A Cystine-Cysteine Intercellular Shuttle Prevents Ferroptosis in xCTKO Pancreatic Ductal Adenocarcinoma Cells

Abstract

In our previous study, we showed that a cystine transporter (xCT) plays a pivotal role in ferroptosis of pancreatic ductal adenocarcinoma (PDAC) cells in vitro. However, in vivo xCTKO cells grew normally indicating that a mechanism exists to drastically suppress the ferroptotic phenotype. We hypothesized that plasma and neighboring cells within the tumor mass provide a source of cysteine to confer full ferroptosis resistance to xCTKO PDAC cells. To evaluate this hypothesis, we (co-) cultured xCTKO PDAC cells with different xCT-proficient cells or with their conditioned media. Our data unequivocally showed that the presence of a cysteine/cystine shuttle between neighboring cells is the mechanism that provides redox and nutrient balance, and thus ferroptotic resistance in xCTKO cells. Interestingly, although a glutathione shuttle between cells represents a good alternative hypothesis as a “rescue-mechanism”, our data clearly demonstrated that the xCTKO phenotype is suppressed even with conditioned media from cells lacking the glutathione biosynthesis enzyme. Furthermore, we demonstrated that prevention of lipid hydroperoxide accumulation in vivo is mediated by import of cysteine into xCTKO cells via several genetically and pharmacologically identified transporters (ASCT1, ASCT2, LAT1, SNATs). Collectively, these data highlight the importance of the tumor environment in the ferroptosis sensitivity of cancer cells.

Genetic Disruption of the γ-Glutamylcysteine Ligase in PDAC Cells Induces Ferroptosis-Independent Cell Death In Vitro without Affecting In Vivo Tumor Growth

“…This could be attributed to the lower oxidative pressure that cells experience in these conditions. However, another possibility that became apparent in our previous work with xCT-KO xenografts is cell-to-cell interplay which, in the case of xCT-KO cells, provides them with the source of reduced cysteine [28,38]. Although the degradation of GSH in vivo is just as intensive as in vitro [35,[39][40][41], the continuous flux of GSH from adjacent cells might be a sufficient source of GSH for GCLc-KO cells to survive and thrive in vivo.…”

“…Based on our previous experience with xCT-KO cell growth in vivo, a potential explanation for GCLc-KO tumor cell survival and proliferation in the same conditions could be attributed to GSH exchange between GCLc-KO and the neighboring WT cells in the tumor mass. To investigate this issue, we used co-culture (cc) of Capan-2 or MiaPaCa-2 GCLc-KO cells and LS174T WT cells (cell line chosen based on the results from our previous study [28]). LS174T cells were pre-seeded in the upper well of a Boyden chamber 24 h before GCLc-KO cells, and the viability of the latter was investigated 96 h later (Figure 6C).…”

System Xc−/GSH/GPX4 axis: An important antioxidant system for the ferroptosis in drug-resistant solid tumor therapy

“…However, in vivo SLC7A11 knock out PDAC cells grew normally. Their further study showed that the presence of a cysteine/cystine shuttle between neighboring cells is the mechanism that provides redox and nutrient balance, and thus ferroptotic resistance in SLC7A11 knock out PDAC cells ( Meira et al, 2021 ). Cysteine is required for preventing ferroptosis in pancreatic cancer (2020a), While the raw material for the synthesis of cysteine is mainly provided by System X c − .…”

Meeting report of the 4th biennial Metabolism and Cancer symposium

“…Dr Vucetic explained that xCT plays its fundamental role in cyst(e)ine import exclusively or almost exclusively in an oxidised form ( in vitro conditions), and that in vivo , cysteine‐reduced form can come from different sources. Interestingly, this work demonstrated that neighbouring fibroblasts secrete small ‘rescue agents’ for the xCT‐KO tumours allowing the complete prevention of ferroptosis [6].…”