Metastatic melanoma is an untreatable cancer lacking reliable and non-invasive markers of disease progression. Exosomes are small vesicles secreted by normal as well as tumor cells. Human ...tumor-derived exosomes are involved in malignant progression and we evaluated the presence of exosomes in plasma of melanoma patients as a potential tool for cancer screening and follow-up.
We designed an in-house sandwich ELISA (Exotest) to capture and quantify exosomes in plasma based on expression of housekeeping proteins (CD63 and Rab-5b) and a tumor-associated marker (caveolin-1). Western blot and flow cytometry analysis of exosomes were used to confirm the Exotest-based findings. The Exotest allowed sensitive detection and quantification of exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. Plasma levels of exosomes in melanoma-engrafted SCID mice correlated to tumor size. We evaluated the levels of plasma exosomes expressing CD63 and caveolin-1 in melanoma patients (n = 90) and healthy donors (n = 58). Consistently, plasma exosomes expressing CD63 (504+/-315) or caveolin-1 (619+/-310) were significantly increased in melanoma patients as compared to healthy donors (223+/-125 and 228+/-102, respectively). While the Exotest for CD63+ plasma exosomes had limited sensitivity (43%) the Exotest for detection of caveolin-1+ plasma exosomes showed a higher sensitivity (68%). Moreover, caveolin-1+ plasma exosomes were significantly increased with respect to CD63+ exosomes in the patients group.
We describe a new non-invasive assay allowing detection and quantification of human exosomes in plasma of melanoma patients. Our results suggest that the Exotest for detection of plasma exosomes carrying tumor-associated antigens may represent a novel tool for clinical management of cancer patients.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, ...sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigenspecific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4⁺ and CD8⁺ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelinreactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance.
Proton pumps like the vacuolar-type H+ ATPase (V-ATPase) are involved in the control of cellular pH in normal and tumor cells. Treatment with proton pump inhibitors (PPI) induces sensitization of ...cancer cells to chemotherapeutics via modifications of cellular pH gradients. It is also known that low pH is the most suitable condition for a full PPI activation. Here, we tested whether PPI treatment in unbuffered culture conditions could affect survival and proliferation of human B-cell tumors. First, we showed that PPI treatment increased the sensitivity to vinblastine of a pre-B acute lymphoblastic leukemia (ALL) cell line. PPI, per se, induced a dose-dependent inhibition of proliferation of tumor B cells, which was associated with a dose- and time-dependent apoptotic-like cytotoxicity in B-cell lines and leukemic cells from patients with pre-B ALL. The effect of PPI was mediated by a very early production of reactive oxygen species (ROS), that preceded alkalinization of lysosomal pH, lysosomal membrane permeabilization, and cytosol acidification, suggesting an early destabilization of the acidic vesicular compartment. Lysosomal alterations were followed by mitochondrial membrane depolarization, release of cytochrome c, chromatin condensation, and caspase activation. However, inhibition of caspase activity did not affect PPI-induced cell death, whereas specific inhibition of ROS by an antioxidant (N-acetylcysteine) significantly delayed cell death and protected both lysosomal and mitochondrial membranes. The proapoptotic activity of PPI was consistent with a clear inhibition of tumor growth following PPI treatment of B-cell lymphoma in severe combined immunodeficient mice. This study further supports the importance of acidity and pH gradients in tumor cell homeostasis and suggests new therapeutic approaches for human B-cell tumors based on PPI.
Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has ...some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co‐localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti‐tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype.
Work from the Lozupone lab shows that TM9SF4, the human homologue of phg1A, which is involved in phagocytosis in Dictyostelium discoideum, is a new tumor marker expressed by metastatic melanoma cells. The study provides evidence that TM9SF4 is involved in phagocytic activity and in derangement of intracellular pH gradients; phenomena that are typically related to the malignant phenotype of several tumors.
