Fucoidan potentiates anti-tumor efficacy of CAR-T cells against non-Hodgkin lymphoma by activation of STAT3 pathway

Abstract

Background

Despite advances in chimeric antigen receptor T-cell (CAR-T) therapy, significant challenges remain, including progressive T-cell exhaustion and poor in vivo persistence. Current strategies to enhance CAR-T function—such as cytokine co-expression—often lead to severe adverse effects, most notably cytokine release syndrome (CRS). Therefore, there is a pressing need to develop safer and more sustainable approaches, particularly through nutritional interventions, to improve the antitumor efficacy of CAR-T therapy. Fucoidan (FO), a bioactive polysaccharide derived from marine plants, has demonstrated immunomodulatory properties and synergistic potential in combination with conventional chemotherapy. However, its role in cellular immunotherapy, including CAR-T therapy, has not yet been explored. This study aims to elucidate the function of FO in CAR-T therapy for non-Hodgkin lymphoma (NHL) and to provide a foundational basis for its clinical translation in the field of cellular immunotherapy.

Methods

The current study used a combination of in vitro and in vivo assays to explore the role of FO in CAR T therapy. The anti-CD19 CAR-T cells were constructed by lentivirus containing CAR-CD19 structure. For phenotype analysis of CAR T cells, different cell populations, such as memory and exhausted CAR T cells, were stained by cell marker and identified by flow cytometry. CAR-T cells from different treatment groups were co-cultured with target cells with different E: T ratio to detect the cytotoxicity and cytokine release of CAR-T cells. We also evaluate FO’s supportive role for function of CAR-T cells in the tumor-bearing mouse models. The underlying mechanism of activated signaling pathway were also investigated and confirmed by the inhibitor.

Results

In this study, we systematically investigated the role of FO in anti-CD19 CAR-T cell-mediated treatment of NHL. Specifically, CAR-T cells’ memory maintenance and exhaustion resistance were enhanced by FO. FO not only improved CAR-T cell’s antioxidant capacity and proliferation, but also prevented apoptosis of activated CAR-T cells, collectively contributing to the improved and sustained anti-tumor efficacy in both in vitro assays and xenograft models. Our mechanistic studies also revealed FO served as a potentiator for CAR-T cells’ function through enhancing the activation of STAT3 signaling pathway.

Conclusions

These findings elucidate the supportive role of FO in enhancing CAR-T cell function, which indicates FO’s clinical potential as immunomodulatory supplement to potentiate CAR-T therapy against NHL.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-07548-2.

Introduction

The bioactive polysaccharides can potentiate immune responses, thereby eliciting immunomodulatory effects [1–3]. Substantial scientific validation also supports the therapeutic potential of marine-origin polysaccharides as immunostimulatory supplements, particularly in modulating pathogen-specific immune recognition and enhancing antitumor cellular defenses [4]. Fucoidan, a sulfated polysaccharide isolated from brown algal species, exhibits pronounced anticancer efficacy through strategic modulation of oncogenic signaling pathways while mitigating chemotherapy- and radiotherapy-induced cytotoxic sequelae. This polysaccharide can also suppress malignant cell survival by inhibiting phosphorylation-dependent activation of PI3K/AKT and ERK1/2 signaling cascades, thereby promoting apoptotic pathways [5]. This compound’s exceptional safety profile has prompted multiple investigations into its synergistic potential with conventional cancer therapies. Pre-clinically relevant demonstrations include its capacity to augment therapeutic outcomes of FOLFOX regimens (5-fluorouracil/leucovorin combined with oxaliplatin) in metastatic colorectal carcinoma management [6]. Despite these advances, critical knowledge gaps persist regarding fucoidan’s immunotherapeutic applications, particularly in the emerging paradigm of engineered cellular therapies, such as CAR-T cell immunotherapy.

