Characterization of surface markers on extracellular vesicles isolated from lymphatic exudate from patients with breast cancer.

BACKGROUND: Breast cancer is the most common cancer, and the leading cause of cancer-related deaths, among females world-wide. Recent research suggests that extracellular vesicles (EVs) play a major role in the development of breast cancer metastasis. Axillary lymph node dissection (ALND) is a procedure in patients with known lymph node metastases, and after surgery large amounts of serous fluid are produced from the axilla. The overall aim was to isolate and characterize EVs from axillary serous fluid, and more specifically to determine if potential breast cancer biomarkers could be identified. METHODS: Lymphatic drain fluid was collected from 7 patients with breast cancer the day after ALND. EVs were isolated using size exclusion chromatography, quantified and detected by nanoparticle tracking analysis, electron microscopy, nano flow cytometry and western blot. The expression of 37 EV surface proteins was evaluated by flow cytometry using the MACSPlex Exosome kit. RESULTS: Lymphatic drainage exudate retrieved after surgery from all 7 patients contained EVs. The isolated EVs were positive for the typical EV markers CD9, CD63, CD81 and Flotillin-1 while albumin was absent, indicating low contamination from blood proteins. In total, 24 different EV surface proteins were detected. Eleven of those proteins were detected in all patients, including the common EV markers CD9, CD63 and CD81, cancer-related markers CD24, CD29, CD44 and CD146, platelet markers CD41b, CD42a and CD62p as well as HLA-DR/DP/DQ. Furthermore, CD29 and CD146 were enriched in Her2+ patients compared to patients with Her2- tumors. CONCLUSIONS: Lymphatic drainage exudate retrieved from breast cancer patients after surgery contains EVs that can be isolated using SEC isolation. The EVs have several cancer-related markers including CD24, CD29, CD44 and CD146, proteins of potential interest as biomarkers as well as to increase the understanding of the mechanisms of cancer biology.

Heat inactivation of foetal bovine serum performed after EV-depletion influences the proteome of cell-derived extracellular vesicles.

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.

Cancer stem cells are prevalent in the basal-like 2 and mesenchymal triple-negative breast cancer subtypes in vitro.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with the most unfavorable clinical outcomes, in part due to tumor heterogeneity, treatment resistance, and tumor relapse. The TNBC subtypes [basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M), and luminal androgen receptor (LAR)] are biologically and clinically distinct entities that respond differently to local and systemic therapies. Therefore, we need to have a better understanding of cancer stemness relating to drug-resistant populations in the TNBC subtypes. Methods: Breast cancer stem cell (BCSC) distribution was investigated using an integrated flow cytometry approach with the ALDEFLUOR™ assay (ALDH) and CD24/CD44 antibodies. In total, 27 commercially available cell lines derived from normal and malignant mammary tissue were characterized into differentiated tumor cells and/or BCSC subpopulations (ALDH-CD44+CD24-/low enriched mesenchymal-like BCSCs, ALDH+non-CD44+CD24-/low enriched epithelial-like BCSCs, and highly purified ALDH+CD44+CD24-/low BCSCs). Results: BCSCs were not only enriched in estrogen receptor (ER) negative (mean, 49.6% versus 6.9% in ER+) and TNBC cell lines (51.3% versus 2.1% in Luminal A), but certain BCSC subpopulations (e.g., enriched mesenchymal-like BCSCs) were also significantly more common in the M (64.0% versus 6.2% in BL1; 64.0% versus 0% in LAR) and BL2 (77.4% versus 6.2% in BL1; 77.4% versus 0% in LAR; 77.4% versus 10.4% in TNBC UNS) TNBC subtypes. In contrast, ALDH status alone was not indicative of ER status or BC subtype. Conclusion: Taken together, these findings demonstrate the enrichment of potentially treatment-resistant BCSC subpopulations in the M and BL2 triple-negative breast cancer subtypes.

Anti-PD-1 checkpoint blockade improves the efficacy of a melphalan-based therapy in experimental melanoma.

INTRODUCTION: The induction of adaptive cellular immunity in patients with in-transit melanoma metastasis treated with hyperthermic isolated limb perfusion (ILP) with melphalan has been shown to contribute to the effectiveness of the therapy. Activated CD8+ T cells appear to be of particular importance for the efficacy of melphalan-based ILP therapy, as observed in both patients and animal models. In this study, we explored the possible synergistic effects of combining melphalan-based therapy with the checkpoint inhibitor anti-PD-1 on tumours in a mouse melanoma model. METHODS: A murine vaccination model that utilized melphalan-exposed melanoma cells was used to mimic certain immunological features of melphalan-based ILP. The effects of the vaccine on tumour growth and PD-1 expression on CD8+ tumour-infiltrating T cells were analyzed. The melphalan-based vaccine was then combined with an anti-PD-1 antibody and tumour growth was assessed. RESULTS: Treatment with melphalan-based therapy significantly induced the expression of PD-1 on CD8+ tumour-infiltrating lymphocytes. Combination therapy using melphalan-based therapy followed by treatment with PD-1 antibodies significantly reduced early-stage tumour growth relative to monotherapies and no treatment. CONCLUSIONS: This study thus suggests that the addition of PD-1 blockade to melphalan-based therapies, such as ILP, may be therapeutically beneficial.

