MMP1/PAR1/SP/NK1R paracrine loop modulates early perineural invasion of pancreatic cancer cells

Abstract

The molecular mechanism of perineural invasion (PNI) is unclear, and insufficient detection during early-stage PNI in vivo hampers its investigation. We aimed to identify a cytokine paracrine loop between pancreatic ductal adenocarcinoma (PDAC) cells and nerves and established a noninvasive method to monitor PNI in vivo. Methods: A Matrigel/ dorsal root ganglia (DRG) system was used to observe PNI in vitro, and a murine sciatic nerve invasion model was established to examine PNI in vivo. PNI was assessed by MRI with iron oxide nanoparticle labeling. We searched publicly available datasets as well as obtained PDAC tissues from 30 patients to examine MMP1 expression in human tumor and non-tumor tissues. Results: Our results showed that matrix metalloproteinase-1 (MMP1) activated AKT and induced protease-activated receptor-1 (PAR1)-expressing DRG to release substance P (SP), which, in turn, activated neurokinin 1 receptor (NK1R)-expressing PDAC cells and enhanced cellular migration, invasion, and PNI via SP/NK1R/ERK. In animals, hind limb paralysis and a decreased hind paw width were observed approximately 20 days after inoculation of cancer cells in the perineurium. MMP1 silencing with shRNA or treatment with either a PAR1 or an NK1R antagonist inhibited PNI. MRI detected PNI as early as 10 days after implantation of PDAC cells. PNI also induced PDAC liver metastasis. Bioinformatic analyses and pathological studies on patient tissues corroborated the clinical relevance of these findings. Conclusion: In this study, we provided evidence that the MMP1/PAR1/SP/NK1R paracrine loop contributes to PNI during the early stage of primary tumor formation. Furthermore, we established a sensitive and non-invasive method to detect nerve invasion using iron oxide nanoparticles and MRI.

Keywords: MR imaging; iron oxide nanoparticles; metastasis; pancreatic ductal adenocarcinoma; perineural invasion.

Figure 1

MMP1 was a key factor during the invasion of pancreatic carcinoma cells. (A) PANC-1 and Mia PaCa-2 cells were co-cultured with TAMs for 24 h and reseeded into Transwell plates. TAMs enhanced pancreatic carcinoma cell migration and invasion. (B) Nerve invasion of pancreatic carcinoma cells in a Matrigel/dorsal root ganglion (DRG) model. Eight days after implantation, cancer cells pre-co-cultured with TAMs exhibited extensive nerve invasion. (C) Microarray, qPCR, and ELISA analyses revealed that MMP1 was the most upregulated cytokine in pancreatic carcinoma cells after co-culture with TAMs. (D) Upregulation of MMP1 enhanced PANC-1 and Mia PaCa-2 cell migration and invasion, which was inhibited by shRNA targeting MMP1. (E) MMP-1 overexpression enhanced PNI, which was inhibited by PAR1 antagonist or shRNA targeting MMP1. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 2

The MMP1/PAR1 axis induced DRG-releasing SP by activating Akt. (A) Thirty minutes after MMP1 (5 nM) stimulation, the SP concentration in the DRG supernatant began to increase and the peak concentration was detected at 2 h. The concentration of SP protein decreased almost to baseline levels at 6 h. (B) DRG were incubated in DMEM with MMP1 (5 nM) and different concentrations of PAR1 antagonist SCH79797 (0, 25, 50, and 100 nM) for 2 h, and the SP concentration in the supernatant was measured by ELISA. The MMP1-induced SP release was inhibited by SCH79797. (C) MMP1 induces DRG-releasing SP via MMP1/PAR1/AKT. After MMP1 stimulation, p-Akt increased in the DRG, whereas the PAR1 antagonist SCH79797 attenuated the phosphorylation of Akt. LY2940021 (Akt pathway inhibitor) was used as a positive control. The expression of ERK1/2 and p-ERK1/2 remained unchanged. (D) ELISA showed that the upregulation of SP induced by MMP1 was attenuated by treatment with the PAR1 antagonist or the Akt pathway inhibitor. Data are shown as the mean ± SD. * P < 0.05 and ** P < 0.01 compared to the control. ^Δ P < 0.05 compared to the SP+ SCH79797 group.

