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WO2018160699A1 - Biomarqueurs pour le diagnostic, la prédiction et/ou le pronostic du cancer du pancréas et utilisations correspondantes - Google Patents

Biomarqueurs pour le diagnostic, la prédiction et/ou le pronostic du cancer du pancréas et utilisations correspondantes Download PDF

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Publication number
WO2018160699A1
WO2018160699A1 PCT/US2018/020233 US2018020233W WO2018160699A1 WO 2018160699 A1 WO2018160699 A1 WO 2018160699A1 US 2018020233 W US2018020233 W US 2018020233W WO 2018160699 A1 WO2018160699 A1 WO 2018160699A1
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mir
pancreatic cancer
microrna
extracellular vesicles
cells
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PCT/US2018/020233
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English (en)
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Reginald HILL
Katherine Richards
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University Of Notre Dame Du Lac
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present disclosure relates to methods and compositions for treating, inhibiting, or ameliorating diseases and/or conditions associated with a cancer, such as pancreatic cancer.
  • a cancer such as pancreatic cancer.
  • the present disclosure relates methods for treating pancreatic cancer in a subject, including measuring a quantity of microRNA in extracellular vesicles, and administering an agent for inhibiting extracellular vesicle secretion and a pancreatic cancer chemotherapy.
  • the disclosure also relates to combination therapeutics and therapies, including administration of extracellular vesicle secretion inhibitors in combination with a pancreatic cancer chemotherapy.
  • Pancreatic cancers such as pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of only 8%. Poor response to available therapies is a major factor contributing to this dismal prognosis.
  • Exosomes secreted membrane vesicles that range in size from 30-100 nm in diameter, released from epithelial cancer cells can promote drug resistance.
  • fibroblasts not epithelial cells, make up the majority of the tumor bulk in PDAC.
  • the mechanisms through which fibroblast-derived exosomes may contribute to chemoresistance following exposure to chemotherapy have not been studied.
  • a molecular-level understanding of possible fibroblast driven mechanisms of chemoresistance is essential for the development of more effective treatment strategies.
  • MicroRNAs are short, single stranded, RNA nucleotide sequences ranging from approximately 18-25 nucleotides long. MicroRNAs that have a complementary base sequence to that of an mRNA sequence of at the 3' untranslated region (UTR) have the ability to bind to the 3' UTR and inhibit translation of the mRNA into an amino acid sequence. Further, if the microRNA is a strong complementary match, it may assist with the degradation of the mRNA strand upon binding to the mRNA strand. Therefore, microRNAs can be regulators of genetic translation that can alter cell behavior, such as proliferation, cell growth, cell senescence, or cell migration. MicroRNAs can be secreted from cells through extracellular vesicles, such as microvesicles or exosomes.
  • pancreatic cancer diagnosis/prognosis is highly invasive and costly, requiring CT or MRI scans and surgery in order to obtain a piece of pancreatic tissue for biopsy. Symptoms associated with pancreatic cancer usually do not arise in patients until late stages of disease when the cancer cells have metastasized. Due to a lack of symptoms with early stage pancreatic cancer, patients often do not seek medical attention until symptoms arise during late stage disease, at which time invasive diagnostic procedures are performed. A non-invasive test for early stage pancreatic cancer diagnosis/prognosis would, therefore, be highly valuable.
  • pancreatic cancer it is therefore an aspect of this disclosure to provide improved treatment, diagnosis, and prognosis of pancreatic cancer by quantifying microRNAs associated with extracellular vesicles and administering a compound for inhibition of extracellular vesicle secretion and a pancreatic cancer therapy agent. It is another aspect of this disclosure to provide methods for treating and/or ameliorating diseases and/or conditions associated with a cancer, such as pancreatic cancer, using compositions and combination therapies provided herein.
  • Some embodiments provided herein relate to a method for treating pancreatic cancer in a subject.
  • the method includes obtaining a sample from a subject having or suspected of having pancreatic cancer, lysing the extracellular vesicles to release a microRNA therefrom, measuring a quantity of microRNA released from the extracellular vesicles, and treating the subject when the quantities of microRNA released from the extracellular vesicles are elevated compared to a reference value of microRNA from a sample obtained from an individual or from a population of individuals not having pancreatic cancer, wherein treating pancreatic cancer comprises inhibiting extracellular vesicle secretion and administering pancreatic cancer chemotherapy.
  • the sample includes extracellular vesicles.
  • the sample includes blood, lymphatic fluid, saliva, urine, pancreatic fine needle aspiration sample, or breast milk.
  • the extracellular vesicles are secreted from pancreatic cancer-associated fibroblasts.
  • the extracellular vesicles are isolated prior to the lysing step.
  • lysing the extracellular vesicles comprises subjecting the sample to one or more of an electric current, sonication, an amphipathic agent, and heating to 50°C or greater.
  • the quantity of microRNA is measured using one or more of RNA extraction, reverse transcription, quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RTPCR), fluorescent measurement, ion-exchange current measurement, microRNA sequencing, and microarray analysis.
  • the microRNA is hsa-miR-21, hsa-miR-221, hsa-miR-222, hsa-miR-181a, hsa-miR-146a, or hsa-miR-92a.
  • inhibiting extracellular vesicle secretion comprises administration of a neutral sphingomyelinase (N-SMase) inhibitor.
  • N-SMase neutral sphingomyelinase
  • the N-SMase inhibitor is GW4869.
  • the pancreatic cancer chemotherapy comprises gemcitabine, fluorouracil (5-FU), irinotecan, oxaliplatin, paclitaxel, capecitabine, cisplatin, or docetaxel, or analogues or combinations thereof.
  • Some embodiments provided herein relate to a method for identifying a subject having pancreatic cancer.
  • the method includes obtaining a sample from a subject having or suspected of having pancreatic cancer, lysing the extracellular vesicles to release a microRNA therefrom, measuring a quantity of microRNA released from the extracellular vesicles, wherein the subject is identified as having pancreatic cancer when the measured quantity of microRNA released from the extracellular vesicles is elevated compared to a reference value of microRNA from a sample obtained from an individual or from a population of individuals not having pancreatic cancer.
  • the sample comprises extracellular vesicles.
  • the sample comprises blood, lymphatic fluid, saliva, urine, pancreatic fine needle aspiration sample, or breast milk.
  • the extracellular vesicles are secreted from pancreatic cancer-associated fibroblasts.
  • the extracellular vesicles are isolated prior to the lysing step.
  • lysing the extracellular vesicles comprises subjecting the sample to one or more of an electric current, sonication, an amphipathic agent, and heating to 50°C or greater.
  • the quantity of microRNA is measured using one or more of RNA extraction, reverse transcription, quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RTPCR), fluorescent measurement, ion-exchange current measurement, microRNA sequencing, and microarray analysis.
  • the microRNA is hsa-miR-21, hsa-miR-221, hsa-miR-222, hsa-miR-181a, hsa-miR-146a, or hsa-miR-92a.
  • FIG. 1 illustrates a schematic overview of cancer- associated fibroblast (CAF)-derived exosome signaling during gemcitabine (GEM) treatment.
  • CAF cancer- associated fibroblast
  • GEM gemcitabine
  • CAFs treated with gemcitabine have upregulation of SNAIL and miRD 146a as well as exosome hypersecretion. This leads to increased cell proliferation, tumor growth, and chemoresistance of adjacent cancer epithelial cells.
  • GW4869 suppresses exosome release and exosomal transfer of SNAIL and miRD 146a.
  • FIG. 2 shows representative micrographs of hematoxylin and eosin (H&E) stained sections of pancreata from human pancreatic ductal adenocarcinoma (PDAC) samples and the Kras; Pten; CoxD2 overexpression mouse model of PDAC. Arrows indicate tumor cells in in the larger field of stromal cells (stars).
  • H&E hematoxylin and eosin
  • Figures 3A and 3B illustrate results of patient derived xenograft (PDX) model of PDAC.
  • Figure 3A illustrates tumor volume over time of mice implanted subcutaneously with PDX samples that were sensitive to GEM treatment or relapseable.
  • Figure 3B illustrates representative H&E staining micrographs and Ki67 staining of GEM treated samples compared to PBS treated controls.
  • Figure 3B illustrates in vivo effects of a pharmaceutical composition containing Compound I and pharmaceutically acceptable Carrier A administered orally at dosages of 0.1 mg/kg (open circles), 0.3 mg/kg (closed triangles), and 1 mg/kg (open triangles) in combination with castration on tumor size, as compared with castration alone (solid circles).
  • Figure 4 schematically illustrates identification of miRNAs that are expressed at lower levels in exosomes of WT fibroblasts (left) and untreated CAFs (middle), but in high levels in GEM D treated CAFs (right).
  • FIG. 5 graphically depicts patient D derived, cancer D associated fibroblasts (CAFs) were treated with ⁇ gemcitabine for 48 hours. Their survival rate was compared with that of GEM D sensitive epithelial cells (L3.6) and gemcitabine resistant epithelial cells (PANC1).
  • Figures 6A and 6B graphically illustrate that GTDCAF exosomes increase cell number and survival of epithelial cells.
  • Figure 6A illustrates live cell counts for L3.6 cells treated with L3.6, GTDPANC1, or GTDCAF1 exosomes for 6 days and 1 ⁇ GEM for 3 days, followed by determination of cell count.
