Immune cell-based therapies for solid tumors, current challenges and therapeutic advances

Abstract

Solid tumors remain difficult to treat due to antigen heterogeneity, physical barriers that limit immune-cell trafficking, and a profoundly immunosuppressive tumor microenvironment (TME). Over the past decade, cancer immunotherapy advanced considerably through innovative strategies, including macrophage reprogramming and CAR-macrophages, dendritic-cell (DC) vaccines, natural killer (NK) and natural killer T (NKT) cell approaches, tumor-infiltrating lymphocyte (TIL) therapy, TCR-engineered and CAR-T cells, emerging B-cell engineering, and cell-derived extracellular vesicles (EVs). Here we summarize how each modality interacts with the TME, highlight key clinical milestones (e.g., FDA approval of a TIL product for melanoma in 2024), and outline bioengineering strategies—multi-antigen targeting, cytokine armoring, trafficking cues, and safety switches—that aim to overcome resistance and toxicity. We also review EV-based, cell-free strategies that retain tumor specificity with potentially improved safety and manufacturability. Finally, we discuss remaining barriers—standardized manufacturing, on-target/off-tumor effects, limited persistence—and propose rational combinations with checkpoint blockade, radiotherapy, and targeted agents. This overview positions immune cell–based therapy as a rapidly maturing, transformative approach for solid tumors.

Graphical abstract

Background

Cancer arises from dysregulated cell and tissue growth driven by a series of cellular and molecular events, leading to the uncontrolled proliferation of transformed cells [1, 2]. In the last decades, cancer treatment, particularly solid tumors, continues to be a considerable challenge due to their intricate structures and heterogenicity [3, 4]. Solid tumors are characterized by abnormal tissue proliferations forming a compact mass, without cystic or fluid-filled regions that pose significant challenges for surgical resection and conventional treatments. Moreover, tumor cells form a genetically and phenotypically diverse subpopulation, with a small but substantial group of cancer stem cells (CSCs). CSCs are particularly resistance to typical cancer treatments, which allows them to survive and contribute to tumor repopulation and disease recurrence [5].

Solid tumors also have a highly complex tumor microenvironment (TME) consists of a dynamic network between tumor cells, fibroblasts, mesenchymal stromal cells, immune cells, and non-cellular fractions such as metabolites, chemokines, cytokines and extracellular matrix (ECM) components.

High levels of immunosuppressive cytokines such as TGF-β, IL-10, and IL-35, metabolic restrictions including hypoxia, glucose depletion, amino acid starvation, and releasing anti-inflammatory mediators and inhibitory molecules are other hallmark features of the TME [6].

Thus, it appears that solid tumors exhibit considerable heterogeneity due to the presence of CSCs and their complex, adaptive TME that effectively coordinate various steps of tumor development, such as immune escape, drug resistance, and metastasis [7–10].

Given these obstacles, developing innovative therapies that can target both CSCs and modulate the dynamic nature of TME at the same time is increasingly recognized as an essential need for effective cancer treatment. Novel approaches like immune cell-based therapies have revolutionized the landscape of cancer treatment, offering unprecedented opportunities to overcome the physical and biochemical limitations which imposed by the TME. Over the last decade, various adoptive cell therapy (ACT) and innate immune cell-based therapies are being applied to a range of oncological conditions and gained attraction due to their potential advantages and outweighing associated risks [11, 12]. (Table 1) This review summarizes the various immune cell therapy approaches for solid tumors and evaluates their advantages and limitations, shedding light on challenges in their future clinical translation.

Table 1.

Summary of the functional roles of immune cells in solid tumor therapy

Cell Type	Key Characteristics	Advantages	Disadvantages	Challenges	References
TIL	Naturally derived from tumor tissue; personalized; polyclonal T cells	Broad antigen recognition; tumor infiltration; minimal toxicity	Time-consuming; T cell exhaustion; non-standardized product	Expensive production; inconsistent quality; weak in vitro expansion	[13]
TCR-T cells	Genetically engineered T cells expressing tumor-specific TCRs; MHC-restricted	Recognizes intracellular antigens; sensitive to low antigen levels	Risk of off-target toxicity; HLA restriction	Poor persistence; TME inhibition; antigen selection challenges	[14]
CAR-T cells	Engineered T cells with synthetic receptors; MHC-independent recognition	Strong in hematologic cancers; specific targeting; durable response	CRS and neurotoxicity; poor solid tumor efficacy	Antigen escape; TME barriers; manufacturing complexity	[15]
CAR-γδT cells	T cells with innate and adaptive functions; MHC-independent	Target stress ligands; less exhausted	Limited clinical data; unknown long-term safety	Expansion methods; CAR compatibility	[16]
Macrophages	Phagocytic myeloid cells; antigen-presenting; innate sensors	TME remodeling; stimulate adaptive immunity	Polarization complexity; immunosuppressive potential	Polarization control (M1/M2); safety; scalability	[17]
CAR-Macrophages (CAR-M)	Macrophages engineered to express CARs; promote phagocytosis and antigen presentation	Remodel TME; recruit adaptive immunity	Low transduction efficiency; limited lifespan	Efficient CAR gene delivery; controlling M1/M2 bias	[18]
NK Cells	Innate cytotoxic lymphocytes; recognize “missing self” targets	MHC-independent killing; no GvHD; off-the-shelf possibility	Weak expansion; short persistence	Improving persistence; CAR optimization	[19]
CAR-NK cells	NK cells modified with CARs; innate immune features	Low GvHD risk; allogeneic potential; broad cytotoxicity	Short persistence; complex expansion	Improving CAR delivery and in vivo survival	[19]
NKT Cells	Express both TCR and NK markers; recognize glycolipids	Unique tumor reactivity; immunomodulatory potential	Rare in circulation; limited protocols	Expansion and in vivo persistence	[20]
CAR-NKT cells	Hybrid T/NK phenotype; recognize glycolipid antigens via CD1d	Bridge innate and adaptive immunity; tumor-homing potential	Low abundance; difficult to isolate	Expansion and persistence optimization	[20]
B Cells	Antibody-producing cells; antigen-presenting capacity	Long-term memory; vaccine potential	Tumor-supporting roles; less direct cytotoxicity	Exploring B-cell ACT or regulatory roles	[21]
Dendritic Cells (DCs)	Professional APCs; activate naïve T cells	Induce tumor-specific T cells; suitable for cancer vaccines	Short half-life; technically demanding	Improving migration and antigen loading	[22]

Macrophage Cell-Based immunotherapy in solid tumors

Macrophages and TME

Macrophages (MQs) are a type of white blood cell that plays a vital role in the immune system, serving as the first line of defense against foreign substances, infection, and disease [23].

MQs have historically been classified into two types: M1 and M2, according to their unique morphology, phenotype, and function. M1 MQs exhibit pro-inflammatory and anti-tumor properties, whereas M2 MQs display anti-inflammatory and pro-tumorigenic features. M2 MQs can be further divided into four subtypes (M2a, M2b, M2c, and M2d) according to their surface marker expression, cytokine production, and response to specific stimuli [24]. Among these, the M2d subtype, also known as tumor-associated MQs (TAMs), represents the most abundant stromal cell population within the TME. Development of TAM is orchestrated by a complex interplay between the signaling pathways and transcription factors. For example, the STAT3 and NF-κB signaling pathways are activated in response to tumor-derived factors, such as IL-6 and TNF-α, leading to the polarization of macrophages towards an M2 phenotype. Moreover, studies of histone modifications showed that lactate accumulation in the TME can epigenetically shift macrophages toward M2-like and immunosuppressive phenotypes via certain modifications such as H3K18la [25]. Recent single-cell RNA sequencing (scRNA-seq) atlases (e.g., in breast cancer) have revealed multiple TAM subtypes that co-express both M1- and M2-associated genes, instead of falling cleanly into one category (for example, C1QC⁺, TUBA1B⁺, and VCAN⁺ TAM populations) [26]. The presence of TAM has been linked to tumor progression, metastasis, and resistance to therapy [27].

MQs are recruited to the TME through various mechanisms, including chemotaxis mediated by chemokines (e.g., CCL2/CCR2, CXCL12/CXCR4), adhesion molecules, and growth factors. Once recruited, they are activated by tumor-derived signals such as colony-stimulating factor-1 (CSF-1) [28]. Activated TAMs produce immunosuppressive cytokines, such as IL-10 and transforming growth factor-beta (TGF-β), which can suppress T cell activation and proliferation [29]. They also express immune checkpoint molecules such as PD-L1, which inhibit T cell function and eliminate anti-tumor immune cells through phagocytosis of T cells and Natural killer (NK) cells. In addition, recent studies demonstrate that TAMs can directly engulf and eliminate anti-tumor immune cells, including CD8⁺ T cells and NK cells, thereby exacerbating immune evasion [26] TAMs further promote the accumulation of regulatory T cells (Tregs) and secrete growth factors, such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which can stimulate tumor cell proliferation and angiogenesis [30]. By releasing matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, TAMs remodel the extracellular matrix, facilitating tumor invasion and metastasis [25, 31]. (Fig. 1)

Fig. 1.

