cancers

Article The CCR2/MCP-1 Pathway and Lung Adenocarcinoma

1,2, 2 2 1 1, Payal Mittal †, Liqing Wang , Tatiana Akimova , Craig A. Leach , Jose C. Clemente †, Matthew R. Sender 1, Yao Chen 1, Brandon J. Turunen 1 and Wayne W. Hancock 2,* 1 Chemical Biology, Medicinal Science Technology, GlaxoSmithKline, Collegeville, PA 19426, USA; [email protected] (P.M.); [email protected] (C.A.L.); [email protected] (J.C.C.); [email protected] (M.R.S.); [email protected] (Y.C.); [email protected] (B.J.T.) 2 Division of Transplantation Immunology, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; [email protected] (L.W.); [email protected] (T.A.) * Correspondence: [email protected]; Tel.: +1-215-590-8709 Current address: Janssen Pharmaceutical Companies of Johnson & Johnson, Philadelphia, PA 19034, USA. †  Received: 22 September 2020; Accepted: 8 December 2020; Published: 11 December 2020 

Simple Summary: Myeloid-derived suppressor cells (MDSCs) are present at the sites of many solid tumors and dampen host immune responses. We studied how these MDSCs accumulate and how this process might be interrupted using lung cancer cell lines growing in immunocompetent mice. We found that tumor cells release a chemokine, MCP-1, that attracts MDSCs as a result of their expression of the MCP-1 receptor, CCR2. We showed that mice lacking CCR2, or the use of a small-molecule inhibitor of CCR2, prevented MDSC recruitment to the tumors and derepressed host T cell responses, allowing immune activation and inhibition of tumor growth. These data suggest that CCR2 inhibitors may be a new tool for cancer immunotherapy.

Abstract: Host anti-tumor immunity can be hindered by various mechanisms present within the , including the actions of myeloid-derived suppressor cells (MDSCs). We investigated the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine lung cancer models. Phenotypic profiling revealed maximal expression of CCR2 by tumor-resident MDSCs, and MCP-1 by transplanted TC1 tumor cells, respectively. Use of CCR2-knockout (CCR2-KO) mice showed dependence of tumor growth on CCR2 signaling. Tumors in CCR2-KO mice had fewer CCR2low MDSCs, CD4 T cells and Tregs than WT mice, and increased infiltration by CD8 T cells producing IFN-γ and granzyme-B. Effects were MDSC specific, since WT and CCR2-KO conventional T (Tcon) cells had comparable proliferation and production of inflammatory , and suppressive functions of WT and CCR2-KO Foxp3+ Treg cells were also similar. We used a thioglycolate-induced peritonitis model to demonstrate a role for CCR2/MCP-1 in trafficking of CCR2+ cells to an inflammatory site, and showed the ability of a CCR2 antagonist to inhibit such trafficking. Use of this CCR2 antagonist promoted anti-tumor immunity and limited tumor growth. In summary, tumor cells are the prime source of MCP-1 that promotes MDSC recruitment, and our genetic and pharmacologic data demonstrate that CCR2 targeting may be an important component of cancer immunotherapy.

Keywords: MDSC; CCR2; lung cancer; anti-tumor immunity; immunotherapy

1. Introduction T cells are the major drivers of anti-tumor immune responses but their functions within the tumor microenvironment can be impaired by immune cells with inhibitory properties, as well as

Cancers 2020, 12, 3723; doi:10.3390/cancers12123723 www.mdpi.com/journal/cancers Cancers 2020, 12, 3723 2 of 19 through the actions of fibroblasts and endothelial cells [1]. One important immunosuppressive mechanism involves the actions of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment [2]. Once recruited, various cytokines, including vascular endothelial growth factor (VEGF) [3], granulocyte colony stimulating factor (G-CSF) and granulocyte/ colony stimulating factor (GM-CSF) [4], are implicated in the expansion and maintenance of MDSCs within the tumor microenvironment. MDSCs blunt anti-tumor immunity through multiple, sometimes surprising mechanisms. For example, they can directly contribute to tumor growth and vascularization by producing MMP9 and differentiating into endothelial cells [5]. They can also induce cross-talk between STAT3 and NOTCH signaling in cancer cells to confer properties of stemness [6]. Additionally, MDSCs can exert direct suppressive effects on various immune cells, including T cells, through their production of arginase (Arg1) and Arg1+ exosomes, nitric-oxide (NO) synthase enzymes, IL-10, TGF-β and indolamine 2,3-dioxygenase (IDO) [7,8]. They can also restrict CD8 T cell activity following antigenic stimulation by secreting reactive oxygen species [9,10]. In addition to suppressing T cell functions, MDSCs facilitate tumor progression through cell–cell contact with and inhibiting their IL-12 secretion [11], inhibiting NK cell-mediated cytotoxicity against tumors [12], and recruiting and inducing Foxp3+ T-regulatory (Treg) within the tumor microenvironment [13,14]. MDSCs are generated from common myeloid progenitor cells in bone marrow and are found abundantly in murine tumors [15] and human cancers such as glioblastoma [16], pancreatic adenocarcinoma [17] and breast cancer [18]. GM-CSF, G-CSF and IL-6 cytokines induce rapid generation of MDSCs in both murine and human bone marrow [19]. Tumor production of GM-CSF plays a crucial role in generation of MDSCs in mice [20] and in humans [21] by acting upon CD33+ peripheral blood mononuclear cells. The chemokine MCP-1 (CCL2) is important for the recruitment of bone marrow generated myeloid cells into the blood, and for the trafficking of MDSCs to tumor sites, by binding to the G -coupled receptor, CCR2 [22–25]. These observations provided a rationale for us to further assess the effects of MCP-1/CCR2 targeting on MDSC recruitment to tumors and concomitant effects on anti-tumor immunity and tumor growth.

2. Results

2.1. Baseline Studies of Myeloid and T Cell Lineages in CCR2RKO Mice We planned to investigate the role of the CCR2/MCP-1 pathway in MDSC-associated tumor progression in murine syngeneic lung cancer models but undertook some basic characterization of the main mouse strain to be used, namely mice lacking the for CCR2 (CCR2KO). We found that CCR2 deletion did not affect thymic T cell development (Figure S1) or the frequencies of peripheral CD4+ and CD8+ T cells, including CD4+Foxp3+ T-regulatory (Treg) cells (Figure S2). Among myeloid cells, the frequency of splenic CD11b+Ly6Chigh inflammatory was significantly compromised in CCR2KO vs. WT mice, whereas neutrophil populations were comparable (Figure S3). This led us to consider a developmental defect in the production of CD11b+Ly6Chigh inflammatory monocytes but we found their frequency in bone marrow was comparable to WT mice (Figure S4). Hence, compared to WT mice, CCR2RKO mice appeared to have normal T cell populations but a marked decrease in the peripheral levels of CD11b+Ly6Chigh monocytes. Previous studies have shown that MCP-1/CCR2 stimulation causes desensitization of the CXCL12–CXCR4 signaling that normally promotes bone marrow cell sequestration [26]. These findings are consistent with a requirement for CCR2 expression for egress of CD11b+Ly6Chigh monocytes from the bone marrow [27].

