The intratumoral microbiota, comprising bacteria, fungi, and viruses within the tumor microenvironment, actively influences carcinogenesis. Key mechanisms include the induction of host DNA damage, modulation of critical oncogenic signaling pathways such as WNT-β-catenin, NF-κB, and PI3K, and the orchestration of inflammatory processes. The microbiome's interaction with the host immune system is complex and bidirectional. On one hand, specific microbes can foster a pro-tumorigenic niche by suppressing the activity of cytotoxic T cells and natural killer (NK) cells or by promoting the accumulation of immunosuppressive cell types like tumor-associated macrophages (TAMs). On the other hand, microbial components can serve as neoantigens for T cell recognition or produce metabolites that reprogram the immune landscape to enhance anti-tumor responses. The composition of this microbiome is emerging as a crucial factor influencing the outcomes of immunotherapies. Prospective investigations in cancer immunotherapy ought to prioritize mechanistic inquiry employing integrative multi-omics methodologies. The execution of meticulously designed clinical trials for the validation of microbial biomarkers, and the systematic, evidence-based development of microbiome-targeted therapeutic interventions aimed at enhancing antitumor immune responses.

Messenger RNA (mRNA)-based transient expression of chimeric antigen receptors (CARs) results in optimal safety profiles and provides promising opportunities to address existing challenges associated with viral vector-based CAR-T-cell therapies and to meet emerging medical needs for noncancerous indications. Conventional linear mRNAs, however, are intrinsically unstable and typically support short-lived protein expression, which can constrain therapeutic activity. Here, we engineered a high-efficiency permuted intron exon (PIE) platform to synthesize scarless circular mRNAs (cmRNAs) that drive robust CAR expression with extended durability. The scarless design avoids extraneous junction sequences, streamlining manufacturability and potentially reducing innate immune sensing. Compared with linear mRNAs, cmRNAs significantly increased both the magnitude and duration of anti-CD19 CAR and anti-GPRC5D CAR expression in primary human T cells. Functionally, cmRNA-based CAR-T cells elicited superior antitumor efficacy over their linear mRNA counterparts, as demonstrated by parallel lines of evidence, including in vitro antigen-specific cytotoxicity, cytokine release, and transcriptomics patterns consistent with sustained activation and absence of exhaustion signatures, as well as in vivo models demonstrating tumor elimination and prolonged survival benefits. Collectively, these findings position cmRNA as a next-generation mRNA modality for potent and controllable CAR expression, thereby providing a robust platform to unleash the full potential of mRNA technologies in cellular immunotherapy and precision medicine.

In recent years, the incidence of B-cell non-Hodgkin lymphoma (B-NHL) in China has shown a steady increase, accounting for approximately 85%-90% of all lymphomas. Although standard immunochemotherapy regimens such as R-CHOP have led to long-term remission in some patients, approximately 30%-40% still experience relapse or refractory disease, with dismal prognosis and a median survival of less than one year. For patients who fail multiple lines of therapy, conventional options such as chemotherapy, radiotherapy, or hematopoietic stem cell transplantation offer limited benefits, highlighting an urgent need for innovative treatments. Chimeric antigen receptor T-cell (CAR-T) therapy, a breakthrough form of adoptive cellular immunotherapy, modifies autologous T cells to specifically recognize and eliminate malignant B cells, thereby achieving significant survival improvement in patients with relapse or refractory B-NHL. The clinical research and clinical application of CAR-T in the treatment of hematological tumors in China are in a state of rapid development. At present, there are two targeting CD19 CAR-T cells for the treatment of B-cell lymphoma, which gradually changed the diagnosis and treatment practice of lymphoma in China. At the same time, the clinic is actively exploring and improving the whole-process management experience of CAR-T therapy in lymphoma patients. So the Lymphoma Quality Control Expert Committee of the National Cancer Quality Control Center organized experts to form the consensus through discussion and revision many times, aiming to provide better guidance for clinicians to standardize the whole-process management of CAR-T cell therapy for B-cell Lymphoma, and further improve the survival benefits of patients.

