CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8+ T cells

Abstract

Increased numbers of tumor-infiltrating macrophages correlate with poor disease outcome in patients affected by several types of cancer, including breast and prostate carcinomas. The colony stimulating factor 1 receptor (CSF1R) signaling pathway drives the recruitment of tumor-associated macrophages (TAMs) to the neoplastic microenvironment and promotes the differentiation of TAMs toward a pro-tumorigenic phenotype. Twelve clinical trials are currently evaluating agents that target the CSF1/CSF1R signaling pathway as a treatment against multiple malignancies, including breast carcinoma, leukemia, and glioblastoma. The blockade of CSF1R signaling has been shown to greatly decrease the number of macrophages in a tissue-specific manner. However, additional mechanistic insights are needed in order to understand how macrophages are depleted and the global effects of CSF1R inhibition on other tumor-infiltrating immune cells. Using BLZ945, a highly selective small molecule inhibitor of CSF1R, we show that CSF1R inhibition attenuates the turnover rate of TAMs while increasing the number of CD8⁺ T cells that infiltrate cervical and breast carcinomas. Specifically, we find that BLZ945 decreased the growth of malignant cells in the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis. Furthermore, we show that BLZ945 prevents tumor progression in the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis. Our results demonstrate that TAMs undergo a constant turnover in a CSF1R-dependent manner, and suggest that continuous inhibition of the CSF1R pathway may be essential to maintain efficacious macrophage depletion as an anticancer therapy.

Keywords: CSF1R; M-CSF; breast cancer; cervical cancer; transgenic mouse models; tumor immune evasion; tumor immunology; tumor-associated macrophages.

Figure 1. The CSF1/CSF1R pathway promotes rapid turnover of regulatory TAMs in MMTV-PyMT mammary tumors. (A–F) Spontaneous mammary tumors in 63 to 77 d old MMTV-PyMT transgenic mice by antibody staining and flow cytometry. (A) Gating strategy to identify CD45⁺CD11b⁺Ly-6G/C(Gr-1)^−/loF4/80⁺ macrophages. Cell populations were gated sequentially from left to right. (B) MHCII, CD206 and intracellular IL-10 expression in CD45⁺CD11b⁺Ly-6G/C(Gr-1)^−/loF4/80⁺ cells (TAMs) identified in (A). Isotype control-stained cells are shown as solid histograms. (C) Mean percentages of IL-10⁺ and IL-10⁻ TAM subpopulations of total CD45⁺ leukocytes isolated from vehicle-treated tumors. (D) Histogram overlay of CSF1R expression in (CD45⁺CD11b⁺Ly-6G/C(Gr-1)^−/loF4/80⁺) TAMs and CD45⁻ tumor cells. Unstained control cells are represented by a solid histogram. (E–F) Flow cytometry data of CD45⁺CD11b⁺F4/80⁺ cells (TAMs) from transgenic MMTV-PyMT mice dosed with 200 mg/kg BLZ945, a CSF1R inhibitor, daily or with 5A1, an anti-CSF1 neutralizing antibody (n > 5 per group) at 10 mg/kg every 5 d. TAMs are gated in magenta and values are graphed in (F). (G) Time course of TAM populations in response to 1–8 d of continuous treatment with BLZ945 (n = 4 per group). (H) MMTV-PyMT mice were first dosed with BLZ945 for 5 d (red bar) and then switched to vehicle dosing to provide a recovery period of up to 6 d (gray bars; n = 4 per group). All graphs represent mean values ± SEM *P < 0.05 vs. vehicle by unpaired t test, 2-tailed. (I–L) Spontaneous tumor pieces from CD45.1⁺ FVB/n MMTV-PyMT mice were implanted into a mammary fat pad in CD45.2⁺ BALB/C nude mice. Donor and recipient mice were treated with 200 mg/kg BLZ945 or vehicle for 5 d prior to resection and implantation. Five days after implant, tumors were re-isolated and analyzed by flow cytometry. (J) Percentage of tumor-derived CD45.1⁺CD11b⁺Ly-6G/C(Gr-1)^−/loF4/80⁺ TAMs and host-derived CD45.2⁺CD11b⁺Ly-6G/C(Gr-1)^−/loF4/80⁺ TAMs in vehicle-treated tumors implanted into mice dosed with vehicle control. (K) Expression of intracellular IL-10 and cell-surface expression of MHCII in CD45.1⁺ (green) and CD45.2⁺ (blue) TAM populations. Unstained control cells are shown as solid histograms. (L) Infiltration of CD45.2⁺ TAMs into vehicle-treated tumors implanted into mice dosed with vehicle or BLZ945 (n > 11 per group), and BLZ945-treated tumors implanted into mice dosed with vehicle or BLZ945 (n > 7 per group). Bar graphs represent mean values ± SEM. Statistical analyses were performed by 2-tailed unpaired Student t test; *P < 0.05 vs. vehicle; data shown are representative of at least 2 experiments.