2.2. Expression of CCR2 and CCL2 at the Tumor Site We began our tumor studies by assessing the expression of CCR2 and MCP-1/CCL2 within tumors that developed in syngeneic C57BL/6 mice after subcutaneous injection of a murine lung adenocarcinoma cell line, TC1. While there are differences in the flow cytometric phenotypic markers of relevant tumor-associated cell types between humans and mice, and even within murine Cancers 2020, 12, x 3 of 20

2.2. Expression of CCR2 and CCL2 at the Tumor Site We began our tumor studies by assessing the expression of CCR2 and MCP‐1/CCL2 within tumorsCancers 2020 that, 12 developed, 3723 in syngeneic C57BL/6 mice after subcutaneous injection of a murine 3lung of 19 adenocarcinoma cell line, TC1. While there are differences in the flow cytometric phenotypic markers of relevant tumor‐associated cell types between humans and mice, and even within murine studies, high low instudies, the current in the current work workwe wedefined defined MDSCs MDSCs as as CD11b+Ly6C CD11b+Ly6ChighLy6GLy6Glow cells,cells, tumor tumor-associated‐associated intermediate high neutrophilsneutrophils (TAN) asas CD11bCD11b++ Ly6CLy6CintermediateLy6GLy6Ghigh cells,cells, and and tumor tumor-associated‐associated macrophages macrophages (TAM) (TAM) low low asas CD11b+Ly6C CD11b+Ly6ClowLy6GLy6Glow cells.cells. Using Using these these definitions definitions and and the the gating gating strategy strategy outlined outlined in in Figure Figure S5, S5, wewe found found that that MDSCs MDSCs expressed expressed significantly significantly more more CCR2 CCR2 than than tumor tumor-associated‐associated CD4 CD4 or or CD8 CD8 T T cells, cells, TAN,TAN, TAM or or tumor tumor cells cells (Figure (Figure1A,B), 1A,B), whereas whereas the tumorthe tumor cells cells themselves themselves expressed expressed the highest the highest levels high levelsof MCP-1 of MCP (Figure‐1 1(FigureC,D). Likewise, 1C,D). Likewise, in peripheral in peripheral blood, CD11b blood,+ Ly6C CD11b+inflammatory Ly6Chigh inflammatory monocytes monocytesexpressed higherexpressed levels higher of CCR2 levels than of CCR2 anti-inflammatory than anti‐inflammatory monocytes ormonocytes neutrophils or neutrophils (Figure S6). (FigureAnalogous S6). dataAnalogous were obtained data were using obtained Lewis using lung adenocarcinomaLewis lung adenocarcinoma cells (Figure cells S7). (Figure S7).

FigureFigure 1. Myeloid-derivedMyeloid‐derived suppressor suppressor cells cells (MDSCs) (MDSCs) derived derived from from subcutaneously subcutaneously transplanted transplanted mouse mouselung epithelial lung epithelial cell tumor cell TC1tumor expressed TC1 expressed the highest the highest levels of levels chemokine of chemokine CCR2 whereas CCR2 whereas tumor cells tumor are cellsthe major are the source major of source CCL2 (MCP-1).of CCL2 (MCP WT C57BL‐1). WT/6 mice C57BL/6 were mice injected were subcutaneously injected subcutaneously with 1 million with TC1 1 millioncells and TC1 tumors cells and were tumors harvested were 21 harvested days post-injection 21 days post and‐injection were analyzed and were for analyzed CCR2 ( forA,B CCR2) and ( CCL2A,B) and(C,D CCL2) expression (C,D) expression by flow cytometry; by flow **cytometry;p < 0.01 and ** p *** < p0.01< 0.001, and ***n = p5 /

2.3. Lack of Tumor Growth in CCR2KO Mice

Given the prominent MDSC expression of CCR2, we assessed TC1 tumor growth in C57BL/6 mice lacking CCR2 (CCR2KO). Compared to WT controls, tumor growth in CCR2RKO mice was Cancers 2020, 12, x 4 of 20

2.3. Lack of Tumor Growth in CCR2KO Mice Cancers 2020, 12, 3723 4 of 19 Given the prominent MDSC expression of CCR2, we assessed TC1 tumor growth in C57BL/6 mice lacking CCR2 (CCR2KO). Compared to WT controls, tumor growth in CCR2RKO mice was significantly inhibited (Figure2A), with reductions in tumor volumes (Figure2B) and tumor masses significantly inhibited (Figure 2A), with reductions in tumor volumes (Figure 2B) and tumor masses (Figure2C). (Figure 2C).

FigureFigure 2. 2. GrowthGrowth of of subcutaneously subcutaneously transplanted transplanted TC1 TC1 mouse mouse lung lung tumors tumors is is dependent dependent on on CCR2 CCR2 signaling.signaling. TC1 TC1 tumor tumor cells cells (1 (1× 10610) were6) were injected injected subcutaneously subcutaneously into intoWT WTand andCCR2KO CCR2KO mice. mice. (A) × Tumors(A) Tumors were were measured measured biweekly biweekly until untilthey were they wereharvested harvested on day on 14 day post 14‐tumor post-tumor inoculations, inoculations, when (whenB) tumor (B) volumes tumor volumes and (C) tumor and (C weights) tumor were weights determined; were determined; ** p < 0.1 and ** p***< p 0.1< 0.001, and n *** = 10/group.p < 0.001, Datan = 10 are/group. representative Data are representativeof three independent of three experiments. independent experiments. 2.4. Host CCR2 Deletion Led to Markedly Reduced MDSCs but Increased Activated CD8+ T Cells at 2.4.Tumor Host Sites CCR2 Deletion Led to Markedly Reduced MDSCs but Increased Activated CD8+ T Cells at Tumor Sites We next assessed what effects CCR2 deletion had on anti-tumor immune responses by harvesting We next assessed what effects CCR2 deletion had on anti‐tumor immune responses by tumors at 14 days post-injection, preparing single-cell suspensions and flow cytometric analysis harvesting tumors at 14 days post‐injection, preparing single‐cell suspensions and flow cytometric (Figure3). The reduced tumor growth in CCR2KO mice (Figure3A) was associated with significantly analysis (Figure 3). The reduced tumor growth in CCR2KO mice (Figure 3A) was associated with decreased numbers of MDSCs but significantly increased numbers of TAN (Figure3B–D). As expected, significantly decreased numbers of MDSCs but significantly increased numbers of TAN (Figure 3B– expression of CCR2 on MDSCs in CCR2KO was markedly reduced compared to MDSCs in WT mice D). As expected, expression of CCR2 on MDSCs in CCR2KO was markedly reduced compared to (Figure3E,F). CCR2 deletion also led to increased CD8 + T cell infiltration but reduced accumulation MDSCs in WT mice (Figure 3E,F). CCR2 deletion also led to increased CD8+ T cell infiltration but of CD4 T cells (Figure3G–I), including reduced numbers of CD4 +Foxp3+ T-regulatory (Treg) cells reduced accumulation of CD4 T cells (Figure 3G–I), including reduced numbers of CD4+Foxp3+ T‐ (Figure3J,K). Analysis of intracellular expression showed that compared to corresponding regulatory (Treg) cells (Figure 3J,K). Analysis of intracellular cytokine expression showed that WT mice, the tumor-infiltrating CD8+ T cells of CCR2KO mice produced increased amounts of IFN-γ compared to corresponding WT mice, the tumor‐infiltrating CD8+ T cells of CCR2KO mice produced and granzyme-B (Figure3L,M). increased amounts of IFN‐γ and granzyme‐B (Figure 3L,M).

Cancers 2020, 12, 3723 5 of 19 Cancers 2020, 12, x 5 of 20

FigureFigure 3. 3. GeneticGenetic ablationablation ofof CCR2CCR2 reducedreduced MDSCMDSC andand TregTreg infiltration infiltration and and increased increased intratumoral intratumoral 6 accumulationaccumulation ofof activated CD8+ CD8+ TT cells. cells. (A (A) TC1) TC1 tumor tumor cells cells (1 × (1 106)10 were) were injected injected subcutaneous subcutaneous (s.c.) × (s.c.)in WT in WTand andCCR2KO CCR2KO mice mice and and tumors tumors were were measured measured until until day day 14. 14. Analysis Analysis of thethe intratumoralintratumoral CD11bCD11b++ myeloidmyeloid populationspopulations showedshowed aa significantsignificant decreasedecrease inin MDSCsMDSCs ((BB,,CC)) andand anan increaseincrease inin TANTAN ((BB,D,D).). MDSCs MDSCs from from CCR2KO CCR2KO mice mice expressed expressed negligible negligible levels levels of of CCR2 CCR2 (E ,(FE),F as) as compared compared to MDSCsto MDSCs in tumorsin tumors from from WT WT mice. mice. Tumors Tumors in CCR2KO in CCR2KO mice mice also showed also showed a higher a higher frequency frequency of CD8 of+ TCD8+ cells T (G cells,H) and(G,H fewer) and CD4 fewer+ T CD4+ cell (G T,I cell) and (G Tregs,I) and (J, KTregs) as compared (J,K) as compared to WT control. to WT Intratumoral control. Intratumoral CD8+ T cells CD8+ from T CCR2KOcells from mice CCR2KO expressed mice significantly expressed significantly higher levels higher of IFN- levelsγ (L,M of) and IFN GzmB‐γ (L,M (N) )and upon GzmB restimulation; (N) upon * p < 0.05, ** p < 0.01, **** p < 0.001. Data are representative of three independent experiments.

