Tumor-derived granulocyte-macrophage colony-stimulating factor regulates myeloid inflammation and T cell immunity in pancreatic cancer

Abstract

Cancer-associated inflammation is thought to be a barrier to immune surveillance, particularly in pancreatic ductal adenocarcinoma (PDA). Gr-1(+) CD11b(+) cells are a key feature of cancer inflammation in PDA, but remain poorly understood. Using a genetically engineered mouse model of PDA, we show that tumor-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) is necessary and sufficient to drive the development of Gr-1(+) CD11b(+) cells that suppressed antigen-specific T cells. In vivo, abrogation of tumor-derived GM-CSF inhibited the recruitment of Gr-1(+) CD11b(+) cells to the tumor microenvironment and blocked tumor development-a finding that was dependent on CD8(+) T cells. In humans, PDA tumor cells prominently expressed GM-CSF in vivo. Thus, tumor-derived GM-CSF is an important regulator of inflammation and immune suppression within the tumor microenvironment.

Figure 1. Gr-1⁺ CD11b⁺ cells accumulate in the spleen and tumor of KPC mice bearing PDA

(A) Gr-1⁺ CD11b⁺ cells from the spleen and pancreas of tumor-bearing KPC mice vs. normal control mice were evaluated by flow cytometry. Representative flow plots for one mouse in each category (left) and summary data (right) are shown. Cells were gated for CD45 expression and then analyzed. Numbers in top right corner of the plots represent the percentage of Gr-1⁺ CD11b⁺ cells among total CD45⁺ cells. For summary data, each symbol represents an individual mouse: spleen (light gray), pancreas (dark gray), normal mice (triangles) and PDA mice (diamonds). Black horizontal bars represent the mean for each group (n = 10 for normal spleen, n = 15 for PDA spleen, n = 3 for normal pancreas, and n = 13 for PDA pancreas). Nml = normal. *** indicates p < 0.001 for the comparisons shown. (B) H&E stain (left) and immunohistochemistry for CD45 (middle) or Gr-1 (right) on a representative PDA lesion. Scale bars, 50 μM. Higher magnification images are shown in the top right corner of each panel. (C) Metastatic lesions on the liver and diaphragm of a representative KPC mouse were evaluated by flow cytometry for Gr-1⁺ CD11b⁺ cells (after gating for CD45 expression), compared to salivary gland cells as a control. (D) Diff-Quick stain of cytospin preparation of sorted CD45⁺ Gr-1⁺ CD11b⁺ cells from the spleen of a tumor-bearing KPC mouse. Scale bar, 20 μM.

Figure 2. Gr-1⁺ CD11b⁺ cells isolated from tumor-bearing KPC mice suppress antigen-specific T cell responses in vitro

(A) Gr-1⁺ CD11b⁺ cells isolated from either the spleen (light gray bars) or pancreas (dark gray bars) of tumor-bearing KPC mice were cocultured with peptide-stimulated OT-1 splenocytes and proliferation was evaluated by ³[H]-thymidine incorporation. Assay was performed in triplicate; data are mean ± SD, representative of 5-10 independent experiments. ** indicates p < 0.01, *** p < 0.001, compared to OT-1 alone. (B) IFN-γ in supernatant from coculture of Gr-1⁺ CD11b⁺ cells with OT-1 T cells (1:1 ratio) was measured by cytokine bead array. Data are representative of 2 independent experiments. (C) Arginase activity was evaluated in the Gr-1⁺ fraction of normal spleens and tumor-bearing KPC spleens. Assay was performed in triplicate; data are mean ± SD, representative of 2 independent experiments. *** indicates p < 0.001. (D) Nitrite concentration in supernatant from coculture of Gr-1⁺ CD11b⁺ cells with OT-1 T cells (1:1 ratio) was measured by the Griess reaction as an indicator of iNOS activity. Data are mean ± SD, representative of 3 independent experiments. *** indicates p < 0.001. (E) Small molecule inhibition of iNOS relieves antigen-specific suppression of T cell proliferation mediated by Gr-1⁺ CD11b⁺ cells. Proliferation assay was performed in triplicate at 1:1 ratio of Gr-1⁺ CD11b⁺ cells to OT-1 cells; data are mean ± SD. nor-NOHA (arginase inhibitor) = N^ω-Hydroxy-nor-L-arginine; L-NMMA (iNOS inhibitor) = N^G-Monomethyl-L-arginine. ** indicates p < 0.01, compared to OT-1 alone. See also Figure S1.

