Disruption of cellular plasticity by repeat RNAs in human pancreatic cancer

Abstract

Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications on tumor cell state and the response of the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we performed spatial molecular imaging at single-cell resolution in 46 primary tumors, revealing correlations of high repeat RNA expression with alterations in epithelial state in PDAC cells and myofibroblast phenotype in cancer-associated fibroblasts (CAFs). This loss of cellular identity is observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs of PDAC and CAF cell culture models pointing to cell-cell intercommunication of these viral-like elements. Differences in PDAC and CAF responses are driven by distinct innate immune signaling through interferon regulatory factor 3 (IRF3). The cell-context-specific viral-like responses to repeat RNAs provide a mechanism for modulation of cellular plasticity in diverse cell types in the PDAC microenvironment.

Keywords: cancer-associated fibroblast; cellular plasticity; extracellular vesicles; pancreatic cancer; repeat RNA; spatial transcriptomics; tumor microenvironment.

Figure 1.. Spatial transcriptomic analysis of repeat RNA expression profiles in different cell types of the PDAC TME

(A) Schema of experimental design using Nanostring CosMx SMI in total 46 PDAC patients with custom repeat RNA probes combined with a 1000 plex RNA panel. (B) Uniform Manifold Approximation and Projection (UMAP) plot showing a diversity of cell types in TME analyzed by CosMx SMI. (C) Histogram depicting the cell type composition for each FOV, determined by spatial transcriptomics. (D) Whole tissue images with coloring of identified cell types from three FFPE tumors (B10, C4, and D10). (E) Raw mean expression of repeat RNAs per cell type with 95% confidence interval (CI). See also Figure S1 and Table S1–S2.

Figure 2.. Diverse repeat RNA species influence cell plasticity response in CAFs.

(A-D) Volcano plots showing differential gene expression of LINE-1 ORF1 high versus low expression in cancer cells (N=87,181), CAFs (N=105,028), macrophages (N=18,746), and endothelial cells (N=13,807) using CosMx SMI. (E) Schematic diagram of the calculation of scaled mean effect size in a single CAF to the number of PDAC cells at different radii ranges. Details are described in the method section. (F-G) The aggregated scaled effect size of RNA expression of all genes in CAFs as a function of with number of nearby PDAC cells at 20–50, 50–100, 100–200 μm (123 FOVs from 3 FFPE and 33 TMA samples). Error bars=95% CI. (H) UMAP plots representing the COL3A1, LINE-1 ORF1 and ORF2 expression in iCAF/myCAF subtypes. (I) Plot showing myCAF-iCAF transition states analyzed by likelihood ratio of myCAFs and iCAFs as a relationship to total non-repeat counts. (J) Mean effect size of applicable iCAF and myCAF marker genes (Table S3) is highlighted. (K) Dot plot of regional LINE-1 ORF1 transcript levels versus distance from tumor in mm with annotation of myCAF (blue) and iCAF (red). (L) Representative spatial localization of CAF subtypes with LINE-1 ORF1 molecular density surrounding tumor glands in PDAC FFPE (C4). (M) Forest plot of each PDAC tumor plotted with a single point. The y-axis represents LINE-1 ORF1 expression in each tumor vertical position. The x-axis shows estimate and 95% CI for the mean change in LINE-1 ORF1 expression between myCAF and iCAF at a distance of <0.05 mm compared to >0.1 mm from tumor glands. Tumors with p<0.05 are given color. See also Figure S2 and Table S2–S3.

Figure 3.. EVs can infect the TME and neighboring PDAC cells inducing loss of cellular identity.

(A) Graphical abstract of cellular dysregulation by repeat RNA-containing EVs. (B-C) Schema of EV isolation from CAF-1 and PDAC cell lines and characterization of purified EVs by NTA and TEM imaging. Scale bar, 200 nm. (D) Immunoblots showing high expression of exosome surface markers (CD9, CD63 and Flotillin-1) in CAF-1 and PDAC-released EVs. (E) RT-qPCR of IFNB1 and ISGs in CAF-1, PDAC3, and PDAC6 treated with CAF-1, PDAC3, and PDAC6-derived EVs or PBS. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test; significance is shown between EV treatment and PBS. Error bar represented as mean ± s.d. derived from N=2 biological replicates. *p<0.05, **p<0.01, ***p<0.005. (F) ISGs expression in THP-1 and HUVEC cell lines upon PDAC3 and 6-derived EVs exposure for 2 days. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.005. (G-I) RT-qPCR of indicated gene expression in CAF-1 (G), PDAC3 (H) and PDAC6 (I) following PBS and PDAC3 and PDAC6-derived EV treatment. Statistical significance was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test; significance is shown between EV treatment and PBS. *p<0.05, **p<0.01, ***p<0.005. (J) Heatmap representing the relative gene expression in CAF-1 upon PDAC3-derived EV treatment after pre-incubation with or without CD9 antibody (5 μg ml⁻¹). Error bar represented as mean ± s.d. derived from N=3 technical replicates. *p<0.05, **p<0.01, ***p<0.005. See also Figure S3.

