A Cholecystokinin B Receptor-Specific Aptamer Does Not Activate Receptor Signaling

Targeted nanoparticles which deliver effective doses of chemotherapeutic drugs directly to pancreatic tumors could improve treatment efficacy without the toxicities associated with systemic drug administration. One protein on tumor cells that can be targeted by nanoparticles is a G-protein coupled cell surface receptor, the cholecystokinin B receptor (CCKBR). Previously, we had shown that attaching the CCKBR ligand gastrin to the surface of nanoparticles can enhance their up-take by tumors. The drawback of using gastrin is that it can also activate the receptor, causing tumor cell growth. This study shows that a DNA aptamer that binds to the CCKBR and enhances nanoparticle up-take by tumors does not activate this receptor. PANC-1 cells, a cultured human pancreatic cancer cell line, were treated for 24 h with CCKBR aptamer 1153. Cell lysates were run on Bis-Tris gels, transferred to membranes, blocked in 5% BSA and incubated overnight with primary antibodies, including antibodies directly against phosphorylated-Akt (Ser473), total Akt, and beta-actin, a protein loading control. Although the CCKBR aptamer 1153 is internalized by pancreatic cancer cells in a receptor-mediated fashion, it does not stimulate cell proliferation. Because of this, we anticipate that it will not activate CCKBR signaling. If aptamer 1153 does not activate downstream receptor signaling, our future work will test whether the aptamer could be used to specifically direct drug-containing nanoparticles to tumors, making chemotherapy treatments for pancreatic cancer patients more effective with fewer off-target effects and toxicity

Utilizing Peptide Ligand GPCRs to Image and Treat Pancreatic Cancer

It is estimated that early detection of pancreatic ductal adenocarcinoma (PDAC) could increase long-term patient survival by as much as 30% to 40% (Seufferlein, T. et al., Nat. Rev. Gastroenterol. Hepatol. 2016, 13, 74–75). There is an unmet need for reagents that can reliably identify early cancerous or precancerous lesions through various imaging modalities or could be employed to deliver anticancer treatments specifically to tumor cells. However, to date, many PDAC tumor-targeting strategies lack selectivity and are unable to discriminate between tumor and nontumor cells, causing off-target effects or unclear diagnoses. Although a variety of approaches have been taken to identify tumor-targeting reagents that can effectively direct therapeutics or imaging agents to cancer cells (Liu, D. et al., J. Controlled Release 2015, 219, 632–643), translating these reagents into clinical practice has been limited, and it remains an area open to new methodologies and reagents (O’Connor, J.P. et al., Nat. Rev. Clin. Oncol. 2017, 14, 169–186). G protein–coupled receptors (GPCRs), which are key target proteins for drug discovery and comprise a large proportion of currently marketed therapeutics, hold significant promise for tumor imaging and targeted treatment, particularly for pancreatic cancer

Expression of the Human Gastrin Receptors CCKBR and CCKCR in PANCO2 Murine Pancreatic Cancer Cells

Pancreatic cancer is the fourth leading cause of cancer mortality. The gastrointestinal hormone, gastrin, stimulates human pancreatic cancer growth via its receptor, CCKBR, and a splice-variant of CCKBR (termed CCKCR) detected only in cancer cells. This cancer-associated variant retains the fourth intron and exhibits increased cell signaling. Recently, a single nucleotide polymorphism (SNP; C\u3eA) in the fourth intron was identified. Presence of SNP(A) was correlated with CCKCR protein expression in pancreatic cancer specimens and decreased patient survival. We hypothesize expression of human CCKCR in murine pancreatic cancer cells will increase tumor growth and metastasis relative to expression of human CCKBR. The aim of this study was to engineer PANC02 murine pancreatic cancer cells to express hCCKBR or hCCKCR (each SNP form). While vectors encoding hCCKBR and hCCKCR-SNP(C) existed, a version mutated to form the SNP(A) required sub-cloning into a vector capable of transfection and selection in mammalian cells. The gene was excised and ligated into pCAGEN.neo. Resulting clones were screened by restriction analysis to confirm insertion and correct orientation of the gene. DNA sequencing confirmed the status of the SNP in each vector. Next, hCCKBR, hCCKCR-SNP(C), hCCKCR-SNP(A) and empty-vector (pCAGEN.neo) were transfected in parallel into PANC02 cells. Resulting neomycin-resistant clones were isolated and RNA was harvested. Clones were screened for up-regulation of corresponding receptor mRNA by endpoint RT-PCR (GAPDH , loading control). Up-regulation was evident in most clones: hCCKBR (11 of 11 clones), hCCKCR-SNP(C) (7 of 10), and hCCKCR-SNP(A) (11 of 11). Real-time RT-PCR analysis is currently ongoing to more precisely quantify mRNA expression relative to empty-vector controls. The results of this study will permit in vivo tumor growth and immunotherapy studies in an immune-competent syngeneic murine model

Tumor-promoting effects of pancreatic cancer cell exosomes on THP-1-derived macrophages.

Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14hi CD163hi CD206hi). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1β, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression

Role of Endogenous Cholecystokinin on Growth of Human Pancreatic Cancer

Cholecystokinin (CCK) and gastrin stimulate growth of pancreatic cancer. Although down-regulation of gastrin inhibits growth of pancreatic cancer, the contribution of endogenous CCK to tumor growth is unknown. The purpose of this study was to evaluate the role of endogenous CCK on autocrine growth of pancreatic cancer. Pancreatic cancer cell lines were analyzed for CCK mRNA and peptide expression by real-time RT-PCR and radioimmunoassay, respectively. The effect of endogenous CCK on growth was evaluated by treating cancer cells with CCK neutralizing antibodies and by down-regulating CCK mRNA by RNAi. Wild-type pancreatic cancer cells expressed significantly lower CCK mRNA and peptide levels than gastrin. Neither treatment of pancreatic cancer cells with CCK antibodies nor the down-regulation of CCK mRNA and peptide by shRNAs altered growth in vitro or in vivo. Conversely, when gastrin mRNA expression was down-regulated, the same cells failed to produce tumors in spite of having sustained levels of endogenous CCK. Pancreatic cancer cells produce CCK and gastrin; however, the autocrine production of gastrin is more important for stimulating tumor growth