METHODS FOR PREDICTING OUTCOME AND TREATMENT OF PATIENTS SUFFERING FROM PROSTATE CANCER OR BREAST CANCER
20210164984 · 2021-06-03
Inventors
Cpc classification
G01N33/57484
PHYSICS
A61K31/00
HUMAN NECESSITIES
International classification
Abstract
The invention relates to methods for predicting the outcome of a patient suffering from prostate cancer or breast cancer and methods for the treatment of prostate cancer or breast cancer. The inventors show that Doublecortin-expressing (DCX.sup.+) neural precursors from the central nervous system (CNS) enter the bloodstream, infiltrate prostate tumours and metastasis and differentiate into neo-neurons that contribute to tumour development. In human primary prostate tumours and transgenic mouse cancer tissues, the density of DCX.sup.+ neural progenitors is strongly associated with tumour aggressiveness, invasion and recurrence. In transgenic cancer mice, oscillations of DCX.sup.+ neural stem cells in the subventricular zone (SVZ), a neurogenic area of the CNS, were associated with egress of DCX.sup.+ cells from the SVZ to the bloodstream. These cells then reach the tumour where they initiate neurogenesis. Selective genetic depletion of DCX.sup.+ cells in mice inhibits the early phases of prostate cancer development, whereas ortho topic transplantation of DCX.sup.+ cells purified from prostate tumour or brain tissues promotes tumour growth and cancer cell dissemination. These results unveil a unique crosstalk between the CNS and the tumour that drives a process of neurogenesis necessary for prostate cancer development, and indicate a novel neural element of the tumour microenvironment as a potential target for cancer treatment. Thus, the invention relates to a method for predicting the outcome of a patient suffering from prostate cancer and compound targeting DCX.sup.+ neural progenitor cells for use in the treatment of prostate cancer.
Claims
1. A method for predicting the outcome of a patient suffering from prostate cancer or breast cancer comprising the steps of: i) determining the quantity of DCX.sup.+ neural progenitor cells in a biological sample obtained from the patient, ii) comparing the quantity determined at step i) with a corresponding predetermined reference value, wherein detecting a differential between the quantity determined at step i) and the corresponding predetermined reference value indicates the outcome of the patient.
2. The method of claim 1, wherein the DCX.sup.+ neural progenitor cells are characterized by expressing at least one of the neural markers selected from the group consisting of Doublecortin (DCX), Polysialylated-neural cell adhesion molecule (PSA-NCAM), Internexin (INA), Sca-1, CD24, EGFR and Nestin.
3. The method of claim 1, comprising a step of concluding that the patient has a good prognosis when the quantity determined at step i) is lower than the corresponding predetermined reference value or concluding that the patient has a poor prognosis when the quantity determined at step i) is higher than the corresponding predetermined reference value.
4. The method of claim 1, comprising a step of concluding that the patient has a non-aggressive, a non-invasive, and/or a non-recurrent prostate cancer when the quantity determined at step i) is lower than the corresponding predetermined reference value or concluding that the patient has an aggressive, an invasive, and/or a recurrent prostate cancer or breast cancer when the quantity determined at step i) is higher than the corresponding predetermined reference value.
5. The method of claim 1, comprising a step of concluding that the patient have a long survival time when the quantity determined at step i) is lower than the corresponding predetermined reference value or concluding that the patient has a short survival time when the quantity determined at step i) is higher than the corresponding predetermined reference value.
6. The method of claim 1, comprising a step of administering an anti-cancer treatment when it is concluded that the patient has a poor prognosis, an aggressive, an invasive, and/or a recurrent prostate cancer or breast cancer, or a short survival time.
7. A method for treating prostate cancer or breast cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound targeting DCX neural progenitor cells.
8. The method of claim 7, wherein the compound targeting DCX.sup.+ neural progenitor cells is selected from the group consisting of DCX inhibitor, CD24 inhibitor, EGFR inhibitor, Nestin inhibitor, Sca-1 modulator and PSANCAM modulator.
9. The method of claim 7, wherein the the compound targeting DCX.sup.+ neural progenitor cells is administered in combination with a classical treatment of prostate cancer or breast cancer.
10. A kit or device, comprising means for determining the level of DCX.sup.+ neural progenitor cells in a biological sample.
11. A kit or device, comprising means for determining at least one of the neural markers selected from the group consisting of Doublecortin (DCX), Polysialylated-neural cell adhesion molecule (PSA-NCAM), Internexin (INA), Sca-1, CD24, EGFR and Nestin.
12. The method of claim 7, wherein the patient has a poor prognosis, an aggressive, an invasive, and/or a recurrent prostate cancer or breast cancer, or a short survival time.
Description
FIGURES
[0175]
[0176]
[0177]
EXAMPLE
[0178] Material & Methods
[0179] Mouse Strains
[0180] Balb/c nu/nu (B6.Cg-Foxn1.sup.nu) and cMyc mice (FVB-Tg(ARR2/Pbsn-MYC)7Key (21), called Hi-Myc hereafter) were obtained from Charles River laboratories and the National Cancer Institute, respectively.
