Use of parvovirus for eliminating cancer stem cells (CSCs)

Abstract

Described is the use of a parvovirus, preferably H-1PV, for the therapeutical elimination of cancer stem cells (CSCs), preferably neuroblastoma stem cells and glioblastoma stem cells.

Claims

1. A method of combating cancer stem cells (CSCs) in a subject having metastatic cancer disease, comprising administering to said subject a composition comprising parvovirus H-1 (H-1PV) effective for therapeutically destroying said CSCs which are characterized by the expression of stem cell markers CD133, nestin, and/or SOX2.

2. The method of claim 1, wherein said CSCs are selected from the group consisting of CSCs resistant to chemotherapy or radiotherapy, potentially relapsing CSCs and neuroblastoma stem cells.

3. The method of claim 1, wherein said composition is administered by intravenous (i.v.) or intratumoral administration.

4. The method of claim 1, wherein said composition further comprises a pharmaceutically acceptable carrier.

5. A method of combating cancer stem cells (CSCs) in a subject suffering cancer relapse, comprising administering to said subject a composition comprising parvovirus H-1 (H-1PV) effective for therapeutically destroying said CSCs which are characterized by the expression of stem cell markers CD133, nestin, and/or SOX2.

6. The method of claim 5, wherein said CSCs are selected from the group consisting of CSCs resistant to chemotherapy or radiotherapy, potentially relapsing CSCs or neuroblastoma stem cells.

7. The method of claim 5, wherein said composition is administered by intravenous (i.v.) or intratumoral administration.

8. The method of claim 5, wherein said composition further comprises a pharmaceutically acceptable carrier.

Description

FIGURE LEGENDS

(1) FIG. 1:

(2) (A) NB124, 72 h after infection with MOI 1 H-1EGFP.

(3) (B) NCH 421, 36 h after infection with MOI 50 H-1EGFP.

(4) (C) Kelly, 48 h after infection with MOI 1 H-1EGFP.

(5) (D) IMR-32, 48 h after infection with MOI 1 H-1EGFP.

(6) FIG. 2:

(7) (A) NS1 Western Blot of NB 124 neuroblastoma progenitor cells.

(8) (B) NS1 Western Blot of three glioblastoma stem-like cells.

(9) FIG. 3: Infection of neuroblastoma initiating cells and glioblastoma stem-like cells with wtH-1PV

(10) See Example 4 for details.

(11) FIG. 4:

(12) (A) Cytomorphology of NB 124, three weeks after infection with wtH-1PV.

(13) (B) Cytomorphology of NCH 421, three weeks after infection with wtH-1PV.

(14) FIG. 5: H-1PV infection significantly reduces cell viability in neuroblastoma initiating and glioblastoma stem-like cells within 15 days after infection

(15) left panel: MTT NB 124 day 15 after infection with H-1PV

(16) right panel: MTT NCH 421 day 15 after infection with H-1PV

(17) FIG. 6: H-1PV induces cytostatic effects on neuroblastoma initiating cells and on three glioblastoma stem-like cell lines in a dose-dependent manner

(18) See Example 7 for details.

(19) The below examples explain the invention in more detail.

EXAMPLE 1

Materials and Methods

(20) (A) Cell Culture

(21) The human neuroblastoma neurosphere culture NB124 was obtained from Dr. Hedwig E. Deubzer (Clinical Cooperation Unti Pediatric Onocology, German Cancer Research Center). The human glioma stem-like cell lines NCH 421, NCH 441, NCH 620, and NCH 644 were obtained from PD Dr. Christel Herold-Mende from the Department of Neurosurgery (Campos et al., 2010). Basis medium (for stem cells): DMEM (Sigma Aldrich, Munich), 1% penicillin-streptomycin, 1% L-glutamine. Stem cell medium: Basis medium, 20% BIT 100 supplement (provitro GmbH, Berlin), 0.02% bFGF (RELIA Tech GmbH, Wolfenbttel), 0.02% EGF (RELIA Tech GmbH). Trypsin-blocking medium: DMEM (Sigma Aldrich, Munich), 10% heat-inactivated foetal bovine serum, 1% penicillin-streptomycin. Stem-like cells were grown as neurosphere cultures as previously published and cultured at 37 C., 5% CO.sub.2 in the respective growth medium (Wan et al., 2010).

(22) (B) Virus Production and Infection

(23) Wild-type H-1PV was produced by infecting NB K-324K human embryonic kidney cells, and purified by filtration (maximal diameter of particles 0.2 m) and iodixanol gradient centrifugation. The contamination of virus stocks with endotoxins was <2.5 EU/ml. Cells were infected with H-1PV as single cell suspensions after typsin treatment in their respective growth medium at 37 C.

(24) (C) Detection of Infectious H-1PV Particles

(25) Virus titres were determined as described previously (Angelova et al., 2009). Briefly, NB-324K cells (7.610.sup.3 cells/well) were seeded in 96-well plates 24 h prior to the assay. Cells were infected by 10-fold serial dilutions of the supernatant of the previously infected neurosphere cultures and incubated for 72 h at 37 C., 5% CO.sub.2.

