WO2013069010A1 - Méthodes pour optimiser individuellement un traitement du cancer - Google Patents

Méthodes pour optimiser individuellement un traitement du cancer Download PDF

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WO2013069010A1
WO2013069010A1 PCT/IL2012/050442 IL2012050442W WO2013069010A1 WO 2013069010 A1 WO2013069010 A1 WO 2013069010A1 IL 2012050442 W IL2012050442 W IL 2012050442W WO 2013069010 A1 WO2013069010 A1 WO 2013069010A1
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ascites
inhibitors
cancer cells
cells
culture
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PCT/IL2012/050442
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Talia GOLAN
Raanan BERGER
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Tel Hashomer Medical Research Infrastructure And Services Ltd.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of personalized medecine. More particularly, the invention relates to methods for individually optimizing a treatment for cancer patients, in particular for patients suffering from pancreatic cancer.
  • PC pancreatic cancer
  • PDA pancreatic ductal adenocarcinoma
  • PNETs pancreatic neuroendocrine tumors
  • PC Cell-line based in vitro model systems of PC are the most common tool for discovery of molecular targets for novel therapeutics as well as for preclinical evaluation of drug candidates.
  • the dependency of the cancer research community on established cell lines as the exclusive model for drug development has many limitations. Firstly, in PC there are a limited number of cancer cell lines, which have been cultured in vitro for a long time and may have altered their phenotypes and genotypes. Secondly, cell lines differ substantially from the clinical situation. The use of animal models in further drug development is mandatory. In vivo models are based on cell-line xenografts which are introduced into immune deficient mice. The limitations of such models are obvious, and these limitations impose unacceptable constraints on any type of drug development effort (Feldmann G, 2009).
  • the sampling of the ascitic fluid at different stages of the patient's tumor progression and the development of a "personalized primary cell culture” helps to establish a "tailor" model for each specific patient, in which it is possible to evaluate in vitro the response of the patient to a specific drug.
  • the establishment of such a model is of unique relevance as it may allow further research relating to pancreatic cancer along the course of the disease.
  • anti- tumoral efficacy of individual drugs may be tested, and combinations of biologically targeted drugs and/or cytotoxic chemotherapeutic drug on primary cultures of ascitic - derived pancreatic cells.
  • an in vitro method for assessing the efficacy of a pharmaceutical composition comprising at least one anti-tumorogenic agent, in the treatment of a hyperproliferative disorder in a subject comprising:
  • an in vitro method for determining an individually-optimized treatment for a subject suffering from a hyperproliferative disorder comprising:
  • a method for treating a hyperproliferative disorder in a subject in need thereof comprising:
  • the hyperproliferative disorder is pancreatic cancer, more particularly pancreatic ductal adenocarcinoma (PDA).
  • the ascites-derived primary cancer cells are primary pancreatic cancer cells.
  • the ascetic fluid comprises cells producing one or more of interleukin-1, interleukin-6, TNFa, CCL2, CCL3, CCL4, CCL5, CCL8, and CCL22 cytokines.
  • anti-tumorogenic agents are selected from the group consisting of Gemcitabine, erlotinib (Tarceva), oxaliplatin, 5-fluorouracil (5-FU), Capecitabine, Ftorafur (l-tetrahydrofuryl-5- fluorouracil), S-l, Thymidylate synthase inhibitors such as Raltitrexed (Tomudex), ZD9331, and Pemetrexed (Alimta, LY231514), cytosine analogs such as Gemcitabine (2',2'-difluorudeoxycytidine, dFdC), Troxacitabine (Troxatyl) and Tezacitabine[(E)- 2'-deoxy-2'-(fluoromethylene) cytidine (FMdC)], platinum analogs such as cisplatin and Oxaliplati, topoisomerase inhibitors such as Irinotecan (CPT-11, Camp
  • C Comparison of epithelial (CK7) and non epithelial (CK 5/6) markers immunostaining.
  • CK7 and CK 5/6 markers immunostaining.
  • PC tissue obtained from patient with pancreatic cancer during curative resection
  • Ascites-derived PC cells obtained from the same patient several months later. Similar staining between the two specimens is demonstrated.
  • D FACS staining of epithelial/epithelial origin marker cytokeratin- FITC supporting the presence of homogeneous epithelial PC enriched cell culture.
  • Fig. 2 Ascites-derived primary PC cultures functional characteristics.