[0181] Hi-Myc mice were intercrossed with C57BL/6-Gt(ROSA)26Sortm1 (EYFP)Cos or C57BL/6-Gt(ROSA)26Sortm1 (HBEGF)Awai/J mice which were previously crossed with Tg(DCX-cre/ERT2)1Mul mice to generate Cre.sup.ERT2-inducible expression of the enhanced yellow fluorescent protein (EYFP) or simian Diphtheria Toxin Receptor (DTR; from simian Hbegf) under the control of a doublecortin (DCX) promoter (all obtained from the Jackson laboratory). The resulting offsprings DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc or DCX-Cre.sup.ERT2/loxp-HBEGF/Hi-Myc express EYFP or DTR, respectively, in DCX-expressing cells after administration of tamoxifen to the animals. Cells expressing DTR can be ablated following diphtheria toxin administration. Respective controls were also generated by intercrossing the three strains.
[0182] Immunodeficient B6.Cg-Foxn1.sup.nu+/− heterozygous nude mice were also intercrossed with Tg(DCX-cre/ERT2)1Mul bred with Gt(ROSA)26Sortm1(HBEGF)Awai/J to deplete cells that express DCX in nu/nu mice.
[0183] Pten transgenic mice were generated by a specific Pten deletion (Pten.sup.loxp/loxp) in prostate epithelial cells under the control of the probasin promoter (ARR2Pbsn-Cre, PB-cre4). PyMT transgenic mice express the polyomavirus PyV middle T antigen specifically in mammary epithelial cells under the control of mouse mammary tumor virus (MMTV).
[0184] Animal Procedures
[0185] All in vivo experiments were approved by the Animal Care and Use Committee of CEA (Fontenay-aux-Roses, France) as referred to the authorisation 2015022617149597.
[0186] For DCX-Cre.sup.ERT2-mediated recombination, tamoxifen was prepared in corn oil (100 mg/kg, twice a day for 5 consecutive days) and was injected by intraperitoneal injection (Sigma-Aldrich, Saint-Louis, Mich.). For DCX.sup.+ cell depletion experiments, diphtheria toxin (4 μg/kg, Sigma-Aldrich) was injected once daily, intraperitoneally, 48 h after the last tamoxifen injection for 3 consecutive days.
[0187] For experiments in transgenic model, DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice were injected with tamoxifen at different time points between week 3 and week 24 after birth, and sacrificed 2 weeks after the last injection of tamoxifen. For inducible genetic tracing experiments, DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice were injected at week 3 after birth and animals were euthanized at 8, 12, 16 and 20 weeks after birth. For histological analyses after DCX.sup.+ cell depletion, DCX-Cre.sup.ERT2/loxp-HBEGF/Hi-Myc animals were injected with tamoxifen (at week 3, week 4 and week 8 or week 12 and week 16 after birth) and killed at week 20.
[0188] For orthotopic xenogeneic model, human prostate tumours were induced by orthotopic surgical implantations of 1×10.sup.5 PC-3luc cells into 6 weeks-old Balb/c nu/nu mice. Three weeks after cell injection, the animals were randomized into the different groups and received, orthotopically, a vehicle or the appropriate transplant of purified Lin.sup.− EYFP.sup.+ cells isolated from prostate, OB or SVZ tissues. For DCX.sup.+ cell depletion experiments, 1×10.sup.5 PC-3luc cells were implanted orthotopically into tamoxifen-injected DCX-Cre.sup.ERT2/loxp-HBEGF/ nu/nu mice (or control littermates), 2 days after the last injection of diphtheria toxin, with or without the co-transplantation of purified sub-populations of Lin.sup.− EYFP.sup.+ cells isolated from DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc prostate tumours.
[0189] Microdissection of the SVZ, OB and DG from the Adult Mouse Brain
[0190] After euthanasia, the head of mice was cut off above the cervical spinal cord region, and a medial caudal-rostral cut was made to remove the skin of the head. The skull was peeled outward to expose the brain. The olfactory bulbs were first removed and collected in RPMI medium supplemented with 10% FBS (Life technologies, Carlsbad, Calif.). Then, the brain was rotated to expose its ventral surface and the SVZ was isolated, under a dissecting microscope, by making a first coronal cut at the level of the base of the optic chiasm and a second cut just before the hippocampus (data not shown), resulting to a coronal section from which the subventricular zone was harvested and collected in RPMI medium supplemented with 10% FBS as described by Guo et al., 2005. The hippocampus containing the dentate gyms was dissected from the remaining caudal part of the brain (37). The medial surface of cerebral hemisphere was placed side up to expose the medial side of the hippocampus. A needle-tip was inserted at the boundary of the dentate gyms and Ammon's horn and slipped superficially along the septo-temporal axis of the hippocampus to isolate the DG which was collected in RPMI medium supplemented with 10% FBS.
[0191] Dissociation of Brain and Prostate Tissues
[0192] Prior to be dissociated, prostate tissues were minced with a scalpel blade, and then placed into C tube for enzymatic dissociation. Brain and prostate tissues were enzymatically dissociated to single-cell suspensions into gentleMACS C tubes using the respective neural (T) or tumor tissue dissociation kits and the gentleMACS octo dissociator with heaters. The gentleMACS programs NTDK_1 or m_TDK_1 were applied, respectively, as recommended by the manufacturer (Miltenyi biotec, Bergisch Gladbach, Germany). After dissociation, the cell suspension was applied to a MACS smartstrainer (70 μm), centrifuged and resuspended in MACS running buffer.
