The early diagnosis of cancer is the critical element in successful treatment and long term favorable patient prognoses. The high rate of mortality is mainly attributed to the tendency for late diagnoses as symptoms may not occur until the disease has metastasized, as well as the lack of effective systemic therapies. Late diagnosis is often associated with the lack of timely sensitive imaging modalities. The promise of nanotechnology is presently limited by the inability to simultaneously seek, treat and image cancerous lesions. This study describes the design and synthesis of fluorescent calcium phosphosilicate nanocomposite particles (CPNPs) that can be systemically targeted to breast and pancreatic cancer lesions. The CPNPs are a ~20nm diameter composite composed of an amorphous calcium phosphate matrix doped with silicate in which a near infra-red imaging agent indocyanine green (ICG) is embedded. In the present studies, we describe and validate CPNP bioconjugation of human holotransferrin, anti-CD71 antibody, and short gastrin peptides via an avidin-biotin-or a novel PEG-maleimide-coupling strategy. The conjugation of biotinylated human holotransferrin (diferric transferrin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to avidin conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors, which are highly expressed on breast cancer cells. Similarly, the conjugation of biotinylated pentagastrin to AvidinCPNPs and decagastrin (gastrin-10) to PEG-CPNPs via PEG-maleimide coupling permits targeting of gastrin receptors, which are over-expressed in pancreatic cancer lesions. These bioconjugated CPNPs have the potential to perform as a theranostic modality, simultaneously enhancing drug delivery, targeting and imaging of breast and pancreatic cancer tumors. Keywords bioconjugation; transferrin receptor; gastrin receptor; breast cancer; pancreatic cancer; calcium phosphate; whole animal imaging Calcium phosphate nanoparticles (CPNPs) have been engineered to be a non-toxic vehicle for the delivery of a diverse range of therapeutic and imaging agents in biological systems. [1][2][3][4] Previous studies have shown that encapsulation within CPNPs improved the lifetime and RESULTS AND DISCUSSION Physical Characterization of CPNPsCitrate functionalized CPNPs were utilized as a platform for functionalization, which allowed the characterization of bioconjugation via zeta potential analysis (Figure 1). Figure 1 shows the zeta potential distribution of Citrate-CPNPs prior to bioconjugation (blue line), and the zeta (violet). Prior to bioconjugation, the Citrate-CPNPs display a negative mean zeta potential value of −16 ± 1.3 mV, which is consistent with previous reports. 1 However, after bioconjugation, the Avidin-CPNPs displayed a relatively high positive mean zeta potential value of +29 ± 8.7 mV. The isoelectric point for avidin is pH 10. Thus, the shift from a negative zeta potential to a positive zeta potential distribution is strong evidence of avidin bioconjugation on...

Leukemia is one of the most common and aggressive adult cancers, as well as the most prevalent childhood cancer. Leukemia is a cancer of the hematological system and can be divided into a diversity of unique malignancies based on the onset of the disease as well as the specific cell lineages involved. Cancer stem cells, including recently identified leukemia stem cells (LSCs), are hypothesized to be responsible for cancer development, relapse, and resistance to treatment, and new therapeutics targeting these cellular populations are urgently needed. Nontoxic and nonaggregating calcium phosphosilicate nanoparticles (CPSNPs) encapsulating the near-infrared fluoroprobe indocyanine green (ICG) were recently developed for diagnostic imaging and drug delivery as well as for photodynamic therapy (PDT) of solid tumors. Prior studies revealed that specific targeting of CPSNPs allowed for enhanced accumulation within breast cancer tumors, via CD71 targeting, or pancreatic cancer tumors, via gastrin receptor targeting. In the present study, ICG-loaded CPSNPs were evaluated as photosensitizers for PDT of leukemia. Using a novel bioconjugation approach to specifically target CD117 or CD96, surface features enhanced on leukemia stem cells, in vitro ICG-CPSNP PDT of a murine leukemia cell line and human leukemia samples were dramatically improved. Furthermore, the in vivo efficacy of PDT was dramatically enhanced in a murine leukemia model by utilizing CD117-targeted ICG-CPSNPs, resulting in 29% disease-free survival. Altogether, this study demonstrates that leukemia-targeted ICG-loaded CPSNPs offer the promise to effectively treat relapsing and multidrug-resistant leukemia and to improve the life of leukemia patients.

Here we describe isolation and characterization of macrophage-tumor cell fusions (MTFs) from the blood of pancreatic ductal adenocarcinoma (PDAC) patients. The MTFs were generally aneuploidy, and immunophenotypic characterizations showed that the MTFs express markers characteristic of PDAC and stem cells, as well as M2-polarized macrophages. Single cell RNASeq analyses showed that the MTFs express many transcripts implicated in cancer progression, LINE1 retrotransposons, and very high levels of several long non-coding transcripts involved in metastasis (such as MALAT1). When cultured MTFs were transplanted orthotopically into mouse pancreas, they grew as obvious well-differentiated islands of cells, but they also disseminated widely throughout multiple tissues in “stealth” fashion. They were found distributed throughout multiple organs at 4, 8, or 12 weeks after transplantation (including liver, spleen, lung), occurring as single cells or small groups of cells, without formation of obvious tumors or any apparent progression over the 4 to 12 week period. We suggest that MTFs form continually during PDAC development, and that they disseminate early in cancer progression, forming “niches” at distant sites for subsequent colonization by metastasis-initiating cells.