  • Figure 6B illustrates PANC1 cells were treated with PANC1 or GTDCAF1 exosomes for 6 days, and AsPCl cells were treated with AsPCl or GTDCAF1 exosomes for 6 days. All cells were then treated with 1 ⁇ GEM for 3 days, and live cells were counted. *pDvalue ⁇ 0.05; **pDvalue ⁇ 0.01.
  • Figure 7 illustrates a schematic method for determining influence of exosomes from chemotherapy treated fibroblasts in proliferation, apoptosis, and sternness in PDAC cells.
  • Exosomes from treatment naive CAFs (D GEM) and GEM treated CAFs (+GEM) will be collected and administered to epithelial tumor cells or mice orthotopically implanted with tumor cells to test the impact of chemotherapy ⁇ treated fibroblast ⁇ derived exosomes on tumor growth and chemoresistance.
  • Figures 8A-8D graphically illustrate an ability of gemcitabine treatment to alter miRNA and RNA expression in pancreatic fibroblast cells.
  • Figure 8A illustrates RTPCR and shows that both SNAIL and miRD 146a were increased in CAFs that were GEM treated (GT) compared to not treated (NT).
  • Figure 8B illustrate that SNAIL and miRD 146a were found increased in exosomes ⁇ derived from CAFs that were GEM treated (GT) compared to not treated (NT).
  • Figure 8C and 8D illustrate epithelial cells were treated with exosomes from their own cell line (control) or GEM treated CAFs (CAF1).
  • Figure 9 illustrates a schematic method for determining a role of SNAIL/mirR-146a in chemotherapy-treated fibroblasts' ability to induce chemoresistance PDAC cells.
  • Exosomes are collected from CAFs with genetically altered miRD 146a or SNAIL expression and administered to epithelial tumor cells or mice orthotopically implanted tumors. Cells/mice are treated with chemotherapy and the role of miRD 146a and SNAIL in cell D extrinsic chemoresistance is determined.
  • FIG. 10 illustrates that expression of miRs linked to PTEN function increased in CAFD derived exosomes following GEM treatment.
  • RTPCR shows that expression of miRs linked to PTEN function were found increased in exosomes that were GEM treated (GT) compared to not treated (NT). miR relative normalized expression was quantified via RTPCR. **p ⁇ 0.01. *p ⁇ 0.05.
  • FIG 11 illustrates that GEM increases exosome secretion.
  • Cancer associated fibroblasts CAF1 or CAFs
  • WT wild type fibroblasts
  • L3.6 GEM sensitive EpCCs
  • PANCl GEM resistant EpCCs
  • Figure 12 illustrates inhibition of exosome secretion.
  • CAFs were treated with GEM or GEM and 20 ⁇ GW4869 for 3 days. Media was collected and exosomes were isolated and relative fluorescent units were quantified. **p ⁇ 0.01.
  • Figure 13 illustrates GW4869 treatment of GEMDtreated CAFs reduced chemoresistance.
  • Epithelial cells were coD cultured with DMSOD treated epithelial cells, DMSOD treated CAFs, or GW4869D treated CAFs (20 ⁇ ) plated on 0.4 ⁇ pore inserts for 3 days. The bottom coD cultured epithelial cells were then treated with gemcitabine for 3 days during coculture. Live coD cultured epithelial cells at the bottom of the plate were quantified. HPAFII are GEM resistant cancer cells. *pDvalue ⁇ 0.05.
  • Figure 14 illustrates GW4869 and GEM combination treatment inhibit tumor growth in vivo.
  • NOD/SCID mice were subcutaneously implanted with AsPCl cells CAF cells.
  • Two weeks post implantation mice were treated intraperitoneally with DMSO+PBS, DMSO+gemcitabine (GEM), or GEM+GW4869 twice weekly for ten days. Tumor growth over the course of the ten day post drug treatment.
  • FIG. 15 illustrates a schematic method for determining a therapeutic potential of inhibiting exosome release to combat fibroblast-mediated chemoresistance.
  • Epithelial cancer cells (EpCCs) cells are subcutaneously co- injected into NOD/SCID JL2y knockout mice with CAFs. Mice are given GEM with or without treatment with GW4869 to block exosome secretion. Tumor weight, proliferation, and apoptosis are measured. In addition, exosome uptake and miR- 146a and SNAIL expression in tumor cells are determined.
  • Figure 16 illustrates combination therapy on autochthonous mouse model of PDAC.
  • Mice are treated with I) GW4869 alone, II) chemotherapy alone; and ⁇ ) gemcitabine plus chemotherapy. All treatments start at 3 months (when all animals have developed PDAC). Samples are collected 1, 3, and 6 months following treatment as indicated (arrows). Three separate chemotherapy regimens are tested using the outlined combination dosing strategy.
  • Gemcitabine (GEM), Folfirinox (FOL), and Gemcitabine plus nabDpaclitaxel (NPT).
  • Figures 17A and 17B illustrate microRNA derived from extracellular vesicles.
  • Figure 17A shows microRNA derived from extracellular vesicles secreted from 1) gemcitabine-treatedPCAFs (Exo GEM) and 2) PBS-treated PCAFs (Exo Cont), quantified via RT-PCR. MicroRNA levels were normalized to number of cultured cells and volume of cell-conditionedmedia.
  • Figure 17B shows microRNA levels quantified via RT-PCR utilizing extracellular vesicle RNA secreted from a second patient-derived PCAF cell line (CAF2).
  • CAF2 patient-derived PCAF cell line
  • Figures 18A and 18B illustrate microRNA-92a levels.
  • Figure 18A shows microRNA-92a levels artificially elevated in pancreatic cancer epithelial cells by transfecting said cells with microRNA-92a mimic nucleotides. Elevated levels of microRNA-92a in transfected cells compared to cells transfected with a negative scramble control siRNA was verified via RT-PCR.
  • Figure 18B shows cells transfected with miR- 92a mimic having reduced levels of PTEN mRNA, as established by RT-PCR, validating that miR-92a binds to and degrades PTEN mRNA.
  • Figure 19A and 19B show Western blots of protein levels.
  • Figure 19A shows epithelial cancer cells (AsPCl) grown in the presence of control media, PCAFconditioned media, or exosome depleted (ED) PCAF-conditioned media. Upon growth in PCAFconditioned media PTEN protein levels decreased. Depletion of exosomes from PCAFconditioned media restored PTEN protein levels.
  • Figure 19B shows that upon growth in PCAF-conditioned media, phospho-AKT protein levels increased. Depletion of exosomes from PCAF-conditioned media restored phospho-AKT protein levels.
  • Figure 20 depicts pancreatic cancer epithelial cells (AsPCl) grown in PCAF-conditioned media expressed higher levels of microRNA-92a compared to control media, as established via RT-PCR.
  • the present disclosure relates in general to methods and compositions for treating pancreatic cancer in a subject.
  • the method includes obtaining a sample from a subject, isolating extracellular vesicles in the sample, lysing the extracellular vesicles, and measuring the quantity of microRNAs associated with the extracellular vesicles.
  • the method further includes administration of a compound for inhibiting extracellular vesicle secretion and administering a pancreatic cancer therapy when a measured quantity of microRNAs in the sample is elevated compared to a quantity of microRNAs in a reference sample from individuals not having pancreatic cancer.
  • the disclosure relates to combination therapies for treatment of pancreatic cancer, wherein the combination therapy includes a compound for inhibiting extracellular vesicle secretion in combination with a pancreatic cancer therapy.
  • the disclosure relates to methods of identifying a subject having pancreatic cancer, including the steps of obtaining a sample from a subject, isolating extracellular vesicles in the sample, lysing the extracellular vesicles, and measuring the quantity of microRNAs associated with the extracellular vesicles.
  • a subject is identified as having pancreatic cancer when the quantity of measured microRNAs in the sample is elevated compared to a quantity of microRNAs in a reference sample from individuals not having pancreatic cancer.
  • salt refers to a salt of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound.
  • the salt is an acid addition salt of the compound.
  • Pharmaceutical salts can be obtained by reacting a compound with inorganic acids such as hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid and/or phosphoric acid.
  • compositions can also be obtained by reacting a compound with an organic acid such as aliphatic or aromatic carboxylic or sulfonic acids, for example formic, acetic, succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic or naphthalenesulfonic acid.
  • organic acid such as aliphatic or aromatic carboxylic or sulfonic acids
  • Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C ⁇ - C 7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine, and/or salts with amino acids such as arginine and/or lysine.
  • a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C ⁇ - C 7 alkylamine,
  • each center may independently be of R-configuration or S -configuration or a mixture thereof.
  • the compounds provided herein may be diastereometrically pure, diastereometrically enriched, or may be stereoisometric mixtures.
  • each double bond may independently be E or Z a mixture thereof.
  • all tautomeric forms are also intended to be included.
  • compositions refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers.
  • the pharmaceutical composition facilitates administration of the compound to an organism.
  • Pharmaceutical compositions can also be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and/or salicylic acid.
  • Pharmaceutical compositions will generally be tailored to the specific intended route of administration.
  • physiologically acceptable defines a carrier, diluent or excipient that does not abrogate the biological activity and properties of the compound.
  • a "pharmaceutically acceptable carrier” refers to a substance, not itself a therapeutic agent, which may facilitate the incorporation of a compound into cells or tissues.
  • the carrier may be a liquid for the dissolution of a compound to be administered by ingestion.
  • the carrier may be a vehicle for delivery of a therapeutic agent to a subject.
