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. 2017 Sep;5(9):718-729.
doi: 10.1158/2326-6066.CIR-16-0311. Epub 2017 Aug 3.

Development of Aggressive Pancreatic Ductal Adenocarcinomas Depends on Granulocyte Colony Stimulating Factor Secretion in Carcinoma Cells

Michael W Pickup  1 , Philip Owens  2 , Agnieszka E Gorska  2 , Anna Chytil  2 , Fei Ye  3 , Chanjuan Shi  4 , Valerie M Weaver  1 , Raghu Kalluri  5 , Harold L Moses  2 , Sergey V Novitskiy  6
Affiliations

Development of Aggressive Pancreatic Ductal Adenocarcinomas Depends on Granulocyte Colony Stimulating Factor Secretion in Carcinoma Cells

Michael W Pickup et al. Cancer Immunol Res. 2017 Sep.

Abstract

The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFβRIIKO), we discovered an effect of TGFβ signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718-29. ©2017 AACR.

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Conflict of interest statement

The authors whose names are listed above have NO conflict of interest to report

Figures

Figure 1
Figure 1. Expansion of Ly6G+ cells in aggressive PDAC
A, Flow cytometry analysis of CD45+ cells in normal, Kras/p48, and KRas/p48TGFβRII-KO tissue and percent to DAPI- live cells quantification. Plots are gated as DAPI-. 10 mice per group were analyzed.* P < 0.05. B, IHC for Gr1 (RB6-8C5) of normal pancreas, pancreas isolated from Kras/p48 mice and Kras/p48TGFβRII-KO mice at 5-6 weeks of age. C, representative flow cytometry plots of CD11b+Gr1+ cells in pancreatic tissue, spleen and bone marrow from normal, Kras/p48, and KRas/p48TGFβRII-KO mice. Plots are gated as CD45+DAPI-. 10 mice per group were analyzed. D, quantitative data of Gr1-high (solid line) and Gr1-low (dotted line) cells in tumor tissue showed on panel C, top. * P < 0.05, ** P < 0.01. 10 mice per group were analyzed. E, ELISA data of G-CSF levels in pancreatic tissue (left) and basal secretion in cultured cell lines (right). Data corresponds to the mean ± SEM of three individual mice from two experiments. Pancreatic cell lines from Kras/p48 mice and Kras/p48TGFβRII-KO mice were cultured in 6 wells plate in 3 ml of DMEM/10% FBS for 24 hr and level of G-CSF was measured in conditioned media. n = 3. * P < 0.05, ** P < 0.01. F, flow cytometry analysis of CD11b+ cells and Ly6G vs. Ly6C cells (gated as CD11b+) after differentiation of bone marrow progenitors (Lin- cells) for 5 days in presence of 25% conditioned media from Kras/p48 and Kras/p48TGFβRII-KO pancreatic carcinoma cell lines. Data correspond to the mean ± SEM of three individual mice from two experiments. * P < 0.05.
Figure 2
Figure 2. Functions of myeloid cells
A, Cell sorting strategy. B, T-cell (CD3+) gene expression profile. Cells were sorted as shown in (A), top row: gene expression for Th2 cytokines presented as ΔΔCt normalized to Gapdh. Bottom row: gene expression for Th1 cytokines presented as ΔΔCt normalized to Gapdh. Data correspond to the mean ± SEM of three individual mice from two experiments. * P < 0.01. C, Gene expression analysis of pro-tumorigenic cytokines previously identified as cytokine mediators of MDSC immune suppression. top row: Gene expression measured by qPCR on sorted Ly6G; middle row: Gene expression analysis of Gr1+ cells, from the spleen of 4T1 tumor bearing mice, treated with conditioned media from Kras/p48 (open bars) or from Kras/p48TGFβRII-KO (closed bars) carcinoma cells. bottom row: Gene expression analysis of Gr1+ cells, from the spleen of 4T1 tumor-bearing mice, treated with G-CSF, 20 ng/ml for 6 hr. Data correspond to the mean ± SEM of three individual mice from two experiments. ** P < 0.05, * P < 0.01. D, T cells (CD3+) were isolated by negative selection from spleen of naïve BALB/c mice, mixed with CD11b cells isolated from pancreatic tissue by using CD11b magnetic microbeads (Miltenyi Biotec, San Diego, CA), anti–G-CSF was used in concentration 40 ng/ml. Data correspond to the mean ± SEM of three individual mice. * P < 0.05.