Presence of tumor-infiltrating CD8+ T cells and macrophages correlates to longer overall survival in patients undergoing isolated hepatic perfusion for uveal melanoma liver metastasis.

Uveal melanoma is a malignant tumor of the eye that often metastasizes to the liver conferring poor prognosis. When comparing immune profiles in peripheral blood of untreated patients with uveal melanoma liver metastasis and healthy blood donors, it was observed that immune cells of uveal melanoma patients carried immunosuppressive features. Patient blood contained an increased content of CD14+HLA-DR-/low M-MDSCs and inflammatory CD16+ monocytes, while their dendritic cells expressed lower levels of activation markers. Melanoma patients also harbored an enhanced fraction of CD4+Foxp3+ regulatory T cells, while their effector T cells expressed lower levels of the activation marker HLA-DR. Biopsies from liver metastases were obtained from patients with uveal melanoma that subsequently underwent hyperthermic isolated hepatic perfusion (IHP) with melphalan. There were trends indicating a positive correlation between a high infiltration of CD8+ T cells in metastases and an activated immune cell profile in blood. High metastatic infiltration of CD8+ T cells and CD68+ macrophages, but not of immunosuppressive CD163+ macrophages, correlated to a longer overall survival in patients treated with IHP. Hence, while the immune system of patients with uveal melanoma shows signs of immunosuppression, the presence of activated immune cells may correlate to a longer survival, at least following IHP treatment.

Isolated limb perfusion with melphalan activates interferon-stimulated genes to induce tumor regression in patients with melanoma in-transit metastasis.

Hyperthermic isolated limb perfusion (ILP) with high-dose melphalan is a treatment option for melanoma patients with metastasis confined to limbs (in-transit metastasis). The therapy entails a complete response (CR) rate of 50-70%. Cellular immunity is proposed to impact on the clinical efficacy of ILP, but the detailed aspects of ILP-induced immune activation remain to be explored. For this study, we explored the potential role of interferon-stimulated gene (ISG) products, including CXCL10, CCL2, PD-L2 and IFN-γ along with expression of their cognate receptors CXCR3, CCR4, CCR5 and PD-1 on lymphocytes, for the clinical efficacy of ILP. Patients with high serum levels of CXCL10, CCL2, PD-L2 and IFN-γ were more likely to achieve CR after ILP. Additionally, the expression of CXCR3, CCR4 and CCR5 on T cells and/or natural killer (NK) cells was enhanced by ILP. Peripheral blood mononuclear cells (PBMCs) secreted high levels of CXCL10, CCL2 and IFN-γ in response to co-culture with melphalan-exposed melanoma cells in vitro. Activated T cells migrated toward supernatants from these co-cultures. Furthermore, melphalan-exposed melanoma cells triggered upregulation of CXCR3, CCR4, CCR5 and PD-1 on co-cultured T cells and/or NK cells. Our results suggest that constituents released from melphalan-exposed melanoma cells stimulate the ISG axis with ensuing formation of chemokines and upregulation of chemokine receptor expression on anti-neoplastic immune cells, which may contribute in ILP-induced tumor regression.

Isolated Limb Perfusion With Melphalan Triggers Immune Activation in Melanoma Patients.

Hyperthermic isolated limb perfusion with melphalan (M-ILP) is a treatment option for melanoma patients with metastases confined to the limbs. This study aimed at defining the role of cellular immunity for the clinical response to M-ILP in melanoma patients. It was observed that patients with enhanced cytotoxic CD8+ T cell reactivity to common antigens (HCMV/EBV/influenza virus) prior to M-ILP were more likely to achieve a complete disappearance of macroscopic tumors (complete response). Following M-ILP treatment, the proportions of CD16+ intermediate and non-classical monocytes in peripheral blood were significantly enhanced along with induction of HLA-DR on CD4+ and CD8+ T cells. For further studies of the mechanism behind melphalan-induced immune activation an in vitro model, aiming at mimicking the clinical M-ILP protocol, was established, where PBMCs were co-cultured with melanoma cells, which had been pre-exposed to melphalan under mild hyperthermia. Upon exposure to melphalan, melanoma cells showed increased expression of immune-related markers including MHC class I and Hsp70. Moreover, when the melphalan-treated melanoma cells were co-cultured with PBMCs, this triggered an increased proportion of CD33+CD14+CD16++ non-classical monocytes among the PBMCs. Furthermore, the melphalan-treated melanoma cells stimulated the expansion of CD8+ T cells in the co-cultured PBMCs. These cells produced enhanced levels of IFN-γ and granzyme B and were capable of killing melanoma cells. To further verify an immunogenic role of melphalan, mice were vaccinated with melphalan-exposed murine melanoma cells. When challenged with live melanoma cells, vaccinated mice showed reduced tumor growth and enhanced infiltration of tumor-specific T cells into tumors. We conclude that melphalan-exposed melanoma cells trigger expansion of CD16+ monocytes and activate cytotoxic T cells and that these events may contribute to the antitumoral efficacy of M-ILP.

