Cytokine fusion proteins are engineered biologics that combine functional modules, such as cytokines, antibodies, or albumin-binding domains, into a single molecular entity. These agents have emerged as promising therapeutic platforms for treating solid tumors. However, their clinical translation remains constrained by the need to achieve potent antitumor activity while mitigating dose-limiting systemic toxicities. Here, we dissect the mechanistic foundations of cytokine fusion protein function by examining key cytokine signaling pathways within the tumor microenvironment. We also provide a comprehensive overview of current engineering strategies and clinical developments, focusing on approaches to optimize pharmacokinetics, enhance tumor-targeted delivery, and achieve therapeutic synergy. Collectively, this Review summarizes the evolving landscape of cytokine fusion protein development and discusses emerging opportunities and challenges that may inform future advances in this field.
Recent advances in immunotherapy have significantly revolutionized cancer treatment landscape and prompted extensive research into novel vaccine-based anti-tumor therapy. As the primary defense against external infections, the mucosal immune response plays a pivotal role in maintaining normal physiological functions and preventing pathogen invasion. Mucosal tissues are among the most common sites of tumor initiation, and therefore harnessing mucosal immune to fight against malignant cells represents a feasible option for optimizing and improving current antitumor therapies. Over the past few decades, numerous efforts have been devoted to designing ideal and effective mucosal vaccine. Some clinical trials using mucosal vaccine for cancer therapy have been also launched continually. In this review, we introduce the mechanisms of mucosal immunity, with a focus on the respiratory, gastrointestinal, and urogenital tracts. We also summarize recent research and clinical trials on the application of mucosal vaccines in anti-tumor therapy, aiming to provide new perspectives and directions for future studies.
Distinguishing indolent from aggressive tumors remains a key challenge in the clinical management of prostate cancer (PC), highlighting the need for better tools for accurate risk stratification. A defining feature of aggressive PC is its propensity for perineural invasion (PNI), a pathological finding that is associated with poor prognosis. Despite its clinical significance, very little is known about the spatial and molecular determinants of PNI in PC. To address this, we used high-definition spatial transcriptomics (Visium HD) to profile the PNI-associated tumor microenvironment (TME) of a representative PC patient at near single-cell resolution. Spatial mapping of the expressed genes and inferred cell types revealed transcriptionally divergent malignant cell states spatially linked to PNI within this patient. Nerve-invasive PC cells were organized within a distinct spatially localized niche that exhibited altered TME characteristics, including increased proportions of macrophages, CD4 T-cells, and endothelial cells, suggesting coordinated changes in the tumor- and immune microenvironments. The PNI-associated niche in this patient further displayed enrichment of pathways involved in immune regulation and extracellular matrix remodeling, consistent with PNI-associated niche remodeling. APP-CD74 was identified as a potential signaling axis associated with tumor-nerve, nerve-macrophage, and nerve-endothelial cell interactions, suggesting PNI in this patient may be associated with distinct microenvironmental signaling programs. We further revealed a PNI-associated PC signature that held biomarker potential at the early-localized and advanced-metastatic disease stages. Although based on a single patient, these results contribute to our understanding of the spatial and molecular features of PNI in PC and may help guide personalized treatment choices for PC patients in the future.
