Introduction

Recent studies using single-cell transcriptomic analysis have reported several distinct clusters of neoplastic epithelial cells and cancer-associated fibroblasts in the pancreatic cancer tumor microenvironment. However, their molecular characteristics and biological significance have not been clearly elucidated due to intra- and inter-tumoral heterogeneity.

Aims & Methods

We performed single cell RNA sequencing using enriched epithelial cells and fibroblasts from 17 pancreatic cancer tissues. Then, to resolve the spatial information of heterogeneous cancer subclusters and cancer-associated niches in human pancreatic cancer tissues, we performed RNA in situ hybridization and spatial transcriptome analysis using 10X Visium technique in paired tumor tissues.

Results

We identified five distinct functional subclusters of pancreatic cancer cells and six distinct cancer-associated fibroblast subclusters. We deeply profiled their characteristics, and we found that these subclusters successfully deconvoluted most of the features suggested in bulk transcriptome analysis of pancreatic cancer. Among those subclusters, we identified a novel cancer cell subcluster, Ep_VGLL1, showing intermediate characteristics between the extremities of basal-like and classical dichotomy, despite its prognostic value. Molecular features of Ep_VGLL1 suggest its transitional properties between basal-like and classical subtypes. In the spatial transcriptome analysis, we constructed an adjacency network graph reflecting the neighborhood enrichment relationships across diverse cell types and draw a putative cancer progression axis from Ep_FXYD2, ADM population, to Ep_TRIM54, Ep_VGLL1, and Ep_KRT6A, supporting our model of pancreatic cancer cell dynamics. In addition, we also discovered that the spatial distributions of Fb_LRRC15, representing the myCAF population, were highly correlated with pancreatic cancer cell distributions, whereas Fb_SFRP1, representing the iCAF population, were located distal to the cancer-associated niche.

Conclusion

This integrative analysis not only provides a comprehensive landscape of pancreatic cancer and fibroblast population, but also suggests a novel insight to the dynamic states of pancreatic cancer cells and unveils potential therapeutic targets.

References

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Collisson, E. A. et al. Molecular subtypes of pancreatic cancer. Nat. Rev. Gastroenterol. Hepatol. 16, 207-220, doi:10.1038/s41575-019-0109-y (2019).
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Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal human cancers. In recent years, bacteria resembling gut microbiota have been found within pancreatic cancer tissues, suggesting their potential influence on tumor growth and prognosis. However, the details remain insufficiently elucidated. Therefore, we clarified the characteristics of the tumor microbiota using resected pancreatic cancer specimens from our institution and to analyze their relationship with the tumor microenvironment and prognosis.

Aims & Methods

We analyzed formalin-fixed paraffin-embedded (FFPE) sections from 162 PDAC patients who underwent surgery between April 2008 and March 2017 in our institution.
DNA were extracted from FFPE using Quantitative polymerase chain reaction (qPCR) and in situ hybridization (ISH) targeting 16S rRNA. Microbial community was explored by using 16S metagenomics from FFPE. The profile of intratumoral bacteria was compared with clinical information, pathological findings including tumor-infiltrating T cells, tumor-associated macrophage, fibrosis, and alterations in four main driver genes (KRAS, TP53, CDKN2A/p16, SMAD4) in tumor genomes.

Results

Among 162 cases, 52 showed amplification in qPCR targeting 16S rRNA and positive ISH reactions targeting 16S rRNA within tumor tissues. The presence of bacteria commonly found in gut microbiota such as Proteobacteria and Firmicutes was observed within tumor tissues of these 52 cases. Comparison with paired normal tissues (N=26) revealed differences in αdiversity (P<0.01) and βdiversity (P<0.01), suggesting the presence of a unique tumor microbiota. Subsequently, we divided the 52 cases into two groups based on the median survival period. We identified one bacterial species was dominant in the long-term survival group and ten were in the long-term survival group using Linear Discriminant Analysis Effect Size. Comparison of bacterial profiles between cases with good and poor prognosis revealed a significant positive correlation between a shorter survival time and the presence of anaerobic bacteria such as Bacteroides, Lactobacillus, and Peptoniphilus. 24 cases which were positive for at least one of the three prognostic bacteria were compared with 28 cases negative for all of them, there were not signicfficant correlation with tumor size, pathological stage, fibrosis,and alterations in four main driver genes,but signicfficant correlation with the lower degree of tumor-infiltrating T cells positive for CD4, CD8, and CD45RO (P<0.01, P<0.01, P<0.01, respectively)

Conclusion

We identified multiple bacterial species within pancreatic cancer tumors associated with prognosis. Increased abundance of Bacteroides, Lactobacillus and Peptoniphilus along with the progression of pancreatic cancer suggests their involvement in worsening prognosis through tumor immunity.