Figure 3
Figure 3. Anti-G-CSF treatment
A, B, Schematic of treatment with either control (open bars) or anti G-CSF (10 ug/mouse, MAB414; closed bars) and KRas/p48TGFβRII-KO or Kras/p48, tumor weight 4 weeks after s.c. injection of cells (5×105), n = 5. C, IHC for Ki-67, CD3, and CD31 of tumor tissue isolated from mice treated IgG (control group, left column) and mice treated with anti G-CSF (right column) 4 weeks after tumor injection. D, Representative flow cytometry plots of tumor infiltrated immune cells. Cells are gated as CD45+. F, representative photograph of tumor tissue isolated from mice 3 weeks after s.c. injection of shRNA control (clone 1) and shRNA G-CSF (clone 1) carcinoma cells (left) and tumor weight (right), n = 5. G, Representative flow cytometry plots of Ly6G/Ly6C analysis of CD45+CD11b+ cells from tumor tissue shown in F. H, Cytokine array data analysis. CD11b+ cells were isolated from tumor tissue showed on F for collection of conditioned media. Data shows fold changes in shRNA G-CSF tumors vs. shRNA control. Graph shows only proteins with statistically significant changes from total 111 proteins (see details in Material and Methods section), n = 3. I, IHC for F4/80 and CD3 of tumors shown in F. J, Weight of tumor tissue isolated from mice 4 weeks after s.c. injection of shRNA control (clone 2) and shRNA G-CSF (clone 5) carcinoma cells treated in parallel with IgG control (open bars) or depletion due to use of CD4 and CD8 antibody (closed bars). K, Scheme of mice treatment with gemcitabine (GEM) and anti G-CSF. Tumor tissue isolated from mice 3 weeks after s.c. injection of Kras/p48TGFβRII-KO carcinoma cell line and treatment during last 2 weeks (left) and tumor weight (right), n = 5. Scale bars indicate 100 um.
Figure 4
Figure 4. Increased number of CD15+ cells and expression of G-CSF correlates with Grade 1/Stage II in human pancreatic cancer
A, B, TCGA data set analysis of pancreatic cancer patient survival for Smad4 High and low expressing patients and analysis of Smad4 expression segregated by metastatic incidence, tumor grade, and tumor stage. C, Representative tissue section from TMA stained for CD15 and G-CSF by IHC. D, CD15 and F, G-CSF analysis of TMA staining grouped by grade and tumor stage. Normal, n = 17; tumor, n = 76; grade 1, n = 17; grade 2, n = 28; grade 3, n = 31; stage I, n = 43; stage II, n = 30; stage III, n = 12; stage IV, n = 4. E, Epithelial scoring of G-CSF in TMA used for C, D, F. Scale bars indicate 100 um.
Figure 5
Figure 5. Schematic summarizing data and working hypotheses
The following numbers refer to the number in the schematic. 1, Tgfbr2KO pancreatic epithelial cells secrete abundant CXCL1/CXCL5 due to lack of TGFβ suppression of chemokine expression (6). 2, The CXCL chemokines recruit to the tumor microenvironment myeloid cells (CD11b+). 3, In parallel with chemokines carcinoma cells secrete increased level of G-CSF. 4, G-CSF promotes differentiation of myeloid cells to Ly6G+ cells, upregulates immunosuppressive genes such as Arg, iNOS, VEGF and upregulate secretion of IL10 making (5) myeloid cells that are immunosuppressive with pro-tumorigenic properties. 6, The net result is enhanced tumor growth and decrease survival.
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