Cancer remains a major therapeutic challenge due to drug resistance and metastasis, processes driven by oxidative stress and redox imbalance. Targeting this vulnerability through ferroptosis (iron-dependent lipid peroxidation) and cuproptosis (copper-driven mitochondrial dysfunction), two ROS-mediated cell death pathways, offers a promising therapeutic strategy. However, clinical translation is hindered by incomplete understanding of their redox regulation and limited immunogenicity. A genome-wide CRISPR knockout screen was performed to identify key regulators of ferroptosis. Genetic depletion or pharmacological inhibition of candidate genes was evaluated across multiple cancer cell lines for sensitivity to ferroptosis inducer RSL3 and the cuproptosis inducer elesclomol (Es). Antitumor efficacy was assessed in xenograft, orthotopic, metastatic, and syngeneic mouse models, alone or combined with immune checkpoint inhibitors. Mechanistic studies also examined ROS production, mitochondrial stress, mitochondrial DNA release, cGAS-STING activation, and immune responses within the tumor microenvironment. Glutathione reductase (GSR), a central enzyme maintaining reduced glutathione (GSH) homeostasis, was identified as the top suppressor of ferroptosis. GSR knockout or pharmacological inhibition markedly sensitized diverse cancer cell lines to RSL3-induced ferroptosis, while GSR overexpression conferred resistance. Strikingly, GSR depletion also enhanced sensitivity to cuproptosis triggered by the copper ionophore Es. In multiple in vivo tumor models, GSR inhibition synergizes with RSL3 or Es to suppress tumor growth, inhibit lung metastasis, and prolong survival. Mechanistically, GSR deficiency amplified ROS production, induced mitochondrial stress, and triggered the cytosolic mitochondrial DNA release under ferroptotic or cuproptotic stress, activating the cGAS-STING pathway in vitro and in vivo. This increased inflammatory cytokine production, promoted immunogenic cell death, and enhanced the release of damage-associated molecular patterns (DAMPs), including HMGB1. Together, GSR inhibition combined with a ferroptosis or cuproptosis inducer transformed the tumor microenvironment into a highly immune stimulatory state, thereby enhancing the efficacy of immune checkpoint blockade through increased dendritic cell activation and T-cell infiltration and activation. GSR represents a key molecular node connecting and modulating ferroptosis and cuproptosis through redox regulation. Targeting GSR amplifies ROS-mediated immunogenic cell death, triggers cGAS-STING activation in cancer cells, and enhances the efficacy of cancer immunotherapy, providing a promising redox-based therapeutic strategy.
Survival in metastatic pancreatic ductal adenocarcinoma (mPDAC) has long been limited by a dismal second-line therapeutic ceiling dictated by conventional chemotherapy. However, breakthrough data from the ASCO 2026 Annual Meeting and the publication of the phase III RASolute-302 trial mark a definitive shift toward targeted KRAS inhibition. This correspondence highlights how the first-in-class pan-RAS (ON) inhibitor daraxonrasib (RMC-6236) virtually doubled median overall survival (13.2 vs. 6.6 months) and progression-free survival compared to chemotherapy in second-line mPDAC, establishing a new standard of care. Concurrently, we evaluate emerging allele-specific strategies from ASCO 2026 designed to optimize target engagement and safety. These include the selective KRAS G12D inhibitor DN022150 and promising horizontal combinations pairing the G12D inhibitor HRS-4642 with either the anti-PD-L1 antibody adebrelimab or a Nectin-4-targeted antibody-drug conjugate (ADC). Furthermore, we address the KRAS G12C cohort where farnesyl transferase co-inhibition (darlifarnib plus adagrasib) successfully bypasses adaptive resistance. Ultimately, the therapeutic landscape of mPDAC is transitioning toward tailored genomic frameworks. Future success will rely on optimizing the clinical sequencing or combination of pan-RAS and allele-specific agents, guided by real-time liquid biopsies, to permanently dismantle resistance and transform mPDAC into a manageable molecular entity.
