Because of improvements in the treatment of patients with metastatic breast cancer, the development of brain metastases (BM) has become a major limitation of life expectancy and quality of life for many breast cancer patients. The improvement of management strategies for BM is thus an important clinical challenge, especially among high-risk patients such as human epidermal growth factor receptor 2-positive and triple-negative patients. However, the formation of BM as a multistep process is thus far poorly understood. To grow in the brain, single tumor cells must pass through the tight blood–brain barrier (BBB). The BBB represents an obstacle for circulating tumor cells entering the brain, but it also plays a protective role against immune cell and toxic agents once metastatic cells have colonized the cerebral compartment. Furthermore, animal studies have shown that, after passing the BBB, the tumor cells not only require close contact with endothelial cells but also interact closely with many different brain residential cells. Thus, in addition to a genetic predisposition of the tumor cells, cellular adaptation processes within the new microenvironment may also determine the ability of a tumor cell to metastasize. In this review, we summarize the biology of breast cancer that has spread into the brain and discuss the implications for current and potential future treatment strategies.

Background: Brain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC. Methods: Immunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice. Results: ALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors. Conclusions: Our findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM. Key Points: 1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC. 2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium. 3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.

Alterations in protein glycosylation have been related to malignant transformation and tumour progression. We recently showed that low mRNA levels of Golgi alpha-mannosidase MAN1A1 correlate with poor prognosis in breast cancer patients. We analysed the role of MAN1A1 on a protein level using western blot analysis (n=105) and studied the impact of MAN1A1-related glycosylation on the prognostic relevance of adhesion molecules involved in breast cancer using microarray data (n=194). Functional consequences of mannosidase inhibition using the inhibitor kifunensine or MAN1A1 silencing were investigated in breast cancer cells in vitro. Patients with low/moderate MAN1A1 expression in tumours showed significantly shorter disease-free intervals than those with high MAN1A1 levels (P=0.005). Moreover, low MAN1A1 expression correlated significantly with nodal status, grading and brain metastasis. At an mRNA level, membrane proteins ALCAM and CD24 were only significantly prognostic in tumours with high MAN1A1 expression. In vitro, reduced MAN1A1 expression or mannosidase inhibition led to a significantly increased adhesion of breast cancer cells to endothelial cells. Our study demonstrates the prognostic role of MAN1A1 in breast cancer by affecting the adhesive properties of tumour cells and the strong influence of this glycosylation enzyme on the prognostic impact of some adhesion proteins.

Aberrant sialylation of glycoproteins has been detected in many tumors, and upregulation of the beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6GAL1) has been implicated with tumor aggressiveness and chemoresistance in experimental models. In our present study, we aimed to study the prognostic or predictive role of ST6GAL1 in ovarian carcinoma, using two independent ovarian cancer cohorts. ST6GAL1 mRNA levels were retrieved from a publicly available database (n = 517), and ST6GAL1 protein levels were analyzed by western blot analysis in a cohort of 204 ovarian tumor samples. The results were correlated with clinical and histological tumor parameters and follow-up information. High ST6GAL1 mRNA levels significantly correlated with lymphovascular invasion and shorter survival, whereas high ST6GAL1 protein expression was associated with advanced stage, distant metastasis and shorter recurrence-free intervals. In both cohorts the prognostic role was most pronounced in tumors without macroscopically visible residual tumor after surgery. In these cases, ST6GAL1 expression levels might help to identify cases with a higher risk of chemoresistance and metastatic relapse that might require an adapted therapeutic regime.

Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from n = 18 OvCA patients and PBL from age-matched healthy donors (HD, n = 14). Multicolor flow cytometry was performed to evaluate the expression of inhibitory receptors (TIGIT, PD-1 and TIM-3), stimulatory receptors (Ox40), and purinergic ectoenzymes (CD39 and CD73) on γδ T cell subsets. We identified an abundant infiltration of Vδ1 T cells in the MALs and TILs. These cells varied in their differentiation: The majority of Vδ1 TILs displayed an effector memory (EM) phenotype, whereas Vδ1 MALs had a more mature phenotype of terminally differentiated effector memory cells (TEMRA) with high CD45RA expression. TIGIT and TIM-3 were abundantly expressed in both MALs and PBLs, whereas Vδ1 TILs exhibited the highest levels of PD-1, CD39, and Ox40. We also observed specific clusters on mature differentiation stages for the analyzed molecules. Regarding co-expression, Vδ1 TILs showed the highest levels of cells co-expressing TIGIT with PD-1 or CD39 compared to MALs and PBLs. In conclusion, the Vδ1 T cell population showed a high prevalence in the MALs and primary tumors of OvCA patients. Due to their (co-)expression of targetable immune receptors, in particular TIGIT with PD-1 and CD39 in TILs, Vδ1 T cell-based approaches combined with the inhibition of these targets might represent a promising strategy for OvCA.