Gene Expression Comparison between the Lymph Node-Positive and -Negative Reveals a Peculiar Immune Microenvironment Signature and a Theranostic Role for WNT Targeting in Pancreatic Ductal Adenocarcinoma: A Pilot Study

Abstract

Over the past several years there has been much debate with regards to the prognostic and clinical significance of pancreatic ductal adenocarcinoma (PDAC) with lymph nodes metastasis. The PDAC gene expression knowledge and the biologic alterations underlying the lymph node involvement convey a clinical implication in dealing with the theranostic window. To this end, we provide an original bioinformatic dissection of the gene expression differences of PDAC according to the nodal involvement from a large public available dataset. Comprehensive transcriptomic analysis from 143 RNA-seq patient's derived samples indicated that WNT increased activation and a peculiar immune microenvironment identify subjects with nodal involvement. In frame of this thinking, we validated the WNT pathway role in increasing the likelihood of lymphatic dissemination in vitro. Moreover, we demonstrated for the first time in a PDAC model the potential therapeutic window that XAV-939-a specific WNT pathway inhibitor-has in re-educating a tumor-permissive immune system. Finally, we outline the potential implication on bystander molecular drivers exerted by WNT molecular inhibition, providing a picture of the proteomic oncogenic landscape changes elicited by XAV-939 on PDAC cells and their clinical implication. Our findings hold the promise to identify novel immune-based therapeutic strategies targeting WNT to enhance PDAC cytotoxicity and restore anti-PDAC immunity in node-positive disease.

Keywords: ANGPTL4; M2 macrophages; MST-1; PDAC; WNT; XAV-939; dendritic cells; lymph node metastases; pancreatic cancer; tumor immune microenvironment.

Figure 1

Volcano plot of differentially expressed genes (DEGs), highlighting the top 5 DEGs. Sig: significant.

Figure 2

Gene ontology (GO) graph related to the top 10 most enriched terms. Significant GO accession codes are reported in rectangles and statistical significance is shown in color scale that ranges from yellow to red (low to high significance). At the right side, the descriptions related to the significant terms are reported.

Figure 3

(a) Biological network including significant enriched terms related to KEGG, GO Biological Process, Reactome pathway, and GO immune system process databases. (b) Bar plot showing the grouping of the terms showed in the network, see text and Supplementary File 1 for details.

Figure 4

(a) Enrichment score graph of GSEA analysis. (b) Genes mostly contributing to the enrichment of “KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION” and their log2fold-change values.

Figure 5

Bar plot displaying the number of cases which have enrichment value above the selected cut-off (median of enrichment scores) relative to N^pos and N^neg cases. * p < 0.05; ** p < 0.05; and *** p < 0.001.

Figure 6

(a) Median effect plot showing the inhibition of cell proliferation of PANC-1 and MIAPaCa-2 cells after 48 h treatment with XAV-939 (0.001–100 µM) as log (fraction affected/fraction unaffected) vs. log(dose). (b) Effect of XAV-939 on PDAC cells migration was assessed in wound healing assay: XAV-939 treatment restrains PDAC migration function in vitro. WNT-β-catenin inhibition reduced the capacity of tumor cell migration. Differences in wound closure were assessed by ImageJ Lab 1.51 Software. Graph bars represent the percentage of surface area in three independent experiments. Values are expressed as mean ± SD of three independent experiments; **** p-value < 0.0001, by t-test. (c) The invasion of PANC-1 and MIAPaCa-2 cells was performed with or without 10 µM XAV-939 by Boyden chambers assay showing the high capability of the drug in reducing PANC-1 cell invasion.

Figure 6

(a) Median effect plot showing the inhibition of cell proliferation of PANC-1 and MIAPaCa-2 cells after 48 h treatment with XAV-939 (0.001–100 µM) as log (fraction affected/fraction unaffected) vs. log(dose). (b) Effect of XAV-939 on PDAC cells migration was assessed in wound healing assay: XAV-939 treatment restrains PDAC migration function in vitro. WNT-β-catenin inhibition reduced the capacity of tumor cell migration. Differences in wound closure were assessed by ImageJ Lab 1.51 Software. Graph bars represent the percentage of surface area in three independent experiments. Values are expressed as mean ± SD of three independent experiments; **** p-value < 0.0001, by t-test. (c) The invasion of PANC-1 and MIAPaCa-2 cells was performed with or without 10 µM XAV-939 by Boyden chambers assay showing the high capability of the drug in reducing PANC-1 cell invasion.

Figure 7

(a) Evaluation of β-catenin expression in PDAC cells treated with XAV-939 (10 μM) for 48 h by quantitative PCR (qPCR), * p-value < 0.05. (b) Evaluation of β-catenin expression in PDAC cells treated with 10 μM XAV-939 for 24 h by Western blotting and analysis of the bands by Image Lab software. C: control cells; T: 10 µM XAV-939 treated cells. β-actin was used to normalize protein expression levels. *** p-value < 0.001.

Figure 7

(a) Evaluation of β-catenin expression in PDAC cells treated with XAV-939 (10 μM) for 48 h by quantitative PCR (qPCR), * p-value < 0.05. (b) Evaluation of β-catenin expression in PDAC cells treated with 10 μM XAV-939 for 24 h by Western blotting and analysis of the bands by Image Lab software. C: control cells; T: 10 µM XAV-939 treated cells. β-actin was used to normalize protein expression levels. *** p-value < 0.001.

Figure 8

(a) Killing of PDAC cells in presence of 10 µM XAV-939 and PBMC. (b) Effect on cell proliferation by XAV-939 (10 µM) on PDAC cells in presence and absence of mDCs alone or PBMCs comprising mDCs. Results are expressed as fold change (*** p-value < 0.001 for PANC-1 treated cells vs. untreated ones).

Figure 9

Immunofluorescent detection of CD40 in mDCs kept in contact with PDAC cells (MiaPaCa-2 in the upper panel and PANC-1 in the lower one) and XAV-939 (10 µM). The specimens were examined using a Leica (DMi8) immunofluorescence microscope. The images reported were recorded with 20× magnification and the quantification of the immunofluorescence was performed by ImageJ software.

Figure 10

(a) A human XL oncology array of 84 human cancer-related protein was performed on PANC-1 and MIAPaCa-2 CM before and after treatment with XAV-939 (10 µM) for 48 h. Array spots were analyzed with ImageJ Lab 1.51 Software and normalized to positive control signal intensities. Graph bars represent the pixel density of the detected cancer-related proteins in two independent experiments. Values are expressed as mean ±SD of ten independent experiments. * p < 0.05; ** p < 0.05; and *** p < 0.001; **** p < 0.0001, versus control. (b) Survival function of ANGPTL4^high versus ANGPTL4^low PDAC patients.