Ernst Waelti

Delivery to cancer cells of antisense L- myc oligonucleotides incorporated in fusogenic, cationic-lipid-reconstituted influenza-virus envelopes (cationic virosomes)

Antisense oligodeoxy‐nucleoside phosphorothioates (OPTs) of L‐myc were encapsulated into reconstituted influenza‐virus‐A envelopes (virosomes). The envelopes of the virosomes consisted of a single positively charged (cationic) lipid bilayer. Binding of cationic virosomes to cellular receptors that are membrane glycoproteins or glycolipids containing terminal sialic acid is mediated by the hemagglutinin glycoprotein (HA) of the influenza virus. After internalization through receptor‐mediated endocytosis, cationic virosomes fuse efficiently with the membranes of the endosomal‐cell compartment, and as a consequence the encapsulated OPT are delivered to the cell cytoplasma. Examination by fluorescence microscopy of the cellular uptake of cationic virosomes containing fluorescein‐labeled OPT showed rapid and efficient incorporation of virosomes. Addition of cationic virosomes (75–150 μl) containing antisense L‐myc OPT in the picomolar range to small‐cell‐lung‐cancer (SCLC) cell cultures that expressed highly the L‐myc oncogene led to strong inhibition of thymidine incorporation in a concentration‐dependent manner. Virosome‐entrapped sense L‐myc OPT and random‐order OPT had only minimal effects on the thymidine uptake. Cells of SCLC cell line NCI‐H82 expressing a very low level of L‐myc were not affected by antisense‐L‐myc virosomes. In Western‐blot analysis, expression of L‐myc protein was suppressed in the antisense‐virosome‐treated NCI‐H209 cells but not in untreated control NCI‐H209 cells. These results suggest that cationic virosomes may have great potential as an efficient delivery system for antisense oligonucleotides in cancer therapy. Int. J. Cancer 77:728–733, 1998.

Soluble factors from human hair papilla cells and dermal fibroblasts dramatically increase the clonal growth of outer root sheath cells

Depending on environmental influences, follicular outer root sheath (ORS) cells in vivo can differentiate either towards interfollicular keratinocytes or, as demonstrated in the rat vibrissa, hair matrix cells. Crucial regulators of both their proliferation and differentiation are the mesenchymal cells of the respective tissues. The interactions of human ORS cells with human hair papilla cells (HPC) or human dermal fibroblasts (HDF) were studied using a two-chamber model separating the two cell types either by a microporous membrane or additionally by a medium layer. The results of 3H-thymidine incorporation studies indicated that ORS cell growth was markedly enhanced in co-culture with either HPC or HDF, the highest stimulatory effect resulting when ORS cells were in close association with the mesenchymal cells. No correlation was found between ORS cell proliferation and IL-6 production in the co-culture system, thus pointing to the secretion by HPC and HDF of growth-promoting soluble factors that are different form IL-6 as well as from EGF, bFGF and insulin present in the culture medium.

Endocrine Manipulation of Meningiomas with Medroxyprogesterone Acetate

Intracranial meningiomas from patients treated with medroxyprogesterone acetate as well as from untreated patients were studied in monolayer tissue culture with trials of in vitro hormonal modulation with medroxyprogesterone acetate. The following conclusions were drawn from investigations which comprise 37 cell culture assays: (a) tissue cultures of meningiomas inherit the disadvantages of loss of the progesterone receptor and frequent transformation to cells resembling fibroblasts after three to four passages. For these reasons, drug testing as well as the establishment of cell cultures that exhibit the characteristics of meningioma are impeded; (b) the progesterone receptor-content of the solid tumors does not reflect the response to medroxyprogesterone acetate-therapy in vitro; (c) medroxyprogesterone acetate-pretreated meningiomas showed sufficient in vitro growth in 38%, and untreated meningiomas grew well in 56% of the cases; (d) medroxyprogesterone acetate-induced inhibition or delay of growth was observed in 35%. These findings have resulted in criticism with respect to the value of meningioma tissue cultures for trials of hormonal manipulation and it is thought that another method, which consists of immunostaining of cycling cells, and has been tested in another study, may be superior to cell culture assays with respect to evaluation of the effect of hormonotherapy in meningiomas. Medroxyprogesterone acetate holds an interesting position because it reduces cell growth in some meningiomas in vitro.

Hormonotherapy of meningiomas with medroxyprogesterone acetate: Immunohistochemical demonstration of the effect of medroxyprogesterone acetate on growth fractions of meningioma cells using the monoclonal antibody Ki-67

The effect of medroxyprogesterone acetate (MPA) on growth fractions of ex vivo meningiomas is demonstrated in using the Ki-67 monoclonal antibody in three cases of meningiomas operated on in two stages and in meningioma specimens from a group of eight patients operated on in one single stage after MPA therapy. Growth fractions in samples from five meningioma patients not treated with MPA were determined for comparison. In the three cases of two-stage operation of the tumors, the percentage of Ki-67-positive cells in meningioma tissue was lower by a factor of 6, 5, and 3, respectively, after MPA therapy. In meningioma specimens from patients receiving no MPA therapy, Ki-67-positive cells were present in 1.02 +/- 0.48%; in samples from MPA-treated tumors the percentage of Ki-67-positive cells was 0.41 +/- 0.40 (different at p less than 0.02 [Wilcoxons test]). In comparison to our previously published data on untreated meningiomas analyzed for progesterone receptors (PR), MPA significantly reduced the PR activity. There was no obvious correlation between PR activity and potential suppression of the tumor growth fraction. It is concluded that MPA is attractive because it reduces the growth fractions of most meningiomas and might be suitable for adjuvant hormonotherapy.

Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab′ fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Students t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the “angiogenic switch” of the tumours.

Endocrine manipulation of meningiomas with medroxyprogesterone acetate: effect of MPA on receptor status of meningioma cytosols

Fifteen patients with intracranial or spinal meningiomas have been treated with the semisynthetic progestational agent medroxyprogesterone acetate (MPA, Depo-Provera) prior to surgical removal of the tumors in order to investigate the influence of MPA on the progesterone receptor (PR) status of meningioma cytosols. MPA acted as a competitive binder to meningioma-PR: The mean PR values were 15.6 fmol/mg protein (range 0-69) and 338.3 fmol/g tumor (range 0-1190), respectively. In comparison, mean PR values of our untreated meningioma series (n = 58) were 54.9 fmol/mg protein (range 0-586) and 2813 fmol/g tumor (range 0-17,168), respectively. In cases of two-stage resection of meningiomas MPA significantly decreased PR activity in the cytoplasm of meningioma cells. We conclude that MPA binds to meningioma PRs, however, its effect on the growth rate of meningiomas has still to be elucidated.

Estrogen and progestin receptors in acoustic and spinal neurilemmomas.: Clinicopathologic correlations

Estradiol and progestin receptors were studied in 20 patients with neuraxial Schwann cell tumors, and their presence was correlated to the clinicopathologic features and the amount of preoperative corticosteroid therapy. Based on an arbitrary cutoff value of 200 fmol per gram of tumor as indicative of a positive receptor value in breast cancer, 4 and 13 of the neurilemmoma tissue samples could be considered as positive for estrogen and progesterone receptors, respectively. Whereas there was no convincing correlation between the estrogen and progestin receptor activity and the age, sex, or menopausal status of the patients, overweight patients had significantly higher estrogen and progestin binding values. The correlation between the amount of preoperative prednisone therapy and the amount of [3H]estradiol and [3H]promegestone binding revealed no dose relationship. Correlating [3H]estradiol and [3H]promegestone content with the histologic type of the schwannomas (Antoni types A and B, respectively), we were not able to draw conclusions, because of the predominance of Antoni type A over Antoni type B tissues in our material. The necessity of nuclear receptor assays, ligand specificity testing, and in vitro studies is stressed as a prerequisite for answering the questions whether neurilemmomas contain genuine sexual steroid hormone receptors and whether these receptors are regulated via an estrogen-estrogen-receptor system as is the case in classical sexual steroid hormone target tissues.

Virosomes as new carrier system for cancer vaccines

HER-2/neu, a tumor-associated antigen (TAAg), plays a critical role in oncogenesis of various tumor types, and its selective overexpression by malignant tumor cells makes it an ideal target for immunotherapy. A prerequisite for clinical vaccines is the construction of safe and highly immunogenic reagents able to generate efficient immune responses against TAAg. Previous protein vaccines, consisting of the extracellular domain of HER-2/neu (pNeuECD), were shown to elicit an immune response that did not provide protection from transplantable tumors expressing HER-2/neu. Here we showed that virosomes, which consist of reconstituted viral envelopes without viral genetic material, can act as a carrier and an adjuvant for a truncated protein pNeuECD . Mice vaccinated with pNeuECD either encapsulated in virosomes or bound to the virosomal membrane (Vir-pNeuECD), generated rNeu-specific humoral and cytotoxic immune responses. In addition, Vir-pNeuECD induced significant tumor rejection and additionally did not lead to delayed tumor formation when compared with free pNeuECD in complete Freund’s adjuvant. There was no difference between the virosomal constructs. Taken together these results suggest that virosomes, as clinically approved safe vaccines, can be used to elicit both humoral and cell-mediated responses against TAAg and induce tumor rejection. Our model is providing important preclinical data to design human vaccination trials for patients with tumors overexpressing HER-2/neu, either as a primary vaccination or as a boost in combination with other vaccines in a context of an adjuvant treatment plan.

Soluble IL‐1 receptor type I binds to human dermal fibroblasts and induces calcium flux

Soluble cytokine receptors appear to modify ligand concentrations by stabilizing ligands or by specifically inhibiting interactions of ligands with their membrane‐bound receptors. Here we describe a new function of the soluble interleukin‐1 receptor type I (IL‐1sR I). This receptor induced a transient rise of intracellular free calcium concentration in human dermal fibroblasts in a dose‐dependent fashion. Mobilization of calcium by IL‐1sR I was abolished in the presence of an equimolar concentration of IL‐1 receptor antagonist (IL‐1ra). Neutralizing antibodies against IL‐1β also abolished calcium mobilization stimulated with IL‐1sR I indicating that IL‐1β is involved. IL‐1sR I bound with high affinity (K d 1–2 nM) to the fibroblasts. In addition, IL‐1sR I enhanced expression of IL‐6 and IL‐8 mRNA. The observation that IL‐1sR I can act as a ligand and agonist for membrane IL‐1 extends the concept of the ligand‐receptor functions of both IL‐1 and IL‐1sR I and adds a new dimension to the cytokine network.

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2×10−10 mol/l) or by pharmacological doses of biotin (10−5 mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.