Role of macrophages in tumor progression and therapy. Cancer and stromal cells secrete cytokines (e.g., CSF-1, IL-34), chemokines (e.g., CCL2, CXCL4) and growth factors (e.g., CSF) that recruit circulating monocytes and induce expansion of tissue-resident macrophages, driving their differentiation into pro-tumor (M2-like) tumor-associated macrophages (TAMs). TAMs produce immunosuppressive cytokines including TGF-β and IL-10 that suppress cytotoxic T lymphocyte and NK cell activity, while also expressing immune checkpoint ligands like PD-L1 that contribute to the expansion and recruitment of regulatory T cells (Tregs). The predominant strategies in MQ-based cancer therapy focus on inhibiting macrophage recruitment, enhancing TAMs phagocytosis, and depleting or reprogramming TAMs

Strategies to manipulate MQs phenotype and number in the TME

The manipulation of MQ phenotype and number in the TME has emerged as a promising strategy for cancer therapy. Strategies aimed at targeting TAMs, such as MQ-depletion therapies or promoting the polarization of MQs towards an anti-tumor phenotype, may provide new opportunities for cancer treatment [32]. These approaches primarily aim to reduce the number of M2 MQs in the TME, thereby reducing the immunosuppression and creating an environment more conducive to effective antitumor immunity [33].

CSF-1R is a key regulator of MQ survival, proliferation, and differentiation. Inhibition of CSF-1R reduces the number of TAMs and promotes an anti-tumoral M1 phenotype [34]. Immune checkpoint molecules, such as PD-1 and PD-L1, play a crucial role in regulating MQ activation [35]. Checkpoint blockade not only enhances T-cell activity but has also been shown to reprogram TAMs toward an M1-like state in preclinical models. Modulation of cytokine levels, such as increasing IL-12 or inhibiting IL-10, has been shown to favor M1 polarization and improve anti-tumor responses [36]. Nanoparticle-mediated delivery of immunomodulatory agents – including siRNA, miRNA mimics, or small-molecule inhibitors – can further promote M1 polarization and enhance anti-tumoral activity [37]. Depletion of MQs using clodronate liposomes or other agents has also been used to inhibit tumor growth and metastasis [38]. Moreover, the use of combination therapies, such as integrating cytokine therapy with nanoparticle-based approaches and checkpoint inhibitors, are emerging as particularly promising strategies [39].

Different biomaterials or tissue-engineering scaffolds have also been designed to deliver specific signals that modulate MQ polarization. For instance, biomaterials with a high degree of hydrophilicity and negatively charged surfaces promoted M2 polarization, whereas biomaterials with a high degree of hydrophobicity and positively charged surfaces tend to induce M1 polarization [40]. Biomaterials with a porous structure can foster M2 polarization by supporting cell–cell interactions and tissue remodeling, whereas dense scaffolds often induce M1 polarization and enhance pro-inflammatory cytokine release [41]. The incorporation of bioactive molecules, such as growth factors and cytokines, into biomaterials or tissue engineering scaffolds is another approach that could modulate MQ polarization. For example, incorporation of granulocyte-macrophage colony-stimulating factor (GM-CSF) or IFN-γ promotes M1 polarization, whereas inclusion of TGF-β drives M2 polarization and fibrosis [42, 43]. (Fig. 1). Analyzing current and previous clinical evaluations of TAM-reprogramming in cancer therapies also revealed that over 200 agents were studied across more than 700 clinical trials targeting TLR, CD47–SIRP axis, IDO1, and STAT signaling pathways. The strategies most often explored are monoclonal antibodies, small molecules, antibody–drug conjugates, pH-dependent target-binding antibodies, bi-specific antibodies, and fusion proteins. However, transforming preclinically studied agents used for TAM-reprogramming into clinically active therapeutics faces several challenges including limited efficacy of monotherapies and complexity of combination strategies, and low specificity in TAM-targeted agents. Addressing these obstacles requires identifying reliable biomarkers for patient selection, applying data-driven designs for combination regimens, developing TAM-specific targets and TME-focused delivery systems. In addition, deepening biological insights through high-dimensional analyses of TAM changes post-therapy is inevitable [44].

Bioengineered MQ therapy

In recent years, engineered MQs generated through genetic modification or small molecule-mediated reprogramming, have garnered significant attention for their potential to overcome the limitations of MQ therapy in the TME [45].

The purpose of engineered MQs therapy in solid tumors is twofold. First, these cells can be used directly as anti-tumor agents, infiltrating the TME and exerting cytotoxic effects on cancer cells [46]. Second, engineered MQs can act as vaccine platforms by presenting tumor-associated antigens to the immune system and eliciting durable, anti-tumor immune response [47]. For example, MQs can be genetically modified to produce therapeutic proteins, such as IL-12, GM-CSF, or TNF-α, which stimulate adaptive and innate anti-tumor immunity. They can be used as a delivery platform for nanoparticles, which can selectively target tumor cells and release therapeutic payloads within tumors [48].

CAR-MQ therapy is an innovative strategy that combines the tumoricidal capabilities of MQs with the precision of CAR-mediated antigen recognition to target and eliminate tumor cells [49]. CAR-MQ strategies involve the use of viral vectors, such as lentivirus or retrovirus, to transduce MQs with CAR genes or mRNA-based approaches, which enable the transient expression of CAR on MQs. This approach offers several advantages, including the ability to avoid the risks associated with viral vector-mediated gene transfer and the potential for rapid and flexible manufacturing [50].

CAR-modified MQs recognize and bind tumor cells expressing specific antigens, such as CD19, CD20, or HER2 [51]. This recognition triggers the activation of MQs, leading to the production of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 that recruit and activate other immune cells, including T cells and NK cells [52]. Activated MQs also exhibit enhanced cytotoxicity and phagocytic activity, allowing them to eliminate tumor cells through direct cell-to-cell contact and the engulfment of cancer cells. CAR-MQ cell therapy offers several potential benefits. First, they enhance the activity of CAR-T cells through antigen cross-presentation and cytokine secretion, leading to improved anti-tumor responses [53]. Second, CAR-MQs reduce the risk of cytokine release syndrome (CRS) by modulating the production of pro-inflammatory cytokines [54]. Third, CAR-MQs can improve the safety of CAR-T cell therapy by reducing the incidence of neurotoxicity. In a mouse model of acute lymphoblastic leukemia, the co-administration of engineered MQs with CAR-T cells led to improved anti-tumor responses and reduced toxicity. In another study engineered MQs were shown to enhance the activity of CAR-T cells and reduce the risk of CRS in a humanized solid mouse model [55].

However, several challenges must be addressed before engineered MQs can be translated into clinical practice. Engineered MQs may elicit immune-related adverse events, such as CRS or autoimmune reactions. Solid tumors exhibit significant heterogeneity, which may limit the efficacy of engineered MQs by restricting the availability of uniform target antigens [56]. Large-scale production of engineered MQs faces significant regulatory and technical hurdles, particularly in ensuring product consistency, potency, and safety [57]. Finally, the source of MQs for CAR-MQ production is a critical factor influencing their efficacy; While peripheral blood mononuclear cells (PBMCs) are readily accessible, their limited expansion potential and donor-to-donor heterogeneity pose potential challenges in translational research. (Fig. 1)

MQ derived EVs in solid tumor therapy

MQs, as key players in the TME, have been shown to release high amounts of extracellular vesicles (EVs) that can modulate the immune response and influence tumor growth [58].

The use of MQ-derived EVs (MQ-EVs) in solid tumor therapy is an emerging field that has garnered significant attention in recent years. One of the primary advantages of using MQ-EVs is their ability to traverse biological barriers within tumors (often termed the “blood–tumor barrier”), enabling localized delivery of therapeutic cargos to the TME. For example, EVs can be loaded with pro-inflammatory molecules or nucleic acid cargos (e.g., siRNA/miRNA mimics) to suppress immunosuppressive signaling and reduce pro-tumor cytokine output, which can promote tumor growth and metastasis [59]. M1MQs-EVs delivering doxorubicin to breast cancer cells increased cytotoxicity and reduced tumor growth, and M1MQ-EVs delivering sorafenib to hepatocellular carcinoma cells reduced cell proliferation and increased apoptosis [60, 61].

Despite the promising results of these studies, the main challenges of using MQ-EVs in clinical practice include large-scale production of EVs with consistent quality and purity, targeted delivery of EVs to the tumor site and efficient uptake of them by cancer cells. To address these challenges, researchers are developing new methods [62, 63]. For example, microfluidic isolation platforms have been used to enrich and purify EVs from MQ culture supernatants with improved yield and standardization [64]. Nanoparticle-assisted targeting and surface engineering (e.g., peptide or antibody decoration of EV membranes) are being evaluated to enhance homing to tumor tissue. In addition to these technical challenges, there are also several regulatory hurdles that must be addressed before clinical application of MQ-EVs [65]. The U.S. Food and Drug Administration (FDA) has not yet issued finalized product-class guidance specific to therapeutic EVs, so expectations for release testing (e.g., identity, purity, potency, sterility) are evolving [66].

Dendritic Cell-Based immunotherapy in solid tumors

DCs and the TME

Dendritic cells (DCs) are known as a professional and heterogeneous class of antigen-presenting cells (APCs) capable of identifying danger signals and engulfing antigenic materials. DCs are involved in both innate and adaptive immune reactions and are exposed to complex alterations in phenotypes and function in response to signals, antigens, and some environmental stimuli. Tumor-infiltrating DCs (TIDCs) often acquire altered phenotypes in the TME, supporting tumor growth, impairing immune surveillance, and contributing to resistance to therapies, including immunotherapies [67, 68].

