High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor

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BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) is treated with transurethral resection of bladder tumor (TURBT) followed by intravesical instillation of chemotherapy or Bacillus Calmette-Guérin therapy. However, these treatments have a high recurrence rate and side effects, emphasizing the need for alternative instillations. Previously, we revealed that expanded allogeneic human natural killer (NK) cells from peripheral blood are a promising cellular therapy for prostate cancer. However, whether NK cells exhibit a similar killing effect in bladder cancer (BCa) remains unknown. METHODS: Expansion, activation, and cryopreservation of allogeneic human NK cells obtained from peripheral blood were performed as we previously described. In vitro cytotoxicity was evaluated using the cell counting kit-8. The levels of perforin, granzyme B, interferon-γ, tumor necrosis factor-α, and chemokines (C-C-motif ligand [CCL]1, CCL2, CCL20, CCL3L1, and CCL4; C-X-C-motif ligand [CXCL]1, CXCL16, CXCL2, CXCL3, and CXCL8; and X-motif ligand 1 and 2) were determined using enzyme-linked immunosorbent assay. The expression of CD107a, major histocompatibility complex class I (MHC-I), MHC-I polypeptide-related sequences A and B (MICA/B), cytomegalovirus UL16-binding protein-2/5/6 (ULBP-2/5/6), B7-H6, CD56, CD69, CD25, killer cell Ig-like receptors (KIR)2DL1, KIRD3DL1, NKG2D, NKp30, NKp46, and CD16 of NK cells or BCa and normal urothelial cells were detected using flow cytometry. Cytotoxicity was evaluated using lactate dehydrogenase assay in patient-derived organoid models. BCa growth was monitored in vivo using calipers in male NOD-scid IL2rg-/- mice subcutaneously injected with 5637 and NK cells. Differential gene expressions were investigated using RNA sequence analysis. The chemotaxis of T cells was evaluated using transwell migration assays. RESULTS: We revealed that the NK cells possess higher cytotoxicity against BCa lines with more production of cytokines than normal urothelial cells counterparts in vitro, demonstrated by upregulation of degranulation marker CD107a and increased interferon-γ secretion, by MICA/B/NKG2D and B7H6/NKp30-mediated activation. Furthermore, NK cells demonstrated antitumor effects against BCa in patient-derived organoids and BCa xenograft mouse models. NK cells secreted chemokines, including CCL1/2/20, to induce T-cell chemotaxis when encountering BCa cells. CONCLUSIONS: The expanded NK cells exhibit potent cytotoxicity against BCa cells, with few toxic side effects on normal urothelial cells. In addition, NK cells recruit T cells by secreting a panel of chemokines, which supports the translational application of NK cell intravesical instillation after TURBT from bench to bedside for NMIBC treatment.

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