  • the carrier may improve the stability, handling, or storage properties of a therapeutic agent.
  • the carrier may facilitate formation of a dose unit of a composition into a discrete article such as a capsule, tablet, film coated tablet, caplet, gel cap, pill pellet, or bead, and the like suitable for oral administration to a subject.
  • a "diluent” refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable.
  • a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and/or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation.
  • a common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that is physiologically compatible with human cells and tissues.
  • an “excipient” refers to an inert substance that is added to a pharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, or disintegrating ability etc., to the composition.
  • a “diluent” is a type of excipient.
  • a "subject” refers to an animal that is the object of treatment, inhibition, or amelioration, observation or experiment.
  • Animal includes cold- and warm-blooded vertebrates and/or invertebrates such as fish, shellfish, or reptiles and, in particular, mammals.
  • “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and/or apes, and, in particular, humans. In some embodiments, the subject is human.
  • a patient is selected who is suspected of having cancer, such as pancreatic cancer.
  • a patient is selected who is in need of treatment of cancer, such as a pancreatic cancer.
  • a patient is selected who has previously been treated for cancer, such as pancreatic cancer.
  • a patient is selected who has previously been treated for being at risk of cancer, such as pancreatic cancer.
  • a patient is selected who has developed a recurrence of cancer, such as pancreatic cancer.
  • a patient is selected who has developed resistance to therapies for cancer, such as pancreatic cancer.
  • a patient is selected who may have any combination of the aforementioned selection criteria.
  • treating do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and/or therapy.
  • the term “inhibit” refers to the reduction or prevention of the growth of a cancer, such as pancreatic cancer.
  • the reduction can be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values.
  • the term “delay” refers to a slowing, postponement, or deferment of an event, such as the growth of a cancer, such as pancreatic cancer, to a time which is later than would otherwise be expected.
  • the delay can be a delay of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values.
  • the terms inhibit and delay are not to be construed as necessarily indicating a 100% inhibition or delay. A partial inhibition or delay may be realized.
  • a therapeutically effective amount is used to indicate an amount of an active compound, or pharmaceutical agent, that elicits the biological or medicinal response indicated.
  • a therapeutically effective amount of compound can be the amount needed to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being administered the therapy. This response may occur in a tissue, system, animal, or human and includes alleviation of the signs or symptoms of the disease being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, in view of the disclosure provided herein.
  • the therapeutically effective amount of the compounds disclosed herein required as a dose will depend on the route of administration, the type of animal, including human, being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • the term "derivative” refers to a chemically modified compound wherein the modification is considered routine by the ordinary skilled chemist, such as an ester or an amide of an acid, or protecting groups such as a benzyl group for an alcohol or thiol, or a tert-butoxycarbonyl group for an amine.
  • analogue refers to a compound, which includes a chemically modified form of a specific compound or class thereof and which maintains the pharmaceutical and/or pharmacological activities characteristic of said compound or class.
  • biosimilar of an approved reference product/biological drug, such as a protein therapeutic, antibody, etc. refers to a biologic product that is similar to the reference product based upon data derived from (a) analytical studies that demonstrate that the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components; (b) animal studies (including the assessment of toxicity); and/or (c) a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference product is licensed and intended to be used and for which licensure is sought for the biological product.
  • the biosimilar biological product and reference product utilize the same mechanism or mechanisms of action for the condition or conditions of use prescribed, recommended, or suggested in the proposed labeling, but only to the extent the mechanism or mechanisms of action are known for the reference product.
  • the condition or conditions of use prescribed, recommended, or suggested in the labeling proposed for the biological product have been previously approved for the reference product.
  • the route of administration, the dosage form, and/or the strength of the biological product are the same as those of the reference product.
  • the facility in which the biological product is manufactured, processed, packed, or held meets standards designed to assure that the biological product continues to be safe, pure, and potent.
  • pancreatic cancer therapy refers to a treatment regime for treating pancreatic cancer.
  • a pancreatic cancer therapy includes a biological agent or therapy, a virus-based agent or therapy, surgery, a chemotherapeutic agent or chemotherapy, such as a taxane-based chemotherapy agent or a platinum-based antineoplastic agent, radiation or radiation therapy, a statin or a statin therapy, a repurposed drug or a repurposed drug therapy, a small molecule inhibitor or a small molecule inhibitor therapy, a therapeutic antibody or a therapeutic antibody therapy, a CAR T cell or a CAR T cell therapy, an immunotherapeutic agent or an immunotherapy, or any combination thereof.
  • chemotherapeutic agent or chemotherapy such as a taxane-based chemotherapy agent or a platinum-based antineoplastic agent, radiation or radiation therapy, a statin or a statin therapy, a repurposed drug or a repurposed drug therapy, a small molecule inhibitor or a small molecule inhibitor therapy, a therapeutic
  • biological agent refers to any chemical or biochemical compound produced by a living organism, which can include a prokaryotic cell line, a eukaryotic cell line, a mammalian cell line, a microbial cell line, an insect cell line, a plant cell line, a mixed cell line, a naturally occurring cell line, or a synthetically engineered cell line.
  • a biologic can include large macromolecules such as proteins, polysaccharides, lipids, and/or nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and/or natural products.
  • a biologic includes Bacillus Calmette-Guerin (BCG) vaccine, sargramostim, filgrastim, pegfilgrastim, recombinant interleukin-12, or interferon alpha, or a combination thereof.
  • BCG Bacillus Calmette-Guerin
  • virus-based therapy refers to the use of virus or virus like particles for use in the treatment, inhibition, or amelioration of a disease or condition.
  • a virus-based therapy includes use of a reo virus, bunyavirus, flavivirus, rubivirus, filovirus, arenavirus, arterivirus, or calicivirus.
  • a virus-based therapy includes a retrovirus, an adenoviral vector, (including the oncolytic adenovirus vector CG0070 (Cold Genesys)), or a Coxsackievirus A21 (CVA21 ; CAVATAK, Viralytics), or a combination thereof.
  • chemotherapeutic agents refers to any therapy that includes natural or synthetic chemotherapeutic agents now known or to be developed in the medical arts.
  • examples of chemotherapeutic agents include the numerous cancer drugs that are currently available.
  • chemotherapy also includes any drug, natural or synthetic, that is intended to treat, inhibit, or ameliorate a disease state, such as cancer e.g., pancreatic cancer.
  • chemotherapy may include the administration of several state of the art drugs intended to treat, inhibit, or ameliorate the disease state, such as cancer e.g., pancreatic cancer.
  • a chemotherapy comprises apaziquone, azacitidine, AZD4877, bleomycin, capecitabine, cyclophosphamide, dacarbazine, decitabine, doxorubicin, epirubicin, eribulin, erlotinib, etoposide, 5-fluorouracil, folinic acid, gemcitabine, ifosfamide, irinotecan, lenalidomide, leucovorin, methotrexate, mitomycin C, mustine, nab-paclitaxel, nanoliposomal irinotecan, oxaliplatin, paclitaxel, pemetrexed, pirarubicine, pralatrexate, prednisolone, procarbazine, rubitecan, sirolimus, temsirolimus, valrubicin, vinblastine, vincristine, vinflunine, or analgues, derivatives
  • Taxanes are a class of diterpenoid drugs that have anti-tumor activity against a wide range of human cancers.
  • Paclitaxel was originally isolated from the bark of the Yew tree, and was known to act by interfering with the normal function of microtubule breakdown. Paclitaxel binds to the ⁇ subunit of tubulin, the building blocks of microtubules, causing hyper-stabilization of the microtubule structures. The resulting paclitaxel/microtubule structure is unable to disassemble, thereby arresting mitosis and inhibiting angiogenesis.
  • a taxane-based chemotherapy comprises docetaxel, paclitaxel, cabazitaxel, larotaxel, ortataxel, milataxel, tesetaxel, or abraxane, or combinations, analogues, derivatives, emulsions, pro-drugs, or lipid conjugates, or polymers thereof.
  • Platinum-based antineoplastic agents are a class of platinum containing agents for use in cancer treatment, and are platinum based alkylating agents. Platinum- based antineoplastic agents inhibit DNA repair and/or DNA synthesis in cells, including cancer cells.
  • the platinum-based antineoplastic chemotherapy comprises cisplatin, carboplatin, dicycloplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, ormaplatin, phenanthriplatin, picoplatin, pyriplatin, or satraplatin, or analogues or derivatives thereof.
  • the pancreatic cancer therapy includes administration of a pancreatic cancer therapy agent.
  • a pancreatic cancer therapy agent is a compound used for the treatment of pancreatic cancer.
  • a pancreatic cancer therapy agent may include, for example, abarelix, actinomycin D, adriamycin, afatinib, aglatimagene besadenovec, aldesleukin, alemtuzumab, algenpantucel-L, alitretinoin, alkylating agents, allopurinol, ALN-PDL, altretamine, amifostine, amphotericin, anastrozole, anthracyclines, apaziquone, arsenic trioxide, asparaginase, atezolizumab, Atu-027, Avastin, avelumab, azacitidine, AZD4877, AZD9291, ba
  • the term "radiation therapy” refers to the medical use of ionizing radiation as part of a cancer therapy designed to kill malignant cells that are progressing through the cell cycle (e.g., in any phase of the cell cycle).
  • the radiation therapy may be internal or external radiotherapy.
  • External radiotherapy involves targeting doses (or "fractions") of high- energy beams of radiation, either X-rays or gamma rays, to the tumor.