CAR-T cells constitute genetically engineered T lymphocytes expressing a synthetic CAR architecture. This molecular construct integrates three critical domains: (1) an extracellular antigen-recognition module comprising a single-chain variable fragment (scFv), (2) a structural hinge domain facilitating spatial orientation, and (3) an intracellular activation complex combining costimulatory signaling elements (typically CD28 or 4-1BB) with the CD3ζ signal transduction component [7]. CAR-T technology has demonstrated remarkable clinical efficacy against hematological malignancies, achieving durable remissions in previously refractory cases [8]. The success of anti-CD19 CAR-T therapies in managing relapsed/refractory CD19 + B-cell neoplasms culminated in FDA approval of multiple commercial products [9]. While great improvement for hematologic cancers, therapeutic challenges still exit, including progressive CAR-T cell exhaustion and impaired in vivo persistence [9]. In addition, within the highly oxidative and immuno-suppressive tumor microenvironment (TME), CAR-T cell proliferation and functional persistence are significantly impaired. Therefore, current researches focus on developing many combinatorial strategies for CAR-T functional potency enhancement, e.g. cytokine co-expression [10–12]. While these strategies demonstrate promising therapeutic potential, they often induce severe adverse effects in patients, particularly cytokine release syndrome (CRS). Consequently, there is an urgent need to develop safer and more sustainable approaches—especially nutritional interventions — to improve the anti-tumor efficacy of CAR-T cell therapy.

Given the immunomodulatory activity of FO, we hypothesized that FO can exerts supportive effects on CAR-T therapy against cancer by enhancing the cytotoxicity of CAR-T cells. Thus, phenotypic and functional alterations in CAR-T cells induced by FO was deeply investigated. Additionally, the study thoroughly verified the FO-mediated augmented tumoricidal activity by in vitro assays and in vivo NHL model, and the mechanisms involved in this functional enhancement was also investigated. Overall, this study aims to elucidate the role of FO in CAR-T therapy for NHL and to establish its application as immunopotentiator for CAR-T therapy. The study lays the groundwork for the clinical application of FO in cellular immunotherapy.

Materials and methods

CAR-T cell construction

The anti-CD19 CAR-T cells were constructed as previous study [10]. T lymphocyte Purification was achieved using the EasySep Human T Cell Isolation Kit (Cat#19051, STEMCELL Technologies, USA). Subsequent T cell activation employed CD3/CD28 magnetic beads (Cat#40203D, Thermo Fisher Scientific, USA) at a 3:1 bead-to-cell ratio for 24 h preceding lentiviral transduction. CAR-T cell cultures were maintained in complete X-VIVO 15 medium (Cat#02-060Q, LONZA Bioscience, Swiss) supplemented with 10% FBS, 200 U/mL recombinant human IL-2 (Cat#200-02, Peprotech, USA), and 1% penicillin-streptomycin antibiotic cocktail.

After T cell activation in vitro for 24 h, CAR-T cells were then transduced with lentivirus containing CD19 CAR structure. Purified CAR-T cells were obtained by the EasySep PE selection kit (Cat#17684, STEMCELL Technologies, USA) along with a PE-conjugated anti-EGFR antibody (Cat#386304, Biolegend, USA).

Reagents

Fucoidan was purchased from MedChemExpress company with purity of 98.15% (Cat#HY-132179, MedChemExpress, USA). According to the manufacturer’s information, FO are mainly characterized as a polymer with backbone structure of repeating (1→4) linked α-l-fucopyranose residues (Fig. 1A), and the Fourier transform infrared spectrometer (FTIR) spectra analysis of FO is in Fig. 1B. The information of FO’s composition and sulfate content is in the supplementary Table S1.

Fig. 1.

FO’s structure and FTIR analysis. (A) Illustration of FO’s structure. (B) FTIR analysis of FO

A stock solution was prepared in PBS and sterilized through 0.22 μm membrane filtration. In the in vitro assays of this study, the concentration of FO treatment is 40 µg/ml. STAT3 inhibitor, BP-1-102 with purity of 99.52% (Cat#HY-100493, MedChemExpress, USA) was dissolved in dimethyl sulfoxide (DMSO) to create stock solutions. All compounds were stored according to manufacturer recommendations until use. The antibodies used in this study were provided in supplementary material - Table S3.

