A PHARMACEUTICAL COMBINATION OF AN ARTEMISININ COMPOUND, 5-AMINOLEVULINIC ACID OR METHYL-5-AMINOLEVULINIC ACID AND A CHEMOTHERAPEUTIC AGENT

Abstract

The present invention relates to a pharmaceutical composition comprising an artemisinin compound, and 5-aminolevulinic acid or methyl-5-aminolevulinic acid and at least one chemotherapeutic agent, preferably at least one anti-glioblastoma drug. This pharmaceutical composition is used for the prophylaxis and/or treatment of hematopoietic cancers, brain cancer, pancreatic cancer, liver cancer, breast cancer, and lung cancer such as non-small cell lung cancer. Preferably, the present application provides a pharmaceutical composition comprising artemisinin (1a) or di-hydroarteminisin (1b) or artesunate (1e) and 5-aminolevulinic acid (2) or methyl-5- aminolevulinic acid (2b) and at least one chemotherapeutic agent, preferably at least one anti-glioblastoma drug for use in prophylaxis and/or treatment of brain cancer, in particular glioblastoma.

Claims

1. A pharmaceutical composition comprising a) an artemisinin compound (1) or a pharmaceutically acceptable salt, cocrystal, or solvate thereof; b) 5-aminolevulinic acid (2), methyl-5-aminolevulinic acid (2b), or a pharmaceutically acceptable salt or solvate thereof; and c) at least one chemotherapeutic agent, preferably at least one anti-glioblastoma drug.

2. The pharmaceutical composition according to claim 1, wherein the artemisinin compound (1) is selected from ##STR00022## ##STR00023## ##STR00024## ##STR00025## ##STR00026## or a pharmaceutically acceptable salt, cocrystal, or solvate thereof.

3. The pharmaceutical composition according to claim 1, wherein the anti-glioblastoma drug is selected from the group comprising or consisting of temozolomide, dexamethasone, lomustine, methotrexate, everolimus, carmustine, cyclophosphamide, cisplatin, carboplatin, 5-fluorouracil, triptolide, homoharringtonin, dactinomycin, doxorubicin, epirubicin, idarubicin, ribavirin, topotecan, flubendazole, itraconazole, vindesine sulfate, cerivastatin, vincristine, vinorebine, nisoldipine, deoxyadenosine, chloro-2′-deoxyadenosine, 5-nonyloxytryptamine, 2(1H)-pyrimidinone, pitavastatin, sertraline, irinotecan, clofazimine, and docetaxel.

4. The pharmaceutical composition according to claim 1, wherein a molar ratio of the artemisinin compound (1) and the 5-aminolevulinic acid (2) or the methyl-5-aminolevulinic acid (2b) is in a range of 1:5 to 1:5000.

5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient and/or diluent.

6. The pharmaceutical composition according to claim 1, wherein the at least one anti-glioblastoma drug is selected from the group consisting of temozolomide, lomustine, cisplatin, and 5-fluorouracil.

7. The pharmaceutical composition according to claim 1 comprising: a) an artemisinin compound (1) selected from ##STR00027## ##STR00028## ##STR00029## or a pharmaceutically acceptable salt, cocrystal, or solvate thereof; b) 5-aminolevulinic acid (2) or a pharmaceutically acceptable salt or solvate thereof; and c) at least one anti-glioblastoma drug selected from the group consisting of temozolomide, lomustine, cisplatin, and 5-fluorouracil.

8. (canceled)

9. A method for the prophylaxis and/or treatment of a cancer selected from hematopoietic cancers, brain cancer, pancreatic cancer, liver cancer, breast cancer, and lung cancer comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising: a) an artemisinin compound (1) or a pharmaceutically acceptable salt, cocrystal, or solvate thereof; and b) 5-aminolevulinic acid (2), methyl-5-aminolevulinic acid (2b), or a pharmaceutically acceptable salt or solvate thereof: and c) at least one chemotherapeutic agent, preferably at least one anti-glioblastoma drug.