  • Internal radiotherapy involves positioning the source of radioactivity inside the body close to the tumor. This can be achieved in two ways: by brachytherapy or by radioisotope therapy.
  • Brachytherapy involves placing a solid source of radiation next to a tumor to give a high dose of radiotherapy.
  • Radioisotope therapy involves administration of a radioactive substance, a radioisotope, either as an intravenous injection, or as an oral capsule or liquid.
  • statin refers to any of a class of lipid- lowering drugs that reduce serum cholesterol levels by inhibiting HMG-CoA reductase, a key enzyme involved in the biosynthesis of cholesterol, the mevalonate pathway or HMG- CoA reductase pathway.
  • statins can comprise atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and/or simvastatin, or combinations thereof, or a combination of a statin and another agent, such as ezetimibe/simvastatin.
  • repurposed drug therapy refers to a strategy by which a new or additional value is generated from a drug by targeting a disease other than those diseases for which the drug was originally intended.
  • a repurposed drug therapy comprises but is in no way limited to eflornithine, indinavir, metformin, or ritonavir, or a combination thereof.
  • small molecule inhibitor therapy refers to small organic molecules, peptides, antibodies, cyclic peptides and/or peptidomimetics that are small molecules, such as less than 10,000 Daltons (but not zero), and that act by inhibition, now known or to be developed in the medical arts.
  • a small molecule inhibitor therapy comprises belinostat, bortezomib, copanlisib, crizotinib, imatinib, dasatinib, dovitinib, rapamycin, everolimus, sirolimus, tipifarnib, pazopanib, alisertib, sapanisertib, lapatinib, lonafarnib, merestinib, olaparib, palbociclib, bosutinib, sorafenib, erlotinib, sunitinib, cabozantinib, gefitinib, ixazomib, vistusertib, vorinostat, entinostat vandetanib, BAY1163877, MLN8054, PLX3397, or BGJ398, or any combination thereof.
  • a therapeutic antibody therapy refers to any antibody, now known or to be developed in the medical arts, which can be administered to a subject as an active agent, including derivatives and fragments thereof, or antigen- specific ligand molecules, such as antibody Fab fragments, or antibody Fc fragments, synthetic receptors, or soluble receptors, which selectively bind a target antigen.
  • a therapeutic antibody therapy comprises cetuximab, ritixumab, bevacizumab, ranibizumab, trastuzumab, or panitumumab, fragments thereof, or any combination thereof, which may be presented on one or more CAR T cells.
  • an immunotherapy refers to a therapy now known or to be developed in the medical arts for a disease that relies on an immune response.
  • an immunotherapy comprises nivolumab, durvalumab, pembrolizumab, atezolizumab, ipilimumab, tremelimumab, CA-170, NEO-PV-01, or a tumor cell-derived vaccine therapy, or any combination thereof.
  • Some embodiments provided herein relate to treatment of pancreatic cancer comprising obtaining a sample, lysing extracellular vesicles, and quantifying an amount of microRNA in the sample, including a quantity of microRNA released from the lysed extracellular vesicles.
  • the extracellular vesicles is secreted from pancreatic cancer associated fibroblasts.
  • the method further includes administration of a combination therapy, wherein the combination therapy includes administration of a pancreatic cancer therapy as described herein in combination with administration of an inhibitor of extracellular vesicles secretion.
  • a compound for inhibiting extracellular vesicle secretion is administered to the subject.
  • a compound for inhibiting extracellular vesicle secretion may include, for example, a neutral sphingomyelinase (N-SMase) inhibitor, such as GW4869 (N,N'-Bis[4- (4,5 -dihydro- 1 H-imidazol-2-yl)phenyl] -3 ,3 '-p-phenylene-bis-acrylamide dihydrochloride) , BCI-137 (N-((2,3-Dihydroxy-6-quinoxalinyl)sulfonyl)alanine), gene targeting of extracellular vesicle protein markers (including, for example CD63), or gene targeting of extracellular vesicle secretion regulators (including for example, Rab GTPases such as Rab27a/b, Rab7, Rab35, or Rabl l).
  • N-SMase neutral sphingomyelinase
  • GW4869 N,N'-Bis[4- (4,5 -dihydro
  • coadministration refers to the delivery of two or more separate therapies or chemical entities, whether in vitro or in vivo. Coadministration refers to the simultaneous delivery of separate therapies or agents; to the simultaneous delivery of a mixture of therapies or agents; as well as to the delivery of one therapy or agent followed by delivery of a second therapy or agent or additional therapies or agents. In all cases, therapies or agents that are coadministered are intended to work in conjunction with each other.
  • a pancreatic cancer therapy agent is provided to the subject in an amount of 0.1 ⁇ to 10 ⁇ , such as 0.1 ⁇ , 0.5 ⁇ , 1.0 ⁇ , 1.5 ⁇ , 2 .0 ⁇ , 2.5 ⁇ , 3.0 ⁇ , 3.5 ⁇ , 4.0 ⁇ , 4.5 ⁇ , 5.0 ⁇ , 5.5 ⁇ , 6.0 ⁇ , 6.5 ⁇ , 7.0 ⁇ , 7.5 ⁇ , 8.0 ⁇ , 8.5 ⁇ , 9.0 ⁇ , 9.5 ⁇ , or 10.0 ⁇ or within a range defined by any two of the aforementioned amounts.
  • a pancreatic cancer therapy agent is provided to the subject in an amount of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg, or within a range defined by any two of the aforementioned amounts.
  • the pancreatic cancer therapy agent is administered to the subject orally or parenterally.
  • the pancreatic cancer therapy agent is administered to the subject twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, twice weekly, three times weekly, once every two weeks, once every three weeks, or once monthly, or a frequency within a range defined by any two of the aforementioned values.
  • an extracellular vesicle secretion inhibitor is provided to the subject in an amount of 1 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 20 ⁇ , 30 ⁇ , 40 ⁇ , 50 ⁇ , 60 ⁇ , 70 ⁇ , 80 ⁇ , 90 ⁇ , 100 ⁇ , 150 ⁇ , 200 ⁇ , 250 ⁇ , 300 ⁇ , 350 ⁇ , 400 ⁇ , or 500 ⁇ or a volume within a range defined by any two of the aforementioned values in a quantity of 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL,
  • the extracellular vesicle secretion inhibitor is administered to the subject orally or parenterally. In some embodiments, the extracellular vesicle secretion inhibitor is administered to the subject twice daily, once daily, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, twice weekly, three times weekly, once every two weeks, once every three weeks, or once monthly, or a frequency within a range defined by any two of the aforementioned values.
  • treatment of pancreatic cancer includes administration of an extracellular vesicle secretion inhibitor and a pancreatic cancer therapy agent provided to a subject in a single formulation or a single dosage.
  • the extracellular vesicle secretion inhibitor and a pancreatic cancer therapy agent are provided to a subject in separate formulations, but simultaneously or sequentially.
  • the extracellular vesicle secretion inhibitor and the pancreatic cancer therapy agent are formulated for oral or parenteral administration.
  • the product combination reduces pancreatic cancer tumor size.
  • treatment of pancreatic cancer comprises administration of a combination of GW4869 and gemcitabine.
  • a pharmaceutical composition comprising an extracellular vesicle secretion inhibitor disclosed herein and an additional therapy described herein can be cyclically administered to a patient. Cycling therapy involves the administration of a first active ingredient for a period of time, followed by the administration of a second active ingredient for a period of time and repeating this sequential administration. Cycling therapy can reduce the development of resistance to one or more therapies, avoid or reduce the side effects of one or more therapies, and/or improve the efficacy of the therapy.
  • a pharmaceutical composition comprising an extracellular vesicle secretion inhibitor disclosed herein and an additional therapy described herein are administered in a cycle of less than 3 weeks, once every two weeks, once every 10 days, or once every week. The number of cycles can be from 1 to 12 cycles, or from 2 to 10 cycles, or from 2 to 8 cycles.
  • the daily dosage regimen for an adult human patient may be the same or different for two active ingredients provided in combination.
  • the active ingredient is an extracellular vesicle secretion inhibitor.
  • the active ingredient is a pancreatic cancer therapy agent as described herein.
  • both an active ingredient including an extracellular vesicle secretion inhibitor and an active ingredient of a pancreatic cancer therapy agent are administered to a subject.
  • an extracellular vesicle secretion inhibitor can be provided at a dose of between 0.001 mg and 3 mg
  • a pancreatic cancer therapy agent can be provided at a dose of between 1 mg and 300 mg.
  • the dosage or each active ingredient can be, independently, a single one or a series of two or more given in the course of one or more days, as is needed by the subject.
  • the active ingredients will be administered for a period of continuous therapy, for example for a week or more, or for months or years.
  • a pharmaceutical composition including an extracellular vesicle secretion inhibitor disclosed herein can be administered one time per day.
  • the pancreatic cancer therapy agent can be administered once a week.
  • human dosages for active ingredients have been established for at least some condition, those same dosages may be used, or dosages that are between 0.1% and 500%, more preferably between 25% and 250% of the established human dosage.
  • a suitable human dosage can be inferred from ED5 0 or ID5 0 values, or other appropriate values derived from in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each active ingredient but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using MEC value.
  • Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.
  • Active ingredients disclosed herein can be evaluated for efficacy and toxicity using known methods.
  • the toxicology of a particular active ingredient, or of a subset of the active ingredients, sharing certain chemical moieties may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans.