[0193] Cell Culture
[0194] PC-3 cells stably transfected with the luciferase 2 gene under the control of human ubiquitin C promoter (Perkin Elmer, Waltham, Mass.), were grown in F12-Glutamax medium supplemented with 10% FBS, 1.5 g/l Bicarbonate sodium (Life technologies, Carlsbad, Calif.).
[0195] After enzymatic dissociation of OB and prostate cells from DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice, cells were washed, resuspended in MACS neuro medium (Miltenyi biotec) supplemented with basic fibroblast growth factor (bFGF, 20 ng/ml) and epidermal growth factor (EGF, 20 ng/ml; Preprotech, Rocky Hill, N.J.), and then 10.sup.6 cells were plated into one well of a 24-well tissue culture plate (Thermofisher scientific, Waltham, Mass.) that was previously coated with Poly-L-ornithine (10 μg/ml, Sigma-Aldrich, Saint-Louis, Mich.) and laminin (10 μg/ml, Corning life sciences, Corning, N.Y.). Half of the medium was replaced every 48 h for 1 week. Then, adherent cells were harvested and passed onto Poly-L-ornithine- and laminin-coated μ-slide 8-well (ibidi, Martinsried, Germany). Cells were incubated in MACS neuro medium supplemented with bFGF (20 ng/ml) and EGF (20 ng/ml) or brain-derived neurotrophic factor (BDNF, 1 μg/ml, Miltenyi biotec) and neurotrophin-3 (NT-3, 0.1 μg/ml; Miltenyi biotec). The medium was replaced every 48 h until neural differentiation of the cells.
[0196] Bioluminescence Imaging
[0197] In vivo and ex vivo bioluminescence imaging was performed and analysed using an IVIS imaging system 200 series (Xenogen, Caliper Life Sciences, Hopkinton, Mass.). Bioluminescent signal was induced by i.p. injection of D-luciferin (150 mg/kg in PBS) 7 min prior to in vivo imaging. For ex vivo imaging, D-luciferin (300 mg/kg) was injected 8 min prior to necropsy. Organs of interest were immersed in a solution of D-luciferin at 150 mg/ml (4).
[0198] Histology and Immunofluorescence For paraffin-embedded sections, human and mouse prostate tissues were previously fixed in formalin or 4% paraformaldehyde, respectively. Blocks were serially sectioned (thickness 5 μm) and H&E staining was performed using standard procedures. Prior to stain, sections were deparaffinized with xylene and rehydrated through graded alcohol washes followed by antigen retrieval in sodium citrate buffer following manufacturer recommendations (Vector laboratories, Burlingame, Calif.).
[0199] For frozen sections, mice were anesthetized with 4% isoflurane before receiving a lethal dose of pentobarbital (60 mg/ml). Animals were then fixed by cardiac perfusion with 0.9% NaCl followed by 4% ice-cold paraformaldehyde (PFA) in 0.01 M PBS. The brain and prostate were collected, post-fixed overnight in 4% PFA at +4° C. and transferred in a 12% sucrose solution in PBS before snap freezing and cryostat sectioning (thickness 12 μm; Leica, Wetzlar, Germany).
[0200] For immunofluoresecnce, nonspecific binding was blocked with goat serum in BSA solution, and sections were incubated overnight with mouse antibodies to DCX (Millipore, Billerica, Mass.), to PSA-NCAM (ABC scientific, Los Angeles, Calif.) or to pan-cytokeratine (Sigma-Aldrich), rabbit antibodies to MAP-2 (Millipore), to βIII-tubulin (Covance, Princeton, N.J.) or to α-Internexin (Millipore), and chicken antibodies to NF-H (Millipore) or to EYFP (Ayes labs, Tigard, Oreg.). Secondary staining was subsquently performed for 1 h at room temperature with the appropriate Alexafluor647-, 568-, 488-conjugated goat antibodies to mouse, rabbit or chicken IgG respectively (Life technologies, Carlsbad, Calif.).
[0201] For dye administration, fluorescein-tagged albumin (65 kDa, 2 mg diluted in 0.1 ml saline; Sigma-Aldrich) and TRITC-tagged dextran (4.4 kDa, 2 mg diluted in 0.1 ml saline, Sigma-Aldrich) were simultaneously IV and IP injected into 5-month-old Hi-Myc cancer mice under anesthesia. After a circulation period of one hour, mice were deeply anesthetized with isoflurane and pentobarbital and euthanized by transcardial perfusion with 0.9% NaCl followed by 4% ice-cold paraformaldehyde (PFA) in 0.01 M PBS. Brains were collected, post-fixed overnight in 4% PFA at +4° C. and transferred in a 12% sucrose solution in PBS before snap freezing and cryostat sectioning (thickness 14 μm; Leica).
[0202] Bright-field images of full Hi-Myc prostate sections were captured and collected with a Zeiss axioscan Z1 (Zeiss MicroImaging, Thornwood, N.Y.) equipped with an Hitachi HV-F202FCL color camera controlled by Zen microscope software.
