---
title: "CXCR4 and PD-1 blockade overcomes stroma-driven T-cell exclusion and exhaustion in PDAC 3D models"
id: "frontiers-in-immunology-17-targeting-stroma-mediated-t-cell-exclusion-and-functional-exhaustion-in"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-17-targeting-stroma-mediated-t-cell-exclusion-and-functional-exhaustion-in"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1844781"
published_at: "2026-07-21T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# CXCR4 and PD-1 blockade overcomes stroma-driven T-cell exclusion and exhaustion in PDAC 3D models
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-17-targeting-stroma-mediated-t-cell-exclusion-and-functional-exhaustion-in
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1844781)
- **Published At:** 2026-07-21T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Pancreatic ductal adenocarcinoma (**PDAC**) features a desmoplastic, immunosuppressive tumor microenvironment (TME) that drives profound **T-cell exhaustion** and resistance to immunotherapy. - The authors developed minimalistic, animal-free 3D models: PANC-1 spheroids, co-cultures with primary pancreatic stellate cells (**PSC**) to mimic stroma, and patient-derived spheroids (**PDS**) containing tumor cells and cancer-associated fibroblasts (CAF). - Primary human T-cells from healthy donors and autologous T-cells from PDAC patients were infiltrated into spheroids to assess infiltration, activation, and checkpoint regulation under controlled conditions. - Infiltrated T-cells upregulated inhibitory receptors including **PD-1**, **LAG-3**, and **CTLA-4**, mirroring tumor-infiltrating lymphocytes isolated from PDAC patient tissue. - Incorporation of PSC produced a fibrotic barrier that substantially restricted T-cell infiltration, modeling stromal immune exclusion observed in PDAC clinical samples. - The stromal chemokine axis **CXCL12–CXCR4** was implicated mechanistically in T-cell exclusion; pharmacologic CXCR4 blockade with **AMD3100** significantly increased T-cell entry into PSC-containing spheroids. - Checkpoint blockade with the anti-**PD-1** antibody **pembrolizumab** partially restored effector functions of infiltrated T-cells within the 3D platform. - The PDS reproduced patient-specific patterns of T-cell suppression and therapeutic responsiveness, supporting translational relevance for personalized testing. - The 3D spheroid platform captures cytokine- and stroma-driven immunomodulation typical of advanced organoid or in vivo systems while remaining scalable and animal-free. - The model offers a mechanistically faithful tool to dissect TME-driven immune suppression and to accelerate functional evaluation of combinatorial or patient-tailored immunotherapies for PDAC.
## Clinical Analysis & Structured Key Points
About us All journals All articles Submit manuscript Submit data Search Frontiers in Immunology Sections Articles Research Topics Editorial board About journal Published in Frontiers in Immunology Cancer Immunity and Immunotherapy 7 impact factor 11.3 citescore Part of a Research Topic Overcoming bottlenecks in cancer immunotherapy: Mechanisms of immune escape and translational strategies Submission open 2537 views 2 articles Editor & Reviewers Edited by L Z Ling Zhong Reviewed by Y S Yizhe Sun X Z Xiao Zhang Outline Abstract 1 Introduction 2 Material and methods 3 Results 4 Discussion Data availability statement Ethics statement Author contributions Funding Acknowledgments Conflict of interest Generative AI statement Publisher’s note Supplementary material References Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Figure 7 View in article Figure 8 View in article ORIGINAL RESEARCH article Front. Immunol., 21 July 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1844781 Targeting stroma-mediated T-cell exclusion and functional exhaustion in pancreatic ductal adenocarcinoma through CXCR4 and PD-1 blockade A D Alina Deipenbrock 1 L H Lina Hofer 1 B E Ben E. Wilmes 1 T C Timur Cetin 2 I E Irene