CC chemokine receptor 2 mediated regulation of macrophages is involved in pancreatic cancer progression in the tumor microenvironment

doi:10.21203/rs.3.rs-5124479/v1

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CC chemokine receptor 2 mediated regulation of macrophages is involved in pancreatic cancer progression in the tumor microenvironment

https://doi.org/10.21203/rs.3.rs-5124479/v1

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Objective: The interaction between pancreatic ductal adenocarcinoma (PDAC) cells and non-tumor cells is important in PDAC. In this study, we investigated the effects of CC chemokines in PDAC.

Design: According to microarray data of cancer-associated fibroblasts (CAFs) stimulated by PDAC cells, the expression of Cc chemokines was analyzed by quantitative RT-PCR. Macrophages were induced from the bone marrow cells of Cc chemokine receptor 2 (Ccr2)-wild-type (WT) and Ccr2-knockout (KO) mice, and their interaction with PDAC cells was examined. Differences in RNA and protein expression between Ccr2 WT and KO macrophages were examined also. Systemic KO of Ccr2 in a geneticallyengineered murine PDAC model was established to analyze the survival impact and histopathological phenotype using immunohistochemistry. The RNA sequences of PDAC cells stimulated with Ccr2-WT or KO macrophages were also examined.

Results:Ccl2 and Ccl7 expression was upregulated in CAFs. Ccr2 is expressed in macrophages in PDAC. Ccr2-WT macrophages promote the invasion of PDAC cells in vitro. Ccr2-KO decreases Cxc chemokine levels and increases interferon-a production in macrophages. Ccr2 KO PDAC mice showed significantly prolonged survival.

Conclusions:CCLs-CCR2 signaling affects the profile and function of macrophages, and Ccr2-KO in macrophages may alter the microenvironment in a tumor-suppressive manner in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a recurrent cancer with a very poor prognosis and a 5-year survival rate of 9.8% ⁽¹⁾. Its incidence and fatalities are increasing, and it is currently the fourth leading cause of cancer-related deaths in Japan ⁽¹⁾. Moreover, it is projected to become the second leading cause of cancer-related deaths in the United States by 2030 ⁽²⁾. Understanding and overcoming the pathogenesis of PDAC presents a critical challenge in this field ⁽³⁾.

KRAS mutation is found in more than 95% of PDAC cases. Other frequently mutated genes include the tumor suppressor genes CDKN2A/p16, TP53, and SMAD4/DPC4, which together with KRAS are referred to as the Big 4 genes ^{(2), (4)−(6)}. We have reported a mouse model with pancreatic-specific Kras mutation and knockout (KO) of TGF-β type 2 receptor (Tgfbr2), (Ptf1a^cre/+; LSL-Kras^G12D/+; Tgfbr2^flox/flox, PKF mice) ⁽⁷⁾. This mouse model develops stromal-rich adenocarcinoma that is very similar to human PDAC histology and dies after a median survival of 59 days due to tumor progression ⁽⁷⁾.

In the stroma-rich tumor microenvironment of PDAC, it has been reported that PDAC cells and various stromal cells infiltrating the tumor microenvironment (TME) interact with each other and contribute to the invasion and proliferation of PDAC cells ⁽⁸⁾. Among the various immune cells infiltrating the TME, tumor-associated macrophages (TAM) have attracted attention ⁽⁹⁾. There are two major types of macrophages (M1 and M2). M1-type macrophages show inflammatory and anti-tumor effects, but on the other hand, M2-type macrophages are reported to be immunosuppressive and promote tumor cell growth. TAMs have been reported to have M2-like properties ^(10)−(14).

Chemokine signals were originally considered important for the chemotaxis of neutrophils and macrophages ⁽¹⁵⁾. Using the PKF mouse model, we have reported that mouse PDAC (mPDAC) cells and mouse cancer-associated fibroblasts (mCAFs) promote each other's invasion and migration via the Cxcls-Cxcr2 axis, and blockade of this axis significantly prolonged the survival of the mice, showing a shift in the infiltrating immune cell profile and decreased angiogenesis ^{(16), (17)}. The tumor-promoting effect of the CXCLs-CXCR2 axis has also been reported previously ⁽¹⁸⁾.