The regulation of DC function by tumors and tumor-associated cells/factors is one of the main mechanisms of developing tumor immune evasion. Although presence of DCs contributes to inducing anti-tumor immunity, impaired performance of TIDCs confers immunosuppression TME negatively modulate DC function through DC-suppressive molecules including TGF-β, M-CSF, VEGF, and IL-6 [69, 70]. These factors change cytokine receptor expression and myeloid differentiation of CD34⁺ progenitors by inhibiting MAPKs, PI3K/Akt, and JAK/STAT signaling pathways, directing them toward an immunosuppressive CD14⁺ monocyte fate that are unable in mediating allogeneic T cell proliferation [71, 72]. Second, the presence of Tregs in TME contribute to tolerogenic DCs and maintain an suppressing anti-tumor immunity [73]. Third, suppressing the function of DCs is mediated by the expression of inhibitory molecules, like PD-L1, PD-L2, Tim3, and LAG3 [74]. Finally, the TME can particularly regulate DCs’ antigen presentation function through influencing the molecules and machinery responsible for antigen presentation. These include reducing the expression of MHC class I and MHC class II molecules and regulators like CIITA, diminishing the expression of genes like transporter associated with antigen processing (TAP) and ER-resident aminopeptidases (ERAP) [75].

DCs as a therapeutic tool in solid tumors

The discovery of DCs as potent APCs led to the development of cellular vaccines that use these cells to present tumor antigens. Manufacturing of DC vaccine initiate with isolation of monocytes or DCs by apheresis, differentiation into immature DCs in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Their activation involves tumor-associated antigens (TAAs) loading to differentiate from immature to mature DCs and finally, the injection of activated DCs to inducing and enhancing multiple arms of the tumor-specific immune response by CD4 + and CD8 + T cells, as well as NK cells. DC-based immunotherapy may be considered a well-tolerated and promising strategy in the cure of patients with refractory malignant tumors. DC vaccines may stimulate immunological memory against tumor antigens, which could lead to long term protection against recurrence and can be customized to target each patient’s unique tumor antigens, developing a personalized therapy [22]. Suminoe et al. added DCs to tumor-specific synthetic peptides or tumor lysates in the presence of the immunogenic protein KLH and found that the resulting mixture inhibited growth of the refractory malignant solid tumors in children by escalating the number of CD8⁺ HLA-DR⁺ lymphocytes and INF-γ⁺CD8⁺ lymphocytes [76]. Jung et al. found that exposing DCs in vitro to photodynamic therapy (PDT)-induced tumor lysates could enhance DC immunotherapy in coping with tumors by improving their function in suppressing solid tumors [77]. Lim et al. reported that although DC immunotherapy may be adequate for suppressing tumor metastasis or recurrence, it failed to eradicate established solid tumors [78] due to active immunosuppression within the TME. Currently, several Phase I/II trials have investigated the effects of factors such as antigen loading and DC subsets on the immunogenic response of DC vaccines and indicated that the efficient activation of DCs is the most crucial step in development of effective DC vaccines. Moreover, FDA approved Provenge in 2010, as the first DC-based therapeutic vaccine for prostate cancer, and in 2017, the Indian government approved APCDEN for treatment resistant solid tumors like ovarian and colorectal cancers [79].

While ongoing progress in DC-based vaccines is promising, the widespread clinical application of DC-based vaccines is still posing multiple limitations related to complexity and variability between patients, antigen selection, antigen-loading methods, and DC subtype choice. Moreover, prior or concurrent anticancer treatments, especially chemotherapy or targeted agents, can impair monocyte quality, reducing DC yield and immunostimulatory capacity. Studies suggest that optimizing the methods for DC generation and antigen loading, overcoming the immunosuppressive nature of TME, increasing the specificity of the immune response through personalized vaccines and treatment sequencing and early monocyte collection to preserve DC functionality can help overcome these challenges. For example combination therapy approaches that mitigate TME suppression hold greater promise [80] (Fig. 2).

Fig. 2.

Role of DCs in cancer immunity and immunotherapy. DCs are considered as the key factors in the initiation and maintenance of effective T-cell-mediated anti-tumor immunity due to their critical roles in antigen presentation. Various TME-generated factors (e.g. IL-10, TGF-β, IL-6, and M-CSF) caused loss of DC function by inhibiting DC recruitment, activation, and antigen presentation. DC-based vaccines stimulate antigen-specific immune responses that activate both cellular and humoral immunity, leading to tumor cell destruction and the development of immunological memory.

Bioengineered DC vaccines

Engineered DC vaccines are a class of strategies according to the manipulating cells for galvanizing the immune system in coping with specific antigens [81]. As an interesting example, DCVax^® (Northwest Biotherapeutics, Inc., MD, USA) is known as a platform technology for presenting DC-based therapeutic vaccines for various cancers, especially glioblastoma multiforme (GBM) [82]. DCVax^®-L, one of the types of DCVax platforms, is known as a personalized active immunotherapy consisting of autologous whole tumor lysate and DCs. Clinical trial studies showed that using DCVax-L creates a good therapeutic paradigm for subjects with GBM [82]. DCs could be genetically engineered to promote their anti-tumoral actions with higher efficiency [83]. The expression of various targets and regulating a variety of pathways of anti-tumor immunity can be facilitated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), RNA interference (RNAi), and viral transduction. There are several strategies by which anti-tumoral functions of DC-based vaccines can be improved. These include (i) increasing tumor-associated antigen (TAA) presentation in which in situ antigen uptake and presentation of related TAAs can be elevated by expression of an engineered receptor [84], (ii) elevation of lymph node migration via up-regulation of CCR7 [85], (iii) Abating immunosuppression by silencing DC-intrinsic immunosuppressive molecules like PD-L1 [86], and (iv) increasing recruitment of immune cells by overexpression of various chemokines at the tumor site [87]. (Fig. 2).

However, the main challenges regarding using engineered DC–based immunotherapies are including poor migration of ex vivo generated DCs to lymphoid organs, variability in patients’ immune status, inconsistent antigen selection and lack of standardized production methods for regulatory approval and routine use. Emerging technologies such as in vivo DC targeting, single-cell transcriptomics, RNA sequencing, and AI-guided antigen prediction can address some limitations and enable more accurate antigen selection and tailored manipulation of specific DC subsets. Biomarker-based patient stratification and standardized manufacturing workflows will also improve consistency and clinical translatability. Combining DC vaccines with cytokine treatments, oncolytic viruses, or checkpoint inhibitors may also boost antitumor responses and promote durable memory T-cell immunity and ultimately improving therapeutic success [88].

DC-Derived EVs in solid tumor therapy

EVs can act as natural vectors to deliver RNA and therapeutic agents targeted to tumor cells, DCs, and T-lymphocytes. Hence, EVs may be considered a promising option for creating novel cell-free anti-tumor vaccines as a good alternative to DC-based vaccines. To create an adequate activation of an anti-tumor immune response, DCs should be capable of delivering 3 types of signals to T cells: (i) presentation of tumor antigens in complexes with MHC class I and II molecules on the DC surface to T-cell receptors, (ii) delivery of signals through interplay between co-stimulatory and adhesion molecules present on the DC surface and their receptors on T cells; and (iii) generating T-cell stimulatory cytokines by DCs [89].

DC-derived EVs (DC-EVs) have shown abilities to deliver all the molecules responsible for activation of anti-tumor T cell-mediated immune responses [90]. There are functionally active complexes including co-stimulatory and adhesion molecules and tumor antigens and MHC molecules class I and II on the surface of DC-derived EVs and they are capable of delivering cytokines, interplay with T lymphocytes and direct initiation of anti-tumor immune responses [91, 92].

Chen et al. found that DC-derived EVs encapsulated with E749–57 peptide could induce the cytotoxic activity of CD8 + T cells on TC-1 tumor cells ex vivo and stimulating CD8 + T cells for the proliferation and IFN-γ excretion. In addition, DC-derived EV vaccine could improve the immune responses of vaccinated mice splenocytes induced by antigen E7 [93]. Similarly, Bu et al. reported that DC-derived EVs loaded with chaperone-rich cell lysates could create a strong T Cell immune response against intracranial glioma in mice [94].

However, DC-derived EVs share several key limitations with other EV types, including low drug-loading and delivery efficiency, limited penetration into dense tumor tissues, and significant heterogeneity arising from differences in DC maturation and production methods. To address these issues, DC- derived EVs can be engineered through membrane modification to enhance targeting, loading of therapeutic or immunostimulatory cargo to strengthen antitumor effects, and incorporation into combination therapies to improve overall efficacy [95].

NK Cell-Based immunotherapy in solid tumors

NK cells and the TME

NK cells are a part of the innate immune system that plays a crucial role in providing a first line of defense against cancer and virus-infected cells [96]. NK cells are characterized by their surface expression of specific receptors and markers, including CD56, CD16, and NKG2D, all of which play a crucial role in the recognition and elimination of target cells. CD56 is used to identify and isolate these cells [97]; CD16 (FcγRIII) is a low-affinity receptor for IgG that enables antibody-dependent cellular cytotoxicity (ADCC); and NKG2D recognizes stress-induced ligands such as MICA and MICB expressed on tumor and infected cells. The interaction between NKG2D and its ligands triggers the activation of NK cells, leading to the production of cytotoxic granules and the elimination of target cells [98].

