Crosstalk between breast cancer and macrophages has potential implications for tumor metastasis. This study investigates macrophage polarization induced by triple-negative breast cancer (TNBC) cell-derived exosomes that promote lymph node (LN) metastasis in orthotopic TNBC models. The MDA-MB-231 cancer cell line expressing the exosomal CD63-red fluorescence (RFP) fusion protein was generated to noninvasively visualize exosome transfer into cancer cells and macrophages. Administration of RFP-tagged exosomes enhanced migration of macrophages and induced macrophage polarization in vitro. In orthotopic TNBC models, noninvasive bioluminescent imaging, ultrasound-guided photoacoustic imaging, and histological analysis revealed that intravenous injection of RFP-tagged exosomes promoted primary tumor growth and axillary LN metastasis in which expression of CD206, a marker or alternatively activated type 2 (M2) macrophages, was significantly higher than expression of NOS2, a marker of classically activated type 1 (M1) macrophages. These results suggest breast cancer cell-derived exosomes stimulate macrophage polarization that creates favorable conditions for LN metastatic processes in TNBC.

The microRNA-200 (miR-200) family is associated with tumor metastasis and poor patient prognosis. We found that miR-200c/141 cluster overexpression upregulated SerpinB2 in the MDA-MB-231 triple-negative (TN) breast cancer cell line. We observed transcription factor (c-Jun, c-Fos, and FosB) upregulation, nuclear localization of c-Jun, and increased SerpinB2 promoter-directed chloramphenicol acetyltransferase activity in miR-200c/141 cluster-overexpressing cells relative to controls. Additionally, miR-124a and miR-26b, which directly target SepinB2, were downregulated compared to controls. In mouse xenograft models, miR-200c/141 cluster overexpression promoted lymph node and lung metastasis, and siRNA-mediated SerpinB2 knockdown decreased lung metastasis, suggesting that SerpinB2 mediates miR-200c/141-induced lung metastasis. We also explored the clinical significance of SerpinB2 protein status through analysis of primary breast tumor samples and The Cancer Genome Atlas (TCGA) data. High SerpinB2 levels were associated with reduced survival and increased lymph node metastasis in breast cancer patients. SerpinB2 was overexpressed in the TN breast cancer subtype as compared to the luminal subtype. The present study demonstrates that SerpinB2 promotes miR-200c/141 cluster overexpression-induced breast cancer cell metastasis, and SerpinB2 overexpression correlates with increased metastatic potential and unfavorable outcomes in breast cancer patients. SerpinB2 may be a useful biomarker for assessing metastasis risk in breast cancer patients.

Although inflammation, oxidative stress, and protease-antiprotease imbalance have been referred to as a pathogenic triad in chronic obstructive pulmonary disease (COPD), little is known about how they interact. The objectives of this study were to elucidate the effect of cigarette smoke extract (CSE) on the neutrophil elastase (NE)-induced inflammatory response and its molecular mechanism in bronchial epithelial cells. We observed that NE activated extracellular signal-regulated kinase (ERK) and induced IL-8 production. Blocking ERK activation using a MEK inhibitor (U0126) suppressed NE-induced IL-8 secretion and knockdown of proteinase-activated receptor 2 (PAR2) using siRNAs inhibited both NE-induced ERK activation and subsequent IL-8 release, suggesting that NE-induced IL-8 production is dependent on PAR2-mediated ERK activation. Interestingly, pre-exposure to CSE markedly enhanced NE-induced IL-8 production. As PAR2 acts as a receptor for NE, we next investigated the effect of CSE on PAR2 expression as a molecular mechanism for the increased IL-8 production induced by NE in CSE exposed cells. CSE, but not NE, increased the expression of PAR2 mRNA and surface membrane protein. Inhibition of p38 MAPK reduced PAR2 expression induced by CSE while inhibition of the ERK and Akt pathway had no effect. Consequently, p38 inhibition significantly abrogated CSE-induced enhancement of IL-8 production in NE-treated cells. Of note, we observed increased PAR2 levels in lung homogenates and lung epithelial cells from CSE-treated mice and from both smokers and patients with COPD. Taken together, these results suggest that CSE upregulates PAR2 in normal human bronchial epithelial cells, thereby enhancing the inflammatory response to NE.

BackgroundThe function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model.MethodsGFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody.ResultsIn the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition.ConclusionsOur findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0867-3) contains supplementary material, which is available to authorized users.

The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R2*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R2*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R2* in both in vivo and ex vivo T2*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines.