated from the tibia and femur bone marrow of C57BL/6 mice following the protocol described in the previous reference (Ren et al., 2012). Cells were maintained in DMEM low-glucose medium (10-014-CVR, CORNING) supplemented with 20% fetal bovine serum (12483020, Gibco), 2% penicillin-streptomycin (SV30010, Invitrogen) and 10 µM ROCK inhibitor (S1049, Selleck) in the adhesive petri dishes. All non-adherent cells were removed after 24 h, and adherent cells were maintained. To obtain MSC clones, cells maintained in 10 cm dishes at 80-90% density were harvested and seeded into 6-well plates at a density of 5 × 10 5 cells/well. Cells were used before the 3rd passage. B16F0 cells were maintained in DMEM high-glucose medium (10-013-CVR, CORNING) supplemented with 10% FBS and 1% penicillin-streptomycin.

RNA Isolation and Gene Expression Assay
Before RNA isolation, BMSCs were incubated with or without cytokines of (50 ng ml −1 IL-17, 10 ng ml −1 IFNγ, and 10 ng ml −1 TNFα) or drugs (100 nM RA and 10 µg ml −1 BetA), respectively, or jointly for 6 h (Han et al., 2014;Song et al., 2015). Total mRNA was isolated with MagZol TM Reagent (R4801-03, Magen) according to the manufacturer's instruction. mRNA purity and quantity were determined with NanoDrop (Thermo Scientific) before qPCR and RNA-seq analysis. For Real-Time qPCR, cDNA was synthesized from mRNA by using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit (AT341, Transgen). Quantitative Real-Time PCR was performed on Bio-Rad CFX96 Touch TM Real-Time PCR Detection system with SYBR Green I Master Mix reagent (11203ES03, YEASEN). Sequences of forward and reverse primer pairs are as follows:

Flow Cytometry
For cell population analysis, cells isolated from peripheral blood and tumors were suspended in staining buffer (PBS, 2% FBS) at a concentration of 2 × 10 6 cells ml −1 and 100 ml of suspension was incubated with fluorescently labeled antibodies for 1 h on ice.
Macrophages were gated as CD11b + F4/80 + . Neutrophils were gated as CD11b + Ly6G + . Monocytes were gated as CD11b + Gr-1 + Ly6G − . MDSCs were gated as CD11b + Gr-1 + . Analyses were performed using a flow cytometer (Attune NxT; Thermo Fisher). The immune cell frequency was calculated as the frequency of each immune cell population in total nucleated cells in peripheral blood or total resident nucleated blood cells from tumor site.

Gene Set Enrichment Analysis (GSEA)
RNA of control MSC and RA pretreated MSCs (100 nM RA for 24 h) were used for RNAseq analysis. Raw data.QZ files were imported into GSEA 3.0 software where background correction and normalization were performed with standard default settings. The.QZ files were combined into one.gct file in GenePattern, then imported into GSEA along with a matching phenotype label file (.cls). GSEA analysis was run with the following parameters: number of permutations = 1,000, collapse dataset to gene symbols = false, permutation type = gene_set, plot graphs for the top sets of each phenotype = 150 (default = 20), gene sets database = h.all.v6.0 symbols.gmt (all hallmarks, version 6), with a phenotype comparison of RA pre-treatment vs. control BMSCs. Leading edge analysis was completed on the Hallmark GSEA output with NF-κB signaling hallmark gene set.

Statistical Analysis
The statistical analysis was performed using GraphPad Prism 8.0 software. Two-tailed Student's t tests were used for the comparison between two groups (*p < 0.05, **p < 0.01, and ***p < 0.001) and the one-way ANOVAs with Tukey's multiple comparison tests were used for the comparison between more than two groups ( ‡ p < 0.05, ‡ ‡ p < 0.01, ‡ ‡ ‡ p < 0.001, and ‡ ‡ ‡ ‡ p < 0.0001). The two-way ANOVAs with Tukey's multiple comparison tests were used for comparison between more than two groups at various time points ( ‡ p < 0.05, ‡ ‡ p < 0.01, ‡ ‡ ‡ p < 0.001, and ‡ ‡ ‡ ‡ p < 0.0001). All data are expressed as mean ± SD.

DATA AVAILABILITY STATEMENT
The accession number for the RNA-seq data reported in our manuscript is GEO: GSE169145.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee, SYSU.

AUTHOR CONTRIBUTIONS
QL, MiZ, and QX designed and performed most of the experiments and analyzed the data. SX, YL, JC, LY, and LW contributed to animal experiments and all the transcriptional assay. LM, DL, and LJ contributed to the discussion. QL, MiZ, and MeZ wrote the manuscript. MeZ supervised the project. All authors contributed to the article and approved the submitted version.