Cancers 2020, 12, x 6 of 20 Cancers 2020, 12, x 6 of 20

restimulation; * p < 0.05, ** p < 0.01, **** p < 0.001. Data are representative of three independent restimulation; * p < 0.05, ** p < 0.01, **** p < 0.001. Data are representative of three independent Cancersexperiments.2020, 12, 3723 6 of 19 experiments. 2.5. Importance of the CCR2/MCP‐1 Pathway for Recruitment of Inflammatory Monocytes 2.5.2.5. Importance Importance of of the the CCR2/MCP CCR2/MCP-1‐1 Pathway Pathway for for Recruitment Recruitment of of Inflammatory Inflammatory Monocytes We next sought to create a system to test and validate the effects of a CCR2 antagonist on WeWe next next sought sought to to create create a asystem system to to test test and and validate validate the the effects effects of a of CCR2 a CCR2 antagonist antagonist on recruitment of inflammatory monocytes, given potential translational significance for tumor therapy. recruitmenton recruitment of inflammatory of inflammatory monocytes, monocytes, given potential given potential translational translational significance significance for tumor for therapy. tumor We first injected WT, CCR2KO and MCP1KO mice with 4% sterile thioglycolate broth and 48 h later, Wetherapy. first injected We first WT, injected CCR2KO WT, and CCR2KO MCP1KO and mice MCP1KO with 4% mice sterile with thioglycolate 4% sterile broth thioglycolate and 48 h later, broth the total numbers of cells in peritoneal lavages were counted and the influx of inflammatory theand total 48 h numbers later, the of total cells numbers in peritoneal of cells lavages in peritoneal were counted lavages and were the counted influx andof inflammatory the influx of monocytes analyzed by flow cytometry. These studies showed potent recruitment of CD11b+Ly6Chigh monocytesinflammatory analyzed monocytes by flow analyzed cytometry. by flow These cytometry. studies showed These studiespotent recruitment showed potent of CD11b recruitment+Ly6Chigh of inflammatory monocytes to the peritoneum in WT mice in response to thioglycolate elicited inflammatoryCD11b+Ly6Chigh monocytesinflammatory to the monocytes peritoneum to the in peritoneum WT mice inin WTresponse mice in to response thioglycolate to thioglycolate elicited , whereas peritoneal recruitment of these cells was largely abolished in CCR2KO or inflammation,elicited inflammation, whereas whereas peritoneal peritoneal recruitment recruitment of these of thesecells was cells largely was largely abolished abolished in CCR2KO in CCR2KO or MCP‐1KO mice (Figure 4). MCPor MCP-1KO‐1KO mice mice (Figure (Figure 4). 4).

Figure 4. The MCP‐1/CCR2 chemokine/ pathway axis has an indispensable role FigureFigure 4. 4. TheThe MCP MCP-1‐1/CCR2/CCR2 chemokinechemokine/chemokine/chemokine receptorreceptor pathway pathway axis axis has has an an indispensable indispensable role role in in the migration of CD11b+Ly6Chigh cells to sites of inflammation. WT, CCR2KO and MCP1KO mice inthe the migration migration of of CD11b CD11b+Ly6C+Ly6Chighhigh cellscells toto sitessites ofof inflammation.inflammation. WT, CCR2KO and MCP1KO mice mice were injected with thioglycolate broth and 48 h later peritoneal lavage cells were analyzed by flow werewere injected injected with with thioglycolate thioglycolate broth broth and and 48 48 h h later later peritoneal peritoneal lavage lavage cells cells were were analyzed analyzed by by flow flow cytometry. (A,B) CD11b+Ly6ChighLy6Glow inflammatory monocytes failed to migrate in response to cytometry.cytometry. (A (A,B,B) )CD11b+Ly6C CD11b+Ly6ChighhighLy6GLy6Glowlow inflammatoryinflammatory monocytes monocytes failed failed to migrate to migrate in response in response to thioglycolate‐elicited inflammation in CCR2KO and MCP‐1KO mice; * p < 0.05 and ** p < 0.01 (n = 3/group). thioglycolateto thioglycolate-elicited‐elicited inflammation inflammation in CCR2KO in CCR2KO and MCP and‐1KO MCP-1KO mice; * p < mice; 0.05 and * p **< p0.05 < 0.01 and (n = ** 3/group).p < 0.01 Data are representative of three independent experiments. Data(n = are3/group). representative Data are of representative three independent of three experiments. independent experiments.

Given our earlier observation ofof aa paucitypaucity ofof splenic splenic CD11b CD11b+Ly6C+Ly6Chighhigh inflammatoryinflammatory monocytes Given our earlier observation of a paucity of splenic CD11b+Ly6Chigh inflammatory monocytes in CCR2KO vs. vs. WT WT mice mice housed housed under under baseline baseline conditions conditions (Figure (Figure S3), S3), their their lack lack of ofrecruitment recruitment to in CCR2KO vs. WT mice housed under baseline conditions (Figure S3), their lack of recruitment to theto the peritoneum peritoneum in response in response to thioglycolate to thioglycolate could could simply simply have have reflected reflected their their relative relative deficiency. deficiency. We the peritoneum in response to thioglycolate could simply have reflected their relative deficiency. We Wetherefore therefore next next checked checked the numbers the numbers of CD11b+Ly6C of CD11b+Ly6Chigh inflammatoryhigh inflammatory monocyte monocyte at baseline at baseline in MCP in‐ therefore next checked the numbers of CD11b+Ly6Chigh inflammatory monocyte at baseline in MCP‐ 1KOMCP-1KO mice. mice. In contrast In contrast to their to their low low numbers numbers in inCCR2KO CCR2KO mice, mice, the the proportions proportions ofof CD11bCD11b+Ly6C+Ly6Chighhigh 1KO mice. In contrast to their low numbers in CCR2KO mice, the proportions of CD11b+Ly6Chigh inflammatory monocytes in MCP‐1KO mice were comparable to that of WT mice (Figure 5). inflammatoryinflammatory monocytes monocytes in in MCP MCP-1KO‐1KO mice mice were were comparable comparable to to that that of WT mice (Figure 55).).

Figure 5.5. AnalysisAnalysis of of baseline baseline CD11b CD11b+Ly6C+Ly6Chighhighinflammatory inflammatory monocytes monocytes in MCP-1KO in MCP mice.‐1KO (Amice.) Similar (A) Figure 5. Analysis of baseline CD11b+Ly6Chigh inflammatory monocytes in MCP‐1KO mice. (A) Similarproportions proportions of CD11b of+ Ly6CCD11b+Ly6Chigh inflammatoryhigh inflammatory monocytes monocytes at baseline at baseline within the within peripheral the peripheral blood of Similar proportions of CD11b+Ly6Chigh inflammatory monocytes at baseline within the peripheral MCP-1KO vs. WT mice (n = 6/group). (B) Quantitative analysis (mean SD). Data are representative ± of three independent experiments. Cancers 2020, 12, x 7 of 20

Cancersblood2020, 12of, 3723MCP‐1KO vs. WT mice (n = 6/group). (B) Quantitative analysis (mean ± SD). Data are7 of 19 representative of three independent experiments.

Lastly, toto assess assess whether whether a potenta potent CCR2 CCR2 small-molecule small‐molecule antagonist antagonist (IC50 (IC50 ~5 nM) ~5 [ 28nM)] could [28] inhibitcould inhibitCCR2-dependent CCR2‐dependent recruitment recruitment of inflammatory of inflammatory cells, we injected cells, thioglycolate we injected into thioglycolate the peritoneal into cavities the peritonealof WT mice cavities and treated of WT mice mice with and CCR2 treated antagonist mice with or CCR2 carrier antagonist alone. We or found carrier that alone. the compound We found high thatdecreased the compound recruitment decreased of CCR2 recruitment+CD11b+Ly6C of CCR2+CD11b+Ly6Cinflammatoryhigh monocytes inflammatory in a dose-dependent monocytes in a dosemanner‐dependent (Figure6 ).manner (Figure 6).