10. The method according to claim 9, wherein the pharmaceutical composition is in a form of a tablet, capsule, syrup, solution, suspension, emulsion, or gel.

11. The method according to claim 9, wherein the pharmaceutical composition is administered by oral, intrathecal, intravenous, subcutaneous, parenteral application or by inhalation.

12. The method according to claim 9, wherein the artemisinin compound (1) is administered in a range of 0.01 to 100 mg/kg per body weight per day and the 5-aminolevulinic acid or the methyl-5-aminolevulinic acid is administered in a range of 0.01 to 200 mg/kg per body weight per day.

13. The method according to claim 9, wherein the at least one anti-glioblastoma drug is administered in a range of 0.01 to 100 mg/kg per body weight per day.

14. The method according to claim 9, wherein the brain cancer is selected from subtypes proneural (PN), mesenchymal (MES), and classical (CL) glioblastoma.

15. The method according to claim 9, wherein the prophylaxis and/or treatment of hematopoietic cancers, brain cancer, pancreatic cancer, liver cancer, breast cancer, and lung cancer and preferably brain cancer is performed in combination with a radiotherapy, immunotherapy, electromagnetic field therapy, hyperthermia therapy, chemotherapy, cancer immunotherapy and/or any other small molecule based therapy.

Description

DESCRIPTION OF FIGURES

[0369] FIG. 1: Haploid screening in mouse stem cells delineates porphyrin biosynthesis as a prerequisite for artemisinin toxicity in mammalian cells. A Schematic of haploid embryonic stem cell screens for compound target identification. B Porphyrin biogenesis pathway. Subcellular location (cytosol, mitochondria) and loss-of-function scores (italic) of major enzymes (bold, capitalised) and co-factors (normal, capitalised) of the screen (retroviral mutagenesis) are indicated. C GO-term analysis reveals porphyrin biogenesis as the major pathway targeted by artemisinin. D Competitive growth assay of artemisinin resistant single cell clones. Wild-type (mCherry+_ Cre) and knockout (GFP+) sister clones were derived from mutant (retrovirus - intron RE, darker shading, Tol2 transposon - intron T2, lighter shading) resistant colonies, treated with artemisinin, analysed with flow cytometry and sanger sequenced for insertion site mapping.

[0370] FIG. 2: Modulation of porphyrin biosynthesis is sufficient to alter artemisinin toxicity in cells via ROS generation. A, B Cell survival of Artemisinin treated mouse embryoni stem cells in combination with A the Ppox inhibitor acifluorfen or B 5-ALA (0.5 mM). Alamar Blue staining was used to assess viability after 48 h of treatment. C Cell viability of artemisinin treated mouse breast cancer cells (4T1) in presence and absence of 5-ALA (0.5 mM). Alamar Blue was used to determine cell survival after 48 h of treatment. D Survival of artemisinin treated primary human glioblastoma cells from cancer patients upon 5-ALA administration. Viability was assessed using CellTiter-Glo after 72 h. Experiments were performed in triplicate. Values are mean ± SD. E ROS levels (DHE staining, PE 582/15 nm - MFI) and F cell survival of artemisinin (0.5 .Math.M), 5-ALA (0.25 mM) or Ppox inhibitor (10 .Math.M) treated neuroblastoma cells (SHSY5Y). Fluorescence of DHE (ROS levels) and cell numbers (survival) were assessed by flow cytometry and automated cell counting. The experiments were performed two times, in triplicate each. Values indicate mean ± SD.