On the other hand, CC chemokine is also associated with several cancers including PDAC ^(19)−(23). CC chemokine receptor 2 (CCR2) is expressed on TAM, tumor-associated neutrophils (TAN), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) ^(24)−(26). Cells infiltrating the TME via CCR2 have been reported to suppress anti-tumor immunity in several cancer types ^(19)−(23). Although it has been reported that TAMs infiltrated into the TME via CCR2 suppress anti-tumor immunity in PDAC, it is not yet clear how CCR2 affects the properties of the macrophages themselves ^(21)−(23).

In this study, we investigated the role of CCR2 in PDAC using a PKF model. We found that Ccr2 is expressed in immune cells, mainly macrophages, but not in PDAC cells. Macrophages promote PDAC cell invasion in vitro in a Ccr2-dependent manner. Comparison of Ccr2-wild type (WT) and Ccr2-KO macrophages, as well as PDAC cells stimulated by Ccr2-WT and -KO macrophages, revealed the signaling involved in promoting and suppressing PDAC invasion. Moreover, systemic Ccr2-KO PKF mice showed significantly prolonged survival with changes in macrophage composition and decreased microvessel invasion in the TME. In addition, Ccr2-KO PDAC cells exhibited increased apoptosis. In vitro and in vivo results suggest that interferon-α production by Ccr2-KO macrophages is involved in enhanced apoptosis. Therefore, the inhibition of CCR2 may be a potent therapeutic strategy for PDAC.

Reagents

The CXCR2 inhibitor SB255002 and the IFN-α agonist RO8191 were purchased from Calbiochem and TOCRIS, respectively.

Mice

PKF mice (Ptf1a^cre/+; LSL-Kras^G12D/+; Tgfbr2^flox/flox) have been previously described ⁽⁷⁾. Ccr2^−/− and C57BL/6 mice were purchased from Jackson Laboratories and CLEA Japan, Inc., respectively. All mice were housed in specific pathogen-free housing with abundant food and water under the guidelines approved by the Ethics Committee for the Care and Use of Laboratory Animals of the University of Tokyo.

Cell line

The mouse PDAC cell line K375 and mouse CAF cell line 311f were previously described ⁽⁷⁾ and were cultured in RPMI (FUJIFILM Wako Pure Chemical Corporation) with the indicated concentration of FBS (SIGMA ALDRICH) as described below. The L929 cells were purchased from Dainippon Pharmaceutical and cultured in 10% FBS-DMEM (FUJIFILM Wako Chemical Corporation).

Study approval

Animal experiments were performed in accordance with the guidelines approved by the Ethics Committee for the Care and Use of Laboratory Animals of the University of Tokyo (approval number: I-P17-136).

cDNA microarray

In the previous study, we evaluated the comprehensive gene expression in mCAFs stimulated with supernatant of mPDAC cell line using cDNA microarray (GeneChip Mouse Genome 430 2.0 array, Affymetrix). The results are reposited on the web (https://figshare.com/articles/dataset/EA1091_Ratio_xls/7092353). We reanalyzed the reposited data in this study.

Additional information is provided in Supplementary Data

Stimulation of pancreatic cancer cells promotes the production of Cc chemokines from CAF

Previously, we performed microarray analysis of mCAFs with or without stimulation by mPDAC cell-conditioned media and found an increase in Cxc chemokine expression upon stimulation of mPDAC ⁽¹⁷⁾. In this study, we reanalyzed the raw data of the repositioned microarray and observed upregulation of several Cc chemokines by stimulation of mPDAC (Fig. 1A) with a much higher signal intensity than Cxc chemokines, which suggested the abundance of CC chemokines in the PDAC TME. qRT-PCR confirmed a significant increase in Ccl2 and Ccl7 expression in mCAFs stimulated with mPDAC (Fig. 1B).

CCR2 is expressed in macrophages in the pancreatic cancer microenvironment

Since we observed increased expression of Ccl2 and Ccl7 when mCAFs were stimulated by mPDAC cells, we hypothesized that CCR2, a major receptor of CC chemokines, might be important in the interaction between mCAF and mPDAC. Therefore, we performed immunostaining for CCR2 to identify the cells that expressed CCR2 in the tumor tissues of PKF mice. There was no CCR2 expression in the tumor epithelium (Fig. 1C). However, CCR2 was strongly expressed in the intralobular and interlobular stroma tissue (Fig. 1C). Thus, we hypothesized that CCR2 may play an important role on the stromal side of the PDAC TME, but not in tumor cells.

