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Lung Cancer Single-Cell RNA-Seq Study Sheds Light on Tumor Microenvironment

NEW YORK (GenomeWeb) – A team led by researchers from the VIB Center for Cancer Biology and the Catholic University of Leuven (KU Leuven) in Belgium has built a catalog of stromal cells in lung cancer and normal lung tissue at the single cell level, providing new insights into lung cancer biology that might be useful for diagnostics and therapy.

In a study published in Nature Medicine today, the researchers reported sequencing the transcriptome of almost 100,000 single cells, about 84,000 of them stromal cells, and identifying 52 stromal subtypes. Of note, they found that certain cellular subtypes correlated with patient survival, pointing to new opportunities for using subtype-specific marker genes as biomarkers for prognosis and therapy response. In addition, certain tumor-surrounding stromal cells could present targets for novel therapies.

Stromal cells surrounding cancer cells — such as macrophages, T cells, and fibroblasts — form the tumor microenvironment, a complex cellular ecosystem. However, the heterogeneity of these stromal cells, and how they might affect the tumor or be affected by it, have not been characterized much, yet.

For their study, the researchers — led by corresponding authors Diether Lambrechts and Bernard Thienpont — performed single-cell RNA-seq on cells from five patients with untreated non-metastatic non-small cell lung cancer. This included three tumor tissue samples for each patient, and one additional non-malignant lung tissue sample for four of the patients. Overall, the team sequenced the transcriptomes of almost 53,000 cells, including about 39,500 cells originating from tumors and 13,500 from normal lung tissue.

Following principal component analysis on about 2,200 genes expressed variably across the cells, they classified the cells into groups of cell types, identifying known cell lineages that included cancer cells, immune cells, fibroblast, endothelial cells, alveolar cells, and epithelial cells.

In parallel, they also performed bulk RNA-seq of a tumor and non-malignant lung sample of another patient and found that the results correlated well with the single-cell RNA-seq data.

Further analysis of the genes expressed by the different cell types revealed a complex cellular ecosystem of 52 stromal cell subclusters and 12 cancer cell subclusters. While the cancer cell subclusters tended to be patient specific, the same stromal cell subclusters occurred across several patients. Comparing results from tumors and matching non-malignant lung tissue, the researchers found that many stromal cell subclusters were enriched for either tumor-derived or lung tissue-derived cells.

To validate the results, they performed RNA-seq on an additional 40,250 single cells from three more NSCLC patients and found 45 of the 52 stromal cell subclusters in those patients. They also assessed marker genes from the cell subtypes in bulk RNA-seq data from 1,572 patients and found that they correlate with survival.

The results revealed novel cell subtypes, marker genes, and altered pathways, and the dataset "will fuel advances in lung cancer diagnosis and therapy," the authors wrote. "It can serve to validate a priori hypotheses from independent data, but also highlight novel targets meriting functional validation."

Specifically, they wrote, the tumor microenvironment appears to be more complex and heterogeneous than previously assumed. Also, some cell subtypes seemed to be influenced by the tumor and its stage, and correlated with patient survival for certain tumor types.

"Hence, in providing a comprehensive catalog of stromal cell types and by characterizing their phenotype and co-optive behavior, this resource provides deeper insights into lung cancer biology that will be helpful in advancing lung cancer diagnosis and therapy," they noted.