[0371] FIG. 3: Cerebral tumor organoids (CNS-PNET) display increased sensitivity to artemisinin and 5-ALA combination therapy. A Human cerebral primitive neuroectodermal tumor organoids for compound profiling. B Schematic workflow representation and analysis of 5-ALA and artemisinin treated organoids. C Representative fluorescence and D brightfield images of DMSO (control), 5-ALA (0.0625 mM), artemisinin (1 .Math.M), or 5-ALA and artemisinin (0.0625 mM and 1 .Math.M) treated tumor organoids (CNS-PNET). Organoids were imaged on day 1, day 5 and day 7. Scale bar 500 .Math.m. E Image analysis and quantification of GFP-positive tumor area of treated organoids. Eight organoids per group were analysed on day 3 and normalized to day 1. Data is shown as box plots (25.sup.th-75.sup.th percentiles, median). F Flow cytometry analysis of dissociated cerebral tumor organoids. Relative percentages of GFP+ tumor cells of at least eight organoids per condition on day 5 normalised to control (DMSO) are shown. Box plots (25.sup.th-75.sup.th percentiles, median) of data are indicated. G ROS/ DHE staining and flow cytometry analysis of dissociated tumor organoids (PE, 582/15 nm). Mean fluorescence intensities (MFI) of DHE stained GFP+ tumor cells and GFP- control cells are indicated. n=2, values are mean ± SD. H Quantification of γH2AX+ cells in cerebral tumor organoids. 6 cryo-sections of 3 organoids each were stained for γH2AX, scanned and 25 regions of interest (ROI 2.500 .Math.m.sup.2) were analysed per condition. Raw numbers were normalised to organoid area (per ROI) and are shown as box plots (25th-75th percentiles, median).

[0372] FIG. 4: Human glioblastoma-like neoplastic organoid models display increased sensitivity to artemisinin and 5-ALA combination therapy. A Representative fluorescence (left panel) and brightfield (right panel) images of 5-ALA and artemisinin treated human cerebral tumor organoids. Organoids were monitored on day 1, day 5, day 8 and day 11. Scale bar 500 .Math.m. B Image analysis and quantification of treated organoids. Area of GFP-positive tumor cells on d11 was normalized to d1. Data is shown as box plots (25.sup.th-75.sup.th percentiles, median) n=4. C Representative brightfield images of 5-ALA and artemisinin treated patient derived glioblastoma spheroids (VBT92). Images were taken on day 3 of culture and treatment..

[0373] FIG. 5 (related to FIG. 1): Artemisinin titration curves and an overview of the porphyrin biosynthesis pathway. A, B Cell survival of artemisinin treated A mouse ESCs and B primary (MEF p3) fibroblasts, as well as mouse (B16F10, 4T1) and human (MDA-MB-231, Mcf7, Panc1) cancer cells. Viability was assessed after 48 h of treatment using Alamar Blue staining. C Porphyrin biosynthesis pathway component integrations sites from artemisinin screens. Genomic locations and targeted introns as well as exons of porphyrin biosynthesis genes (i.e. enzymes, bold), as well as retroviral (upper panel) or Tol2 transposon (lower panel) integrations sites (vertical bars) on the forward (black) and reverse (grey) strand are shown.

[0374] FIG. 6 (related to FIG. 2): Effects of Artemisinin and 5-ALA combination treatment on survival, ROS production and mitochondrial membrane potential in cancer cell lines A Cell survival of artemisinin treated mouse cancer cells (Mcf7 -breast cancer; B16F10 - melanoma) in presence and absence of 5-ALA (0.5 mM). Alamar Blue was used to determine viability after 48 h. B, C Cell survival of artemisinin treated primary human glioblastoma cells in presence or absence of 5-ALA. CellTiter-Glo was used to assess viability, 72 h. Values are mean ± SD. D, E ROS/ DHE staining and flow cytometry analysis of piperlongumine or artemisinin treated D Jurkat and E HL-60 cells (48 h). Values are mean ± SD. F ROS levels (DHE staining, PE 582/15 nm -MFI), G cell survival and H JC-1 levels of artemisinin (0.5 .Math.M), 5-ALA (0.25 mM) or Ppox inhibitor (10 .Math.M) treated Jurkat cells, 48 h. JC-1 negative cells have impaired mitochondrial membrane potential associated with apoptotic cell death. DHE fluorescence, relative cell numbers and percentage of JC-1 negative cells were assessed using high throughput flow cytometry and automated cell counting. All experiments were performed in triplicate and repeated once (JC-1) or twice (DHE, cell survival). Values are mean ± SD.