We focused on macrophages according to many previous reports of CCR2 expression in macrophages in the TME of several cancers, including PDAC ^(19)−(23). Fluorescent double staining of F4/80 and CCR2 in the PDAC TME of PKF mice revealed double positive cells in the tumor stroma, confirming CCR2 expression in macrophages (Fig. 1D).

Macrophages promote invasion of pancreatic cancer cells, which is inhibited by Ccr2 KO in macrophages

We induced bone marrow-derived macrophages (BMDM) from C57BL/6 mice with Ccr2 WT or systemic Ccr2 KO (Ccr2^−/−) to investigate the role of Ccr2 in PDAC (Supplementary Fig. 1A). The induced cells were confirmed to be F4/80 positive by cell immunostaining (Supplementary Fig. 1B). We also confirmed KO of Ccr2 in macrophages from Ccr2 KO mice using western blotting and qRT-PCR (Supplementary Fig. 1C, D).

First, cell proliferation assays were performed on the mPDAC cells and mCAFs. mPDAC cells and mCAFs were cultured in the supernatant of Ccr2 WT or Ccr2 KO BMDM. The supernatants of Ccr2 WT BMDM and Ccr2 KO BMDM showed significantly enhanced proliferation of mPDAC cells after 96 h and of mCAFs after 72 h, respectively, compared to the control (without the supernatant of BMDM); however, no significant difference was observed in the proliferation of Ccr2 WT and KO BMDM (Supplementary Fig. 2). Therefore, we concluded that Ccr2 in BMDM does not directly contribute to the proliferation of mPDAC cells and mCAFs.

Next, we performed an invasion assay to examine whether macrophages affect the invasive ability of mPDAC cells. The supernatant of Ccr2 WT BMDM significantly promoted the invasion of mPDAC cells compared to the control (without the supernatant of BMDM), whereas the supernatant of Ccr2 KO BMDM significantly inhibited invasion compared to the Ccr2 WT BMDM (Fig. 1E). These results indicate that macrophages promote the invasion of mPDAC cells in a Ccr2-dependent manner.

Cxc chemokines are involved in the pancreatic cancer invasion induced by macrophages

To address the underlying mechanism of Ccr2-dependent mPDAC cell invasion induced by macrophages, we performed a cytokine array analysis and comprehensively examined the changes in cytokines produced by macrophages in Ccr2 KO mice. We observed various cytokine changes in the supernatant of Ccr2 KO BMDM compared to that of Ccr2 WT BMDM (Fig. 2A). Among them, we focused on those that were downregulated in Ccr2 KO BMDM compared to that in Ccr2 WT BMDM. The expression of cytokines that were downregulated in the cytokine array was also examined using qRT-PCR. The result, Il-1α, Cxcl2, Cxcl4, Cxcl5, Osteopontin, Osteoactivin, Tgfβ2 and VE-cadherin were downregulated in both analyses (Fig. 2B, 2C).

We focused on Cxc chemokines because we had reported the significance of Cxc chemokines in PDAC progression in previous studies ^{(16), (17)}. Among Cxcl2, Cxcl4, and Cxcl5, which were decreased in Ccr2 KO BMDM in this study, Cxcl2 and Cxcl5 were the primary ligands for CXCR2, suggesting that the CXCLs-CXCR2 axis could play a crucial role in promoting mPDAC cell invasion. We conducted an invasion assay using mPDAC cells exposed to Ccr2 WT BMDM supernatant with the addition of the CXCR2 inhibitor SB225002. The presence of CXCR2 inhibitor significantly inhibited the invasion of mPDAC cells (Fig. 2D), which indicated that Cxcls produced by macrophages contributed to the promotion of mPDAC cell invasion. Therefore, targeting Ccr2 may offer a potential strategy for suppressing PDAC by inhibiting the CXCLs-CXCR2 axis.

