Circulating tumour cells and PD-L1-positive small extracellular vesicles: the liquid biopsy combination for prognostic information in patients with metastatic non-small cell lung cancer.

BACKGROUND: Circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and extracellular vesicles (EVs) are minimally invasive liquid biopsy biomarkers. This study investigated whether they predict prognosis, alone or in combination, in heterogenous unbiased non-small cell lung cancer (NSCLC) patients. METHODS: Plasma samples of 54 advanced NSCLC patients from a prospective clinical trial. CtDNA mutations were identified using the UltraSEEK™ Lung Panel (MassARRAY® technology). PD-L1 expression was assessed in small EVs (sEVs) using an enzyme-linked immunosorbent assay. RESULTS: At least one ctDNA mutation was detected in 37% of patients. Mutations were not correlated with overall survival (OS) (HR = 1.1, 95% CI = 0.55; 1.83, P = 0.980) and progression-free survival (PFS) (HR = 1.00, 95% CI = 0.57-1.76, P = 0.991). High PD-L1+ sEV concentration was correlated with OS (HR = 1.14, 95% CI = 1.03-1.26, P = 0.016), but not with PFS (HR = 1.08, 95% CI = 0.99-1.18, P = 0.095). The interaction analysis suggested that PD-L1+ sEV correlation with PFS changed in function of CTC presence/absence (P interaction = 0.036). The combination analysis highlighted worse prognosis for patients with CTCs and high PD-L1+ sEV concentration (HR = 7.65, 95% CI = 3.11-18.83, P < 0.001). The mutational statuses of ctDNA and tumour tissue were significantly correlated (P = 0.0001). CONCLUSION: CTCs and high PD-L1+ sEV concentration correlated with PFS and OS, but not ctDNA mutations. Their combined analysis may help to identify patients with worse OS. TRIAL REGISTRATION: NCT02866149, Registered 01 June 2015, https://clinicaltrials.gov/ct2/show/study/NCT02866149 .

PD-L1 in circulating exosomes of Merkel cell carcinoma.

Exosomes, as potential circulated biomarkers, have recently become a topic of interest in the field of oncology. Immune checkpoint molecule PD-L1 has recently been detected in circulating exosomes from cancer patients. The purpose of this work was to evaluate PD-L1 levels in circulating exosomes (Exo-PD-L1) isolated from patients' plasma suffering from Merkel cell carcinoma (MCC). We conducted a prospective bicentric cohort study. PD-L1 was analysed in circulating exosomes from plasma samples of patients suffering from MCC stage I to IV (according to the AJCC 8). Exosomes from 34 patients corresponding to 66 samples were analysed. PD-L1 was identified in circulating exosomes of MCC patients. Exo-PD-L1 levels of MCC patients were similar to healthy donors and lower than other cancers such as melanoma. Exo-PD-L1 levels tended to be higher in MCC patients with distant metastases. Furthermore, Exo-PD-L1 levels did not significantly vary over the course of the disease whatever the disease course or the response to treatment. This study assessed the presence of PD-L1 in circulating exosomes of MCC patients. The low levels of Exo-PD-L1 and small changes over the course of the disease may be due to the metastatic dissemination of MCC, which is mainly through the skin and lymph nodes rather than blood. PD-L1 was identified in circulating exosomes of MCC patients and tends to be higher in advanced disease. This preliminary study is a proof of concept of PD-L1 detection in circulating exosomes of MCC patients.

Heat shock proteins and exosomes in cancer theranostics.

Heat shock proteins (HSPs) are a superfamily of molecular chaperones that were discovered through their ability to be induced by different stresses including heat shock. Other than their function as chaperones in proteins homeostasis, HSPs have been shown to inhibit different forms of cell death and to participate in cell proliferation and differentiation processes. Because cancer cells have to rewire their metabolism, they require a high amount of these stress-inducible chaperones for their survival. Therefore, HSPs are unusually abundant in cancer cells where they have oncogene-like functions. In cancer, HSPs have been involved in the regulation of apoptosis, immune responses, angiogenesis, metastasis and treatment resistance. Recently, HSPs have been shown to be secreted through exosomes by cancer cells. These tumor-derived exosomes can be used as circulating markers: HSP-exosomes have been reported as biomarkers of cancer dissemination, response to therapy and/or patient outcome. A new range of functions, mostly in modulation of anticancer immune responses, have been described for these extracellular HSPs. In this review, we will describe those recently reported functions of HSP-exosomes that makes them both targets for anticancer therapeutics and biomarkers for the monitoring of the disease. We will also discuss their emerging interest in cancer vaccines.