Figure 4.. Diverse repeat RNA species alter CAF myofibroblast phenotype and function.

(A) Schematic diagram of repeat RNA transfection with IVT RNAs. IFNB1 expression in CAF-1 followed by various repeat RNA transfection. (B) HSATII, HERV-K (env) and ISG expression in pCW57.1-HSATII CAF-1 by Dox treatment for 48 hrs. (C) Workflow depicting sample processing and scRNA-seq from patients-derived CAF cell lines. UMAP of total 4,595 cells (CAF-1 untreated; N=739, CAF-1 treated; N=613, CAF-2 untreated; N=1,161, CAF-2 treated; N=605, CAF-3 untreated; N=877, CAF-3 treated; N=600). (D) UMAP of CAF cell lines colored by the normalized expression of myCAF and iCAF-associated marker genes. (E) Violin plots showing the myCAF and iCAF representative marker genes expression across CAF cell lines. (F) Bar graph representing the average of Log₂ Fold change of myCAF and iCAF-associated gene expression in HSATII-transfected CAF cells compared to untreated. Statistical significance is analyzed by Pearson’s Chi-squared test (Chi-squared=6.549, df=1). (G) Heatmap showing the gene expression of myCAF and iCAF marker genes in CAF-1 with diverse repeat RNA and LPS (10 μg ml⁻¹) treatment. (H) Graph showing migratory ability of CAF-1 following GFP mRNA and HSATII IVT RNA transfection analyzed by Boyden chamber assay (N=6, biological triplicates with technical duplicates). (I) Stressed matrix contraction (SMC) assay to measure CAF contractile force after HSATII transfection (31.25 fmol). Graph shows % of reduced size compared to original gel diameter (N=3, independent biological replicates). (J-K) CM was collected from GFP (GFP-CAF-1-CM) and HSATII transfected CAF-1 (HSATII-CAF-1-CM). EMT-related gene expression and cell motility by CM treatment derived from HSATII-CAF-1 in PDAC3 and PDAC6 were analyzed by RT-qPCR and Boyden chamber assay. Statistical significance was determined by one-way ANOVA (A, K) followed by Dunnett’s (A) or Tukey’s (K) multiple comparison test, two-way ANOVA followed by Tukey’s (B) multiple comparison test, and unpaired student’s t-test (H, I, J). *p<0.05, **p<0.01, ***p<0.005, n.s., non-significant. See also Figure S4 and Table S3.

Figure 5.. Repeat RNA influences PDAC tumor heterogeneity via crosstalk between CAF and PDAC cells.

(A) RT-qPCR of IFNB1 in PDAC3 cells following IVT repeat RNA and GFP mRNA transfection. (B) Analysis of mesenchymal and epithelial marker gene expression in PDAC3 after repeat RNA transfection for 48 hrs. (C) Mesenchymal marker gene expression by RT-qPCR in PDAC cell lines with GFP and HSATII IVT RNA (N=3, biological replicates). Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, **p<0.01, ***p<0.005, n.s., non-significant. (D) Treatment with CM (extrinsic) from GFP or HSATII transfected CAF-1 into the GFP or HSATII transfected (intrinsic) PDAC cell lines. (E) Workflow of RNA-ISH quantification by Halo software. The color components for cell nuclei (blue, hematoxylin), epithelial markers, mesenchymal cells were extracted using color deconvolution. Details are described in the method section. (F-H) Representative images of PDAC patient tumors untreated (N=137) or after neoadjuvant FOLFIRINOX chemotherapy (N=26), stained for combined RNA-ISH for HSATII (red) and IHC for CD8 or CD163 (brown) and EMT RNA-ISH assay with hematoxylin counterstain. Scale bar, 100 μm. Quantification of % QM of Epi cells in tumor gland and standard deviation (Std) of % QM of Epi cells were performed using the Halo image analysis platform. Statistical significance was assessed by two-sided Wilcoxon rank sum (Mann-Whitney) test to perform pairwise comparisons. See also Figure S5.