  • Fig. 3 Overexpression of EMT characteristics in cells displaying an "epithelial- mesenchymal" morphological appearance.
  • A A similar cytokeratin staining between epithelial morphology cells and the EMT-like morphology cells. However, a significant higher fraction of cancer stem cell marker- CD44+ cells were observed in cells displaying "epithelial mesenchymal” morphology.
  • B Real-Time PCR EMT markers expression: Comparison of cultures with epithelial versus "epithelial mesenchymal" morphology in two patients (I and II). Several EMT markers are upregulated in the cultures displaying "epithelial mesenchymal” morphology.
  • Fig. 3 An cytokeratin staining between epithelial morphology cells and the EMT-like morphology cells. However, a significant higher fraction of cancer stem cell marker- CD44+ cells were observed in cells displaying "epithelial mesenchymal” morphology.
  • B Real-
  • Fig. 5 Ascites-derived primary PC cultures display varying chemotherapeutic responses.
  • A Varied responses in cells obtained from two different patients (patient 33 and patient 42).
  • B Two different cell cultures from the same patient. Cells were isolated before and after treatment with gemcitabine.
  • the present invention provides a method of individually optimizing treatment for a proliferative disorder. Specifically, the present invention can be used to select the most favorable pharmaceutical agents for the treatment of proliferative disorders such as prostate cancer or pancreatic cancer.
  • proliferative disorders such as prostate cancer or pancreatic cancer.
  • Pancreatic cancer refers to a malignant neoplasm of the pancreas.
  • the most common type of pancreatic cancer, accounting for 95% of these tumors is adenocarcinoma, which arises within the exocrine component of the pancreas.
  • the symptoms that lead to diagnosis depend on the location, the size, and the tissue type of the tumor. They may include abdominal pain and jaundice (Wikipedia. Pancreatic Cancer, 2011).
  • Pharmacological treatments of PC is tipically given to patients not suitable for resection with curative intent.
  • Administered treatments include amongst others: Gemcitabine, erlotinib (Tarceva), oxaliplatin, 5-fluorouracil (5-FU), Capecitabine, Ftorafur (l-tetrahydrofuryl-5-fluorouracil), S-l, Thymidylate synthase inhibitors such as Raltitrexed (Tomudex), ZD9331, and Pemetrexed (Alimta, LY231514), cytosine analogs such as Gemcitabine (2',2'-difluorudeoxycytidine, dFdC), Troxacitabine (Troxatyl) and Tezacitabine[(E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC)], platinum analogs such as cisplatin and Oxaliplatin, topoisomerase inhibitors such as Ir
  • Isogai et al. teaches a method to test cis-diamminedichloroplatinum (CDDP) sensitivity in ascites tumor cells from patients with peritonitis carcinomatosa (Isogai, Nagaya, Matsuoka, Watanabe, Tsukikawa, & Kubota, 2007).
  • CDDP cis-diamminedichloroplatinum
  • Brigulova et al. teaches a method to evaluate the cytotoxic effects of selected chemo therapeutics in cells isolated from ovarian tumours and ascites of individual patients (Brigulova, Cervinka, Tosner, & Sedlakova, 2010). Said publications do not teach how to optimize anti- tumorigenic treatments to PC patients.
  • the present inventors devised an in vitro assay wherein a selection of anti-tumorigenic drugs will be added to primary ascites samples of a pancreatic cancer cells and its anti-tumorigenic activity will be tested using engraftment of pancreatic cancer cells in turkey embryos, as by proliferation assay, apoptosis assays, in vivo- in-ova drug response assays, migration and invasion assay, and/or any other method that will be known to a person skilled in the art.
  • the drug showing greatest anti-tumorigenic activity will be selected as being the drug of choice for the particular individual by performing short-term and long-term biomarkers-driven clinical studies including preclinical, phase Ila proof of concept, and randomized phase II and perhaps phase III clinical trials, the present inventors will show that an anti-tumorigenic drug that had been selected according to the above described ex-vivo assay is the most preferable drug for the treatment of that PC patient.
  • the term "hyperproliferative disorder” refers to disorders characterized by an abnormal or pathological proliferation of cells, for example, cancer, psoriasis, hyperplasia and the like.
  • treatment refers to methods of killing, inhibiting or slowing the growth or increase in size of a body or population of hyperproliferative cells or tumor or cancerous growth, reducing hyperproliferative cell numbers, or preventing spread to other anatomic sites, as well as reducing the size of a hyperproliferative growth or numbers of hyperpproliferative cells.