Figure 6. CCR2 antagonist inhibited the migration of CCR2+CD11b+Ly6Chigh inflammatory monocytes Figure 6. CCR2 antagonist inhibited the migration of CCR2+CD11b+Ly6Chigh inflammatory to the site of inflammation. WT mice were injected with 4% sterile thioglycolate broth or vehicle monocytes to the site of inflammation. WT mice were injected with 4% sterile thioglycolate broth or control. Mice were treated with vehicle alone or CCR2 antagonist twice daily at a concentration of 0.4, vehicle control. Mice were treated with vehicle alone or CCR2 antagonist twice daily at a 2 or 10 mg/kg for 48 h post-thioglycolate injections. Total number of cells in peritoneal lavage were concentration of 0.4, 2 or 10 mg/kg for 48 h post‐thioglycolate injections. Total number of cells in counted. (A) Representative histogram plot demonstrating the increased percentage of CCR2+ cells in peritoneal lavage were counted. (A) Representative histogram plot demonstrating the increased the peritoneal cavity in response to thioglycolate induced inflammation and abrogation of this effect percentage of CCR2+ cells in the peritoneal cavity in response to thioglycolate induced inflammation upon CCR2 antagonist treatment in a dose-dependent manner. (B) Quantification of total number of and abrogation of this effect upon CCR2 antagonist treatment in a dose‐dependent manner. (B) Ly6Chigh inflammatory monocytes and (C) CD11b+CCR2+ cells in the peritoneal lavage in response to Quantification of total number of Ly6Chigh inflammatory monocytes and (C) CD11b+CCR2+ cells in the treatments noted; * p < 0.05, ** p < 0.01 and *** p < 0.001; n = 3/group and data are representative of the peritoneal lavage in response to the treatments noted; * p < 0.05, ** p < 0.01 and *** p < 0.001; n = three independent experiments. 3/group and data are representative of three independent experiments. Collectively, these data showed that we could use a commercially available CCR2 antagonist to assessCollectively, the effects these of CCR2data showed inhibition that on we MDSC could recruitmentuse a commercially and syngeneic available tumor CCR2 cell antagonist growth into WTassess mice. the effects However, of CCR2 before inhibition undertaking on MDSC those studies,recruitment given and reports syngeneic of CCR2 tumor expression cell growth by Tin cells, WT mice.including However, Foxp3+ beforeTregs [29undertaking,30], we felt those it prudent studies, to consider given reports whether of CCR2 CCR2 targeting expression might by also T acells,ffect includingthe activation Foxp3+ and proliferationTregs [29,30], of we CD4 felt+ and it prudent CD8+ e fftoector consider T cells, whether as well asCCR2 the suppressivetargeting might functions also affectof CD4 the+Foxp3 activation+ Treg and cells. proliferation of CD4+ and CD8+ effector T cells, as well as the suppressive functions of CD4+Foxp3+ Treg cells.

Cancers 2020, 12, 3723 8 of 19 Cancers 2020, 12, x 8 of 20

2.6.2.6. CCR2KO CCR2KO Mice Mice Had Had Normal Normal CD4+ CD4+ andand CD8+ CD8+ TT cells, cells, Including Including Fully Fully Functional Functional CD4+Foxp3+ CD4+Foxp3 +Treg CellsTreg Cells WeWe assessed the effects effects of CCR2 gene deletion deletion on on T T cell cell alloactivation, alloactivation, proliferation proliferation and and cytokine cytokine production in a parent → F1 model by adoptively transfering CCR2RKO or WT T cells from C57BL/6 production in a parent → F1 model by adoptively transfering CCR2RKO or WT T cells from C57BL/6 mice into F1 (C57BL/6 DBA/2) recipients. We found that CCR2RKO mice had comparable T cell mice into F1 (C57BL/6 × DBA/2) recipients. We found that CCR2RKO mice had comparable T cell proliferativeproliferative and and functional functional responses responses to to WT WT mice, mice, as as well well as as showing showing comparable comparable Treg Treg suppressive suppressive functionfunction (Figure (Figure 77).).

FigureFigure 7. GeneticGenetic ablation ablation of of CCR2 CCR2 did did not not affected affected the the ability ability of of T T cells cells to to proliferate and produce 6 inflammatoryinflammatory cytokines or the suppressive capability ofof Foxp3Foxp3++ Tregs. CFSE-labelledCFSE‐labelled T cells (70 × 10106)) × fromfrom the the spleens spleens and and lymph lymph nodes nodes of of WT WT or or CCR2KO CCR2KO C57BL/6 C57BL/6 mice mice were were injected injected i.v. i.v. into into B6D2/F1 B6D2/F1 mice.mice. After 33 days, days, spleens spleens were were harvested harvested from from the adoptively the adoptively transferred transferred B6D2/F1 B6D2/F1 mice and mice analyzed and analyzedfor the extent for the of proliferationextent of proliferation of transferred of transferred T cells and T theircells and ability their to produceability to inflammatory produce inflammatory cytokines. cytokines.(A–D) In vivo(A–Dproliferation) In vivo proliferation WT and CCR2KO WT and CD8 CCR2KO+ T cells CD8+ and T CD4 cells+ Tand cells CD4+ were T comparable. cells were comparable.(E–H) CD8 and (E– CD4H) CD8 T cells and from CD4 WT T and cells CCR2KO from WT mice and were CCR2KO equally capable mice were of secreting equally inflammatory capable of secretingcytokines inflammatory (TNF-α, IFN- cytokinesγ) upon restimulation (TNF‐α, IFN‐γin) upon vitro.( restimulationI,J) WT and in CCR2KO vitro. (I, CD4J) WT+ andFoxp3 CCR2KO+ Tregs CD4+suppressed Foxp3+ conventional Tregs suppressed CD4 T cellconventional proliferation CD4 to T a similarcell proliferation extent. Data to a are similar representative extent. Data of three are representativeindependent experiments. of three independent experiments.

2.7.2.7. CCR2 CCR2 Antagonist Antagonist Inhibited Inhibited the the Growth Growth of of Syngeneic Syngeneic Tumor Tumor Cells In Vivo SeekingSeeking datadata of of potential potential translational translational significance, significance, we returned we returned to our to original our original immuno-oncology immuno‐ oncologyfocus and focus assessed and theassessed effects the of theeffects CCR2 of the antagonist CCR2 antagonist on the growth on the of growth TC1 tumor of TC1 cells tumor in syngeneic cells in syngeneicWT mice. WeWT foundmice. We the CCR2found antagonistthe CCR2 antagonist markedly impairedmarkedly tumor impaired growth tumor (Figure growth8). (Figure 8).