Background Endogenous opioid peptides have been shown to play a role in the development and/or perpetuation of inflammation. We hypothesize that the endogenous opioid system is involved in inflammatory bowel disease, and antagonism of the opioid–opioid receptor will lead to reversal of inflammation. Aims A randomized double-blind placebo-controlled study was designed to test the efficacy and safety of an opioid antagonist for 12 weeks in adults with active Crohn’s disease. Methods Forty subjects with active Crohn’s disease were enrolled in the study. Randomized patients received daily oral administration of 4.5-mg naltrexone or placebo. Providers and patients were masked to treatment assignment. The primary outcome was the proportion of subjects in each arm with a 70-point decline in Crohn’s Disease Activity Index score (CDAI). The secondary outcome included mucosal healing based upon colonoscopy appearance and histology. Results Eighty-eight percent of those treated with naltrexone had at least a 70-point decline in CDAI scores compared to 40% of placebo-treated patients (p = 0.009). After 12 weeks, 78% of subjects treated with naltrexone exhibited an endoscopic response as indicated by a 5-point decline in the Crohn’s disease endoscopy index severity score (CDEIS) from baseline compared to 28% response in placebo-treated controls (p = 0.008), and 33% achieved remission with a CDEIS score <6, whereas only 8% of those on placebo showed the same change. Fatigue was the only side effect reported that was significantly greater in subjects receiving placebo. Conclusions Naltrexone improves clinical and inflammatory activity of subjects with moderate to severe Crohn’s disease compared to placebo-treated controls. Strategies to alter the endogenous opioid system provide promise for the treatment of Crohn’s disease.

Objectives Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved with the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. Methods The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-KrasG12D transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK-receptor antagonist (proglumide, 0.1mg/ml). Pancreas from mice were removed and examined histologically for number and grade of PanINs after 1, 2 or 4 months of antagonist therapy. Results Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed and progression to advanced lesions arrested in mice treated with proglumide compared to controls (p=0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared to vehicle (pitalic>0.001). Conclusions These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. Use of CCK-receptor antagonists may have a role in cancer prophylaxis in high risk subjects, and may reduce fibrosis in the microenvironment.

Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against “exposed” CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bioconjugation of AP1153 to the surface of fluorescent NPs greatly facilitated delivery of NPs to PDAC tumors in vivo. The selectivity of this AP-targeted NP delivery system holds promise for enhanced early detection of PDAC lesions as well as improved chemotherapeutic treatments for PDAC patients.

Background Obesity and dietary fat are associated with increased risk of several malignancies including pancreatic cancer. The incidence of pancreatic cancer is increased in countries that consume diets high in fat. Aim The purpose of this study was to assess the relationship and mechanism of action between dietary fat and endogenous cholecystokinin (CCK) on pancreatic tumor growth and metastasis in an immunocompetent animal model. Methods C57BL/6 mice were placed on regular, low-fat, or high-fat diets for 8 weeks before establishment of Panc-02 orthotopic pancreatic tumors. Mice were then treated with a CCK-A receptor antagonist, devazepide, or vehicle for an additional 2.5 weeks. Pancreas tumors were weighed and metastases counted. Blood CCK levels were measured by radioimmunoassay (RIA). Tissues were examined histologically and studied for genes associated with metastasis by RT-PCR array. Effects of the CCK antagonist on Panc-02 cells invasiveness was assessed in a Matrigel invasion assay. Results Mice that received the high-fat diet had larger tumors and tenfold higher serum CCK levels by RIA compared to normal diet controls (p < 0.01). Pancreatic tumors in high-fat diet mice treated with the antagonist had fewer intravascular tumor emboli and metastases compared to controls. The reduction in tumor emboli correlated with decreased vascular endothelial growth factor-A (VEGF-A) expression in tumors (p < 6 × 10−9). In vitro invasiveness of Panc-02 cells also was reduced by CCK-A receptor antagonist treatment (p = 1.33 × 10−6). Conclusion CCK is a mediator of dietary fat-associated pancreatic cancer. CCK is also involved in the invasiveness of pancreatic tumors through a mechanism involving VEGF-A.

Objectives This study evaluated the effects of gastrin mRNA down-regulation on growth of human pancreatic cancer. Methods Gastrin expression was examined in human pancreatic cancer cell lines by RT-PCR and peptide expression was assessed by immunocytochemistry. Gastrin was down-regulated using either stable transfection of an antisense gastrin cDNA or one of three shRNA (short hairpin RNA) constructs. Tumor formation was evaluated following either subcutaneous or orthotopic injections into nude mice. The effect of nanoliposomes loaded with gastrin siRNA was tested in mice bearing pancreatic tumors. Results Stable transfection of gastrin antisense or shRNAs into BxPC-3 cells resulted in clones with >90% reduction in gastrin mRNA. Tumor growth rate and incidence of metastases in both wild type and transfected pancreatic cancer cells was directly proportional to the degrees of gastrin mRNA expression. Immunofluoresence analysis confirmed that gastrin peptide levels were decreased in antisense and shRNA tumors. Gastrin knockdown clones had lower Ki-67 and increased cleaved caspase-3 staining, consistent with known effects of gastrin on proliferation and apoptosis. Tumors in mice treated with gastrin siRNA were smaller than controls. Conclusions These results suggest that RNAi targeting of gastrin could serve as an effective treatment for pancreatic cancer.