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Comparative Study
. 2015 Mar 10;6(7):4717-32.
doi: 10.18632/oncotarget.3069.

Stromal-derived factor-1α/CXCL12-CXCR4 chemotactic pathway promotes perineural invasion in pancreatic cancer

Qinhong Xu  1 , Zheng Wang  1 , Xin Chen  1 , Wanxing Duan  1 , Jianjun Lei  1 , Liang Zong  1 , Xuqi Li  2 , Liang Sheng  1 , Jiguang Ma  3 , Liang Han  1 , Wei Li  1 , Lun Zhang  1 , Kun Guo  1 , Zhenhua Ma  1 , Zheng Wu  1 , Erxi Wu  4 , Qingyong Ma  1
Affiliations
Comparative Study

Stromal-derived factor-1α/CXCL12-CXCR4 chemotactic pathway promotes perineural invasion in pancreatic cancer

Qinhong Xu et al. Oncotarget. .

Abstract

Perineural invasion (PNI) is considered as an alternative route for the metastatic spread of pancreatic cancer cells; however, the molecular changes leading to PNI are still poorly understood. In this study, we show that the CXCL12/CXCR4 axis plays a pivotal role in the neurotropism of pancreatic cancer cells to local peripheral nerves. Immunohistochemical staining results revealed that CXCR4 elevation correlated with PNI in 78 pancreatic cancer samples. Both in vitro and in vivo PNI models were applied to investigate the function of the CXCL12/CXCR4 signaling in PNI progression and pathogenesis. The results showed that the activation of the CXCL12/CXCR4 axis significantly increased pancreatic cancer cells invasion and promoted the outgrowth of the dorsal root ganglia. CXCL12 derived from the peripheral nerves stimulated the invasion and chemotactic migration of CXCR4-positive cancer cells in a paracrine manner, eventually leading to PNI. In vivo analyses revealed that the abrogation of the activated signaling inhibited tumor growth and invasion of the sciatic nerve toward the spinal cord. These data indicate that the CXCL12/CXCR4 axis may be a novel therapeutic target to prevent the perineural dissemination of pancreatic cancer.

Keywords: CXCL12/CXCR4 axis; pancreatic cancer; perineural invasion; tumor microenvironment.

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Conflict of interest statement

Conflict of Interest Statement

No potential conflicts of interest were disclosed.