Patients with relapsed acute myeloid leukemia (AML), particularly following allogeneic stem cell transplant (alloHCT), have extremely limited therapeutic options. CD123 is an AML-associated antigen and represents an attractive target for immunotherapy. We report outcomes of a phase 1 trial evaluating CD123-targeting chimeric antigen receptor (CAR) T cells in patients with relapsed/refractory (r/r) AML or blastic plasmacytoid dendritic cell neoplasm (BPDCN). This was a single center, open-label, phase 1 dose escalation study enrolling patients with either CD123-positive r/r AML (Arm 1) or BPDCN (Arm 2). Patients received autologous or donor-derived allogeneic CD123 CAR T cells following lymphodepletion. The co-primary objectives were to examine safety and anti-tumor activity using an activity-constrained for toxicity design and to determine the recommended phase 2 dose. Secondary objectives included assessments of progression-free and overall survival. We enrolled 41 patients, of whom 21 patients (n = 19 on Arm 1 and n = 2 on Arm 2) received CD123 CAR T-cell infusion. The median age of treated AML patients was 45 years (range: 20–71); the two BPDCN patients were aged 24 and 75 years. Among AML patients, 17 (89
While multiple phase III trials have established the survival benefit of adding a programmed cell death protein 1 (PD-1) antibody to first-line chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-negative advanced gastric or gastroesophageal junction (G/GEJ) adenocarcinoma, evidence across different programmed cell death ligand 1 (PD-L1) expression levels remains limited, preventing definitive conclusions about the superiority of combination therapy in certain subgroups. We firstly performed a post-hoc analysis of the RATIONALE-305 trial, finding only marginal benefit in the 1 ≤ tumor area positivity (TAP) < 5 and 1 ≤ combined positive score (CPS) < 5 subgroups. Subsequently, we performed a pooled analysis of individual patient-level data from RATIONALE-305 and four additional phase III trials (CheckMate 649, KEYNOTE-062, KEYNOTE-859, and ORIENT-16). The pooled analysis demonstrated that adding a PD-1 antibody to chemotherapy significantly improved overall survival in patients with intermediate PD-L1 levels (1 ≤ CPS < 10: HR, 0.84; 95
Abstract Background Baseline CD20 expression ≥ 20% in B-cell acute lymphoblastic leukemia (B-ALL) has been associated with poorer outcomes, which improved after rituximab introduction into frontline therapy for CD20-positive cases. We investigated the clinical and biological significance of this threshold by monitoring early dynamics of CD20 expression. Methods In the GMALL 08/2013 trial, adults with B-ALL received a cyclophosphamide/dexamethasone prephase, followed by Induction and Consolidation I, which included four rituximab doses in BCR::ABL1 -negative patients, irrespective of CD20 status. CD20 expression was measured by standardized multiparametric flow cytometry in 274 patients in bone marrow and blood at diagnosis and in blood after prephase. IG/TR based measurable residual disease (MRD) was correlated with baseline bone marrow or the highest CD20-positive blast percentage recorded throughout prephase. The historical GMALL 07/2003 cohort treated without rituximab served for comparison. Results Baseline CD20 expression was significantly higher in blood than in bone marrow and increased further during prephase. In paired baseline samples, 6/76 c-/pre-B ALL cases (7.9%) were CD20-negative by bone marrow (< 20%) but positive in blood. In paired blood samples, 14/106 patients crossed the 20% threshold after prephase (12/86 c-/pre-B ALL, 13.9% and 2/20 pro-B ALL, 10.0%). Among 182 BCR::ABL1 -negative patients, highest recorded CD20 across all time points classified 76 (41.8%) as < 20% and 106 (58.2%) as ≥ 20%. Higher CD20 expression was associated with improved MRD response under rituximab. Among MRD-evaluable patients after Induction I ( n = 161), molecular complete remission (MolCR) was achieved in 10.6% of patients with CD20 expression < 20% compared with 30.5% of those with CD20 expression ≥ 20%. After Consolidation I ( n = 159), corresponding MolCR rates were 50.0% and 72.6%, respectively. Associations were weaker when only baseline CD20 bone marrow status was considered. No association with MRD response was observed in GMALL 07/2003 patients treated without rituximab, suggesting a treatment-driven effect in GMALL 08/2013. Conclusions CD20 expression in adult B-ALL varies from diagnostic bone marrow to blood and post-prephase measurements. The highest recorded CD20% value better predicts early MRD responses under rituximab. Post-prephase CD20 reassessment in blood identifies additional patients with eligibility for rituximab. Registry ClinicalTrials.gov, TRN: NCT02881086 (2016-08-23); NCT00198991 (2005-09-12).