Ccr2 KO prolongs survival of Kras^G12D+Tgfbr2^KO pancreatic cancer mice

Our in vitro experiments showed that CCR2 played an important role in the invasion of mPDAC cells. Next, we attempted to elucidate the role of CCR2 in the TME of PDAC in vivo using systemic KO of Ccr2 in PDAC mice. We crossed PKF mice with Ccr2^−/− mice and established PKF mice with systemic KO of Ccr2 (PKFC mice). Kaplan-Meier analysis of PKFC and PKF mice showed that PKF mice had a median survival time (MST) of 58 days, whereas PKFC mice showed a significantly prolonged survival time of 80.5 days (p = 0.0011) (Fig. 3A). This indicates that the blockade of CCR2 has a suppressive effect on PDAC progression, with a significant prognostic impact.

Ccr2 inhibition alters macrophage composition in the tumor microenvironment of pancreatic cancer and inhibits vascular invasion, while increases apoptosis of cancer cells

PKFC mice showed significantly prolonged survival compared to PKF mice; however, invasive PDAC formation was also observed. Therefore, we intensively examined pancreatic tumors in both mice.

No significant difference was observed in pancreatic tumor weight (Fig. 3B). H&E images showed tumor formation with abundant stroma in PKFC mice, similar to PKF mice (Fig. 3C). No significant difference was observed in the tumor-to-stromal area ratio between PKF and PKFC mice (Fig. 3D). In addition, Ki-67 staining, an index of proliferative capacity, showed no significant difference between the two groups of mice in the tumor epithelia (Fig. 3E).

Next, we performed various immunostaining to compare stromal cells infiltrating the pancreatic tumor tissues. We observed a significant increase in inducible nitric oxide synthase (iNOS)-positive cells, a marker of M1-like inflammatory macrophages, and a significant decrease in Arginase-1-positive cells, a marker of M2-like immunosuppressive macrophages, in the intralobular stroma, although no significant difference was observed in F4/80, the total number of macrophages (Fig. 4A). This suggested that Ccr2 KO altered the macrophage composition in the TME of PDAC. Significant differences were also observed in FoxP3-positive regulatory T cells (Tregs) infiltrating the interlobular stroma (Supplementary Fig. 4); however, the number of infiltrating cells was much larger in the macrophages, suggesting a significant impact of macrophages on the tumor. Therefore, we focused on the macrophages in the interlobular stroma. There were no significant differences in the number of infiltrating neutrophils, CD4- and CD8-positive T cells, B cells, CAFs, and MDSCs (Supplementary Figs. 3 and 4). We performed RNA sequencing to comprehensively compare the differences between the gene expression profiles of Ccr2 WT and Ccr2 KO BMDM. In the gene set enrichment analysis (GSEA), the Prerank method ⁽²⁷⁾ revealed that Ccr2 WT BMDMs showed enriched gene sets reported to be upregulated in M2-like, immunosuppressive macrophages, such as Angiogenesis, TGF-β signaling, and the IL-6 pathway (Supplementary Fig. 5) ^(28)−(30). In contrast, E2F targets reported to be upregulated in M1-like inflammatory macrophages were enriched in Ccr2 KO BMDMs ⁽³¹⁾ (Supplementary Fig. 5). This comparison suggested that Ccr2 WT BMDM contained more M2-like macrophages, whereas Ccr2 KO BMDM contained more M1-like macrophages.

The microinvasion of blood vessels in pancreatic tumor tissues was also examined. Venous and lymphatic invasion were compared in the tumor tissues from PKFC and PKF mice by double staining with K-19 and CD31 or LYVE-1, respectively. Venous microinvasion was significantly suppressed in PKFC mice, whereas lymphatic invasion was suppressed in PKFC mice (Fig. 4B). These results suggest that Ccr2 contributes to vascular invasion in mPDAC, which is consistent with the in vitro invasion assays described above.

Cleaved Caspase-3, an index of cell apoptosis, was significantly increased in PKFC mice in the tumor epithelia (Fig. 4C). The increased levels of cleaved Caspase-3 in PKFC mice suggested that iNOS-positive inflammatory macrophages might have induced apoptosis in the pancreatic tumor epithelia ⁽³²⁾.

