Abstract 6577: ST6GAL1+ exosomes confer treatment resistance in rectal cancer

Abstract Rectal cancer (RC) is a highly morbid and fatal disease. Locally advanced RC is treated with neoadjuvant chemoradiation. Patients with a complete pathologic response have improved outcomes compared with those with incomplete response and may be able to avoid surgical resection. Among incomplete responders, we have found that there is increased tumor ST6GAL1 protein, which is a glycosyltransferase that adds alpha 2, 6-sialic acid to N-glycans. Through knockdown (KD) studies, we have shown that ST6GAL1 causes chemoradiation treatment resistance in RC. Exosomes are small particles that can be transferred between cells. We hypothesized that these exosomes transfer functional ST6GAL1 thereby spreading treatment resistance and can be found in the plasma of a mouse model of RC. In order to test these hypotheses, we cultured colorectal cancer SW620 cells and isolated exosomes by ultracentrifugation from the media. Exosome yield and size were determined using a NanoSight particle analyzer and further characterized using transmission electron microscopy (TEM). Exosomes from ST6GAL1 CV cells were used to treat ST6GAL1 KD. Western blot, immunofluorescence (IF) microscopy, and flow cytometry for alpha 2, 6-sialic acid by SNA lectin were utilized. Colony formation assay and cleaved caspase-3 IF staining were performed. Mice bearing a patient derived xenograft model of rectal cancer were treated with chemoradiation daily for 2 weeks (5 days on, 2 days off) and tumor and mouse plasma exosome samples were tested for ST6GAL1. We found that human colorectal cancer cells in culture produced exosomes containing ST6GAL1. These exosomes were 91.2 ± 6.8 nm in diameter and had an umbilicated appearance on TEM. When ST6GAL1 KD cells were treated with ST6GAL1+ exosomes, the protein was transferred to KD cells as measured by western blot. IF staining showed that transferred ST6GAL1 protein co-localized to the Golgi apparatus, where it is natively found. Additionally, exosome-treated cells became more resistant to chemoradiation-induced apoptosis and showed improved colony formation (CV 21.7 ± 4.6 colonies vs KD 12.7 ± 3.5 colonies, p = 0.03; KD plus exosomes 17.7 ± 3.0 colonies vs CV, p = 0.38). ST6GAL1 protein levels increased in tumors from a patient derived xenograft during treatment and peaked at 2 days. We further found that ST6GAL1 protein in exosomes isolated from the plasma of these mice peaked at the same time as in the tumor. In conclusion, rectal cancer exosomes carrying ST6GAL1 spread treatment resistance to previously sensitive cells and can be detected in the plasma of tumor bearing mice. Interfering in exosome-mediated sharing of ST6GAL1 is a promising and entirely novel potential therapeutic strategy for RC. These exosomes may also serve as a biomarker for identifying patients with treatment resistant rectal cancer. Citation Format: Jeremie M. Lever, Regina Irwin, Vivek Somasundaram, Matthew Stiles, Marshall C. McLeod, Karin Hardiman. ST6GAL1+ exosomes confer treatment resistance in rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6577.