Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells.

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

Epithelial/Mesenchymal Characteristics and PD-L1 Co-Expression in CTCs of Metastatic Breast Cancer Patients Treated with Eribulin: Correlation with Clinical Outcome.

We aimed to evaluate the co-expression of PD-L1 and epithelial-mesenchymal markers in CTCs from metastatic breast cancer (MBC) patients and to determine if there is any relationship with patients' outcome after eribulin treatment. Using cytospin preparations of peripheral blood mononuclear cells (PBMCs) from MBC patients treated with eribulin and a combination of immunocytochemistry and immunofluorescence, we quantified PD-L1, keratins and vimentin in single and cluster CTCs on days 1 and 8 of the first-treatment cycle. CTCs (n = 173) were found in 31 out of 38 patients. At baseline, the presence of cluster CTCs (p = 0.048), cluster mesenchymal CTCs (mCTCs) (p = 0.0003) or cluster PD-L1+mCTCs (p = 0.006) was associated with shorter overall survival (OS). In multivariate cox regression analysis, the detection of cluster mCTCs was the only parameter associated with increased risk of death (p = 0.024). On day 8 post-eribulin administration, PD-L1+mCTCs and especially single PD-L1+mCTCs decreased in 75% and 89% of patients, respectively. The detection of single PD-L1+mCTCs after eribulin treatment was correlated with shorter PFS (p = 0.047) and OS (p = 0.020). In conclusion, our study identified for the first time that cluster and single PD-L1+mCTCs subpopulations are of clinical significance in patients with MBC and highlighted the importance of CTC phenotyping during treatment with eribulin.

Nuclear localization of PD-L1: artifact or reality?

BACKGROUND: The levels of expression and membrane localization of programmed cell death ligand 1 (PD-L1), an immune checkpoint type I transmembrane glycoprotein, are related to the clinical response of anti-PD-L1/PD-1 therapy. Although the biologically relevant localization of PD-L1 is on the plasma membrane of cancer cells, it has also been reported to be in the cytoplasm and sometimes in the nucleus. Furthermore, it has been claimed that chemotherapeutics can modify PD-L1 expression and/or its nuclear localization. RESULTS: Data from our group suggest that the nuclear localization of PD-L1, and other plasma membrane proteins as well, could be an artifact resulting from inadequate experimental conditions during immunocytochemical studies. Mild detergent and rigorous fixation conditions should be used in order to preserve the membrane localization and to prevent an erroneous translocation of PD-L1 and other non-interconnected membrane proteins, such as CD24, into other cellular compartments including the nucleus, of untreated and chemotherapeutically treated breast cancer cells. CONCLUSION: We propose that well-specified and rigorously followed protocols should be applied to immunocytochemical diagnostic techniques, especially to those related to individualized diagnosis and treatment.