Mutant KRAS-induced expression of ICAM-1 in pancreatic acinar cells causes attraction of macrophages to expedite the formation of precancerous lesions

Abstract

Desmoplasia and an inflammatory environment are defining features of pancreatic cancer. Unclear is how pancreatic cells that undergo oncogenic transformation can cross-talk with immune cells and how this contributes to the development of pancreatic lesions. Here, we demonstrate that pancreatic acinar cells expressing mutant KRAS can expedite their transformation to a duct-like phenotype by inducing local inflammation. Specifically, we show that KRAS(G12D) induces the expression of intercellular adhesion molecule-1 (ICAM-1), which serves as chemoattractant for macrophages. Infiltrating macrophages amplify the formation of KRAS(G12D)-caused abnormal pancreatic structures by remodeling the extracellular matrix and providing cytokines such as TNF. Depletion of macrophages or treatment with a neutralizing antibody for ICAM-1 in mice expressing oncogenic Kras under an acinar cell-specific promoter resulted in both a decreased formation of abnormal structures and decreased progression of acinar-to-ductal metaplasia to pancreatic intraepithelial neoplastic lesions.

Significance: We here show that oncogenic KRAS in pancreatic acinar cells upregulates the expression of ICAM-1 to attract macrophages. Hence, our results reveal a direct cooperative mechanism between oncogenic Kras mutations and the inflammatory environment to drive the initiation of pancreatic cancer.

Fig. 1. Depletion of macrophages attenuates progression of Kras-caused abnormal structures

(A–C) At seven weeks of age, p48^cre;LSL-Kras^G12D mice (or control mice; shown are LSL-Kras^G12D) were injected with GdCl₃ or PBS as indicated. GdCl₃ (10 mg/kg) was intravenously-injected through the tail vein every 2 days for 1 week. After a two week rest animals were treated a second time with GdCl₃ every 2 days for 1 week (treatment scheme outlined in Supplemental Figure S1A). At the endpoint (week 13) pancreata were harvested and analyzed. (A) Pancreata were subjected to IHC for H&E, alcian blue and F4/80. A representative area of the pancreas tissue is shown. The scale bar is 50 µm. (B) H&E stained tissue samples from PBS- (n=4) or GdCl₃-treated (n=3) p48^cre;LSL-Kras^G12D mice were evaluated and quantitated for abnormal structures. Bar graph: Quantitation of abnormal structures (isolated ADM regions, ADM-PanIN transition areas and PanIN lesions all combined) per pancreas section. Pie graphs: Percentage of isolated ADM areas, ADM-PanIN transition areas and PanINs. (C) Enlarged sections of an ADM area (C1) and a PanIN1 lesion (C2) from F4/80 staining. The scale bar is 50 µm.

Fig. 2. Mutant Kras induces expression of ICAM-1 in acinar cells

(A–C) Mouse primary pancreatic acinar cells were isolated, infected with lentivirus carrying an oncogenic Kras (Kras^G12V) mutant and seeded in 3D organoid culture. After 5 days, cells were analyzed for expression of ICAM1, CXCL10, CXCL11 or EGFL7 using quantitative PCR (A), for expression of ICAM1 using Western blot (B; silver staining served as loading control). In addition, at days 0, 3 and 5 of Kras^G12V expression, supernatants were analyzed by ELISA for soluble ICAM-1 (C). (D) Pancreata of p48^cre;LSL-Kras^G12D or control (shown is p48^cre) mice at week 14 were analyzed for expression of ICAM1, macrophage infiltration (F4/80) and amylase expression (acinar cell marker) using immunofluorescence labeling. DAPI marks the nuclei. Left side shows composite and right side single channels. The bar indicates 50 µm. (E) Acinar cells were isolated from 6 weeks old p48^cre;LSL-Kras^G12D or control (shown is p48^cre) mice and analyzed for expression of ICAM1 using quantitative PCR. (F) Human tissue samples with regions of ADM or PanIN1A, PanIN1B, or PanIN2 (focally) lesions were stained with H&E (top row) or ICAM1 (IHC, bottom row). The bar indicates 50 µm.

