Peter Jantscheff

Antimetastatic Effects of Liposomal Gemcitabine and Empty Liposomes in an Orthotopic Mouse Model of Pancreatic Cancer

Objectives: Test the efficacy of liposomal gemcitabine (GemLip) on primary tumor and metastases using the pancreatic tumor cell line AsPC1 implanted orthotopically into nude mice. Methods: The efficacy of gemcitabine and GemLip cells was tested on luciferase-transduced AsPC1 cells in vitro as well as implanted orthotopically into the pancreata of nude mice. Results: In vitro, the IC50s for GemLip and gemcitabine were 20 nM and 140 nM, respectively. However, when applied against established tumors, GemLip (8 mg/kg) blocked tumor growth for 5 consecutive weeks according to bioluminescence measurements in vivo. Gemcitabine (240 mg/kg) had no effect on luciferase-monitored tumor growth. When analyzed at the time of necropsy, GemLip strongly reduced tumor size (−64% ± [SD] 27%; ***P < 0.0001), whereas gemcitabine only weakly (−36% ± 37%) affected tumor size. Empty liposomes had no effect on the tumor size. GemLip and empty liposomes both significantly interfered with the metastatic spread to the liver, as measured using luciferase assays (GemLip, *P = 0.01; empty liposomes, *P = 0.036). In addition, they showed effects against spleen, as well as peritoneal metastases. Conclusions: GemLip presents an effective new formulation of gemcitabine, combining the targeting and protective features of the liposomes with their antimetastatic effects to target pancreatic cancer.

Lysophosphatidylcholine Pretreatment Reduces VLA-4 and P-Selectin–Mediated B16.F10 Melanoma Cell Adhesion In vitro and Inhibits Metastasis-Like Lung Invasion In vivo

Lysophosphatidylcholine (LysoPC) is an important intermediate in degradation and biosynthesis of phosphatidylcholine (PC). Reduced plasma LysoPC levels observed in patients with advanced cancer indicate a deregulation of LysoPC metabolism in metastasis. Recent data showed strong antimetastatic effects of liposomes consisting of saturated PC in a murine pancreatic metastasis model. LysoPC, generated from saturated PC after accumulation of the liposomes in tumor tissue, might be contributing to these effects. Examining effects of high local concentrations of saturated LysoPC and investigating potential molecular mechanisms, fast removal of saturated LysoPC from medium by murine B16.F10 melanoma cells and radical shifts in tumor cell membrane fatty acid (FA) composition toward saturated FAs were observed in vitro. Scanning electron microscopy revealed remarkable morphologic surface changes of LysoPC-treated tumor cells, probably causing their impaired migratory ability on fibronectin. A LysoPC concentration exceeding a threshold of about 400 μmol/L, slightly above physiologic levels, strongly reduced VLA-4–mediated binding of B16.F10 cells to VCAM-1 as well as P-selectin–dependent interaction with activated platelets, although expression levels were not altered. These findings were reflected in a syngenic intravenous lung invasion model using repeatedly ex vivo LysoPC-treated (450 μmol/L) B16.F10 cells, resulting in significantly reduced lung metastasis-like lesions (−48.3%, P = 0.006). Prior application of 50 IU unfractionated heparin further reduced lung invasion (−81.6%, P = 0.043). Our work shows for the first time that saturated LysoPC in high concentrations reduces melanoma cell adhesion in vitro and hematogeneous dissemination in vivo by direct ex vivo tumor cell targeting. Mol Cancer Ther; 10(1); 186–97. ©2011 AACR.

Combinatorial synthesis of new cationic lipids and high-throughput screening of their transfection properties.

Here we describe the first synthesis–screening approach for the identification and optimization of new cationic lipids for gene transfer in various cell lines. Combinatorial solid‐phase chemistry was used to synthesize a library of new cationic lipids based on 3‐methylamino‐1,2‐dihydroxypropane as the polar, cationic lipid part. As the nonpolar lipid part, different hydrocarbon chains were bound to the amino group of the scaffold and the amino group was further methylated to afford constantly cationic lipids. Lipids were synthesized in both configurations and as racemates, and the counter ions were also varied. By using a fully automated transfection screening method and COS‐7 cells, the cationic lipid N,N‐ditetradecyl‐N‐methyl‐amino‐2,3‐propanediol (KL‐1‐14) was identified as a candidate lipid for the development of an improved transfection reagent. Screening the transfection properties of KL‐1‐14 in numerous combinations with the helper lipids dioleoylphosphatidylethanolamine (DOPE) and cholesterol (Chol) revealed that Chol is the most suitable helper lipid and the best KL‐1‐14/Chol ratio is 0.5–0.7. Compared to the standard transfection lipid N‐[1‐(2,3‐dioleoyloxy)propyl]‐N,N,N‐trimethylammonium methyl sulfate (DOTAP), transfection efficiency was improved by a factor of about 40. Furthermore, by using R‐ and S‐configured KL‐1‐14, it could be shown that the configuration of the lipids had no significant influence on its transfection efficiency. The highest transfection efficiencies were achieved with chloride as the counter ion. The new lipofection reagent was further tested to transfect the cell lines MDA‐MB‐468, MCF‐7, MDCK‐C7, and primary dentritic cells (DC), which are important for the development of new anticancer gene therapy strategies. Even in these cells, KL‐1‐14/Chol (1:0.6) had improved transfection efficiencies, which were about two to four times higher than for DOTAP.

