Ex vivo micro-tumor testing platform to characterize and predict patient-specific immunotherapy drug responses

Background. Immunotherapies have shown significant clinical benefit, but upfront identification of patients with therapy-sensitive cancers remains a major challenge. We have previously demonstrated that our ex vivo micro-tumor platform, which preserves the tumor microenvironment (TME), is predictive for clinical responses to platinum-based therapy (Koedoot et al., npj precision oncology, 2025). Here, we show that the platform also enables patient-specific profiling of drug sensitivity and immune activity for immune checkpoint inhibitors (ICIs) and bispecific T cell engagers (BiTEs). Methods. Fresh tumor tissues were collected from colorectal cancer (CRC, N=6), non-small cell lung cancer (NSCLC, N=23) and ovarian cancer (OC, N=85) patients with primary or recurrent disease. Micro-tumors including the TME were isolated from resections, ascites or pleural fluid, embedded in hydrogel and exposed to chemotherapeutics, six ICIs and/or CD3xMUC16 BiTE ubamatamab. Drug responses were quantified by extracting morphological features from high-throughput 3D imaging. Treatment sensitivity was defined by statistically significant immune cell expansion and micro-tumor killing. Sensitivity profiles were correlated with immune cell populations. Supernatants collected after treatment were analyzed for secreted cytokines, chemokines and cytotoxic mediators using multiplex immune biomarker profiling. PD-L1 tumor proportion scoring (TPS) reported on original patient tumor tissue was used (NSCLC). Results. Significant ex vivo ICI responses were observed in 50% of OC, 27% of early NSCLC, and 33% of metastatic NSCLC samples, with reproducible results across serial samples. Most OC samples were positive for T-cell, macrophage and NK-cell markers but not for B-cell markers. Stronger immune responses were observed in OC samples with high CD3 (p=0.01) or CD68 (p=0.003) expression. Importantly, immune sensitivity correlated with PD-L1 TPS (p=0.13, NSCLC). Ubamatamab induced significant immune expansion in 42% (8/19) of OC samples, half of which also showed significant tumor killing. When compared to non-responders, ubamatamab responders with effective tumor killing exhibited robust and simultaneous upregulation of T cell effector cytokines (IFN-gamma­, IL-2), cytotoxic mediators (Granzyme B, Perforin) and pro-inflammatory cytokines (CXCL9, CXCL11). Conclusions. This study reports classification, characterization and prediction of autologous patient-specific sensitivity to ICIs and BiTEs by characterizing immune populations and reporting correlation with known clinical markers (PD-L1). Our ex vivo micro-tumor platform enables functional profiling of patient-specific immunotherapy responses, supporting biomarker discovery and selection of patients for emerging immunotherapies. The next step is to correlate the ex vivo sensitivity to actual clinical outcome.