  • the toxicity of particular compounds in an animal model such as mice, rats, rabbits, or monkeys, may be determined using known methods.
  • the efficacy of a particular active ingredient may be established using several recognized methods, such as in vitro methods, animal models, or human clinical trials. When selecting a model to determine efficacy, the skilled artisan can be guided by the state of the art to choose an appropriate model, dose, route of administration and/or regime.
  • the toxicology of a pharmaceutical composition including an extracellular vesicle secretion inhibitor may be established by determining in vitro toxicity towards a cell line, such as a mammalian, and preferably human, cell line. The results of such studies are often predictive of toxicity in animals, such as mammals, or more specifically, humans.
  • the toxicity of a pharmaceutical composition including an extracellular vesicle secretion inhibitor may be established by determining in vivo toxicity in an animal model, such as mice, rats, rabbits, or monkeys.
  • cancer refers to a class of diseases of humans (and animals) characterized by uncontrolled cellular growth.
  • cancer is used interchangeably with the terms “tumor,” “malignancy,” “hyperproliferation” and “neoplasm(s).”
  • cancer cell(s) is interchangeable with the terms “tumor cell(s),” “malignant cell(s),” “hyperproliferative cell(s),” and “neoplastic cell(s)” unless otherwise explicitly indicated.
  • hypoproliferative hyperproliferative
  • hyperplastic neoplastic
  • malignant neoplastic
  • neoplasm(s) are meant to include all types of hyperproliferative growth, hyperplastic growth, neoplastic growth, cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • pancreatic cancer refers to cellular carcinomas characterized by uncontrolled cell growth of pancreatic cells.
  • pancreatic cancer includes pancreatic ductal adenocarcinoma (PDAC), which is a malignancy of the pancreatic ductal epithelium.
  • PDAC pancreatic ductal adenocarcinoma
  • Other forms of pancreatic cancer include acinar cell carcinoma of the pancreas, cystadenocarcinomas, pancreatoblastoma, and pancreatic mucinous cystic neoplasms.
  • Pancreatic ductal adenocarcinoma has a dismal 5 -year survival rate of less than 8%. PDAC is currently the third leading cause of cancer-related deaths in the United States and is predicted to become the second leading cause of cancer- related deaths by 2030. Poor response to available therapies is a major factor contributing to this dismal prognosis.
  • gemcitabine GEM
  • FOL folfirinox
  • folinic acid a nucleoside analog
  • fluorouracil irinotecan
  • oxaliplatin a four-drug combination consisting of folinic acid, fluorouracil, irinotecan, and oxaliplatin is the standard-of-care adjuvant therapy in patients eligible for pancreatic resection.
  • fibroblasts contribute to the microenvironment surrounding cancer cells.
  • the majority of the tumor bulk of PDACs consists of fibroblasts.
  • CAFs cancer- associated fibroblasts
  • GEM-treated fibroblasts show hypersecretion of chemoresistance-promoting exosomes. Accordingly, some embodiments provided herein relate to methods for blocking the release of fibroblast-derived exosomes, resulting in reduced chemotherapy resistance in PDAC cells.
  • chemotherapy-treated, patient-derived fibroblasts hypersecrete exosomes that increase chemoresistance in neighboring pancreatic cancer cells. Furthermore, pharmacological inhibition of exosome hypersecretion reduces chemoresistance in vitro and suppresses tumor growth in vivo.
  • exosomes from fibroblasts exposed to chemotherapy contain miRNAs that help recipient epithelial cells survive chemotherapy.
  • the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least.”
  • the term “comprising” means that the process includes at least the recited steps, but may include additional steps.
  • the term “comprising” means that the compound, composition or device includes at least the recited features or components, but may also include additional features or components.
  • miRNAs refers to post-transcriptional regulators that typically bind to complementary sequences in the three prime untranslated regions (3' UTRs) of target messenger RNA transcripts (mRNAs), usually resulting in gene silencing.
  • miRNAs are short ribonucleic acid (RNA) molecules, for example, 25 nucleotides long or less, such as 24, 23, 22, 21, 20, 19, or 18 nucleotides or less.
  • RNA ribonucleic acid
  • MicroRNAs can be secreted from cells through extracellular vesicles such as microvesicles and exosomes. Therefore, microRNAs can be found in bodily fluids such as blood, lymphatic fluid, saliva, urine, and breast milk where they are mostly found encased in said lipid extracellular vesicles and are protected from degradation by RNases. Cancer cells secrete more exosomes and microvesicles from the cell surface than normal cells.
  • Cancer cells can also secrete more of certain microRNAs within these vesicles compared to normal cells, and they can change their microRNA secretion upon drug treatment. Therefore, quantifying the copy number of certain microRNAs found within bodily fluid extracellular vesicles is of great potential for diagnosing disease and following patients' response to therapy.
  • miRNA expression can mimic the effect of both oncogenes and tumor suppressor genes. miRNA expression serves as an important mechanism to orchestrate tumor/microenvironment interactions. Recent studies have shown that fibroblast-derived miRNAs are key regulators of mechanisms needed for cancer progression including endothelial cell function. Furthermore, in many instances, miRNA transfer between cells is mediated by exosomes.
  • Fibroblasts may inhibit or foster tumor development. Fibroblasts were previously believed to serve merely a passive role in PDAC drug resistance, impeding drug delivery by physically blocking cytotoxic chemotherapeutics from reaching their target epithelial cells. This led to the development of fibroblast-depleting therapies. Unfortunately, these therapies showed either very small increases in survival or more aggressive tumors. These results show a need to better understand how fibroblasts react to chemotherapy and how they may contribute to drug resistance in order to devise effective treatment strategies. Recent studies have shown that exosomes released from fibroblasts have been found to increase invasive behavior and drug resistance pathways in breast cancer cells. Several studies have shown that exosomal-derived miRNAs promote metastases and enhance endothelial cell migration. Yet no studies have examined the effects of exosomes derived from fibroblasts exposed to chemotherapy.
  • pancreatic cancer diagnosis/prognosis is highly invasive and costly, requiring CT or MRI scans and surgery in order to obtain a piece of pancreatic tissue for a biopsy.
  • Symptoms associated with pancreatic cancer usually do not arise in patients until late stages of disease when the cancer cells have metastasized. Due to a lack of symptoms with early stage pancreatic cancer, patients often do not seek medical attention until symptoms arise during late stage disease, at which time invasive diagnostic procedures are performed. A non-invasive test for early stage pancreatic cancer diagnosis/prognosis would, therefore, be highly valuable.
  • stromal tissue comprising fibroblasts increases in volume surrounding sites of hyperplasia, metaplasia, dysplasia, inflammation, and neoplasia.
  • fibroblast cells surround early precursor cellular lesions known as pancreatic intraepithelial neoplasias (PanlNs) and acinar to ductal metaplasias (ADMs), both of which are thought to be the original sites of cancer cell development and tumor formation.
  • Pancreatic intraepithelial neoplasias Pancreatic intraepithelial neoplasias (PanlNs) and acinar to ductal metaplasias (ADMs)
  • Pancreatic ductal adenocarcinoma (PDAC) tumors end up largely consisting of stromal tissue and fibroblasts.
  • PDAC pancreatic ductal adenocarcinoma
  • pancreatic cancer diagnosis and prognosis Because this influx of fibroblasts, known as desmoplasia, occurs during early stages that precede tumor formation, secreted factors, such as microRNAs encased in extracellular vesicles, from pancreatic fibroblasts at sites of abnormal cell growth may be a good source of biomarkers for pancreatic cancer diagnosis and prognosis.
  • secreted factors such as microRNAs encased in extracellular vesicles
  • microRNAs as diagnostic and prognostic markers for pancreatic cancer. Some embodiments relate to methods of quantifying microRNA levels from a sample from a subject having or suspected of having pancreatic cancer and comparing the measured quantity to a quantity of microRNA levels from a sample of an individual or population of individuals without pancreatic cancer to identify microRNAs upregulated in cancerous tissue. Some embodiments provided herein relate to quantifying microRNA levels in serum from healthy individuals not having pancreatic cancer and from a subject having pancreatic cancer in order to identify microRNAs that are more prevalent in serum derived from patients with pancreatic cancer. In some embodiments, the method further includes measuring microRNAs that are found within extracellular vesicles, including extracellular vesicles released from pancreatic cancer-associated fibroblasts (PCAFs).
  • PCAFs pancreatic cancer-associated fibroblasts
  • Some embodiments provided herein relate to a method of pancreatic cancer treatment, diagnosis, or prognosis including quantifying microRNAs derived from a patient sample and comparing the measured quantity of microRNAs to a reference value of microRNA or total RNA.
  • the method includes releasing microRNAs from extracellular vesicles including lysing extracellular vesicles, and thereafter quantifying released microRNAs.
  • microRNAs quantified for the treatment, diagnosis, or prognosis of pancreatic cancer may include, for example, hsa-miR-21, hsa-miR-221, hsa-miR-222, hsa-miR-181a, hsa-miR-146a, or hsa- miR-92a.
  • Extracellular vesicle refers to microscopic particles secreted by cells in the size of a few nm to a few ⁇ .
  • Extracellular vesicles are lipid- based microparticles or nanoparticles, or protein-rich aggregate, present in a sample (e.g., a biological fluid) obtained from a subject.
  • Extracellular vesicles are also referred to in the art and herein as exosomes, ectosomes, micro vesicles, apoptotic bodies, or nanovesicles.