The interplay between NK cells and the TME is a critical determinant of cancer progression and treatment outcomes. NK cells are a heterogeneous population, and different subsets have been identified in TME based on their surface marker expression and functional properties. For example, the CD56^bright subset of NK cells expresses higher levels of NKG2D and has been shown to be more cytotoxic and produce more cytokines than the CD56^dim subset [99]. In addition to their direct cytotoxic effects, NK cells also play a key role in shaping the TME [100].

However, the TME can also suppress NK cell function, leading to immune evasion and cancer progression. Tumor cells can produce immunosuppressive factors, such as TGF-β, IL-10, and PGE2, which can inhibit NK cell activation and cytotoxicity. TGF-β acts mainly via the SMAD3 pathway in NK cells and suppress the IFN-γ production, IL-10 decreases NK cell survival and function through dampening cytokine release pathways and PGE2 inhibit NK cell cytotoxicity via downregulation of NK-activating receptors such as NKp30, NKp44, NKp46, and NKG2D. Additionally, the TME can promote the accumulation of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which suppress NK cell activity through both soluble mediators (TGF-β, IL-10) and cell contact-dependent mechanisms [101]. (Fig. 3)

Fig. 3.

NK cell-based cancer immunotherapies. NK cells play a pivotal role as the first line defenders against tumor cells, recognizing and directly killing target cells with downregulated MHC class I or upregulated stress ligands (e.g., MICA/B, ULBPs) by releasing perforin and granzyme and secreting immunostimulatory cytokines. Infusion of ex vivo-expanded or cytokine-activated (IL-2/IL-15/IL-18) autologous/allogeneic NK cells, NK Cell Metabolic/Genetic Engineering are the most common therapeutic platforms that have been developed to enhance NK-cell cytotoxicity

NK cells as a therapeutic tool for solid tumors

The therapeutic potential of NK cells lies in their ability to target cancer cells while sparing healthy tissues. Unlike T cells, which require antigen presentation and activation, NK cells can recognize and kill tumor cells in an antigen-independent manner. This property makes them an attractive option for cancer treatment, particularly for solid tumors that often exhibit heterogeneous antigen expression [102]. Additionally, NK cells can be easily expanded and activated ex vivo, enabling researchers to scale-up functional effector cells for innate immunotherapy [103].

Several NK cell-based therapies are currently being developed for the treatment of solid tumors including: (i) Autologous NK Cell Therapy: In this approach, NK cells are isolated from a patient’s peripheral blood and expanded ex vivo using cytokines and growth factors; (ii) Allogeneic NK Cell Therapy: NK cells are isolated from a healthy donor and expanded ex vivo then infused into the patient, (iii) NK Cell-Derived Cytokines: NK cells are isolated and expanded ex vivo, and then used to produce cytokines, such as IFN-γ and TNF-α, which can be used to stimulate an anti-tumor response; (iv) NK Cell-Based innate Immunotherapy: in this approach, NK cells are isolated and expanded ex vivo, and then genetically modified to express tumor-specific receptors, such as CARs [104].

Using autologous or allogenic NK cells has been shown to be effective in treating various types of cancer, including leukemia, lymphoma, and solid tumors [105]. For instance, a phase I trial is evaluating the safety and feasibility of autologous NK cell infusion in patients with advanced solid tumors [106]. Another phase I/II trial is assessing the efficacy of allogenic NK cell therapy in combination with chemotherapy in patients with relapsed or refractory acute myeloid leukemia [107]. Additionally, a phase II trial is investigating the use of NK cell therapy in combination with checkpoint inhibitors in patients with metastatic melanoma. NK cell-based immunotherapies, such as bispecific antibodies and CAR-NK cells, are being developed to enhance the anti-tumor activity of NK cells [108].

Despite their potential as a therapeutic approach, NK cells can also develop resistance mechanisms in the TME, leading to reduced anti-tumor activity. For example, tumor cells can downregulate the expression of NKG2D ligands, making them less susceptible to NK cells [109]. Additionally, the TME can promote the upregulation of checkpoint molecules, such as PD-1, on NK cells, leading to NK cell exhaustion [110]. Several strategies are being explored to overcome NK cell resistance in the TME. These include the use of checkpoint inhibitors, such as anti-PD-1 antibodies, to enhance NK cell function, as well as the development of novel immunomodulatory agents that can target the TME and promote NK cell activation [111]. While encouraging, NK cell therapies face challenges including limited in vivo persistence, inefficient tumor trafficking, and manufacturing scalability. Addressing these barriers is essential to fully realize their clinical potential [112]. (Fig. 3)

Bioengineered NK cell therapy

Bioengineered NK cells have been developed to improve their recognition and killing of cancer cells through genetic and molecular modifications. Approaches include expression of CARs, arming with tumor-specific antibodies, and modification of cytokine receptors to enhance proliferation and cytotoxicity [113]. Compared with unmodified NK cells, engineered NK cells demonstrate superior anti-tumor efficacy, making them an attractive option for treatment of solid tumors.

One of the primary advantages of engineered NK cells is their ability to target cancer stem cells (CSCs) [114], which are often resistant to traditional chemotherapy and radiotherapy. In this regard, NK cells are equipped with CARs or T cell receptors (TCRs) that recognize specific CSCs surface antigens such as CD44, CD133, and CD24 and release cytotoxic granules, such as perforin and granzyme, which then induce apoptosis [115, 116]. Beyond antigen recognition, engineered NK cells can also recognize CSCs through their specific cytokines and chemokines. For example, CSC-derived IL-6 can trigger engineered NK cells to produce IFN-γ [117] which in turn activates the immune response against the CSCs. In another mechanism, bioengineered NK cells target CSCs by generating ROS via the activation of specific enzymes like NADPH oxidase [118].

Thus, the use of engineered NK cells has several advantages over traditional cancer therapies: (1) specific targeting of CSCs which reduces the risk of recurrence and resistance [119], (2) feasible ex vivo expansion and the ability to infiltrate solid tumor tissues, which is a major obstacle for many cancer therapies [120], and (3) having the potential to be used in combination with other immunotherapies, such as checkpoint inhibitors, to enhance their anti-tumor activity [121].

Researchers have developed various strategies to enhance the anti-tumor activity of engineered NK cells. One approach is to arm NK cells with immune stimulatory molecules, such as IL-12 or IFN-γ, which can enhance their cytotoxic activity and promote an anti-tumor immune response [122]. Another approach is to engineer NK cells to resist immune suppression by expressing immune checkpoint inhibitors, such as PD-1 or CTLA-4, which can block the inhibitory signals from the TME [123]. A study demonstrated that CAR-NK cells targeting the tumor antigen HER2 showed significant anti-tumor activity in a murine model of HER2-positive breast cancer [124]. Another study demonstrated that bioengineered NK cells expressing IL-12 and a CAR targeting the tumor antigen GD2 showed enhanced anti-tumor activity in a murine model of neuroblastoma [125].

Despite these promising results, several challenges remain that should be addressed before engineered NK cells can be translated into clinical practice. One of the main challenges is the development of scalable and cost-effective methods for the production and expansion of engineered NK cells [126]. Currently, the production of engineered NK cells requires complex and labor-intensive processes, including viral vector transduction, cell sorting, and expansion, which can be time-consuming and expensive [127]. Another challenge is the development of strategies to enhance the in vivo persistence and anti-tumor activity of engineered NK cells [128]. While engineered NK cells have shown promising anti-tumor activity in preclinical studies, their in vivo persistence and activity can be limited by various factors, including immune suppression, tumor heterogeneity, and the lack of tumor-specific antigens [129]. In addition, there are also regulatory and logistical challenges that need to be addressed before engineered NK cells can be translated into clinical practice. There is a need for standardization of the production and characterization of engineered NK cells, as well as the development of guidelines for their clinical use [130].

NK Cell-Derived EVs in solid tumor therapy

Recently, NK cell–derived extracellular vesicles (NK-EVs) have emerged as a cell-free immunotherapy with intrinsic anti-tumor activity through various mechanisms [131]. One of the primary mechanisms is the transfer of anti-tumor proteins and microRNAs from NK cells to tumor cells, which can lead to the inhibition of tumor growth and induction of apoptosis. For example, NK-EVs contain high levels of Fas ligand that induce apoptosis in tumor cells [132]. Additionally, NK-EVs express NKG2D ligands, which can activate the immune response against tumor cells. They also exert anti-tumor effects through the modulation of TME [133]. and inhibition the proliferation and migration of tumor-associated endothelial cells, which are essential for tumor angiogenesis [134]. The use of NK-EVs in solid tumor therapy has several advantages over traditional cancer therapies. NK-EVs can target tumor cells selectively, reducing the risk of toxicity to healthy cells. Moreover, they can penetrate deep into tumors, allowing for the delivery of anti-tumor molecules to areas that are difficult to reach with traditional therapies [135].Finally, NK-EVs stimulate the immune response against tumor cells and lead to the development of long-term anti-tumor immunity, reducing the risk of tumor recurrence [136].