  • treatment is not necessarily meant to imply cure or complete abolition of hyperproliferative growths.
  • a treatment effective amount is an amount effective to result in the killing, the slowing of the rate of growth of hyperproliferative cells, the decrease in size of a body of hyperproliferative cells, and/or the reduction in number of hyperproliferative cells.
  • the potentiating agent (or agents) is included in an amount sufficient to enhance the activity of the first compound, such that the two (or more) compounds together have greater therapeutic efficacy than the individual compounds given alone (e. g., due to synergistic interaction; reduced combined toxicity, etc.).
  • subject in need thereof as used herein typically refers to a human subject.
  • the subject has been diagnosed with the anti-proliferative disorder.
  • the subject may or may not have received treatment for the disorder.
  • ascites cells sample refers to cells obtained from paracentesis from patients.
  • Ascites may be collected at the time of paracentesis for palliation from pancreatic cancer patients.
  • the cells are sedimented by centrifugation, resuspended in 1:1 ascites fluid supernatant and RPMI 1640 medium (Biological industries, Beit HaEmeq, Israel) supplemented 2mM L-glutamine, 0.5% penicillin- streptomycin (100 U/ml), 0.5% sodium pyruvate, 10% FBS (Biological industries, Beit HaEmeq, Israel), then plated in tissue culture dishes and the medium lifted and placed in a fresh dish and incubated at 37°C and 5% C02.
  • Establishment of appropriate concentrations of the pharmaceutical agents can be effected by a comparison of the in-vivo treatment dose for each drug through equations with the in-vitro culture environment.
  • the in-vitro concentration for each drug may be calculated according to the serum levels acquired after in-vivo injections or according to functional equivalence tests. Alternatively or additionally, the appropriate concentrations may be established by in-vitro calibration assays.
  • assays used to measure the anti-tumorigenic activity include:
  • Proliferation assay 2x10 cells are seeded in a 96 wells plate. A pharmaceutical agent in different combinations are be added. After 72h the amount of cells will be measured by XTT cell proliferation kit (Biological industries)
  • Apoptosis assay lxlO 5 cells after different pharmaceutical agent treatment are fixed, pelleted and stained with propidium iodide (PI) for 1 hour before flow cytometry analysis. Cell cycle distribution and cell size are estimated using a standard protocol Engraftment of pancreatic cancer cells in turkey embryos: 3xl0 6 cells are injected to 7/8 days eggs. Eggs are treated with different pharmaceutical agents. On day 14, tumours are dissected and paraffin embedded for immunohistochemical staining.
  • PC Primary Pancreatic Cancer
  • Cells are plated in tissue culture dishes and the medium is lifted and placed in a fresh dish and incubated at 37 °C and 5% C0 2 . The medium is removed and placed in a fresh dish after 20 minutes. Fresh medium (without the ascites fluid supernatant) is changed twice a week. Cultures are monitored for mycoplasma using the EZ-PCR mycoplasma test kit (Biological industries, Israel).
  • the human pancreatic carcinoma cell line PANC-1 was maintained in DMEM.
  • the human breast epithelial cell line MCFIO was maintained in a mixture of DMEM and Coon's modified F12 medium.
  • the human melanoma cell line C8161 was maintained in RPMI. All media were supplemented with 10% FBS. All the cell lines were obtained from the American Type Culture Collection (ATCC, USA).
  • the dilutions of the antibodies were as follows: p53 (Leica Microsystems, U.K., NCL- P53-1801) 1:100, Calretinin (Cell Marque, Rocklin, USA, 232A-76 ) 1:100 , CK 5/6 (DAKO, Denmark, M 7237) 1:50, CK7 (Biogenex Laboratories, USA, MU255-UC) 1:100, CK19 (DAKO, Denmark, M 772) 1:50 and CEA (DAKO, Denmark, A 0115) 1:1000.
  • the slides were warmed to 600C for lhour and then processed using a fully automated protocol. Detection was performed with an Ultra View detection kit and counterstained with hematoxylin (both from Ventana Medical Systems). After the immunostaining was completed, slides were dehydrated rapidly in ethanol, cleared in xylene and covered with a coverslip containing Entellan (Merck, Germany).