Cancers 2020, 12, 3723 9 of 19 Cancers 2020, 12, x 9 of 20

FigureFigure 8.8. Therapy with a CCR2 antagonistantagonist inhibitedinhibited tumortumor growthgrowth inin syngeneicsyngeneic WTWT mice. mice. MiceMice werewere transplantedtransplanted s.c. s.c. with with 1.2 1.210 ×6 10TC16 TC1 tumor tumor cells. cells. On day On 3 post-tumorday 3 post inoculations,‐tumor inoculations, mice were mice randomly were × distributedrandomly distributed to obtain similar to obtain (~20 similar mm3) average (~20 mm tumor3) average sizes across tumor both sizes groups across before both startinggroups before CCR2 antagoniststarting CCR2 treatment. antagonist Mice weretreatment. treated Mice twice were daily treated with a twice 10 mg /dailykg dose with of CCR2a 10 mg/kg antagonist dose or of vehicle CCR2 controlantagonist until or the vehicle day of control harvest until on day the day 15. ( ofA) harvest Tumor measurementson day 15. (A) Tumor in response measurements to CCR2 antagonist in response or vehicleto CCR2 control antagonist treatments. or vehicle (B) Area control under treatments. the curve analysis. (B) Area ( Cunder) Tumor the volumes curve analysis. on the day (C of) Tumor tumor harvest;volumesn on= 10the/group day of and tumor *** p harvest;< 0.001. n Data = 10/group are representative and *** p < 0.001. of three Data independent are representative experiments. of three independent experiments. 2.8. Effects of the CCR2 Antagonist on Tumor-Infiltrating Myeloid Cells 2.8. EffectsGiven of our the data CCR2 on Antagonist how CCR2 on gene Tumor deletion‐Infiltrating reduced Myeloid MDSC Cells and Treg infiltration and increased intratumoral accumulation of activated CD8+ T cells (Figure3), we undertook a corresponding analysis Given our data on how CCR2 gene deletion reduced MDSC and Treg infiltration and increased of cells present within the tumor microenvironment of WT mice receiving vehicle or CCR2 antagonist intratumoral accumulation of activated CD8+ T cells (Figure 3), we undertook a corresponding therapy. Given its efficacy in blocking CCR2+ cell recruitment in the thioglycolate studies (Figure6), analysis of cells present within the tumor microenvironment of WT mice receiving vehicle or CCR2 we expected the compound would decrease MDSC numbers within tumor-bearing WT mice. This was antagonist therapy. Given its efficacy in blocking CCR2+ cell recruitment in the thioglycolate studies not the case but rather the MDSCs that were still present expressed lower levels of CCR2 and had (Figure 6), we expected the compound would decrease MDSC numbers within tumor‐bearing WT lower levels of CD206 and higher levels of inducible nitric oxide synthase (iNOS) than MDSCs in mice. This was not the case but rather the MDSCs that were still present expressed lower levels of control-treated mice (Figure9). Additionally, TAMs in CCR2 antagonist mice had decreased expression CCR2 and had lower levels of CD206 and higher levels of inducible nitric oxide synthase (iNOS) than of CD206, a marker of an M2 phenotype. Hence, use of the CCR2 antagonist induced cells with more of MDSCs in control‐treated mice (Figure 9). Additionally, TAMs in CCR2 antagonist mice had an M1 phenotype compared to the M2 phenotype associated with MDSCs and TAMs of tumor-bearing decreased expression of CD206, a marker of an M2 phenotype. Hence, use of the CCR2 antagonist WT mice. induced cells with more of an M1 phenotype compared to the M2 phenotype associated with MDSCs and TAMs of tumor‐bearing WT mice.

Cancers 2020, 12, 3723 10 of 19 Cancers 2020, 12, x 10 of 20

Figure 9. MDSCs within CCR2 antagonist antagonist-treated‐treated mice expressed significantlysignificantly lower levels of CCR2 and both MDSCs and TAMs expressed an M1 phenotype as opposed to the M2 phenotype of vehicle control-treatedcontrol‐treated mice. ( A) Intratumoral analysis post post-therapy‐therapy with vehicle control or CCR2 antagonist (10 mgmg/kg/d)/kg/d) showed the presence of MDSCsMDSCs within the tumor microenvironment. ( (BB,,CC)) However, these MDSCs MDSCs expressed expressed significantly significantly lower lower levels levels of CCR2 of CCR2 as opposed as opposed to vehicle to vehicle control control-treated‐treated mice. mice.(D) CCR2 (D) CCR2antagonist antagonist treatment treatment also resulted also resulted in a significant in a significant increase increase in the frequency in the frequency of TANs. of TANs.(E–H) (MDSCsE–H) MDSCs and (I,J and) TAMs (I,J) from TAMs CCR2 from antagonist CCR2 antagonist-treated‐treated mice displayed mice displayed an M1 phenotype an M1 phenotype as indicated as indicatedby their downregulation by their downregulation of CD206, and of CD206, the MDSCs and the displayed MDSCs higher displayed iNOS higher transcript iNOS and transcript protein andlevels protein as compared levels asto comparedMDSCs from to MDSCsvehicle control from vehicle‐treated control-treated mice; * p < 0.05, mice; ** p < * 0.01,p < 0.05,and *** ** p < 0.0010.01, and *** p 0.001 (n 10 group). Data are representative of three independent experiments. (n = 10/group).< Data= are /representative of three independent experiments. 2.9. Effects of the CCR2 Antagonist on Tumor-Infiltrating T Cells 2.9. Effects of the CCR2 Antagonist on Tumor‐Infiltrating T Cells Consistent with the effects of CCR2 gene deletion on anti-tumor responses (Figure3), use of a CCR2 antagonistConsistent significantly with the increased effects of CD8 CCR2 T cell gene infiltration deletion and on anti to a‐ lessertumor extent responses also boosted (Figure CD43), use+ T of cell a infiltration,CCR2 antagonist too, and significantly both intratumoral increased T cell CD8 populations T cell infiltration had the phenotype and to a lesser of effector extent/memory also boosted T cells (CD44CD4+ hiT CD62Lcell infiltration,low) (Figure too,10). Inand conjunction both intratumoral with the influx T cell of activatedpopulations T cells, had increased the phenotype expression of hi low ofeffector/memory several pro-inflammatory T cells (CD44 cytokines CD62L was) (Figure observed, 10). In including conjunction IFN- withγand the TNF- influxα, andof activated increased T expressioncells, increased of the expression cytolytic granules,of several granzyme-B pro‐inflammatory and perforin, cytokines commonly was observed, associated including with the IFN actions‐γand ofTNF eff‐αector, and CD8 increased+ T cells expression (Figure 11). of We the also cytolytic assessed granules, effects of granzyme CCR2 antagonist‐B and perforin, therapy ofcommonly memory responsesassociated by with re-challenging the actions mice of effector with a second CD8+ injectionT cells (Figure of tumors 11). 30 We days also after assessed the previous effects experiment of CCR2 antagonist therapy of memory responses by re‐challenging mice with a second injection of tumors 30 had ceased. We found that mice that had rejected the TC1 tumors in response to therapy with a CCR2 days after the previous experiment had ceased. We found that mice that had rejected the TC1 tumors antagonist in the first round of tumor studies had developed potent memory responses and showed in response to therapy with a CCR2 antagonist in the first round of tumor studies had developed complete inhibition of growth of tumors upon re-challenge (Figure S8). potent memory responses and showed complete inhibition of growth of tumors upon re‐challenge (Figure S8).

Cancers 2020, 12, x 11 of 20

Cancers 2020, 12, 3723 11 of 19

Cancers 2020, 12, x 11 of 20

Figure 10. CCR2 antagonist treatment resulted in greater intratumoral infiltration of activated T cells Figure 10.FigureCCR2 10. CCR2 antagonist antagonist treatment treatment resulted resulted in greater in greater intratumoral intratumoral infiltration of infiltration activated T cells of activated as comparedas comparedto vehicle to vehiclecontrol control‐treated‐treated mice. mice. ( A(A,B,B)) TumorsTumors from from CCR2 CCR2 antagonist antagonist‐treated‐ treatedmice mice T cells as compared to vehicle control-treated mice. (A,B) Tumors from CCR2 antagonist-treated exhibited significantlyexhibited significantly increased increased infiltration infiltration of ofCD8+ CD8+ and (A(A,C,)C CD4+) CD4+ T cells, T cells, and also and contained also contained mice exhibited significantly increased infiltration of CD8+ and (A,C) CD4+ T cells, and also increased transcriptincreased transcript levels of levels (D) of CD8 (D) α CD8asα comparedas compared to to vehicle control control‐treated‐treated tumors. tumors. Additionally, Additionally, containedboth increased (E,G) CD8+ transcript and CD4+ levels (E,H) ofT cells (D) from CD8 αCCR2as comparedantagonist‐treated to vehicle mice displayed control-treated a more tumors. both (E,G) CD8+ and CD4+ (E,H) T cells from CCR2 antagonist‐treated mice displayed a more Additionally,activated both phenotype (E,G) CD8 (increased+ and CD4 CD44+ and(E,H decreased) T cells CD62L) from CCR2 compared antagonist-treated to T cells from the mice control displayed a activated phenotypegroup; n = 5–9/group, (increased * p < CD440.05, ** andp < 0.01, decreased *** p < 0.005, CD62L) and **** comparedp < 0.001. Data to are T representativecells from the of control more activated phenotype (increased CD44 and decreased CD62L) compared to T cells from the control group; n = 5–9/group,three independent * p < experiments. 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001. Data are representative of group; n = 5–9/group, * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001. Data are representative of three independent experiments. three independent experiments.