Despite their potential, NK-EVs also have some limitations. One of the main limitations is the difficulty in isolating and purifying EVs from NK cells, which is a time-consuming and expensive process. The stability and half-life of EVs in vivo are still not well understood, and the potential of NK-EVs to induce an immune response against healthy cells is still a risk of autoimmune responses or unintended immune activation [137, 138]. Recently, NK-EVs are also being explored as a combination therapy with other cancer treatments, such as chemotherapy and immunotherapy to enhance the efficacy of these treatments while reducing their toxicity [139]. The role of immune cell-EVs in inhibiting tumor initiation and progression depending on the context and the cell of origin.

NKT Cell-Based immunotherapy in solid tumors

NKT cells and the TME

Natural killer T (NKT) cells are a unique subset of T lymphocytes that share features of both T cells and NK cells. NKT cells arise from a distinct lineage of thymocytes that express the invariant T cell receptor (TCR) α-chain, Vα14-Jα18 in mice and Vα24-Jα18 in humans [135]. This invariant TCR is responsible for the recognition of glycolipid antigens presented by the non-classical major MHC molecule, CD1d. They are also characterized by their unique functional and phenotypic properties and expression a range of cell surface markers, including CD3, CD56, CD161 and exhibit a distinct cytokine profile, producing both Th1- and Th2-associated cytokines, such as IFN-γ and IL-4, respectively. NKT cells play a crucial role in the immune system, particularly in the early stages of immune responses against pathogens and tumors. In the context of cancer, NKT cells have significant role in the regulation of anti-tumor immune responses. In the TME, NKT cells recognize lipid antigens presented by CD1d on tumor or myeloid cells through T-cell receptor (TCR) leading to upregulation of CD40L on their surface which interacts with CD40 on dendritic cells and other myeloid cells. The mentioned interferences include activation and the production of cytokines, such as IL-12, IFN-γ and TNF-α that exert anti-tumor effects by activating other immune cells, such as NK cells and T cells. NKT cells also possess cytotoxic activity, by releasing granzymes and perforin which allows them to directly kill cancer cells. Additionally, NKT cells can produce chemokines, such as CCL3 and CCL4, which can attract other immune cells, such as T cells and MQs, to the TME. The function of NKT cells in the TME is tightly regulated by various mechanisms. One of the key regulators of NKT cells is the presence of cytokines, such as IL-12 and IL-18 that activate NKT cells and enhance their anti-tumor activity. On the other hand, while type I NKT cells mainly enhance antitumor responses, tumor cells produce immunosuppressive cytokines such as TGF-β, IL-10, and IL-13 leading to a transition toward regulatory type II NKT cells that induce immunosuppressive features of TME through cytokine secretion, encouraging the accumulation of regulatory T cells and myeloid-derived suppressor cells.

NKT cells as a therapeutic tool for solid tumors

The anti-tumor activity of NKT cells is attributed to their ability to recognize and kill tumor cells directly, as well as produce cytokines, such as and TNF-α to activate other immune cells, such as DC and cytotoxic T cells [130]. These unique properties of NKT cells make them an attractive target for immunotherapy and vaccine development [140].

Various strategies have been developed to exploit NKT cell therapy for cancer. Lipid-based vaccines consist of lipid antigens such as α-galactosylceramide (α-GalCer), a synthetic glycolipid that is presented by CD1d molecules on APCs leading to the activation of NKT cells in vivo. Preclinical studies have shown that α-GalCer can stimulate the anti-tumor activity of NKT cells, leading to the inhibition of tumor growth and metastasis [141]. Clinical trials are currently underway to evaluate the safety and efficacy of α-GalCer in patients with solid tumors.

The use of NKT cells as a vaccine adjuvant also offers several advantages. NKT cells activation by a wide range of antigens, including those derived from pathogens, tumors, and even self-antigens allow the development of vaccines that can target multiple epitopes and provide broad protection against various diseases [142]. In addition, NKT cells can be expanded and activated ex vivo, enabling the generation of large numbers of cells that can be used as a vaccine adjuvant [143]. However, the mechanisms by which NKT cells exert their adjuvant activity are not fully understood, but several studies have shed light on the underlying processes. One key mechanism is the production of cytokines, such as IFN-γ and TNF-α, which can activate APCs and enhance the immune response [106]. Another mechanism is the activation of DCs, which can lead to the production of pro-inflammatory cytokines and the presentation of antigens to T cells. NKT cells also possess direct antimicrobial and antitumoral activities which involve the transfer of ex vivo expanded NKT cells into patients [144]. Finally, application of checkpoint inhibitors, such as anti-PD-1 and anti-CTLA-4 antibodies could enhance NKT cell activity by blocking inhibitory signals [145].

There are also several challenges that need to be addressed to fully exploit the therapeutic potential of NKT cells. One of the major challenges is the limited understanding of NKT cell biology, particularly in the context of human cancer. Further research is needed to elucidate the mechanisms of NKT cell activation, regulation, and function in the TME [146]. (Fig. 4)

Fig. 4.

Role of NKT cells in the regulation of tumor immunity. NKT cells bridge innate and adaptive immunity by recognizing lipid antigens presented on CD1d molecules expressed by tumor cells, APCs, and other TME components. Upon activation, they rapidly produce cytokines (IFN-γ, TNF-α, IL-4), stimulating NK cells, DCs, CD8⁺ and CD4⁺ T cells to amplify antitumor responses. CAR-NKT cells represent a novel therapeutic approach for solid tumors, combining CAR-directed tumor targeting with the innate-like properties of NKT cells

Bioengineered NKT cell therapy

Engineered NKT cells can target a broad range of tumor cells, including those that are resistant to conventional therapies. NKT cells are capable of recognizing tumor cells in an MHC-unrestricted manner, which eliminates the need for MHC matching between the donor and recipient [147]. This feature makes them a promising approach for allogenic adaptive cell therapy. Furthermore, NKT cells can also recognize and eliminate CSCs [148]. Several engineering strategies have been developed to enhance the anti-tumor activity of NKT cells. One approach is to genetically modify NKT cells to express tumor-specific receptors, such as CARs or T cell receptors (TCRs) [149]. Another approach is to engineer NKT cells to produce cytokines, such as IL-2 or IL-15, which can enhance their anti-tumor activity and promote their proliferation and survival [150].

Preclinical studies have shown the ability of NKT cells engineered to express a CAR to recognize and kill tumor cells [151]. In a study, engineered NKT cells expressing a CAR specific to the tumor antigen GD2 could eradicate established neuroblastoma tumors in mice [152]. Another study showed that engineered NKT cells expressing a TCR specific for the tumor antigen NY-ESO-1 could inhibit the growth of established melanoma tumors in mice [153]. Several clinical trials are also currently underway to evaluate the safety and efficacy of engineered NKT cells in patients with solid tumors. One ongoing phase I clinical trial is assessing the safety and feasibility of autologous engineered NKT cells in patients with advanced melanoma [154]. Another phase I clinical trial is evaluating the safety and feasibility of allogenic engineered NKT cells in patients with relapsed or refractory multiple myeloma [155].

Another promising approach is the use of NKT cells as a platform for bispecific T cell engagers (BiTEs). BiTEs are bispecific antibodies that can engage T cells and tumor cells, leading to the formation of immunological synapses and the killing of tumor cells. NKT cells are an attractive candidate for BiTE therapy due to their ability to recognize and respond to lipid antigens on tumor cells [156]. Preclinical studies have shown that NKT cells can be redirected to tumor cells using BiTEs, leading to the inhibition of tumor growth and metastasis. Clinical trials are currently underway to evaluate the safety and efficacy of BiTE-NKT cells in patients with solid tumors [157].

Despite the promising results of bioengineered NKT cells in preclinical and clinical studies, still several challenges and limitations need to be addressed. One major challenge is the limited availability of NKT cells in peripheral blood, which can make it difficult to obtain enough cells for therapy [158]. Another challenge is the potential risk of bioengineered NKT cells to cause off-target effects or cytokine release syndrome, which can be severe and life-threatening. To overcome the challenges and limitations of engineered NKT cells, several innovative approaches are being explored [159]. One area of research is the development of novel engineering strategies to enhance the anti-tumor activity of NKT cells while reducing their potential for off-target effects [160]. Another area of research is combination therapies that incorporate engineered NKT cells with other immunotherapies, such as checkpoint inhibitors or cancer vaccines [161]. (Fig. 4)

CAR-NKT cells exhibited a superior antitumor efficacy compared with CAR-T cells.

T-Cell-Based immunotherapy in solid tumors

T-Cells in the TME

Antitumor immunity, especially T-cell infiltration, is a major determinant of prognosis in solid tumors [162]. However, the TME imposes strong metabolic and immunologic constraints. Hypoxia, nutrient depletion, and immunosuppressive metabolites such as lactate impair T-cell activation and survival. Through the Warburg effect, tumor cells outcompete T cells for glucose, amino acids, and nucleotides leading to disrupted T-cell metabolism and glycolytic activity, diminished phosphoenolpyruvate (PEP) levels, inhibition of Ca²⁺ flux following TCR engagement, and suppression of the mTOR signaling pathway that are critical for T cell activation, proliferation, and cytokine secretion [163]. This reprogramming also promotes immune escape by enhancing checkpoint pathways and expanding regulatory T cells that dampen immune responses and induce tolerance by secretion of inhibitory cytokines like IL-10, IL-35.