Despite PFS in MM patients extending to 17 years due to contemporary quadruplet induction therapies, there remains a necessity for novel products in the treatment of high-risk patients. BCMA, GPRC5D, FcRH5, SLAMF7, and TACI represent the principal CAR-T target molecules, with dual-targeted treatments under development to enhance their efficacy. Ide-cel and Cilta-cel are CAR-T cells directed against BCMA, having received FDA approval for RRMM based on the Phase 2 KarMMa and CARTITUDE trials, respectively. Research is currently being conducted on the administration of these products in newly diagnosed patients and for maintenance therapy. Additional anti-BCMA targeted medicines, including LCAR-B38M, completely humanized CAR-T (FHVH-T), P-BCMA-ALLO-1, ALLO-715, and anti-BCMA CAR-NK, provide promising treatment options. Moreover, the anti-CD19 Fast-CAR, designed to shorten production time, and PHE885, which possesses in-vivo proliferation capability, are regarded as very efficacious. Arlo-cel, developed for the significant target GPRC5D, has demonstrated efficacy against conventional treatments. The development of academic CAR-Ts such as ARI0002h, HBI0101, eque-cel, zevor-cel, anito-cel, and Sleeping Beauty (utilizing a non-viral vector) have importance due to their accessibility and cost-effectiveness. The real-world data have demonstrated comparable efficacy and safety outcomes in both academic and commercial CAR-T research. CAR-T cell researchs are also being undertaken in SMM and AL amyloidosis. CAR-PRISMM and CAR-HiRiSMM are regarded as extremely effective and safe for patients with high-risk SMM. NXC-201, which targets BCMA, has been developed for AL amyloidosis. Notwithstanding these promising outcomes, numerous difficulties confront CAR-T therapy. These factors may relate to the tumor, the patient, and the CAR-T product. To overcome these issues, strategies are being implemented, including combination therapy, the incorporation of gamma-secretase inhibitors etc. In conclusion, CAR-T treatments have evolved into an effective therapy modality being anticipated to be utilized in earlier phases in the future. Gene editing (CRISPR) method contributes to the future perspective.

Chimeric antigen receptor (CAR) T-cell therapy constitutes a paradigm-shifting advancement for treating refractory malignancies, demonstrating unique therapeutic capabilities beyond conventional cancer treatments, which has brought new hope to many patients with advanced cancer. This cellular immunotherapy has achieved unprecedented success in hematologic cancers, yet its extension to solid tumors confronts fundamental biological constraints. Lifethreatening toxicities such as cytokine release syndrome and immune effector cell-associated neurotoxicity originate from dysregulated immune activation cascades, while T cell exhaustion—characterized by hierarchical epigenetic reprogramming and progressive metabolic failure—severely undermines long-term therapeutic efficacy. These dual limitations demand innovative bioengineering solutions to augment both safety profiles and functional persistence. The present review analyzes core biological mechanisms restricting CAR-T efficacy and surveys cutting-edge approaches designed to circumvent these barriers through integrated genetic reprogramming, metabolic optimization, and microenvironment-responsive receptor engineering. Subsequent discussion focuses on how such multidisciplinary strategies could potentiate cellular immunotherapy’s applicability across diverse oncological contexts, ultimately transforming cancer management paradigms.

Cell-cell interactions play an important role in maintaining tissue homeostasis and regulating physiological processes, involving direct contact, signal molecule transmission, vesicle-mediated communication, and other modes. A profound understanding and precise regulation of cell-cell interactions can help reveal cellular function mechanisms, guide tissue engineering, and promote the development of novel therapeutic strategies. In recent years, functional nucleic acids (FNAs) have become important tools for analyzing and regulating cell-cell interactions, owing to their excellent biocompatibility, structural programmability, and dynamic responsiveness. This review summarizes the progress of FNAs in cell-cell interactions, systematically sorts regulatory strategies of cell-cell interaction based on DNA hybridization, molecular recognition, scaffold construction, and response to environmental stimuli, and highlights their applications in cellular immunotherapy, cellular force monitoring, reconstruction of 3D tissue models in vitro, and cell-cell communication. Finally, current challenges and future development directions are discussed, aiming to provide new perspectives for advancing the application of FNAs in cell-cell interactions and precision medicine.

Tumor-Infiltrating Lymphocyte Therapy Generated from Melanoma Brain Metastasis: Proof-of-Concept for CNS-Derived Cellular Immunotherapy.

