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. 2021 Aug;20(8):1388-1399.
doi: 10.1158/1535-7163.MCT-20-0591. Epub 2021 Jun 4.

CSF1/CSF1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment

Tomohiro Fujiwara  1   2   3 , Mohamed A Yakoub  1   2 , Andrew Chandler  1   2 , Alexander B Christ  1   2 , Guangli Yang  4 , Ouathek Ouerfelli  4 , Vinagolu K Rajasekhar  1 , Aki Yoshida  3 , Hiroya Kondo  3 , Toshiaki Hata  3 , Hiroshi Tazawa  5 , Yildirim Dogan  6   7 , Malcolm A S Moore  6 , Toshiyoshi Fujiwara  5 , Toshifumi Ozaki  3 , Ed Purdue  2 , John H Healey  8   2
Affiliations

CSF1/CSF1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment

Tomohiro Fujiwara et al. Mol Cancer Ther. 2021 Aug.

Abstract

Colony-stimulating factor 1 (CSF1) is a primary regulator of the survival, proliferation, and differentiation of monocyte/macrophage that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). Considering current advances in understanding the role of the inflammatory tumor microenvironment, targeting the components of the sarcoma microenvironment, such as TAMs, is a viable strategy. Here, we investigated the effect of PLX3397 (pexidartinib) as a potent inhibitor of the CSF1 receptor (CSF1R). PLX3397 was recently approved by the Food and Drug Administration (FDA) to treat tenosynovial giant cell tumor and reprogram TAMs whose infiltration correlates with unfavorable prognosis of sarcomas. First, we confirmed by cytokine arrays of tumor-conditioned media (TCM) that cytokines including CSF1 are secreted from LM8 osteosarcoma cells and NFSa fibrosarcoma cells. The TCM, like CSF1, stimulated ERK1/2 phosphorylation in bone marrow-derived macrophages (BMDMs), polarized BMDMs toward an M2 (TAM-like) phenotype, and strikingly promoted BMDM chemotaxis. In vitro administration of PLX3397 suppressed pERK1/2 stimulation by CSF1 or TCM, and reduced M2 polarization, survival, and chemotaxis in BMDMs. Systemic administration of PLX3397 to the osteosarcoma orthotopic xenograft model significantly suppressed the primary tumor growth and lung metastasis, and thus improved metastasis-free survival. PLX3397 treatment concurrently depleted TAMs and FOXP3+ regulatory T cells and, surprisingly, enhanced infiltration of CD8+ T cells into the microenvironments of both primary and metastatic osteosarcoma sites. Our preclinical results show that PLX3397 has strong macrophage- and T-cell-modulating effects that may translate into cancer immunotherapy for bone and soft-tissue sarcomas.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors TF, MY, AC1 AC2, GY, OO, RV, TO, EP disclosed no potential conflicts relevant to this article. JH is a paid consultant for Daiichi Sankyo.