CD8⁺ T-cells exert antitumor activity by releasing perforin and granzymes to directly kill tumor cells and by secreting IFN-γ, which boosts antigen presentation and recruits additional immune cells [164]. CD4⁺T-cells also play a critical role in antitumor immunity through differentiation into Th1 cells that activate cytotoxic T lymphocytes, enhance APC function, and modulate the TME toward an inflammatory state through IL-2 and IFN-γ secretion. Additionally, some CD4⁺T cells have shown direct cytotoxic activity, contributing to tumor cell death directly or through interactions within the TME [165].

Chronic antigen exposure and immunosuppressive cytokines such as IL-10 and TGF-β drive T-cell exhaustion, characterized by reduced cytokine production, high inhibitory receptor expression, and altered transcriptional profiles. Exhausted T cells retain partial pathogen- or tumor-control capacity but may lose all effector functions in tumors. Two subsets of exhausted T cells exist: progenitor cells with recall potential and terminally differentiated cells with limited functionality [166].

Another escape mechanism is resistance, where tumors reduce antigen presentation by downregulating MHC class I, often seen in breast, prostate, and lung cancers [167]. Moreover, several intrinsic and extrinsic factors, including immune cells and soluble mediators, drive reduced T-cell proliferation and responsiveness to tumor antigens. For example, tumor cells express inhibitory ligands, such as PD-L1, PD-L2, and CTLA-4 ligands, which bind to receptors on T-cells (e.g., PD-1, CTLA-4) to suppress their function and promote immune evasion. On the other hand, B cells support T-cell memory, NK cells boost T-cell activation and Tregs suppress them through cytokines, maintaining immune balance and tolerance in both normal and diseased settings [168, 169].

T-Cells as a therapeutic tool in solid tumors

Immune evasion by tumors and the immunosuppressive TME necessitate innovative strategies to improve therapeutic outcomes. As a key component of the adaptive immune system, T cells have revolutionized cancer treatment by their ability to specifically eradicate cancer cells. Tumor-infiltrating lymphocytes (TILs) are a specialized population of T cells that naturally infiltrate into the tumors. The therapeutic potential of TILs lies in their ability to specifically recognize and target tumor-associated antigens. TILs consist predominantly of CD8⁺ cytotoxic T-cells and CD4⁺ helper T-cells, which recognize tumor-specific or neoantigens through their T-cell receptors. This antigen specificity makes TILs a compelling option for solid tumor treatment, where the heterogeneity of antigen expression presents significant challenges for other immunotherapies [170]. TILs are directly derived from resected tumor tissue. The process begins with tumor digestion to isolate lymphocytes, then proceed to their ex-vivo expansion in the presence of high-dose interleukin (IL)-2. Once a sufficient early TIL population is achieved, further amplification is performed through a 14-day rapid expansion protocol (REP) involving stimulation with agonistic anti-CD3/CD28 antibodies, high-dose IL-2, and irradiated allogeneic or autologous PBMCs as feeder cells. After non-myeloablative lymphodepletion (NMA-LD) using cyclophosphamide and fludarabine to promote TIL engraftment, patients are administered TILs accompanied by high-dose IL-2 to enhance in vivo outgrowth of the infused TIL [171].

Several TIL-based therapies are currently being developed for the treatment of solid tumors, including (i) Bulk TIL Therapy which involves the expansion of the entire TIL population without selection, (ii) Selected TIL Therapy is an enrichment method, focused on isolating tumor-reactive T-cells targeting neoantigens or tumor-specific epitopes. This approach enhanced the precision of therapy and improved response rates in solid tumors like non-small cell lung cancer (NSCLC), (iii) Genetically Modified TILs which are engineered to improve their functionality, persistence, or resistance to immunosuppressive factors within the TME [172, 173].

Several studies have highlighted the success of TIL therapy in a variety of solid tumors like melanoma, cervical, ovarian, and NSCLC [174]. In 2024, FDA approved the first TIL therapy product, Lifileucel (Amtagvi) for the treatment of adults with unresectable or metastatic melanoma. A phase II trial with lifileucel, demonstrated overall response rate (ORR) of up to 36% in metastatic melanoma patients with checkpoint inhibitor-resistant [175]. Similar advances have been reported in patients with heavily pretreated cervical cancer, where TIL therapy achieved ORRs exceeding 40% [176].

Despite their potential as a therapeutic approach, TIL therapy faces several significant challenges, limiting its widespread application. The manufacturing process is complex, requiring specialized facilities and skilled staff, making it expensive and logistically difficult, especially in developing countries. Furthermore, the extended duration of in-vitro expansion (up to two months) may not be feasible for patients with rapidly progressing diseases, and prolonged culture can lead to TIL exhaustion, reducing their cytotoxicity and persistence. The therapy’s personalized nature also facilitates regulatory approval and consistency in TIL quality. Additionally, the immunosuppressive TME including regulatory T-cells, MDSCs and inhibitory cytokines such as TGF-β as well as tumor heterogeneity, antigen escape, ineffective TIL infiltration to the tumor site can pose barriers to effective TIL function [177, 178].

Several strategies were developed that can be used to overcome some of these challenges. Using checkpoint inhibitors (e.g., anti-CTLA-4 or anti-PD-1) has shown promise in reversing T-cell exhaustion and boosting anti-tumor activity [179]. Furthermore, selective blockade of TGF-β signaling for blunting Treg function, inducing TAMs repolarization to an anti-tumor phenotype as well as genetically modifications of TILs to express cytokines like IL-15 or resistance to TGF-β could also enhance TIL functionality in the TME [180]. (Fig. 5)

Fig. 5.

T cell-mediated anti-tumor immune response. Both CD4 and CD8 T cells exert antitumor properties either directly by killing tumor cells or indirectly by activating innate immune cells. CD8⁺ T cells serve as the main cytotoxic effectors that eliminate tumor cells by releasing perforin and granzyme and activating death receptor pathways, whereas CD4⁺ T cells support CD8 T cells through cytokine production (e.g., IFN-γ, IL-2) and licensing DCs. Tumor-infiltrating lymphocyte (TIL) therapy and modification of T cell receptors (CAR-T cell therapy and engineered TCR therapy) have emerged as promising approaches in cancer immunotherapy

Bioengineered T-Cells therapy

Genetically modified T cells, including engineered T-cell receptors (TCRs) and especially chimeric antigen receptors (CARs), have transformed immunotherapy, particularly in targeting cancers that resist conventional treatments [181]. CAR T cells are T lymphocytes modified to express CARs, which combine an antibody-derived binding domain with T-cell signaling and co-stimulatory domains. This design allows CAR T cells to recognize antigens independently of MHC molecules, overcoming one of the immune evasion tactics of tumors that downregulate MHC. This binding activates intracellular signaling domains that trigger T-cell proliferation, cytokine release, and direct cytotoxic effects against the targeted cancer cells.

For solid tumors, CAR T-cell designs include elements to improve their penetration and persistence within the TME, such as chemokine receptor modifications for better migration [182]. CAR T-cells are classified into different generations based on their structural and functional advancements, each designed to address specific therapeutic challenges. The first-generation CARs include a single intracellular signaling domain (typically CD3ζ), which can trigger T-cell activation but often lacks potency. This generation is rarely used in clinical settings due to poor durability. The second-generation CARs integrate one co-stimulatory domain (CD28 or 4-1BB), leading to better activation and persistence. The second-generation is widely used in hematologic malignancies such as B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) [183]. For example, Yescarta is the first FDA-approved CD19-targeted second-generation CAR T-cells for B-ALL [184]. The third-generation CARs contain two co-stimulatory domains, further improving T-cell survival and antitumor activity. They have been explored in preclinical and clinical trials for solid tumors like glioblastoma (HER2-targeted CARs) and metastatic melanoma [185]. The fourth-generation CARs or TRUCKs (T cells redirected for universal cytokine killing) combine the CAR with cytokine production capabilities (e.g., IL-12), enhancing immune responses in the TME which are investigated for cancers with immunosuppressive TMEs, including pancreatic cancer and triple-negative breast cancer. The fifth-generation CARs introduce cytokine receptor-based intracellular signaling to augment activation even in immune-suppressive environments, making them promise for solid tumors. This generation was applied to preclinical studies for solid tumors like ovarian cancer and mesothelioma [186].

Currently, CD19 and B cell maturation antigen (BCMA) targeted CAR T-cell therapy has revolutionized treatment of relapsed or refractory B-cell malignancies [183]. By 2024, 11 CAR-T cell therapy products (Yescarta, Breyanzi, Kymriah, Tecartus, Carteyva, Abecma, Fucaso, Zevor-cel, Inati-cel and Aucatzyl) have received marketing approval in the world [187].

However, several challenges have remained for achieving similar success in solid tumors such as tumor heterogeneity, variability in antigen expression, and inherent genetic mutations, as well as the complex structure and barriers of the TME, such as a dense ECM, abnormal tumor vasculature, and immunosuppressive characteristics, which hinder CAR T-cell infiltration and persistence, along with high production costs and scalability issues [15].