In vitro cytotoxicity assays

Cytotoxicity was evaluated using the lactate dehydrogenase (LDH) release assay by CytoTox96^® Non-Radioactive Cytotoxicity Kit (Cat#G1780, Promega, USA) according to the manufacturer’s protocol. Briefly, target cells (1.0 × 10⁴ cells/well) were co-cultured with CAR-T cells at effector-to-target (E: T) ratios of 5:1, 2.5:1 and 1.25:1 in 96-well plates. The plates were incubated at 37 °C. After incubation, supernatants were collected, and LDH activity was quantified colorimetrically by measuring absorbance at 490 nm. Spontaneous LDH release (effector and target cells cultured separately) and maximum LDH release (target cells lysed with 1% Triton X-100) were measured in parallel. Specific cytotoxicity was calculated using the formula: 100 × [(Experimental − Effector spontaneous − Target spontaneous) / (Maximum release − Target spontaneous)].

ELISA

Target cells (1 × 10⁴ cells/well) were co-cultured with CAR-T cells at a 2.5:1 effector-to-target (E: T) ratio for 24 h. Post-incubation, cell-free supernatants were collected and analyzed for secreted IFN-γ, Granzyme B, IL-2, TNF-α using commercially available ELISA kits (Cat# Ek180/158/182/102 MultiSciences, USA) according to manufacturer’s protocols.

Mouse models

Six- to eight-week-old NSG male mice (NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ) were maintained under specific pathogen-free (SPF) conditions at the Experimental Animal Center. All animal studies were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Shenzhen University General Hospital. On Day − 14, mice were intraperitoneally inoculated with 2.5 × 10⁶ Daudi-luciferase cells suspended in Matrigel (Cat#354262, Corning, USA). Following tumor engraftment, animals were randomized into three groups (n = 3–5/group) for weekly intravenous treatments: (1) T cell; (2) CAR-T therapy (CAR-T control group); (3) CAR-T therapy + FO (10 mg/kg, intravenous injection once every three days after CAR-T infusion). Tumor progression was monitored bi-weekly using the IVIS Spectrum Imaging System (IVIS Spectrum, PerkinElmer, USA). Bioluminescence signals were quantified with Living Image software and expressed as total flux (photons/sec).

ROS detection

The ROS levels were quantified employing the fluorescent probe DCFH-DA (Cat#D6883, Sigma-Aldrich, USA). The cells underwent two PBS washes before being incubated with 10 µM DCFH-DA diluted in serum-free medium for 0.5 h at 37 °C in darkness. Subsequent to three PBS washes for excess probe removal, fluorescence intensity measurements were conducted using both: (1) a SpectraMax i3x microplate reader (Molecular Devices) at excitation/emission wavelengths of 480/535 nm, and (2) parallel flow cytometry analysis.

NRF2 and STAT3 activity assays

NRF2 and STAT3 activity was assessed using pSTAT3-TA-Luc and pNRF2-TA-Luc reporter system (Cat#D2259/4236, Beyotime, China). Cells were transiently transfected with the plasmid. At 24 h post-transfection, cells were treated with experimental compounds or vehicle control for the specified durations. Luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega, USA) following the manufacturer’s protocol. Briefly, cells were lysed in passive lysis buffer, and firefly luciferase signals were normalized to co-transfected Renilla luciferase (pRL-TK, Promega, USA) for transfection efficiency correction. Relative luminescence was measured on a microplate reader.

Statistical analysis

All experiments were performed in triplicate or more to ensure reproducibility. Data are presented as mean ± SD and analyzed using Student’s t-test. Statistical significance was defined as P < 0.05, with asterisks denoting specific thresholds: * (P < 0.05), ** (P < 0.01), *** (P < 0.001). “ns” means “not significant”.

Results

FO promoted memory T cell formation and resistance to exhaustion in CAR-T cells

T cells were isolated using anti-CD3/CD28 magnetic beads and transduced with a lentiviral vector encoding a anti-CD19-specific chimeric antigen receptor (CAR) to generate CAR-T cells targeting CD19 + malignancies (Fig. 2A). Following transduction, CAR-T cells were enriched using a purity-sorting protocol and subjected to FO treatment for 3 days prior to flow cytometric analysis (Fig. 2A). The CAR construct, as illustrated in Fig. 2B, consists of an anti-CD19 single-chain variable fragment (scFv), a CD8-derived hinge and transmembrane domain, and intracellular co-stimulatory signaling motifs (4-1BB and CD3ζ). Robust CAR expression was confirmed in transduced T cells (Fig. 2C).