Figure 3

The SP/NK1R axis induced cancer cell migration, invasion, and PNI. (A) After SP stimulation, both PANC-1 and Mia PaCa-2 cells exhibited enhanced migration and invasion, which was attenuated by the NK-1R antagonist SP + L-732,138. (B) In the cancer cells and Matrigel/DRG co-culture system, SP stimulation enhanced PNI. When SP/NK1R was blocked with the NK1R antagonist, PNI was inhibited. (C-D) SP activated p-ERK1/2 and upregulated Vimentin and Twist1 expression in both PANC-1 and Mia PaCa-2 cells, which was attenuated by treatment with the NK1R antagonist or the ERK pathway inhibitor, indicating that SP induced the EMT phenotype of PANC-1 and Mia PaCa-2 cells via SP/NK1R/ERK. Data are shown as the mean ± SD. ** P < 0.01 compared to the control, ^Δ P < 0.05 compared to the SP+L-732,138 group.

Figure 4

MMP1/PAR1 and SP/NK1R contributed to PNI in vivo. (A) Sciatic nerve score and sciatic nerve function index of mice with different treatments. (a) Paralysis occurred approximately 20 days after implantation. (b-d) The most severe bilateral hind limb paralysis was observed in mice treated with PANC-1 + TAMs or PANC-1 shNC + TAMs tumors on day 28. Mice receiving MMP1-shRNA, the PAR1 antagonist, or the NK1R antagonist exhibited much less severe paralysis. (B) Surgical images of sciatic nerve PNI. Cancer cells were injected into perineurium of left sciatic nerves (red arrow), and the right sciatic nerves were injected with 3 μL PBS as controls (black arrow). Mice treated with PANC-1 + TAMs or PANC-1 shNC + TAMs tumors exhibited obvious PNI. Knock down of MMP1 by shRNA or treatment with the PAR1 antagonist or the NK1R antagonist significantly reduced PNI. (C) Histological images of sciatic nerves and liver tissues of mice with different treatments. On day 28, the PBS-treated group exhibited normal proximal sciatic nerve histology and caliber and negative MMP1 IHC staining. Injection of PANC-1 cells only resulted in a slightly expanded nerve caliber with cancer cell infiltration and low MMP1 expression. Injection of PANC-1 + TAMs or PANC-1 shNC + TAMs resulted in extensive PNI and highly positive MMP1 IHC staining. MMP1-shRNA significantly reduced MMP1 expression in cancer cells and treatment with the PAR1 antagonist or the NK1R antagonist did not affect MMP1 protein expression. Less nerve caliber expansion with cancer cell infiltration was observed in these three groups. Liver metastases were detected in all experimental groups except for the PBS group. ** P < 0.01 compared to the control and ^Δ P < 0.01 compared to other groups.

Figure 5

Monitoring of PNI by MRI scans in vivo. PANC-1 cells labeled with IONP, PANC-1^IONP, exhibited low signals upon MRI. Right sciatic nerves were injected with PANC-1 ^IONP (blue arrow) and left sciatic nerves were injected with PANC-1 ^IONP + TAMs (yellow arrow). From day 0 to day 7, no apparent PNI was observed in either sciatic nerve. On day 10, a low-signal area on the left sciatic nerve became enlarged and demonstrated thickened sciatic nerves, indicative of PNI. On day 17, both sciatic nerves exhibited PNI, but the left side was more apparent than the right side. On day 20, left hind limb paralysis was observed and tumor formation was detected on the left side by MRI and surgical images. The right hind limb did not exhibit paralysis and no tumor formation was detected. On day 24, left hind limb paralysis was more severe. MRI clearly showed cancer cell invasion along nerves from the left sciatic nerve injection area (yellow arrow) toward the spinal cord (white arrow).

Figure 6

H&E staining of sciatic nerves at individual time points was consistent with MRI. Cancer cells were restricted to the injection area under the epineurium (black arrow) without contacting a nerve bundle (yellow arrow) on days 0, 3, and 7. From day 10, cancer cells infiltrated into the left sciatic nerve tract and progressed thereafter. Cancer cells infiltrated into the right sciatic nerve tract on day 17 and the progression was much less severe than that on the left side.

Figure 7

MMP1 expression data from public datasets and post-operation PDAC patients. (A) MMP1 mRNA expression levels were higher in PDAC tissues than adjacent non-tumor tissues from the public dataset GSE28735. (B) Immunohistochemical images for MMP1 expression in PDAC and adjacent non-tumor tissues collected from clinical PDAC patient specimens. The positive rate of PDAC tissues was 86.7% (26/30). Negative or very weak positive staining was observed in the most of non-tumor tissues. (C) MMP1 expression positively correlated with PNI (R=0.599, P=0.00046). (D) The number of PNI in PDAC patients with IHC MMP1 positivity was higher than MMP1-negative patients. Data are shown as the mean ± SD. * P < 0.05.