[0375] FIG. 7 (related to FIG. 3): Cancer cell population in CNS-PNET tumor organoids displays high sensitivity to artemisinin and 5-ALA combination therapy. A Cell survival of dissociated CNS-PNET tumor organoids. Quantification of GFP+ tumor cells and GFP- control cells of treated organoids normalised to control (DMSO) are shown. Data is shown as box plots (25.sup.th-75.sup.th percentiles, median). B Representative fluorescence (left panel) and brightfield (right panel) images of control (DMSO), 5-ALA (0.0625 mM), artemisinin (1 .Math.M) or 5-ALA + artemisinin (0.0625 mM and 1 .Math.M) treated cerebral tumor organoids. Scale bar 500 .Math.M. C Image quantification of GFP-positive tumor area on day 5 of treatment, as compared to day 1. Box plots (25.sup.th-75.sup.th percentiles, median) of data are shown. D Representative images of anti-Sox2, anti-GFP and DAPI stained sections of artemisinin and 5-ALA treated organoids. Scale bar 50 .Math.m.

[0376] FIG. 8 (related to FIG. 3): Staining of CNS-PNET tumor organoid sections following artemisinin and 5-ALA treatment. Representative fluorescent (anti-GFP, DAPI) and H&E (haematoxylin & eosin stained) images of fixed cryo-sections of control and treated tumor organoids. Regions of rosette-like strucutres (R) or tumor tissue (T) are indicated and magnified (6.8x). Scale bar 500 .Math.M.

[0377] FIG. 9 (related to FIG. 3): Analysis of CNS-PNET tumor organoids following artemisinin and 5-ALA treatment A Representative FACS plots of ROS/ DHE stained dissociated tumor organoids. Flow cytometry analyses (PE, 582/15 nm) of GFP+ tumor cells and GFP- wild-type cells are shown. B Representative microscopy images of γH2AX, GFP and DAPI stained tumor organoid sections. Scale bar 50 .Math.M. C Representative images and analysis masks of γH2AX stained and scanned tumor organoid slides.

[0378] FIG. 10 (related to FIG. 3): Continued analysis of CNS-PNET tumor organoids following artemisinin and 5-ALA treatment A Quantification of Caspase 3 (Casp3) and B Ki67-positive cells in 5-ALA and artemisinin treated tumor organoids. Per condition and group, 3 organoids, 6 sections each, were stained with Caspase3 or Ki67, imaged using a high-magnification fluorescent scanner, and 25 regions of interest (ROI 2.500 .Math.m.sup.2) were chosen and analysed. The numbers of Casp3 or Ki67 positive cells were normalised to the analysed area (per ROI, GFP+ or GFP-) and are shown as box plots (median, 25th-75th percentiles). C Representative images of anti-Casp3, anti-GFP and DAPI stained sections. Scale bar 50 .Math.m.

[0379] FIG. 11 (related to FIG. 4): Artemisinin and 5-ALA combination treatment of human glioblastoma-like neoplastic organoid models A Fluorescence images of 5-ALA and artemisinin treated cerebral tumor organoids. Organoids were monitored on d1, d5, d8 and d11. Scale bar 500 .Math.m. B Image analysis and quantification of GFP-positive tumor area of treated organoids. Organoids were analysed on d8 and normalized to d1. Data is shown as box plots (25.sup.th-75.sup.th percentiles, median).

[0380] FIG. 12: Artemisinsin (ART) and its derivatives Dihydroartemisinin (DHA) and Artesunate (ARS) exert synergistic antiproliferative effects on glioblastoma cell lines and mouse embryonic stem cells (ESC) when combined with 5-Aminolevulinic acid (5-ALA). Shown are 6-day titration curves of artemisinin compounds and 5-ALA. A-R Cell survival of 5-ALA treated glioblastoma lines (as indicated) and ESC in combination with the indicated A, D, G, J , M P ART doses, or B, E, H, K, N, Q DHA doses, or C, F, I, L, O, R ARS doses compared to untreated controls. Values are Mean ±SEM from ≥ 3 Independent experiments. Dotted lines mark 100% survival.