(26) After alkaline lysis (0.75 M NaOH), DNA was transferred to a nylon membrane, cross-linked, and hybridized with a NS-1 specific probe radiolabeled with P.sup.32. Blots were exposed to X-ray film for autoradiography. Titration experiments were always performed in duplicates. Virus was applied at multiplicities of infection (MOI, expressed in plaque-forming units per cell; pfu) as indicated in the text.

(27) (D) Viral DNA Extraction and Quantitative Real-time PCR

(28) The supernatant of the infected neurosphere cell cultures was collected at different time points after infection. The supernatant was subjected to alkaline lysis in 1 M NaOH in TE buffer for 30 min at 56 C. After neutralization with an equimolar concentration of HCl the samples were diluted 1:100 with sterile water and directly analyzed. Quantification of viral DNA was carried out by real-time qPCR with an NS1-specific TAQMAN dual labeled hydrolysis probe (Applied Biosystems by Life Technologies, Carlsbad (Calif.), U.S.A), using an ABI PRISM 7700 sequence detection system thermal cycler (Applied Biosystems by Life Technologies, Carlsbad (Calif.), U.S.A) and analyzed by means of SDS 2.1 software (Applied Biosystems by Life Technologies, Carlsbad (Calif.), U.S.A) as described elsewhere (Abschuetz et al., 2006). Briefly, a DNA fragment of 141 nt within the NS1 gene of H-1PV was amplified, and detected using probe: 5-6-FAM-ATGCAGCCAG-ACAGTTA-Q-MGB 3. A plasmid that contained the NS1-sequence in serial dilutions in the range of 10.sup.1-10.sup.8 copies/reaction was used to standardize the qPCR. Individual reaction mixtures (20 l) consisted of 1 TAQMAN Universal PCR Master Mix formulation for real-time PCR (Applied Biosystems), 0.3 M labelled NS1-TAQMAN dual labeled hydrolysis probe, 0.3 M of each primer and 3 l template. PCR conditions were 2 minutes at 50 C. (destruction of contaminating template by AMPERASE Uracil N-Glycosylase), then 10 minutes 95 C., followed by 40 cycles of denaturation at 95 C. for 15 seconds and annealing/extension at 60 C. for 60 s.

(29) (E) Microscopy

(30) Phase contrast images were generated using an inverted phase contrast microscope (Olympus; Model CKX41) using Cell B software (Olympus Europa GmbH, Hamburg, Germany). Other phase contrast images were obtained using a Leica DFC350 FX camera (Leica Microsystems, Wetzlar, Germany) and the Leica FIRECAM digital image management software for MACINTOSH digital computers.

(31) (F) Assessment of Cell Viability and Lysis

(32) Proliferation of neuroblastoma cells was tested with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as recommended by the manufacturer (Sigma-Aldrich, St. Louis, Mo., U.S.A.). Cells (2,500 cells per well) were cultured in 96-well plates and infected at MOIs indicated in the Figures. After 15 days, cells were washed in PBS and incubated with 0.5 g/ml MTT-solution for up to 2 hours. After discarding the supernatant and drying the cells, 100 l isopropanol per well were added. Extinction values were photometrically determined at 570 nm (MULTISKAN PLUS microplate reader, Titertek Instruments Inc., Huntsville, Ala., U.S.A).

(33) Cell lysis was determined by measuring the release of lactate dehydrogenase into culture medium by use of the CYTOTOX 96 Cytotox 96 cytotoxicity assay Kit (Promega Corporation, Madison Wis., U.S.A.)) according to the manufacturer's instructions.

EXAMPLE 2

Neuroblastoma Progenitor and Glioblastoma Stem-like Cells are Susceptible to H-1PV Infection

(34) To determine if H-1PV was able to infect neuroblastoma progenitor cells, NB124 cells were subjected to one replication unit per cell of recombinant, replication-deficient H-1 virus (H-1EGFP) virus expressing GFP. NCH 421 glioblastoma neurospheres were infected with MOI 50 of H-1EGFP. The MYCN amplified neuroblastoma cell lines Kelly and IMR-32 were kindly provided by Prof. Dr. Olaf Witt, CCU Pediatric Oncology, German Cancer Research Center, Heidelberg and served as positive controls Immunofluorescence microscopy revealed that GFP expression could be detected in both neuroblastoma cell lines and the NB124 neurosphere culture and after infection with H-1EGFP, indicating successful infection of neuroblastoma cells and gene expression driven by the viral promoter in the neuroblastoma progenitor cells infected (FIG. 1, left panel, phase contrast microscopy, middle panel merge, right panel, fluorescence microscopy).

EXAMPLE 3

H-1PV Protein Expression Persists in Neuroblastoma Progenitor Cells and High Grade Glioma Stem Cells up to 15 Days

(35) In order to demonstrate that the proteins of wtH-1PV were expressed in infected neuroblastoma and high grade glioma progenitor cells, Western blot analysis of NS1 and NS2 proteins in infected cells was performed. NS1 and NS2 are non-structural proteins required for infection of host cells, and VP1/2 are structural viral capsid proteins (Chen et al., 1989; Caillet-Fauquet et al., 1990, Brandenburger et al., 1990). Following infection with 50 pfu per cell wtH-1PV, these viral proteins were expressed in all stem cell lines investigated, at day 9 and day 15 after infection (FIGS. 2A,B). Human neuroblastoma (IMR-32) and glioblastoma (U87) non-stem cell like cell lines served as positive controls. U87 human glioblastoma cells grown under standard conditions served as positive control.

