Expression data from Cysltr1 deficient bone marrow in vitro induced myeloid cells

In established cancer in mice and humans, “emergency” myelopoiesis usually occurs to facilitate malignancy progression. In particular, neutrophils and immature myeloid cells accumulated in the bone marrow, periphery and tumor are potent mediators of immunosuppression and tumor promotion. However, the molecular pathways regulating tumor-induced emergency myelopoiesis remain largely elusive. Here, we demonstrated that Cysteinyl leukotriene receptor 1 (CysLTR1), a key player for asthma, allergic rhinitis and other inflammatory conditons, was expressed selectively in myeloid cells from tumor-bearing hosts with the most significant expression in infiltrating immature neutrophils. Genetic ablation and pharmacological inhibition of CysLTR1 resulted in diminished tumor growth with enhanced antitumor immunity. The anti-tumor effect of CysLTR1 inhibition was linked with transcriptomic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor immune phenotype; this process required the distinct transcriptional factors to specifically dictate neutrophil progenitor commitment and differentiation in association with controlled degranulation. Furthermore, single-cell RNA sequencing analysis provided a comprehensive transcriptional landscape of neutrophil maturation, function and fate decision in tumor bearers, confirming a CysLTR1-dependent transcriptomic mechanism for fine-tuned regulation of tumor-induced emergency granulopoiesis. CysLTR1-mediated changes in the differentiation and degranulation also occurred in human neutrophils and correlated with the survival and immune checkpoint therapy responsiveness in cancer patients. Consistently, targeting CysLTR1 with clinically available antagonists overcame resistance to immune checkpoint PD-1 inhibitors in several mouse tumor models. These findings thus have important implications for understanding the abnormal myelopoiesis during cancer progression and suggest new therapeutic approaches to improve immune checkpoint blockade. Overall design: Bone marrow cells were isolated from BALB/c WT mice and Cysltr1 deficient mice, and cultured in complete RPMI1640 media with GM-CSF (40ng/mL), GM-CSF (40ng/mL) + IL-6 (40ng/mL), or GM-CSF (40ng/mL) + 24h EMT6 conditional media (50% v/v) in vitro to induce myeloid cells. Cells were then gathered for bulk RNA sequence after 4 days. A triplicate of each sample was considered, and each sample was derived from a pool of three mice.

在小鼠与人类的实体瘤中，通常会发生“紧急”髓系造血（emergency myelopoiesis）以促进恶性肿瘤进展。具体而言，中性粒细胞与未成熟髓系细胞在骨髓、外周血及肿瘤组织中蓄积，它们是介导免疫抑制与肿瘤促发的强效因子。然而，调控肿瘤诱导的紧急髓系造血的分子通路在很大程度上仍未阐明。

本研究证实，半胱氨酰白三烯受体1（Cysteinyl leukotriene receptor 1, CysLTR1）——作为哮喘、变应性鼻炎及其他炎症性疾病的关键调控分子——在荷瘤宿主的髓系细胞中选择性表达，其中以浸润性未成熟中性粒细胞的表达水平最为显著。

对CysLTR1进行基因敲除与药理学抑制，可减缓肿瘤生长并增强抗肿瘤免疫。CysLTR1抑制的抗肿瘤效应与粒细胞生成的转录重编程、以及中性粒细胞重编程为抗肿瘤免疫表型密切相关；这一过程需要特定转录因子特异性调控中性粒细胞祖细胞的定向分化，并与受控的脱颗粒过程相关联。

此外，单细胞RNA测序（single-cell RNA sequencing）分析全面描绘了荷瘤宿主中性粒细胞成熟、功能及命运决定的转录图谱，证实了CysLTR1依赖的转录机制可精准调控肿瘤诱导的紧急粒细胞生成。

CysLTR1介导的分化与脱颗粒变化同样存在于人类中性粒细胞中，且与癌症患者的生存预后及免疫检查点治疗响应性相关。一致的是，使用临床可用的拮抗剂靶向CysLTR1，可在多种小鼠肿瘤模型中克服对PD-1免疫检查点抑制剂的耐药性。

因此，本研究结果对于理解肿瘤进展过程中的异常髓系造血具有重要意义，并为优化免疫检查点阻断治疗提供了全新的策略。

【整体实验设计】从BALB/c野生型（WT）小鼠及Cysltr1基因敲除小鼠中分离骨髓细胞，于体外在添加粒细胞-巨噬细胞集落刺激因子（GM-CSF, 40ng/mL）、GM-CSF（40ng/mL）+白细胞介素6（IL-6, 40ng/mL），或GM-CSF（40ng/mL）+24小时EMT6条件培养基（体积占比50%）的完全RPMI1640培养基中培养，以诱导髓系细胞生成。培养4天后收集细胞，进行批量RNA测序。每个样本设置3次生物学重复，且每个样本均来源于3只小鼠的混合细胞。