Figure 6. CAF and PDAC divergent responses to repeat RNAs are IRF3-dependent.

(A) Immunoblot of innate immunity signaling pathway in CAF-1 after HSATII IVT RNA transfection (31.25 fmol; low and 250 fmol; high). (B) Time course response immunoblots of type-I interferon signaling pathway in PDAC3 and PDAC6 (250 fmol of HSATII IVT RNA). (C) Cytoplasmic/nuclear fractionation assay was performed in CAF-1, PDAC3, and PDAC6 upon HSATII transfection to monitor IRF3 cellular localization. Relative IRF3 intensity was normalized to GAPDH (cytosolic marker) and Lamin A/C (nucleus marker), respectively. (D) RT-qPCR of IFNB1 in CAF-1 and PDAC3 transfected with si-Mock, si-TBK1, and si-IRF3. Knockdown efficiency was verified by Immunoblot. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison. **p<0.01, ***p<0.005. (E) Evaluation of HSATII response in a large panel of cell lines. (F-G) RT-qPCR analysis of gene expression in CAF-1 and CAF-3 transfected with control siRNA (si-Mock) or siRNA specific for IRF3 (si-IRF3). Error bars represent mean ± s.d. derived from N=2 or 3 biological replicates. Statistical analysis was performed unpaired student’s t-test. *p<0.05, **p<0.05, ***p<0.005, n.s., non-significant. (H) RT-qPCR of three myCAF genes and IFNB1 in V5-IRF3 overexpressed CAF-1 (N=3, biological triplicates). (I) RT-qPCR of ACTA2, FN1, and SERPINE1 in PDAC3 transfected with si-Mock and si-IRF3. Statistical significance was determined by Two-way ANOVA followed by Tukey’s multiple comparison test. **p<0.05, ***p<0.005. (J) Heatmap representing the relative gene expression in control and cancer cell lines infected with single guide RNA (sgRNA) targeting IRF3 upon HSATII transfection (upper) or HSATII IVT RNA-treated CAF-1 CM stimulation (lower) for 24 hrs. (K-L) Multiplex IF staining with a 3-antibody panel (PanCK, α-SMA, IRF3) with DAPI in five PDAC TMAs (TMA1, 4, 28, 31, 32). Bar graph depicting the percentage of PanCK⁺ or α-SMA⁺ cells correlated with IRF3 (total IRF3; T-IRF3 and nuclear IRF3; Nuc-IRF3), and the aggregated mean percentage of positive cells for each core. PanCK⁺ cells (N=181,536), α-SMA⁺ cells (N=59,326) from 32 cores. Statistical significance was determined by paired Wilcoxon signed-rank test (***p<0.005). Scale bar, 100 μm. (M) IRF3 divergent response to HSATII RNA in CAF and PDAC. See also Figure S6.

Figure 7. Dissecting HSATII RNA-sensing pathways.

(A-B) Lentiviral transduced PDAC3 cells with sgRNA targeting RLRs, and control sgNC were analyzed for RLR activity by immunoblot and RT-qPCR treated with GFP or HSATII IVT RNA. Graphs represent the relative gene expression (N=3, biological replicates) in RLR KO cell lines. Statistical significance was determined by Two-way ANOVA followed by Tukey’s multiple comparison test. ***p<0.005, n.s, not significant. (C) Immunofluorescence staining images of dsRNA in PDAC3 cells following AF488 HSATII ssRNA transfection for 24 hrs. Area of cytoplasmic dsRNA was measured using Halo software (untreated N=101, HSATII treated N=93) and statistical significance was determined by unpaired student’s t-test. ***p<0.005. Scale bar, 20 μm. (D) Graph depicting gene expression of SAMHD1, IFNB1, and IL6 in SAMHD1 knockdown cell lines. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. **p<0.01, ***p<0.005. (E-F) Evaluation of HSATII RNA-mediated gene expression and downstream signaling in SAMHD1 knockdown cell lines. (G-H) Immunoblots of innate immunity- and NF-kB-related pathways in CAF-1 RLR KO cell lines after GFP mRNA or HSATII transfection for 24 hrs. RT-qPCR of IFNB1, iCAF- and myCAF-associated gene expression in CAF-1. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, ***p<0.005, n.s., non-significant. (I) Relative gene expression upon PDAC3-derived EV treatment in WT and MAVS KO CAF-1 cells.

(J-K) Schema of HSATII RNA-induced antiviral response in PDAC and CAFs and crosstalk in PDAC TME. See also Figure S7.