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Tumor Microenvironment Immune Diversity Revealed by Single-Cell Sequencing

NEW YORK (GenomeWeb) – A Memorial Sloan Kettering Cancer Center-led team has published a catalog of immune cell phenotypes in the breast cancer microenvironment, uncovering enhanced lymphoid and myeloid immune cell heterogeneity associated with the tumors relative to normal breast tissue.

As they reported online today in Cell, the researchers did single-cell RNA sequencing on more than 45,000 immune cells from tumor and matched normal blood, breast, and lymph node samples from eight breast carcinoma cases, using their SEQC pipeline and a computational approach dubbed "Biscuit" to cluster and characterize the immune cell populations in these tissues.

"This atlas revealed vast diversity in immune cells of both the adaptive and innate immune systems, with the biggest change linked to the tissue of residence," MSKCC researchers Dana Pe'er and Alexander Rudensky, the study's senior authors, and their colleagues wrote.

In the process, the team identified immune cell clusters with co-varying expression profiles, along with expansions of specific immune cell populations in the microenvironment of breast tumors — results supported by additional T cell receptor sequencing (TCR-seq) on 27,000 T cells.

The findings "have important implications for characterizing tumor-infiltrating immune cells," the authors explained. "The observed continuum of T cell states argues strongly against the classical notion of a few discrete states of differentiation or activation shaping the tumor microenvironment."

Given the progress being made in treating some cancer types with immune checkpoint blockade drugs targeting the CTLA-4 or PD-1 pathways, there is interest in teasing out the immune features found in both tumor and immune cells, the team noted, while exploring potential contributions that regulatory T cells (Tregs), effector T cells targeted by Tregs, and other immune cells make to boosting or curtailing tumor growth.

Using Illumina HiSeq 2500 instruments, Droplet Genomics chips, and a custom-built inDrop single-cell RNA-seq microfluidics platform, the researchers profiled transcriptome patterns in 47,016 immune cells from eight treatment-naïve, primary breast carcinoma tumors. They also analyzed single-cell RNA-seq in immune cells from matched normal blood, breast, and lymph node tissue.

The set included tumors with estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, the team noted, as well as triple-negative tumors lacking ER, PR, and HER2. And within the samples profiled, the group saw a range of anticipated immune cell clusters — from monocytes, macrophages, and mast cells to T cells, B cells, dendritic cells, and neutrophils.

Together, the researchers identified 83 distinct immune cell cluster, including 38 T cell clusters, 27 clusters falling in the myeloid immune cell lineage, nine B cell clusters, and nine clusters of natural killer cells, which could be further subdivided into immune subgroups with Treg, effector, and other roles. All but 10 of these clusters were shared in more than one patient, they reported, though the phenotypes often differed within the tumor samples.

"While T and myeloid lineage cells exhibited considerable overlap between tumor and normal tissue samples, we observed increased phenotypic heterogeneity and expansion of cell populations in the tumor," the authors wrote, noting that a "subset of T cell clusters was present in both tumor and normal tissue, but cytotoxic T cell clusters were more abundant in [the] tumor, as were Treg clusters."

Through a series of follow-up experiments, including analyses of paired TCR-seq and RNA-seq data for 27,000 more T cells assessed with 10X Genomics approaches, the team continued comparing the immune repertoires in normal and breast tumor tissues, highlighting phenotypic expansions that often took place in the breast cancer microenvironment as well as the diverse TCR patterns accompanying them.

"These observations, along with the resulting extensive immune single-cell RNA- and TCR-seq datasets and comprehensive analytical platform, will facilitate better understanding of potential mechanisms behind immune cell contributions to promoting and opposing tumor progression," the authors concluded. 

In a pair of related studies published in Nature Medicine earlier this week, independent University of Melbourne- and Peking University-led teams used single-cell RNA-seq to characterize T cells associated with triple-negative breast cancers and non-small cell lung cancers, respectively — analyses that pointed to immune cell clusters with expression signatures showing potential ties to patient outcomes in these cancer types.