Liposomal gemcitabine (GemLip)-efficient drug against hormone-refractory Du145 and PC-3 prostate cancer xenografts.

Gemcitabine (Gemc) is an efficient chemotherapeutic drug in various cancer types (e.g., pancreas) but has only limited effects on hormone‐refractory prostate cancer (HRPCa). Since HRPCa cells are highly sensitive to even low doses of Gemc in vitro, the lack of clinical effects might be due to rapid degradation of Gemc by deaminases combined with impaired accumulation in tumor tissue and PCa cells. Liposomal formulation (GemLip) is expected to protect the entrapped cytotoxic substance from enzymatic degradation and furthermore augment its accumulation within tumor tissues due to an enhanced permeability of the tumor vessels.

A novel two-stage backpressure-independent micropump: modeling and characterization

A novel design of a piezoelectric silicon micropump is proposed, which provides a constant flow rate over a wide backpressure range of up to 30 kPa. This highly appreciable feature is based on a new serial arrangement of two active valves and relies on both an appropriate electrical actuation sequence of the piezo-actuators and an immanent limitation of the membrane deflection by the valve seats. The design is optimized for the low flow regime ranging from 0.1 to 50 µl min−1. A detailed lumped-parameter model is derived in order to reveal the physics behind this pumping principle and to identify the optimum control scheme. For the fabrication of our device, a comparably simple and robust 2-wafer process is utilized. A thorough experimental investigation demonstrates the high performance of the micropump. The backpressure independence of the flow rate enables high-resolution volumetric dosing within the aforementioned flow range. The stroke volume and hence the resolution of the micropump is adjustable via the upstroke voltage of the actuator between 50 and 200 nl. Depending on this setting typical actuation frequencies range from 0.05 to 5 Hz and the flow rate scales proportional to the frequency within that frequency range.

Pressure-Independent Micropump with Piezoelectric Valves for Low Flow Drug Delivery Systems

We present - for the first time - a novel design of a micropump which enables a backpressure-independent flow rate up to 20 kPa within the low flow regime required for drug delivery systems. Our concept, based on two piezoelectrically actuated diaphragms, allows an accurate dosing in the range of 1 - 50 µ l/min with freely programmable release profiles and offers the potential to minimize chip size and power consumption in comparison to 3-actuator peristaltic micropumps. The stroke volume is adjustable between 50 - 200 nl by means of voltage control which enables a high resolution volumetric dosing. Within the relevant frequency range below 2 Hz the flow rate is proportional to the frequency. Our design also excels in its comparably simple and robust 2-layer fabrication process.

Saturated and mono-unsaturated lysophosphatidylcholine metabolism in tumour cells: a potential therapeutic target for preventing metastases

BackgroundMetastasis is the leading cause of mortality in malignant diseases. Patients with metastasis often show reduced Lysophosphatidylcholine (LysoPC) plasma levels and treatment of metastatic tumour cells with saturated LysoPC species reduced their metastatic potential in vivo in mouse experiments. To provide a first insight into the interplay of tumour cells and LysoPC, the interactions of ten solid epithelial tumour cell lines and six leukaemic cell lines with saturated and mono-unsaturated LysoPC species were explored.MethodsLysoPC metabolism by the different tumour cells was investigated by a combination of cell culture assays, GC and MS techniques. Functional consequences of changed membrane properties were followed microscopically by detecting lateral lipid diffusion or cellular migration. Experimental metastasis studies in mice were performed after pretreatment of B16.F10 melanoma cells with LysoPC and FFA, respectively.ResultsIn contrast to the leukaemic cells, all solid tumour cells show a very fast extracellular degradation of the LysoPC species to free fatty acids (FFA) and glycerophosphocholine. We provide evidence that the formerly LysoPC bound FFA were rapidly incorporated into the cellular phospholipids, thereby changing the FA-compositions accordingly. A massive increase of the neutral lipid amount was observed, inducing the formation of lipid droplets. Saturated LysoPC and to a lesser extent also mono-unsaturated LysoPC increased the cell membrane rigidity, which is assumed to alter cellular functions involved in metastasis. According to that, saturated and mono-unsaturated LysoPC as well as the respective FFA reduced the metastatic potential of B16.F10 cells in mice. Application of high doses of liposomes mainly consisting of saturated PC was shown to be a suitable way to strongly increase the plasma level of saturated LysoPC in mice.ConclusionThese data show that solid tumours display a high activity to hydrolyse LysoPC followed by a very rapid uptake of the resulting FFA; a mechanistic model is provided. In contrast to the physiological mix of LysoPC species, saturated and mono-unsaturated LysoPC alone apparently attenuate the metastatic activity of tumours and the artificial increase of saturated and mono-unsaturated LysoPC in plasma appears as novel therapeutic approach to interfere with metastasis.