Figure 3. c-kit⁺ lineage^neg splenocytes are highly proliferative precursors of Gr-1⁺ CD11b⁺ cells and are driven to proliferate via tumor-derived factors

(A) Tumor-bearing KPC mice exhibit splenomegaly as indicated by spleen size of normal mice and tumor-bearing KPC mice (left) and spleen weight of normal and KPC mice (right). Black horizontal bars represent the mean for each group (n = 5 for normal mice, n = 12 for KPC mice). Nml = normal. ** indicates p < 0.01. (B) H&E of tumor-bearing KPC spleen (left) reveals marked disruption of normal splenic architecture and classic findings of extramedullary hematopoiesis. Note the abundance of megakaryocytes (white arrow) and areas of erythropoiesis (black arrow). Scale bars, 50 μM. Spleen and bone marrow from normal and tumor-bearing KPC mice (right) were evaluated by flow cytometry for c-kit⁺ cells. (C) c-kit⁺ Gr-1^neg CD11b^neg cells (top) and Gr-1⁺ CD11b⁺ cells (bottom) from the spleens of tumor-bearing KPC mice were labeled with CFSE and cultured in the presence of either unconditioned control media (No CM) or PDA conditioned culture media from KPC tumor lines (+ CM). Gr-1⁺ CD11b⁺ phenotype is shown at baseline post-isolation (left). Flow cytometric analysis of CFSE dilution on day 5 (middle) demonstrates that Gr-1^neg CD11b^neg c-kit⁺ cells proliferate in response to tumor conditioned media, but not control media, whereas freshly isolated Gr-1⁺ CD11b⁺ MDSC do not proliferate in either condition. Black horizontal bars indicate undiluted CFSE intensity on day 5. The day 5 output of the proliferative Gr-1^neg CD11b^neg c-kit⁺ culture was evaluated by flow cytometry for surface expression of Gr-1 and CD11b (far right). Data are representative of 5-10 independent experiments. (D) The Gr-1⁺ CD11b⁺ cells generated from c-kit⁺ cells after 5 days with CM were evaluated for arginase (left) and iNOS (right) activity. Data are mean ± SD from one of 2 independent experiments; ** indicates p < 0.01, *** p < 0.001. (E) Suppressive capacity of the Gr-1⁺ CD11b⁺ cells generated from c-kit⁺ cells after 5 days with CM was assessed in the OT-1 T cell suppression assay, performed in triplicate. Data are mean ± SD. ** indicates p < 0.01, *** p < 0.001, compared to OT-1 alone. See also Figure S2.

Figure 4. Tumor-derived GM-CSF induces proliferation and differentiation of c-kit⁺ lineage^neg precursors into functional MDSC

(A) Cytokines produced by primary PDA lines derived from KPC tumors and normal pancreatic ductal cell lines are compared in a heat map. Color scale and corresponding cytokine values (pg/mL) are shown below. Last column indicates whether conditioned media from that cell line was found to drive proliferation of c-kit⁺ lineage^neg cells in a CFSE proliferation assay. Nml = normal; nd = not determined. (B) Recombinant GM-CSF is sufficient to induce proliferation and differentiation of c-kit⁺ lineage^neg cells into MDSC. Day 5 FACS analysis (top) was performed to evaluate Gr-1 and CD11b expression on c-kit⁺ lineage^neg splenocytes cultured with control media (No CM), CM from PDA lines (+ PDA CM), or recombinant GM-CSF (200 pg/ml). Suppressive capacity of culture outputs (bottom) was evaluated in the OT-1 T cell suppression assay. Data are representative of at least 3 independent experiments. (C) CFSE dilution of c-kit⁺ lineage^neg cultures is shown for day 5. Neutralization of GM-CSF by anti-GM-CSF mAb (100 μg/mL) in PDA-1 conditioned media blocks proliferation of c-kit⁺ lineage^neg cells. Black horizontal bars indicate undiluted CFSE intensity on day 5. Data are representative of independent experiments using conditioned media from 3 different PDA cell lines. As a control, the neutralizing capability of this anti-GM-CSF mAb was shown by its effect on recombinant GM-CSF in this assay. See also Figure S3

