Development and In Vitro Characterization of a Gemcitabine-loaded MUC4-targeted Immunoliposome Against Pancreatic Ductal Adenocarcinoma.

BACKGROUND/AIM: Pancreatic Ductal adeno-carcinoma (PDAC) is a devastating disease. Gemcitabine is the standard chemotherapeutic agent against PDAC but has only limited effectiveness. The aim of the study was to develop and study the targeting affinity and in vitro antiproliferative effect of a MUC4-targeted gemcitabine-loaded immuno-liposome for treatment of PDAC. MATERIALS AND METHODS: Gemcitabine-loaded immunoliposomes were developed by grafting anti-MUC4 antibodies to the liposomal surface. Targeting affinity was compared in vitro between immunoliposomes and non-targeted liposomes and anti-proliferative effect was compared in vitro between free drug, non-targeted liposomal gemcitabine and MUC4-targeted immunoliposomal gemcitabine on a MUC4-positive pancreatic cancer cell line, Capan-1. RESULTS: Development of a MUC4-targeted immunoliposome was confirmed and characterized by immunoblots and size characterization. The MUC4-targeted immunoliposome showed a significantly higher targeting affinity compared to the non-targeted liposomes and also showed an improved antiproliferative effect compared to free and non-targeted liposomal drug. CONCLUSION: Successful development and characterization of a MUC4-targeted immunoliposome shows promising results for a targeted treatment and improved retention of gemcitabine for treatment of PDAC.

Low MUC4 expression is associated with survival benefit in patients with resectable pancreatic cancer receiving adjuvant gemcitabine.

BACKGROUND: Previous in vitro studies have shown that mucin 4 (MUC4) confers resistance toward gemcitabine in pancreatic cancer cells. To date, there are few clinical studies corroborating these findings. The aim of this study was to evaluate the predictive impact of MUC4 expression on survival in patients with resectable pancreatic cancer receiving adjuvant gemcitabine. MATERIALS AND METHODS: MUC4 expression was investigated by immunohistochemistry in 78 tissue sections from patients with pancreatic ductal adenocarcinoma undergoing Whipple resection. The H-score was used to evaluate MUC4 expression. The Kaplan-Meier method and Cox proportional hazards regression analysis were used to assess the predictive role of MUC4 expression. RESULTS: The MUC4 protein was expressed in 93.6% (73/78) of pancreatic cancer tissue specimens. None of the normal control pancreatic tissues had any MUC4 expression. Low MUC4 expression (H-score ≤100) was detectable in 42 (53.8%) of tumors and high MUC4 expression (H-score >100) was detectable in 36 (46.2%) of tumors. Low expression of MUC4 was associated with favorable survival (p = .027), whereas high MUC4 expression did not correlate with survival (p = .87) in patients receiving adjuvant gemcitabine treatment. CONCLUSIONS: This is the first study indicating a predictive role of MUC4 expression for gemcitabine treatment in the clinical setting.

Combining gas-phase electrophoretic mobility molecular analysis (GEMMA), light scattering, field flow fractionation and cryo electron microscopy in a multidimensional approach to characterize liposomal carrier vesicles.

For drug delivery, characterization of liposomes regarding size, particle number concentrations, occurrence of low-sized liposome artefacts and drug encapsulation are of importance to understand their pharmacodynamic properties. In our study, we aimed to demonstrate the applicability of nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyser (nES GEMMA) as a suitable technique for analyzing these parameters. We measured number-based particle concentrations, identified differences in size between nominally identical liposomal samples, and detected the presence of low-diameter material which yielded bimodal particle size distributions. Subsequently, we compared these findings to dynamic light scattering (DLS) data and results from light scattering experiments coupled to Asymmetric Flow-Field Flow Fractionation (AF4), the latter improving the detectability of smaller particles in polydisperse samples due to a size separation step prior detection. However, the bimodal size distribution could not be detected due to method inherent limitations. In contrast, cryo transmission electron microscopy corroborated nES GEMMA results. Hence, gas-phase electrophoresis proved to be a versatile tool for liposome characterization as it could analyze both vesicle size and size distribution. Finally, a correlation of nES GEMMA results with cell viability experiments was carried out to demonstrate the importance of liposome batch-to-batch control as low-sized sample components possibly impact cell viability.

Nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) of liposomes: applicability of the technique for nano vesicle batch control.

Liposomes are biodegradable nanoparticle vesicles consisting of a lipid bilayer encapsulating an aqueous core. Entrapped cargo material is shielded from the extra-vesicular medium and sustained release of encapsulated material can be achieved. However, application of liposomes as nano-carriers demands their characterization concerning size and size distribution, particle-number concentration, occurrence of vesicle building blocks in solution and determination of the resulting vesicle encapsulation capacity. These questions can be targeted via gas-phase electrophoretic mobility molecular analysis (GEMMA) based on a nano electrospray (nES) charge-reduction source. This instrument separates single-charged nanoparticles in the gas-phase according to size in a high-laminar sheath-flow by means of an orthogonal, tunable electric field. nES GEMMA analysis enables to confirm liposome integrity after passage through the instrument (in combination with atomic force microscopy) as well as to exclude vesicle aggregation. Additionally, nanoparticle diameters at peak apexes and size distribution data are obtained. Differences of hydrodynamic and dry particle diameter values, as well as the effect of number- and mass-based concentration data analysis on obtained liposome diameters are shown. Furthermore, the repeatability of liposome preparation is studied, especially upon incorporation of PEGylated lipids in the bilayer. Finally, the instruments applicability to monitor mechanical stress applied to vesicles is demonstrated.

Apicidin sensitizes pancreatic cancer cells to gemcitabine by epigenetically regulating MUC4 expression.

BACKGROUND/AIM: Mucin 4 (MUC4) has been linked to resistance to gemcitabine in pancreatic cancer cells. The aim of the present study was to assess whether epigenetic control of MUC4 expression can sensitize pancreatic cancer cells to gemcitabine treatment. MATERIALS AND METHODS: A 76-member combined epigenetics and phosphatase small-molecule inhibitor library was screened for anti-proliferative activity against the MUC4(+) gemcitabine-resistant pancreatic cancer cell line Capan-1, followed by high-content screening of protein expression. RESULTS: Apicidin, a histone deacetylase inhibitor, showed the greatest anti-proliferative activity with a lethal dose 50 (LD50) value of 5.17 µM. Apicidin significantly reduced the expression of MUC4 and its transcription factor hepatocyte nuclear factor 4α. Combined treatment with a sub-therapeutic concentration of apicidin and gemcitabine synergistically inhibited growth of Capan-1 cells. CONCLUSION: Apicidin appears to be a novel anti-proliferative agent against pancreatic cancer cells that may reverse chemoresistance by epigenetically regulating MUC4 expression.

Comparison of MUC4 expression in primary pancreatic cancer and paired lymph node metastases.

UNLABELLED: OBJECTIVE. Mucin 4 (MUC4) is a transmembrane glycoprotein that is expressed in pancreatic ductal adenocarcinoma (PDAC), but not in normal pancreatic tissue. MUC4 has a proposed role in pancreatic tumor progression and metastasis. The purpose of this pilot study was to investigate MUC4 expression during PDAC metastasis by comparing the expression in the primary tumor and paired lymph node metastases from the same patient. MATERIAL AND METHODS. Surgical specimens from 17 cases of primary PDAC and paired lymph node metastases were immunohistochemically analyzed for MUC4 expression. The modified histochemical score (H-score) was used for staining assessment. RESULTS. Positive staining for MUC4 was detected in most primary and metastatic PDAC tumors (15/17 vs. 14/17). The concordance for MUC4 expression in primary tumors and corresponding lymph node metastases was 82%. In two cases, the primary tumor was MUC4-positive and the lymph node metastases were negative, while in one patient with a MUC4-negative primary tumor, the lymph node metastasis was positive. The distribution of H-score for expression of MUC4 significantly correlated (r = 0.615; p = 0.009) between primary tumors and paired metastatic lesions. CONCLUSIONS: MUC4 was observed in both primary and matched metastatic tumors with a high level of concordance, suggesting that MUC4 expression is retained following PDAC metastasis.