Exosomal PD-L1 Research Articles

45P - 5-fluorouracil-induced up-regulation of exosomal PD-L1 causing immunosuppression in gastric cancer patients

45P - 5-fluorouracil-induced up-regulation of exosomal PD-L1 causing immunosuppression in gastric cancer patients

Abstract A35: BRAF inhibition increases exosomal PD-L1 protein expression in melanoma

Abstract Background: Modulation of the PD-1/PD-L1 axis in melanoma is of critical importance in both the setting of targeted therapy treatment, as well as in newer trials combining targeted therapy with checkpoint blocking antibodies. BRAF inhibition has been shown to increase PD-L1 expression in melanoma tumors. Exosomes are circulating microvesicles that contain a subtranscriptome and/or subproteome of their cell of origin, including tumor cells and immune cells. We evaluated melanoma exosomal PD-L1 protein expression and the effect of BRAF inhibitor (BRAFi) treatment on exosomal PD-L1 protein levels. Methods: Melanoma cell lines (BRAFi sensitive [A375] and resistant [RPMI7951; induced resistant A375]) and patient plasma were used for analysis. Exosomal vesicles were isolated using centrifugation. Functional proteomics by reverse phase protein array (RPPA) were performed in cell lines and exosomes. RPPA PD-L1 results were quantified in cells and exosomes treated with control (DMSO) and BRAFi (PLX-4720). Exosomal PD-L1 expression from serial patient plasma with metastatic melanoma were assessed using ELISA prior to and after initiation of anti-PD-1 therapy. Results: Functional proteomic analysis revealed PD-L1 expression on both BRAFi sensitive and resistant cell lines. Cell line derived exosomes demonstrated enrichment of PD-L1 expression compared to their cell of origin, with significantly higher levels of expression in resistant lines and upon treatment with BRAFi therapy. Exosomes derived from patients with melanoma also revealed pre-treatment and on-treatment PD-L1 expression. Furthermore, consistent with cell line data, in a representative patient treated with BRAFi therapy prior to anti-PD-1 therapy, exosomal PD-L1 protein expression increased dramatically upon treatment with BRAFi and preceded a complete response to anti-PD-1 therapy. All other patients were treated only with checkpoint blockade and the majority demonstrated correlation of PD-L1 protein expression with tumor burden. Conclusions: These results confirm that exosomes from cell lines and patient samples express PD-L1 that can be serially monitored, and that treatment with BRAF inhibition results in increased PD-L1 levels, which in vitro are persistently elevated in BRAFi resistant cells. We also noted that exosomal PD-L1 protein expression levels tend to correlate with tumor burden in patient samples. Measuring PD-L1 expression may serve as a potential biomarker of tumor burden and as an inducible response to BRAFi therapy that predicts synergism with checkpoint anti-PD-1 therapy. Citation Format: Gyulnara G. Kasumova, Alvin Shi, Jessica A. Cintolo-Gonzalez, Isabel Chein, Dennie T. Frederick, Roman Alpatov, William A. Michaud, Deborah Plana, David J. Panka, Ryan B. Corcoran, Keith T. Flaherty, Ryan J. Sullivan, Manolis Kellis, Genevieve M. Boland. BRAF inhibition increases exosomal PD-L1 protein expression in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A35.

Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response.

Tumor cells evade the immune surveillance by up-regulating surface expression of PD-L1, which interacts with PD-1 on T cells to elicit the immune checkpoint response1,2. Anti-PD-1 antibodies have shown remarkable promise in treating tumors, including metastatic melanoma2–4. However, patient response rate is low4,5. A better understanding of PD-L1-mediated immune evasion is needed to predict patient response and improve treatment efficacy. Here we report that metastatic melanoma releases a high level of extracellular vesicles (EVs), mostly in the form of exosomes, that carry PD-L1 on their surface. Interferon-γ (IFN-γ) up-regulates PD-L1 on these vesicles, which suppresses the function of CD8 T cells and facilitates tumor growth. In patients with metastatic melanoma, the level of circulating exosomal PD-L1 positively correlates with that of IFN-γ, and changes during the course of anti-PD-1 therapy. The magnitudes of the early on-treatment increase in circulating exosomal PD-L1, as an indicator of the adaptive response of the tumor cells to T cell re-invigoration, stratifies clinical responders from non-responders. Our study unveils a mechanism by which tumor cells systemically suppress the immune system, and provides a rationale for the application of exosomal PD-L1 as a predictor for anti-PD-1 therapy.