Copper, an essential trace element with dual functions in cancer progression, drives tumor growth via oncogenic signaling, metabolic plasticity, and extracellular matrix remodeling. By contrast, copper overload triggers cuproptosis, a form of mitochondrial proteotoxic cell death mediated by the FDX1/LIPT1/DLAT/Fe-S regulatory axis. To date, a unified theoretical framework integrating copper metabolism, tumor microenvironment (TME) remodeling, and antitumor immunity remains lacking. In this review, we reframe the TME as a structured copper ecosystem in which both cellular components and the extracellular matrix are modulated by copper, and propose a contextual copper signaling network, in which copper-mediated tumor cell fate is determined by the labile copper pool, the metabolic state, and tumor cellular heterogeneity. We further delineate a unified causal chain linking cuproptosis-driven immunogenicity and cGAS-STING activation to immune cell activation and PD-L1 modulation. Therapeutically, copper chelation and cuproptosis induction strategies have demonstrated promising efficacy, and combining cuproptosis induction with existing antitumor therapies may reverse therapeutic resistance and enhance treatment efficacy. Future studies need to validate cuproptosis-related signatures as predictive biomarkers for precision oncology and refine copper-targeted therapies to minimize systemic toxicities.
Advances presented at the 2026 American Association for Cancer Research (AACR) Annual Meeting highlight a shift from standalone artificial intelligence (AI) models to integrated, agentic systems across oncology. Platforms such as Synapse enable large-scale data coordination, supporting interoperable and reproducible research. Building on this foundation, conversational and multi-agent AI tools (e.g., DrBioRight, GP CoPilot, Isabl AI Agent) allow natural language interaction with multimodal cancer data, lowering technical barriers. Agentic frameworks for real-world data (RWD) transformation, including clinical document abstraction, cohort extraction, and social determinants of health (SDOH) analysis, demonstrate high accuracy and scalability, while self-critical systems improve reliability. Clinically, AI shows growing impact through validated imaging biomarkers, enhanced trial matching, and scalable cohort identification. In parallel, multi-agent systems are accelerating therapeutic discovery, including CAR-T development and immunotherapy target identification. Collectively, these advances position AI as an active, collaborative partner in cancer research and precision oncology.
Failure after anti-CD19 chimeric antigen receptor (CAR) T-cell therapy in diffuse large B-cell lymphoma (DLBCL) is associated with poor survival and there is no established standard of care. We previously reported the phase 2 LYSA BiCAR trial of short-ramp-up glofitamab after CAR T-cell failure. Here, we present the final survival results and a pre-specified external comparative effectiveness analysis against a contemporary control arm constructed from academic data. BiCAR is a multicenter, single-arm trial in adults with CD20-positive DLBCL refractory to, or in first relapse/progression after anti-CD19 CAR T-cell therapy. Participants received obinutuzumab pretreatment followed by intravenous glofitamab with an accelerated step-up to 30 mg within 8 days, then 30 mg every 21 days for up to 11 cycles. For comparative analyses, we constructed an external control arm from patients included in the French DESCAR-T registry and the ALYCANTE phase 2 trial who experienced CAR T-cell failure, who subsequently started non-bispecific systemic therapy, met key BiCAR eligibility criteria, and initiated treatment within a ±1-month window around the BiCAR treatment period. To minimise datasource bias, both arms, glofitamab (BICAR) and control, were constructed from individual patient data from the DESCAR-T registry and the ALYCANTE trial. A propensity score including major prognostic variables was estimated and applied using stabilized inverse probability of treatment weighting with multiple imputation. Additional weighting schemes and restrictions were applied in sensitivity analyses, and restricted mean survival time (RMST) was evaluated by treatment arm. Among 47 enrolled BiCAR patients, 46 received glofitamab. At a median follow-up of 30.4 months, median overall survival (OS) was 17.3 months, and the 2-year OS rate was 38.3
CAR-T therapy is effective in hematologic cancers but faces challenges in solid tumors due to antigen heterogeneity and an immunosuppressive tumor microenvironment (TME). Systemic CTLA-4 blockade enhances immunity but often causes severe adverse events. To overcome these limitations, we developed a dual-modular nanobody-based CAR-T platform targeting fibroblast activation protein (FAP) on cancer-associated fibroblasts and locally releasing an anti-CTLA-4 nanobody within the tumor stroma. FAP/CTLA-4 dual-module CAR-T cells were generated and assessed in vitro for antigen-specific cytotoxicity, cytokine release, and exhaustion. Antitumor efficacy was evaluated in xenograft models, measuring tumor growth, survival, and T-cell infiltration (Ethics Approval Number: 202001011). One patient with refractory glioblastoma received intrathecal infusion; clinical response, cerebrospinal fluid (CSF) cytokines (Ethics Approval Number 2022-0553-01), and safety were monitored. Tumor and immune microenvironment changes were analyzed via transcriptomic sequencing and multiplex immunofluorescence staining. In vitro, engineered CAR-T cells showed potent cytotoxicity, cytokine production, and reduced exhaustion. In vivo, they induced tumor regression, prolonged survival, and increased T-cell infiltration. In the glioblastoma patient, intrathecal administration resulted in disease stabilization, elevated CSF cytokines, and a favorable safety profile. Transcriptomic sequencing and multiplex immunofluorescence staining indicated TME remodeling toward an immunologically active state. FAP/CTLA-4 DMN CAR-T overcomes the immunosuppressive solid tumor microenvironment through localized immunomodulation, demonstrating promising efficacy in preclinical models and a patient with refractory glioblastoma.