Fig. 2.

FO contributes the maintenance of central memory subset and resistance to exhaustion in CAR-T cells. (A) Schematic illustration of the research model. The prepared CAR-T cells were treated with PBS or FO for 3 days. CAR-T cell phenotype: the status of differentiation (CD45RO, CD62L, CD4, and CD8), exhaustion (PD-1, TIM-3), were detected by flow cytometry. (B) Schematic representation of CD19-CAR structure. (C) Relative expressions of CAR mRNA in CAR T cells were detected by qPCR (n = 3). (D) The percentage of CD8 and CD4 subset in each group (n = 3). CAR expression and FO treatment exert no effect on the ratio of CD8 and CD4 T cells. (E&F) Flow-cytometric analysis of central memory subsets (CD45RO+ & CD62L + cells, marked by the red box) in CAR-T cells from each group. FO treatment enhances the frequency of memory cells. (G) The level of exhaustion-related receptors (PD-1, TIM-3) in control or FO-treated CAR-T cells, (n = 3). All data are shown as the mean ± SD. ***p < 0.001

Emerging clinical evidence has established that the enrichment of memory-like CAR-T cells correlates with durable functional persistence and sustained remission in recipients of CAR-T therapy [13]. Here, the memory phenotype was identified by measuring the proportion of CD62L and CD45RO double-positive (CD45RO+&CD62L+) subsets in CAR-T cells, typically named as central memory T cells (TCM). Dose-response screening revealed FO treatment induced dose-dependent enrichment of TCM populations, with maximal enhancement observed at 40 µg/ml FO concentration (supplementary Fig. 1). This dose was also applied in the following in vitro experiments. Flow cytometry revealed that FO treatment did not alter the baseline CD4/CD8 ratio (1:1; Fig. 2D, supplementary Fig. 2). Notably, TCM population was significantly enriched in both CD4 + and CD8 + subsets following FO exposure (Fig. 2E&F). Exhaustion-associated surface receptors (PD-1 and Tim-3) were analyzed to evaluate CAR-T cell functional status [14]. FO treatment substantially reduced the frequency of exhausted CAR-T cells, particularly the PD-1+&Tim-3 + double-positive population (Fig. 2G). These phenotypic shifts indicate that FO enhances the formation of memory-like CAR-T cells while mitigating exhaustion.

FO strengthened CAR T cells’ antioxidant defense

The tumor microenvironment (TME) is inherently characterized by pronounced oxidative stress, with elevated ROS level constituting a pivotal immunosuppressive niche [15]. NRF2, a master transcription factor, controls the expression of antioxidant genes essential for neutralizing reactive oxygen species (ROS). In FO-treated CAR-T cells, we observed enhanced Nrf2 activity, accompanied by significant upregulation of downstream antioxidant genes, including HO-1, SOD2, CAT, GPX1, and NQO1 (Fig. 3A-C). Consistent with this transcriptional reprogramming, FO treatment markedly increased intracellular levels of reduced glutathione (GSH), a key antioxidant metabolite critical for ROS detoxification (Fig. 3D). These findings demonstrated that FO reinforces the antioxidant defense system in CAR-T cells.

Fig. 3.

FO enhances CAR-T cell’s antioxidant ability. (A) FO increases the activity of NRF2 in CAR-T cells (n = 3). (B&C) QPCR and western blot of NRF2 target genes (n = 3). FO increases the expression of NRF2 target genes, CAT, NQO1, HO-1, SOD2, and GPX1. (D) GSH level were raised in the CAR-T cells with FO treatment (n = 3). (E) ROS level was quantified in each group (n = 3). FO decreased ROS level in CAR-T cells. (F) ROS staining by DCFH-DA of each group were examined by flow cytometry. FMO control as a reference for gating. All data are shown as the mean ± SD. *p < 0.05, **p < 0.01

To assess intracellular reactive oxygen species (ROS) levels in CAR-T cells, we employed the fluorescent ROS-sensitive probe 2’,7’-dichlorofluorescein diacetate (DCFH-DA). Upon cellular uptake, DCFH-DA is deacetylated to DCFH, which undergoes oxidation by ROS to form the fluorescent compound dichlorofluorescein (DCF) for quantification. DCFH-DA staining revealed that FO-treated CAR-T cells exhibited significantly reduced ROS levels compared to untreated controls, confirming FO’s capacity to suppress ROS accumulation (Fig. 3E). This inhibitory effect was further validated by flow cytometric analysis (Fig. 3F).