[0203] Fluorescence images were captured and analyzed using a Leica TCS SP8 X confocal microscope equipped with White Light Laser, a PMT SP confocal detector coupled with a Leica hybrid detector (HyD) for super-sensitive confocal imaging (Leica, Wetzlar, Germany). Images were obtained as three dimensional (3D) stacks scanning through the whole thickness of the tissue controlled by LAS X 2.0.1.14392 software and analysed using high performance 3D imaging Volocity 6.3.1 software (Perkin Elmer, Waltham, Mass.).
[0204] Human Prostate Samples
[0205] Radical prostatectomies were obtained for staining after institutional review board approval at the department of pathology and biological resources platform at Henri Mondor hospital (Créteil, France; CPP n°16169). Human prostate tissues were collected, fixed in formalin and embedded in paraffin as part of routine care at Henri Mondor hospital. For each block, a section was stained with H&E to evaluate tissue viability, to localize normal areas among cancer, and to map the different Gleason grade areas. All patient had histologically confirmed and clinically localized prostate cancer or benign hyperplasia, and did not received prior treatment at the institution. Patient characteristics including age, preoperative PSA levels, date of surgery, pathological stages and Gleason score are shown in Table 1. PSA recurrence was defined as a single PSA value at >0.2 ng/ml, two values at 0.2 ng/ml, or secondary treatment for a rising PSA. Recurrence might be local or distant, although no metastasis has been documented thus far in this cohort of patients. Extraprostatic extension was defined as disease involving one or more of extracapsular, ganglion, or seminal vesicle extension and positive surgical margins. Quantification of DCX.sup.+ cells was conducted blind, without knowledge of clinical data, in prostate tumour or hyperplastic areas and in remaining normal prostate tissues surrounding cancer areas. For each marker defined above, the average of 10 representative fields (one field=0.15 mm.sup.2) was calculated from normal areas and for tumour grade captured as described above. A total of 1040 z-stack images were acquired and converted in 2D maximum projections that were analyzed with the Volocity software to quantify DCX.sup.+/DAPI.sup.+ cells per field (DAPI, 4′,6-diamidino-2-phenylindole).
TABLE-US-00001 TABLE 1 Clinical and Pathological characteristics of men with benign hyperplasia or prostate cancer Patient characteristics Tumor characteristics Age PSA Date of Gleason Pathological #invaded Recurrence # (years) Risk (ng/ml) the surgery score stage 0−/1+ 1 67 BPH 17 2013 — — — — 2 72 BPH 7.42 2013 — — — — 3 58 BPH 1.42 2013 — — — — 4 85 BPH 3.3 2013 — — — — 5 63 BPH 4.5 2014 — — — — 6 85 BPH 6 2013 — — — — 7 72 BPH — 2013 — — — — 8 55 BPH 0.92 2013 — — — — 9 71 BPH 5.37 2013 — — — — 10 68 BPH 4.9 2013 — — — — 11 62 BPH 27 2013 — — — — 12 69 BPH 3.6 2013 — — — — 13 63 BPH 6.6 2013 — — — — 14 83 BPH — 2014 — — — — 15 76 BPH 6 2014 — — — — 16 65 Low 6.25 2007 6 T2c 0 0 17 63 Low 5.31 2007 6 T2b 0 0 18 68 Low 5.75 2007 6 T2c 0 0 19 69 Low 6.4 2007 6 T2a 0 0 20 59 Low 5 2007 6 T2a 1 0 21 72 Low 10.23 2006 6 T2c 0 0 22 58 Low 6.09 2006 6 T2c 0 0 23 64 Low 3.9 2007 6 T2c 0 0 24 55 Low 4.34 2007 6 T2c 0 0 25 59 Low 3.83 2006 6 T2c 0 0 26 63 Low 5.3 2007 6 T3a 1 0 27 60 Low 4.2 2007 6 T2c 1 0 28 59 Low 1.7 2008 6 T2b 0 0 29 53 Low 4.6 2008 6 T2c 0 0 30 73 Low 6.37 2008 6 T2a 1 0 31 76 Low 4.54 2008 6 T2c 0 0 32 60 Low 3.5 2009 6 T2c 1 1 33 60 High 13 2006 7 T2a 0 0 34 66 High 9.98 2007 7 T2a 0 0 35 71 High 9.6 2006 8 T3b 2 0 36 71 High 3.9 2006 8 T3a 2 1 37 70 High 13.22 2006 8 T3a 2 1 38 63 High 10.32 2008 8 T3a 2 1 39 75 High 12.26 2008 8 T4 4 1 40 70 High 8.7 2008 8 T3b 3 1 41 53 High 2.9 2009 8 T3b 2 1 42 60 High 8.91 2009 8 T4 3 1 43 60 High 16 2009 8 T3a 1 1 44 66 High 15.95 2009 8 T3a 2 1 45 62 High 7.11 2007 9 T3a 1 0 46 70 High 45 2008 9 T3b 4 1 47 71 High 8.7 2008 9 T3b 4 0 48 50 High 12 2009 9 T3b 4 1 49 72 High 7.6 2007 9 T3b 2 0 50 74 High 4.5 2007 9 T3b 3 1 51 72 High 77.1 2006 9 T4 2 0 52 68 High 17.55 2006 9 T3a 2 0 BPH, Benign prostate hyperplasia; Low, Low-risk tumour; High, High-risk tumour Low- and High-risk prostate cancers were defined as Gleason score <7 and Gleason score ≥7, respectively
indicates data missing or illegible when filed
[0206] Flow Cytometry
[0207] The brain and prostate of DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice were dissected and dissociated as described above. Cellularity of SVZ, OB, DG and prostate were manually counted with viability in trypan blue using a Neubauer chamber before flow analysis. Then cells were incubated for 10 min. with FcR blocking reagent (Miltenyi biotec). Subsequently, fluorochrome-conjugated monoclonal antibodies specific to mouse CD45 (clone 30F11), TER119 (clone Ter-119), CD31 (clone 390), CD326 (clone caa7-9G8), CD49f (clone REA518), Sca-1 (clone REA422), PSA-NCAM (clone 2-2B), CD24 (clone M1/69) were used for 30 min at the concentration recommended by the manufacturer (Miltenyi biotec). Cells were also incubated with biotinylated EGF complexed with BV785-streptavidin (Invitrogen by Thermo Fischer Scientific). Analyses of stained cell suspensions were performed on a 5-laser SORP LSR II (355/408/488/561/640; BD BioSciences, San Jose, Calif.) and data were analysed with FlowJo software (Tree Star, Ashland, Oreg.). Cell populations were purified using a Becton Dickinson SORP ARIA II.
