964 Single-cell spatial characterization of microenvironmental impacts on immunotherapy discrepancies between adult and pediatric nasopharyngeal carcinoma patients

Background

Nasopharyngeal carcinoma (NPC) is an EBV-related and highly inflamed malignancy of strategic importance in Asia and Africa. Only 0.1–1% NPC incidences are diagnosed in children, and pediatric NPC exhibits superior prognosis and immunotherapy outcomes. Microenvironmental characteristics might contribute to such discrepancies between adult and pediatric NPC. Thus, we apply scRNA-seq and Visium spatial RNA-seq to primary pediatric NPC with paired blood, and incorporate our data with multi-central NPC cohorts. We develop a computational framework to infer spatial cellular constitutes and signaling. We report low lipid metabolism, weak interaction between NPC cells and Tregs/CD8+ T cells, and enriched T memory stem cells (Tscm) collectively result in stronger and long-lasting immunity in pediatric NPC and as new immunotherapeutic vulnerabilities (figure 1).

Methods

We applied scRNA-seq to 5 primary pediatric NPC tissues with paired blood samples, and spatial-seq to 4 paired pediatric and 7 adult NPC tissues. We established a multi-central NPC cohort with 69 patient samples by integrating our scRNA-seq data with three adult NPC cohorts.^1–3 A personalized pipeline incorporated with Seurat,⁴ Spacexr⁵ and SpaCET⁶ was used to analyze single-cell spatial data. Key findings were validated by multiplex staining, flow cytometry, and blood tests on patient samples.

Results

We established a systematic NPC scRNA-seq and spatial-seq cohort containing 503,021 cells from 69 samples, and 15,222 spatial spots from 11 samples. We developed a personalized computational framework to characterize spatial co-localization and regionally enriched signaling. We unveiled that fatty acid (FA)/cholesterol metabolism were elevated in the tumor-T cell core in adult NPC (p=2.12e-5), and such metabolic aberrance was validated by Oil Red O staining and blood cholesterol/FA tests (p=0.012). Strong co-localizations between NPC cells and Tregs (r=0.47, p<2.2e-22)/exhausted T cells (r=0.29, p<2.2e-22) were found in adult NPC, with spatially enriched CD70-CD27, LGALS9-TIM3 and Nectin-4 interactions. This finding was corroborated by multiplex staining of KRT19+ NPC cells, CD4+/FOXP3+ Tregs, and CD8+/PD-1/TIM-3+ exhausted T cells. We identified novel IL7R+/ANXA1+ Tscm populations in pediatric NPC, with strong resilience to immunosuppressive cues, including TGF-β, PEG2 and adenosine, and had a positive impact on long-term immunity and immunotherapeutic outcomes (p=7.49e-9).

Conclusions

We demonstrate that higher immunosuppression and exhaustion caused by lipid metabolism and tumor-intrinsic interactions, and a lower age-associated Tscm pool, are the vital drivers of immunotherapeutic discrepancies in adult and pediatric NPC patients. Pre-treatment screening of Tscm abundance in NPC patients might help stratify immunotherapy responders, and targeting metabolic and interacting vulnerabilities might generate therapeutic benefits.

References

Chen YP, Yin JH, Li WF, Li HJ, Chen DP, Zhang CJ, Lv JW, Wang YQ, Li XM, Li JY, et al. Single-cell transcriptomics reveals regulators underlying immune cell diversity and immune subtypes associated with prognosis in nasopharyngeal carcinoma. Cell Res. 2020;30(11):1024–1042. Gong L, Kwong DL, Dai W, Wu P, Li S, Yan Q, Zhang Y, Zhang B, Fang X, Liu L, et al. Comprehensive single-cell sequencing reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of nasopharyngeal carcinoma. Nat Commun. 2021;12(1):1540. Liu Y, He S, Wang XL, Peng W, Chen QY, Chi DM, Chen JR, Han BW, Lin GW, Li YQ, et al. Tumour heterogeneity and intercellular networks of nasopharyngeal carcinoma at single cell resolution. Nat Commun. 2021;12(1):741. Hao Y, Hao S, Andersen-Nissen E, Mauck WM, 3rd, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, et al. Integrated analysis of multimodal single-cell data. Cell. 2021;184(13):3573–3587 e3529. Cable DM, Murray E, Zou LS, Goeva A, Macosko EZ, Chen F, Irizarry RA. Robust decomposition of cell type mixtures in spatial transcriptomics. Nat Biotechnol. 2022;40(4):517–526. Ru B, Huang J, Zhang Y, Aldape K, Jiang P. Estimation of cell lineages in tumors from spatial transcriptomics data. Nat Commun. 2023;14(1):568.

Ethics Approval

The study was approved by the ethics committee at the University of Hong Kong. We complied with all related ethical regulations. Written informed consent was obtained from adult and pediatric NPC patients for their tissues and blood samples to be used in the study.

Consent

Written informed consent was obtained from adult and pediatric NPC patients for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.