CAR-T cell therapy has shown remarkable success in hematologic malignancies but remains limited in solid tumors such as liver cancer due to antigen heterogeneity, low target antigen density, and an immunosuppressive tumor microenvironment (TME). Cytokine engineering can enhance CAR-T persistence and effector function; however, the optimal cytokine payload may vary depending on tumor type, target antigen expression level, and microenvironmental context, making systematic experimental comparison time-consuming and labor-intensive. Here, we applied a large language model (LLM)–based CAR-T in silico platform to systematically evaluate cytokine engineering strategies, including IL-2, IL-7, IL-12, IL-15, and IL-18, in glypican-3 (GPC3)–targeted CAR-T cells for liver cancer. We used cytokine selection as a biologically grounded benchmark to test whether the platform could recover known CAR-T cell-relevant cytokine biology and support future novel predictions. Computational predictions identified IL-15 as the most effective enhancer, particularly against tumor cells with low GPC3 expression. Guided by these results, we generated cytokine-armored GPC3 CAR-T cells and performed in vitro and in vivo validation. IL-15-engineered CAR-T cells exhibited superior proliferation, persistence, and serial cytotoxicity against GPC3-low liver cancer cells. In human liver cancer xenograft models, IL-15-enhanced CAR-T cells achieved improved tumor control compared with conventional and other cytokine-engineered CAR-T cells. The recovery of IL-15 served as a positive benchmark supporting the validity of the LLM-guided CAR-T in silico workflow. Collectively, this study establishes an LLM-guided framework, schema-constrained for rational cytokine selection in CAR-T engineering and identifies IL-15 as a potent enhancer for targeting antigen-low liver cancers.
Gastric cancer with peritoneal metastasis has a poor prognosis and limited treatment options. Claudin 18.2 (CLDN18.2) CAR T therapy has shown activity in advanced gastric cancer, but its role in peritoneal disease remains insufficiently characterized. Here, we report three patients with gastric cancer and peritoneal metastasis from a phase I trial of satri-cel (NCT03874897), all with high CLDN18.2 expression. All three patients experienced clinical and radiologic benefit after treatment and achieved durable peritoneal disease control. After CLDN18.2-targeted CAR T-cell therapy, one patient underwent conversion surgery 8 months after infusion. After resection of subsequent bilateral ovarian metastases, no further disease progression was observed, corresponding to long-term control of peritoneal metastases. The other two patients achieved overall survival of 44 and 35 months, respectively. Although circulating CAR T-cells became nearly undetectable in peripheral blood by around day 30 after infusion, CAR T-cell infiltration was still detected in resected gastric or ovarian tumor tissues months later. These findings support substantial and durable activity of CLDN18.2 CAR T-cell therapy in gastric cancer with peritoneal metastasis.
