Abstract 5676: Digital spatial profiling for determination of pancreatic stromal components and correlation with survival
Authors: Tushoski Gerik W , Fu Dongtao Ann , Meng Lingsong , Herremans Kelly , Riner Andrea N , Forsmark Christopher E , Huo Zhiguang , Hughes Steven J , Han Song 
Published: June 2022

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death in the United States. Recently, intensive research has revealed the remarkable heterogeneity and complexity of the different components in the pancreatic cancer tumor microenvironment. However, our knowledge regarding active stroma (dominated with activated cancer-associated fibroblasts), infiltrated immune cells and actual tumor cells interactions in the tumor microenvironment, and how these interactions impact patient outcomes, remains limited. Digital spatial profiling (DSP) technology now allows us to quantitatively analyze transcript and protein expression on intact tissue, offering an opportunity to study the intra-tumor cell-to-cell communication. Formalin-fixed paraffin-embedded (FFPE) pancreatic tumors from patients with shorter survival (DFS &lt; 12 months, n=2), and longer survival (DFS &gt; 36 months, n=2) were recruited in this study. Immunofluorescent histochemistry staining of tissue morphology identification uses antibodies to pan-cytokeratin (panCK+) for PDAC cells, alpha-smooth muscle actin (aSMA+) for cancer-associated fibroblasts (CAFs). Six replicated regions of interest (ROI) of tumor, CAFs adjacent and distal to tumor area were annotated from each tissue sample and a panel of 78 transcripts (73 target genes and 5 reference genes) were profiled using GeoMx DSP (Nanostring). Person’s correlation (R2) was computed for regression of GeoMx DSP data. All statistical analyses were performed using the GraphPad Prism software. p-values were calculated on two-sided t-tests and p &lt; 0.05 was considered statistically significant.
As expected, the morphologic selection successfully separates panCK+ PDAC cells from CAFs demonstrated by the correspondent expression of epithelial genes of EpCam and KRT, allowing single cell profiling. Within the panel of 78 genes, 9 genes (approximal 12%) were detected with significantly higher expression levels in PDAC cells than CAFs. Five genes (BATF3, IL12b, ITGB8, CD4 and IFNAR1) were found increased expression levels in CAFs-adj comparing to CAFs-dis, regulating molecular functions of Organismal Death (p value = 3.88E-03), Maturation of Cells (p value = 2.94E-06), and Migration of Cells (p value = 2.37E-03). When comparing the differential transcriptomes in PDAC cells between the longer and shorter survival groups, higher expression of antigen presentation gene HLA-E and integrin alpha- and beta- family genes (ITGAV, ITGAX, ITGB2, and ITGB8) was seen in longer-lived patients. Proliferation genes (CCND1 and MKI67) were also found to be increased in CAFs in patients with longer survival. Our results suggest that further exploration of gene and protein expression and interactions within the pancreatic tumor microenvironment may improve our understanding of tumor and stromal communication and how it impacts on patient outcomes.
Citation Format: Gerik W Tushoski, Dongtao Ann Fu, Lingsong Meng, Kelly Herremans, Andrea N Riner, Christopher E Forsmark, Zhiguang Huo, Steven J Hughes, Song Han. Digital spatial profiling for determination of pancreatic stromal components and correlation with survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5676.