Chemoradiation treatment increases extracellular vesicle release causing treatment resistance in rectal cancer cell lines

Introduction: Colorectal cancer (CRC) is the 3 rd most prevalent form of cancer in the U.S., with younger Americans comprising an increasing fraction of case numbers. Current treatment includes chemoradiation treatment (CRT) followed by surgical resection in incomplete responders. Only about 25% of patients have a complete response to CRT due to the presence of treatment resistance in most patients. ST6GAL1 is a sialyltransferase, that has been shown to be upregulated in various cancer types, including CRC. Our group has previously shown that it confers treatment resistance to CRT in vitro and in vivo . We have also shown that ST6GAL1 is packaged into extracellular vesicles (ECVs) by human CRC cel lines, and that these ECVs can transfer treatment resistance between cells. We hypothesized that CRT would increase ECV release, thereby, spreading treatment resistance and that these ECVs could be measured in the plasma of a patient derived xenograft model of rectal cancer. Methods: We cultured SW620 CRC cells, treated with or without 5-Flourouracil and 5 Gy of radiation (CRT), and isolated ECVs from the cells' conditioned medium using differential centrifugation. ECV size and concentration were determined using a Nanosight with NTA software. Our previously validated SW620 ST6GAL1 Control vector (CV) and knockdown (KD) cell line was used to assess transfer of resistance to apoptosis after CRT. We added ECVs isolated from untreated or CRT treated SW620 cells to ST6GAL1 KD cells, treated with 5 Gy of CRT, and performed immunofluorescence for cleaved caspase-3 (CC3). We also co-cultured KD cells with CV cells (+/- CRT) and assessed CC33 staining after CRT. We assessed tumor and plasma ECV levels of ST6GAL1 before, during, and after CRT in a PDX model of rectal cancer. Results: SW620 cells undergoing CRT had a greater than twofold higher release of ECVs/cell compared to untreated cells ( 2.57 ± 0.84 p=0.033, n=4) . No significant differences were detected in mean ECV size between CRT(+) and CRT(-) emitting cells. ST6GAL KD cells directly treated with SW620 ECVs cells from either CRT treated or untreated donor cells showed a significantly reduced CC3 production after CRT compared to untreated KD cells. Co-culture of KD cells with radiated CV cells reduced CC3 levels after CRT (1.59± 0.32, n=2). ST6GAL1 could be measured in ECVs isolated from the plasma of the PDX model and the number of ECVs and amount of ST6GAL1 both increased during CRT. Conclusion: We have shown that exogenously added ECVs containing ST6GAL1 confer treatment resistance in vitro. Our co-culture data additionally validates this finding by showing that ST6GAL1 containing cells can transfer ECVs to neighboring cells and confer treatment resistance. Our data reveals that ECV release is increased in response to CRT in vitro and in vivo . If seen in patients, this increase could be spreading resistance, potentially worsening treatment outcomes.