[0381] FIG. 13: ART or DHA combined with 5-ALA increases the antiproliferative effect of Temozolomide (TMZ) treatment in glioblastoma cell lines and ESC. A-L 5-ALA and TMZ treated glioblastoma lines (as indicated) and ESC in combination with the indicated A, C, E, G, I, K ART doses or B, D, F, H, J, L DHA doses compared to untreated controls. Values are Mean +SEM from ≥ 5 Independent experiments. Dotted lines mark 100% survival and the survival upon TMZ treatment only.

[0382] FIG. 14: ARS combined with 5-ALA or Methyl-5-ALA increases the antiproliferative effect of Temozolomide (TMZ) treatment in glioblastoma cell lines and ESC. 5-ALA is more potent than Methyl-5-ALA in this setup. A-F 5-ALA and TMZ treated glioblastoma lines (as indicated) and ESC in combination with the indicated ARS doses compared to untreated controls. Values are Mean +SEM from ≥ 3 Independent experiments. Dotted lines mark 100% survival and the survival upon TMZ treatment only.

[0383] FIG. 15: ART combined with 5-ALA increases the antiproliferative effect of Lomustine (CCNU) treatment in glioblastoma cell lines and ESC. A-F 5-ALA and CCNU treated glioblastoma lines (as indicated) and ESC in combination with ART doses compared to untreated controls. Values are Mean +SEM from ≥ 4 Independent experiments. Dotted lines mark 100% survival and the survival with CCNU treatment only.

[0384] FIG. 16: ART, DHA, or ARS combined with 5-ALA increases the antiproliferative effect of cisplatin (CP) and 5-Fluorouracil (5-FU) treatment in two lung cancer cell lines. A-L Cell survival of 5-ALA treated lung cancer lines (as indicated) in combination with the indicated A, D, G, J ART doses, or B, E, H, K, DHA doses, or C, F, I, L, ARS doses and either cisplatin (A-F) or 5-FU (G-L) conditioning compared to untreated controls. Values are Mean +SEM from ≥ 4 Independent experiments. Dotted lines mark 100% survival and the survival upon chemotherapeutic (cisplatin or 5-FU) treatment only.

[0385] FIG. 17: ART, DHA, or ARS combined with 5-ALA increases the antiproliferative effect of cisplatin (CP) and 5-Fluorouracil (5-FU) treatment in HepG2 cells (liver cancer). A-F Cell survival of 5-ALA treated HepG2 cells in combination with the indicated A, D, ART doses, or B, E, DHA doses, or C, F, ARS doses and either cisplatin (A-C) or 5-FU (D-F) conditioning compared to untreated controls. Values are Mean +SEM from ≥ 4 Independent experiments. Dotted lines mark 100% survival and the survival upon chemotherapeutic (cisplatin or 5-FU) treatment only.

[0386] FIG. 18: ART, DHA, or ARS combined with 5-ALA increases the antiproliferative effect of cisplatin and 5-fluorouracil (5-FU) treatment in MD-MBA-231 cells (breast cancer). A-F Cell survival of 5-ALA treated MD-MBA-231 cells in combination with the indicated A, D, ART doses, or B, E, DHA doses, or C, F, ARS doses and either cisplatin (A-C) or 5-FU (D-F) conditioning compared to untreated controls. Values are Mean +SEM from ≥ 4 Independent experiments. Dotted lines mark 100% survival and the survival upon chemotherapeutic (cisplatin or 5-FU) treatment only.