BackgroundMucosal melanoma is a rare subtype of melanoma originating from mucosal tissues (1), metastases are very aggressive and respond poorly to therapy, including immune checkpoint inhibitors ...(ICI) such as anti-CTLA4 and anti-PD1 antibodies (2–5). CD8+ T cells constitute the most abundant immune infiltrate in metastatic melanoma, of which the Tissue Resident Memory subset (TRM) is of particular interest (6). CD8+ TRM cells express the highest levels of immune checkpoint receptors, proliferate in response to ICI and correlate with longer disease-free and overall survival (6–8). The immune landscape in mucosal melanoma remains poorly characterized. We aimed to: 1) phenotype CD8+ T cells and TRM infiltrating metastatic mucosal melanoma, 2) characterize the clonality of TRM in relation to other CD8+ T cell subsets and 3) define the capacity of CD8+ T cells and TRM to respond to melanoma cells and to in vivo and in vitro anti-PD1 treatment.MethodsWe investigated the CD8+ T and TRM cells infiltrating two temporally- and spatially-distant subcutaneous metastases, these originated from a primary vaginal mucosal melanoma. One metastasis was excised prior to anti-PD1 treatment and one was anti-PD1 refractory, having progressed on treatment. We used mass cytometry and single-cell RNA and TCR sequencing to characterise the phenotype and clonality of the T cells, multiplex immunohistochemistry to define their spatial relationship with tumour cells and other T cells, and functional assays to determine TRM response to tumour cells (figure 1).ResultsCD8+ TRM frequency increased with time and anti-PD1 treatment, forming clusters at the tumour margin. T cells in the anti-PD1 refractory lesion were more activated than T cells in the first tumour and were bound by anti-PD1 antibody in vivo. T cells could not be stimulated by anti-PD1 directly ex vivo. Both metastatic lesions shared common T cell clusters including TRM. Furthermore, TRM in each tumour shared T cell clones, suggesting the presence of common antigens between metastatic sites. Indeed, the two metastases had a similar mutational profile. In vitro expanded tumour infiltrating lymphocytes from both lesions recognized tumour cells from both lesions and the same neoantigen generated from a single point mutation in the gene CDKN1C. Finally, tumour cells stimulated TRM cells more robustly than other T cells subsets.Abstract 548 Figure 1Graphical depiction of the methods used to characterise T cells in mucosal metastatic melanomaConclusionsIn this patient with vaginal mucosal melanoma, subsequent melanoma metastases of clonal origin attracted CD8+ T cells of similar specificity, among which TRM cells responded more vigorously to tumour cells than other T cells subsets.AcknowledgementsThe authors would like to acknowledge imCORE La Hoffmann- Roche Ltd. for funding.Ethics ApprovalPatients diagnosed with stage 3 or 4 metastatic melanoma and undergoing clinically indicated surgery were enrolled in prospective studies approved by the Peter MacCallum Cancer Centre human ethics research committee (13/141). All experimental protocols have been approved and clinical data has been collected prospectively.ReferencesCarvajal RD, Hamid O, Ariyan C. Mucosal Melanoma. cited 2020 Apr 1; Available from: https://www.uptodate.com/contents/mucosal-melanomaDel Vecchio M, Di Guardo L, Ascierto PA, Grimaldi AM, Sileni VC, Pigozzo J, et al. Efficacy and safety of ipilimumab 3 mg/kg in patients with pretreated, metastatic, mucosal melanoma. Eur J Cancer Oxf Engl 1990; 2014 Jan;50(1):121–7.Postow MA, Luke JJ, Bluth MJ, Ramaiya N, Panageas KS, Lawrence DP, et al. Ipilimumab for patients with advanced mucosal melanoma. The Oncologist 2013 Jun;18(6):726–32.D’Angelo SP, Larkin J, Sosman JA, Lebbé C, Brady B, Neyns B, et al. Efficacy and safety of nivolumab alone or in combination with ipilimumab in patients with mucosal melanoma: a pooled analysis. J Clin Oncol Off J Am Soc Clin Oncol. 2017 Jan 10;35(2):226–35.Hamid O, Robert C, Ribas A, Hodi FS, Walpole E, Daud A, et al. Antitumour activity of pembrolizumab in advanced mucosal melanoma: a post-hoc analysis of KEYNOTE-001, 002, 006. Br J Cancer 2018;119(6):670–4.Boddupalli CS, Bar N, Kadaveru K, Krauthammer M, Pornputtapong N, Mai Z, et al. Interlesional diversity of T cell receptors in melanoma with immune checkpoints enriched in tissue-resident memory T cells. JCI Insight Internet. 2016 Dec 22 cited 2019 Apr 24;1(21). Available from: https://insight.jci.org/articles/view/88955Edwards J, Wilmott JS, Madore J, Gide TN, Quek C, Tasker A, et al. CD103+ Tumor-resident CD8+ T cells are associated with improved survival in immunotherapy-naïve melanoma patients and expand significantly during anti-PD-1 treatment. Clin Cancer Res Off J Am Assoc Cancer Res 2018 Jul 1;24(13):3036–45.Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, et al. Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis. Nat Med 2018 Jul;24(7):986–93.
Minimally invasive approaches to detect residual disease after surgery are needed to identify patients with cancer who are at risk for metastatic relapse. Circulating tumour DNA (ctDNA) holds promise ...as a biomarker for molecular residual disease and relapse
. We evaluated outcomes in 581 patients who had undergone surgery and were evaluable for ctDNA from a randomized phase III trial of adjuvant atezolizumab versus observation in operable urothelial cancer. This trial did not reach its efficacy end point in the intention-to-treat population. Here we show that ctDNA testing at the start of therapy (cycle 1 day 1) identified 214 (37%) patients who were positive for ctDNA and who had poor prognosis (observation arm hazard ratio = 6.3 (95% confidence interval: 4.45-8.92); P < 0.0001). Notably, patients who were positive for ctDNA had improved disease-free survival and overall survival in the atezolizumab arm versus the observation arm (disease-free survival hazard ratio = 0.58 (95% confidence interval: 0.43-0.79); P = 0.0024, overall survival hazard ratio = 0.59 (95% confidence interval: 0.41-0.86)). No difference in disease-free survival or overall survival between treatment arms was noted for patients who were negative for ctDNA. The rate of ctDNA clearance at week 6 was higher in the atezolizumab arm (18%) than in the observation arm (4%) (P = 0.0204). Transcriptomic analysis of tumours from patients who were positive for ctDNA revealed higher expression levels of cell-cycle and keratin genes. For patients who were positive for ctDNA and who were treated with atezolizumab, non-relapse was associated with immune response signatures and basal-squamous gene features, whereas relapse was associated with angiogenesis and fibroblast TGFβ signatures. These data suggest that adjuvant atezolizumab may be associated with improved outcomes compared with observation in patients who are positive for ctDNA and who are at a high risk of relapse. These findings, if validated in other settings, would shift approaches to postoperative cancer care.
