The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1⁺ myofibroblasts with prognostic values and potential biological significance. CST1⁺ myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology.

Dysplasia and intramucosal esophageal squamous cell carcinoma (ESCC) frequently go unnoticed with white-light endoscopy and, therefore, progress to invasive tumors. If suitable targets are available, fluorescence molecular endoscopy might be promising to improve early detection. Microarray expression data of patient-derived normal esophagus (n = 120) and ESCC samples (n = 118) were analyzed by functional genomic mRNA (FGmRNA) profiling to predict target upregulation on protein levels. The predicted top 60 upregulated genes were prioritized based on literature and immunohistochemistry (IHC) validation to select the most promising targets for fluorescent imaging. By IHC, GLUT1 showed significantly higher expression in ESCC tissue (30 patients) compared to the normal esophagus adjacent to the tumor (27 patients) (p < 0.001). Ex vivo imaging of GLUT1 with the 2-DG 800CW tracer showed that the mean fluorescence intensity in ESCC (n = 17) and high-grade dysplasia (HGD, n = 13) is higher (p < 0.05) compared to that in low-grade dysplasia (LGD) (n = 7) and to the normal esophagus adjacent to the tumor (n = 5). The sensitivity and specificity of 2-DG 800CW to detect HGD and ESCC is 80% and 83%, respectively (ROC = 0.85). We identified and validated GLUT1 as a promising molecular imaging target and demonstrated that fluorescent imaging after topical application of 2-DG 800CW can differentiate HGD and ESCC from LGD and normal esophagus.

Gene fusions generated by chromosomal rearrangements function as oncogenic drivers in human cancers. We previously showed that EWSR1-ETS oncofusions of Ewing sarcoma (EwS) directly induce surface expression of IL1 receptor accessory protein (IL1RAP), which along with limited expression in healthy tissues except placenta nominate IL1RAP as a promising EwS immunotherapy target. We therefore engineered antibody-drug conjugates (ADCs) with different cytotoxic payloads to target IL1RAP. ADCs potently blocked tumor growth and induced durable regression of EwS xenografts in mice, and diminished metastatic dissemination in vivo. Moreover, we show that other oncofusions also induce IL1RAP expression in diverse cancers, including NPM-ALK in anaplastic large cell lymphoma (ALCL), and ETV6-NTRK3 in multiple tumor types. IL1RAP expression rendered these malignancies similarly vulnerable to IL1RAP-targeting ADCs, which effectively blocked growth of ALCL xenografts and syngeneic ETV6-NTRK3+ sarcomas. Lack of detectable normal tissue toxicity, including in non-human primates, support the further clinical translation of IL1RAP-targeting ADCs.

Supplementary Table S2. qPCR primers.

Supplementary Figure S9. Identification and characterization of AB030, a humanized IgG1 anti-IL1RAP binder for immunotherapy.