Tryptophan metabolism drives dynamic immunosuppressive myeloid states in IDH-mutant gliomas [CEL-Seq2]

Intratumoral microglia and MF constitute up to 70% of the tumor mass of high-grade gliomas (HGG) with profound impact on hallmarks of malignancy such as angiogenesis and immunosuppression. The dynamics and functional states of intratumoral myeloid cells during tumor progression and the molecular mechanisms controlling them are poorly understood. Here we define homeostatic and antigen-presenting myeloid cellular states in experimental and human HGG by longitudinal single-cell RNA-sequencing and combined transcriptome and proteome profiling, respectively. During glioma progression, myeloid cells gradually shift from a homeostatic to a tumor-associated effector state. We show that these dynamics are under strict control by early changes in resident microglia and the tumor genotype: In gliomas with mutations in isocitrate dehydrogenase (IDH), a disease-defining driver mutation, differentiation of invaded myeloid cells was blocked resulting in an immature, immunosuppressive phenotype. In late-stage IDH-mutant gliomas, monocyte-derived MF drive a tolerogenic remodeling of the glioma microenvironment thus preventing T-cell response. We define the molecular mechanism responsible for blocking functional differentiation in IDH-mutant gliomas to be causally related to enhanced metabolization of tryptophan to kynurenine, an endogenous ligand of the aryl hydrocarbon receptor (AHR), leading to a time-dependent uptake of extracellular tryptophan via LAT1-CD98 by intratumoral myeloid cells. Consequently, the immunosuppressive phenotype in IDH-mutant glioma models was reversed by pharmacologic inhibition of LAT1-CD98 or AHR. Thus, we provide evidence for a tumor genotype-dependent dynamic network of resident and recruited intratumoral myeloid cells shaping the immune microenvironment of IDH-mutant HGG and identify tryptophan metabolism as a viable therapeutic target for the immunotherapy of IDH-mutant tumors. Overall design: CD45+ Lin- cells were FACS sorted from cell suspensions of control patients or cases wirh IDHwt or IDHmut glioblastoma multiforme. CEL-Seq2 protocol was used for single cell sequencing (Hashimshony et al. 2016).

瘤内小胶质细胞与巨噬细胞（MF）在高级别胶质瘤（HGG）的肿瘤组织中占比最高可达70%，并对血管生成、免疫抑制等恶性肿瘤标志产生深远影响。目前，肿瘤进展过程中瘤内髓系细胞的动态变化与功能状态，以及调控这些过程的分子机制仍尚不明确。
本研究分别通过纵向单细胞RNA测序与联合转录组-蛋白质组分析，在实验模型与人类HGG样本中界定了稳态型与抗原呈递型髓系细胞亚群。研究发现，胶质瘤进展过程中，髓系细胞会逐渐从稳态状态转向肿瘤相关效应状态。
进一步研究表明，上述动态变化严格受驻留小胶质细胞的早期改变与肿瘤基因型调控：在携带疾病标志性驱动突变的异柠檬酸脱氢酶（IDH）突变型胶质瘤中，浸润性髓系细胞的分化过程被阻断，最终形成未成熟的免疫抑制表型。在晚期IDH突变型胶质瘤中，单核细胞来源的巨噬细胞会驱动胶质瘤微环境发生耐受型重塑，进而抑制T细胞应答。
本研究明确，IDH突变型胶质瘤中阻断髓系细胞功能分化的分子机制，与色氨酸向犬尿氨酸的代谢增强存在因果关联——犬尿氨酸是芳香烃受体（AHR）的内源性配体，可介导瘤内髓系细胞通过LAT1-CD98呈现时间依赖性的细胞外色氨酸摄取。实验证实，通过药物抑制LAT1-CD98或AHR，可逆转IDH突变型胶质瘤模型中的免疫抑制表型。
综上，本研究证实了驻留与招募的瘤内髓系细胞所构成的、依赖于肿瘤基因型的动态网络，可塑造IDH突变型HGG的免疫微环境，并明确色氨酸代谢可作为IDH突变型肿瘤免疫治疗的潜在治疗靶点。
实验整体设计：从对照患者或IDH野生型（IDHwt）、IDH突变型（IDHmut）多形性胶质母细胞瘤患者的细胞悬液中，通过荧光激活细胞分选术（FACS）分选出CD45阳性、谱系阴性（CD45+ Lin-）细胞；采用CEL-Seq2方案完成单细胞测序（Hashimshony等人，2016）。