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- Crescitelli, Rossella, 1985, et al.
(författare)
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Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation
- 2020
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs in vivo. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small and large EVs were isolated with differential ultracentrifugation, and these were further separated into high and low-density (HD and LD) fractions. All EV subpopulations were then analysed in depth using electron microscopy, Bioanalyzer (R), nanoparticle tracking analysis, and quantitative mass spectrometry analysis. Subpopulations of EVs with distinct size, morphology, and RNA and protein cargo could be isolated from the metastatic melanoma tissue. LD EVs showed an RNA profile with the presence of 18S and 28S ribosomal subunits. In contrast, HD EVs had RNA profiles with small or no peaks for ribosomal RNA subunits. Quantitative proteomics showed that several proteins such as flotillin-1 were enriched in both large and small LD EVs, while ADAM10 were exclusively enriched in small LD EVs. In contrast, mitofilin was enriched only in the large EVs. We conclude that enzymatic treatments improve EV isolation from dense fibrotic tissue without any apparent effect on molecular or morphological characteristics. By providing a detailed categorization of several subpopulations of EVs isolated directly from tumour tissues, we might better understand the function of EVs in tumour biology and their possible use in biomarker discovery.
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| 2. |
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| 3. |
- Johansson, Junko, 1989, et al.
(författare)
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Isolated limb perfusion with melphalan activates interferon-stimulated genes to induce tumor regression in patients with melanoma in-transit metastasis
- 2020
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Ingår i: Oncoimmunology. - 2162-402X. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- 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-gamma 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-gamma 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-gamma 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.
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| 4. |
- Johansson, Junko, 1989, et al.
(författare)
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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
- 2020
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Ingår i: OncoImmunology. - : Informa UK Limited. - 2162-402X. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Kiffin, Roberta, et al.
(författare)
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Anti-PD-1 checkpoint blockade improves the efficacy of a melphalan-based therapy in experimental melanoma
- 2021
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Ingår i: Ejso. - : Elsevier BV. - 0748-7983. ; 47:9, s. 2460-2464
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Tidskriftsartikel (refereegranskat)abstract
- 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 mono-therapies 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. (C) 2021 Published by Elsevier Ltd.
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