CAR T cells with FO treatment exhibited enhanced proliferation and survival

Following antigen engagement, CAR-T cells undergo activation-induced proliferation to eliminate target cells, subsequently entering apoptosis to terminate the immune response [16]. Enhancing proliferative capacity and delaying apoptotic pathways are therefore critical for sustaining CAR-T cell functionality. To evaluate FO’s impact on these processes, we analyzed proliferation and apoptosis in activated CAR-T cells.

FO-treated CAR-T cells displayed significantly elevated frequencies of Ki67 + cells in both CD4 + and CD8 + subsets compared to controls after 3 days of activation, indicative of enhanced proliferative activity (Fig. 4A). This finding was corroborated by increased total cell counts in FO-treated groups, confirming robust cellular expansion (Fig. 4B). Furthermore, Annexin V/7-AAD staining revealed a marked reduction in apoptotic rates in FO-treated CAR-T cells, compared to the control (Fig. 4C&D). Collectively, these data demonstrated that FO potentiated CAR-T cell proliferation and prolonged survival upon activation.

Fig. 4.

FO enhanced the proliferation of CAR T cells and prolonged the survival of CAR-T cells. (A) Frequency of Ki67 + cells in CD4 + and CD8 + CAR-T cells stimulated with αCD3 and αCD28 Abs for 3 days in each group (n = 3). (B) CAR-T cell counts of each group after activation (n = 3). FO enhanced the proliferation of CAR-T cells. (C&D) Representative flow plots (C) and summary (D) of 7AAD and Annexin V staining of apoptotic cells after simulation (n = 3). All data are shown as the mean ± SD. *p < 0.05

FO enhanced cytotoxicity of CAR T cells against NHL cells

To evaluate FO’s impact on CAR-T cell-mediated cytotoxicity, we performed in vitro killing assays using CD19-CAR T cells co-cultured with NHL cells (Daudi). FO-treated CAR-T cells exhibited significantly enhanced lytic activity against cancer cells, compared to untreated controls in primary killing assays (Fig. 5A). To assess functional persistence, CAR-T cells were isolated after the initial co-culture and rechallenged with fresh target cells in secondary and tertiary killing assays. Strikingly, the superiority of FO-treated CAR-T cells became more pronounced with successive rounds of killing. As to low effector-to-target (E/T) ratios (1.25:1 and 2.5:1), the prolonged co-culture duration amplified the differential cytotoxicity between FO-treated and control groups (Fig. 5A-B).

Fig. 5.

CAR-T cells with FO treatment showed strong antitumor activity against NHL cells in vitro. (A) Cytotoxicity of control or FO-treated CAR-T cells against Daudi cells (NHL cells) at the Effector (CAR-T) / Target (Daudi) (E/T) ratio of 1.25:1, 2.5:1, and 5:1 for 8 h (n = 3). The first, second and third round assay showed FO enhanced the cytotoxcity of CAR-T cells. (B) Cytotoxicity assay of the Effector/Target ratio of 1.25:1, 2.5:1 at different co-culture time (n = 3). (C&D) The cytotoxicity effect of CAR-T cells were detected by flow cytometry. After 48 h co-culture of CAR-T and Daudi at E/T ratio 1:1(C, 0 h) and 1:2(D, 0 h), Daudi cells were almost eliminated in FO group. 16.3% and 31.8% of Daudi cell still survival in control group. (E) ELISA analysis of TNF-α, IFN-γ, IL-2, and Granzyme B in control or FO-treated group of CAR-T cells (n = 3). All data are shown as the mean ± SD. *p < 0.05, **p < 0.01

To evaluate CAR-T cell cytotoxicity directly, we employed flow cytometry to detected the population change of CAR-T and target cells. Target cancer cells were co-cultured with CAR-T cells at effector-to-target (E/T) ratios of 1:1 and 1:2 (Fig. 5C, amp and D, 0 h). After 48 h incubation period, near-complete elimination of target cells was observed in FO-treated groups, whereas control groups still maintained significantly higher target cancer cell population (Fig. 5C, amp and D, 48 h). Additionally, consistent with the observed cytotoxic enhancement, FO-treated CAR-T cells secreted elevated cytokines, including interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and granzyme B (Fig. 5E).

