Single-cell RNA sequencing reveals localized tumour ablation and intratumoural immunostimulant delivery potentiate T cell mediated tumour killing

Abstract

Background: Metastatic breast cancer poses great challenge in cancer treatment. N-dihydrogalactochitosan (GC) is a novel immunoadjuvant that stimulates systemic immune responses when administered intratumourally following local tumour ablation. A combination of photothermal therapy (PTT) and GC, referred to as localized ablative immunotherapy (LAIT), extended animal survival and generates an activated B cell phenotype in MMTV-PyMT mouse mammary tumour microenvironment (TME). However, how T cell populations respond to LAIT remains to be elucidated.

Methods: Using depletion antibodies, we studied the contributions of CD8⁺ and CD4⁺ T cells to the therapeutic effect of LAIT. Using single-cell RNA-sequencing (scRNAseq), we analysed tumour-infiltrating T cell heterogeneity and dissected their transcriptomes upon treatments of PTT, GC, and LAIT (PTT+GC).

Results: Loss of CD8⁺ T cells after LAIT abrogated the therapeutic benefits of LAIT. Ten days after treatment, proportions of CD8⁺ and CD4⁺ T cells in untreated TME were 19.2% and 23.0%, respectively. Upon LAIT, both proportions were increased to 25.5% and 36.2%, respectively. In particular, LAIT increased the proportions of naïve and memory cells from a resting state to an activated state. LAIT consistently induced the expression of co-stimulatory molecules, type I IFN responsive genes, and a series of antitumor cytokines, Ifng, Tnf, Il1, and Il17 in CD8⁺ and CD4⁺ T cells. LAIT also induced immune checkpoints Pdcd1, Ctla4, and Lag3 expression, consistent with T cell activation. Relevant to clinical translation, LAIT also upregulated genes in CD8⁺ and CD4⁺ T cells that positively correlated with extended survival of breast cancer patients.

Conclusions: Overall, our results reveal that LAIT prompts immunological remodelling of T cells by inducing broad proinflammatory responses and inhibiting suppressive signalling to drive antitumour immunity.

Keywords: N-dihydrogalactochitosan (GC); antitumour immune response; localized ablative immunotherapy (LAIT); mouse mammary tumour virus-polyoma middle T (MMTV-PyMT); photothermal therapy (PTT); single-cell RNA sequencing (scRNAseq); tumour-infiltrating T cells.