Interferon-α signaling is enhanced in pancreatic cancer cells stimulated with Ccr2 KO BMDM

As apoptosis was significantly increased in the PDAC tissue of PKFC mice, we used RNA sequencing to investigate the differences in transcriptional profiles between mPDAC cells stimulated by Ccr2 WT and KO macrophage supernatants to determine the factors involved in apoptosis due to changes in the composition of M1-like and M2-like macrophages. The gene ontology (GO) and GSEA analyses showed that the interferon-α (IFN-α) pathway was predominantly upregulated in mPDAC cells stimulated with Ccr2 KO BMDM supernatant (Fig. 5A, 5B). Interferon regulatory factor 9 (IRF9) and signal transducer and activator of transcription 1 (STAT1) were found to be significantly upregulated among the expression variation genes associated with IFN-α. Western blotting revealed that phospho-STAT1 (pSTAT1) and STAT1 expression levels were elevated in mPDAC cells stimulated with Ccr2 KO BMDM (Fig. 5C). Since it has been reported that type 1 IFN in human macrophages is regulated via CCR2 ⁽³³⁾, we investigated the expression of type 1 IFN in BMDM using qRT-PCR. Ccr2 KO significantly increased the levels of type 1 IFNs (IFN-α and IFN-β) in BMDM (Fig. 5D).

Ccr2 KO significantly increases Stat1 expression in mouse pancreatic cancer cells and interferon-α production in macrophages in the tumor tissues of PKFC mice, resulting in cell death of mouse pancreatic cancer cells

As type 1 IFN-related factors were significantly elevated in mPDAC cells stimulated by Ccr2 KO BMDM in vitro, we evaluated this phenomenon in the tumor tissues of PDAC mice. In PKFC mice, STAT1 expression was significantly upregulated in the pancreatic tumor epithelia (Fig. 6A). In addition, fluorescent double staining of F4/80 and IFN-α showed that macrophages in the tumors of PKFC mice produced IFN-α significantly more frequently than in PKF mice (Fig. 6B). An in vitro apoptosis assay using IFN-α agonists and macrophage supernatants revealed that IFN-α and Ccr2 KO BMDM supernatants both significantly induced apoptosis in mPDAC cells (Fig. 6C, D).

These results suggest that blockade of CCR2 induced IFN-α production from macrophages and STAT1 signal activation in tumor cells, contributing to the increased apoptosis of PDAC cells.

In this study, we showed that CCR2 regulates the production of CXC chemokines and IFN-α in macrophages, thereby inducing PDAC invasion and apoptosis, and that inhibition of CCR2 can improve the prognosis of PDAC.

CCR2, a receptor of the major chemokine family, CC chemokines, has been reported to play important roles in many malignancies, including PDAC ^(19)−(22). The efficacy of CCR2 inhibitors against PDAC has also been reported in humans and mice ^{(23), (34), (35)}; however, clinical trials on human PDAC are still in phase 1, and the mouse models previously used were transplanted models. In this study, we investigated the phenotype of whole-body Ccr2 KO mice using a genetically engineered PDAC model, PKF mice, which can most closely recapitulate clinical PDAC histology. We also performed in vitro assays to understand the underlying mechanisms of action of Ccr2 in macrophages.

The possible TME of PDAC, based on the present results, is shown in Fig. 7. In the tumor-stromal interaction, PDAC cells stimulate CAFs to produce CCL2 and CCL7, which stimulate macrophages via CCR2. Macrophages produce CXCL2 and CXCL5, which promote PDAC cell invasion (Fig. 7A). CCR2 inhibition also increases IFN-α production in macrophages, which induces apoptosis of PDAC cells. In the TME, CCR2 inhibition changes the phenotype of macrophages from M2-dominant to M1-dominant and upregulates IFN-α expression. As a result, inhibition of vascular invasion and an increase in apoptosis of PDAC cells may suppress PDAC progression and improve prognosis (Fig. 7B). Although there been reported that TAMs migrate into the TME via CCR2 and contribute to tumor promotion ^(20)−(22), there are no reports on the CCR2-dependent production of CXCLs in macrophages. With regard to CXCL production by macrophages, it has been reported that CXCL9 and CXCL10 produced by macrophages play a cancer-promoting role in the TME of breast cancer ⁽³⁶⁾, but there are no reports on CXCL2 or CXCL5.

