Supplementary MaterialsSupplementary Information 41598_2018_30437_MOESM1_ESM. mediator in the gemcitabine-induced infiltration of macrophages
Supplementary MaterialsSupplementary Information 41598_2018_30437_MOESM1_ESM. mediator in the gemcitabine-induced infiltration of macrophages within the pancreatic tumor microenvironment and suggest the requirement of additional mechanism(s) for macrophage polarization. Introduction Pancreatic cancer (PC) is the third leading cause of cancer-related death in the United States, and remains one of those cancers that have seen no significant improvements in their clinical outcome over past several decades1,2. More upsettingly, it is expected to become the second leading cause of cancer-related death by the year 2030 or even earlier considering the continued increases in its incidence and mortality3. According to the American Cancer Society, approximately 55, 440 patients are expected to be diagnosed with PC this year and about 44, 330 people will succumb to this disease4. Gemcitabine, a nucleoside analogue, is used either as a single agent or in combination with other chemotherapeutic agents to treat PC, but these therapies provide marginal benefits only to the PC patients5. The poor outcomes of current therapies are largely associated with inherent or acquired chemoresistance of PC cells6C8. Furthermore, unique properties of pancreatic tumor microenvironment (TME) are also believed to play an important role in the unusual chemoresistance of PC9C11. Regardless of their Rabbit Polyclonal to CAMK5 curative efficacy, most chemotherapies are associated with wide range of adverse effects on nontarget tissues. Chemotherapeutic treatment is associated with a significant negative impact on the immune system including increased recruitment of the tumor supportive immune cells in the TME. More importantly, in the context of PC, tumor-infiltrated or tumor-associated macrophages (TAMs) have been shown to promote cancer stemness and chemoresistance12,13. Therefore, the present study was undertaken to examine the effect of gemcitabine treatment on pancreatic tumor immune-microenvironment, especially on macrophages. Our data demonstrate that orthotopic human pancreatic tumor xenografts from gemcitabine-treated mice have greater infiltration of macrophages of the M2 phenotype. Further, our data show Nocodazole kinase activity assay that the conditioned media from gemcitabine-treated human Nocodazole kinase activity assay PC cells (MiaPaCa-2 and Colo-357) promotes migration, invasion, growth, and M2 polarization of RAW264.7 macrophages. Mechanistically, we have identified IL-8 to be a crucial factor in gemcitabine induced growth, migration and invasion of macrophages, but it did not appear to be involved in their M2 polarization. Together, these significant findings could be useful in developing approaches for better clinical management of PC by overcoming unintended immunosuppressive effect of chemotherapy. Results Gemcitabine-treated pancreatic tumors exhibit greater infiltration of macrophages with M2 phenotype To examine the effect of chemotherapy on immune microenvironment, we studied orthotopically-grown pancreatic tumors from either vehicle- or gemcitabine-treated mice. Total RNA and protein were isolated from frozen pancreatic tumor xenografts, and expression of immune cell-specific biomarkers was examined. Our data from the RT-PCR analysis showed an elevated expression of the common leukocyte marker, CD45 (2.2-fold) and CD68 macrophage marker (5.2-fold) in xenograft tumors from gemcitabine-treated mice as compared to vehicle treated group (Fig.?1A). We next examined the expression of Arg-1 and TGF-1, classical markers of the M2 phenotype of macrophages, and observed their elevated levels in gemcitabine-treated tumor tissues (Fig.?1A). Consistent to this, we also observed enhanced expression of CD45, CD68, Arg-1 and TGF-1 at the protein level as evident by our immunoblot analyses (Fig.?1B). We subsequently conducted immunohistochemical analyses on formalin-fixed tumor slices and recorded an increased presence of CD45+/ CD68+ cells having an elevated expression of Arg-1 and TGF-1 in tumors from gemcitabine-treated mice, compared to those treated with vehicle only (Fig.?1C). We also analyzed pancreatic tumor sections for F4/80, a marker specific for mouse macrophages by immunohistochemistry staining. Increased staining of F4/80+cells was observed in tumor sections from gemcitabine-treated mice as compared to those of vehicle-treated mice (Supplementary Fig.?1). Together, these findings suggest that gemcitabine treatment triggers an increased infiltration of immune cells, specifically, M2 macrophages in pancreatic Nocodazole kinase activity assay tumors. Open in a separate window Figure 1 Gemcitabine induces a specific increase in macrophage infiltration in pancreatic tumors. (A) cDNA was prepared, and qRT-PCR was performed for transcripts of CD45 (all leukocytes), CD68 (macrophages), Arg-1 and TGF-1 using the total RNA from tumor xenografts of either vehicle or gemcitabine-treated mice. GAPDH Nocodazole kinase activity assay was used as internal control. Bars represent mean??SD. *p? ?0.05. (B) Western blot analyses of whole-tumor lysate to analyze the expression of CD45, CD68, Arg-1 and TGF-1 protein detection. -actin was used as an internal control. Fold change indicates the level of expression after normalization with -actin. (C) Representative images (20X and 100X) of tumor sections (tumors were resected from.