Esposito 3 D W Dirk Weyhe 2 J S Johannes Stegmaier 4 N E Nicole E. Teusch 1* 1. Heinrich-Heine-Universität Düsseldorf, Institute of Pharmaceutical Biology and Biotechnology, Düsseldorf, North Rhine-Westphalia, Germany 2. Carl von Ossietzky Universität Oldenburg, University Hospital for Visceral Surgery, Pius-Hospital Oldenburg, Oldenburg, Lower Saxony, Germany See more Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies to date and characterized by a unique immunosuppressive and highly desmoplastic tumor microenvironment (TME). These features drive profound T-cell dysfunction and maintain high resistance to current immunotherapy. By recapitulating the complex 3D architecture of human PDAC, we demonstrate the key immunosuppressive mechanisms that drive T-cell dysfunction within the tumor microenvironment. Method: 3D PDAC spheroids—generated from PANC-1 cells alone or together with primary pancreatic stellate cells (PSC)—were infiltrated with primary human T-cells from healthy donors, allowing controlled analysis of T-cell infiltration, activation, and checkpoint regulation. Furthermore, patient-derived spheroids (PDS) composed of primary tumor cells and cancer-associated fibroblasts were infiltrated with autologous T-cells. Results: Infiltrated T-cells exhibited a pronounced exhaustion signature, including strong upregulation of PD-1, LAG-3, and CTLA-4, closely mirroring the phenotype of tumor-infiltrating lymphocytes (TIL) isolated from PDAC patient samples. Incorporation of pancreatic stellate cells (PSC) generated a fibrotic barrier around the tumor cells that markedly restricted T-cell infiltration, modeling the desmoplastic TME characteristic of PDAC. From a mechanistic perspective, stromal CXCL12–CXCR4 enhanced T-cell exclusion: pharmacological CXCR4 blockade with AMD3100 significantly enhanced T-cell infiltration into PSC-containing spheroids. Furthermore, treatment with the anti-PD-1 monoclonal antibody pembrolizumab partially restored the effector cell function of T-cells within the 3D system. These results demonstrate that this minimalistic platform is capable of capturing complex, cytokine- and stroma-driven immunomodulation typically observed only in advanced organoid or in vivo systems. Crucially, key immunological features—including T-cell exhaustion, stromal exclusion, and therapeutic responsiveness—were fully reproduced in PDS. PDS reproduced patient-specific T-cell suppression patterns and therapeutic responses, underscoring the translational relevance of the platform. Conclusion: Together, our findings identify critical determinants of T-cell dysfunction in PDAC and introduce a versatile, animal-free 3D model that powerfully captures hallmark immune-evasion mechanisms in PDAC. This system provides a scalable and mechanistically faithful tool for dissecting TME-driven immune suppression and for accelerating the functional evaluation of immunotherapeutic strategies, including patient-tailored approaches. 1 Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a five-year survival rate of only about 13% (1, 2). Late diagnosis, a high metastatic potential, and a heterogeneous tumor microenvironment (TME) are key contributors to this poor prognosis (1, 3). The PDAC TME consists of various cell types, including immune cells, endothelial cells, neuronal cells and cancer-associated fibroblasts (CAF) (4–6). CAF promote the tumor progression through the release of immunosuppressive cytokines and chemokines like TGF-β, CXCL10, and CXCL12, and produce large amounts of extracellular matrix (ECM), resulting in a dense and desmoplastic stroma (4, 7). In addition to CAF, the PDAC TME contains diverse immune cell populations, however, these are predominantly immunosuppressive, including tumor associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and regulatory T-cells (Treg). TAM are mainly polarized towards the immunosuppressive M2 phenotype contributing to tumor progression by enhancing immunosuppression and chemoresistance (8, 9). MDSC impair T-cell