[0387] FIG. 19: ART, DHA, or ARS combined with 5-ALA increases the antiproliferative effect of cisplatin and 5-fluorouracil (5-FU) treatment in MiaPaca-2 cells (pancreas cancer). A-F Cell survival of 5-ALA treated MiaPaca-2 cells in combination with the indicated A, D, ART doses, or B, E, DHA doses, or C, F, ARS doses and either cisplatin (A-C) or 5-FU (D-F) conditioning compared to untreated controls. Values are Mean +SEM from ≥ 4 Independent experiments. Dotted lines mark 100% survival and the survival upon chemotherapeutic (cisplatin or 5-FU) treatment only.

EXAMPLES

Materials and Methods

Mammalian Tissue Culture

[0388] Mouse embryonic stem cell clones (clone AN3-12) {Elling:2011gla} were cultured in DMEM supplemented with 10% fetal bovine serum (FCS), penicillin-streptomycin, non-essential amino acids, sodium pyruvate (1 mM), I-glutamine (2 mM), β-mercaptoethanol (0.1 mM), and LIF (20 .Math.g ml-1). SH-SY5Y cells were cultured in DMEM/F12 1:1 supplemented with 10% FCS (fetal calf serum), penicillin-streptomycin, and L-glutamine. 4T1 cells were cultured in IMDM supplemented with 10% fetal calf serum (FCS), penicillin-streptomycin and L-glutamine. MEFs, Mcf7, MDA-MB-231, Panc1, LN229, A549, MiaPaca-2, B16F10 and PlatE cells were cultured in DMEM supplemented with 10% FCS, penicillin-streptomycin and L-glutamine. HepG2-, T98G-, and U87MG cells were cultured in EMEM, and SHP77-, VBT92-, and VBT281 cells were cultured in RPMI, each supplemented with 10% FCS, penicillin-streptomycin and L-glutamine. All cells were cultured at 37° C., on f 20%O.sub.2 and 5%CO.sub.2.

Cell Lines

[0389] Mouse AN3-12 ESC lines were generated in our laboratory and characterized and authenticated as previously described {Elling, U. et al. Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell stem cell 9, 563-574 (2011)}. Haploid murine ESCs were used for insertional mutagenesis and derivation of gene trap knockout cell lines. SH-SY5Y were obtained directly from the supplier (Sigma Aldrich) and used for growth assays and cellular stainings. Jurkat cells as used in in vitro viability and DHE assays were obtained from an in-house source and are functionally described elsewhere {Reikerstorfer, A., Holz, H., Stunnenberg, H. G. & Busslinger, M. Low affinity binding of interleukin-1 beta and intracellular signaling via NF-kappa B identify Fit-1 as a distant member of the interleukin-1 receptor family. The Journal of biological chemistry 270, 17645-17648 (1995)}. Mcf7, MDA-MB-231, 4T1, Panc1 and B16F10 cancer cell lines were obtained in house. MEFs were generated and obtained in our laboratory. PlatE cells were used for recombinant retrovirus and lentivirus production as previously described {Taubenschmid, J. et al. A vital sugar code for ricin toxicity. Cell Research 27, 1351-1364 (2017). Stadlmann, J. et al. Comparative glycoproteomics of stem cells identifies new players in ricin toxicity. Nature 549, 538-542 (2017)}. All cell lines were tested negative for mycoplasma. No cell line listed by ICLAC was used.

Competitive Growth Assays

[0390] Haploid ESCs harbouring gene traps in nomic introns were seeded at low density in normal ESC growth medium and infected for 12 h with two viruses, one encoding mCherry plus Cre recombinase and the other coding for GFP, both together with puromycin (Invivogen, ant-pr-1). Infected cells were selected (final concentration of puromycin, 1 .Math.g/ml) after 24 h and expanded. Ratios of GFP- to mCherry/Cre-expressing cells in the presence and absence of artemisinin were determined using high-throughput flow cytometry (BD LSRFortessa HTS Cell Analyzer).

Compound Profiling in Cell Lines

[0391] For dosage responses, cells were seeded in 96-wells (25.000/ 96-well, in triplicate -where indicated) and subjected to compounds for 48 h. Cell viability was assessed using automated cell counting (high-throughput flow cytometry), Alamar Blue staining (Invitrogen, DAL1100) or CellTiter-Glo Luminescent Assay (Promega, G7570, according to the manufacturer’s protocol) respectively.

