Paracrine Activation of Chemokine Receptor CCR9 Enhances The Invasiveness of Pancreatic Cancer Cells

Abstract

Chemokine receptors mediate cancer progression and metastasis. We have previously examined chemokine receptor CCR9 expression in pancreatic cancer. Here, our objective was to evaluate pancreatic stellate cells (PSCs) as a source of CCL25, the CCR9 ligand, and as an activator of CCL25-CCR9 signaling in pancreatic cancer cells. CCL25 and CCR9 expression levels in human pancreatic cancer tissues and normal human pancreas were assessed by immunohistochemsitry. In vitro secretion of CCL25 in PSCs and PANC-1 cells was verified by enzyme-linked immunosorbent assay. Pancreatic cancer cell invasion was measured using a modified Boyden chamber assay with CCL25, PSC secreted proteins, and PANC-1 secreted proteins as the chemoattractant. There was immunostaining for CCR9 expression in human pancreatic tumor tissues, but not in normal pancreatic tissue. CCL25 expression was absent in the normal pancreatic tissue sample, but was observed in cancer cells and in the stromal cells surrounding the tumor. In vitro, both PANC-1 cells and PSCs secreted CCL25. In an invasion assay, exposure to CCL25, PSC- and PANC-1-conditioned media significantly increased the invasiveness of PANC-1 cells. Inclusion of a CCR9-neutralizing antibody in the invasion assay blocked the increase in invading cells elicited by the chemoattractants. Our studies show that pancreatic cancer invasiveness is enhanced by autocrine and paracrine stimulation of CCR9. PSCs in the tumor microenvironment appear to contribute to paracrine activation of CCR9. Investigations into CCR9 as a potential therapeutic target in pancreatic cancer must consider cancer cell autocrine signaling and also paracrine signaling from interactions in the tumor microenvironment.

Fig. 1

CCR9 and CCL25 are expressed in pancreatic cancer tissue samples. Formalin-fixed paraffin-embedded human normal pancreatic tissue and pancreatic tumor tissue were stained with anti-CCR9 or anti-CCL25 antibodies. The normal pancreatic tissue sample shows tall columnar cells with basally located nuclei. There is an absence of CCR9 staining throughout the tissue a. Malignant ductal adenocarcinoma lesion demonstrates nuclear crowding and strong immunostaining for cytoplasmic CCR9 protein b. There is an absence of CCL25 staining within the normal architecture of the pancreatic duct and stroma c. In the malignant pancreatic lesion, with characteristic nuclear crowding, enlarged nuclei, and rare mitoses, there is moderate heterogeneous immunostaining for CCL25 protein in the nuclei. Moderate CCL25 staining throughout the stroma is also seen d. (Magnification 100x and 200x)

Fig. 2

PSC and PANC-1 cells secrete CCL25. PSC and PANC-1 cells were incubated for 24 h after which time cell culture media was collected. An ELISA was utilized to measure the amount of secreted CCL25 present in the media. Over a 24-hour incubation period PSCs secreted 67.4 pg/ml and PANC-1 secreted 77.6 pg/ml. Data shown represent the average of two independent experiments, error bars show standard deviation

Fig. 3

PANC-1 cells show increased invasion towards CCL25, PSC and PANC-1 conditioned media. CCL25 or PSC and PANC-1 cells were placed in the bottom of a matrigel chamber. 24 h later, PANC-1 cells were seeded on top of the matrigel +/− a CCR9-neutralizing antibody and allowed to invade for 24 h prior to fixation and quantification. There was an average increase in invasion towards CCL25 and the PSC and PANC-1 cells from 121 cells in the control condition to 185 and 339, respectively. Data shown represent the average of two independent experiments, error bars show standard deviation