function directly by upregulating programmed death-ligand 1 (PD-L1) to inhibit T-cell activation, and indirectly via an IL-10 dependent release of TGF-β, driving the expansion of Treg (10, 11). Treg (FoxP3+, CD4+, CD25+) secrete the anti-inflammatory cytokines IL-10 and TGF-β and express the checkpoint molecule cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), thereby inhibiting effector T-cell function and polarization of TAM towards M2 (12). Treg inversely correlate with CD8+ T-cell infiltration and are associated with poor clinical outcomes (13). Treg activate a tolerogenic phenotype in dendritic cells, characterized by downregulation of the costimulatory molecules CD86 and CD40, MHC class II, and IFN-y, leading to suppressed activation of CD8+ T-cells (10). Accordingly, elevated Treg frequencies are considered a negative prognostic biomarker in PDAC (12, 13). Besides Treg, CD4+ T helper (Th) cells are largely polarized toward a Th2 phenotype, promoting immunosuppression through the release of anti-inflammatory cytokines such as IL-4 and IL-13, which polarize anti-inflammatory M2 macrophages and support tumor cell metabolism through increased glycolysis via JAK-STAT signaling (14, 15). In addition, CD4+ Th17 cells have also been described in the TME of PDAC patients, where they are associated with a poorer prognosis by promoting the formation of pancreatic intraepithelial neoplasia (PanIN) and enhancing the stem cell properties of tumor cells (10, 16, 17). Th17 secrete IL-17, which increases fibrosis, neovascularization and myeloid cell recruitment, enhancing survival and growth of tumor cells through the activation of the gp130-JAK2-STAT3 pathway (17–19). Cytotoxic lymphocytes (CD8+ T-cells, (CTL)) are crucial for the elimination of tumor cells, mediating killing through the secretion of granzyme B, perforin, IFN-γ, and TNF-α, or of mediating FasL-induced apoptosis (12). In PDAC, however, effector T-cells are largely excluded from the TME and confined to the periphery within the stroma-rich areas surrounding the tumor (12). This exclusion is driven by the CAF-derived chemokine CXCL12, which acts as a specific ligand for the G protein-coupled receptor CXCR4 in T-cells, promoting their chemotactic migration into stroma-rich areas (20, 21). Furthermore, T-cells in the PDAC TME exhibit an exhausted phenotype characterized by increased expression of checkpoint molecules, reduced proliferative capacity, and diminished effector function (12, 22, 23). Key checkpoint molecules elevated in these T-cells include programmed cell death protein-1 (PD-1), lymphocyte-activation gene 3 (LAG-3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (24, 25). PD-1 engages its ligands PD-L1 and PD-L2, expressed on tumor cells and other TME-resident T-cells, attenuating T-cell activation and promoting T-cell apoptosis (26). CTLA-4 competes with CD28 for binding to B7 ligands on antigen-presenting cells, thereby preventing co-stimulatory signaling required for full T-cell activation and driving an immunosuppressive state (27, 28). LAG-3 interacts with MHC class II molecules, interfering with effective T-cell receptor (TCR) signaling and dampening T-cell activation (29). Collectively, these mechanisms reduce effector function of CD4+ T-cells and impair the cytotoxic activity of CD8+ T-cells against tumor cells (28, 29). Advances in immunotherapy have focused on reactivating tumor-infiltrating T-cells, by blocking checkpoint molecules with monoclonal antibodies (11, 30). The first FDA approved immune checkpoint inhibitor (ICI) was the CTLA-4 blocker ipilimumab for metastatic melanoma in 2011 (31). Since then, numerous ICI targeting CTLA-4, PD-1, or PD-L1, including pembrolizumab, nivolumab, durvalumab, atezolizumab, and avelumab, have been approved for various malignancies, such as non-small cell lung cancer, urothelial carcinoma, Hodgkin lymphoma, and hepatocellular carcinoma (32–37). In PDAC, several ICI have been or are currently under clinical investigated, including