Tumor-Derived Exosomes: Hidden Players in PD-1/PD-L1 Resistance.

Recently, immunotherapy has garnered increasing importance in cancer therapy, leading to substantial improvements in patient care and survival. By blocking the immune checkpoints-protein regulators of the immune system-immunotherapy prevents immune tolerance toward tumors and reactivates the immune system, prompting it to fight cancer cell growth and diffusion. A widespread strategy for this is the blockade of the interaction between PD-L1 and PD-1. However, while patients generally respond well to immunotherapy, a certain proportion of patients present tumors that resist these treatments. This portion can be very high in some cancers and hinders cancer curability. For this reason, current efforts are focusing on combining PD-1/PD-L1 immunotherapy with the targeting of other immune checkpoints to counter resistance and achieve better results. Exosomes, small vesicles secreted by almost any cell, including tumor cells, have proven to be key actors in this resistance. The exosomes released by tumor cells spread the immune-suppressive properties of the tumor throughout the tumor microenvironment and participate in establishing metastatic niches. In this review, we will describe immune checkpoints and immune modulators whose presence in tumor-derived exosomes (TEXs) has been established. We will focus on the most promising proteins under scrutiny for use in combination with PD-1 blockade therapy in a clinical setting, such as PD-L1, CTLA-4, TIM-3, CD73/39, LAG-3, and TIGIT. We will explore the immunosuppressive impact of these exosomal proteins on a variety of immune cells. Finally, we will discuss how they can change the game in immunotherapy and guide therapeutic decisions, as well as the current limits of this approach. Depending on the viewpoint, these exosomal proteins may either provide key missing information on tumor growth and resistance mechanisms or they may be the next big challenge to overcome in improving cancer treatment.

Fluorescence In Situ Hybridization of Small Non-Coding RNAs.

The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor® 488 conjugate. A protocol allowing efficient coupling of RNA-FISH and protein detection by immunofluorescence is also described as well as the bioinformatics pipeline, Substructure Analyzer, we recently developed to automate fluorescence signal analysis.

Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5' Adaptor Ligation.

Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.

Membrane-anchored heat-shock protein 70 (Hsp70) in cancer.

Hsp70 is a highly conserved and inducible heat shock protein that belongs to the HSP70 family of molecular chaperones and plays a central role in protein homeostasis. The main function of Hsp70 is to protect cells from physiological, pathological and environmental insults, as it assists an ATP-dependent manner the process of protein folding. Since Hsp70 provides critical cell survival functions, cancer cells are assumed to rely on this chaperone. Strong evidence suggests that Hsp70 is upregulated in different type of cancers and is involved in tumor growth, invasion, migration and resistance to anti-cancer therapy. Interestingly, this Hsp70 upregulation induces Hsp70 re-location into plasma membrane. In this review, the role of Hsp70 in cancer will be discussed focusing particularly on the extracellular membrane-bound Hsp70. The mechanism by which Hsp70 is translocated to plasma membrane of tumor cells and the recent discoveries of drugs targeting this Hsp70 in cancer therapy will be also highlighted.

Exosomal miRNA: Small Molecules, Big Impact in Colorectal Cancer.

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths worldwide. Tumor microenvironment (TME) contains many cell types including stromal cells, immune cells, and endothelial cells. The TME modulation explains the heterogeneity of response to therapy observed in patients. In this context, exosomes are emerging as major contributors in cancer biology. Indeed, exosomes are implicated in tumor proliferation, angiogenesis, invasion, and premetastatic niche formation. They contain bioactive molecules such as proteins, lipids, and RNAs. More recently, many studies on exosomes have focused on miRNAs, small noncoding RNA molecules able to influence protein expression. In this review, we describe miRNAs transported by exosomes in the context of CRC and discuss their influence on TME and their potential as circulating biomarkers. This overview underlines emerging roles for exosomal miRNAs in cancer research for the near future.

Increased Levels of Interleukin-17A Exosomes in Psoriasis.