FO enhances the antitumor activity of CAR T cells against NHL in vivo

To evaluate the antitumor efficacy of CAR-T cells in vivo, we established NHL xenograft model by intraperitoneal injection of Daudi-luciferase cells into NSG mice. Fourteen days post-tumor engraftment, mice were intravenously injected CAR-T or T cells. In CAR-T injected mice, FO were intravenously injected periodically, with PBS as control. Tumor burden was monitored periodically via bioluminescence imaging (Fig. 6A). In CAR-T treated mice, FO-conditioned group showed enhanced antitumor activity, as evidenced by a significant reduction in tumor burden (Fig. 6B, C) and prolonged survival (Fig. 6D) compared to controls. Meanwhile, the absence of significant body weight loss in mice underscores the systemic safety of FO (supplementary Fig. 3). Furthermore, FO exerted no detectable effect on the anti-tumor function of non-CAR T cells in vivo, ruling out any intrinsic antitumor activity of FO itself for the process of CAR-T treatment (supplementary Fig. 4). To assess CAR-T cell dynamics, peripheral blood samples were collected at days 7, 14, and 21 post-infusion. FO treatment robustly enhanced CAR-T cell expansion in vivo (Fig. 6E). Increased number of CAR-T cells was detected in bone marrow of FO-conditioned mice (Fig. 6F). Furthermore, FO-treated CAR-T cells in bone marrow exhibited reduced expression of exhaustion-associated surface receptors (Fig. 6G). These findings collectively underscore FO’s ability to enhance CAR-T cell antitumor functionality and persistence in vivo.

Fig. 6.

FO improves anti-tumor activity of CAR T cells in vivo. (A) The animal experiments to assess anti-tumor activity of CAR T cells in vivo were schematically illustrated. On day − 14, 2.5 × 10⁶ Daudi-luciferase cells were injected into NSG mice. On day 0, 2 × 10⁶ CAR-T cells were injected intravenously into NSG mice with NHL tumor burden. On day 14, 28, and 42, bioluminescence imaging was conducted. (B) Bioluminescence imaging of mice in each group. FO group showed super tumor clearance, compared to the control. “X” represents dead subject (endpoint). (C) Statistical analysis of tumor burden by bioluminescence imaging (n = 3 or 5). (D) Survival curve of NHL NSG mice in each group (n = 3 or 5). (E) The percentage of CD3 + T cells in peripheral blood of each mouse (n = 5). This was used to evaluate the expansion of CAR T cells in vivo. (F) Numbers of CAR-T cells remained in bone marrow of each group (n = 3). (G) Frequency of exhausted CAR-T cells remained in bone marrow of each group (n = 3). All data are mean ± S.D. *p < 0.05

FO mediated functional enhancement against NHL through STAT3 pathway

The activity of CAR T cells has been strongly associated with the activation STAT3 pathway [16, 17]. To investigate FO’s impact on this critical signaling pathway, we systematically analyzed STAT3 activation status in CAR T cells with and without FO treatment. Our findings demonstrated enhanced STAT3 phosphorylation in FO-treated CAR-T cells, accompanied by upregulated expression of STAT3-regulated genes (SOX2, SOCS3, Survivin, ABCA1) (Fig. 7A ~ C). Pharmacological inhibition of STAT3 in FO-treated CAR T cells effectively abolished these transcriptional changes induced by FO (Fig. 7C). NRF2 activity is tightly associated with STAT3 activation [18, 19], and STAT3 inhibition also lead to the repression of NRF2 activity (Fig. 7D).

Fig. 7.

