{
  "abstract": "Background Nivolumab (Nivo) is an anti-PD-1 immune checkpoint inhibitor approved for colorectal cancer (CRC), yet responses are highly variable between patients, and the dynamics of immune activation are not well understood. Patient-derived organotypic tumor spheroids (PDOTS) are an ex vivo platform that preserves the multicellular architecture and immune microenvironment of individual tumors, allowing for functional assessment of drug response. In this longitudinal study, we used colorectal cancer (CRC) PDOTS to evaluate the time-dependent effects of Nivolumab on tumor cytotoxicity and immune modulation, offering insight beyond single timepoint assays or animal models.Methods Tumors resected from CRC patients (n=4) were processed into PDOTS, loaded into microfluidic devices, and treated with Nivo monoclonal antibody (100 µg/ml) or media as a control. Cytotoxicity was evaluated on days 1, 2 and 3 post-treatment using Hoechst/Propidium Iodide staining, quantified by automated image analysis, and analyzed relative to the media control. RNA was next isolated from the PDOTS and the gene expression profiles generated using the NanoString IO360 Panel. A change in tumor cell abundance was determined by measuring differences in tumor associated RNA counts between treatment and control groups. Gene expression was analyzed across multiple timepoints to resolve kinetics.Results Nivolumab induced three distinct response kinetics across the CRC patient-derived tumor spheroids: an early response phenotype, a delayed response phenotype, and a non-responsive phenotype. In the early responsive patient (P3), both cytotoxicity and immune activation were detected as early as Day 1. NanoString profiling showed elevated immune pathways including IFN-γ and TCR signaling pathways relative to control on day 1, which remained sustained through Day 3. In contrast, the delayed response phenotype in patients P1 and P2 displayed depressed immune activation at Day 1, followed by a rise in immune activation on Days 2 and 3, culminating in measurable cytotoxicity by Day 3. The third phenotype, patient P4 showed no evidence of immune activation at any timepoint. Factors potentially underlying these differences, including immune cell frequencies at baseline are presented.Conclusions This study highlights the value of longitudinal profiling in PDOTS to uncover dynamic patterns of response to immune checkpoint inhibitors. Reliance on terminal gene expression alone can miss transient or delayed immune activity, potentially leading to inaccurate conclusions. Time-resolved functional analysis offers a more complete view of immunotherapy response and resistance, supporting its utility in preclinical evaluation and translational research. Ongoing efforts aim to expand cohort size and further refine response kinetics in CRC.Ethics Approval This study was approved by the WCG Institutional Review Board (study 1280260). All patients gave and signed an informed consent form to release resected tumor tissue for research. All patient information was anonymized.",
  "authors": [
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Julia Vail"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Ketki Bhise"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Danielle Hagee"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Sinal Patel"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Anthony Attardo"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Kirti Khardenavis"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Chunxiao Cui"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Alyssa Martin"
    },
    {
      "affiliations": [
        "Xsphera Biosciences Inc., Cambridge, MA, USA"
      ],
      "name": "Michael A Perricone"
    }
  ],
  "title": "487 Divergent kinetics of nivolumab response in colorectal cancer revealed by longitudinal analysis of patient-derived organotypic tumor spheroids (PDOTS)",
  "uid": "1100f6eb-70a1-5e26-a4c4-bdacfddad6fa"
}