Figure 11. CCR2 antagonist treatment resulted in greater intratumoral expression of pro-inflammatory compared to that of tumors in vehicle-treated mice; qPCR data, ** p < 0.01, *** p < 0.001,

and n = 5–10 mice/group. Data are representative of three independent experiments. Cancers 2020, 12, x 12 of 20

Figure 11. CCR2 antagonist treatment resulted in greater intratumoral expression of pro‐inflammatory Cancersgenes2020 ,compared12, 3723 to that of tumors in vehicle‐treated mice; qPCR data, ** p < 0.01, *** p < 0.001, and n = 5–1012 of 19 mice/group. Data are representative of three independent experiments.

2.10. Increased Cytotoxicity of T Cells from CCR2 Antagonist-TreatedAntagonist‐Treated MiceMice In furtherfurther studies,studies, the the e ffeffectsects of of therapy therapy with with the the CCR2 CCR2 antagonist antagonist on anti-tumoron anti‐tumor cytotoxicity cytotoxicity was determinedwas determined by isolating by isolating T cells T fromcells drainingfrom draining lymph lymph nodes nodes at day at 14 day post-TC1 14 post injection.‐TC1 injection. We found We thatfound the that CCR2 the antagonist CCR2 antagonist markedly markedly impaired impaired tumor growth tumor (Figure growth 12 (FigureA,B) and 12A,B) significantly and significantly increased theincreased T cell cytotoxicitythe T cell cytotoxicity (Figure 12 C,D).(Figure 12C,D).

Figure 12.12.T T cells cells derived derived from from tumor-draining tumor‐draining lymph lymph nodes ofnodes CCR2 of antagonist-treated CCR2 antagonist mice‐treated displayed mice greaterdisplayed cytotoxic greater potential cytotoxic against potential TC1 against tumor TC1 cells tumor as compared cells as to compared the tumor-draining to the tumor lymph‐draining node cellslymph of vehicle-treatednode cells of mice.vehicle Transplanted‐treated mice. TC1 Transplanted tumors were TC1 treated tumors with CCR2were antagonisttreated with or vehicle CCR2 controlantagonist when or vehicle they reached control ~50 when mm they3 until reached the day~50 mm of harvest3 until the (Day day 14). of harvest On the (Day day 14). of harvest, On the lymphday of nodeharvest, cells lymph were coculturednode cells withwere 1cocultured105 CFSE-labelled with 1 × 10 TC15 CFSE tumor‐labelled cells at TC1 indicated tumor ecellsffector: at × targetindicated ratios. effector: (A) Tumor target growth ratios. curves(A) Tumor in response growth to curves vehicle in control response or CCR2to vehicle antagonist control treatment. or CCR2 (antagonistB) Area under treatment. the curve (B analysis,) Area under (C) representative the curve analysis, histograms (C) and representative (D) cumulative histograms data showing and ( theD) greatercumulative cytotoxic data potentialshowing the of CCR2 greater antagonist-treated cytotoxic potential tumor-draining of CCR2 antagonist lymph‐ nodetreated T cells tumor against‐draining TC1 tumorlymph cellsnode at T indicated cells against target:e TC1ff tumorector ratios cells asat indicated compared target:effector to T cells from ratios tumor as lymph compared nodes to in T micecells receivingfrom tumor vehicle lymph control; nodes *inp mice< 0.05, receiving *** p < 0.001,vehicle and control;n = 3 /*group. p < 0.05, Data *** arep < 0.001, representative and n = 3/group. of three independentData are representative experiments. of three independent experiments.

2.11. Production of MCP MCP-1‐1 by Tumor CellsCells RatherRather ThanThan HostHost CellsCells PromotesPromotes TumorTumor Growth Growth Lastly, givengiven thethe importanceimportance ofof CCR2CCR2++ MDSC recruitment to promoting tumor growth in our studies, wewe assessed assessed whether whether production production of MCP-1of MCP/CCL2‐1/CCL2 was was host host derived derived or tumor or tumor derived derived by testing by syngeneictesting syngeneic tumor growth tumor ingrowth WT vs. in MCP-1KO WT vs. MCP mice.‐1KO We mice. found We comparable found comparable growth in WTgrowth vs. MCP-1KO in WT vs. miceMCP‐ (Figure1KO mice 13A,B), (Figure as 13A,B), well as comparableas well as comparable recruitment recruitment of CD4+ andof CD4+ CD8 +andT cellsCD8+ (Figure T cells 13(FigureC–E), and13C–E), MDSCs and andMDSCs TAN and (Figure TAN 13 (FigureF–H). Hence, 13F–H). we Hence, conclude we that conclude CCR2-depenednt that CCR2‐ edepenedntffects observed effects in ourobserved studies in likely our studies reflect likely the production reflect the of production MCP-1 by of tumor MCP cells‐1 by rather tumor than cells host rather cells. than host cells.

Cancers 2020, 12, 3723 13 of 19 Cancers 2020, 12, x 13 of 20

FigureFigure 13.13.Tumor-derived Tumor‐derived MCP-1 MCP appears‐1 appears indispensable indispensable for the for growth the ofgrowth TC1 tumors of TC1 and tumors recruitment and ofrecruitment MDSCs to of the MDSCs tumor microenvironment.to the tumor microenvironment. TC1 tumors were TC1 transplanted tumors were in WTtransplanted or MCP-1KO in WT mice or andMCP monitored‐1KO mice until and day monitored 13 post-injection, until day when13 post they‐injection, were harvested when they and were analyzed harvested by flow and cytometry. analyzed (byA) flow Tumor cytometry. growth curves (A) Tumor in WT growth and MCP-1KO curves in mice WT and and (B MCP) area‐1KO under mice the and curve (B analysis.) area under Intratumoral the curve Tanalysis. cell analysis Intratumoral from WT T andcell analysis MCP-1KO from mice WT revealed and MCP comparable‐1KO mice (revealedC,D) CD4 comparable and CD8 ( C(C,E,D) T) CD4 cell populations.and CD8 (C,E Myeloid) T cell populations. lineage analysis Myeloid also displayedlineage analysis similar also overall displayed frequencies similar of overall (F,G) MDSCs frequencies and (ofF, H(F),G TANs) MDSCs in tumors and (F grown,H) TANs in WT in vs.tumors MCP-1KO grown micein WT (n vs.= 5 MCP/group‐1KO and mice data (n are = 5/group representative and data of threeare representative independent experiments).of three independent experiments). 3. Discussion 3. Discussion Monocytes express four chemokine receptors relevant to their migration to inflammatory Monocytes express four chemokine receptors relevant to their migration to inflammatory sites, sites, CCR1, CCR2, CCR3 and CCR5 [31]. From an evolutionary perspective, CCR2 is the CCR1, CCR2, CCR3 and CCR5 [31]. From an evolutionary perspective, CCR2 is the oldest of these oldest of these receptors and it is thought that it gave rise to the other three as a result of gene receptors and it is thought that it gave rise to the other three as a result of gene duplication [32]. Such duplication [32]. Such duplication has led to decades of analysis in efforts to unravel the complexity of duplication has led to decades of analysis in efforts to unravel the complexity of chemokine/chemokine receptor expression and signaling and has often resulted in frustrations over chemokine/chemokine receptor expression and signaling and has often resulted in frustrations over what was interpreted as chemokine/chemokine receptor redundancy [33,34]. However, recent studies what was interpreted as chemokine/chemokine receptor redundancy [33,34]. However, recent studies have highlighted CCR2 as the primary driver of myelomonocytic recruitment during inflammation [35]. have highlighted CCR2 as the primary driver of myelomonocytic recruitment during inflammation In addition, the importance of and their receptors in regulating anti-tumor immunity and [35]. In addition, the importance of chemokines and their receptors in regulating anti‐tumor tumor metastases were recently reviewed [36–38], including the dominant role of CCR2 in promoting immunity and tumor metastases were recently reviewed [36–38], including the dominant role of recruitment of monocytes with pro-tumorigenic and pro-metastatic activities. Consistent with this, CCR2 in promoting recruitment of monocytes with pro‐tumorigenic and pro‐metastatic activities. ongoing clinical trials have reported encouraging results of CCR2 inhibitors, especially as combination Consistent with this, ongoing clinical trials have reported encouraging results of CCR2 inhibitors, therapies, in patients with pancreatic cancers, hepatocellular carcinomas and ovarian cancers [37]. especially as combination therapies, in patients with pancreatic cancers, hepatocellular carcinomas The current study arose from a desire to further understand the complex interactions occurring and ovarian cancers [37]. within the tumor microenvironment and how these events might be targeted therapeutically. We focused The current study arose from a desire to further understand the complex interactions occurring within the tumor microenvironment and how these events might be targeted therapeutically. We