  • lxlO 5 cells are seeded in triplicate on Trans well ThinCerts PET 8- ⁇ membranes (Greiner-bio-one) in RPMI 1640 supplemented with 0.5% FBS.
  • the upper well content was removed using cotton swabs and the number of cells that migrated was estimated using an XTT cell proliferation kit (Biological- Industries, Israel) according to manufacturer's instructions. Cell numbers were normalized to OD values of known numbers of cells.
  • 3x104 cells were seeded in Matrigel (BD Biosciences, USA) pre-coated inserts. C8161 cells (a highly invasive cell line) were used as a positive control.
  • the lower well contains the same medium with 10% FBS.
  • the upper well content which contained non-migrated cells, is removed using cotton swabs.
  • the amount of cells that migrates through the membranes is measured by standardized XTT staining (Biological-Industries), according to manufacturer's instruction. Cell numbers are normalized to OD values of known numbers of cells. Percent of invasion is calculated out of the number of cells seeded. For invasion assays, 3xl0 4 cells are seeded in Matrigel pre-coated inserts (BD Biosciences, USA). The matrigel is diluted 1:20 in serum free media. C8161 cells (a highly invasive cell line) are used as a positive control.
  • Cytokines composition in ascites' supernatant Cell-free supernatants are harvested and stored at -80°C until needed. The cytokines' composition is assessed using a commercially- available kit. In-ovo. Fertile chicken (Gallus Gallus) eggs are obtained from a local farm. On embryonic day 8 a window is made in the shell exposing the chorioallantoic membrane (CAM). l-3xl0 6 ascites-fluid-derived primary PC cells mixed with Matrigel (BD Biosciences, USA) in a total volume of 100 ⁇ are transplanted onto the CAM onto a 1cm diameter polypropylene ring, the eggs are then sealed and incubated for an additional 8 days.
  • CAM chorioallantoic membrane
  • Embryonic liver tissue and the mass in the ring on the CAM are dissected for PCR and histological analysis.
  • Matrigel alone and Matrigel with MCF10 cells are used as controls .
  • DNA is extracted from plated cultures using a DNeasy Blood & Tissue kit (Qiagen, Germany). Human DNA is detected using primers to the human specific alpha- satellite sequences [Grinberg et al., Leuk. Res. 33:1417-1426 (2009)]. The number of cycles is 35. Human DNA isolated from patient cells (positive control) and chicken DNA (negative control) are run with each PCR reaction. The alpha- satellite amplification product is 476 bp. The primers used are as follows: forward- GGG ATA ATT TCA GCT GAC TAA ACA and reverse- AAA CGT CCA CTT GCA GAT TCT AG [Grinberg et al., Leuk. Res. 33:1417-1426 (2009)].
  • RNA is isolated using TRIZOL reagent (Invitrogen, USA) and is used as a template in reverse transcription reaction using the ReadyMix PCR Master Mix (Thermo Scientific, USA) according to the manufacturer's protocol.
  • QRT-PCR is performed using SYBR-Green Master Mix (Applied-Biosystems, USA) on a 7500 Real-Time PCR system (Applied-Biosystems, USA). Gene expression is normalized to GAPDH. All primers (SEQ ID NOs: 1-20) were purchased from Sigma-Aldrich (USA).
  • Chemotherapeutic assays 2x10 ascites-derived primary PDA cells are seeded in 96- well plates and cultured for 4 days. The cells are treated with different chemotherapeutic agents: gemcitabine 10 ⁇ (medac, Germany), erlotinib 10 ⁇ (Roche, Switzerland), cisplatin ⁇ (Pharma-Chem, Holland), 5FU 5 ⁇ and oxaliplatin 6 ⁇ (Ebewe, Austria), irinotecan 10 ⁇ (Hospira, Australia) and paclitaxel ⁇ (Teva, Israel) for 72h.
  • FOLFIRINOX is a combination of 5FU, irinotecan and oxaliplatin.
  • Cell proliferation is measured using the XTT cell proliferation kit (Biological-Industries) according to the manufacturer's protocol.
  • Patients clinical characteristic Data on patient demographics, clinical history, surgery, systemic treatment, and responses to treatment and germ-line mutation of BRCA 1/2 were collected from the Chaim Sheba Medical Center cancer database and patient records.
  • Clinical stages were classified according to the TNM AJCC staging system after review of the patients medical, operative, and pathology reports. Responses to treatment are categorized according to imaging results.