  • Extracellular vesicles are secreted or shed from a variety of different mammalian cell types.
  • Exosomes are secreted membrane vesicles that range in size from 30-100 nm in diameter that are enriched with miRNA.
  • Studies examining the miRNA, RNA, and protein contents of melanoma exosomes show that the difference of miRNA signals between normal melanocytes and melanoma cells is much larger than that of mRNA signals or protein.
  • studies have shown that exosomal-derived miRNAs promote metastases and enhance endothelial cell migration. Accordingly, the present disclosure relates to exosomal miRNAs derived from fibroblasts for regulating chemoresistance.
  • Fibroblasts exposed to gemcitabine exhibit increased expression of SNAIL and miR-146a and hypersecretion of chemoresistance-promoting exosomes (Figure 1). Cancer cells dependent on the exosomes from fibroblasts for survival may be susceptible to chemotherapy if release of these exosomes is blocked.
  • Embodiments provided herein relate to method of treating, diagnosis, or prognosing pancreatic cancer in a subject.
  • the method includes obtaining a sample from a subject.
  • a sample can include peripheral blood, sera, plasma, pancreatic fine needle aspiration (FN A) sample, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus
  • the extracellular vesicles are secreted from pancreatic cancer- associated fibroblasts (PCAFs).
  • PCAFs pancreatic cancer- associated fibroblasts
  • the extracellular vesicles are isolated. In some embodiments, the extracellular vesicles are not isolated. In some embodiments, the extracellular vesicles are lysed. Lysis of extracellular vesicles can be performed, for example, by subjecting the sample to an extracellular vesicle lysis buffer, to an electric current, to sonication, to an amphipathic agent, or to heating to a temperature of 50°C or greater.
  • PCAFs are innately chemoresistant, and PCAF-conditioned cell media increases proliferation and chemoresistance of a chemosensitive PDAC cell line.
  • gemcitabine increases secretion of exosomes from cells, most prominently from PCAFs, and PCAF exosomes increases proliferation and chemoresistance of pancreatic epithelial cancer cells.
  • exosomes derived from gemcitabine-treated PCAFs increases proliferation and chemoresistance of pancreatic epithelial cancer cells more than exosomes derived from untreated PCAFs (Richards et al., 2016). Therefore, we identified that factors within PCAF-secreted extracellular vesicles that elicit heightened chemoresistance in cancer cells and may be reliable biomarkers for prognosis.
  • lysis of extracellular vesicles released microRNAs from the extracellular vesicles.
  • the method further includes quantifying microRNAs in the sample after lysis of extracellular vesicles.
  • MicroRNAs in the sample may be quantified by various techniques, including, for example, RNA extraction, reverse transcription, quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RTPCR), fluorescent measurement, ion-exchange current measurement, microRNA sequencing, or microarray analysis.
  • quantifying the microRNAs may be performed by subjecting the microRNAs to fluorescent oligonucleotide probes with at least 70% complementation to said microRNAs and thereafter measuring fluorescence or subjecting the microRNAs to oligonucleotide probes with at least 70% complementation to said microRNAs wherein said probes are attached to an ion-exchange membrane, and measuring the difference in the current-voltage characteristic of the ion-exchange membrane compared to a control.
  • microRNAs to be quantified that are identified as being present in pancreatic cancer- associated fibroblast extracellular vesicles that may promote chemoresistance and serve as both prognostic and diagnostic markers are selected from a group comprising hsa-miR-21, hsa-miR-221, hsa-miR-222, hsa-miR-181a, hsa-miR-146a, and hsa-miR-92a.
  • One microRNA quantified and compared to a control may be any one of said microRNAs alone.
  • any combination of said microRNAs may be used.
  • the level of microRNAs in a sample can be compared to the microRNA levels in a patient-matched sample or to the average microRNA levels within fluid samples from a control population without pancreatic cancer.
  • the quantification of hsa-miR-21 and hsa-miR-92a within a patient blood sample may be normalized to RNU6 microRNA or total RNA relative to a control blood sample, which is an average level of miR-21 and miR-92a in patients without pancreatic cancer.
  • treatment of pancreatic cancer is initiated when a quantity of measured microRNA in the sample is elevated compared to a reference value of microRNA from a sample obtained from an individual or from a population of individuals not having pancreatic cancer.
  • treatment of pancreatic cancer includes administration of a compound that inhibits extracellular vesicle secretion and administration of a pancreatic cancer therapy agent.
  • administration of the compound that inhibits extracellular vesicle secretion and administration of the pancreatic cancer therapy agent take place simultaneously or sequentially.
  • a method of identifying a subject having pancreatic cancer by measuring a quantity of microRNA in a sample, including the quantity of microRNA in extracellular vesicles.
  • a quantity of measured microRNA in the sample is elevated compared to a reference value of microRNA from a sample obtained from an individual or from a population of individuals not having pancreatic cancer, and the subject is identified as having pancreatic cancer.
  • diagnosis and prognosis of pancreatic cancer is made based on the level of microRNAs in the sample compared to a reference value, wherein elevated quantities of microRNA in a sample is indicative of pancreatic cancer.
  • Kras Pten mouse model of PDAC. Animal models that faithfully recapitulate both the histology and molecular profile of the human disease have become essential tools in the dissection of the cellular and molecular pathogenesis of PDAC. Mutations in KRAS, resulting in its constitutive activation, are observed early in tumorigenesis and are found in over 90% of PDACs. However, mice expressing KrasG12D from its endogenous locus progress to invasive cancers only after a prolonged latency unless coupled with other genetic alterations found in human PDAC. Loss of PTEN function in the context of mutant KrasG12D activation resulted in accelerated pancreatic intraepithelial neoplasia (PanIN) development and full progression to PDAC. This significantly decreased survival in the KrasG12D mouse model to 6 months in the case of heterozygous Pten deletion, suggesting that concurrent dysregulation of the PTEN and RAS pathways act synergistically to promote pancreatic cancer initiation and progression.
  • KRAS Mutations in KRAS
  • the Kras; Pten; Cox-2 OE mouse model This model accelerates tumor progression and has pronounced desmoplasia.
  • Fibroblasts are one of the major components of the inflammatory tumor microenvironment that is a hallmark of PDAC. Inflammation contributes to tumor development by overexpressing cyclooxygenase-2 (COX-2) in the mouse model of PDAC.
  • Cyclooxygenases, COX-1 and COX-2 are enzymes that are essential for production of prostaglandins. While COX-1 is a constitutively expressed housekeeping enzyme, COX-2 expression is upregulated in pancreatitis and pancreatic cancer.
  • Cox-2 overexpression (Cox-2 OE) leads to tumors accelerated PDAC development in the Kras; Pten mouse model.
  • this model mimics the pronounced desmoplastic (stromal cell presence) reaction that is a hallmark of PDAC.
  • Hematoxylin and eosin (H&E) stained sections of human PDAC samples demonstrate that pancreatic cancer cells make up only a minor portion of the tumor field.
  • This pronounced presence of fibroblasts is mimicked in the Kras; Pten; Cox-2 OE mouse model ( Figure 2).
  • this novel mouse model allowed testing of the effectiveness of the combination therapies on a model that has an abundance of fibroblasts.
  • a patient-derived, orthotopic xenograft (PDX) model was established.
  • PDX patient-derived, orthotopic xenograft
  • cells from each PDX line were implanted subcutaneously into NSG mice and allowed to grow to about 5 mm in diameter.
  • Tumor bearing NSG mice were then randomly separated into two cohorts with 5-10 mice per cohort, including: 1) control group (PBS treated); and 2) GEM treatment group (100 mg/kg GEM twice weekly).
  • Group 2 was then further separated into two subgroups, one with continuous treatment and the other with treatment release after the initial 3-4 week treatment course; both subgroups were monitored for up to a month and the slopes of their tumor growth were calculated as shown in Figures 3A and 3B. Seven patient samples were tested in this way, yielding two unique response groups.
  • the first group includes two PDX lines that are very sensitive to GEM treatment, hereafter termed as “Sensitive” (PDX-S1 and PDX-S2). These tumors shrank throughout the treatment course, as observed by the negative growth slope ( Figure 3A, top). Interestingly, even after withdrawal of GEM, tumors did not grow back after a month of drug release ( Figure 3A, top). Histologically, whereas placebo treated tumors were highly proliferative, tumor epithelia seemed to almost completely stop cycling after three weeks of GEM treatment, as seen by Ki67 staining ( Figure 3B).
  • the second group includes five PDX lines, hereafter termed “Relapseable" (PDX-R1-5).
  • Patient-derived fibroblasts were grown from tumor samples obtained from patients who had undergone surgical resection. Briefly, patient-derived PDAC tumor tissue was minced into l-3mm fragments, trypsinized for 30 minutes, washed in DMEM with 10% FBS, plated in a petri dish with DMEM containing 10% FBS, and fibroblasts were allowed to grow out of tumor fragments for 2-3 weeks. Cells were infected and immortalized with hTERT. Cells were authenticated by IDEXX RADILTM and were found to be mycoplasma free and did not genetically match any cell line in the DSMZ database. Cells used in experiments ranged from passage 2-10.
  • Exosomes derived from chemotherapy-treated, patient- derived, cancer-associated fibroblasts (CAFs) cells promote chemoresistance in epithelial cells through inhibition of apoptosis and increase sternness.