[0392] Cell survival assays (FIGS. 12-19) were performed in 96-well plates in technical duplicates. Treatments started 24 hours post cell plating and lasted for 6-days prior to cell viability assessment with the Cell Titer Glo 2.0 Luminescent assay (Promega, G9242).

Dihydroethidium (DHE) Staining

[0393] Treated cells were collected, washed with 1x HBSS (without Ca2+ and Mg2+), incubated with 1mM DHE (Dihydroethidium (Hydroethidine), Invitrogen, D11347) in 1x HBSS for 45 min at 37° C., washed twice and counterstained with DAPI or a viability dye (eBioscience™ Fixable Viability Dye eFluor™ 780, 65-0865-18) for 10 min on ice. Cells were then collected, strained and analysed using flow cytometry for DHE in the red fluorescent spectrum (PE channel).

JC-1 Staining

[0394] Cells were collected, washed with 1xPBS, incubated with 2 .Math.M JC-1 in 1PBS (MitoProbe JC-1 Assay Kit-1, Invitrogen, M34152) for 35 min at 37° C., washed twice, and analysed using flow cytometry in the red (PE channel) and green (FITC channel) fluorescence spectrum.

Cerebral Organoid Formation

[0395] Cerebral organoids were generated as described before { Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373-379 (2013)}. Human embryonic stem cells (feeder-free H9, WiCell) were transferred into low-attachment 96-well-plates (Corning) in a density of 9.000 cells per well and incubated in human stem cell medium. After 6 days, the medium was changed to neural induction media, containing Dulbecco’s modified eagle medium DMEM/F12, N2 supplement (Invitrogen), Glutamax (Invitrogen), minimum essential media-nonessential amino acids (MEM-NEAA) and 1 .Math.g/ml heparin (Sigma), to promote growth of ectodermal tissue. On day 11, embryonic bodies (EBs) were embedded in droplets of Matrigel and transferred to differentiation medium, containing DMEM/F12: Neurobasal 1:1, N2 supplement (Invitrogen), B27 supplement (without vitamin A) (Invitrogen), 50 .Math.M 2-mercaptoethanol, 1:4,000 insulin (Sigma), Glutamax (Invitrogen), penicillin-streptomycin, MEM-NEAA onto 10 cm plates. 5 days later, organoids were transferred to an orbital shaker and maintained in differentiation media containing vitamin A (in B27 supplement).

Cerebral Tumor Organoid Formation + Nucleofection

[0396] Tumor initiation was induced by either oncogene amplification, using a Sleeping Beauty (SB) transposase, or mutation of tumor suppressor genes, using the CRISPR-Cas9 system in day 10 old embryoid bodies (EBs). Plasmids carrying the transposase as well as GFP and the desired oncogenes or expressing Cas9 nuclease were introduced by electroporation. In brief, a mixture of 1 .Math.g DNA and 100 .Math.l nucleofector solution was added to 10 EBs and transferred to a nucleofection cuvette. The Lonza Nucleofector 2b and the A-023 program were used for electroporation/ nucleofection. Thereafter, EBs were transferred into 10 cm dishes containing differentiation media containing vitamin A and embedded into Matrigel 24 hours later. Two different types of tumors were initiated {Bian:2018gs}: Central nervous system primitive neuroectodermal tumors (CNS-PNET) were induced by the overexpression of Myc. Glioblastoma-like tumor group 2 (GBM-2) was caused by mutagenesis of the tumor suppressor genes p53, NF1 and PTEN. All plasmids were designed by Shan Bian {Bian, S. et al. Genetically engineered cerebral organoids model brain tumor formation. Nat. Methods 15, 631-639 (2018)}.

