Abstract
Cancer metastasis to the brain is a significant clinical problem. Metastasis is the consequence of favorable interactions between invaded cancer cells and the microenvironment. Here, we demonstrate that cancer-activated astrocytes create a sustained low-level activated type I interferon (IFN) microenvironment in brain metastatic lesions. We further confirm that the IFN response in astrocytes facilitates brain metastasis. Mechanistically, IFN signaling in astrocytes activates C-C Motif Chemokine Ligand 2 (CCL2) production, which further increases the recruitment of monocytic myeloid cells. The correlation between CCL2 and monocytic myeloid cells is confirmed in clinical brain metastasis samples. Lastly, genetically or pharmacologically inhibiting C-C Motif Chemokine Receptor 2 (CCR2) reduces brain metastases. Our study clarifies a pro-metastatic effect of type I IFN in the brain even though IFN response has been considered to have anti-tumor effects. Moreover, this work expands our understandings on the interactions between cancer-activated astrocytes and immune cells in brain metastasis.
Introduction
Brain metastasis is the most ominous form of relapse in cancer patients, which is associated with poor prognosis and almost invariably lethal. The most common sources of brain metastasis are lung, breast carcinoma, and melanoma. Despite significant improvements in cancer treatment, current therapies have limited efficacy in brain metastases1,2,3,4,5. Consequently, brain metastasis becomes a significant clinical challenge in patients who have survived the primary tumor and extracranial metastases. Therefore, there is an urgent need to overcome this challenge by identifying mechanistic insights, prognostic markers, and novel therapeutic targets in brain metastasis studies.
Upon arrival in the distal organs, invaded cancer cells passively adapt to the new microenvironment. Meanwhile, the cancer cells actively modify the stromal cells to create a metastasis-specific microenvironment6,7. In the brain, astrocytes are the most abundant stromal cells. Metastatic cancer cells induce astrogliosis by activating the surrounding astrocytes, marked by increased glial fibrillary acidic protein (GFAP) expression and cellular processes8,9. Of note, the versatile astrocytes have diverse functions. On one hand, astrocytes release the killing factor in the microenvironment to induce cancer cell apoptosis10. On the other hand, astrocytes have been shown to facilitate cancer cell survival, growth, and migration at different stages of metastatic outgrowth10,11,12,13,14,15,16,17. Interactions between cancer cells and astrocytes in the brain microenvironment are dynamic, complex, and far more inextricably linked.
Immune cells are the most studied microenvironmental cells in cancer. Multiple myeloid subpopulations have been shown to facilitate carcinogenesis and metastasis. Myeloid-derived suppressor cells (MDSC), including monocytic MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC), are currently defined as pathologically activated monocytes and neutrophils, respectively18,19,20. Tumor-associated macrophages are either differentiated from recruited monocytes and M-MDSC from circulation or modified from tissue residential macrophages21,22,23. Modified by the tumor microenvironment, these flexible myeloid cells elicit immunosuppressive functions to promote tumor growth18,19,20,21,22. Our knowledge on the immune cells in brain metastatic lesions is very limited. The specialized brain–blood barrier (BBB) provides a highly selective permeability barrier to the entrance of immune cells24. Thus, the brain used to be defined as an immune-privileged organ. The immunotherapies were expected to be ineffective in brain tumors due to the limited drug delivery and immune responses in the brain. Patients with active brain metastatic lesions were invariably excluded from immunotherapy clinical trials. However, this concept has been revised. The brain has an immune-specialized rather than immune-privileged environment under pathological conditions25. Retrospective studies indicate that multiple immunotherapies improved overall survival in brain metastasis patients26,27,28,29,30. Therefore, immune cells are an important component in the brain microenvironments that regulate metastatic outgrowth.
Our current study detects chronic low-level type I interferon (IFN) activation in the cancer cells and the reciprocal astrocytes throughout the brain metastatic process. IFN responses in astrocytes promote brain metastasis by enhancing the recruitment of monocytic myeloid cells in both breast cancer and melanoma models. We further delineate that IFN signaling activates the production of C-C motif chemokine ligand 2 (CCL2) in reactive astrocytes. Genetically or pharmacologically targeting C-C motif chemokine receptor 2 (CCR2), the paired receptor of CCL2 on monocytic myeloid cells, decreases brain metastasis.
Results
Sustained low-level activation of type I IFN in brain metastasis
Once extravasated into the brain parenchyma, the invaded cancer cells not only passively adapt to the new microenvironment but also actively modify the surrounding brain stromal cells. To better understand the mutual interactions between astrocytes and brain metastatic cancer cells, we set up in vitro coculture experiments and performed RNAseq in both cell types (Fig. 1a and Supplementary Fig. 1a, b). We used primary human astrocytes and MDA231-BrM cells, the brain metastatic derivatives (BrM) of human breast cancer MDA-MB-231 cells31,32, in the coculture experiments. BrM cells were stably labeled with fluorescent protein which allowed us to sort out cancer cells and astrocytes after coculture (Supplementary Fig. 1b, c). Compared to cultured alone condition, we unbiasedly screened the BrM cell-induced changes in astrocytes as well as the astrocyte-induced changes in BrM cells. Ingenuity Pathway Analysis (IPA) of significantly differentially expressed genes (false discovery rate (FDR) < 5%) allowed us to identify the signaling pathways activated by coculture. Of note, in both astrocytes and BrM cells, IFN signaling pathway was the top activated pathway (Fig. 1b, c)….