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Background: MODUL is an adaptable, phase 2, signal-seeking trial testing novel agents as first-line therapy for predefined subgroups of patients with metastatic colorectal cancer ...(mCRC). Previously reported findings demonstrated that adding atezolizumab to fluoropyrimidine (FP)/bevacizumab as first-line maintenance treatment after induction with FOLFOX + bevacizumab did not improve efficacy outcomes in BRAF
wt
mCRC. Given these efficacy results, exploratory assessments on tumour samples were conducted to provide insights into factors that might affect efficacy of maintenance treatment and provide guidance on appropriate therapeutic strategies for BRAF
wt
mCRC. Methods: In patients with BRAF
wt
tumours (Cohort 2), experimental treatment was FP/bevacizumab + atezolizumab. Primary efficacy endpoint: progression-free survival (PFS). Overall survival (OS) was a secondary endpoint. Archival tissue samples from 104 patients were analysed by immunohistochemistry (IHC) at HistoGeneX (PD-L1; CD8/GrB/FoxP3). SP142 antibody was used for PD-L1 IHC analysis, which evaluated PD-L1
low
(IC 0–1) vs PD-L1
high
(IC > 1) in correlation with PFS and OS in the control and experimental arms. CD8/GrB/FoxP3 triplex staining was also performed to evaluate potential correlations with efficacy. Results: 445 patients with BRAF
wt
mCRC were randomised (2:1 ratio) to maintenance treatment in Cohort 2. Archival samples from 104 patients were analysed: FP/bevacizumab + atezolizumab (n = 82); FP/bevacizumab (n = 22). The biomarker evaluable population (BEP) for PD-L1 was n = 81 for FP/bevacizumab + atezolizumab PD-L1
low
n = 35 (43%); PD-L1
high
n = 46 (57%) and n = 22 for FP/bevacizumab PD-L1
low
n = 16 (72%); PD-L1
high
n = 6 (28%). The BEP for CD8/GrB was n = 50 for FP/bevacizumab + atezolizumab and n = 16 for FP/bevacizumab. No difference in PFS or OS was observed in the FP/bevacizumab + atezolizumab vs FP/bevacizumab arms for PD-L1
high
PFS: HR = 1.5 (95% CI 0.45−4.8), p = 0.51; OS: HR = 1.3 (95% CI 0.38−4.1), p = 0.71 or PD-L1
low
PFS: HR = 0.92 (95% CI 0.47−1.8), p = 0.81; OS: HR = 0.78 (95% CI 0.4−1.5), p = 0.48. Similar results were observed with CD8/GrB
high
PFS: HR = 0.73 (95% CI 0.27−2.0), p = 0.55; OS: HR = 0.66 (95% CI 0.24−1.8), p = 0.44, CD8/GrB
low
PFS: HR = 1.0 (95% CI 0.42–2.5), p = 0.96; OS: HR = 0.73 (95% CI 0.3–1.8), p = 0.5, FoxP3
high
PFS: HR = 0.97 (95% CI 0.37−2.5), p = 0.95; OS: HR = 0.95 (95% CI 0.36−2.5), p = 0.91 and FoxP3
low
PFS: HR = 0.73 (95% CI 0.29−1.9), p = 0.53; OS: HR = 0.5 (95% CI 0.19−1.3), p = 0.18. Conclusions: These findings suggest that PD-L1, CD8/GrB and FoxP3 might not be predictive biomarkers in BRAF
wt
mCRC. Further analyses are needed to further evaluate potential predictive and prognostic factors of response in this setting. Clinical trial information: NCT02291289.
The increased presence of sialylated glycans on the tumor surface has been linked to poor prognosis, yet the effects on tumor-specific T cell immunity are hardly studied. We here show that ...hypersialylation of B16 melanoma substantially influences tumor growth by preventing the formation of effector T cells and facilitating the presence of high regulatory T cell (Treg) frequencies. Knock-down of the sialic acid transporter created "sialic acid low" tumors, that grew slower in-vivo than hypersialylated tumors, altered the Treg/Teffector balance, favoring immunological tumor control. The enhanced effector T cell response in developing "sialic acid low" tumors was preceded by and dependent on an increased influx and activity of Natural Killer (NK) cells. Thus, tumor hypersialylation orchestrates immune escape at the level of NK and Teff/Treg balance within the tumor microenvironment, herewith dampening tumor-specific T cell control. Reducing sialylation provides a therapeutic option to render tumors permissive to immune attack.
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