Recent studies have focused on overcoming these barriers with innovative strategies, including (1) Utilizing multi-specific CARs, such as TanCARs and LoopCARs that target multiple antigens to enhance specificity and prevent antigen escape [188]. For instance, dual-targeted CARs like CD19/CD22 for ALL and HER2/IL13Rα2 for glioblastoma have shown promising results in clinical and preclinical studies [189]; (2) Engineering chemokine receptors (CXCR1 and CXCR2) to enhance CAR T-cell trafficking and tumor infiltration; (3) Arming CAR-T cells with natural or synthetic cytokine signals like TGF-β and secreting stimulatory cytokines such as IL-12 or IL-18, to improve survival, persistence and activity; (4) Integrating CAR T-cells with checkpoint inhibitors targeting PD-1 or CTLA-4 to counteract T-cell exhaustion within the TME [188]; (5) Targeted CAR T-cell delivery, such as intraventricular injection in brain tumors, and tumor-specific post-translational modification targets to enhance precision and reduce off-tumor toxicity [188]; (6) Combining CAR T-cells with oncolytic viruses, radiotherapy, and using immune checkpoint blockade to induce synergistic clinical efficacy. These combination therapies not only enhanced anti-tumor activity but also facilitated to overcome barriers like antigen escape and limited persistence [190].

CAR T-cells engineered with “off-switches” or humanized antibody fragments are being developed to mitigate toxicities such as cytokine release syndrome (CRS) and neurotoxicity, ensuring safer applications, especially in pediatric settings [191]. (Fig. 5)

T-Cell or CAR-T Cell-Derived EVs

CAR T-cell-derived EVs (CAR T-EVs) have emerged as a promising tool in cancer therapy due to their ability to deliver therapeutic molecules, such as proteins, lipids, and genetic material, directly to tumor cells while minimizing immune-related adverse effects. Their small size allows them to penetrate tumor barriers effectively, and their low immunogenicity suggests the potential for off-the-shelf therapeutic applications for a broad range of patients [192]. Furthermore, CAR T-EVs carry bioactive components such as perforin, granzymes, cytokines, and T-cell receptors, enabling them to induce apoptosis and modulate tumor cells effectively.

Compared with live CAR T-cell therapies, CAR T-EVs are non-proliferative and have a short half-life, minimizing the risk of severe immune reactions, such as cytokine release syndrome (CRS), and other adverse events. CAR T-EVs have also shown some promise in targeting solid tumors, where dense and fibrotic TME inhibit CAR T cells infiltration and activity. In preclinical models, CAR T EVs targeting EGFR or HER2 antigens have shown selective tumor cell killing and surpassing immune evasion mechanisms like PD-L1 overexpression [193].

Despite the promising results, CAR T-EVs therapies in solid tumors face several challenges and limitations. Technical challenges and scalability are significant obstacles to large-scale production of CAR T-EVs with consistent quality. Though targeting efficiency appears promising, further refinement is needed to ensure specificity and to avoid off-target effects. Strategies such as surface modifications are being examined. The lack of standardized guidelines for the clinical application of EVs is another major challenge. Finally, optimizing in-vivo EV stability and half-life is crucial for optimizing their therapeutic efficacy [194]. Recent advancements, such as microfluidic devices and nanoparticle-based systems, are being explored to address these limitations. Researchers are also focusing on integrating CAR T EVs into combination therapies with checkpoint inhibitors, chemotherapy, and radiotherapy. Finally, even though CAR T EVs face some challenges, their unique properties make them an excellent alternative to traditional CAR T-cell therapies, including their ability to bypass immune suppression, overcome immune checkpoints, and minimize systemic toxicities [195, 196].

B Cell-Based immunotherapy in solid tumors

B-Cells in the TME

In the past decade, the role of immune cells within the TME has garnered increasing attention, with much focus placed on T lymphocytes. However, B lymphocytes have also emerged as crucial contributors to antitumor immunity, operating through diverse mechanisms beyond their classical role in antibody production [12]. Depending on the context of the TME, B cells function both to promote and inhibit tumor progression.

Antitumor roles include antibody dependent mechanisms such as complement-dependent cytotoxicity (CDC) where antibodies bind to tumor cell, recruits and activates the complement system to form pores that lead to target cell lysis and death. Antibody-dependent cellular cytotoxicity (ADCC) involves NK cells detecting the Fc region of antibodies coated tumor cells via their CD16 receptor and releasing cytotoxic granules containing perforin and granzymes. B cells also produce cytokines such as IL-6, TNF-α, and IFN-α, which recruit and activate DCs and cytotoxic T cells [197]. Besides secreting cytokines and producing antibodies, B cells also act as effective APCs, activating CD4⁺ and CD8⁺ immune cells in the TME. This role is particularly important when DCs are reduced or rendered dysfunctional in tumors [198]. Furthermore, in tumors containing tertiary lymphoid structures (TLSs), B cells contribute to local immune responses by facilitating T-cell activation and effector function. TLSs, which resemble secondary lymphoid organs, are associated with favorable clinical outcomes in several cancers and may enhance responses to immune checkpoint blockade therapies [199].

Conversely, B cells can promote tumor progression. Tumor cells and associated stromal cells release cytokines such as IL-6, IL-1β, IL-35, and TGF-β, which induce differentiation of infiltrating B cells into a regulatory phenotype. Once formed, regulatory B cells (Bregs) produce IL-10 and TGF-β, suppressing effector T cells and inducing Tregs, thereby sustaining immunosuppressive TME [200, 201]. Furthermore, the nutrient-deficient TME, driven by tumor consumption of glucose and amino acids, also decreases B cells’ survival, activation, and ability to produce antibodies [202]. Activation of B cells rely mainly on glycolysis and oxidative phosphorylation; thus, a deficiency in glucose and amino acids directly restricts ATP synthesis, impairs protein translation, and disrupts mTOR signaling, the mentioned factors are vital for B-cell growth, survival, and their differentiation into plasma cells. Moreover, tumor cells express PD-L1, which inhibits B cell proliferation and drives them towards a regulatory phenotype. Tumor cells also attract immunosuppressive cells such as Tregs and MDSCs to the TME, which further dampen B cell activity and antibody production through cytokine-mediated suppression.

Understanding the precise mechanisms governing the dual roles of B cells in tumor immunity will be essential for developing targeted therapies that leverage their antitumor potential while mitigating pro-tumorigenic effects [199, 200]. Recent findings suggest that therapeutic strategies targeting immunosuppressive pathways, reversing PD-1/PD-L1 interactions, or promoting the formation and maintenance of TLSs could significantly enhance B cell-mediated antitumor immunity. Furthermore, addressing metabolic deficiencies within the TME may further support B cell activity and enabling a more robust immune response against tumors.

B-Cells as a therapeutic tool in solid tumors

B cells, critical components of the adaptive immune system, are increasingly recognized as therapeutic targets in cancer treatment. These cells play multifaceted roles, ranging from antibody production to modulation of TME. Over the decades, advances in technology, particularly monoclonal antibody (mAb) engineering, have greatly expanded the scope of B-cell-targeted therapies, offering new hope in both hematological malignancies and solid tumors. The advent of mAb technology in the 1980s revolutionized our understanding of B-cell biology, leading to the identification of key surface molecules such as CD20, CD19, and CD79 [203]. These discoveries paved the way for targeted therapies like rituximab, a CD20-specific antibody that depletes B cells via apoptosis, CDC, and ADCC [204].

Combination therapies also have highlighted the power of integrating mAbs with other immunotherapies. For example, patients with lymphoma and lung cancer concurrently treated with rituximab and bendamustine achieved complete remissions, and Pembrolizumab, an anti-PD-1 immune checkpoint inhibitor, maintained long-term remissions [205, 206]. Antibody-drug conjugates (ADCs) such as polatuzumab-vedotin, that target CD79b in B-cell lymphomas, also showed remarkable potential by delivering cytotoxic payloads directly to cancer cells, enhancing specificity and reducing systemic toxicity [207].

TLSs further highlights the therapeutic role of B cells. TLS-positive tumors (e.g., NSCLC, colorectal cancer) correlate with better outcomes and increased responsiveness to immunotherapy [208]. Strategies that promoted TLS formation or enhance their functionality could transform “cold” tumors into “hot” ones, making them more amenable to immunotherapy [209].

Despite these advances, B cell-based immunotherapy still has limitations such as varying prognostic value across different cancer types, heterogeneity of B-cell populations and tumors’ immune evasion by downregulating target antigens or creating an immunosuppressive TME [210]. However, high densities of CD20⁺ B cells in HER2⁺ breast cancer cells were linked to better outcomes; On the other hand, in certain melanoma subtypes, CD138⁺ plasma cells were associated with poor survival. This duality complicates the development of universal B-cell-targeted therapies and highlights the need for precision approaches that selectively enhance beneficial subsets while mitigating harmful ones [211].

Ongoing research and technological innovations such as bispecific antibodies, pairing of mAb and immune checkpoint inhibitors, and integration of B-cell-targeted therapies with other immunotherapies can help address some of the current limitations. For example, pairing engineered B cells with checkpoint inhibitors or certain vaccines could amplify anti-tumor responses [212]. Advances in bispecific antibodies and CAR technology are also expanding therapeutic possibilities, offering precision approach and adaptability in targeting diverse cancer types [213]. Moreover, advanced techniques like single-cell RNA sequencing and spatial transcriptomics are beginning to unravel the complexities of B-cell subsets, offering novel insights into their roles in the TME [214]. (Fig. 6)

Fig. 6.