  • Clinical Characteristics The model is based on the linkage and correlation between the ascites-derived PDA cells and the clinical course of the correspondent patient. Clinical characteristics of 22 patients for which primary cells cultures have been successfully established from the fluid obtained at palliative paracentesis/pleurocentesis, are summarized in Table 1 below. The clinical characteristics include: demographics, stage at diagnosis, metastatic sites, types and duration of treatments and germ line mutation carrier status. The personalized therapeutic response was correlated with the clinical characteristics of the patient.
  • Morphology of primary PC cells Morphological changes were observed at different stages of the patients disease.
  • the most common morphology of the ascitic-derived primary PC cell cultures was a cobblestone epithelial monolayer, tight cell-to-cell junctions, characteristic of epithelial cells (Fig. 1A).
  • Fig. 1A cobblestone epithelial monolayer, tight cell-to-cell junctions, characteristic of epithelial cells
  • Fig. 1A In four patients cultures were obtained during the progression of their disease; in three of these patients morphological changes in the cultures between the initial harvesting to later harvesting (ranging from weeks to months) were observed. These changes included elongated cells with a fibroblast spindle-like appearance and a gain of mesenchymal morphology characteristics and loss of epithelial morphology characteristics (Fig. IB).
  • IHC Immunohistochemistry
  • Cytokeratin staining by FACS was observed in 90-95% of the isolated cells in all samples supporting the presence of homogeneous epithelial PC enriched cell culture. Tumorigenicity and metastatic potential was further demonstrated by positive CD44 staining in 20-30% of selected CK positive isolated cells.
  • EMT Epithelial-mesenchymal-transition
  • EMT characteristics were examined by real-time PCR in two patients after mesenchymal morphology characteristics were observed. Up-regulation of EMT markers including Zeb-1, Zeb-2, Twist, Slug and Dab2 were observed in cells displaying 'epithelial- mesenchymal' morphology (Fig. 3B).

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Abstract

La présente invention concerne une méthode in vitro qui permet de déterminer un traitement optimisé individuellement pour un patient souffrant d'un trouble hyperprolifératif, qui comprend les étapes suivantes consistant : (a) à prélever une quantité de liquide d'ascite sur le patient; (b) à isoler et à mettre en culture des cellules cancéreuses primaires issues de ladite quantité du liquide d'ascite; (c) à mettre en contact des échantillons de la culture de cellules cancéreuses primaires dérivées d'ascite avec une pluralité de compositions pharmaceutiques, chacune desdites compositions pharmaceutiques comprenant au moins un agent anti-tumorogène; (d) à analyser l'activité anti-tumorogène de chacune desdites compositions pharmaceutiques sur la culture de cellules cancéreuses primaires dérivées de l'ascite; et (e) à identifier la composition pharmaceutique provoquant la plus forte activité anti-tumorogène à partir de ladite pluralité de compositions pharmaceutiques, ladite composition pharmaceutique constituant le traitement optimisé individuellement pour traiter ledit trouble hyperprolifératif chez ledit patient.
PCT/IL2012/050442 2011-11-10 2012-11-06 Méthodes pour optimiser individuellement un traitement du cancer WO2013069010A1 (fr)

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Cited By (3)

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WO2016154082A3 (fr) * 2015-03-23 2016-11-17 Tang Yao Procédés de culture de tissus primaires et de criblage de médicaments utilisant un sérum autologue et des fluides
WO2016181114A1 (fr) 2015-05-08 2016-11-17 Imagen Therapeutics Limited Milieu personnalisé
US10639313B2 (en) 2017-09-01 2020-05-05 Ndsu Research Foundation Compound for inhibition of delta-5-desaturase (D5D) and treatment of cancer and inflammation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016154082A3 (fr) * 2015-03-23 2016-11-17 Tang Yao Procédés de culture de tissus primaires et de criblage de médicaments utilisant un sérum autologue et des fluides
US10745667B2 (en) 2015-03-23 2020-08-18 Yao Tang Methods of primary tissue culture and drug screening using autologous serum and fluids
WO2016181114A1 (fr) 2015-05-08 2016-11-17 Imagen Therapeutics Limited Milieu personnalisé
US10639313B2 (en) 2017-09-01 2020-05-05 Ndsu Research Foundation Compound for inhibition of delta-5-desaturase (D5D) and treatment of cancer and inflammation

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