[0208] RNA Extraction, RT-PCR and Q-PCR
[0209] Gene expression levels were analysed from RNA extracted, using the RLT solution (Qiagen, Calsbad, Calif.), from purified cells isolated from the 8-week-old brain or 4-month- and 12-month-old prostate of DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice by quantitative real-time PCR. Reverse transcriptase (Superscript VILO; Invitrogen) was performed in accordance with the manufacturer's instructions. qPCR was performed with Fast SYBR Green (ABI Applied biosystems by Thermo Fischer scientific). Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a standard.
[0210] RNA-sequencing
[0211] Lin.sup.− EYFP.sup.+ cells were isolated from 8-week-old brain or 16-week-old prostate of DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice, and were collected in Qiazol (Qiagen). RNA was extracted using the miRNeasy microkit (Qiagen) and mRNA libraries were prepared using Smart-Seq v4 Ultra Low Input RNA (Takara, Otsu, Shiga). Briefly, cDNA was synthesized by using the locked nucleic acid (LNA) technology integrated with SMART (Switching Mechanism at 5′ End of RNA Template) technology. For each library, post RT-PCR cycle number was adjusted according to the number of cells. Libraries were individually adapted and indexed using the Illumina Nextera XT kit and then, were controlled on the Agilent bioanalyzer (Tapestation 2200, Agilent Technologies). Identical librairies were pooled before sequencing at an average read depth of 70 millions reads per sample. Final libraries were controlled on Tapestation 2200 (Agilent Technologies) and were quantified with fluorimetric intercalant. All RNA-seq libraries were sequenced using the Illumina Nextseq 500 with HighOutPut cartridge to generate about 2×400 millions of 75 bases reads.
[0212] Sequenced reads were trimmed using trimmomatic version 0.36 based on a quality threshold of 33. Reads were aligned on the Mus Musculus genome release GRCm38.p6 using ultrafast RNA-seq aligner STAR. Quantification of gene expressions was performed using HTSeq version 0.10 based on default parameters and transcriptomic analyses were performed with the EdgeR package. The GO term id #GO:0043005 and #GO:0030182 were selected to study a list of genes associated to neuron projection and neuron differentiation. Hierarchical clustering in the heatmap representations were generated based on the Ward 2 distance and the complete linkage method. Correlation analyses restricted to the selected lists of genes belonging to Gene Ontologies were done using the Spearman coefficient of correlation and the statistical significance was determined based on the associated P-value. Expression analysis of genes in ImmGen populations was performed using the ImmGen web portal (based on the MyGeneSet service). Statistical comparisons between SVZ, OB, or prostate samples and ImmGen populations were performed based on the Spearman coefficient of correlation restricted to the lists of 200 most expressed genes in each sample.
[0213] Stereotaxic Injection
[0214] TdTomato cDNA was cloned under the control of the CAG promoter in a lentiviral shuttle plasmid that contains a self-inactivating HIV-1-derived genome (38). Recombinant lentiviral particles were produced by transient transfection of HEK-293T cells, and tittered by ELISA quantification of the p24 capsid protein (HIV-1 p24 antigen ELISA, Gentaur, France). Prior to inject in the subventricular zone of 5-week-old mice, viral particles were diluted in Phosphate-Buffered Saline to a final concentration of 50 ng of p24. μl.sup.−1 and delivered in a volume of 2 μl by stereotaxic injection at the following coordinates: 0.7 mm anterior and 1.3 mm lateral relative to bregma; and 2.5 mm below the skull surface. Viral solution was injected at a speed of 0.2 μl min.sup.−1 and the injection cannula was left in place for 5 minutes before being slowly removed.
[0215] Statistical Analyses
[0216] For mouse analyses, all values are reported as means ± SEM. Statistical significance was assessed by a nonparametric unpaired Mann-Whitney test. Significance was set at P<0.05.
