Dados do Trabalho

Título

Characterization of Gastric Adenocarcinoma Immune Environment

Introdução

The spatial organization of infiltrating lymphocytes throughout the tumor microenvironment can shape their functional state, rendering them antitumoral characteristics when well-organized. Tertiary lymphoid structures (TLSs) are special aggregates of lymphoid cells, formed under chronic inflammatory conditions, creating niches for B and T cells interaction. These lymph node-like structures may predict favorable clinical responses.

Objetivo

We aimed to assess, both in silico and ex vivo, the cellular composition and organization of TLSs in gastric adenocarcinoma (GA) patients.

Métodos

We collected peripheral blood, peritumoral and tumoral tissue samples from 23 patients with GA that underwent resection surgery at the A.C.Camargo Cancer Center and at the Santa Casa de Misericordia de São Paulo (CEP approval 2847/20). Patients were all adults, non-metastatic, treatment-naïve, without comorbidities and without anti-inflammatory therapy taken after the diagnosis. All tissues were processed and analyzed by multiparametric flow cytometry using antibody panels for detecting specific cellular populations and activation/exhaustion phenotypes. For the in silico analyses, we utilized RNAseq data from The Cancer Genome Atlas (TCGA, 315 stage I-III GA patients), as well as publicly available datasets of RNAmicroarray (68 patients with dyspepsia and intestinal metaplasia) and single cell RNAseq (scRNAseq, 8 patients with GA). The immune abundance in RNAseq and RNAmicroarray data was estimated by the MCPcounter algorithm. The scRNAseq data was analyzed using the R package Seurat.

Resultados

Hierarchical clustering of MCPcounter estimates for samples in the TCGA dataset identified 4 immune groups. The highest immune-infiltrated group displayed increased abundance of B and T cells, as well as the greatest expression of immune-checkpoints (e.g., PDCD1), TLS-related genes (e.g., CXCL13 and CXCR5) and a TLS signature. This signature was particularly enriched in genomically stable (GS) and EBV+ patients. We hypothesize that such process may also be seen in chronic gastritis. Accordingly, analysis of RNAmicroarray samples revealed the existence of a TLS-like signature in the reactive gastric mucosa. However, these signals disappear in preneoplastic stages. Using GA scRNAseq, we observed that the abundance of CD8 T cells expressing the B cell chemoattractant CXCL13 correlated with the abundance of B cells expressing its receptor CXCR5. These CD8 T cells exhibited a signature related to a tissue resident tumor reactive state, that and involves the expression of ENTPD1 (CD39) and ITGAE (CD103). Finally, flow cytometry analysis showed an accumulation of antitumor CD8 T cells co-expressing CD39 and CD103 in the tumor compared to the other sites.

Conclusões

Our results demonstrate that the TLS signature correlates with higher immune abundance scores, in which immune checkpoints and TLS-related chemokines are found. Altogether, we observed that the correlation between B and T cells detected in the bulk RNAseq could be translated into a more direct interaction through the expression of CXCL13 by CD8 T cells and its receptor CXCR5 by B cells in the tumor compartment. Ultimately these CD8 T exhibit a profile related to a tissue resident tumor reactive state, which was demonstrated by scRNAseq and flow cytometry.

Palavras-chave

Gastric Adenocarcinoma, Tertiary Lymphoid Structures, CD8 T cells

Financiador do resumo

FAPESP and CAPES