背景:非肌肉浸润性膀胱癌(NMIBC)采用经尿道膀胱肿瘤切除术(TURBT),然后膀胱内滴注化疗或卡介苗治疗。然而,这些治疗有很高的复发率和副作用,强调需要替代灌输。以前,我们发现,从外周血扩增的同种异体人类自然杀伤(NK)细胞是前列腺癌的一种有前途的细胞疗法。然而,NK细胞在膀胱癌(BCa)中是否表现出类似的杀伤作用尚不清楚。
方法:扩展,激活,和冷冻保存从外周血中获得的同种异体人NK细胞如我们先前所述。使用细胞计数试剂盒-8评估体外细胞毒性。穿孔素的水平,颗粒酶B,干扰素-γ,肿瘤坏死因子-α,和趋化因子(C-C-基序配体[CCL]1,CCL2,CCL20,CCL3L1和CCL4;C-X-C-基序配体[CXCL]1,CXCL16,CXCL2,CXCL3和CXCL8;以及X-基序配体1和2)使用酶联免疫吸附测定。CD107a的表达,主要组织相容性复合体I类(MHC-I),MHC-I多肽相关序列A和B(MICA/B),巨细胞病毒UL16结合蛋白-2/5/6(ULBP-2/5/6),B7-H6,CD56,CD69,CD25,杀伤细胞Ig样受体(KIR)2DL1,KIRD3DL1,NKG2D,流式细胞术检测NK细胞或BCa和正常尿路上皮细胞的NKp30、NKp46和CD16。在患者来源的类器官模型中使用乳酸脱氢酶测定法评估细胞毒性。在皮下注射5637和NK细胞的雄性NOD-scidIL2rg-/-小鼠中使用卡尺在体内监测BCa生长。使用RNA序列分析研究差异基因表达。使用transwell迁移测定法评估T细胞的趋化性。
结果:我们发现在体外,NK细胞对BCa系具有更高的细胞毒性,产生更多的细胞因子,通过脱颗粒标记CD107a的上调和干扰素-γ分泌增加来证明,由MICA/B/NKG2D和B7H6/NKp30介导的活化。此外,NK细胞在患者来源的类器官和BCa异种移植小鼠模型中表现出对BCa的抗肿瘤作用。NK细胞分泌趋化因子,包括CCL1/2/20,以在遇到BCa细胞时诱导T细胞趋化性。
结论:扩增的NK细胞对BCa细胞表现出有效的细胞毒性,对正常尿路上皮细胞几乎没有毒副作用。此外,NK细胞通过分泌一组趋化因子来招募T细胞,它支持在TURBT从工作台到床边进行NMIBC治疗后NK细胞膀胱内滴注的翻译应用。
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.