Acute myeloid leukemia (AML) with persistent measurable residual disease (MRD) and relapsed/refractory myelodysplastic syndromes (MDS) are low-blast myeloid diseases for which there are few effective therapeutic options. CD123 represents an attractive target in these diseases. Vibecotamab is a bispecific antibody that binds to CD123 on malignant blasts and to CD3 on T-cells, to recognize and eliminate CD123-positive malignant cells. This single-center phase II study evaluated the efficacy of vibecotamab in patients AML with detectable MRD (AML-MRD cohort) or with MDS or chronic myelomonocytic leukemia (CMML) after hypomethylating agent failure (MDS/CMML cohort). In cycle 1, patients received vibecotamab IV on day 1 (0.43 µg/kg), day 3 (0.75 µg/kg), day 5 (1.1 µg/kg), and days 8, 15 and 22 (1.7 µg/kg). In subsequent cycles, patients received vibecotamab IV on days 1, 8, 15, and 22 (1.7 µg/kg). The primary outcomes were MRD negativity rate (AML-MRD cohort) and overall response (MDS/CMML cohort). Between May 2022 and April 2025, 48 patients were enrolled (21 AML-MRD cohort, 27 MDS/CMML cohort). The median ages of the AML-MRD and the MDS/CMML cohorts were 70 and 76 years, respectively. In the AML-MRD cohort, the median MRD level by flow cytometry was 0.64
Systemic mastocytosis (SM) is a spectrum of hematologic disorders characterized by accumulation of atypical mast cells (MCs) in extracutaneous organs. SM with an associated hematologic neoplasm (SM-AHN), the most frequent subtype of advanced SM, is predominantly associated with myeloid neoplasms, consistent with shared clonal architecture. Because of its rarity and heterogeneity, robust outcome data aligned with contemporary classifications are needed to inform risk stratification. We analyzed the 10th data wave of the European Competence Network on Mastocytosis registry (34 European centers and 1 US center). SM and AHN diagnoses followed the 2022 World Health Organization classification. Baseline characteristics and overall survival (OS) were compared between patients with myeloid SM-AHN and SM without AHN (SM-no-AHN). Within SM-AHN, outcomes were analyzed by SM component (advanced: aggressive SM [ASM] or MC leukemia [MCL] vs. non-advanced: bone marrow mastocytosis, indolent SM, or smoldering SM) and AHN subtype. Among 3,925 patients with SM, 467 (11.9
Ovarian cancer is characterized by an immunosuppressive “cold” tumor microenvironment, which poses a major challenge to effective therapy. Inducing immunogenic cell death through PANoptosis represents a promising strategy for remodeling the tumor microenvironment. Z-DNA binding protein 1 (ZBP1), an interferon (IFN)-stimulated gene, is a key sensor of Z-conformation nucleic acids (Z-NA) driving PANoptosis. While ZBP1 upregulation is traditionally attributed to transcriptional induction, its early non-transcriptional regulatory mechanisms remain elusive. ZBP1 expression and its prognostic value were analyzed using public databases and clinical cohorts. APEX2 proximity labeling, PLA and Co-IP identified the E3 ubiquitin ligase regulating ZBP1. The IFN-mediated ZBP1 post-translational modification pathway was delineated utilizing PLA, Co-IP, in vitro phosphorylation, mutagenesis assays, an intestine-specific conditional knockout model. DCAF1−/− ovarian cancer cells and immunocompetent ID8 peritoneal models were generated to evaluate tumor growth and cell death. The therapeutic efficacy of combining the DCAF1 inhibitor (B32B3) with the Z-NA inducer (CBL0137) was assessed in an immunocompetent ID8 murine peritoneal tumor model and two chemoresistant patient-derived xenograft (PDX) models. ZBP1 is significantly downregulated in ovarian cancer, whereas its elevated expression predicts a favorable prognosis and correlates with high IFN responsiveness. Type I IFN triggers a rapid accumulation of ZBP1 protein prior to its transcriptional upregulation. Mechanistically, we identified the E3 ligase substrate receptor DCAF1 as a negative regulator that targets ZBP1 for proteasomal degradation. Interferon signaling activates the kinase DAPK3, which phosphorylates DCAF1 at S1328. This phosphorylation event compromises the assembly of the CRL4DCAF1 complex, thereby abrogating DCAF1-mediated degradation of ZBP1. In ovarian cancer models, genetic ablation of DCAF1 restored ZBP1 levels and significantly restrained tumor progression. Therapeutically, combining B32B3 with CBL0137 elevated intracellular Z-NA and stabilized ZBP1, driving PANoptosis and suppressing ovarian tumor growth across. Our study identifies the IFN-DAPK3-DCAF1 pathway as a critical post-translational mechanism that ensures rapid ZBP1 stabilization. This highlights a fundamental strategy to bypass transcriptional latency for the rapid activation of immune responses, offering a strong clinical rationale to harness this pathway to induce ZBP1-dependent PANoptosis for the treatment of refractory tumors.