FO improved CAR-T cell function by activating STAT3 signaling pathway. (A) FO increased the phosphorylation of STAT3 (n = 3). (B) The FO-induced STAT3 activity enhancement was abolished by STAT3 inhibitor BP-1-102 (8µM) (n = 3). (C) QPCR assay of STAT3 target genes of each group (n = 3). (D) NRF2 activity of each group (n = 3). (E) CD45RO+&CD62L + CAR-T cells in each group were determined by flow cytometry (n = 3). (F) The exhausted CAR-T cells in each group (n = 3). (G) The cytotoxicity assays of CAR-T cell in each group (n = 3). STAT3 inhibitor blocked the FO-induced cytotoxic enhancement. (H) The secreted cytokines (IL-2, IFN-γ) of CAR-T cells (n = 3). All data are mean ± S.D. *p < 0.05, **p < 0.01

Functional characterization revealed that STAT3 blockade reversed FO-mediated cellular advantages. After adding STAT3 inhibitor, the memory T cell population reduced significantly, while exhausted cell populations expanded in FO-treated group (Fig. 7E&F). This phenotypic shift on CAR-T cells was accompanied by the diminished cytotoxic capacity against NHL cells, as evidenced by reduced killing efficiency and attenuated cytokine release (Fig. 7G&H). Collectively, these mechanistic insights established STAT3 pathway activation as a critical mediator of FO’s supportive role on CAR-T cell functionality. Our data suggest that FO enhances CAR T cell performance through STAT3-dependent modulation of memory maintenance, exhaustion resistance, and effector function.

Discussion

This study represents a new standpoint and a paradigm shift in nutritional intervention for cellular immunotherapy by functional food factor. FO, a polysaccharide abundantly present in brown marine algae, shows a broad spectrum of biological activities including antioxidant, anticoagulant, antitumor, antiviral, and anti-inflammatory effects, along with immune-modulatory properties [20]. Our current study uncovers that FO enhanced CAR-T cell functionality through STAT3 pathway. Specifically, FO improved memory formation and resistance to exhaustion of CAR-T cells, and meanwhile FO also exerted protection against oxidative stress and augmented activated CAR-T cell proliferation and survival. Importantly, we demonstrated FO-induced enhancement of CAR-T cell cytotoxicity both in vitro assay and in vivo animal experiments. This study is the first demonstration of food functional factor -fucoidan’s supportive role in CAR-T cell immunotherapy.

Marine-derived polysaccharides have garnered significant attention in recent decades as a promising class of biomaterials for therapeutic applications. Fucoidan (FO), a sulfated polysaccharide predominantly isolated from brown algae, demonstrates multifaceted bioactivities in oncological interventions, particularly through its immunomodulatory and antitumor properties [21]. Multiple studies revealed FO’s ability to suppress tumor progression [22]. Despite robust preclinical evidence supporting FO’s anticancer potential [23], its clinical translation in cancer therapy still faces substantial challenges. The therapeutic efficacy of FO in cancer management requires administration of supraphysiological doses, which are neither achievable through dietary intake nor feasible via conventional intravenous delivery due to pharmacokinetic limitations. Furthermore, the inherent complexity of cancer, characterized by heterogeneous molecular mechanisms and adaptive resistance pathways, complicates the direct application of FO monotherapy. To address these limitations, many studies propose to leverage FO’s properties in combination therapies. The synergistic anticancer functions of FO with cancer chemotherapy agents have been proved, such as oxaliplatin and 5-fluorouracil/leucovorin [24]. While the synergistic potential of polysaccharides in cellular immunotherapy remains underexplored, our study provides pioneering evidence that FO potentiates the cytotoxic efficacy of CAR-T cells against NHL. This finding positions FO as a novel immunological supplement capable of enhancing cellular therapy, thereby expanding its translational potential in clinical cancer immunotherapy.

