Abstract 160: Identification of the carcinoma and immune cells in the breast cancer by single-cell RNA sequencing

Abstract

Abstract Summary: Breast cancer is a heterogeneous type of cancer accompanying extensive immune cell infiltration. Although its mortality rate has been decreasing with early diagnosis and introduction of molecular targeted therapies, it is still one of the world leading cause of cancer death in women. In breast cancer, molecular subtyping is used for the patient stratification and treatment decisions, emphasizing the importance of inter-patient tumoral heterogeneity in the tumor cell behavior. Tumor cells within a patient may also manifest intra-tumoral heterogeneity, which may cause treatment resistance. Single cell gene expression profiling would reveal both inter- and intra-tumoral heterogeneity in breast cancer encompassing tumor cells and associated stromal and immune cells. Experimental Process: From 4 different subtype primary breast cancer and 2 lymph node metastases, single cells were captured and amplified through C1™Single-cell Auto Prep System (Fluidigm) with the SMARTer Ultra Low RNA Kit. Sequencing libraries were constructed with the Nextera XT DNA sample Prep Kit and sequenced through a Hiseq2500 (Illumina) as 100-bp paired end mode of the TruSeq Rapid PE Cluster kit and Truseq Rapid SBS kit. Computational Process: RNA sequencing reads were aligned to the modified human genome reference using 2-pass mode of STAR_2.4.0b, and transcript per million (TPM) values were obtained as the relative gene expression levels of single cells through RSEM v1.2.17. Results: As most breast cancers originate from the mammary epithelium, we distinguished carcinoma cells from associated non-tumor cells by epithelial cell adhesion molecule (EPCAM) signature genes. We validated the distinction with chromosomal gene expression patterns and gene set enrichment analyses using tumor, stromal, or immune genesets. With or without removal of non-tumor cells, TNBC tumor cells showed the highest gene expression heterogeneity, recapitulating the known inter-patient heterogeneity. TNBC tumor cells from a single patient were categorized into 6 different TNBC-subtypes, further demonstrating intra-tumoral heterogeneity. In addition, immune cells identified in the TNBC tumor were naïve B cells and regulatory T cells, suggesting non-activated status of the immune system. Conclusions: The high resolution gene expression profiling revealed features of breast cancer transcriptome which may provide clues for molecular directed therapies targeting the tumor or the immune compartment. Citation Format: Woosung Chung, Hye Hyeon Eum, Kyu-Tae Kim, Kyung-Min Lee, Arum Jo, WonShik Han, Hae-Ock Lee, Woong-Yang Park. Identification of the carcinoma and immune cells in the breast cancer by single-cell RNA sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 160.