Immune checkpoint regulators such as PD-L1 have become exciting new therapeutic targets leading to long lasting remissions in patients with advanced malignancies. However, in view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non-responders are urgently needed. In the present paper, we provide evidence that PD-L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor-positive, HER2-negative breast cancer patients. We performed western blot, flow cytometry and immunocytochemical analyses to demonstrate the specificity of the PDL1 antibody used in our study and established immunoscores for PDL1 expression on single tumor cells. We then selected sixteen patients with circulating tumor cells (CTCs) using the CellSearch(®) system and found PD-L1((+)) CTCs in 11 patients (68.8%). The fraction of PD-L1((+)) CTCs varied from 0.2 to 100% in individual patients. This is the first report demonstrating the expression of PD-L1 on CTCs. The established CTC/PD-L1 assay can be used for liquid biopsy in future clinical trials for stratification and monitoring of cancer patients undergoing immune checkpoint blockade.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry. Low expression of miR-30 in primary tumors from patients with breast cancer were associated with poor relapse-free survival. In addition, estrogen receptor (ER)-negative/progesterone receptor (PR)-negative breast cancer cells expressed lower miR-30 levels than their ER/PR-positive counterparts. Overexpression of miR-30 in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden , miR-30 did not affect tumor cell proliferation, but did inhibit tumor cell invasion. Furthermore, overexpression of miR-30 restored bone homeostasis by reversing the effects of tumor cell-conditioned medium on osteoclastogenesis and osteoblastogenesis. A number of genes associated with osteoclastogenesis stimulation (), osteoblastogenesis inhibition (), tumor cell osteomimicry (), and invasiveness () were identified as targets for repression by miR-30. Among these genes, silencing or in ER-/PR-negative breast cancer cells recapitulated inhibitory effects of miR-30 on skeletal tumor burden Overall, our findings provide evidence that miR-30 family members employ multiple mechanisms to impede breast cancer bone metastasis and may represent attractive targets for therapeutic intervention. These findings suggest miR-30 family members may serve as an effective means to therapeutically attenuate metastasis in triple-negative breast cancer. .

Metastases arise from disseminated tumor cells (DTC) that colonize secondary organs. However, DTC survival strategies to start metastatic outgrowth are unclear. The hostile (hypoxic, hypoglycemic) microenvironmental conditions of the bone marrow serve as an ideal model environment for investigation of DTC survival strategies under environmental stress. We investigated the breast cancer DTC cell line BC-M1 established from the bone marrow of a cancer patient by 2-D DIGE and MS analysis. We observed specific overexpression of the unfolded protein response (UPR) proteins Grp78, Grp94, and protein disulfide-isomerase in breast, lung, and prostate cancer DTC cell lines from the bone marrow. The UPR contributes to survival under adverse environmental conditions including chemotherapy. We show in cellular models that Grp78 expression of the UPR is regulated by tyrosine 1248 of ErbB-2. The breast cancer DTC cell lines shared stem/progenitor cell cancer phenotypes (CD44(high)/CD24(low)). Immunocytochemical staining of bone marrow samples from breast cancer patients confirmed in situ high expression of Grp78 and Grp94 in DTC of breast cancer patients, indicating the potential of both proteins as novel markers for DTC detection. Our results suggest the presence of a previously not recognized stress resistant DTC population that combines stem/progenitor attributes with an UPR phenotype.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC‐derived breast cancer cell line, designated CTC‐ITB‐01, established from a patient with metastatic estrogen receptor‐positive (ER+) breast cancer, resistant to endocrine therapy. CTC‐ITB‐01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC‐ITB‐01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA‐sequencing data indicate that CTC‐ITB‐01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC‐ITB‐01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC‐ITB‐01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.