**1. Introduction**

EBV is an oncogenic virus that infects about 95% of the worldwide adult population. After primo-infection, it remains hidden in nuclei of memory B-cells, resulting in life-long persistent infection. During infection, including transient reactivation, some infected Bcells enter the lytic cycle or EBV latency III program (also called proliferation program), with transcription of the full range of latent genes [1]. In an immunocompetent host, the balance established between the immune system and the virus avoids development of cancers. However, a rupture in equilibrium can occur, causing EBV lymphoproliferative disorders, such as Hodgkin lymphomas (HL), Burkitt lymphomas (BL) or DLBCLs [2], which is the most common non-Hodgkin lymphoma. This aggressive tumor affects Blymphocytes and has two major biologically distinct subtypes: germinal center B-cell (GCB) and activated B-cell (ABC) [3]. ABC-DLBCL is associated with worse outcomes when treated with chemo-immunotherapy, the standard clinical care for this pathology.

Inhibitory immune checkpoints exert inhibitory effects on adaptive and innate immune systems. They are crucial for self-tolerance but also mediate immune evasion of

**Citation:** Saliba, J.; Manseur, C.; Groult, H.; Akil, H.; Tannoury, M.; Troutaud, D.; Maugard, T.; Feuillard, J.; Arnaudin, I.; Jayat-Vignoles, C. Anti-Proliferative and Pro-Apoptotic vLMW Fucoidan Formulas Decrease PD-L1 Surface Expression in EBV Latency III and DLBCL Tumoral B-Cells by Decreasing Actin Network. *Mar. Drugs* **2023**, *21*, 132. https:// doi.org/10.3390/md21020132

Academic Editors: Barbara De Filippis, Alessandra Ammazzalorso and Marialuigia Fantacuzzi

Received: 26 January 2023 Revised: 13 February 2023 Accepted: 15 February 2023 Published: 18 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cancer cells, contributing to tumor emergence and development [4]. PD-L1 (also known as B7-H1 or CD274) is expressed on a sizeable fraction of tumor types and is one of the most critical [5]. It can interact with its receptor, PD-1, which is expressed on numerous cells involved in anti-tumor response (such as activated T-cells, dendritic cells or NK cells) and leads to inhibitory signals [6]. We have previously shown that PD-L1 is also overexpressed on EBV latency III B-cells and strongly inhibits anti-tumoral T-cell response in an autologous B/T model; expansion of anergic conventional and unconventional CD4+ Tregs (regulatory T-cells) leads to inhibition of CD4+ and CD8<sup>+</sup> effector T-cells proliferation [7]. The PD-L1/PD-1 axis is also critical for numerous B-cell lymphomas, such as HL and non-Hodgkin lymphomas, among them follicular lymphoma and DLBCL (which variably express PD-L1) [8,9]. PD-L1 overexpression in many cancer types and strong immune response inhibition following interaction with PD-1 have led to development of anti-PD-L1 and anti-PD-1 immunotherapies to block the PD-L1/PD-1 axis and restore immune response [10,11]. However, immune checkpoint inhibitors have some limitations since tolerance breakdown is not limited to tumors; related adverse events can affect multiple organ systems (gut, skin, endocrine glands, liver or lung) [12–14]. While keeping the advantage of immunotherapy, new therapeutic strategies can be considered, such as use of adjuvants capable of decreasing PD-L1 membrane expression and consequently lowering antibody doses [15].

Fucoidans are non-allergenic, non-irritating, biodegradable and biocompatible sulfated fucose-based polysaccharides constituents of brown seaweeds [16,17]. Extracts from *Fucus vesiculosus* algae are approved by the FDA (Food and Drug Administration) as GRAS (generally recognized as safe). In Europe, preparations that contain fucoidans are registered by the EMA (European Medicines Agency) for use as ingredients in food categories [17,18]. Numerous studies report bioactive properties with health benefits, predominantly dependent on the natural source extraction method, sulfate groups content and molecular weight of polysaccharide. For instance, fucoidans possess antioxidant, anticoagulant, anti-pathogenic and anti-inflammatory activities [19–21]. They also exhibit *in vitro* and *in vivo* anticancer properties, almost exclusively studied on solid tumor cells (colon, breast, lung, bladder, hepatoma or melanoma). The mechanisms of action described are generally cell cycle arrest, apoptosis, anti-metastatic effects and stimulation of macrophages, T-cells and NK cells. Nevertheless, scarce studies indicate decrease in PD-L1 checkpoint [22,23], which can be consistent with the fact that fucoidan impairs pathways responsible for PD-L1 expression: PI3k/AKT, NF-κB or RAS/ERK1/2 [24–29]. Fucoidan extracts can also protect against side effects associated with chemotherapeutic drugs and radiation-induced damages [16,30]. As suggested by some authors who evaluated pre-clinical safety of fucoidan extracts [31], they may become an appropriate and natural anticancer therapeutic as an adjunctive antitumor drug. However, more information is needed, particularly in the field of hematological malignancies and immune checkpoints, such as PD-L1, which is the overall objective in this study. We evaluated opportunity to use vLMW-F to decrease PD-L1 membrane expression and associated molecular mechanisms.

#### **2. Results**