Figure 5. PDA cells produce GM-CSF and other factors in vivo in KPC mice

(A) Cytokines in fresh PDA supernatant (directly ex vivo) vs. normal pancreas supernatant were compared by cytokine bead array. y-axes are on log scales; each symbol represents an individual mouse: normal mice (light gray diamonds) and PDA mice (dark gray diamonds). Black horizontal bars represent the mean for each group (n = 3 for normal mice, n = 7 for PDA mice). ** indicates p < 0.01. (B) Transcriptional analysis was used to evaluate GM-CSF mRNA expression of YFP⁺ (epithelial) cells vs. YFP^neg (stromal) cells sorted from tumors of Kras^G12D; p53^fl/+; Pdx-1-Cre; RosaYFP mice (n=3) or normal pancreata from lineage labeled wild type mice (Pdx-1-Cre; RosaYFP) (n=3). Data are mean ± SD. * indicates p < 0.05, *** p < 0.001. (C) Immunohistochemistry for GM-CSF was performed on surgical pancreas samples from 20 patients with PDA. PDA tumors from three representative patients are shown (upper left, upper right, lower left), and adjacent pancreatic tissue without invasive tumor is shown for one patient (lower right). The lower left panel is shown at higher power magnification. Scale bars, 50 μM.

Figure 6. GM-CSF neutralization with anti-GM-CSF antibody limits tumor growth and recruitment of Gr-1⁺ CD11b⁺ cells in vivo

(A) Normal control mice (6-14 weeks) were implanted subcutaneously with the GM-CSF-producing cell line PDA-1 in matrigel with either a neutralizing anti-GM-CSF mAb (αGM) or isotype control antibody, with or without tumor cell irradiation (IR), and with or without intraperitoneal administration of a depleting CD8 mAb. On day 6, tumor growth was evaluated by H&E (top; images of one representative mouse per group are shown) and tumor weight (bottom; data are mean ± SD). n = 3 to 11 mice per group. Scale bars, 50 μM. ** indicates p < 0.01, *** p < 0.001. (B) The immune infiltrate was evaluated by immunohistochemistry (left) and immunofluorescence (right) for CD45 and Gr-1. Images of one representative mouse per group are shown (3 to 11 mice per group). Scale bars, 50 μM. (C) Absolute numbers of CD45⁺ cells (left) and Gr-1⁺ CD11b⁺ cells (right) were quantified by flow cytometry. Data are mean ± SD. * indicates p < 0.05, ** p < 0.01, *** p < 0.001. See also Figure S4.

Figure 7. Genetic knockdown of GM-CSF demonstrates that tumor-derived GM-CSF regulates tumor growth and recruitment of Gr-1⁺ CD11b⁺ cells in vivo

(A) Normal control mice or Rag2^-/- mice (6-14 weeks) were implanted subcutaneously with either shGM-CSF PDA-1 cells (shGM) or vector only PDA-1 cells (Mock) in matrigel with or without tumor cell irradiation (IR), and with or without intraperitoneal administration of a depleting CD8 mAb. On day 6, tumor growth was evaluated by H&E (top; images of one representative mouse per group are shown) and tumor weight (bottom; data are mean ± SD). n = 3 to 7 mice per group. Scale bars, 50 μM. * indicates p < 0.05, ** p < 0.01, *** p < 0.001. (B) The immune infiltrate was evaluated by immunohistochemistry (left) and immunofluorescence (right) for CD45 and Gr-1. Images of one representative mouse per group are shown (3 to 7 mice per group). Scale bars, 50 μM. (C) Absolute numbers of CD45⁺ cells (left) and Gr-1⁺ CD11b⁺ cells (right) were quantified by flow cytometry. Data are mean ± SD. * indicates p < 0.05, ** p < 0.01, *** p < 0.001. See also Figure S5.

Figure 8. Working model links tumor-derived GM-CSF, MDSC generation, and CD8⁺ T cell suppression in PDA