Abstract 4651: Comparison of the tumor and lymph node immune microenvironment in early non-small cell lung cancer through multimodal single cell sequencing
Authors: Xi Zhanhao , Koga Yusuke , McDermott Shannon , Beane Jennifer , Mazzilli Sarah A. , Suzuki Kei , Campbell Joshua D. 
Published: April 2023

Abstract
Background/Purpose: Previous studies have analyzed the tumor and local immune microenvironments in lung cancers and suggest immune modulation is associated with worse clinical outcome. However, the tumor-immune microenvironment in early stage lung tumors and lymph nodes (LNs) have not been fully described. We aim to compare cell states in the immune microenvironments between lung tumors and LNs through multi-modal profiling of the transcriptome and surface proteins.
Methods: Needle biopsy samples were taken from 10 treatment-naive early stage lung cancer patients undergoing lung cancer resections. Tissues were obtained from normal lung, lung tumor, and multiple mediastinal LNs, and processed for scRNA-seq including labeling with Total-Seq C CITE-seq panel to quantify the levels of 130 cell surface proteins. In total, 76,721 cells (4,462 normal lung; 39,019 tumor; 33,240 LN) were identified with a median of 1,673 genes and 92 protein features detected per cell. Protein expression was decontaminated through the decontX algorithm. Weighted-Nearest Neighbor analysis from the Seurat R package was applied to integrate the CITE-seq and RNA-seq level data for clustering cells into subpopulations.
Results: Six broad cell populations were identified including T/NK, myeloid (CD14+), B (CD19+), mast (TPSAB1+), pDC (IRF8+), and epithelial (EPCAM+) cells. Among 8 CD4+ T lymphocyte subpopulations and 11 CD8+ T lymphocyte subpopulations observed through clustering, a naïve CD4+ and a CD8+ T subpopulation (LEF1+, TCF7+) was observed respectively. These naïve T lymphocyte populations displayed increased proportions in LNs in comparison to tumors. In addition, 5 of the 8 observed CD4+ T lymphocyte populations were enriched in LNs. Immune populations enriched in LNs were largely shared and uniform across different patients. In contrast, a single CD4+ and CD8+ T lymphocyte subpopulation displayed expression of T lymphocyte exhaustion markers (TIGIT+, LAG3+, PD-1+) and were enriched in tumors. 6 of the 11 observed CD8+ T lymphocyte populations were enriched in tumor samples in comparison to LNs. Two alveolar macrophage populations (MARCO+) were enriched in normal lung tissue, in which one showed a heightened stress response.
Conclusion: Single-cell profiling reveals diversity in immune cell populations between LNs, tumor, and adjacent normal tissue in early-stage LUAD. The results suggest the composition of immune cell type is fairly consistent across LNs but more heterogeneous in the tumor and adjacent normal tissue in early-stage patients. In the future, we aim to determine if these immune subpopulations are associated with survival, recurrence, aggressiveness, and predict responses for neoadjuvant treatments, which could improve prognosis and patient quality of life.
Citation Format: Zhanhao Xi, Yusuke Koga, Shannon McDermott, Jennifer Beane, Sarah A. Mazzilli, Kei Suzuki, Joshua D. Campbell. Comparison of the tumor and lymph node immune microenvironment in early non-small cell lung cancer through multimodal single cell sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4651.