CAR T cell therapy has emerged as a transformative modality in immunotherapy, achieving unprecedented clinical responses in B-cell malignancies through CD19-targeted constructs. However, durable remission remains elusive in most patients [25], largely due to progressive CAR T cell dysfunction (e.g., exhaustion, senescence) and tumor-intrinsic escape mechanisms (e.g., antigen loss, immunosuppressive microenvironment with oxidative properties) [26]. Critical determinants of therapeutic efficacy include the proliferative capacity, stem-like memory phenotype, and polyfunctional cytokine secretion profile of infused CAR T cells, which collectively underscore the imperative to engineer next-generation constructs with enhanced fitness. Current optimization strategies focus on following points: (1) CAR structural refinement – Incorporating divergent costimulatory domains (e.g., 4-1BB vs. CD28) and mutated signaling motifs (e.g., truncated CD3ζ) to fine-tune activation thresholds and persistence [27]; (2) Combinatorial engineering –co-expressing immunomodulatory factor (e.g., IL-15 superagonists, 4-1BBL) to counteract suppressive microenvironments [28, 29]. Paradoxically, these enhancements often exacerbate toxicity risks, including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity (ICANS), and on-target/off-tumor effects, thereby limiting clinical adoption. In this context, fucoidan (FO), a marine sulfated polysaccharide with established antioxidative and immunoregulatory properties, presents a compelling adjunctive strategy. Distinguished by its favorable safety profile in nutritional supplementation contexts, FO circumvents the toxicity of synthetic immunomodulators and enhances the cyto-toxicity of CAR-T cells against cancer cells, similar to metformin (supplementary Fig. 5), a drug known to improve CAR-T efficacy [30]. Our findings revealed FO’s capacity to potentiate CAR-T cell therapy, resulting in the enhanced in vivo proliferative kinetics and tissue residency, sustained effector functionality against NHL xenografts. For example, while FO-induced increase in cytokines release is correlated with enhanced anti-tumor efficacy, it also warrants careful monitoring for potential risks, such as Cytokine Release Syndrome (CRS). Whether FO can induce CRS in human remains unclear, and it is important to note that mouse models are limited in their ability to fully simulate human CRS, which should be considered as a potential risk of FO in the future clinical trial. This study positions FO as a first-in-class natural supplement capable of amplifying CAR T cell therapeutic efficacy.

Nevertheless, this study has limitations. The translational relevance of our findings to human populations requires further validation, which highlights directions for future research. These findings were derived from preclinical models, and the direct applicability to human patients remains to be established. The significant immunological differences between murine and human systems necessitate further validation in humanized mouse models or early-phase clinical trials. For example, while FO-induced increase in cytokines release is correlated with enhanced anti-tumor efficacy, it also warrants careful monitoring for potential risks, such as Cytokine Release Syndrome (CRS). Whether FO can induce CRS in human remains unclear, and it is important to note that mouse models are limited in their ability to fully simulate human CRS, particularly in detecting key CRS indicators IL-6, which should be considered as a potential risk of FO in the future clinical trial. Furthermore, we did not mechanistically resolve the direct molecular target(s) through which FO potentiates STAT3 pathway activation. While our data implicate higher STAT3 phosphorylation in FO-mediated CAR T cells, the precise upstream interactome remains undefined. To address these gaps, future studies should be conducted to delineate the hierarchical signaling events driving this phenomenon. Additionally, the pharmacokinetics, optimal dosing regimen, and long-term safety profile of fucoidan in the context of CAR-T therapy warrants deeper investigation. Such investigations are critical for translating FO’s supplementary potential in CAR-T cellular immunotherapy.

Conclusion

In summary, our findings provided the compelling evidences that FO supplement can enhance anti-cancer efficacy of CAR-T cells against NHL, with maintenance of memory phenotype and reduced exhaustion in CAR-T cells through activation of STAT3 signaling pathway. The combination of CAR-T cell therapy and FO supplement shows significant promise for achieving better clinical outcome.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CAR

Chimeric antigen receptor

DCFH-DA

2’,7’-dichlorofluorescein diacetate

E/T ratio

The ration of effector cell (CAR-T)/target cells (Daudi)

FO

Fucoidan

GSH

Reduced glutathione

ROS

Reactive oxygen species

scFv

single-chain Fragment of Variable region

Author contributions

Qingzheng Kang and Liang Zhang: Conceptualization, Methodology, Data curation, Formal analysis, Writing – original draft. Xiaoqing Wu: Methodology, Formal analysis, Writing – review & editing. Xuanren Shi: Supervision, Conceptualization, Writing – review & editing.

Funding

This work was supported by Shenzhen Natural Science Foundation (Basic Research Project, No. JCYJ20240813180400002).

Data availability

The data supporting the conclusions of this article are included within the article and its additional file.

Declarations

Ethics approval and consent to participate

All animal studies were conducted in accordance with the guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Shenzhen University General Hospital (Approval IDs: 202311-35).

Consent for publication

Not applicable.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.