Figures

Fig. 1
Fig. 1. Expression levels of CXCR4 and CXCL12 in pancreatic cancers
The expression level of the CXCR4 protein (A) and mRNA (B) in six PCa cell lines (BxPC-3, CFPAC-1, Panc-1, SW1990, AsPC-1, MiaPaCa-2) and Schwann cell RSC96; (C) MiaPaCa-2, Panc-1 cells and BxPc-3 cells were labeled with a fluorescence-conjugated CXCR4-specific antibody (green). Nuclei were stained with DAPI (blue; 200× magnification).
Fig. 2
Fig. 2. Expression of CXCR4 and CXCL12 in pancreatic cancer tissues
(A) HE staining (a), immunohistochemical staining of S100 (b) served as a nerve tissue marker and CK19 (c) served as a cancer cell marker in PCa tissues with PNI; (B) The representative immunohistochemical staining for CXCR4 and CXCL12 in the normal pancreas (d and g) and the resected PCa specimens accompanied without PNI (e and h) or with PNI (f and i) (200× magnification); the peripheral nerve (red arrow) was infiltrated by PCa cells (black arrow). Scale bar, 100μm.
Fig. 3
Fig. 3. CXCL12 promoted pancreatic cancer cells metastasis and invasion via the receptor CXCR4
(A) The effects of the CXCL12/CXCR4 axis on PCa cell migration capability were assessed using a cell migration assay. The PCa cells were seeded into an uncoated chamber co-cultured with or without RSC96 for 24 h. (B) The number of migrated cells was quantified by counting the number of cells from 10 random fields at 200× magnification. (C) The effect of CXCL12/CXCR4 axis on PCa cell invasion capability was assessed using a Matrigel invasion assay. PCa cells were seeded into the Matrigel-coated invasion chamber pretreatment with or without CXCL12 for 48 h. (D) The number of invaded cells was quantified by counting the number of cells from 10 random fields at 200× magnification. The data are representative of 3 independent experiments. Column: mean (n=10); bar: SD; *P<0.05. (E) Western blot assays were performed to evaluate the effect of CXCL12/CXCR4 axis on the expression of CXCR4, MMP-2, uPA and NGF at protein level. (F) qRT-PCR was performed to evaluate the effect of CXCL12/CXCR4 axis on the expression of MMP-2, uPA and NGF at mRNA level. Column: mean; bar: SD; *P< 0.05.
Fig. 4
Fig. 4. CXCL12/CXCR4 signaling pathway mediates the development of DRG
The expression of CXCR4 and CXCL12 in DRGs and RSC96 cells was detected. (A) DRGs were labeled with fluorescence-conjugated NF-200 (red) with CXCL12 or CXCR4 specific antibodies. Nuclei were stained with DAPI (blue) (200× magnification, scale bar, 200μm). a and b represent 400× magnification pictures in the white border (scale bar, 100 μm); (B) The protein levels of CXCL12 and CXCR4 in DRGs and RSC96 cells were detected by western blot assays; (C) ELISA was performed to detect the expression of CXCL12 in conditioned media from pancreatic cancer cells, DRGs and RSC96 cells grown under serum-free condition for 72 h using a commercial kit. New-born rat DRGs were isolated and cultured in the medium containing CXCL12 (100 ng/ml) or both CXCL12 and the CXCR4 antagonist AMD3100 (2 μg/ml). (D) Five days after implantation, the DRGs were labeled with a fluorescence-conjugated NF-200 (red). Nuclei were stained with DAPI (blue). The cells were observed by an inverted light microscope imaging system 100× magnification (*P<0.05, scale bar, 500μm). (E) The number of outgrowth neurites was quantified (100× magnification). Column: mean; bar: SD; *P< 0.05.
Fig. 5
Fig. 5. CXCL12/CXCR4 axis is involved in the interaction between pancreatic cancer cells and DRG
(A) In the co-culture model, PCa cell clusters gradually migrated to the DRG, and neurite outgrowths extended from the DRG to the clusters, providing an invasive pathway for the clusters (a). This trend was significantly enhanced by adding CXCL12 (100 ng/mL) (b). AMD3100 impaired the promoting effect of CXCL12 on cancer cell cluster migration and neurite outgrowth (c). sh-CXCR4 blocked the effect of neurite-produced (d) or the exogenous CXC12 (e). Panels f, g, h, i and j display the entire shape of the DRG in each group after 7 days culture (100× magnification). The yellow line represents the initial frontier of the PCa cell colony at day 0. Scale bar, 500μm. (B) An illustration showing the calculation of nerve invasion index (α/γ) and DRG outgrowth index (β/γ). (C) The PCa cells infiltrated and migrated along the neurite. Different magnification (100×, 200×, and 400×) of the invaded cells in Fig. 5A (b) with a red dotted line was observed by fluorescent microscope under the light scope (blue arrow) and PCa cells transduced by green fluorescent protein vector (white arrow). Scale bar, 500μm; Black bar, 200μm; Red bar, 100 μm. (D) Invasion index (E) DRG outgrowth index, in the CXCL12 group were significantly increased compared with the control group. This increase was inhibited by pretreatment with the CXCR4 blocker AMD3100 and sh-CXCR4 (*P<0.05 vs. sh-Control, #P<0.05 vs. sh-Control with CXCL12).
Fig. 6
Fig. 6. Relationship between CXCL12/CXCR4 axis and PNI of pancreatic cancer in vivo
(A) Microscopic findings in mouse PNI models. Immunohistochemical staining of S100 (nerve tissue marker), CK19 (cancer cell marker) and CXCR4 were used to evaluate PNI in vivo neural invasion model. The tumor cells of sh-Control were arranged around nerves (the nerve indicated by red arrowheads). In contrast, sh-CXCR4 derived tumors exhibited no interaction between the tumor cells and nerves (Scale bar, 1mm); (B) Western blot of the resected tumors in mouse PNI models revealed a stable CXCR4 gene knockdown effect in sh-CXCR4 compared with sh-Control. MiaPaCa-2-shControl (n=6) and MiaPaCa-2-shCXCR4 (n=6) cells were implanted in a distal part of the left sciatic nerve, the right sciatic nerve served as a sham operation control (injected with saline, n=6). (C) Representative images of the spread length (in mm) between the first and fifth toes of the mouse hind limbs (a-c), xenograft tumor in situ of the sciatic nerve (d-f) and corresponding HE staining (g-i, 40× magnification) in different treatment groups (Scale bar, 1mm). (D) The mean left sciatic nerve scores in different groups were measured weekly for 7 weeks (*P< 0.05 vs. sh-Control). The sciatic nerve indexes (paw span in mm) (E) in sham operation group; (F) MiaPaCa-2-shControl group and (G) MiaPaCa-2-shCXCR4 group (P=0.018 vs. sh-Control) were measured at 1, 4, and 7 weeks after injection. The tumor volume (H) and invasion nerve diameter (I) were measured in sh-Control and the sh-CXCR4 groups (P values were determined by ANOVA, *P<0.05 vs. sh-Control).
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