Tumor immunity is shaped not only by the cellular composition of the tumor microenvironment (TME), but also by how stromal and immune elements are spatially organized within it. Among these regulators, cancer-associated fibroblasts (CAFs) and tertiary lymphoid structures (TLS) have emerged as two major determinants of the spatial niche of anti-tumor immunity. CAFs comprise heterogeneous and plastic stromal populations that can remodel the extracellular matrix, restrict lymphocyte infiltration, sustain chronic inflammation, and promote therapeutic resistance, while selected subsets may also support antigen presentation and lymphoid organization. In contrast, TLS function as ectopic immune hubs that coordinate local T- and B-cell priming, clonal expansion, and B-cell maturation, and are increasingly associated with favorable prognosis and improved responses to immune checkpoint blockade. In this review, we synthesize recent advances from single-cell and spatial multi-omics studies to examine how CAF states and TLS maturation programs arise, interact, and remodel the immune landscape across tumor types. We propose that the balance between CAF-driven stromal restriction and TLS-associated immune organization is a central determinant of immune exclusion versus immune activation. We discuss how this spatial framework may refine tumor classification, improve patient stratification, and guide therapeutic strategies aimed at stromal reprogramming, TLS induction or maturation, and rational combination immunotherapy, while highlighting innate immune-stromal circuits, including IL-33-activated ILC2s, NCR⁺ ILC3s, LTi-like programs, and myeloid organizer cells, as upstream regulators of TLS neogenesis. By integrating CAF biology, TLS dynamics, and spatial context, this review provides a conceptual framework for understanding and targeting the stromal architecture of tumor immunity.
Primary central nervous system large B-cell lymphoma (PCNS-LBCL) exhibits the worst prognosis among all extranodal LBCLs, with the enriched genetic mutations in MyD88L265P, CD79B and PIM1. However, the upfront incorporation of targeted therapies remains an unmet need in newly diagnosed (ND) patients. In this study, we found that in the presence of MyD88L265P context, B-cell receptor downstream bruton’s tyrosine kinase (BTK) was significantly overexpressed, which subsequently enhanced the stability of PIM1 oncoprotein. To evaluate the intracranial delivery of BTK inhibitor(BTKi), patient-derived PCNS-LBCL xenografted mice models were treated with highly-selective BTKi orelabrutinib, either monotherapeutically or in combining with methotrexate. Interestingly, orelabrutinib showed excellent efficiency crossing BBB with the functional BTK blockade and promoted PIM1 degradation, providing the molecular basis of incorporating BTKi into PCNS-LBCL therapy. Thus, we determined the efficacy and toxicity of orelabrutinib in combination with high-dose methotrexate in a prospective dose-escalating cohort and real-world practice. The patients being treated with orelabrutinib-included ORMD regimen showed better response and more prolonged survival compared to those with RMD regimen. We further investigated the genetic mutations of PCNS-LBCL across tumor tissue, cerebrospinal fluid (CSF) and plasma. The mutations in CSF circulating tumor DNA (ctDNA) rather than plasma-ctDNA were more consistent to those in tumor tissue, indicating that CSF-ctDNA is a useful tool for monitoring PCNS-LBCL. In summary, our data provided the molecular rationale as well as clinical evidences that incorporation of BTKi into frontline induction therapy is a promising strategy for ND PCNS-LBCL.
