Brain metastasis is a serious clinical manifestation in cancer patients and develops in approximately 20–30% of patients with solid cancers (1–3). Although management of brain metastasis has improved with multimodal therapies, effective management remains a challenge and the outcome of brain metastases is uniformly poor, with less than 10% of patients with brain metastasis surviving more than 2 years (2, 4). Cancer immunotherapies, i.e. immune-checkpoint inhibitors (ICIs), have enhanced the overall survival of cancer patients and dramatically changed therapeutic strategies for metastatic and other advanced stage of certain types of cancers (5–7). Furthermore, clinical trials have provided evidence that ICIs or ICI combined with radiation therapy could have sustained treatment efficacy for brain metastases (1, 2). However, these treatments can increase the risk of adverse effects, i.e. neurologic toxicity or radiation necrosis, in patients with brain metastases (1, 8). Moreover, no definitive biomarkers have been identified that can differentiate patients with brain metastases who may benefit from ICIs from those at risk for adverse effects (8).

¹⁸F-Fluorodeoxyglucose (FDG) positron emission tomography (PET) is one of the fundamental imaging modalities for pre-therapeutic and therapeutic evaluation as well as end-of-treatment evaluations in clinical practice of many cancers (9–11). ¹⁸F-FDG uptake is associated with elevated glycolysis in cancer cells. However, ¹⁸F-FDG uptake can also be related to inflammation or immune reactions due to the consumption of glucose by immune cells (9, 11–13). Thus, ¹⁸F-FDG uptake in cancer can reflect the tumor microenvironment, including not only the metabolic activity of cancer cells but also local immune reactions (11, 14–16). Since the response to immunotherapy can be associated with tumor infiltrating immune cells (17–20) and immune cell response can be visualized by ¹⁸F-FDG PET (11, 14, 16), research has been conducted on the relationship between ¹⁸F-FDG uptake and immunological features of the tumor microenvironment. In certain types of primary cancers, ¹⁸F-FDG uptake is an additional biomarker that is predictive of immunological features and responses to ICIs (15, 21–24).

Since the microenvironment in brain metastases is different from the primary tumor (2, 25), to improve the efficiency of immunotherapy in brain metastases, a better understanding of the microenvironment in brain metastases, especially immune cell infiltration, is mandatory. However, few studies have used ¹⁸F-FDG uptake for evaluation of immune cell infiltrate in brain metastases (26). Therefore, here we investigated the correlation between ¹⁸F-FDG uptake and infiltration of various immune cells in brain metastases.

The tumor microenvironment of brain metastases is unique and distinct from other sites of the body not only in terms of cellular components but also in metabolism (25, 31, 32). The cellular components of brain include astrocytes, microglia, oligodendrocytes, and neurons that are not present elsewhere in the body (2, 25, 32). In addition, parenchymal cells of the normal brain show high levels of glucose metabolism (33) and this metabolic characteristic of normal brain hampers the delineation of tumors from normal brain by ¹⁸F-FDG compared with amino acid tracers, such as ¹¹C-methionine (MET) and 6-[18F]-L-fluoro-L-3, 4-dihydroxyphenylalanine (FDOPA) (34). Nevertheless, ¹⁸F-FDG is clinically preferred because commercially available ¹⁸F-FDG is the easiest to - perform in facilities without cyclotrons and costs are mostly covered by health insurance, though this can vary from country to country, but also shows cost benefits. In addition, previous studies reported that ¹⁸F-FDG PET provides valuable information on the metabolic status of the tumor microenvironment as well as local immune reactions (14, 15, 21–24, 35). To overcome the weakness of ¹⁸F-FDG in the brain and enhance the delineation of tumor from normal brain, we defined the reference value; the ROI was circularly drawn on the frontal white matter area of the contralateral brain without any abnormal findings on MRI based on previous studies (27–29). We found a wide range of ¹⁸F-FDG uptake in metastatic brain lesions.

¹⁸F-FDG can be taken up by many tumor-associated immune cells, such as tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs) and granulocytes such as neutrophils (11, 14, 16). ¹⁸F-FDG uptake is correlated with PDL-1 expression and TILs, especially CD8+ cytotoxic T cells in primary cancers (14, 22–24). Furthermore, abundant infiltration of TILs including CD8+ T cells in primary cancers is associated with better response to immunotherapy (18, 20, 36, 37). Here we investigated the correlation between ¹⁸F-FDG uptake and immune cell infiltration with various immune cell markers in brain metastases. In contrast to previous reports with primary cancers (14, 22–24), there were no significant correlations between ¹⁸F-FDG uptake and T cell infiltration grade in brain metastases.

¹⁸F-FDG uptake of immune cells in brain metastases can differ from uptake in other sites of the body. Immune cells including lymphocytes, neutrophils and the monocyte/macrophage family express high levels of glucose transporters and hexokinase activity with increased ¹⁸F-FDG uptake (12, 13). However, immune cells in metastatic brain lesions compete to utilize glucose not only with tumor cells but also brain parenchymal cells, because brain parenchymal cells have also high glucose metabolism (33, 38). This difference of the metabolic environment in brain makes the mechanism of ¹⁸F-FDG uptake in brain metastases highly complex. We suggest the possibility that such a unique tumor microenvironment may be one of possible explanations of our negative results.

