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. 2019 Jun 4;10(1):2450.
doi: 10.1038/s41467-019-10424-5.

CaMKK2 in Myeloid Cells Is a Key Regulator of the Immune-Suppressive Microenvironment in Breast Cancer

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Free PMC article

CaMKK2 in Myeloid Cells Is a Key Regulator of the Immune-Suppressive Microenvironment in Breast Cancer

Luigi Racioppi et al. Nat Commun. .
Free PMC article

Abstract

Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8+ T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2-/- mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2-/- macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8+ T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.

Conflict of interest statement

The authors declare the following competing interests: L.R., E.R.N., W.H., N.C. and D.P.M. have applied for a patent covering the use of CaMKK2 inhibitors alone or in combination with immunotherapy for the treatment of cancer. Title: “CaMKK2 inhibitor compositions and methods of using the same. Racioppi, L., Nelson, E.R., Huang, W., Chao, N. and McDonnell, D.P. Provisional Patent Application No.: 62/371,309; August 5, 2016. The remaining authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
CaMKK2 is expressed in mammary tumor-associated stromal cells. a Expression of CaMKK2 in human breast cancer. Representative images (×400 magnification) of malignant mammary tissue samples stained with an anti-CaMKK2 antibody. T and S refer to tumor and stromal cells, respectively. Scale bar = 100 μm. b CaMKK2 staining intensity is correlated between cancer and stromal cells of the same human breast tumors. c The Camkk2 promoter is active in myeloid cells associated with mammary tumors. E0771 cells (4 × 105 cells/mouse) were inoculated into the mammary fat pad of (Tg)-Camkk2-EGFP reporter mice. Subsequently, tumors were removed and digested and single-cell suspensions were stained for markers of myeloid cells. The gating strategy used to identify myeloid subsets and lymphoid cells is shown in Supplementary Fig. 1B. (Left) FACS profiles report the expression of EGFP reporter in tumor-associated immune cells. The vertical line refers to negative control (EGFP-negative splenocytes). (Right) Mean fluorescence intensity (MFI) of EGFP expression in immune cell subsets. Bar graph reports the mean ± SEM (standard error of the mean; N = 3 independent tumors in each group). *p < 0.05, ****p < 0.005, respectively. A t test was used to calculate p-values. Similar results have been observed in three independent experiments
Fig. 2
Fig. 2
The growth of mammary tumors is attenuated in mice lacking CaMKK2. a Mammary tumors from MMTV-PyMT mice propagated in a C57BL/6 background were harvested, diced, and orthotopically grafted into syngeneic C57BL/6 mice that were WT or knockout for Camkk2 (WT and Camkk2−/−, respectively; mean ± SEM; N = 10 in each group). b Murine E0771 cells (4 × 105 cells/mouse) were orthotopically grafted in WT and Camkk2−/− mice, and subsequent tumor volume measured as indicated (N = 5 in each group; this experiment was replicated with a comparable number of mice at least three times). A two-way ANOVA test was used to calculate p-values. c Representative images of E0771 tumors from WT and Camkk2−/− mice. Sections were stained with anti-CD3 or anti-F4/80 antibodies (scale bar = 50 μm). d Quantitation of CD3+ and F4/80+ cells in high-power optic (×400 magnification) fields in stained sections (six fields for each section; N = 3 individual tumors in each group). Bar graph reports the mean ± SEM. N = 18 data points/genotype. *p < 0.05, **p < 0.01, respectively. A t test was used to calculate p-values. e E0771 tumors of comparable size (500–700 mm3) were removed from WT and Camkk2−/− mice, digested with collagenase and DNase I to obtain a single-cell suspension. Immune cells were identified by flow cytometry, according to the gating strategy reported in Supplementary Fig. 2C. Pie charts show the mean percentages of immune cell subsets detected in tumors removed from WT and Camkk2−/−. Values reported under each pie chart refer to the percentages of tumor-infiltrating CD45+ cells. N = 8 and 4 tumors from WT and Camkk2−/− mice were analyzed. A t test was used to calculate statistical significance. p-values color code: blue and red values indicate a statistically significant increase or decrease of the indicated cell types in tumors from WT compared with KO host, respectively. f Gene expression analysis in E0771 tumors removed from WT and Camkk2−/− mice (N = 5 and 5, respectively). A t test was used to calculate p-values. Bar graph reports the mean ± SEM. *p < 0.05, ***p < 0.005