the PD-1 inhibitors nivolumab or pembrolizumab, the PD-L1 inhibitor durvalumab, and the CTLA-4 inhibitor ipilimumab (38–41). However, as monotherapies, these antibodies have failed to demonstrate an increase in overall survival (OS) and progression free survival (PFS) compared to chemotherapy, and led to grade 3 treatment-related adverse events in up to 55% of the patients (26, 42–44). To overcome this limited efficacy, multiple clinical trials have evaluated ICI in combination with other therapeutics. These include combination with standard chemotherapy regimens (FOLFIRINOX or gemcitabine plus nab-paclitaxel) alongside pembrolizumab, nivolumab, ipilimumab, or durvalumab (45–47), as well as combinations with PARP-inhibitors (48, 49), or TME-modulating agents such as the CXCR4 antagonist motixafortide (BL-8040), an indoleamine 2,3-dioxygenase 1 [IDO1] inhibitor (50, 51), and a focal adhesion kinase (FAK) inhibitor (52). To date, no broadly effective immunotherapeutic strategy is available for the vast majority of PDAC patients with microsatellite-stable tumors, although pembrolizumab is FDA-approved and effective for the rare (1.2%) microsatellite instability-high (MSI-H) and deficient mismatch repair (dMMR) patient subset (26, 53, 54). To elucidate mechanisms underlying T-cell dysfunction in the PDAC TME, we examine the composition, spatial localization and functional state of tumor-infiltrating lymphocytes in PDAC patients. Clinical analyses have frequently reported an altered CD4+/CD8+ T-cell ratio in both the tumor microenvironment and in peripheral blood from PDAC patients (12, 55). The TME is often characterized by a relative enrichment of CD4+ T-cell subsets, including Th2-polarized and regulatory Treg, alongside a reduced cytotoxic CD8+ T-cell compartment (12). In the peripheral blood, an increased expansion of regulatory and memory CD4+ T-cell populations has been described (55). The overall increased CD4/CD8 ration is less consistent compared to the TME and the shift is associated more with an increase of specific CD4+ subpopulations (e.g. Treg) rather than a reduction of CD8 T-cells (55). This suggest a systemic shift towards a CD4 dominant immunity, however it remains unclear how the peripheral immune alterations reflect the intratumoral T-cell composition. In addition, tumor-infiltrating T-cells commonly exhibit elevated expression of multiple inhibitory receptors, including PD-1, CTLA-4, and LAG-3, indicative of an exhausted phenotype (24). We therefore investigate the effect of infiltration into PDAC spheroids on T-cell checkpoint expression. Beyond functional exhaustion, spatial immune exclusion represents another hallmark of the PDAC microenvironment. Cytotoxic T-cells are frequently restricted to stromal regions or the tumor periphery, while the tumor core remains poorly infiltrated (20). This phenomenon has been attributed in part to the dense desmoplastic stroma and the activity of CAF, which can promote T-cell exclusion via the CXCL12–CXCR4 axis (21). By mimicking the spatial composition of the TME we analyzed the role of CAF on the infiltration of T-cells. While these immunologic alterations have been well documented, they have been predominantly studied in isolation in patient tumor samples, mouse models and in vitro models (56–58). Conventional 2D models and classic patient-derived organoids lack the spatial organization and cellular composition of the TME and are therefore of limited utility for investigating of T-cell infiltration and immunotherapeutic responses. As a result, the interplay between checkpoint molecule upregulation, the accumulation of Treg, and the stroma-mediated exclusion of T-cells, and how these collectively contribute to immunotherapy resistance in PDAC remains poorly understood. Therefore, we aim to recapitulate these key immunosuppressive mechanisms simultaneously to drive T-cell dysfunction within the TME using the complex 3D architecture of human PDAC. This model was specifically designed to recapitulate the key mechanisms of immune evasion in PDAC, thereby providing a more physiologically relevant and predictive platform for the assessment of immunotherapeutic treatments and patient-specific responses than current in vitro models. 