Overcoming barriers in solid tumor immunotherapy remains challenging, with adoptive T cell therapies limited by antigen loss, poor tumor infiltration, and T cell exhaustion. Here, we present a nanoengineered tumor-infiltrating lymphocyte (TIL) therapy using anti-PD-1 antibody (αPD-1)-conjugated ZIF-8 nanoparticles to effectively suppress pancreatic tumor growth. These nanoparticles release hyaluronidase (HAase) and decitabine (DEC) in acidic tumor microenvironments, promoting stroma degradation and C-C motif chemokine ligand 5 (CCL5) secretion, while retaining αPD-1 on TILs to prevent exhaustion. CCL5 recruits additional nanodrug-loaded TILs for further release of HAase and DEC, establishing a self-reinforcing infiltration loop. This approach increases TIL infiltration by 12-fold in immunodeficient mice and, in immunocompetent settings, mobilizes both exogenous TILs and endogenous CD8+ T cells, enabling tumor eradication and metastasis suppression with 10-fold lower TIL doses than conventional therapies. Collectively, this nanoengineered TIL therapy offers a potential strategy for addressing immune-resistant tumors, showing distinct benefits in stromal-rich settings.

Glioblastoma multiforme (GBM) remains one of the most aggressive and treatment-resistant human cancers, characterized by highly infiltrative growth, extensive cellular heterogeneity, and a profoundly immunosuppressive microenvironment. Although CAR-T cell therapy has revolutionized the treatment of hematologic malignancies, its clinical impact in GBM has been limited by antigenic escape, poor tumor infiltration, T-cell exhaustion, and significant toxicity risks. Recent advances in CRISPR-Cas9 genome editing offer unprecedented opportunities to overcome these barriers by enabling precise, multiplex genetic reprogramming of T cells. In this review, we synthesize current progress in CRISPR-enhanced CAR-T engineering for GBM, focusing on strategies to overcome immune checkpoint suppression, optimize metabolic fitness, enhance trafficking across the blood–brain barrier, reduce neuroinflammation-associated toxicities, and generate universal allogeneic CAR-T products. We also compare the genomic target spaces of candidate guide RNAs (crRNA, d10r10, X37) and highlight their predicted off-target profiles relevant to GBM therapeutic design. Preclinical studies demonstrate that CRISPR-edited CAR-T cells targeting EGFRvIII, IL-13Rα2, HER2, and B7-H3 significantly enhance survival in murine GBM models, while emerging clinical trials indicate acceptable safety and early evidence of anti-tumor activity. We further discuss technological innovations—including base editing, prime editing, CRISPRi/a, non-viral delivery platforms, and precision-medicine–guided CAR design—as well as regulatory, ethical, and manufacturing considerations required for clinical translation. Collectively, these advances underscore the transformative potential of CRISPR-engineered CAR-T therapies to reshape GBM treatment and pave the way toward more effective and accessible cellular immunotherapies. Keywords. Glioblastoma multiforme (GBM), CAR-T cell therapy, CRISPR-Cas9 genome editing, T-cell engineering, Immune checkpoint resistance, Tumor microenvironment, Guide RNA design, EGFRvIII, IL-13Rα2, Universal allogeneic CAR-T cells, Preclinical models, Precision immunotherapy

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. 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. 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. 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.

The immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34⁺ HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours.

Advancements in cellular immunotherapy demanded efficient immune cell delivery. To meet this need, we introduced hydrogel-based immune millirobots designed for high immune cell loading and precise tumor targeting. These ultrasoft robots, embedded with magnetic nanoparticles, exhibited adaptable locomotion: walking, rolling, climbing, and undulating, enabling navigation through complex biological environments and alignment with varied tumor morphologies. They responded to magnetic fields and ionic or pH changes, facilitating propulsion, grasping, and localized delivery. In vitro, the millirobots eradicated three-dimensional tumor models in four days; in vivo, they notably reduced tumor growth in HepG2-luc tumor-bearing nude mice within 15 days. Bioluminescence imaging confirmed enhanced natural killer cell activity at tumor sites. The robots demonstrated excellent biocompatibility and biodegradability and caused no adverse effects postimplantation. This work showcased a responsive, soft robotic system with potential for advancing immune cell delivery and exploring tumor-immune dynamics in cancer therapy.