Figures

Figure 1.
Figure 1.
Profiling of cytokine/chemokine produced from CMG, LM8, and NFSa cell lines. A. Cytokine/chemokine production in the culture supernatant tested by cytokine array. a. G-CSF; b., GM-CSF; c. I-309 (CCL-1); d. Eotaxin (CCL11); e. IFN-γ; f. IL-6; g. IL-7; h. IL-17; i. IP-10 (CXCL-10); j. I-TAC (CXCL-11); k. KC (CXCL-1); l. CSF-1; m. JE (CCL-2); n. MIG (CXCL-9); o. MIP-2 (CXCL-2); p. RANTES (CCL5); q. SDF-1 (CXCL12); r. TIMP-1. B. Concentration of cytokines/chemokines in CMG, TCM/LM8, and TCM/NFSa, evaluated by the signal intensity shown in A.
Figure 2.
Figure 2.
Cellular features of TAMs produced by culture supernatant of bone and soft tissue sarcoma cells. A. Morphology of BMDM-TAMs after expansion of BMDMs with culture supernatant of CMG, LM8, and NFSa cell lines. Bars, 100 μm. B. Western blots showing pERK/2 levels in BMDM-TAMs with M-CSF, TCM/LM8, and TCM/NFSa at the time points indicated. Enhanced pERK1/2 was detected with a peak stimulation after five, 10, and 15 minutes, respectively. C. Surface marker profile of BMDM-TAMs produced by CSF-1/CMG, TCM/LM8, and TCM/NFSa, assessed by flow cytometry. Right, cellular distribution of CD45+/CD11b+ and CD45+CD11b+CD206+/CD45+CD11b+CD80+. Left, the MFI of CD206+/CD80+. N.S., not significant. D. Trans-well assay to evaluate the chemotaxis of BMDMs in response to M-CSF, TCM/LM8, and TCM/NFSa. Left, photographs of BMDMs passed through the trans-well chamber. Scale bar, 200 μm. Right, the numbers of BMDMs passed through the trans-well chamber. Data are presented as mean ± SD (n = 3 per group). ***p < 0.001, Mann-Whitney U test.
Figure 3.
Figure 3.
CSF-1R inhibition reduces M2 polarization and deplete tumor-associated macrophages in bone and soft-tissue sarcoma model. A. Eradication of pERK1/2, stimulated with M-CSF, TCM/LM8, and TCM/NFSa, at 100 nM and 250 nM of PLX3397. B. Depolarization of BMDM-CSF-1, BMDM-LM8, and BMDM-NFSa against M2-like phenotype. Upper, decreased percentage of CD45+/CD11b+/CD206+ populations by PLX3397 treatment (0 nM, 100 nM, and 500 nM). Lower, decreased MFI of CD206+/CD80+ by PLX3397 treatment (0 nM, 100 nM, and 500 nM). C. Quantification of mRNA expression of M1 and M2 genes, including IL-1β, iNOS, CD80, CD206, and CCL-2, after PLX3397 treatment (0 nM, 100 nM, and 500 nM). HPRT was uses as an internal control. Data are presented as mean ± SD (n = 3 per group). D. Survival of TAMs in response to PLX3397 treatment. Right, phase-contrast micrograph of BMDMs in the presence of 100 nmol/l and 500 nmol/l PLX3397. Scale bar, 100 μm (lower) and 25 μm (upper). Light, relative proliferation rates of BMDM-CSF-1, BMDM-LM8, and BMDM-NFSa after 24 hour treatment with increasing dose of PLX33897 (0, 50, 100, 200, and 500 nM). Data are presented as mean ± SD (n = 3 per group). *p < 0.05, ***p < 0.001; one-way analysis of variance with Sidak’s multiple comparisons test. E. Trans-well chemotaxis. Macrophage migration was reduced by PLX3397 in a dose-dependent manner (0, 100, 500, 1000 nmol/l). At the time periods of one-hour post-treatment, cells were seeded and cultured on the trans-well chamber for six hours. The number of migrated cells were photographed (upper) and counted (lower). Scale bar, 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Student’s t-test.
Figure 4.
Figure 4.
CSF-1R inhibition by PLX3397 blocks tumor growth and improves metastasis-free survival in orthotopic osteosarcoma mouse model. A. Macroscopic appearance of LM8-Luc tumors in C3H/JeJ mice on Days 0 and 19 after tumor cell inoculation. Mice were inoculated intratibially with LM8-Luc cells (1 × 106 cells/site) and were systemically treated with control PBS, low-dose PLX3397 (5 mg/kg), or high-dose PLX3397 (10 mg/kg) on Day seven and 14. Tumor masses are outlined by a dotted line. B. Tumor growth in an orthotopic LM8 osteosarcoma xenograft model of each treatment group (n = 5 per group). Data was expressed as mean tumor volume ± SD. *p < 0.05, as compared with high-dose PLX3397 and PBS group; one-way ANOVA corrected for multiple comparisons. C. Luminescence intensity from the primary tumors of each treatment group measured on Day 19 using an IVIS. D. Monitoring of luminescence intensity from the primary tumors of each treatment group. Data was expressed as mean tumor volume ± SD. *p < 0.05, as compared with high-dose PLX3397 and PBS group; one-way ANOVA corrected for multiple comparisons. E. Lung metastases measured on Day 19 using an IVIS. F. Lung metastases validated by H&E staining. Black arrow represents metastatic foci in the lung. Scale bar, 200 μm (left). Scale bar, 40 μm (right). G. Kaplan-Meier curves showing metastasis-free survival for each group of mice. Log-rank test was performed between PBS control group (black line) and low-dose PLX3397 group (blue line; p = 0.353) or high-dose PLX3397 group (red line; *p = 0.0014).
Figure 5.
Figure 5.
Systemic treatment of PLX3397 depletes TAMs and increases lymphocyte infiltration into LM8 osteosarcoma. A. Composition of tumor cells and TAMs evaluated by flow-cytometric analysis using the dissociated LM8 tumor cells. Data were represented as mean ± SEM; n = 3; Mann–Whitney test: *p < 0.05. B. Flow cytometry analysis of TAMs (CD45+CD11b+CD206+; left) and CD8 T cells (CD45+CD3+CD8+; right) within the dissociated LM8 tumor cells and representative flow data. C. Composition of infiltrating immune cells (CD3+, CD4+, and CD8+ cells) evaluated by flow-cytometric analysis using the dissociated LM8 tumor cells. Data were represented as mean ± SEM; n = 3; Mann – Whitney test: *p < 0.05. D. Composition of infiltrating FOXP3+ regulatory T cells evaluated by immunohistochemistry using the dissociated LM8 tumor cells. Data were represented as mean ± SEM; n = 3; Mann–Whitney test: *p < 0.05. E. Distribution of infiltrating CD68+ macrophages, CD8+ T cells, and FOXP3+ regulatory T cells in PLX3397- or PBS-treated tumors (green). Nuclei were stained with DAPI (blue). Left, H&E staining images. Scale bars, 50 μm (lower) and 10 μm (upper).
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