  • CAFs cancer-associated fibroblasts
  • This example demonstrates that chemotherapy-treated fibroblast derived exosomes exhibit a pro-survival effect when incubated with PDAC cells.
  • Exosomes are collected from CAFs grown in vitro either in the presence or absence of the cytotoxic chemotherapy, gemcitabine.
  • CAFs PDAC patient-derived fibroblast lines
  • GEM gemcitabine
  • PANC1 cells GEM resistant
  • CAF lines survived GEM treatment at a rate comparable to GEM-resistant PANC1 cells ( Figure 5).
  • wild-type fibroblasts also demonstrated the same survival rate, indicating that CAFs are intrinsically resistant to GEM.
  • CD63 is a well-established exosomal marker and has been used previously in exosome studies.
  • a CAF line was modified to fluorescently tag CD63-positive exosomes with Green Fluorescent Protein (GFP) (CAF-CD63GFP).
  • GFP Green Fluorescent Protein
  • CAFs were grown in GEM for three passages in order to ensure GEM resistance.
  • Exosomes were collected from media of CAF cells that were exposed to GEM treatment (GT-CAFs), and added to epithelial cells that had been grown in exosome depleted media. After a 1-week incubation, the cells were treated with 100 nM to 1 ⁇ GEM (depending on the IC 50 for the cell line) for 72 hours, and then tumor cell viability was assessed. Tumor cell uptake of stromal cell derived exosomes was confirmed using immunofluorescence for GFP.
  • GTCAF-derived exosomes induced a significant increase in cell survival in all recipient cell lines (Figure 6). This data show that exosomes from GT-CAFs increase survival in both GEM sensitive and resistant cell lines.
  • Exosomes were collected from WT fibroblasts, treatment naive CAFs, and GEM treated CAFs (GT-CAFs) and added to epithelial cancer cell lines (EpCCs). Three drug resistant cell lines were tested (PANC1, AsPC-1, HPAFII) and three drug sensitive cell lines were tested (BXPC3, L3.6, CFPAC-1). Cells were incubated with exosomes for a 1-week incubation and then treated with GEM for 72 hours. Cell apoptosis and proliferation was assessed via Caspase 3 activation and MTT assay.
  • Exosomes derived from the aforementioned CAF lines were analyzed to determine whether they increase pancreatic cancer sphere formation. This in vitro assay, where sphere number and size determine "sternness", allows a determination of whether GT- CAF exosomes increase the development of cells that possess tumor initiating ability.
  • L3.6 or AsPC-1 cells (which have sphere forming ability) were cultured in Corning ultra- low attachment plates in the presence of exosomes (10 ⁇ g/day each for 1-3 days) from WT fibroblasts, treatment naive CAFs, and GT-CAFs, respectively. The number and average size of "pancreaspheres" formed were recorded daily after the second day of exosome treatment.
  • EpCCs were implanted orthotopically in NOD/SCID JL2y knockout mice using established protocols (Figure 6).
  • Orthotopic xenografts were established using three drug resistant cell lines (PANC1, AsPC-1, HPAFII) and three drug sensitive cell lines (BXPC3, L3.6, CFPAC-1) to ensure that the results are widely applicable to PDAC.
  • Tumor cells were infected by a lenti-virus vector containing luciferase to allow noninvasive monitoring of tumor growth.
  • Exosomes from treatment naive CAF-CD63GFP cells and GEM treated CAF-CD63GFP cells were collected and administered to the mice every other day by intra-orbital injections one week after tumors engrafted (approx. 4-8 weeks).
  • GEM 100 mg/kg twice-a-week for 3 weeks
  • Tumor growth was followed using an IVIS Lumina.
  • Mice from each cohort were sacrificed two weeks, one month, and two months after the beginning of GEM treatment and tumor size and volume was measured to determine increase in tumors in mice exposed to exosomes isolated from conditioned media of GEM treated fibroblasts, indicating a resistance to chemotherapy.
  • the following example demonstrates a method of quantifying microRNAs obtained from a PCAF sample. Chemotherapy treatment of fibroblast induces hypersecretion of survival-promoting exosomes through a SNAIL mediated mechanism that increases miR-146a levels. This example demonstrates the role that the SNAIL/miR- 146a signaling axis plays in CAF-derived, exosomal regulation of chemoresistance.
  • Chemotherapy treatment of fibroblasts induces release of survival-promoting exosomes through a SNAIL mediated mechanism that increases miR-146a levels.
  • miR-146a is known to suppress invasion in PDAC cells, its role in chemoresistance was previously unknown. Recent data show that miR-146a is significantly overexpressed in hepatocellular carcinoma cells resistant to chemotherapy and it regulates sensitivity to interferon-based therapy in hepatocellular carcinoma cells.
  • exosomal miRNAs derived from chemotherapy-treated fibroblasts regulate chemoresistance in PDAC.
  • miR-146a was recently shown to be induced by SNAIL through the -catenin-TCF4 complex in colorectal cancer; however it has not been determined if SNAIL-miR-146a signaling plays a critical role in chemoresistance in PDAC and through what mechanisms.
  • RTPCR revealed that miR-146a and SNAIL are 5 -fold upregulated in CAFs following GEM treatment (Figure 8A). Moreover, SNAIL and miR-146a are increased in GT-CAF-derived exosomes ( Figure 8B). Increased SNAIL and miR-146a expression by GT-CAFs cells resulted in elevated SNAIL and miR-146a levels in cells receiving GT- CAF exosomes ( Figures 8C and 8D). To fully elucidate this mechanism it is important to first validate the hypothesized role of exosome-delivered miR-146a and SNAIL.
  • CAFs were generated with miR-146a or SNAIL overexpression (OVE) or knockdown (KD) by transducing CAFs with lentiviruses that overexpress SNAIL or siRNA lentiviruses against SNAIL (Applied Biological Materials). miR-146a expression was knocked down using anti-miR-146a lenti virus. miR-146a was activated using a miR- 146a mimic lentivirus. RTPCR and Western blot was used to validate the success of these genetic alterations.
  • exosome treatment results showing a change in levels of miR-146a and SNAIL in recipient cells was validated via RTPCR and Western blotting.
  • the cells were treated with 1 ⁇ GEM and cell viability was assessed after three days.
  • the ability of the exosomes from the modified CAF cells to alter proliferation, apoptosis, and survival in recipient cells was determined as described in Example 1.
  • Elevated SNAIL expression plays a critical role in chemoresistance promoting sternness in cancer cells.
  • Exosomes collected from the genetically modified CAF lines were collected and analyzed to determine altered pancreasphere formation in EpCCs using the methodology described in Example 1.
  • SNAIL-miR-146a signaling was determined necessary for GT-CAF exosomes to promote the acquisition of sternness in recipient cells, aiding chemoresistance.
  • miR-146a has numerous targets. However, the targets responsible for miR-146a's contribution to PDAC chemoresistance, and the mechanism through which they act, were previously unknown. As provided herein, miR-146a is shown to be important for exosomal-mediated chemoresistance, and we identified the target of miR-146a in these cells. Variants of the epithelial cancer cell lines were generated, and were engineered to overexpress miR-146a (mOE-L3 and mOE-PANCl, respectively). RNA was collected from these cell lines for microarray analysis. Gene expression patterns were analyzed using the Human Genome U133 Plus 2.0 chip (Affymetrix).
  • RNAs found in the analysis were also down-regulated in a majority of the three GEM sensitive and three GEM resistant cell lines following addition of exosomes from GEM-treated CAFs. Verification of miR-146a regulation of the identified target genes was performed using 3'-UTR clones via luciferase-based assays.
  • the 3'-UTR of the putative targets were cloned downstream of the firefly luciferase gene.
  • the chimeric transcript level was regulated by its interaction with miR- 146a, which resulted in quantifiable luciferase activity.
  • a decrease in luciferase expression in cells transfected with lentiviral miR-146a indicated that miR-146a directly regulates the putative target through its 3'-UTR binding.
  • Knockdown or overexpression of these target genes in the EpCCs was performed and the effect on cell proliferation and chemoresistance was evaluated as described in Example 1 to validate that miR-146a's mechanism of action require these target genes.
  • Exosomes from GEM-treated CAFs, SKO-CAFs, mKO-CAFs, and untreated SOE-CAFs and mOE-CAFs cells were collected and administered via intra-orbital injection to mice with orthotopic tumors as described in Example 1.
  • the same GEM sensitive and GEM resistant cell lines described in Example 1 were used. Mice were left untreated or treated with GEM. Mice from each cohort were sacrificed two weeks, one month, and two months after the beginning of GEM treatment and proliferation rate, apoptosis, and tumor size were determined. Tumors were sectioned and immunohistochemistry for SNAIL was performed to determine changes in SNAIL expression. RTPCR of frozen samples were utilized to determine changes in miR-146a expression.
  • SNAIL/miR-146a plays a critical role in the pro- survival effect observed when GT-CAF-derived exosomes are incubated with cancer cells.
  • miRNA-SEQ analysis identified miRs that were significantly increased in the exosomes of GEM-treated CAFs compared to treatment naive CAFs and WT pancreatic fibroblast. Five out of the top 10 hits were miRs that have been linked to the PTEN pathway. miR-92a, miR-221, miR-181a, miR-222, and miR-21 were all found to be significantly increased in the MIRSEQ analysis (Table 1).
  • Statistical analyses for this example include: the normalized expression levels of RNA will be compared using analysis of variance (ANOVA). Statistical analyses were performed by unpaired Students t-test. Significance was defined as *p ⁇ 0.05.