B-cell-mediated immunity in tumor microenvironment. B cells exert anti-tumor effects through antibody-dependent cell cytotoxicity (ADCC) which mark tumor cells by tumor-specific antibodies (e.g., IgG) and recruit effector cells like NK cells, macrophages, and neutrophils via Fcγ receptors to lyse target cells. Moreover, B cells present tumor antigens on MHC class II molecules to CD4⁺ and CD8 + T cells, a process especially important when DCs are depleted or dysfunctional. Engineered B represents a novel modality for solid tumor therapy, modified to express chimeric antigen receptors (CAR-B), bispecific antibodies, or cytokine payloads (e.g., IL-12, IFN-α) that target tumor-associated antigens and secrete therapeutic proteins

Bioengineered B-Cells therapy

Engineering B cells represent a transformative advance in immunotherapy, leveraging their natural abilities for antibody production, immune modulation, and adaptability to therapeutic applications. This approach integrates genetic engineering, viral transduction, and in-vivo delivery systems, enabling B cells to function as precise and durable therapeutic agents. These advancements hold promises for treating solid tumors, infectious diseases, and beyond.

In preclinical studies, B cells engineered to express tumor-specific antigens enhanced T-cell activation and promoted robust anti-tumor immunity, demonstrating precise targeting and with fewer off-target effects and systemic side effects [215]. Additionally, CAR technology is now being adapted for B cells. By equipping B cells with CARs, researchers have enabled these cells to target antigens directly while harnessing their ability to produce therapeutic antibodies. CAR-B cells have shown promise in delivering sustained therapeutic agents in preclinical models, offering a platform for precision medicine in solid tumors [216].

Recently, genome editing tools like CRISPR/Cas9 and mRNA technology have enabled precise genetic modifications in B cells, allowing the introduction of therapeutic payloads such as cytokines, antibodies, or bispecific molecules. For example, CRISPR/Cas9 has been used to engineer plasma cells to produce bispecific antibodies like anti-CD19/CD33, activating T cells against leukemia in preclinical studies. Similarly, mRNA-based approaches provide a safer alternative for transient modifications, minimizing risks of permanent off-target effects. While these tools offer precision and versatility, challenges such as delivery efficiency, off-target activity, and scalability require further refinement [217, 218]. Emerging in-vivo genome editing technologies, such as lipid nanoparticles (LNPs) and in-vivo transduction systems also provide exciting alternatives to traditional ex-vivo methods. These approaches deliver CRISPR components or mRNA directly to circulating B cells, bypassing the need for labor-intensive culture processes. Several in-vivo delivery systems have also been employed to modify B cells to secrete therapeutic proteins continuously [219].

Antibody engineering further enhances the therapeutic potential of B cells. Engineered B cells have been developed to produce monoclonal, bispecific, or antibody-drug conjugates, allowing precise tumor targeting [220]. For example, antibody-cytokine fusion proteins have been used to simultaneously target tumors and modulate immune responses [221]. While these strategies reduce systemic toxicity, ensuring long-term and consistent antibody production remains a significant challenge within the immunosuppressive TME [222]. Engineered B cells secreting interleukin-12 (IL-12), for instance, have shown potential in enhancing T cell activation within the TME [223]. However, regulatory B cells (Bregs), which produce immunosuppressive cytokines like IL-10, can inhibit these therapeutic effects, necessitating strategies to suppress Bregs while preserving effector B cell function [224].

B cell-based vaccines are another innovative application, particularly for diseases requiring robust, long-term immune responses. Engineered B cells expressing antigens or antibodies have been explored in vaccines for cancer and chronic infections, such as HIV. These approaches provided adaptability to antigenic variation and long-lasting protection but require strategies to induce memory B cell responses and improve efficacy across diverse patient populations. The long-lived plasma cells engineered to produce therapeutic proteins continuously could also revolutionize treatments requiring sustained efficacy.

However, significant challenges remain in optimizing efficacy and translating these therapies into clinical practice. For example, antigen heterogeneity, immune suppression in the TME, and limited persistence of CAR-B cells remain significant hurdles. Lentiviral-transduced CAR-B cells targeting HER2⁺ or CD19 cells have shown preclinical efficacy in cancer models. However, potential risks like insertional mutagenesis and the complexity of large-scale production remain potential obstacles to clinical implementation [204, 205]. Furthermore, the immunosuppressive nature of many TMEs, inefficient targeting, immune evasion, production scalability and ensuring therapeutic safety remain remarkable challenges for widespread clinical utility [225]. By leveraging B cell natural properties and integrating cutting edge technologies, researchers are uncovering novel ways to harness these cells for therapeutic benefit, paving the way for next-generation cellular therapies [226]. (Fig. 6)

Conclusion

According to ClinicalTrials.gov, CAR-T, NK, TIL, and (TCR)-based therapies have dominated the landscape of immune cell-based therapies with potential efficacy against multiple solid tumors, including lungs, skin, breast, colorectal and gastric cancers (Fig. 7). As solid malignancies account for more than 90% of all new cases of cancer worldwide with vast majority of cancer-related morbidity and mortality, the successful translation of immune cell based strategies into effective treatments could significantly improve clinical outcomes [227].

Fig. 7.

Immune cell-based therapeutic in solid tumor clinical trials. (A) Number of current clinical trials of different immune cell-based therapies for solid tumors. (B) Species of solid tumors studied in clinical trials. (C) The clinical development milestones for immune cell-based therapeutic in solid tumor. Tumor-infiltrating lymphocytes (TIL) [257], chimeric antigen receptor T cells (CAR T) [258], T cell receptor-engineered T cells (TCR T) [259], Dendritic Cells (DC) [260], natural killer cells (NK) [261], CAR natural killer cells (CAR NK) [262], natural killer T cells (NKT) [263], CAR natural killer cells (CAR-NKT) [264], CAR macrophage (CAR-MQ) [18]

However, the application of these promising strategies toward solid tumors is inherently dependent upon addressing several critical challenges. A primary obstacle is tumor heterogeneity, wherein solid tumors exhibit significant variability in their cellular composition and antigen expression, hindering the ability to effectively target al.l malignant cells [228–230]. Furthermore, the immunosuppressive TME poses a formidable barrier, characterized by the presence of Tregs, MDSCs, and inhibitory molecules such as PD-L1 and TGF-β, all of which impede the efficacy of cell therapies [231–233]. Another significant hurdles are limited tumor infiltration of immune cells due to physical barriers like the dense ECM as well as short-term persistence and exhaustion of cells within the TME which can impair their therapeutic efficacy [234–236] Additionally, clinical application of genetically modified immune cell-based therapies is associated with particular toxicities which can sometimes be life-treating such as cytokine release syndrome (CRS), immune effector cell–associated neurotoxicity syndrome, and on-target off-tumor toxicity [237, 238].

CRS, also known as a cytokine-associated toxicity, is a systemic inflammatory response caused due to production of a wide range of proinflammatory cytokines from activated CAR-NK, CAR-NKT, TCR-T and CAR-T-cells as well as tumor-resident myeloid cells leading to monocyte recruitment and a feedback loop that causes a cytokine storm. Subsequently, the cytokine release causes the disruption of the blood-brain barrier (BBB), allowing immune cells and cytokines to pass into the cerebrospinal fluid and brain, and immune effector cell–associated neurotoxicity syndrome (ICANS). Utilizing innovative contributory strategies such as refining target antigen selection, engineering armored and programmable CARs, advancing gene transfer techniques, optimizing lymphodepleting chemotherapy regimens, and developing innovative toxicity management strategies are essential for enhancing function, persistence, and infiltration in solid tumors.

Finally, the complexity and cost of manufacturing cell-based therapies as well as efficient and safe gene editing and delivery methods are essential for generating engineered immune cells with desired properties [239]. To address these challenges, several strategies are being explored. Combination therapies, which combine cell therapies with other cancer treatments like checkpoint inhibitors, chemotherapy, and radiation therapy, can enhance therapeutic efficacy [240–243]. Engineering cells to overcome immunosuppression, such as by expressing cytokines, chemokine receptors, or inhibitory molecule antagonists, can enhance their activity within the TME [244–246]. Improving tumor infiltration can be achieved through local delivery of cells, engineering cells to express matrix-degrading enzymes, or using ultrasound or magnetic fields to guide cells to the tumor [235, 247–249]. Developing safer engineered cells, such as by incorporating suicide genes, using inducible promoters, or developing bispecific or Tri-specific CARs, can reduce toxicity [250–252]. Optimizing manufacturing processes through the development of closed and automated systems, bioreactors, and optimized cell culture conditions can reduce costs and increase scalability [253, 254]. Finally, identifying novel targets and biomarkers to predict response and monitor toxicity can improve patient selection and treatment outcomes [255, 256].

The field of immune cell-based therapies for solid tumors is rapidly evolving. Advances in gene editing, synthetic biology, and immunology are paving the way for the development of more effective and safer therapies. Continued research and clinical trials are essential to translate these promising strategies into clinical practice.

Authors’ contributions

Conception, manuscript design and writing: SHT. Collection of data and writing: EY, NT, RKH, ST and MS. All authors contributed to the manuscript writing. Visualization: AK. SHT, AZ and MV made the main edition. All authors read and approved of the final manuscript.

Funding

No grants, funds, or other financial support were received.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.