Compound Profiling in Cerebral Tumor Organoids

[0397] Cerebral tumor organoids were treated with different compounds and the survival and growth of transformed as well as untransformed neurons was monitored. Fluorescence labeling of tumor cells allowed clear distinguishability of transformed cells as compared to non-labeled throughout the experiment using brightfield microscopy, as well as fluorescence imaging. The transformed tumor tissue and untransformed neurons within the same organoid was monitored using fluorescence imaging, as well as brightfield microscopy throughout the experiment. At the endpoint of the treatment (d5 or d7 respectively), the number of GFP-positive cells was assessed by flow cytometry.

Flow Cytometry Analysis of Cerebral Tumor Organoids

[0398] Cerebral (tumor) organoids were enzymatically and mechanically dissociated using 1x trypsin, 35-45 min incubation at 37° C., and gentle shaking. Organoids were singularised by careful resuspension, addition of differentiation medium and straining (Falcon Round-Bottom Tubes with Cell Strainer Cap, 5 mL, 35 .Math.m nylon mesh cell strainer snap cap). Single cells in suspension were counterstained with DAPI or a viability dye (eBioscience™ Fixable Viability Dye eFluor™ 780, 65-0865-18), and analysed using flow cytometry (BD LSRFortessa HTS Cell Analyzer). The amounts of GFP-positive cells were analyzed.

Imaging of Cerebral Tumor Organoids

[0399] 5-ALA or Artemisinin treated cerebral organoids were imaged at the indicated time points using the Axio Vert.A1 Inverted Microscope system (Zeiss Objective EC Plan-Neofluar 2.5x/0.085 Pol M27, 0.5 camera adapter). Brightfield and green fluorescence images were taken from the same area. Additionally, for image analyses, the Celldiscoverer 7 (Zeiss), a fully integrated high-end automated live cell imaging system, was used.

Immunohistochemistry Staining

[0400] Cerebral organoids were fixed in 4% PFA (room temperature, 1 h), incubated with 30% sucrose (4° C., o/n), OCT (Tissue-tek OCT Compound, SANOVA PHARMA GESMBH, 4583) embedded and 20 .Math.m sections were cryostat cut (-12/-14° C.). For staining, sections were blocked and permeabilized for 1 h on RT in 0.25% Triton X-100, 4% donkey serum in PBS, stained with primary antibodies diluted in in 0.1% Triton X-100, 4% donkey serum in PBS at RTovernight, incubated with secondary antibodies diluted in in 0.1% Triton X-100, 4% donkey serum in PBS for 2 h at RT and counterstained with DAPI (4′,6-Diamidino-2-Phenylindole, Dilactate, Invitrogen, D3571) at RT for 20 min. Fluorescence Mounting Medium (Dako, S302380) was used for mounting the sample slides. The organoids were imaged on an LSM780 Axio Imager (point laser scanning confocal microscope, GaAsP (Gallium Arsenide) detectors with QE of 45% and up to 2x SNR) with a standard filter set (CH1: 371-735, CH2: 479-735, CH3 Quasar (GaAsP): 416-690) through a 20 x/0.8 plan-Apochromat objective (Carl Zeiss) using laser illumination (Laser Diode 405 - 25 mW, Argon 458, 488, 514 - 30 mW, DPSS 561 -15 mW, HeNe 633 - 5 mW).

Statistics and Reproducibility

[0401] All values in FIGS. 1-11 are given as means ± SD, unless stated otherwise. All experiments were reproduced at two to seven independent times, with similar results.

[0402] GraphPad Prism was used to generate figures and perform statistical analyses (GraphPad Software). An a priori sample size estimation was not performed. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Data were analysed by using the unpaired two-tailed Student’s t-test, as indicated. P < 0.05 was accepted as statistically significant. Box and whisker plots depict the median and ranges from the first to the third quartile.

[0403] For data represented in FIGS. 12-19, all values are given +SEM. Data were analyzed by using the paired two-tailed Student’s t-test in line with the experimental setup. Significance is indicated as follows: * p<0.05, **p<0.01, ***p<0.001.