2 Material and methods 2.1 Cell culture The PANC-1 cell line was obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; #41965039, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; #10270-106, Gibco) and 1% penicillin–streptomycin (Pen-Strep; #15140122, Gibco). Pancreatic stellate cells (PSC) were kindly provided by Dr. Erkan (Koç University Hospital, Turkey). Ethical approval for the use of PSC was obtained from the Biomedical Sciences Ethics Committee of Koç University, and written informed consent was received from all participating patients. PSC were isolated from individuals diagnosed with pancreatic ductal adenocarcinoma and cultured in DMEM/F12 medium (#11320074, Gibco) containing 20% FBS and 1% Pen-Strep under sterile conditions. All cell cultures were maintained at 37 °C and 5% CO2. 2.2 Isolation of T-cells Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats obtained from anonymized healthy donors (Düsseldorf University Hospital, Germany) who had provided material approved for in vitro research. Autologous T-cells from PDAC patients were isolated in the same manner from patient’s PBMC. The isolation was performed using a Ficoll-Paque density gradient centrifugation method. Briefly, buffy coats were diluted with D-PBS and carefully layered over Histopaque (#10771, Sigma-Aldrich, St. Louis, MO, USA). Following centrifugation at 850 × g for 20 min with minimal acceleration and deceleration, the PBMC layer was collected. To remove residual erythrocytes, cells were incubated for 10 min in ammonium chloride lysis buffer, then washed with Dulbecco’s Phosphate Buffered Saline (D-PBS), and the cell count was determined. T-cells were subsequently isolated from PBMC using Magnetic-Activated Cell Sorting (MACS) with biotinylated CD4 (#130-113-224, Miltenyi Biotec, Bergisch Gladbach, Germany) and CD8 (#130-110-676, Miltenyi Biotec) antibodies, followed by anti-biotin microbeads (#130-090-485, Miltenyi Biotec) according to the manufacturer’s instructions, employing the MidiMACS separator (#130-042-302, Miltenyi Biotec). The purified T-cells were maintained in RPMI medium (#21875-034, Gibco) supplemented with 10% FBS, 1% penicillin–streptomycin, and 50 ng/mL IL-2 (#11340025, ImmunoTools, Friesoythe, Germany). For T-cell activation and expansion, CD3/CD28 Dynabeads (#11161D, Thermo Fisher Scientific, Waltham, MA, USA) were added at a 1:2 bead-to-cell ratio. 2.3 Isolation and cultivation of patient-derived tumor cells, T-cells and CAF PDAC tissue and full blood were obtained during resection at Pius-Hospital Oldenburg, Germany. The use of human tissue samples was approved by the ethics committee at Carl von Ossietzky University Oldenburg and confirmed by Heinrich Heine University Düsseldorf (study number 2022-058). All patients provided written informed consent prior to tissue collection. Clinicopathological characteristics of the included PDAC patients are summarized in Supplementary Table 1. All patients underwent surgical resection without prior neoadjuvant treatment. For tissue dissociation, the tumor specimen was washed in DPBS supplemented with 2% (v/v) FBS, and carefully trimmed to remove residual adipose tissue. The remaining tumor tissue was finely minced into fragments smaller than 1 cm³ and enzymatically digested in 15 mL of digestion solution, containing 0.6 mg/mL Collagenase IV (#17104–019 Gibco), 0.1 mg/mL DNAse (#10104159001 Roche, Basel, Switzerland) and 2% FBS in RPMI (#21875–034 Gibco) at 37 °C with constant agitation for 45–60 min. The resulting suspension was filtered through a 70 µm cell strainer, and remaining tissue fragments were mechanically dissociated using a syringe plunger. The filter was rinsed with DPBS containing 2% FBS, and the cell suspension was centrifuged at 550 g for 7 min. The cell pellet was