HighlightsWhat are the main findings?Provision of a phenotypic analysis of γδ T cells at the time of isolation and after ten days of stimulation using a protocol based on zoledronate and interleukin 2.Identification of potential phenotypic markers predictive of donor suitability for allogeneic cell therapies.What are the implications of the main findings?Contribution to the understanding of γδ T cell biology and their therapeutic potential.Basis for future clinical trials.Due to their anti-tumor activity and non-major histocompatibility complex (MHC) binding T cell receptor, γδ T cells are suitable candidates for allogeneic cellular immunotherapy in cancer. Recently, we developed a new protocol called Ko-Op for stimulation of γδ T cells (specifically Vy9Vδ2 T cells) that generates a cell product consisting mainly of γδ T cells with preserved anti-tumor activity targeted for clinical-grade application. In this study, we investigated the phenotype of stimulated γδ T cells and correlated this with results of functional assays to obtain a deeper understanding of the characteristics of stimulated γδ T cells. Additionally, an intensive analysis of surface molecules of unstimulated and stimulated γδ T cells is presented. Since heterogeneous results regarding the response to therapy with γδ T cells observed in earlier clinical trials could be a consequence of various extents of γδ T cell adhesion and migration ability, we addressed surface molecules associated with cellular activity and adhesion and migration functions as well. By investigating correlations between the phenotype of unstimulated γδ T cells and cellular cytotoxicity, as well as the degranulation ability of stimulated γδ T cells, we could draw conclusions about optimal donors for further allogeneic cellular therapies. Finally, we demonstrated that the phenotype varies over the time of culture and is clearly modifiable by changing the stimulation protocol.

Designing immunity with cytokines: A logic-based framework for programmable CAR therapies.

Long-Term Efficacy of Epstein-Barr Virus-Specific T Cells for Epstein-Barr Virus-Associated Post-Transplantation Lymphoproliferative Disorder after Allogeneic Stem Cell Transplantation: A Real-World Study.

The impact of anticoagulant used for peripheral blood collection on NK cell yield, function, and proliferation.

CAR-T cell therapy for the treatment of lung cancer: Current challenges and emerging therapeutic strategies.

Development and qualification of a sensitive method for residual feeder cell detection in NK cell therapy products.

Embryonic germ layer origin fundamentally shapes cancer therapy response patterns, with mesoderm-derived malignancies showing responsiveness to cellular immunotherapy, endoderm-derived epithelial cancers demonstrating sensitivity to protein signaling inhibitors, and ectoderm-derived tumors exhibiting immunogenicity enabling breakthrough responses to checkpoint blockade and mRNA vaccine strategies. To investigate these patterns, we conducted a systematic review, using a guided review method and large language learning models, following the Preferred Reporting Items for Systematic Reviews and Meta-analyses 2020 guidelines, and searched PubMed, Embase, and Web of Science through October 2025. Evidence synthesis incorporated National Comprehensive Cancer Network Clinical Practice Guidelines, American Society of Clinical Oncology publications, American Cancer Society statistics, and landmark Phase 3 and Phase 4 clinical trials. Given extreme clinical heterogeneity, we performed narrative synthesis with megatrend analysis, employing triple-checking verification methodology of all clinical outcome data using independent search strategies, with explicit documentation of large language model-assisted abstract screening followed by an exclusive human reviewer completion of eligibility assessment, data extraction, and synthesis. Three major megatrends emerged from our analysis: mesoderm-derived hematologic malignancies achieved relevant response rates of 82–97% with chimeric antigen receptor T-cell therapies across multiple pivotal trials; endoderm-derived adenocarcinomas demonstrated vulnerability to targeted therapies, with median overall survival extending to 19–47 months with matched protein pathway inhibitors; and ectoderm-derived melanoma showed immune control with checkpoint blockade achieving approximately 34% 10-year overall survival and 49% risk reduction with personalized mRNA neoantigen vaccines. These findings suggest that embryonic lineage provides a potentially valuable exploratory context for understanding therapeutic response patterns, complementing molecular biomarker-driven precision oncology to guide treatment selection, trial design, and drug development prioritization.

A growing assortment of cellular immunotherapies targeting CD19, CD20, BCMA, and GPRC5D are being used across the spectrum of hematologic malignancies to treat acute lymphoid leukemia, various types of lymphomas, and multiple myeloma. Although T-cell–engaging and CAR T-cell therapies are yielding benefits for patients with these types of blood cancers, they have been associated with various unique toxicities, which require recognition and attention from all members of the treatment team—including medical oncologists, clinical pharmacists, and advanced practice providers—especially as an increasing number of these patients are being treated in the ambulatory setting.