Role of NOX2-Derived Reactive Oxygen Species in NK Cell-Mediated Control of Murine Melanoma Metastasis.

The NADPH oxidase of myeloid cells, NOX2, generates reactive oxygen species (ROS) to eliminate pathogens and malignant cells. NOX2-derived ROS have also been proposed to dampen functions of natural killer (NK) cells and other antineoplastic lymphocytes in the microenvironment of established tumors. The mechanisms by which NOX2 and ROS influence the process of distant metastasis have only been partially explored. Here, we utilized genetically NOX2-deficient mice and pharmacologic inhibition of NOX2 to elucidate the role of NOX2 for the hematogenous metastasis of melanoma cells. After intravenous inoculation of B16F1 or B16F10 cells, lung metastasis formation was reduced in B6.129S6-Cybbtm1DinK (Nox2-KO) versus Nox2-sufficient wild-type (WT) mice. Systemic treatment with the NOX2-inhibitor histamine dihydrochloride (HDC) reduced melanoma metastasis and enhanced the infiltration of IFNγ-producing NK cells into lungs of WT but not of Nox2-KO mice. IFNγ-deficient B6.129S7-Ifngtm1Ts /J mice were prone to develop melanoma metastases and did not respond to in vivo treatment with HDC. We propose that NOX2-derived ROS facilitate metastasis of melanoma cells by downmodulating NK-cell function and that inhibition of NOX2 may restore IFNγ-dependent, NK cell-mediated clearance of melanoma cells. Cancer Immunol Res; 5(9); 804-11. ©2017 AACR.

Mixture effects between different azoles and ß-naphthoflavone on the CYP1A biomarker in a fish cell line.

The cytochrome P450 1A (CYP1A) biomarker response was studied in the Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) cell line, which represents a good model for studies on aryl hydrocarbon receptor (AhR) - CYP1A signaling. The PLHC-1 cells were exposed to the prototypical CYP1A inducer and AhR agonist ß-naphthoflavone (BNF) in combination with different azoles. Two imidazoles (clotrimazole and prochloraz) and two benzimidazoles (nocodazole and omeprazole) were used. Exposure to clotrimazole, prochloraz and nocodazole resulted in 2-4 fold induction of the CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activities at 24 and 48h, whereas exposure to the omeprazole for 48h had no effect on the EROD activity. Clotrimazole, nocodazole and prochloraz also acted as inhibitors of EROD activities in situ in PLHC-1 cells (IC50=1.3-7.7µM), whereas omeprazole had no effect on this activity (IC50=72µM). Exposure to 10µM prochloraz resulted in 3-fold induction of CYP1A mRNA and exposure to 10µM nocodazole resulted in 16-fold induction of CYP1A mRNA levels at 24h compared to controls. In the mixture experiments, more-than-additive mixture effects between BNF and the azoles clotrimazole, prochloraz and nocodazole on EROD activities were evident, with nocodazole showing the strongest mixture effect. The presence of nocodazole increased the response to BNF up to 200-fold on CYP1A mRNA and up to 16-fold on EROD activities and prolonged the effect of BNF exposure on EROD activities by 24h or longer. This suggests that azoles that are inhibitors and/or competing substrates for the CYP1A enzymes can cause increased sensitivity to exposures to chemicals that depend on CYP1A metabolism for their elimination in situations of mixed chemical exposures. The results also suggest that the EROD biomarker response can be significantly affected in azole-contaminated areas. The responsiveness of the EROD biomarker to BNF exposure was studied in PLHC-1 that had been pre-treated with nocodazole for 5 or 24h at concentrations that are known to disassemble microtubules at 24h in these cells. Pre-treatment of PLHC-1 cells with nocodazole for either 5 or 24h had no effect on the responsiveness to BNF exposure, which implies that the EROD activity can be induced in cells with disassembled microtubules.