Salivary gland and oral cancer immune cell interaction: a tumour-cell based initiation of immune evasion

<p dir="ltr"><i>Background: </i>Adenoid cystic carcinoma (ACC) is a rare salivary gland cancer of the head and neck (HN) region. Current treatment is surgery with optional radiation therapy, resulting predominantly in tumour recurrence and/or metastasis. Immunotherapy could therefore be the first-line treatment of HN cancer in a neoadjuvant setting. While oral squamous cell carcinoma (OSCC) and mucoepidermoid carcinoma (MEC) have been shown to respond well to immune checkpoint therapy, ACC is unresponsive. The ACC microenvironment exhibits low immunogenicity, with few tumour-infiltrating lymphocytes and dendritic cells, that partially explains its poor prognosis.</p><p dir="ltr">Gipie is a microtubule-associated protein involved in protein trafficking and also immune cells activation. Gipie is present in ACC cells. Therefore, I investigated oral and salivary gland cancer cell proliferation, apoptosis, and morphologic changes caused by Gipie in the presence of immune cells. Additionally, to understand the different responses of oral and salivary gland cancers to immunotherapy, I examined the differential expression of immunosuppressive proteins in HN cancer cells during cancer–immune cell interactions.</p><p dir="ltr"><i>Methods: </i>I assayed UM-HACC-2A (ACC), A-253 (MEC), SCC4, SCC9, SCC25, Cal 27 (OSCC) and OKF6 (normal oral) cells co-cultured with NK-92 and Jurkat E6.1 (acute T-cell leukaemia) cells to construct multiple salivary gland and oral cancer–immune cell co-culture models. Gipie-silenced cancer cells (for 48 hours) were compared with their unaltered subsets. Cancer–immune cell interactions were maintained for 16 hours. Overall, 336 co-culture models were examined to determine apoptosis, immune activation, morphology, transmigration, and protein expression. Additionally, mass spectrometry was performed to identify changes in protein profiles.</p><p dir="ltr"><i>Results: </i>ACC cells exhibited pseudonormal morphology after interacting with immune cells in the three-dimensional co-culture model. Gipie-silenced ACC cells transformed to a lymphoblast-like morphology. Compared with unaltered ACC cells, Gipie-silenced ACC cells (n = 24) showed a significant increase in apoptosis, a decrease in T-regulatory cells (FoxP3⁺/IL-2Rα⁺/CD25⁺), and an increase in activated NK cells (NKp30⁺/IFN-γ⁺), with a significantly higher release of granzyme (n = 12) and perforin (n = 12). The MEC cell responses were significantly lower compared with ACC cells following Gipie silencing, while OSCC and OKF6 cell responses were negligible under identical conditions.</p><p dir="ltr">Analysis of the differential protein expressions of ACC, MEC and OSCC, and normal oral cells showed that the significantly overexpressed proteins in the OSCC cells participated more in metabolic processes compared with normal oral cells, while the significantly overexpressed proteins in the salivary gland cancer cells had a greater effect on the immune response to cancer cells compared with OSCC cells. Compared with MEC cells, the significantly overexpressed proteins in the ACC cells showed a strong correlation with the cytokine-mediated signalling pathway, negative regulation of leukocyte cell adhesion, and immune checkpoint pathway proteins such as the PD-1/PD-L1 pathway and CTLA-4 inhibitory signalling.</p><p dir="ltr"><i>Conclusions: </i>The presence of Gipie confers pseudonormal morphology to ACC cells, thereby reducing immune attack. Thus, trafficking proteins may affect the interactions between ACC cells and immune cells during conventional chemotherapy or monoclonal immunotherapy. The immunosuppressive proteins found in ACC may be responsible for lowering the anti-tumour reactivity of immune cells.</p>