Figure 2. Treatment with BLZ945 decreases macrophage content in tumor and liver, but does not affect lung macrophages, circulating monocytes or tumor cell proliferation. (A–C) 56–63 d old female MMTV-PyMT transgenic mice were randomized by tumor volume and dosed with BLZ945 or vehicle control for 16 d (n > 3 per group). Tissues were formalin-fixed and analyzed by immunohistochemistry. Stained slides were scanned using a ScanScope XT and analyzed by ImageScope v11.2 using the positive pixel count algorithm (Aperio Technologies). (A) Tumor, liver and lung sections were stained using an anti-CSF1R antibody to identify macrophages (brown staining). (B) Time course of circulating monocyte numbers in peripheral blood of individual mice (n = 4 per time point). Complete blood counts were performed by IDEXX Laboratories. (C) Tumor tissue was stained using an anti-Ki67 antibody as a measure of proliferation. All graphs represent mean values ± SEM.

Figure 3. Pharmacological blockade of CSF1R signaling increases infiltration of T cells and decreases tumor growth but does not affect pulmonary metastasis in PyMT mice. (A–E) 63- to 70-d old MMTV-PyMT transgenic mice were randomized by total tumor burden and dosed with 200 mg/kg BLZ945 or vehicle (n = 9 per group) at the indicated time points. Individual tumor volumes were calculated by caliper measurements with total tumor burden being the sum of these measurements. (A) Cumulative tumor burden of vehicle- and BLZ945-dosed mice. (B) Lungs from MMTV-PyMT mice were formalin-fixed and serially sectioned to histologically evaluate the number of individual metastases per mm² of lung. (C) The total metastatic tumor area as a percentage of lung tissue. (D) The average area (mm²) of lung metastatic spread (E) Representative graph showing individual tumor volumes taken from a vehicle-treated mouse. (F–I) Spontaneous tumors from naïve MMTV-PyMT mice were pooled and digested to form a single-cell suspension. Cells were injected into mammary fat pads of syngeneic mice. PyMT allograft-recipient mice with average tumor volumes ~280 mm³ were randomized into 2 groups and dosed with 200 mg/kg BLZ945 or vehicle control 21 d post-implantation. (F) Caliper measurements of tumor volumes (n = 6 per group) were taken every 3–4 d. In a separate study, tumors (n = 4 per group) were analyzed by flow cytometry to determine infiltration of (G) CD45⁺CD11b⁺Ly-6G/C(Gr-1)^−/loF4/80⁺ TAMs and (H and I) CD45⁺CD3⁺CD4⁺ and CD45⁺CD3⁺CD8⁺ T cells in tumor allografts . Graphs display mean values ± SEM. Statistical analyses were performed by 2-tailed unpaired Student t test; *P < 0.05 vs. vehicle; data shown are representative of at least 2 experiments.

Figure 4. Treatment with BLZ945 reduces macrophages, enhances T cell infiltration, and prevents tumor growth in the K14-HPV16 transgenic mouse model of cervical carcinoma. (A–D) Estrogen pellets were administered to 1-mo old K14-HPV16 transgenic mice every 2 mo. Age-matched control mice that did not receive estrogen pellets are referred to as “T0.” At 6 mo of age, mice were dosed with 200 mg/kg BLZ945 or vehicle control for 1 mo, after which time whole cervixes were formalin-fixed for histological analyses. (B) Serial sections of cervical tissue were hematoxylin and eosin (H&E) stained and tumor volumes determined by multiplying the tumor area by the depth of serial sections. Cervical tumors from 6-mo old estrogen-naïve mice (T0) served as a baseline control. (C and D) Cervical tissues were stained with an anti-CSF1R antibody to label macrophages by immunohistochemistry. Magnified views of tumor (t) and stroma (s) regions within the cervix are boxed. Scale bar, 100 µm. (D) Quantification of CSF1R staining in cervical tumor and stroma regions. (E–H) Pharmacodynamic study to monitor changes in tumor-infiltrating and stromal immune cells after 5–7 d of treatment with BLZ945. Whole cervixes were frozen, serially sectioned, and stained with H&E to identify the transformation zone. Tumor and stroma regions within the cervix were scored separately for (F) CSF1R⁺ macrophages, (G) CD8⁺ T cells, and (H) CD4⁺ T cells. Bar graphs represent mean values with SEM. Statistical analyses were performed by Mann–Whitney nonparametric test; *P < 0.05 vs. vehicle.