Characterization Of Colorectal Liver Metastasis At Single-Cell Resolution Reveals Dynamic Interplay In The Tumor Microenvironment

Colorectal cancer (CRC) metastasizes to the liver in over half of patients. Compared to primary tumors, the biology of CRC liver metastasis (CLM) is poorly characterized. We performed high-throughput microfluidics-based single-cell RNA sequencing (scRNA-Seq) to define the epithelial and stromal components of CLMs. Using the 10X Genomics platform, scRNA-Seq was performed on single-cell suspensions generated from dissociation of fresh surgical excisions. We detected 1,551 cells from 2 CLMs and 5,407 cells from paired normal liver. Cells were sequenced at an average depth of 1 million reads/cell using Illumina sequencing. Graph-based clustering and differential expression analysis was performed using the Seurat algorithm. Tumor epithelial cells (EPCAM+, CDH1+, TFF3+) consisted of heterogeneous subpopulations with diverse transcriptomes and activation of varying signaling pathways. The stroma consisted of myofibroblasts, endothelial cells and immune cells. Myofibroblasts consisted of multiple subtypes indicative of a range of differentiation between smooth muscle cells and fibroblasts (ACTA2+, THY1+, COLA1+). These cells were absent in normal liver and could be confirmed as a desmoplastic stroma on histology. The immune infiltrate was rich in monocyte-derived macrophages, with few dendritic cells, CD4 and CD8 T cells. We identified 2 macrophage subpopulations in both CLMs. These cells expressed markers belonging to both the M1 and M2 classes (IL1A+, IL1B+, TNF+, MARCO+, MSR1+, CD68+) suggestive of a spectrum of polarization states. Macrophages showed increased expression of the immune checkpoint inhibitor TIM3, while CD4 and CD8 cells showed upregulation of TIGIT. Immune cell populations also expressed a variety of cytokines such as chemokines and interleukins capable of regulating functional effector states. Tumor epithelial cells expressed receptors for various growth factors of the EGF, VEGF, PDGF, FGF and TGFGB families. Stromal cells expressed several heterologous growth factors that can act as receptors ligands and influence tumor cell growth. This included BMPs and INHA expressed in myofibroblasts that can modulate TGFB signaling, as well as FGFs, HGF and VEGF. Endothelial cells secreted PDGF and macrophages expressed AREG and EREG that can activate EGF signaling. These features of the cellular landscape of CLMs can be used for devising novel therapeutic strategies for patients unfit for surgery or for those with unresectable tumors. We will test inhibition of the identified heterologous growth factors and improving the antitumor potential of macrophages in an ex vivo air liquid interphase organoid system from a cohort of CLMs. Citation Format: Anuja Sathe, Jiamin Chen, Christina Wood-Bouwens, Alison Almeda, Billy Lau, Sue M. Grimes, George A. Poultsides, Hanlee Ji. Characterization of colorectal liver metastasis at single-cell resolution reveals dynamic interplay in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2126.