It has been reported that IFN-α production in macrophages is downregulated via CCR2 ⁽³⁷⁾, which is consistent with our results that IFN-α production is increased in Ccr2-KO macrophages. Fourteen subtypes of IFN-have been reported α in mice, and IFN-α4, IFN-α11, and IFN-α12 are reported to be highly bioactive ⁽³⁸⁾. In this study, we observed elevated levels of IFN-α4 and IFN-α12 in macrophages following CCR2 inhibition.

The Anti-tumor effects of type I IFNs, including IFN-α, have been confirmed in several cancers ^(38)−(42), but they are not currently the standard therapy due to their adverse effects. However, in recent years, owing to advances in cancer immunotherapy, a combination of immunotherapy and type I IFNs has been explored ^(43)−(46).

Almost all cells in the body, including macrophages, are considered to produce type I IFN ⁽⁴²⁾; therefore, it is unclear which cells in the TME primarily produce type I IFN. The present study indicates that Ccr2-mediated regulation of IFN-α in macrophages may play an important role in PDAC. CCR2 inhibition increases the production of IFN-α in macrophages, and the activated IFN-α signaling pathway in PDAC cells may induce apoptosis in PDAC cells.

IFN-α binds to its receptor and activates various downstream signals. Among these, there are many reports on the JAK-STAT pathway ⁽⁴⁰⁾. IFN-α inhibits proliferation and angiogenesis in cancer cells ⁽⁴²⁾. It has also been reported to induce apoptosis and inhibit cancer cell invasion by suppressing EMT induction ^{(47), (48)}. In the present study, an increase in IFN-α production by macrophages and STAT1 expression in PDAC cells were observed in PKFC mice. Taken together, IFN-α may be involved in the underlying mechanisms of PDAC control by inducing apoptosis.

The combination of CCR2 inhibitors and FOLFIRINOX has been demonstrated in humans and mice, and it has been reported that in human PDAC, the combination with FOLFIRINOX suppressed the induction of monocytes from the bone marrow and reduced TAM infiltration into the TME ⁽³⁴⁾. In an orthotopic transplantation mouse model, CCR2 inhibition also suppressed TAM infiltration; however, an increase in TAN infiltration has also been reported ⁽³⁵⁾. In the present study, there was no change in the infiltration of neutrophils and macrophages into the TME, but CCR2 inhibition caused a shift from M2-like dominant to M1-like dominant change in the macrophage phenotype, which was considered important for increased apoptosis, suppression of vascular invasion, and prolonged survival observed in this study. There might be differences, especially in the immune microenvironment, between the transplantation and genetically engineered models. In contrast, in comparison to our previous study in which Cxcr2 heterozygous KO was performed in PKF mice, both observed increased apoptosis of cancer cells, suppression of cancer microinvasion, and prolonged survival of mice, as well as increased M1/M2 ratio of macrophages, while Cxcr2 heterozygous KO mice showed decreased neutrophil infiltration and increased macrophage infiltration into the TME ⁽¹⁶⁾. This may also be a difference between Ccr2 KO and Cxcr2 KO mice. It is also likely that CCR2 KO and CXCR2 KO macrophages differ in their altered gene expression profiles. The possibility that simultaneous knockout of CCR2 and CXCR2 may synergistically inhibit PDAC progression remains an issue for future investigation.

This study has some limitations. First, this study examined mouse PDAC, but not human PDAC, although the TME was intact compared to the transplantation models. In mouse pancreatic tumor tissues, we found Ccr2-expressing cells other than macrophages, and we were unable to examine the role of these cells in the PDAC TME. In this study, we observed significant changes in lymphoid cells reported to have CCR2, such as Tregs, although the number of infiltrated cells in the TME was very small. We observed that CXCLs produced by macrophages promoted PDAC cell invasion; however, other cytokines detected in the cytokine array might also contribute to PDAC cell invasion.