[0217] For patient studies, statistical analyses were performed using R software. Statistical comparisons of the different groups were performed using Wilcoxon rank-sum tests. The non-influence of patient ages on statistical comparisons was verified using regression analyses. Correlation between the number of DCX.sup.+ cells and the number of invaded zones was identified using the Spearman coefficient of correlation. Survival curves were modeled using Kaplan-Meier estimates. Statistical significance of the difference between survival curves was then assessed using the log-rank (Mantel-Cox) test. P-values lower than 0.05 were considered as significant.
[0218] Results
[0219] Stromal DCX+ Cells and Tumour Aggressiveness in Human
[0220] Doublecortin (DCX) is a classical marker of neural precursors which are located in developing and adult neurogenic regions of the CNS (14-16). Analysis of the stroma of human prostate primary tumours revealed DCX.sup.+ cells that also expressed specific markers of neural precursors (i.e. Polysialylated-neural cell adhesion molecule, PSA-NCAM (17); Internexin, INA (18,19); data not shown), but did not express markers of epithelial cells (Pancytokeratin, PanCK, data not shown) or mature nerve fibres (i.e. Neurofilament-Heavy, NF-H (20); data not shown). To assess a potential clinical relevance of the neo-development of a neuronal network in prostate cancer, we quantified DCX.sup.+ cells in low- and high-risk prostate cancer specimens from patients (37 treatment-naïve cancer patients versus 15 patients with benign prostate hyperplasia; Table 1). The density of DCX.sup.+ cells was highly associated with tumour aggressiveness (
TABLE-US-00002 TABLE 2 Statistical comparison between the number of DCX.sup.+ cells for each type of human tissue Tissue type P-value 95% lower CI 95% lower CI Low (Normal) vs BPH 0.1678 −0.3000 1.8000 High (Normal) vs BPH 0.0000 2.7000 11.4000 High (Normal) vs Low 0.0001 2.3001 10.5000 (Normal) Low (Tumour) vs BPH 0.4499 −3.5001 1.1999 High (Tumour) vs BPH 0.0001 6.1001 14.0000 High (Tumour) vs Low 0.0000 7.7999 14.8001 (Tumour) Abbreviations: DCX, Doublecortine; BPH, Benign prostate hyperplasia; Low, Low-risk tumour; High, High-risk tumour; vs, versus; CI, Confidence intervals
TABLE-US-00003 TABLE 3 Statistical comparison between the number of DCX.sup.+ cells and the number of invaded zones # Invaded zones versus 0 zone P-value 95% lower CI 1 zone 0.3507 −2.4 2 zones 0.0001 8.3 3 zones 0.0033 10.0 4 zones 0.0008 20.6 Extra-prostatic extension was defined as a disease involving the invasion of one or more zones (extracapsular extention, seminar vesicle, ganglion and positive surgical margins)
TABLE-US-00004 TABLE 4 Correlation between the number of DCX+ cells and the number of invaded zones Spearman Tissue type coefficie P-value 95% lower CI 95% upper CI Normal 0.5825 1.55E−04 0.2870 0.8076 Tumour 0.7342 2.32E−07 0.5403 0.8540 Combined 0.7797 7.97E−09 0.5862 0.8832
indicates data missing or illegible when filed
[0221] DCX.sup.+ Neural Progenitors Initiate Tumour Neurogenesis
[0222] DCX.sup.+ cells were also present in the stroma of Hi-Myc mouse prostate cancer tissues (Hi-Myc mice express c-Myc specifically in prostate epithelial cells under the control of the probasin promoter (21); data not shown). Thus, to easily track, isolate and characterize DCX.sup.+ cells in Hi-Myc tumours, we generated triple-transgenic cancer mice (DCX promoter/enhancer driving tamoxifen-inducible Cre; DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice, data not shown). The presence of DCX:EYFP.sup.+ cells in the neurogenic areas (i.e. SVZ/OB and DG) of the brain showed the recombination efficiency (22) (data not shown). In prostate tumours, DCX:EYFP.sup.+ cells were found in the stromal compartment (data not shown) and characterized with the antigenic profile Lin.sup.− (Lineage negative: CD45, TER119, CD31, CD326, CD49f) Sca-1.sup.+ (prostate stromal cells are defined as Lin.sup.− Sca-1.sup.+) DCX:EYFP.sup.+ (hereafter called EYFP.sup.+) and PSA-NCAM.sup.+ (data not shown) (17,23,24). Lin.sup.− EYFP.sup.+ cells were found in prostate tumours but not in prostate tissues from littermates without the c-Myc transgene (data not shown). This population expressed similar specific neural markers (i.e. PSA-NCAM, CD24, EGFR) than neural precursors isolated from the SVZ, OB and DG in the CNS (data not shown) (25,26). This result suggested the presence, in tumours, of neural precursors, which we decided to further characterize. Lin.sup.− EYFP.sup.+ cells purified from tumours did not exhibit the activated neural stem cell signature (GFAP.sup.+ GLAST.sup.+ CD133.sup.+ EGFR.sup.+ MASH1.sup.+/− Nestin.sup.+/− CD24.sup.−/lo) of Lin.sup.− EYFP.sup.+ cells isolated from the brain, but rather, a neural progenitor signature (GFAP.sup.− GLAST.sup.− CD133.sup.−/lo EGFR.sup.−/lo Mash1.sup.−/lo Nestin.sup.+ CD24.sup.+; data not shown) (25,26). Transcriptomic analysis of Lin.sup.− EYFP.sup.+ cells purified from tumours did not show any similarities with gene expression profiling of immune or endothelial cells (data not shown) but showed neuron differentiation (data not shown) and neuron projection (data not shown) signatures that were significantly similar to the ones of Lin.sup.