Integrating small RNA-seq and single-cell multiomics to investigate tumor-T-cell communication.

Connecting exosomal miRNAs with T-cell responses

The researchers studied exosomes from 17 molecularly diverse TNBC cell lines. Activated human T cells from healthy donor blood were exposed to these exosomes in vitro for 48 hours and then analyzed at single-cell resolution.

Using CITE-seq, the team profiled 179,169 T cells and identified distinct CD4+, CD8+, regulatory T-cell, T follicular helper, NKT and γδ T-cell populations. Exosome exposure caused transcriptional reprogramming within existing T-cell populations, rather than major changes in overall cell composition. Regulatory and dysfunction-associated phenotypes increased, while exosome-treated T cells showed reduced tumor-cell killing in functional assays.

To investigate potential regulators of these effects, the researchers profiled exosomal non-coding RNAs from the 17 TNBC cell lines and from plasma samples collected from 28 patients with TNBC and seven healthy donors. Bulk exosomal small RNA libraries were prepared using the NEXTFLEX™ Small RNA-Seq Kit v4 NEXTFLEX Small RNA Sequencing Kit V4 , and the miRNA data were computationally integrated with the separately generated single-cell T-cell transcriptomic results.

The researchers focused on miRNAs that were consistently dysregulated in both TNBC cell-line-derived exosomes and patient plasma exosomes and linked them to genes whose expression changed in T cells after exosome exposure.

This analysis identified 20 upregulated miRNAs linked through previously validated miRNA–mRNA interactions curated in miRTarBase to ten downregulated T-cell genes, including FOS, HBEGF, HMGCS1, IER3 and TNFSF9. These genes are involved in T-cell activation, co-stimulation, signaling and metabolism. The network analysis used T-cell differential expression independent of clustering. The broader single-cell analysis resolved changes within T-cell populations, while small RNA-seq identified exosomal miRNAs that could contribute to the observed T-cell responses.

miR-98-5p emerges as a regulatory hub

Among the identified miRNAs, hsa-miR-98-5p emerged as a highly connected node associated with several downregulated targets, including HBEGF, TNFSF9 and IER3.

The researchers tested this finding by introducing a miR-98-5p mimic into T cells. Increased miR-98-5p was associated with a higher proportion of CD4+CD25+CD127− regulatory T cells and an increased PD-1+ fraction within this population. HB-EGF and TNFSF9 protein levels decreased, while STAT3 phosphorylation increased. These results supported the integrated miRNA–mRNA analysis, although the authors note that further work is needed to establish direct causal mechanisms.

Other datasets added further context. Single-cell V(D)J sequencing identified T-cell receptor clonotypes detected specifically after exosome exposure, while bulk and single-cell cytokine profiling revealed changes in T-cell secretory function. Together, these approaches provided a multi-layer view of exosomal RNA cargo alongside transcriptional, phenotypic and functional changes in T cells.

Adding regulatory context through small RNA-seq

This study illustrates how small RNA-seq can help connect extracellular regulatory signals with downstream immune-cell responses. By integrating exosomal miRNA profiles with single-cell RNA-seq, patient plasma data and functional assays, the researchers moved beyond cataloguing miRNAs to identify regulatory networks that may contribute to tumor–immune communication, highlighting how small RNA-seq can complement single-cell transcriptomics and functional profiling to provide a more complete view of immune regulation and cell-to-cell communication.