{
  "abstract": "Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and treatment-resistant solid tumors, with a 5-year survival rate below 13%. 1 While immunotherapy has transformed outcomes in several cancers,2–3 PDAC remains largely refractory due to its immunologically ‘cold’ tumor microenvironment (TME), marked by dense desmoplasia, immune exclusion, and immunosuppression. A major barrier is the poor infiltration and functional exhaustion of T cells within the tumor. To address this, we developed an ex vivo strategy to generate and assess tumor-specific lymphocytes using a physiologically relevant organoid model that recapitulates key immunologic features of the TME.Methods We engineered a personalized immunocompetent organoid model (iPTO) by co-culturing resected PDAC tumor cells with autologous tumor-draining lymph node (TDLN) cells. Autologous peripheral blood mononuclear cells (PBMCs) were circulated through the system using a microfluidic ‘tumor-on-a-chip’ platform, generating tumor-reactive cytotoxic lymphocytes, termed Organoid Interacting Lymphocytes (OILs) ( figure 1a). To model stromal influence, multicellular PTOs were created by incorporating patient fibroblasts without hydrogel scaffolds, better simulating the PDAC TME. Each patient sample was expanded for 2 weeks before analysis (figure 1b). Real-time tumor-immune interactions were captured using high-resolution time-lapse microscopy via the Ramona Optics multi-camera imaging system, assessing lymphocyte infiltration, tumor engagement, spatial dynamics, and cytotoxicity.Results Organoid Interacting Lymphocytes (OILs) exhibited significantly enhanced colocalization with tumor cells compared to control immune cells, indicating improved homing and immune synapse formation within PTOs. High-resolution time-lapse imaging captured dynamic tumor cell killing by OILs, along with tumor cell clustering and directional migration suggestive of active immune evasion. Representative stills from the time-lapse series ( figure 2a) highlight immune-tumor interactions, while single-cell tracking (figure 2b) quantitatively confirmed movement dynamics. On average, OILs migrated 6.1 pixels closer to tumor cells over 24 hours versus 0.6 pixels for control immune cells (P < 0.05; figure 2c). Notably, in fibroblast-enriched organoids designed to mimic the tumor stromal barrier, OILs retained their superior infiltration and cytotoxic capabilities. These findings align with prior results in appendiceal and mesothelioma PTO models, where OILs outperformed tumor-infiltrating lymphocytes (TILs) in inducing cancer cell apoptosis.4 Conclusions Our findings indicate OILs as a potent, patient-specific adoptive cell therapy capable of overcoming PDAC’s immunosuppressive TME. By leveraging advanced organoid models and live-cell imaging, we captured dynamic tumor-immune interactions and demonstrated functional immune activation. This platform provides a robust preclinical tool for evaluating personalized immunotherapy strategies and supports further clinical development of OIL-based therapies in pancreatic cancer.References Siegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. CA: A Cancer Journal for Clinicians 2025;75(1):10–45.Votanopoulos KI, Forsythe S, Sivakumar H, Mazzocchi A, Aleman J, Miller L, Levine E, Triozzi P, Skardal A. Model of patient-specific immune-enhanced organoids for immunotherapy screening: feasibility study. Annals of Surgical Oncology 2020;27(6):1956–1967.Forsythe SD, Erali RA, Sasikumar S, Laney P, Shelkey E, D’Agostino R, Jr, Miller LD, Shen P, Levine EA, Soker S, Votanopoulos KI. Organoid platform in preclinical investigation of personalized immunotherapy efficacy in appendiceal cancer: feasibility study. Clinical Cancer Research 2021;27(18):5141–5150.Hutchins D, Schaaf C, et al. (2025). Enhancing patient lymphocyte response to peritoneal malignancies using a personalized immunocompetent microfluidic co-culture platform. [Manuscript Submitted for Publication]. Nature Communications.Ethics Approval This study was conducted in accordance with Human Research Protection Program standards and approved by the Wake Forest University Institutional Review Board as #IRB0043947.Abstract 328 Figure 1Ex vivo lymph node mediated tumor antigen primingAbstract 328 Figure 2Representative patient timelapse, cell tracking, and localization",
  "authors": [
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Damian C Hutchins"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Nicholas Edenhoffer"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Tiefu Liu"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Cecilia Schaaf"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Nadeem Wajih"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Adam Hall"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Shay Soker"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Konstantinos Votanopoulos"
    },
    {
      "affiliations": [
        "Wake Forest University School of Medicine, Winston-Salem, NC, USA"
      ],
      "name": "Eleftherios Makris"
    }
  ],
  "title": "328 Personalized organoid-based immunotherapy for pancreatic cancer: real-time imaging of cytotoxic lymphocyte-tumor dynamics in an ex vivo model",
  "uid": "37cdb387-b920-5c69-8267-fe27c11fdd78"
}