Single-cell analysis predicts that mesenchymal ovarian cancer cells promote CD8+ T cell exhaustion through the LGALS3 - LAG3 axis

Cancer cells often metastasize by undergoing an epithelial-mesenchymal transition (EMT). Although abundance of CD8+ T-cells in the tumor microenvironment correlates with improved survival, mesenchymal cancer cells acquire greater resistance to antitumor immunity in some cancers. We hypothesized the EMT modulates the immune response to ovarian cancer. Here we show that cancer cells from infiltrated/inflamed tumors possess more mesenchymal cells, than excluded and desert tumors. We also noted high expression of LGALS3 is associated with EMT in vivo, a finding validated with in vitro EMT models. Dissecting the cellular communications among populations in the tumor revealed that mesenchymal cancer cells in infiltrated tumors communicate through LGALS3 to LAG3 receptor expressed by CD8+ T cells. We found CD8+ T cells express high levels of LAG3, a marker of T cell exhaustion. The results indicate that EMT in ovarian cancer cells promotes interactions between cancer cells and T cells through the LGALS3 - LAG3 axis, which could increase T cell exhaustion in infiltrated tumors, dampening antitumor immunity. Overall design: OVCA420 cells (10,000/well) were plated into in 6-well plates. Cells were treated with 10ng/mL TGF-ß1 (R&D Systems, #240-B-010), with treatment timed in such a way that all time-points were synchronized at the time of collection. Cells were passaged as needed to avoid confluence, and fresh TGF-ß1 was added every two days with refreshed media. Cells were not passaged in the 2 days prior to final collection to avoid artifacts during sequencing. Single-cell suspensions were processed using the 10x Genomics Single Cell 3' RNA-seq kit v3. Final libraries were sequenced on an Illumina sequencer after gene expression libraries were prepared according to the manufacturer's protocol.