TIM-3 as a prospective target to eliminate pancreatic cancer stem cells

Background : Pancreatic cancer (PDAC) is nonresponsive to conventional therapies and poor survival is the norm. A small percentage of malignant cells undergo epithelial-mesenchymal transition to evolve into pancreatic cancer cells stem cells (PCSCs) that evade therapy and subsequently give rise to residual/recurrent tumors and also drive therapy resistance. While radiotherapy (RT), is a cornerstone of PDAC management, it promotes EMT via activation of TGF-beta, NF-kB and Akt/GSK-3beta pathways. TIM-3 (HAVCR-2) a cell surface marker traditionally designates exhausted T-cells. However TIM-3 overexpression is being increasingly reported in solid tumors. We report RT induced TIM-3 overexpression in PDAC and propose that RT triggered TIM-3, on pancreatic cancer cells could serve as a novel target in selectively eliminating PCSCs. Methods : GEO datasets were analyzed with the Seurat package in R to evaluate TIM-3 expression across tissue types (benign pancreatic tissue, non metastatic primary PDAC/Pm0, primary of metastatic PDAC/Pm1 and matched liver metastases/Lm). TIM-3 expression between treatment naive and chemo-RT treated tumors was also compared. In vitro, murine (Panc02-ova, KPC) and human (PANC-1, MIA PaCa-2) pancreatic cancer cells were treated with increasing doses of RT alone or in combination with NRF-2 / ATM inhibitor to either simulate a high oxidative stress environment or inhibit DNA repair pathways. Following treatments, TIM-3 expression in these samples were evaluated using RT-qPCR, western blotting, flow cytometry and immunofluorescence. In vivo, Panc-02 xenografts in C57BL/6 mice were irradiated (10Gy RT at 320Kv & 13.2 mA). Tumors were collected, 24 hrs post RT, and dissociated. Single cells thus obtained were assessed for the expression of TIM-3, using flow cytometry. Results : Advanced stages of cancer (Pm1 and Lm tumors) demonstrated greater TIM-3 expression compared to Pm0 tumors. Further, increased TIM-3 expression was noted in chemo-RT treated tumors compared to untreated tumors. In vitro, RT dose dependent increase in TIM-3 mRNA and protein levels was recorded and cells subjected to 10Gy RT showcase maximal TIM-3 expression (Control < 3Gy < 6Gy < 10Gy). Addition of NRF2 inhibitor or ATM inhibitor before exposing cells to RT further increased TIM-3 levels.The TIM-3+ cells exhibited higher CD24+ and CD44+ positivity, indicating its association with PCSC phenotype. Conclusion : Our results indicate that TIM-3 can be employed as a specific marker to designate PCSCs in addition to the classical stem cell phenotype (ESA+ CD24+ CD44+). Increased mRNA, total protein and TIM-3 levels post RT are possibly driven by elevated oxidative stress in addition to RT induced ds-DNA breaks. The novel role of TIM-3 as a PCSC marker, establishes it as an ideal candidate for targeting both immune-suppressive T cells and therapy resistant PCSCs.