PD-L1 mRNA expression in plasma-derived exosomes is associated with response to anti-PD-1 antibodies in melanoma and NSCLC

Background:PD-L1 expression in tumour tissues is widely used to select patients to receive anti-PD-1/PD-L1 antibodies, but data are lacking on the correlation of plasma PD-L1 levels with the effect of treatments.Methods:To investigate the association between PD-L1 mRNA in plasma-derived exosomes and response to nivolumab and pembrolizumab in patients with melanoma (n=18) and NSCLC (n=8), blood was obtained at time point 0 and after 2 months. Exosomal PD-L1 mRNA was measured by digital droplet PCR.Results:The mean±s.e.m. PD-L1 levels in patients with complete and partial responses were 830.4±231.3 and 242.5±82.5 copies per ml at time 0 vs 2 months, respectively (P=0.016). In patients with stable disease the mean±s.e.m. values were 298.8±97.2 vs 247.5±29.8 copies per ml (P=0.586), while in progressive disease, PD-L1 mRNA levels were 204.0±68.8 vs 416.0±87.8 copies per ml at time 0 vs 2 months, respectively (P=0.001).Conclusions:This study demonstrates that exosomal PD-L1 is significantly associated with response to treatment.

Association of PD-L1 mRNA levels in plasma-derived exosomes with response to nivolumab and pembrolizumab in melanoma and NSCLC.

210 Background: The anti-PD-1 monoclonal antibodies (moAb) nivolumab and pembrolizumab have improved the survival of melanoma and non-small cell lung cancer (NSCLC) patients. However, treatment selection is based on tumor PD-L1 expression by immuno-histochemistry and no specific approaches are available to monitor treatment response. The aim of this study was to investigate the association between PD-L1 mRNA levels in plasma-derived exosomes and response to nivolumab and pembrolizumab in patients affected by melanoma (n = 18) and NSCLC (n = 8). Methods: Blood (6 ml) was obtained at 1) baseline (before initiation of anti-PD1 moAb or at the time of last available radiological evaluation of disease response) and 2) after two months of treatment (at the time of first response evaluation or disease re-assessment). Exosomes were extracted from plasma and PD-L1 mRNA expression was measured by digital PCR and expressed as copies/ml. Results: Overall, the number of copies of mRNA PD-L1/ml plasma varied according to tumor response; in particular, an increase was found in patients with PD and a decrease was observed in patients who achieved a CR/PR. The mean±SEM values of PD-L1 in patients responding to treatment (CR+PR) were 830.4±231.3 and 242.5±82.5 copies/ml (baseline vs. 2 months, p = 0.016), respectively. In patients with stable disease the mean±SEM values were as expected 298.8±97.2 vs. 247.5±29.8 copies/ml (p = 0.586), while in progressive disease PD-L1 expression levels were 204.0±68.8 vs. 416.0±87.8 copies/ml (p = 0.001), respectively. Conclusions: In the present study, we demonstrate for the first time that changes in exosomal PD-L1 expression occur in melanoma and NSCLC patients treated with nivolumab or pembrolizumab and may correlate with the radiological tumor response. This proof-of-concept study demonstrates the feasibility of detecting PD-L1 in plasma and its relationship with response to treatment.

The association of an exosomal form of PD-L1 with immunosuppressive activity and gastric cancer prognosis.

47 Background: The greatest challenge in cancer immunotherapy is to identify efficient predictive biomarkers to select patients for treatment. Though tumor cell membrane PD-L1 has been most anticipated, tumor cell membrane PD-L1 does not correlate with higher response rates and predict for clinical benefit. Subsequently studies showed that the prognostic value of tumor cell membrane PD-L1 in cancer patients remains controversial. In addition to membrane-associated PD-L1, tumor cell also secreted extracellular soluble PD-L1 in the microenvironment. However, even if extracellular soluble PD-L1 was detected, it did not solve the problem mentioned above. In the present study, we discovered another form of PD-L1 in extracellular microenvironment in cancer cells, that is exosomal PD-L1. However, the predictive role and the effect of tumor-derived exosomal PD-L1 is unclear. Methods: We evaluated the prognostic value of exosomal PD-L1 in the plasma of gastric cancer patients by ELISA and the effect of exosomal PD-L1-derived from gastric cancer cell lines by Western blot and Flow cytometry analysis. Results: Exosomal PD-L1 was detected in plasma samples from 69 gastric cancer patients, and exosomal PD-L1 content was significantly associated with T stage (P = 0.012). Overall Survival was significantly lower in the high exosomal PD-L1 group (p = 0.021). Additionally, gastric cancer cells also secreted exosomal PD-L1, with the amounts positively associated with PD-L1 amounts in the corresponding gastric cancer cell lines. Besides, exosomal PD-L1 was more stable and showed stronger immunosuppressive effects in the microenvironment compared with soluble PD-L1. Conclusions: Exosomal PD-L1 might predict the survival in gastric cancer, and induces higher T-cell apoptosis levels compared with soluble PD-L1.

