In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer

Abstract

Brain metastases frequently occur in lung cancer and dramatically limit prognosis of affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized. We established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intravital 2-photon laser scanning microscopy. This allowed in vivo tracking of fluorescence-expressing tumor cells and TAM/M on a single-cell level over weeks. Intracarotid injection of red tdTomato-fluorescent Lewis lung carcinoma cell was performed in transgenic mice either proficient or deficient for CX3CR1. After intracarotid cell injection, intravascular tumor cells extravasated into the brain parenchyma and formed micro- and mature macrometastases. We observed potential phagocytosis of extravasated tumor cells by TAM/M. However, during later steps of metastasis formation, these anti-tumor effects diminished and were paralleled by TAM/M accumulation and activation. Although CX3CR1 deficiency resulted in a lower number of extravasated tumor cells, progression of these extravasated cells into micro metastases was more efficient. Overall, this resulted in a comparable number of mature macrometastases in CX3CR1-deficient and -proficient mice. Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic option to prevent dissemination of lung cancer cells to the brain. Given the close interaction between TAM/M and tumor cells during metastasis formation, other therapeutic approaches targeting TAM/M function may warrant further evaluation. The herein established orthotopic mouse model may be a useful tool to evaluate such concepts in vivo .

Tumor Immune Microenvironment and Immunotherapy in Brain Metastasis From Non-Small Cell Lung Cancer

“…In BM, the proportion of most immune cell subpopulations decreases, but with a few exceptions, such as M2-macrophages, whose ratio in the proportion is strongly associated with BM ( 65 ). Therefore, the change of TAM/m from M1 to M2 phenotype may be one of the reasons for the shift of TME from anti-tumor to pro-tumor ( 66 ). Recent in-depth studies of lung cancer BM have revealed that around 10% of TAM/m contain peripheral-derived macrophages in early metastases, rising to 20% in massive metastases ( 67 ).…”

“…In contrast to primary NSCLC, BM microenvironment macrophages infiltrate more and show elevated monocyte-derived macrophages ( 47 ). The phenomena may reflect the antitumor effect of TAM/m in the early stages of BM formation and the accumulation and activation of TAM/m in the later stages ( 66 ).…”

“…C-X3-C motif chemokine receptor 1/C-X3-C motif chemokine ligand 1 axis is a signaling pathway which regulates TAM/m ( 66 ). This pathway has been much studied in brain inflammation, and its activation regulates microglia activation in a quiescent state ( 68 ), thereby inhibiting the release of IL-6, TNF-α, etc.…”

“…The relationship between this pathway and BM is inconclusive and warrants investigation. Considering the complex involvement of C-X3-C motif chemokine receptor 1 in the metastatic cascade, its nonspecific inhibitors may not be an appropriate therapeutic option ( 66 ). In addition, colony-stimulating factor 1 receptor inhibitors acting on TAM have shown promise in melanoma and enhancing chemotherapy response and might be applied in BM ( 70 ).…”

In Vivo Motility Patterns Displayed by Immune Cells Under Inflammatory Conditions

“…The next step to perform a MP-IVM protocol is to select the proper surgical model, to enable the exposure and immobilization of the targeted organ (Figure 1A) (8,9,55). Although this typically requires minimally invasive surgery, more advanced surgical setups can be employed for long-term imaging of internal organs, including gut (56), brain and spinal cord (57), primary tumors and metastasis (58,59) amongst others (60)(61)(62)(63).…”