EXAMPLE 4

H-1PV Actively Replicates in Neuroblastoma Progenitor Cells and Glioblastoma Stem-like Cells

(36) In order to address the issue whether H-1PV was able to multiply in neuroblastoma progenitor cells, cells were infected with wtH-1PV. Viral genome copy numbers in the supernatant were determined by real-time PCR in a time period ranging from 3 days up to 21 days after infection. In NB 124 neuroblastoma cells, the viral genome copy numbers increased up to 1,000 fold and the titer of infectious particles even increased up to 100,000 fold indicating highly efficient viral multiplication in these cells. The efficiency of replication of fully infectious viral progeny in these neuroblastoma progenitor cells even exceeded that observed in standard neuroblastoma cell lines which displayed a significant increase in viral copy numbers varying from a 10.sup.2 to 10.sup.4 fold increase within 72 to 144 h after infection (Lacroix et al., 2010).

(37) In NCH 421 glioblastoma stem-like cells viral genome copy numbers increased up to 10,000 fold during 21 days after infection, which is comparable to the replication efficiency in other human glioma cell lines without stem-cell properties (Geletneky et al., 2005).

(38) In order to quantify the generation of infectious progeny H-1PV, infectious particle assays with supernatants of the same infected neuroblastoma progenitor cell line in culture were additionally performed. The progeny H-1 viruses were biologically active, i.e. able to infect NBK-324K cells. In the infection unit assay, a 4,000-fold increase in the neuroblastoma neurosphere culture NB124 and a 3,500-fold increase of infectious particles in the glioma stem-like cell line NCH 421 compared to the input virus could be determined (FIG. 3; upper curve: Vg/ml; lower curve: IU/ml).

(39) Taken together, H-1PV could be proven to productively infect neuroblastoma progenitor cells and glioblastoma stem-like cells. Infection of these cells could be demonstrated to induce the expression of essential viral proteins, efficient viral replication and production of infectious H-1PV progeny.

EXAMPLE 5

H-1PV Induces Lytic Infection in Neuroblastoma and High Grade Glioma Stem Cell Lines

(40) In order to test to what extent infection of neuroblastoma cells with wild type H-1PV was lytic, the cytomorphology of the infected cells was documented by phase contrast microscopy. Three weeks after infection H-1PV could be shown to induce significant cytopathic effects on cultured NB124 cells applying an MOI of 0.01 p. f. u. per cell or more (FIG. 4A). Three weeks after infection H-1PV could be shown to induce significant cytopathic effects on cultured NCH 421 cells applying an MOI of 1 p. f. u. per cell or more (FIG. 4B).

EXAMPLE 6

H-1PV Infection Significantly Reduces Cell Viability in Neuroblastoma Progenitor and Glioblastoma Stem-Like Cells within 15 Days after Infection

(41) H-1PV could be shown to induce significant cytopathic effects on cultured neuroblastoma neurospheres. In order to quantify cytopathic effects with regard to metabolic activity and cellular integrity, MTT test was performed in NB124 and NCH 421 cells 15 days after infection with increasing MOIs of wtH-1PV. Applying an MOI of 1 pfu/cell reduced viable neuroblastoma progenitor cells by 80% and in glioblastoma stem-like cells by 60% within 15 days after infection (FIG. 5).

EXAMPLE 7

H-1PV Induces Cytostatic Effects on Neuroblastoma Progenitor Cells and on Three Glioblastoma Stem-like Cell Lines in a Dose-dependent Manner

(42) NB 124 neuroblastoma neurosphere cultures and glioblastoma stem-like cells NCH 421, NCH 620 and NCH 644 were infected with either 1 pfu per cell (FIG. 6, left panel) or 50 pfu per cell (FIG. 6, right panel) and subsequently viable cells were counted in multiplicates of 6 wells per time point. Starting from day 12 to 15 after infection a significant difference in the number of viable cells could be demonstrated for each tumor stem cell line. Applying an MOI of 1 the effect was less pronounced than after application of the higher MOI of 50 p. f. u. per cell (FIG. 6; upper curve: mock; lower curve: H-1PV).

EXAMPLE 8

Cytostatic Effect on Tumor Stem Cells Does Not Seem to be Mediated by Apoptosis

(43) Neuroblastoma and high-grade glioma neurosphere cell cultures were analyzed by propidium idodine staining and subsequent flow cytometry for the presence of a sub-G1 DNA content (less that 2n) cell population that is indicative of DNA fragmentation and apoptosis. In parallel Western Blots for the detection of viral proteins such as NS1 were performed. However induction of NS1-expression in NB124 neuroblastoma stem cells nor in high grade glioma stem cells neither correlated with G2-arrest nor with the appearance of a sub-G1 population.

REFERENCES

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