Fig. 3
Fig. 3
Attenuated tumor growth in Camkk2 KO host is mediated by CD8+ T cells. Murine E0771 (4 × 105) cells were orthotopically grafted in WT and Camkk2−/− mice, and subsequently tumors of comparable sizes (500–700 mm3) were harvested and digested with both collagenase and DNase I. Single-cell suspensions were then stained for lymphoid and myeloid markers, and cell subsets were identified according with gating strategy reported in Supplementary Fig. 3A–C. a Increased accumulation of CD8+ T cells in E0771 cell-derived tumors propagated in Camkk2−/− mice compared with WT. Bar graph reports the mean ± SEM from two combined independent experiments. b Percentages of tumor-infiltrating lymphocyte subsets in E0771 tumors growing in Camkk2−/− mice compared with WT. (upper left panel) Intracellular expression of Granzyme B+ (GzmB+). CD69+, PD-1+, and LAG-3+ expression was detected on an independent set of eight and four tumors removed from WT and Camkk2−/− mice, respectively. Bar graph reports the mean ± SEM. A t test was used to calculate p-values. c Representative FACS dot plots of tumor single-cell suspensions stained with myeloid markers (left). Bar graph reports the mean ± SEM of CD11b+ MHC II+ subset from combined independent experiments. A t test was used to calculate p-values. N = 13 and 15 tumors removed from WT and Camkk2−/− mice, respectively. d Mammary tumor growth in Camkk2−/− mice depleted of CD8+ T cells. Camkk2−/− mice were treated with anti-CD8 antibody or control isotype every 3 days starting a week before tumor cell inoculation. At day 0, E0771 cells were orthotopically grafted, and mice were treated with antibodies every 4 days. Graph depicts tumor size (mean ± SEM; N = 6 in each group). e Mammary tumors display retarded growth in mice devoid of Camkk2 in myeloid cells. E0771 cells were orthotopically grafted into LysMCre+ Camkk2wt/wt and LysMCre+ Camkk2fl/fl mice. Tumor volume was measured (mean ± SEM; N = 4 for each group). This experiment was replicated with similar results. A two-way ANOVA test was used to calculate p-values. *p < 0.05, ***p < 0.005
Fig. 4
Fig. 4
CaMKK2 regulates gene transcription in BMDM. Bone marrow-derived macrophages (BMDM) were generated from WT and Camkk2−/− mice in the presence or absence of E0771-conditioned medium (RM and TCM, respectively). Subsequently, BMDM was harvested and analyzed for gene expression. N = 4 biological replicates/group. a, b Volcano plots of differentially expressed genes (DEGs) in WT (left) and Camkk2−/− (right) generated in the presence of RM or TCM. Open circles indicate genes showing comparable expression levels in WT and Camkk2−/− BMDM. DEGs between WT and Camkk2−/− BMDM are shown as green filled circles. Yellow open circles refer to the Camkk2 gene. c Heatmaps of DEGs affiliated with M1, M1 and dendritic cells (M1&DC), or M2 signatures. The color key for the heatmap indicates (row-wise) scaled RPKM values (z-score). d Real-time quantitative PCR (qPCR) analysis of genes associated with M1 (Nos2) and M2 (Arg1, Chil3/Ym1 and Retnla/Fizz1) gene signatures (four biological replicates were analyzed for each genotype). A t test was used to calculate p-values. The data are graphed as the mean ± SEM. Asterisks refer to *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. This experiment was replicated at least three times with similar results
Fig. 5
Fig. 5
CaMKK2 links tumor factor signaling to AMPK activation. WT and Camkk2−/− BMDM were generated in regular differentiation medium in the presence or absence of E0771-conditioned medium (RM and TCM, respectively). a CaMKK2 and phospho-CaMK1 immunoblots. b Expression of phosphorylated (T-172; p-AMPK) and the total AMPKα was assessed by immunoblot. c Quantification of p-AMPK/AMPK ratio (N = 3 biological replicates; mean ± SEM). Each lane refers to protein lysate extracted from a single biological replicate. A t test was used to calculate p-values. Asterisks refer to *p < 0.05, **p < 0.01, and ***p < 0.005, respectively
Fig. 6
Fig. 6