Metastasizing, Luciferase Transduced MAT‑Lu Rat Prostate Cancer Models: Follow up of Bolus and Metronomic Therapy with Doxorubicin as Model Drug

The most fatal outcomes of prostate carcinoma (PCa) result from hormone-refractory variants of the tumor, especially from metastatic spread rather than from primary tumor burden. The goal of the study was to establish and apply rat MAT-Lu prostate cancer tumor models for improved non-invasive live follow up of tumor growth and metastasis by in vivo bioluminescence. We established luciferase transduced MAT-Lu rat PCa cells and studied tumor growth and metastatic processes in an ectopic as well as orthotopic setting. An intravenous bolus treatment with doxorubicin was used to demonstrate the basic applicability of in vivo imaging to follow up therapeutic intervention in these models. In vitro analysis of tissue homogenates confirmed major metastatic spread of subcutaneous tumors into the lung. Our sensitive method, however, for the first time detects metastasis also in lymph node (11/24), spleen (3/24), kidney (4/24), liver (5/24), and bone tissue (femur or spinal cord - 5/20 and 12/20, respectively). Preliminary data of orthotopic implantation (three animals) showed metastatic invasion to investigated organs in all animals but with varying preference (e.g., to lymph nodes). Intravenous bolus treatment of MAT-Lu PCa with doxorubicin reduced subcutaneous tumor growth by about 50% and the number of animals affected by metastatic lesions in lymph nodes (0/4), lung (3/6) or lumbar spine (0/2), as determined by in vivo imaging and in vitro analysis. Additionally, the possible applicability of the luciferase transduced MAT-Lu model(s) to study basic principles of metronomic therapies via jugular vein catheter, using newly established active microport pumping systems, is presented.

An Implantable Active Microport Based on a Self-Priming High-Performance Two-Stage Micropump

This paper reports on significant progress in the development of an implantable active microport system for an automated administration of aqueous drug suspensions. A novel piezoelectric two-stage micropump ensures the controlled release of minute amounts of fluid with flow rates between 0.1 mul/min and 50 mul/min. A modification of the chamber design reduces the detrimental effect of entrapped air bubbles. Due to an increased compression ratio the micropump has now a full capability to pump gas which enables a reliable self-priming. Moreover, the absence of air bubbles in the pump chamber yields a significantly enhanced accuracy of the delivered fluid volumes.

Abstract 1156: New spontaneous and carcinogen-induced mouse-derived isograft (MDI) tumor models for immune therapeutic approaches

First findings in the late 1990s and the early 2000s, that blockade of immune checkpoint inhibitors (ICI) by antibodies could induce rejection of established tumors and induce immunity also to secondary exposure with these tumor cells, led again to a stronger focus of experimental studies on syngeneic tumor models in immunocompetent animals. The availability of such models, however, is mainly limited by the small number of genetically-modified (GEM) or long-term passaged cell line-derived tumor models. We here describe establishment and first characterization of a new type of animal tumor models, named Mouse-Derived-Isografts (MDI), from spontaneously appearing or carcinogen-induced syngeneic tumors in both sexes of various mouse strains (C3H/HeN, CBA/J, BALB/C, DBA/2N and C57BL/6N). The tumors were obtained from animals during long-term observation (≥ 1-2 years) of normally fed, otherwise untreated animals (spontaneous - sMDI) or during short-term observation (3-9 month) of once subcutaneously, intramuscularly, intraperitoneally or orally methylcholanthrene (MCA) or methyl-nitroso-urea (MNU) treated animals (carcinogen - cMDI). Criteria to perform a primary necropsy were critical weight loss, bad general conditions or externally observable tumor growth in monitored animals. At this first step, tissues were assessed only macroscopically, and conspicuous ones were transplanted (in PDX-like manner) as small pieces into sex-matched syngeneic animals. If possible tissue/tumor pieces were also frozen and stored in 10% DMSO freezing medium at -80°C. In a second step these re-transplanted, outgrowing tumors were amplified once again in syngeneic mice for freeze storing, and then the models were finally established by re-transplantation and testing outgrowth of the frozen stored tumor pieces in the syngeneic animals. The tumors appear phenotypically stable, as far as routine standard HE stained sections could show. General characteristics of these models are; they are primary spontaneous or carcinogen-induced tumors, of low passage number ( Citation Format: Peter Jantscheff, Janette Beshay, Thomas Lemarchand, Cynthia Obodozie, Christoph Schaechtele, Holger Weber. New spontaneous and carcinogen-induced mouse-derived isograft (MDI) tumor models for immune therapeutic approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1156.