Company Profile: Nanosight: Delivering Practical Solutions for Biological Nanotechnology

NanomedicineVol. 7, No. 8 News & ViewsCompany Profile: NanoSight: delivering practical solutions for biological nanotechnologyBob Carr & Jeremy WarrenBob CarrNanoSight Limited, Minton Park, London Road, Amesbury, Wiltshire, SP4 7RT, UKSearch for more papers by this author & Jeremy Warren* Author for correspondenceNanoSight Limited, Minton Park, London Road, Amesbury, Wiltshire, SP4 7RT, UK. Search for more papers by this authorEmail the corresponding author at jeremy.warren@nanosight.comPublished Online:30 Aug 2012https://doi.org/10.2217/nnm.12.43AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleReferences1 Gardiner C, Dragovic R, Brooks A, Alvarez L, Harrison P, Sargent I. Visualisation, sizing and counting of cellular microparticles and exosomes using nanoparticle tracking analysis. Presented at: Oxford Biomedical Imaging Festival. Oxford, UK, 16 September 2009.Google Scholar2 Sargent I. Microparticles in pregnancy and pre-eclampsia. Presented at: Micro and Nanovesicles in Health and Disease Conference. Oxford, UK, 23–24 September 2010.Google Scholar3 Malloy A. Count, size and visualize nanoparticles. Mater. Today14(4),170–173 (2011).Crossref, CAS, Google Scholar4 Filipe V, Hawe A, Jiskoot W. Critical evaluation of nanoparticle tracking analysis (NTA) by nanosight for the measurement of nanoparticles and protein aggregates. Pharm. Res.27(5),796–810 (2010).Crossref, Medline, CAS, Google Scholar5 Ghonaim HM, Li S, Blagbrough IS. N1,N12-diacyl spermines: SAR studies on non-viral lipopolyamine vectors for plasmid DNA and siRNA formulation. Pharm. Res.27(1),17–29 (2010).Crossref, Medline, CAS, Google Scholar6 Moser M. Emerging analytical techniques to characterize vaccines. Proc. Intl. Conf. Vaccines Europe. Brussels, December 2008.Google Scholar7 Domingos RF, Peyrot C, Wilkinson KJ. Aggregation of titanium dioxide nano particles: role of calcium and phosphate. Environ. Chem.7,61 (2010).Crossref, CAS, Google Scholar8 Howard AG. On the challenge of quantifying man-made nanoparticles in the aquatic environment. J. Environ. Monit.12,135–142 (2010).Crossref, Medline, CAS, Google Scholar9 Gallego-Urrea JA, Tuoriniemi J, Pallander T, Hassellöv M. Measurements of nanoparticle number concentrations and size distributions in contrasting aquatic environments using nanoparticle tracking analysis. Environ. Chem.7(1),67–81 (2010).Crossref, CAS, Google Scholar10 Farkas J, Christian P, Urrea JA et al. Effects of silver and gold nanoparticles on rainbow trout (Oncorhynchusmykiss) hepatocytes. Aquatic Toxicol.96(1),44–52 (2010).Crossref, Medline, CAS, Google Scholar101 NanoSight directors. www.nanosight.com/about/nanosight-directorsGoogle Scholar102 Official Journal of the European Union. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2011:275:0038:0040:EN:PDFGoogle ScholarFiguresReferencesRelatedDetailsCited ByCHMP2A regulates tumor sensitivity to natural killer cell-mediated cytotoxicity7 April 2022 | Nature Communications, Vol. 13, No. 1Isolation of Extracellular Vesicles from Leishmania spp.28 March 2020Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and MemoryCell, Vol. 177, No. 2Insights into the Proteome of Gastrointestinal Stromal Tumors-Derived Exosomes Reveals New Potential Diagnostic BiomarkersMolecular & Cellular Proteomics, Vol. 17, No. 3An In Vitro and In Vivo Comparison of Solid and Liquid–Oil Cores in Transdermal Aconitine NanocarriersJournal of Pharmaceutical Sciences, Vol. 103, No. 11 Vol. 7, No. 8 Follow us on social media for the latest updates Metrics Downloaded 140 times History Published online 30 August 2012 Published in print August 2012 Information© Future Medicine LtdFinancial & competing interests disclosureThe authors are directors, employees and shareholders of NanoSight, while Bob Carr is also the Founder and Chief Technical Officer. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.The article was prepared in conjunction with Jezz Leckenby, Talking Science Limited, NanoSight’s UK-based PR agency.PDF download