Exosomes are involved in modulating the immune system and mediating communication between cells. The aim of this study was to investigate the involvement of exosomes in psoriasis. Exosomes from patients with psoriasis were analysed by nanoparticle tracking analysis and protein expression was analysed by western blotting. The concentration of HSP70 was determined by an enzyme-linked immunosorbent assay, and concentrations of interleukin (IL)-1ß, IL-2, IL-6, IL-10, IL-17A and tumour necrosis factor alpha (TNF-α) were determined by flow cytometry. Based on the severity of psoriasis, evaluated by body surface area (≤ 10% vs. > 10%), 2 groups of patients were compared (49 with mild psoriasis and 71 with moderate-to-severe psoriasis). The number (2.52×1011 ± 2.29×1010 vs. 1.79×1011 ± 1.93×1010, p = 0.19) and size (94.44 ± 22.00 nm vs. 96.87 ± 28.30 nm, p = 0.72) of exosomes and the concentration of HSP70 in the exosomes were not significantly different in the 2 groups of patients. IL-17A exosome levels were significantly higher in patients with moderate-to-severe psoriasis compared with those with mild psoriasis (p = 0.02). There were no significant differences in levels of TNF-α, IL-1, IL-2, IL-6 and IL-10. This study shows, for the first time, the presence of circulating exosomes in patients with psoriasis. These data confirm the involvement of circulating exosomes in psoriasis, in particular in moderate-to-severe psoriasis, through IL-17A-producing exosomes.

Enhanced SRSF5 Protein Expression Reinforces Lamin A mRNA Production in HeLa Cells and Fibroblasts of Progeria Patients.

The Hutchinson Gilford Progeria Syndrome (HGPS) is a rare genetic disease leading to accelerated aging. Three mutations of the LMNA gene leading to HGPS were identified. The more frequent ones, c.1824C>T and c.1822G>A, enhance the use of the intron 11 progerin 5'splice site (5'SS) instead of the LMNA 5'SS, leading to the production of the truncated dominant negative progerin. The less frequent c.1868C>G mutation creates a novel 5'SS (LAΔ35 5'SS), inducing the production of another truncated LMNA protein (LAΔ35). Our data show that the progerin 5'SS is used at low yield in the absence of HGPS mutation, whereas utilization of the LAΔ35 5'SS is dependent upon the presence of the c.1868C>G mutation. In the perspective to correct HGPS splicing defects, we investigated whether SR proteins can modify the relative yields of utilization of intron 11 5'SSs. By in cellulo and in vitro assays, we identified SRSF5 as a direct key regulator increasing the utilization of the LMNA 5'SS in the presence of the HGPS mutations. Enhanced SRSF5 expression in dermal fibroblasts of HGPS patients as well as PDGF-BB stimulation of these cells decreased the utilization of the progerin 5'SS, and improves nuclear morphology, opening new therapeutic perspectives for premature aging.

Fluorescence in situ hybridization of small non-coding RNAs.

RNA FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA FISH protocol that we developed in order to study the expression and localization of satellite III RNAs. This specific class of non-coding RNAs is expressed in response to various cellular stresses including heat shock. This protocol is based on the use of a biotinylated LNA probe subsequently detected by a streptavidin-Alexa Fluor(®) 488 conjugate. A protocol allowing efficient coupling of RNA FISH and protein detection by immunofluorescence is also described in this chapter.

A conserved splicing mechanism of the LMNA gene controls premature aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. Most cases of HGPS are due to a heterozygous silent mutation (c.1824C>T; p.Gly608Gly) that enhances the use of an internal 5' splice site (5'SS) in exon 11 of the LMNA pre-mRNA and leads to the production of a truncated protein (progerin) with a dominant negative effect. Here we show that HGPS mutation changes the accessibility of the 5'SS of LMNA exon 11 which is sequestered in a conserved RNA structure. Our results also reveal a regulatory role of a subset of serine-arginine (SR)-rich proteins, including serine-arginine rich splicing factor 1 (SRSF1) and SRSF6, on utilization of the 5'SS leading to lamin A or progerin production and a modulation of this regulation in the presence of the c.1824C>T mutation is shown directly on HGPS patient cells. Mutant mice carrying the equivalent mutation in the LMNA gene (c.1827C>T) also accumulate progerin and phenocopy the main cellular alterations and clinical defects of HGPS patients. RNAi-induced depletion of SRSF1 in the HGPS-like mouse embryonic fibroblasts (MEFs) allowed progerin reduction and dysmorphic nuclei phenotype correction, whereas SRSF6 depletion aggravated the HGPS-like MEF's phenotype. We demonstrate that changes in the splicing ratio between lamin A and progerin are key factors for lifespan since heterozygous mice harboring the mutation lived longer than homozygous littermates but less than the wild-type. Genetic and biochemical data together favor the view that physiological progerin production is under tight control of a conserved splicing mechanism to avoid precocious aging.