In brain metastases, T cell infiltration tends to be less frequent than in peripherally located primary lesions whereas infiltration of microglia and monocytes can be abundant (39–41). We found that among immune cells, macrophages most frequently showed diffuse strong infiltration in brain metastases (14.7%, 5/34 cases) followed by neutrophils (5.9%, 2/34 cases). We observed a significantly positive correlation between grades of macrophages markers (CD68 and CD163) and T cell markers (CD3 and CD8). Interestingly, infiltration of macrophages (CD68+) was significantly associated with increased ¹⁸F-FDG uptake in metastatic lesions from the breast, although the number of patients was as small at 10. Contrary to our expectation, the majority of metastatic lesions from the breast revealed only focal mild or multifocal mild infiltration of immune cell infiltration except for neutrophils. In addition, different from other types of metastatic tumors, infiltration of macrophages (CD68) in metastatic breast tumor showed no significant correlation with T cell markers but revealed a negative correlation with Ki-67 proliferative index, suggesting that a less complex immune environment and a low proliferation rate of tumor cells could explain the result. The association of ¹⁸F-FDG uptake with immune cell infiltration in primary breast cancers is controversial. Kajary et al. (42) reported no correlation between TILs and kinetic parameters using whole-body ¹⁸F-FDG PET. However, other studies revealed a significant correlation between ¹⁸F-FDG uptake and TILs in breast cancers (21, 22, 43). Furthermore, all of these studies have focused only on TILs in primary tumors and did not investigate other immune cells such as macrophages. In brain metastases, similar to brain tumors, the majority of immune cells are macrophages that may hinder the cell mediated immune response in metastasis (39–41). Macrophages can polarize as either M1 macrophages or M2 macrophages. M1 macrophages can produce inflammatory mediators directed against pathogens and tumor cells, while M2 macrophages are involved in immunosuppression and repair. Tumor associated macrophages (TAMs) take on a pro-tumoral M2 phenotype involved in growth, extracellular matrix remodeling, angiogenesis and immunosuppression (32, 39, 40, 44). However, such an oversimplification of macrophage phenotype has been disputed because the status of macrophage activation reveals a much wider range in vivo (32, 39). In addition to CD68, we also analyzed macrophages using CD163, which is one of the markers suggesting M2 macrophages. Infiltration of CD163+ macrophages was slightly higher than that of CD68+ macrophages. However, we could not find a significant correlation between the infiltration of CD163+ macrophages and ¹⁸F-FDG uptake.

Pukrop T et al. (45) reported that microglia/macrophages can be identified in brain metastases from the breast, ranging from only few to up to 50% of all cells. TAMs within the brain tend to be pro-tumorigenic and TAM depletion strategies may provide a survival advantage in several types of cancer (32). Activated TAMs promoted cancer cell invasion and colonization of the brain tissue in vitro whereas blocking microglia function reduced cancer cell invasion. However, TAMs can be activated by cancer cells without polarization to M2 macrophages (45). We found that infiltration of CD68+ macrophages in brain metastases was only significantly correlated with maximum and mean ¹⁸F-FDG uptake ratios at the metastatic lesions (p < 0.001 and p = 0.005, respectively). Since TAM density is associated with poor prognosis in breast cancer patients and eliminating macrophages from the tumor site in mouse models of breast cancer induced a delay of tumor progression, targeting TAM in brain metastases from the breast may provide a new therapeutic strategy (25, 40, 41, 46).

In addition to immune cell infiltration in brain metastases, we also analyzed additional biologic makers related to ¹⁸F-FDG uptake. ¹⁸F-FDG uptake in cancer tissues from primary malignant lesions is commonly associated with high levels of HK and GLUT (47–50). In addition, the Ki-67 proliferation index, which indicates the growth rate of tumor cells, is also associated with tumor ¹⁸F-FDG uptake (51). However, we could not find associations of ¹⁸F-FDG uptake in brain metastases with GLUT1 or HK2 or the Ki-67 proliferation index. We cannot explain these negative results. However, we suggest that the mechanism involving ¹⁸F-FDG uptake in the brain metastases may be more complex than in the primary cancer due to a unique tumor microenvironment in which not only cancer cells but also immune cells and brain parenchymal cells compete to utilize glucose for survival (25, 52). The complex mechanisms that influence ¹⁸F-FDG uptake in brain metastasis remain to be determined. Therefore, our study may provide reference data for subsequent studies to address the mechanism of ¹⁸F-FDG uptake in brain metastases.

The small number of patients included in this study may be a limitation to our study. It was not easy to find cancer patients available for brain ¹⁸F-FDG PET and with pathological data of metastatic brain lesion. Although we confirmed that the number of patients in this study satisfied the statistically meaningful sample size, we also acknowledge that the sample number is small. In particular, the number of samples in subgroups according to primary cancer site was very small. Therefore, future studies including large samples are needed to validate our study results.

In conclusion, we investigated the degree of ¹⁸F-FDG uptake in brain metastases and its correlation with immune cell infiltration and several biologic markers. ¹⁸F-FDG uptake was not correlated with immune cells and other biologic markers. However, in certain types of metastatic cancer, ¹⁸F-FDG uptake may be a non-invasive tool for predicting immunological features of brain metastases.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study was conducted retrospectively and was approved by the Institutional Review Board of Ajou University (AJIRB-MED-MDB-19-244). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Y-SA and J-HK conceived of the presented idea. Y-SA and J-HK developed the theory and performed the computations. S-HK, TR, SP, and T-GK verified the analytical methods. J-HK encouraged SP and T-GK to investigate histologic aspect and supervised the findings of this work. All authors contributed to the article and approved the submitted version.

This work was supported by the faculty research fund of Ajou University School of Medicine to S-HK and J-HK, National Research Foundation of Korea to J-HK (NRF-2016R1D1A1B02010452), and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2020R1A6A1A03043539) to J-HK.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.