O-03.04 Exploring immunotherapy resistance in primary and metastatic oral cancer using organoid technology

<h3>Background</h3> Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer, with a survival rate of less than 50%. Immune checkpoint inhibitor (ICI) therapy has recently been approved for the treatment of recurrent or metastatic OSCC. However, only 1 in 5 OSCC patients respond to ICI treatment. To explore the mechanisms of resistance to ICI therapy, we aimed to develop a novel <i>ex vivo</i> autologous co-culture model system of cancer organoids and T cells. <h3>Materials and Methods</h3> We collected tissue and blood samples from primary, recurrent and metastatic OSCC patients. After generating a biobank of patient-derived organoids (PDOs), we established co-cultures of PDOs and autologous peripheral blood mononuclear cells (PBMCs) to expand tumor-reactive T cells. After evaluating T cell expansion and anti-tumor reactivity by immunophenotyping, we performed organoid killing assays to validate the tumor specificity of the expanded cytotoxic T cells. Then, we performed bulk RNA sequencing on PDOs and single-cell RNA/TCR sequencing on the expanded T cells to investigate the possible resistance mechanisms. <h3>Results</h3> After successful generation of 18 PDO lines, characterization of the PDOs revealed the expression of immunomodulatory molecules, and immune checkpoints such as PD-L1. Although interferon-gamma (IFN-γ) stimulation of PDOs increased the expression of these markers, metastatic PDOs showed decreased IFN-γ signaling and antigen presentation compared to primary tumor PDOs. Furthermore, we found that activated CD8a⁺ T cells in the PBMC fraction were reduced in metastatic OSCC. Next, we combined PDOs with PBMCs in suspension co-culture. Comprehensive flow cytometry revealed that CD3⁺ T cells were enriched in the immune cell fraction over 2 weeks, although the proportion of CD8a⁺ and CD4⁺ T cells showed a heterogeneous response to Pembrolizumab (anti-PD-1). In our sets of patient-matched PDOs from primary and metastatic OSCC, we found T cell-mediated tumor reactivity and cytotoxicity against a primary tumor PDO line, whereas CD8a⁺ T cells co-cultured with the patient-matched metastasis-derived PDOs did not show robust reactivity and the level of active caspase-3 did not increase in the PDOs. Single-cell RNA/TCR sequencing revealed a CD4⁺ T cell cluster with a regulatory phenotype that expanded exclusively in the co-culture with the metastatic PDO line, whereas co-culture with the primary tumor line resulted in a higher proportion of activated CD8⁺ T cells upon anti-PD-1 treatment. <h3>Conclusions</h3> By adapting cancer organoids as an immuno-oncology model, we show for the first time that tumor-reactive T cells can be expanded <i>ex vivo</i> from OSCC patient material. The model system can ultimately be used to better understand why treatment fails and to predict the outcome of ICI treatment. L. Szabó: None. L. Wilde: None. N. Afrin: None. C. Peralta Viteri: None. N. Harnischfeger: None. A. Mader: None. M. Krafft: None. V. Buck: None. A. Kübler: None. S. Hartmann: None. K. Kretzschmar: None.