Cancers 2020, 12, 3723 14 of 19 on analyzing the role of CCR2-MCP-1 signaling in the recruitment of CCR2+ MDSCs into the tumor microenvironment using syngeneic, immunocompetent mice. Tumor cells recruited CCR2+ MDSCs through their expression of high levels of MCP-1/CCL2, the ligand for chemokine CCR2. Genetic and pharmacological intervention of CCR2-MCP-1 signaling significantly decreased tumor growth in conjunction with increased effector cytotoxic CD8+ T cells. These effects were MDSC dependent as both WT and CCR2KO CD4+ and CD8+ T cells displayed comparable ability to proliferate and produce effector cytokines. Additionally, the ability of Tregs to suppress effector T cell proliferation was comparable between WT and CCR2KO mice. Surprisingly, the frequency of tumor-associated MDSCs was not decreased in CCR2 antagonist-treated mice, but the MDSCs now displayed significantly lower levels of CCR2 expression and had an M1 phenotype. Taken together, ablation of CCR2-MCP1 signaling boosts anti-tumor efficacy by preventing the tumor infiltration of immunosuppressive CCR2+ M2 MDSCs and by promoting cytotoxic T cell responses. CCR2 targeting may be of considerable translational significance given that a second CCR2+ cell population, CCR2+ TAMs, can mediate immune evasion by inducing PD-L2 expression by tumors [39]. Hence, there may be clinical utility by combining currently approved anti-PD-1 mAb with targeting of CCR2/MCP-1 so as to overcome multiple mechanisms of tumor immune evasion [27]. The basis for the CCR2 antagonist leading to an M1 phenotype of MDSCs present at the tumor site in our studies is currently unknown, though there are some clues from the literature. Constitutive MCP-1 production by monocytes, dendritic cells and endothelial cells is counteracted by ongoing uptake and internalization by CCR2+ cells [40]. Hence, in CCR2RKO mice or during treatment with a CCR2 antagonist, including the compound used in the current study, blood levels of MCP-1 are markedly elevated [40]. MCP-1 is typically linked with M2 responses but its role in polarization may vary by context, given its association with M1 macrophages in inflammatory bowel disease, , multiple sclerosis and type 2 diabetes [41]. Further studies are required to ascertain whether raised levels of MCP-1 contributed to the M1 phenotype of CCR2+ MDSCs present within tumors of mice treated with the CCR2 antagonist, or whether simply CCR2 blockade induced this phenotype, as has been shown can occur in vitro using murine macrophage cell lines cultured in the presence of CCR2 antagonists [42]. Likewise, additional studies are required to determine whether insufficient inhibition of CCR2-mediated migration likely explains the persistence of CCR2+ cells within tumors or whether additional chemokine/chemokine receptor pair(s) were responsible for recruitment of these cells to tumors despite use of the CCR2 antagonist. In addition to a change to an M1 phenotype of the CCR2+ MDSCs in mice treated with the CCR2 antagonist, we were surprised to find considerable CCR2 expression by the tumor cells themselves (Figure1). Relatively little is known concerning the functions of CCR2 on tumor cells, though one group reported that the binding of MCP-1 to CCR2 can induce the survival and migration of breast cancer cells [43]. In addition, just prior to submission of our paper, CCR2 expression by murine breast cancer cells was shown to support immune evasion by inhibiting dendritic cell infiltration and maturation, suppressing cytotoxic T cell activity, decreasing MHC class I expression and upregulating PD- expression [44]. If these data can be confirmed beyond breast cancer models in mice, the utility of targeting CCR2 may well extend beyond its effects on immunosuppressive cells in the tumor microenvironment to encompass direct effects on tumor cells themselves.

4. Materials and Methods

4.1. Animals We purchased C57BL/6 (H-2b), CCR2KO mice (B6.129S4-CCR2tm1Ifc/J, Stock No. 004999) and MCP-1KO mice (B6.129S4-CCL2tm1Rol/J, Stock No. 004434) on a C57BL/6 background, and B6D2F1/J (H-2b/d, Stock No. 100006) from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed under pathogen-free conditions and were used at 5–8 weeks of age, unless specified. All studies Cancers 2020, 12, 3723 15 of 19 were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and used protocols approved by the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia (IAC 2017-001047, 12 December 2019, Waltham, MA, USA).

4.2. Antibodies We purchased directly conjugated monoclonal antibodies for flow cytometry from Invitrogen (Waltham, MA, USA): CD4-PE, Clone GK1.5; CD11b-ef450, Clone M1/70; CD44-FITC, Clone IM7; CD45AF700, Clone 30-F11; CD206-APC, Clone MR6F3; FoxP3-ef450, Clone FJK-16S, IFN-γ-PE, Clone XMG1.2; GzmB-APC, Clone GB11; iNOS-PE, Clone CXNFT; live dead fixable dead cell stain , Catalogue No. L34959. BD Pharmingen (San Jose, CA, USA): CD3-AF700, Clone 17A2; CD4-Pacblue and perCPcy5.5, Clone RM4-5; CD8-FITC, perCPcy5.5, and PECY7, Clone 53-6.7; CD62L-PECY7, Clone MEL-14; H2Kd-PE, Clone 17A2; Ly6G-PE and percpcy5.5, Clone 1A8; Ly6C-APCCY7, Clone-AL-21; TNF-α PECY7, Clone MP6-XT22. Biolegend (San Diego, CA, USA): CD3-PECY7 Clone 17A2; B220-percpcy5.5, Clone RA3-6B2. R&D Systems: CCR2-APC, Clone 475301; CCL2-APC, Clone 123616. eBioscience (San Diego, CA, USA): H2Kd/Dd-ef450 Clone 34-1-25; CD4-APC, Clone GK1.5.

4.3. Cell Lines and Tumor Models For mouse tumor studies, the TC1 tumor cell line was derived from C57BL/6 mouse lung epithelial cells, immortalized with HPV-16E6 and E7 and transformed with the c-Ha-ras oncogene [45]. TC1 tumor cells (1 106) were transplanted subcutaneously in a total volume of 100 µL in WT, CCR2 KO or MCP-1 × KO mice using the right flank of each mouse except for the re-challenged experiment when mice were re-challenged with 1 106 TC1 tumor cells on the left flank 30 days post-primary TC1 tumor rejection. × Additional studies were performed using Lewis lung carcinoma cells (ATCC). Tumor volumes were measured by using a standard formula (long diameter short diameter short diameter/2) and × × recorded in mm3 until the indicated day of tumor harvest or experimental endpoint.

