GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy.

Fig. 1

Immunization with GM-CSF-secreting tumor cells leads to increased survival compared to irradiated tumor cells co-administered in combination with rGM-CSF in tumor-bearing mice. a C57BL/6 mice were immunized with HBSS, 1 × 10⁶ unmodified B16F10 cells, GM-CSF-secreting (∼200 ng/1 × 10⁶ cells/24 h) B16F10 cells (F10GM 200), or B16F10 cells in combination with 200 ng of rGM-CSF (B16F10/rGM 200). The rGM-CSF was co-administered at the immunization site by subcutaneous injection of 200 ng once per day for 4 days. Mice (n = 10/group) were challenged 7 days post-immunization with 1 × 10⁶ live B16F10 tumor cells. Animals were assessed for tumor growth twice weekly, and sacrificed if tumors became necrotic or exceeded 1,500 mm³ in size. Data is presented as a Kaplan–Meier survival curve with n = 10 per group. b To evaluate the aforementioned groups in a treatment setting, C57BL/6 mice were challenged with 2 × 10⁵ live B16F10 tumor cells. Three days later mice were immunized with HBSS, 1 × 10⁶ unmodified B16F10, F10GM 200, or B16F10/rGM 200. Tumor assessment and data analysis was performed as described above. c GM-CSF serum levels following immunization with B16F10 cells and rGM-CSF or GM-CSF-secreting tumor cells. C57BL/6 mice (n = 10/group) were immunized with 1 × 10⁶ B16F10 cells in combination with a single injection of 800 ng rGM-CSF, or with 1 × 10⁶ irradiated GM-CSF-secreting (∼800 ng/1 × 10⁶ cells/24 h) F10GM 800 tumor cells. Serum samples were collected 6, 24, 48, and 72 h post-immunization and GM-CSF levels were determined by ELISA. d Mice immunized with F10GM 800 tumor cells had sustained levels of GM-CSF at the injection site. Animals (n = 6/group) were treated as described in panel c. The immunization site was collected at the time points indicated, processed as described in Sect. “Materials and methods”, and evaluated for GM-CSF levels by ELISA. The background of this assay was ∼70 pg/ml based on the levels of GM-CSF obtained from the immunization site of mice treated with B16F10 (non GM-CSF-secreting) tumor cells

Fig. 2

Tumor cells persist and efficiently recruit dendritic cells to the injection site following immunization with GM-CSF-secreting tumor cells. On day 0, C57BL/6 mice were inoculated subcutaneously with 2 × 10⁵ B16F10 live tumor cells. Three days later, mice were immunized with 3 × 10⁶ irradiated B16F10 cells, B16F10 cells and four subcutaneous injections (days 3, 4, 5, and 6) of 200 ng of rGM-CSF, or GM-CSF-secreting (∼200 ng/1 × 10⁶cells/24 h) F10GM cells. All tumor cells expressed GFP. Injection sites were collected for histological analysis on days 1, 7, 14, and 21 post-immunization, and stained with DAPI (blue, nuclear staining) and CD11c (red, dendritic cells). GFP-positive tumor cells are shown in green. Stained 10 μm sections were analyzed by fluorescence microscopy using a Zeiss Axioplan microscope and are shown at ×20 magnification. Photomicrographs presented are representative of five mice

Fig. 3

Immunization with GM-CSF-secreting tumor cells increased the number of DCs at the injection site. C57BL/6 mice were inoculated subcutaneously with 2 × 10⁵ live B16F10 tumor cells. Three days later, mice were immunized with 3 × 10⁶ irradiated B16F10 or F10GM tumor cells. At 1, 4, and 8 days post-immunization, the injection site was collected and stained with CD11c, MHC class II (I-Ab), and langerin. a Histograms demonstrating the qualitative increase in the percentage of CD11c+ and MHC class II+/Langerin+ (gated on CD11c+) cells at the immunization site. The data shown are from day 4 post-immunization. b Qualitative analysis of the total number of cells (top panels), total number of dendritic cells (based on a CD11c+ gate; middle panels), and total number of MHC class II+/Langerin+ cells at the immunization site (bottom panels)

Fig. 4

Increased apoptosis was observed at the immunization site of F10GM treated mice. Animals were treated and evaluated as described in Fig. 2. a The injection sites were collected for histological analysis and stained with DAPI (blue, nuclear staining) and caspase 3 (red, apoptotic cells). GFP-positive tumor cells are shown in green. On days 4 and 10 post-immunization, increased numbers of caspase 3 positive cells were observed in the F10GM immunized mice compared to animals treated with B16F10. Sections are at ×20 magnification. b The dense infiltrate of apoptotic cells observed in F10GM mice appeared to be comprised of both neutrophils and tumor cells. The injection sites of F10GM treated mice were collected for histological analysis and stained with DAPI (blue, nuclear staining) and either caspase 3 (red) or neutrophils (red). GFP-positive tumor cells are shown in green. Enhanced neutrophil infiltration was only observed in F10GM treated mice. Sections are at ×10 magnification. c Tumor cells efficiently recruit CD4+ T-cells to the injection site following immunization with GM-CSF-secreting tumor cells. Injection sites were collected for histological analysis on days 1, 7, 14, and 21 post-immunization, and stained with DAPI (blue, nuclear staining) and CD4 (red). GFP-positive tumor cells are shown in green. Stained 10 μm sections were analyzed by fluorescence microscopy using a Zeiss Axioplan microscope and are shown at ×20 magnification. Photomicrographs presented are representative of five mice

Fig. 5

T-cell responses specific for multiple tumor antigens were elicited in mice immunized with a GM-CSF-secreting cancer immunotherapy. Mice were immunized bi-weekly with HBSS, or with 3 × 10⁶ irradiated B16F10 or F10GM tumor cells. On day 4 (a) and 12 (b) post the first or second (multiple) immunization, splenocytes were collected and stimulated in vitro for 5 days with irradiated B16F10 tumor cells. Antigen specific responses were assayed by ⁵¹Cr release for lysis of peptide pulsed RMAS cells. The peptides used for these studies were TRP2_180–188 (SVYDFFVWL), GP100_25–33 (EGSRNQDWL), and MAGE-AX_169–176 (LGITYDGM)

Fig. 6

Granulocyte-macrophage colony-stimulating factor-secreting tumor cell immunotherapies lead to broad antigen-specific immune response. On day 0, mice were immunized with HBSS, 1 × 10⁶ irradiated B16F10 (B16F10) or GM-CSF-secreting tumor cells (F10GM). Animals were challenged 7 days later with 1 × 10⁶ live cells of the indicated B16 subclones, including B16F10 (a), B16 (b), B16F0 (c), and B16F1 (d). The treatment regimen and median survival time for each group is indicated in the upper box of each panel (n = 10/group). Subgroups of mice from the groups described in panel A were sacrificed 14 days post-immunization. The lower charts show IFNγ ELISPOT analysis of the splenocytes performed using irradiated B16F10 (a), B16 (b), B16F0 (c), and B16F1 (d) tumor cells as stimulators