  • the following example demonstrates a method of quantifying microRNAs obtained from a PCAF sample. Inhibition of exosome release attenuates tumor chemoresistance.
  • GW4869 a compound that inhibits exosome secretion, was used to inhibit exosome secretion.
  • Autochthonous and patient-derived xenograft PDAC mouse models were contacted with GW4869 and/or current standard-of-care chemotherapy, and the benefits of combination therapy using this novel approach was determined by measuring survival.
  • This example demonstrates the benefits of utilizing exosome secretion blocking therapies in combination with currently approved chemotherapy to overcome chemoresistance.
  • GEM significantly increases the number of exosomes secreted by EpCCs, CAFs, and wild type fibroblasts (Figure 11).
  • CAF cells were treated with GEM or GEM and GW4869. The number of exosomes released was quantified. GW4869 treatment decreased exosome secretion by -70% in GEM treated CAFs ( Figure 12).
  • depletion of exosomes from GT-CAF-conditioned media, using GW4869 treatment or centrifugation significantly reduced expression of both SNAIL and miR-146a in recipient epithelial cells receiving the GT-CAF-conditioned media.
  • cancer cells co-cultured with GT-CAFs showed a significantly increased survival rate following exposure to GEM, blocking GT-CAF exosome hypersecretion using GW4869 treatment significantly reduced this survival benefit in multiple cell lines (Figure 13).
  • mice In vivo: to accurately recapitulate human PDAC, which has a high prevalence of fibroblasts, both epithelial tumor cells and CAFs were co-injected subcutaneously into mice. Both GEM-resistant AsPC-1 and CAFs were implanted due to CAFs having the most exosome hypersecretion during GEM treatment and, therefore, are target cells for therapies targeting exosome-secretion.
  • the mice Two weeks post tumor cell injection, the mice were treated via IP injections with PBS alone (control), 200 0.3 mg/ml GW4869 and GEM, or GEM alone twice weekly for two weeks. Tumors of control mice and mice treated with GEM steadily increased in size over time, while tumors of mice given combination therapy (GW4869 and GEM) remained relatively the same size, displaying significantly reduced growth rate after treatment compared to control mice ( Figure 14).
  • Subcutaneous xenografts using three GEM-resistant EpCC cell lines (AsPC-1 and, HPAFII, PANC1) and two GEM-sensitive cell lines (L3.6, BXPC3) were established to ensure that the results are widely applicable to PDAC.
  • a lentivirus vector containing luciferase to allow non-invasive monitoring of tumor growth was used to infect all tumor cell lines.
  • the EpCCs and fibroblasts were co-inoculated at a ratio of 1:5 to model the high stromal cell content seen in human PDAC, particularly after treatment with cytotoxic chemotherapy.
  • the same two-week dosing strategy was tested as previously performed (Figure 15). Mice were sacrificed at 6 weeks post cell injection and tumor weight was measured. Tumors were paraffin embedded, sectioned, and immunohistologically stained for Ki67 to quantify proliferating tumor cells and TUNEL to quantify tumor cells undergoing apoptosis.
  • mice were sacrificed from each cohort to determine the frequency of cancer cells showing uptake of GFP expressing exosomes from the CAF-CD63GFP cells. FACS analysis was used to determine the number of GFP-positive EpCC cells in a million cells from each tumor using a previously described method.
  • the Pdxl-Cre + ; Kras G12D/+ ; Pten lox/+ ; Cox-2 OE mouse model recapitulates the inflammation seen in human PDAC, making it an ideal model to test the efficacy of exosome hypersecretion inhibition in combination with chemotherapy. Furthermore, this model has well-defined tumor kinetics, providing reliable determinations for development of tumors. Therefore, this animal model was used to evaluate the combination therapy treatments described herein (schematic shown in Figure 16). Tumors develop at 3 months in this mouse model. Pdxl- Cre + ; Kras + ; Pten lox/+ ; Cox-2 OE mice were treated with 1) GEM, 2) GW4869; and 3) GEM, plus GW4869.
  • GEM 50 mg/kg, twice per week
  • GW4869 0.3 mg/ml, twice per week
  • All treatments began at 3 months when all animals developed PDAC.
  • Mice were analyzed at 1, 3 and 6 months post treatment. Histological samples were collected and lesion presence, lesion severity, cell proliferation rate, and cell death rate were assessed as previously described. All mice were genetically engineered so that their tumors express luciferase. Tumor growth was followed using an IVIS Lumina. Altered expression of SNAIL and miR-146a were used in the cancer cells, or other alternative miRs identified, as evidence that the combination treatment successfully inhibited exosome hypersecretion.
  • GEM+GW4869 combination therapy resulted in decreased chemoresistance, leading to reduced tumor growth/burden since GEM was more effective at killing PDAC cells in all the tumor models.
  • the tumors of mice receiving GEM+GW4869 exhibited less GFP+ exosomes and a lower expression of miR-146a and SNAIL.
  • the additional miRs identified in Table 1 also exhibited decreased expression in GEM+GW4869 treated tumors. While GEM is still the most widely used adjuvant treatment for PDAC, GEM+Nab-paclitaxel (NPT) or Folfirinox (FOL) were also examined.
  • GW4869 increased the efficacy of these chemotherapies as well.
  • CAFs treated with NPT show a 5 -fold increase in exosome release compared to a 7-fold increase in exosome released observed in GEM-treated CAFs.
  • GW4869 enhanced the efficacy of GEM+NPT and FOL ( Figure 16).
  • the combination of GW4869 plus GEM+NPT or FOL on human tumors in xenograft model was also efficacious.
  • mice in each group provide a minimum detectable difference between two group proportions not larger than 0.68 units using a two-sided Fisher's exact test at 0.05 significance and 80% power.
  • 20 mice in each group provide a minimum detectable difference between two group means of 1.32 standardized units using a two- sided two sample t-test at 0.05 significance and 80% power, and assuming estimated standard deviations of 1.0 in both groups.
  • the disclosure and methods provided herein thus relate to the use of exosome release inhibition to improve the management of patients with chemoresistant PDACs while elucidating critical information about the underlying molecular mechanisms response for the failure of efficacy of current standard-of-care chemotherapy.
  • the following example demonstrates a method of quantifying microRNAs obtained from a PCAF sample.
  • PCAFs were treated with either PBS as a control, or gemcitabine.
  • Secreted extracellular vesicles were collected from the PCAFs.
  • the extracellular vesicles were lysed, and the microRNA contained therein was released.
  • the released microRNA was quantified.
  • MicroRNA-Seq results show that microRNAs hsa-miR-21, hsa-miR-221, hsa- miR-222, hsa-miR-181a, and hsa-miR-92a were more prevalent in exosomes derived from gemcitabine-treated PCAF derived exosomes compared untreated PCAF derived exosomes (Table 1).
  • microRNAs identified in Table 1 may alter cellular pathways involved in tumorigenesis and can target tumor suppressor gene, PTEN, as shown in Table 2 and Table 3.
  • Table 2 Signaling pathways manipulated by identified microRNAs
  • Endoplasmic Reticulum UBE2E3, SAR1B, YOD1, HSPA5, UBE2J1, SKP1, EDEM1, Protein Processing EDEM3, MAN1A2, SVIP, SEC62, SEC24A, LMAN1,
  • B Cell Receptor FOS GSK3B, PIK3CB, PIK3AP1, PIK3R3, PIK3R1, AKT3, Signaling PIK3CA, MALT1, MAP2K1, CARD11, NFATC3
  • Certain microRNAs were transfected into pancreatic epithelial cancer cells and were validated to target a tumor suppressor gene, PTEN via RT-PCR ( Figures 18A and 18B).
  • PTEN acts as a tumor suppressor in part by inhibiting phosphorylation of AKT.
  • AKT is phosphorylated it becomes activated and promotes cell proliferation, protein synthesis, cell growth, cell metastasis, and chemoresistance.
  • pancreatic cancer epithelial cells When pancreatic cancer epithelial cells were cultured in PCAF-conditioned media, phosphorylated AKT protein levels were increased compared to control, and phosphorylated-AKT protein levels were reduced when PCAF-conditioned media was depleted of exosomes ( Figures 19A and 19B). Further, when pancreatic epithelial cancer cells were grown in PCAF- conditioned media, expression level of certain microRNAs was increased ( Figure 20), suggesting that the microRNAs are more prevalent within PCAF derived exosomes after gemcitabine treatment, can be transferred to pancreatic cancer cells, can inhibit translation of PTEN, and can promote chemoresistance and proliferation.
  • fibroblasts exposed to chemotherapy play an active role in promoting proliferation and chemoresistance of cancer cells through exosome hypersecretion.
  • exosome-derived miRNAs released by CAFs regulate chemoresistance and direct the design of therapeutic interventions.
  • chemotherapy-induced exosomal miRNAs may be a target of therapies given in combination with cytotoxic drugs, leading to significantly improved patient response.

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Abstract

L'invention concerne un procédé pour le traitement, le diagnostic ou le pronostic du cancer du pancréas chez un sujet, comprenant l'obtention d'un échantillon contenant des vésicules extracellulaires en son sein, les vésicules extracellulaires étant sécrétées par des fibroblastes associés au cancer du pancréas. Le procédé comprend en outre la lyse des vésicules extracellulaires et la libération des micro-ARN contenus en leur sein et la quantification des micro-ARN libérés.
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