Abstract P5-06-10: Tumor microenvironment modulates ganglioside expression leading to immunosuppression in triple negative breast cancer
Authors: Anand Vivek , El-Dana Fouad , Ly Stanley , Andreeff Michael , Battula Venkata Lokesh 
Published: March 2022

Abstract
Triple-negative breast cancer (TNBC) is one of the most aggressive human malignancies, characterized by a strikingly poor prognosis linked to high rates of distant metastases and disease relapse. Among several mechanisms that contribute to TNBC aggressiveness is the ability of TNBC cells to evade the anti-tumor immune response. Numerous efforts have been directed to identify and target molecules and signaling pathways that contribute to TNBC immune evasion. A considerable proportion of TNBCs are resistant to immune-checkpoint therapy, implying that additional molecules are involved in immune suppression in these malignancies. We have previously reported that ganglioside GD2 identifies breast cancer stem-like cells (BCSCs) and promotes tumorigenesis. GD3 synthase (GD3S), a key enzyme that regulates GD2 biosynthesis, is significantly upregulated in TNBCs. Gangliosides have been found to promote an immunosuppressive tumor microenvironment. In this study, we hypothesize that tumor-microenvironment influences expression of gangliosides downstream of GD3S, including GD2/GD3, suppressing the anti-tumor immune response while inhibiting GD3S expression enhances immune-mediated killing of TNBC cells. We performed immunohistochemistry (IHC) on primary TNBC patient samples (N=89) and measured GD2 expression using an automated imaging system (Vectra-Polaris). Immunohistochemistry analysis revealed that approximately 60% of samples had detectable GD2 expression of different intensity and was associated with poor overall survival of patients with TNBC (p=0.002). Our IHC data also demonstrated that GD2 is expressed not only in tumors but also in the tumor-associated stroma in ~60% of TNBC samples, suggesting the effect of crosstalk between TME and TNBC cells on GD2 expression. Additionally, to substantiate the role of TME on GD2 expression, we cultured bone marrow-derived mesenchymal stromal cell (BM-MSCs) with MDA-MB-231 cells and found a 2-fold increase in GD2 expression in BM-MSC and MDA-MB-231 cells (p&lt;0.001), suggesting microenvironmental factors contribute to GD2 expression on both tumor and stromal cells. To examine the immunomodulatory role of gangliosides we used the TCGA dataset to analyze the co-expression of GD3S mRNA with T-cell exhaustion markers in all breast cancer patients. We found a significant (p&lt;0.001) positive correlation between ST8SIA1 and CTLA-4 (pearson= 0.4), LAG-3 (pearson= 0.33), and PD1 (pearson=0.3) in breast cancer patients. Furthermore, we overexpressed GD3S in TNBC and non-TNBC cells and observed its effect on T-cell mediated killing of breast cancer (BC) cells by a live-cell imaging system (IncuCyte). Overexpression of GD3S caused upregulation of GD2 in BT549, HIM3 (TNBC), and MCF7 (non-TNBC) cells, and their co-culture with T-cells resulted in a significant reduction (p&lt;0.001) in cell death compared to the controls. In conclusion, GD2 is a tumor-specific marker in patients with TNBC and that GD3S overexpression is associated with T-cell exhaustion and maintenance of an immunosuppressive microenvironment in breast cancer. This immunosuppressive microenvironment in TNBCs fostered by gangliosides downstream of GD3S is also a manifestation of the cross-talk between tumor and tumor-associated stromal cells.
Citation Format: Vivek Anand, Fouad El-Dana, Stanley Ly, Michael Andreeff, Venkata Lokesh Battula. Tumor microenvironment modulates ganglioside expression leading to immunosuppression in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-10.