In conclusion, in the PDAC microenvironment, macrophages promote PDAC invasion in a CCR2-dependent manner partly by producing CXC chemokines. CCR2 inhibition changes the macrophage profile from M2-dominant to M1-dominant and increases IFN-α production, thereby inhibiting invasion and increasing apoptosis in PDAC, resulting in prolonged survival. Thus, CCR2 inhibition may be a potent treatment option for PDAC that contributes to prognosis.

aSMA: a-smooth muscle actin, BMDM: Bone marrow-derived macrophage, CAF: Cancer-associated fibroblasts, CCL: CC chemokine ligand, CCR2: CC chemokine receptor 2, CXCL: C-X-C motif ligand, CXCR: C-X-C motif chemokine receptor, DMSO: dimethyl sulphoxide, FBS: Fetal bone serum, FDR: False discovery rate, GO: Gene Ontology, GSEA: gene set enrichment analysis, HRP: Horse radish peroxidase, IFN: Interferon, IFN-a: Interferon-a, IRF: Interferon regulatory factor, iNOS: inducible nitric oxide synthase, KO: Knock out, M-CSF: Macrophage colony-stimulating factor, MDSC: Myeloid-derived suppressor cell, MPO: Myeloperoxidase, NRS: Normalized enrichment score, PanIN: Pancreatic intraepithelial neoplasia, PDAC: Pancreatic ductal adenocarcinoma, PDGFR-a: Platelet-derived growth factor receptor-a, PFA: Paraformaldehyde, PKF; Ptf1a^cre/+;Lox-Stop-Lox(LSL)-Kras^G12D/+;Tgfbr2^flox/flox, PKFC; Ptf1a^cre/+;LSL-Kras^G12D/+;Tgfbr2^flox/flox;Ccr2^-/-, SD: Standard deviation, STAT1: Signal transducer and activator of transcription 1, TAM: Tumor-associated macrophage, TAN: tumor-associated neutrophil, TGF-b; transforming growth factor-b, TME: Tumor microenvironment, Treg: Regulatory T cells, WT: Wild type

Conflict of interest

The authors have declared that no conflict of interest exists.

Authors’ contributions

GK, HI, KM, and RT designed the experiments; GK and HI acquired and analyzed the data; GN and TK supported the experiments; GK and HI wrote the manuscript; MS, HI, KM, and RT supported the pathological analysis; and GK, HI, RT, KM, and MF discussed the data.

Acknowledgments

We thank Christopher VE Wright (Vanderbilt University) for Ptf1a^cre/+ mice, Tyler Jacks (Massachusetts Institute of Technology) for LSL-Kras^G12D/+ mice, and Harold L Moses (Vanderbilt-Ingram Cancer Center) for Tgfbr2^flox/flox mice. We also thank Sayaka Ito (The University of Tokyo) for technical assistance, and laboratory members for helpful discussions. This work was supported by the Japanese Society for the Promotion of Science Kakenhi Grant 20K08278, and Research Grant of the Princess Takamatsu Cancer Research Fund to HI.

Data Transparency Statement

The data, analytical methods, and research materials will be made available to other researchers. We are considering making the data available at the same location as that of the cDNA microarray.

Foundation for Promotion of Cancer Research: CANCER STATISTICS IN JAPAN 2021（https://ganjoho.jp/public/qa_links/report/statistics/2021_jp.html）
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There is NO conflict of interest to disclose.

qRTPCRdata.xlsx
qRT-PCR data
CCR2KnockoutcheckBactin.tif
CCR2 Knockout check Bactin
CCR2IFNpathway48hrBactin.tif
CCR2 IFN pathway 48hr Bactin
CCR2IFNpathway48hrpStat1.tif
CCR2 IFN pathway 48hr pStat1
CCR2KnockoutcheckCCR2.tif
CCR2 Knockout check of Macrophage
CCR2IFNpathway48hrStat1.tif
CCR2 IFN pathway 48hr Stat1
CCR2IFNpathway15minStat1.tif
CCR2 IFN pathway 15min Stat1
SupplementaryData.docx

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CC chemokine receptor 2 mediated regulation of macrophages is involved in pancreatic cancer progression in the tumor microenvironment

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Abstract

Figures

Introduction

Material and Methods

Reagents

Mice

Cell line

Study approval

cDNA microarray

Additional information is provided in Supplementary Data

Results

Stimulation of pancreatic cancer cells promotes the production of Cc chemokines from CAF

CCR2 is expressed in macrophages in the pancreatic cancer microenvironment

Macrophages promote invasion of pancreatic cancer cells, which is inhibited by Ccr2 KO in macrophages

Cxc chemokines are involved in the pancreatic cancer invasion induced by macrophages

Interferon-α signaling is enhanced in pancreatic cancer cells stimulated with Ccr2 KO BMDM

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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