4.4. Single-Cell Preparations TC1 tumors were harvested from mice post-euthanization and placed in 1XHBSS media (Gibco, Gaithersburg, MD, USA, Catalogue No. 24020-117). Tumors were chopped into small pieces (2–4 mm) using scissors and forceps and transferred into gentleMACS tube (Miltenyi Biotech, San Diego, CA, USA, Catalogue No. 130-096-334) in a total volume of 5 mL comprised of 2.5 mL 1XHBSS + 2.5 mL tumor dissociation kit (Miltenyi Biotech, Catalogue No. 130-096-730). Dissociated tumor tissues were incubated at 37 ◦C at 200 rpm for 40 min. Post-incubation, a single-cell suspension was prepared by passage through 70 µM strainers (ThermoFisher, San Diego, CA, USA, Catalogue No. 22363548). Cells were pelleted and RBCs were lysed by adding 3 mL of 1X RBS lysis buffer (eBioscience, Catalogue No. 00-4333-57). Cells were finally resuspended in 1 mL PBS and further processed for flow cytometric analysis. To obtain single-cell suspensions from spleen and lymph nodes, tissues were harvested, directly crushed through the strainers, RBCs were lyzed and cells processed for flow cytometry. Peripheral blood cell populations were analyzed by retro-orbital bleeding of mice, lysis of RBCs and processing the remaining cells for flow cytometry.

4.5. Thioglycolate-Induced Peritonitis Model A volume of 1 mL sterile of freshly prepared 4% thioglycolate broth (Sigma Aldrich, St. Loius, MO, USA, Catalogue No. 70157) in double distilled water was injected intraperitoneally in WT, CCR2KO or MCP-1KO mice. At 48 h post-thioglycolate injections, 10 mL of ice-cold sterile PBS was injected into the peritoneal cavity of mice utilizing a 26-G needle. Peritoneal lavage was retrieved using an 18-G needle connected to a 10 mL syringe. The total volume of retrieved lavage was recorded and processed for flow analysis. To evaluate the effective dose response studies, a commercially available small-molecule CCR2 antagonist [28], was injected (10 mg/kg) 30 min before thioglycolate broth injections at the indicated doses. Cancers 2020, 12, 3723 16 of 19

4.6. Suppression, Proliferation, and Cytokine Release Assays Tregs and conventional non-Treg CD4 T cells were isolated from WT or CCR2 KO mice using a Treg isolation kit (Miltenyi Biotech, Catalogue No. 130-091-041). The 5 104 CFSE labelled non-Treg × conventional CD4 T cells were stimulated with CD3 mAb (1 µg/mL) in the presence of 5 104 irradiated × T cell-depleted splenocytes and varying ratios of Tregs, as indicated, for 72 h. The suppression ability of WT and CCR2 KO Tregs cell was compared by analyzing CFSE dilution. In vivo proliferation of WT and CCR2 KO T cells was determined in a GVHD model that was induced by adoptively transferring 70 million CFSE-labelled total cells derived from spleen and lymph nodes of WT or CCR2 KO C57BL/6 mice into B6D2/F1 mice that were heterozygous for B6 and D2 alleles at all genomic loci. In vivo T cell proliferative of WT and CCR2 KO T cells were compared by CFSE dilution using flow cytometry. Pro-inflammatory cytokine (IFN-γ and TNF-α) secretion capability of WT and CCR2KO T cells was compared by re-stimulating the splenocytes (1 106) of B6D2/F1 mice post-adoptive × transfer of WT or CCR2KO cells using a stimulation cocktail (eBioscience, Cell Stimulation Cocktail 500 , Catalogue No. 00-4975-93, plus protein transport inhibitor Monensin, 1000, Cat# 420701, × × Biolegend) for 5 h at 37 ◦C. This was followed by surface staining, fixing and permeabilizing the cells using a fixation/permeabilization solution kit (BD Pharmingen, Catalogue No. 554714) and eventually performing intracellular staining. Additionally, proinflammatory cytokine production by CD8 T cells derived from tumors extracted from WT and CCR2KO mice was also compared using above mentioned method.

4.7. Cell Cytotoxicity Analysis Against TC1 Tumor Cells Cells derived from tumor-draining lymph nodes of mice treated with CCR2 antagonist (10 mg/kg, twice daily) or vehicle control were co-cultured with in vitro-cultured and CFSE-labelled (2.5 µM) 1 105 TC1 tumor cells in U-bottom 96-well plate at the indicated ratios for 24 h at 37 C. Percentage × ◦ tumor cell killing was determined using flow cytometry.

4.8. Real-Time qPCR Tumors were dissected from tumor bearing mice that were treated with either CCR2 antagonist or vehicle control and were flash frozen in liquid nitrogen. RNA was isolated using a RNeasy kit (Qiagen, San Diego, CA, USA) and its integrity and quantity was analyzed by NanoDrop (ND-1000) and Nanochip 2100 Bioanalyzer (Agilent Technologies, San Diego, CA, USA). RNA was reverse transcribed to cDNA (Applied Biosystems, San Diego, CA, USA) and Taqman primer and probe sets were utilized for qPCR execution. Data were normalized to endogenous 18 s rRNA and relative fold expression was determined.

4.9. Statistical Analyses Data in this study were analyzed using GraphPad Prism 7 software and presented as the mean SD. ± The tumor growth rate over a period of time was calculated using area under the curve (AUC) analysis and differences between multiple groups were compared by one-way ANOVA with Turkey’s post-hoc test; p < 0.05 was considered a significant difference.

5. Conclusions This study analyzed the role of CCR2-MCP-1 signaling in the recruitment of CCR2+ MDSCs into the tumor microenvironment in syngeneic, immunocompetent mice. Tumor cells recruited CCR2+ MDSCs through their expression of high levels of MCP-1/CCL2, the ligand for chemokine CCR2. Genetic and pharmacological intervention of CCR2-MCP-1 signaling significantly decreased tumor growth in conjunction with increased effector cytotoxic CD8+ T cells. These effects were MDSC dependent as both WT and CCR2KO CD4+ and CD8+ T cells displayed comparable ability to proliferate Cancers 2020, 12, 3723 17 of 19 and produce effector cytokines. Additionally, the ability of Tregs to suppress effector T cell proliferation was comparable between WT and CCR2KO mice. Interestingly, though the frequency of MDSCs was not compromised in a CCR2 antagonist-treated group, MDSCs displayed significantly lower levels of CCR2 expression and attained an M1 phenotype. Taken together, ablation of CCR2-MCP1 signaling boosts anti-tumor efficacy by preventing the tumor infiltration of immunosuppressive CCR2+ M2 MDSCs and by promoting cytotoxic T cell responses, and may serve as a new therapeutic component in immuno-oncology.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/12/3723/s1, Figure S1: CCR2 gene deletion did not affect thymic T cell development, Figure S2: CCR2 gene deletion did not affect the frequencies of CD4+ or CD8+ T cells, including CD4+Foxp3+ Treg cells, in the periphery, Figure S3: CCR2 gene deletion significantly reduced the frequency of splenic Ly6Chigh inflammatory monocytes, Figure S4: The frequency of CD11b+Ly6Chigh inflammatory monocytes was comparable in the bone marrows of WT and CCR2KO mice, suggesting that the compromised frequency of these cells in the periphery was not attributable to a developmental defect in their production in CCR2KO mice, Figure S5: Gating strategy used to evaluate T cells and myeloid lineage populations in TC1 tumors, Figure S6: Blood CD11b+ Ly6Chigh inflammatory monocytes expressed higher levels of CCR2 than anti-inflammatory monocytes or neutrophils, Figure S7: MDSCs associated with subcutaneously transplanted Lewis lung carcinoma cells expressed the highest levels of CCR2 whereas tumor cells were the main source of CCL2 (MCP-1), Figure S8: Mice that rejected the TC1 tumors in response to therapy with a CCR2 antagonist also developed memory responses. Author Contributions: Conceptualization, P.M., C.A.L. and W.W.H.; methodology, P.M., C.A.L. and W.W.H.; formal analysis, P.M., C.A.L. and W.W.H.; investigation, P.M., C.A.L., L.W., T.A. and W.W.H.; resources, C.A.L., J.C.C., M.R.S., Y.C. and B.J.T.; data curation, P.M., C.A.L. and W.W.H.; writing—original draft preparation, P.M. and W.W.H.; writing—review and editing, P.M. and W.W.H.; supervision, C.A.L. and W.W.H.; project administration, C.A.L. and W.W.H.; funding acquisition, C.A.L. and W.W.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded in part by a sponsored research agreement with GlaxoSmithKline. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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