− EYFP.sup.+ cells isolated from the SVZ or OB. In accordance with this phenotype, Lin.sup.− EYFP.sup.+ progenitors, isolated from prostate tumours, could differentiate into newly born neuron ex vivo (data not shown). To explore the neurogenic capacity of the Lin.sup.− EYFP.sup.+ progenitors in vivo (data not shown), and to determine the different stages of Lin.sup.− EYFP.sup.+ neural progenitor cell lineage (data not shown), we performed inducible tissue-specific genetic tracing in prostate tumours. Activation of genetic recombination by tamoxifen at week3 after birth resulted in the presence of Lin.sup.− EYFP.sup.+ progenitors in tumours but not in healthy tissues surrounding the prostate at week 8, 12 and 16 (Hi-Myc tumour areas begins to develop 12 weeks after birth, data not shown) and the emergence of EYFP.sup.+ INX.sup.+ nerve fibres from EYFP.sup.+ neuroblasts 8 months after birth, suggesting that neo-neurons arise and develop, in situ, in the TME from Lin.sup.− EYFP.sup.+ neuroblasts (data not shown). To document this neuronal differentiation, 2 stromal sub-populations of EYFP.sup.+ progenitor cells were purified from tumours, based on the antigenic profile Lin.sup.− EYFP.sup.+ Sca-1.sup.+ PSA-NCAM.sup.+ (hereafter called Sca.sup.lo PSAN.sup.+ and Sca.sup.huh PSAN.sup.+; data not shown). The Sca.sup.hi PSAN.sup.+ population upregulated the expression of markers used for the isolation of activated precursors from the SVZ, such as Nestin, EGFR and MASH1, highlighting a potential activated state of these progenitors (such as transit-amplifying cells) (26). By contrast, the Sca.sup.lo PSAN.sup.+ populations had lower neural marker expression, suggesting a less activated status, but exhibited expression of the Neuro-D1 transcription factor required for neuronal differentiation (27) (i.e neuroblasts (26); data not shown). The frequencies of the 2 sub-populations fluctuated over time, reflecting that different stages of differentiation occur along with tumour development (data not shown). These results suggested that activated Sca.sup.hi PSAN.sup.+ progenitors may give rise to Sca.sup.lo PSAN.sup.+ neuroblasts during the early phases of tumour development (week 16 and week 20).
[0223] DCX.sup.+ Neural Progenitors Egress from the SVZ during Tumorigenesis
[0224] During the development of Hi-Myc prostate cancer, the number of Lin.sup.− EYFP.sup.+ neural precursors in SVZ/OB areas, but not in the DG, oscillated significantly over time (data not shown). These oscillations were not associated with any change of the cellularity of the neurogenic areas of the brain or with any increase of cell death, excluding a non-specific toxicity of tamoxifen (data not shown). Then, we identified 3 sub-populations of Lin.sup.− EYFP.sup.+ neural precursors (Sca.sup.− PSAN.sup.−, Sca.sup.−/lo PSAN.sup.+, Sca.sup.lo PSAN.sup.int) in the SVZ areas, and found that only the Sca.sup.− PSAN.sup.− population significantly oscillated during cancer development (data not shown), reaching a low level at week 4, 12 and 20 after birth, when this population was high in the tumour (Sca.sup.− PSAN.sup.− green population in prostate). Lineage tracing experiments confirmed oscillations of Lin.sup.− EYFP.sup.+ progenitors in the SVZ at 6 weeks after birth, and highlighted the presence of Lin.sup.− EYFP.sup.+ cells in the blood of 6, 12 and 16-week-old Hi-Myc cancer mice (data not shown). These results suggested a potential egress of Lin.sup.− EYFP.sup.+ Sca.sup.− PSAN.sup.− neural stem/progenitor cells, from the SVZ, that might give rise to Lin.sup.− EYFP.sup.+ Sca.sup.− PSAN.sup.− neural progenitors homing in the tumour. Further characterizations of Sca.sup.− PSAN.sup.− populations, in SVZ and tumour, showed that central populations were GFAP.sup.+ GLAST.sup.+ neural stem cells while Sca.sup.− PSAN.sup.− cells in tumours did not express the stem cell markers (data not shown). These results suggested a potential egress of GFAP.sup.− GLAST.sup.− Lin.sup.− EYFP.sup.+ Sca.sup.− PSAN.sup.− neural stem cells, from the SVZ, that might give rise to GFAP.sup.− GLAST.sup.− Lin.sup.− EYFP.sup.+ Sca.sup.− PSAN.sup.− neural progenitors present in the tumour. In addition, we found CD45.sup.− EYFP.sup.+ cells in the blood of 4-month-old Hi-Myc cancer mice that were not found in healthy littermates (data not shown). To evidence the potential egress, stereotaxic injections of a tdTomato-expressing (tdTom.sup.+) lentiviral vector into the SVZ of DCX-Cre.sup.ERT2/loxp-EYFP/Hi-Myc mice were performed to track precursor cells that could emigrate from the SVZ towards the prostate tumour (data not shown). Lin.sup.− Sca.sup.− PSAN.sup.− tdTom.sup.+ EYFP.sup.− cells were found in the blood at the 4-month tumour stage, indicating the release of this population from the SVZ into the circulation (data not shown), and Lin.sup.− tdTom.sup.+ EYFP.sup.+ and Lin.sup.− tdTom.sup.+ EYFP.sup.− cells were found in the tumour of 5-month-old mice, showing migration of Lin.sup.− Sca.sup.− PSAN.sup.− tdTom.sup.+ EYFP.sup.+ and tdTom.sup.+ EYFP.sup.− cells from the SVZ through the bloodstream towards prostate tumours (data not shown) where they could differentiate in neurons (data not shown). Mouse Pten (39) prostate or PyMT (40) breast cancer models underwent similar stereotaxic injections of a tdTomato-expressing lentiviral vector into the SVZ, and displayed a significant accumulation of Lin.sup.− tdTom.sup.+ cells in tumours, indicating that migration of neural progenitors is not restricted to Hi-Myc tumours but might be a more general feature of cancer development (