Phenotype of WT and Camkk2−/− BMDM generated in the presence or absence of tumor-conditioned medium. WT and Camkk2−/− BMDM were generated in the presence of RM or TCM. a Left: CD206 and MHC II expression on CD11b+ F4/80+ gated BMDM. Right: percentages of CD206+/MHC II and CD206/MHC II+ subsets (mean ± SEM; N = 3 biological replicates). b Expression of MHC II on CD11b + F4/80 + gated BMDM (mean of MFI ± SEM; N = 3 biological replicates). c Cxcl9 expression (mean ± SEM; N = 3 biological replicates). These experiments were replicated at least three times. d Tumors of comparable size (500–700 mm3) were removed from WT and Camkk2−/− mice, and myeloid cells were then identified and sorted by flow cytometry. Bar graph reports mean ± SEM of CD206+/MHC II and CD206/MHC II+ TAM percentages gated on “Mac” subset in the Supplementary Fig. 2 (N = 5 and 4 tumors removed from WT and Camkk2−/− mice, respectively). e Cxcl9 expression in TAM sorted from E0771 tumors removed from WT and Camkk2−/− mice (mean ± SEM; N = 3 and 3 tumors for each genotype)
Fig. 7
Fig. 7
CaMKK2 mediates the immune-stimulatory ability of BMDM generated in tumor-conditioned medium. a CFSE-labeled T cells isolated from wild-type mice were cultured with anti-CD3 antibody, in the presence or absence of WT or Camkk2−/− BMDM generated in regular medium or the tumor-conditioned medium (RM and TCM, respectively). Dot plots and histograms of T cells recovered after co-culture for 72 h (Left and right, respectively). T cells were identified according to the gating strategy reported in the Supplementary Fig. 3A. b Percentage of CFSE-low CD8+ T cells (gate is displayed in the panel A dot plots). N = 3 biological replicates for each genotype. t test was used to calculate p-values. The experiment was replicated with similar results. c BMDM were generated from WT and Camkk2−/− mice (WT and KO, respectively) in the presence of TCM. After 5 days of culture, BMDM supernates were collected and tested in a chemotaxis transwell assay for the ability to recruit T cells isolated from lymph nodes of WT mice or CD8+ OTI T cells (left and right panels, respectively). Bar graphs report mean ± SEM of T cells migrating toward BMDM supernates after 12 h and percentages of CD8 + OTI T cells migrating toward BMDM supernates at 6 and 12 h (mean ±SEM; N = 3 replicates for each genotype). t test was used to calculate p-values. These experiments were replicated with similar results. d A 3D microfluidic tumor on-a-chip model was used to test the effects of BMDM in E0771 tumor microenvironment (schematic of 3D on-chip model is shown in Supplementary Fig. 9). Graph reports kinetics of OTI CD8+ T cells migrating toward 3D E0771-microenvironment infiltrated by WT or Camkk2−/− BMDM (mean± SEM; N = 3 low magnification fields). Two-way ANOVA was used to calculate p-values. Asterisks refer to *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001, respectively
Fig. 8
Fig. 8
Pharmacological inhibition of CaMKK2 attenuates mammary tumor growth in immunocompetent mice. a E0771 (4 × 105) cells were orthotopically grafted into syngeneic WT mice. Starting at day 2 after grafting, mice were treated three times/week with vehicle or STO-609 (IP, 100 μmoles/kg body weight), and subsequently tumor volumes measured (mean ± SEM; N = 6 in each group); two-way ANOVA was used to calculate p-values. b, c T lymphocytes and myeloid cells within E0771 mammary tumors treated with STO-609 or vehicle. Tumors of comparable size (500–700 mm3) were removed, digested and single-cell suspensions were stained for myeloid and lymphoid markers, and analyzed using the gating strategy reported in Supplementary Fig. 3C and 11. Treatment with STO-609 resulted in the accumulation of CD8+ T cells and CD11b+ MHC II+ myeloid cells (b and c, respectively). Bar graph shows mean ± SEM; N = 5 and 4 tumors in Veh and STO-609 groups, respectively. A t test was used to calculate p-values. d STO-609 failed to affect mammary tumor growth in CD8+ T cell-depleted Camkk2−/− mice. Camkk2−/− mice were treated with anti-CD8 or control isotype antibodies. Subsequently, E0771 cells were orthotopically grafted, and mice treated with STO-609 or vehicle. Tumor volumes were measured (mean ± SEM; N = 3, 7, and 8 in isotype/Veh, CD8/Veh and CD8/STO groups, respectively). Two-way ANOVA test was used to calculate p-values. Asterisks refer to *p < 0.05

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