Fig. 3. Kras induced chemoattractants mediate attraction of macrophages

(A) 7 × 10⁴ HeLa cells stably-expressing either GFP or GFP-tagged full-length transmembrane ICAM-1, and 7 × 10⁴ DiI-labeled Raw264.7 cells were plated as indicated in an ibidi removable 2 well silicone culture insert that was placed in a cell culture µ-Dish. 24 hours after seeding the culture inserts were carefully removed. After 24 hours, migration of macrophages towards HeLa-GFP or HeLa-GFP-ICAM-1 cells was assessed by fluorescent microscopy in indicated areas 1 and 2. The bar indicates 200 µm. (B, C) Freshly-isolated primary mouse macrophages (B), or M1 or M2 polarized primary mouse macrophages (C) were seeded in a Transwell chamber and migration towards recombinant ICAM1 (0, 50, 500 ng/ml) was determined (t = 16 hours). Shown are numbers of migrated macrophages per field. The asterisk indicates statistical significance as compared to the control. (D, E) Primary mouse pancreatic acinar cells from LSL-Kras^G12D mice were isolated and infected with adeno-null virus (control) or adenovirus harboring cre recombinase to induce expression of oncogenic Kras. Cells were then seeded in bottom wells of transwell chambers. Live dye-labeled freshly-isolated primary mouse macrophages (D), or M1 or M2 polarized primary mouse macrophages (E) were added into the top chambers of the Transwell plates and migration towards the acinar cells was determined either in presence of isotype-matched control antibody (Ctrl-AB) or an ICAM-1 neutralizing antibody (NAB). Shown are numbers of migrated macrophages per field. * indicates statistical significance as compared to the adeno-null control; ** indicates statistical significance as compared to Adeno-cre. (F) At three weeks of age, p48^cre;LSL-Kras^G12D mice (or control mice; see Supplemental Fig. S5A) were injected every other day with ICAM-1 neutralizing antibody (NAB), isotype-matched control antibody (Control AB) or vehicle control (PBS) for a total of 11 weeks. Pancreata were subjected to IHC for H&E and F4/80. A representative area of the pancreas tissue under each condition is shown. Scale bar is 50 µm. (G) H&E stained tissue samples from control (n=6) or mICAM-1 NAB-treated (n=3) p48^cre;LSL-Kras^G12D mice were evaluated and quantitated for abnormal structures. Bar graph: Quantitation of abnormal structures (isolated ADM regions, ADM-PanIN transition areas and PanIN lesions all combined) per section. Pie graphs: Percentage of isolated ADM areas, ADM-PanIN transition areas and PanINs.

Fig. 4. Presence of TNF and ECM degradation are consequences of abundance of macrophages in regions of ADM

(A) Pancreata of p48^cre;LSL-Kras^G12D treated with PBS or mICAM-1 NAB (see Figure 3D) were analyzed for expression of TNF in regions of macrophage infiltration using immunofluorescence labeling. F4/80 marks macrophages and DAPI marks the nuclei. Left side shows composite and right side single channels. The bar indicates 100 µm. (B) Primary mouse pancreatic acinar cells from LSL-Kras^G12D mice were isolated and infected with adeno-null virus (control) or adenovirus harboring cre recombinase to induce expression of mutant Kras (labeled: Kras^G12D). Cells then were seeded in 3D explant organoid culture in absence (control) or presence of recombinant mouse TNF (50 ng/ml). ADM events were determined at day 5. Bar graph: * indicates statistical significance as compared to the control (adeno-null + control treatment), ** indicates statistical significance to control and to TNF-treated adeno-null group; *** indicates statistical significance to all other groups. Photos show representative areas of the explant culture. The bar is 200 µm. (C) Pancreata of p48^cre;LSL-Kras^G12D mice were subjected to in situ zymography followed by DAPI staining as described in materials & methods. The scale bar is 25 µm. (D) Pancreata of p48^cre;LSL-Kras^G12D mice (or control mice; shown are LSL-Kras^G12D) injected with GdCl₃ or PBS as indicated in Supplemental Figure S1A were subjected to H&E staining and immunofluorescence analysis for F4/80 and MMP9. DAPI stain was added to visualize nuclei. A representative area of the pancreas tissue is shown. The scale bar is 50 µm. (E) Scheme of how Kras^G12D-expressing acini and M1-polarized macrophages may crosstalk to potentiate acinar-to-ductal metaplasia.