[0225] DCX.sup.+ Neural Progenitors Regulate Tumour Development
[0226] To characterize the role of DCX.sup.+ progenitors in cancer development, we intercrossed DCX-Cre.sup.ERT2 mice with an inducible diphtheria toxin receptor (iDTR) line in a nude or Hi-Myc background to study, respectively, tumour growth of luciferase-expressing xenogeneic orthotopic (called later, PC-3luc) or transgenic tumours (data not shown). Selective depletion of DCX.sup.+ cells after tamoxifen and diphtheria toxin treatment significantly reduced the incidence of neoplastic lesions and inhibited the engraftment of PC-3luc tumour cells, suggesting a critical role for DCX.sup.+ cells in the early stages of tumour development (data not shown). Further, selective depletion of progenitor cells in the SVZ by stereotaxic injection of diphtheria toxin within the SVZ induced a significant inhibition of tumour development (data not shown). Conversely, orthotopic transplantation of purified Lin.sup.− EYFP.sup.+ cells, isolated from prostate tumour or brain tissues, into established PC-3luc xenografts enhanced tumour growth (data not shown), invasion of lymph nodes (data not shown) and metastasis (data not shown). To study the activity of the 3 sub-populations of neural progenitors (Sca.sup.− PSAN.sup.−, Sca.sup.lo PSAN.sup.+ and Sca.sup.hi PSAN.sup.+) in tumour development and progression, we performed selective depletion experiments in DCX-Cre.sup.ERT2/iDTR nude mice before grafting PC-3luc cells mixed with each of the 3 purified sub-populations (data not shown). As the transplantation of activated stem/progenitor cells induces faster kinetics of neuron formation than quiescent cells (28), only the activated Sca.sup.hi PSAN.sup.+ population promoted tumour growth 7 weeks after transplantation (data not shown). These results indicated that DCX.sup.+ neural progenitors contribute to prostate tumour development and dissemination.
[0227] Discussion
[0228] Numerous studies have now established that cancer development depends on nerves (4-9). Ingrowth of newly formed autonomic nerve fibres into the tumour contributes to prostate cancer initiation and progression through the respective activation of the β-adrenergic and muscarinic cholinergic signalling (4,7), and also, prostate cancer cells may invade large nerves surrounding the tumour to metastasize, a process called perineural invasion (29). The present study unveils an unprecedented process of tumour-associated neo-neurogenesis by which neural stem/progenitor cells leave the SVZ and reach, through the blood, the primary tumour where they differentiate into neo-nerves that support cancer development and progression. While clinical oncology studies clearly point out the long-term cognitive decline of cancer patients treated by chemotherapies that damage neural progenitor cells in the brain, our study raises the intriguing possibility that the tumour itself might deplete the neurogenic niches in the brain by attracting neural precursor cells to support its own development, and suggest that treatment-naïve cancer patients may, also, develop cognitive impairment (30,31).
[0229] The CNS has been shown to regulate the function of peripheral organs, such as leptin-dependent bone formation through modulation of the sympathetic nervous system (32,33), and gut function in healthy and pathological conditions through regulation of the autonomic nervous system (34). Similarly, the CNS can regulate cancer development and progression. Under stress conditions, the CNS can activate the autonomic nervous system or the hypothalamic-pituitary-adrenal axis, and this results to the secretion of divers mediators, such as glucocorticoids and catecholamines, that favour tumour initiation and progression (35). The present study uncovers a novel type of crosstalk between CNS and prostate tumours, as it reveals a unique migration of central neural precursors that nurture tumour development. Together with a recent study that showed that lung tumours could drive distant granulopoiesis in bone, leading to the egress and migration of a neutrophil population to foster the tumour (36), our results also showed how a tumour could have a dialogue with a distant organ to recruit cells that are required for its growth and dissemination. Further studies will be necessary to characterize the molecular events that control the egress of neural stem cells from the brain.
[0230] Altogether, these results open new avenues to diagnose and monitor cancer development and to uncover novel therapies targeting neural progenitors in the tumour microenvironment.
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