resuspended in DMEMF12 containing 10% FBS for tumor cell cultivation and 20% FBS for CAF cultivation. Pure cell lines were obtained via differential trypsinization (59). Characterization of patient-derived cells was performed via immunofluorescence staining. Cancer cell were stained for vimentin (#sc-6260, Santa Cruz Biotechnology, Dallas, TX, USA), pan-cytokeratin (#130-133-439, Miltenyi Biotec), cytokeratin 7 (#130-115-446, Miltenyi Biotec), E-cadherin (#130-111-992, Miltenyi Biotec), CD44 (#130-113-900, Miltenyi Biotec), Epcam (#14-9326-82, Thermo Fisher), Ki67 (#MAB7617, R&D Systems, Minnneapolis, MN, USA), and N-cadherin (#ab98952, Abcam, Cambridge, England). Patient-derived CAF were immunofluorescence labeled for αSMA (#19245S, Cell Signaling, Danvers, MA, USA). For flow cytometric analysis T-cells from the tumor tissue were isolated from the digested tumor tissue using MACS with a biotinylated CD45 antibody (#130-110-630, Miltenyi Biotec), followed by anti-biotin microbeads (#130-090-485, Miltenyi Biotec) according to the manufacturer’s instructions. Isolated immune cells were stained for FACS analysis as described below. 2.4 Spheroid formation For the generation of mono-culture spheroids composed of PANC-1 cells, further referred to as PANC-1 spheroids, 1500 PANC-1 cells in 50 µL DMEM with 2.5% (v/v) Matrigel (#356230, Corning, NY, USA) were added into an ultra-low attachment coated 96-well plate (#650970, Greiner Bio-One, Kremsmünster, Austria) and centrifuged at 1500 rpm for 5 min. Co-culture spheroids were generated according to our previous protocol (60). Patient-derived spheroids (PDS) composed of patient-derived tumor cells and primary CAF accordingly. 2.5 Immunofluorescence staining of tumor slices Formalin-fixed, paraffin-embedded (FFPE) PDAC tumor sections (1.5 µm) were kindly generated at the Institute of Pathology from the University Hospital Düsseldorf. All samples were anonymized and their use was approved by the ethics committee of the Heinrich Heine University Düsseldorf (study number 2022-2170). Tissue sections were deparaffinized using xylene and a graded ethanol series, followed by antigen retrieval in Tris(hydroxymethyl)aminom
## Related Clinical Research

- [Source page lacked article content for immune checkpoint inhibitor strategies in lung cancer](https://medichelpline.com/clinical-feed/frontiers-in-immunology-16-immune-checkpoint-inhibitor-based-combinatory-and-alternative-strategies-for.md)
- [CT-guided intratumoral immunotherapy for advanced solid tumors: safety and systemic effects](https://medichelpline.com/clinical-feed/frontiers-in-immunology-3-ct-guided-intratumoral-immunotherapy-for-advanced-solid-tumors-a-prospective.md)
- [Challenging FDA-Approved Cancer Drug Dosages: A Patient and Research Perspective](https://medichelpline.com/clinical-feed/kff-health-news-2-how-much-of-a-cancer-drug-is-too-much-patients-researchers-challenge-fda.md)
- [Translational modelling questions receptor‑occupancy‑based dosing for pembrolizumab (PD‑1 inhibito](https://medichelpline.com/clinical-feed/british-journal-of-cancer-0-translational-modelling-challenges-receptor-occupancy-based-dosing-of-pd-1.md)
- [PD-L1 expression and survival in unresectable or recurrent gastric cancer treated with first-line](https://medichelpline.com/clinical-feed/british-journal-of-cancer-0-pd-l1-expression-and-survival-in-unresectable-recurrent-gastric-cancer-treated.md)

## Navigation
- [← Back to Infectious Disease Feed](https://medichelpline.com/clinical-feed/infectious-disease.md)
- [← All Clinical Specialties](https://medichelpline.com/clinical-feed.md)
## Medical & Regulatory Disclaimer

> [!CAUTION]
> MedicHelpline content is structured for research, educational, and professional discovery purposes. It does not constitute individual medical advice, clinical diagnosis, or treatment recommendations.
> Always verify dosing, contraindications, and regulatory alerts against official product labeling and primary regulatory sources before clinical decision-making.