Entity

Time filter

Source Type

Houston, TX, United States

Armstrong T.S.,UTHSC SON | Gilbert M.R.,MDACC
Current Treatment Options in Oncology | Year: 2014

Opinion statement Symptom occurrence impacts primary brain tumor patients from the time of diagnosis and often heralds recurrence. In addition, the therapy can also result in symptoms that may compound tumor-associated symptoms, further impacting the patient's function and overall quality of life. There is increasing recognition that clinical studies evaluating tumor response using only measures of tumor size on imaging or survival are inadequate in brain tumor patients. Many symptoms can only be assessed from the patient, and patient reported outcome measures have been developed and have adequate reliability and validity. These measures are beginning to be incorporated into clinical trials. Guidelines on their use and meaning are needed to standardize assessment across trials and facilitate measurement of clinical benefit. © 2014 Springer Science+Business Media New York. Source


Suidan R.S.,Sloan Kettering Cancer Center | Ramirez P.T.,Anderson Cancer Center | Sarasohn D.M.,MSKCC | Teitcher J.B.,MSKCC | And 14 more authors.
Gynecologic oncology | Year: 2014

To assess the ability of preoperative computed tomography (CT) scan of the abdomen/pelvis and serum CA-125 to predict suboptimal (>1cm residual disease) primary cytoreduction in advanced ovarian, fallopian tube, and peritoneal cancer. This was a prospective, non-randomized, multicenter trial of patients who underwent primary cytoreduction for stage III-IV ovarian, fallopian tube, and peritoneal cancer. A CT scan of the abdomen/pelvis and serum CA-125 were obtained within 35 and 14 days before surgery, respectively. Four clinical and 20 radiologic criteria were assessed. From 7/2001 to 12/2012, 669 patients were enrolled; 350 met eligibility criteria. The optimal debulking rate was 75%. On multivariate analysis, three clinical and six radiologic criteria were significantly associated with suboptimal debulking: age ≥ 60 years (p=0.01); CA-125 ≥ 500 U/mL (p<0.001); ASA 3-4 (p<0.001); suprarenal retroperitoneal lymph nodes >1cm (p<0.001); diffuse small bowel adhesions/thickening (p<0.001); and lesions >1cm in the small bowel mesentery (p=0.03), root of the superior mesenteric artery (p=0.003), perisplenic area (p<0.001), and lesser sac (p<0.001). A 'predictive value score' was assigned for each criterion, and the suboptimal debulking rates of patients who had a total score of 0, 1-2, 3-4, 5-6, 7-8, and ≥ 9 were 5%, 10%, 17%, 34%, 52%, and 74%, respectively. A prognostic model combining these nine factors had a predictive accuracy of 0.758. We identified nine criteria associated with suboptimal cytoreduction, and developed a predictive model in which the suboptimal rate was directly proportional to a predictive value score. These results may be helpful in pretreatment patient assessment. Copyright © 2014 Elsevier Inc. All rights reserved. Source


Characterization of disease progression and genotyping for the Pdx1-cre;LSL-KrasG12D;P53R172H/+ (herein referred to as KPC) and Ptf1a (P48)-cre;LSL-KrasG12D;Tgfbr2L/L (herein referred to as KTC) mice were previously described31, 32, 33. These mice were bred to Snai1L/L (herein referred to as SnailcKO), Twist1L/L (herein referred to as TwistcKO), and R26-LSL-EYFP33. SnailcKO mice were kindly provided by S. J. Weiss. TwistcKO mice were kindly provided by R. R. Behringer via the Mutant Mouse Regional Resource Center (MMRRC) repository. The resulting progeny were referred to as KPC, KPC;SnailcKO, KPC;TwistcKO, KTC and KTC;SnailcKO mice and were maintained on a mixed genetic background. Both males and females were used indiscriminately. Mice were given gemcitabine (G-4177, LC Laboratories) via intraperitoneal injection (i.p.) every other day at 50 mg kg−1 of body weight. Hypoxyprobe was injected in a subset of mice i.p. at 60 mg kg−1 of body weight 30 min before euthanasia. For in vivo colonization assays, one million KPC, KPC;TwistcKO and KPC;SnailcKO tumour cells in 100 μl of PBS were injected intravenously via the retro-orbital venous sinus. Four to eleven mice were injected per cell line. All mice were euthanized at 15 days post injection. All mice were housed under standard housing conditions at MD Anderson Cancer Center (MDACC) animal facilities, and all animal procedures were reviewed and approved by the MDACC Institutional Animal Care and Use Committee. Tumour growth met the standard of a diameter less than or equal to 1.5 cm. Investigators were not blinded to group allocation but were blinded for the assessment of the phenotypic outcome by histological analyses. No statistical methods were used to predetermine sample size and the experiments were not randomized. Histology, histopathological scoring, Masson’s trichrome staining (MTS), and Picrosirius Red have been previously described19, 33. Formalin-fixed tissues were embedded in paraffin and sectioned at 5 μm thickness. MTS was performed using Gomori’s Trichome Stain Kit (38016SS2, Leica Biosystems). Picrosirius red staining for collagen was performed using 0.1% picrosirius red (Direct Red80; Sigma) and counterstained with Weigert’s haematoxylin. Sections were also stained with haematoxylin and eosin (H&E). Histopathological measurements were assessed by scoring H&E-stained tumours for relative percentages of each histopathological phenotype: normal (non-neoplastic), PanIN, well-differentiated PDAC, moderately-differentiated PDAC, poorly-differentiated PDAC, sarcomatoid carcinoma, or necrosis. When tumour histology was missing or of poor quality, the mice were excluded from primary tumour histological analysis and this was determined blinded from genotype information. A histological invasion score of the tumour cells into the surrounding stroma was scored on a scale of 0 to 2, with 0 indicating no invasion and 2 indicating high invasion, where invasion is defined as tumour cell dissemination throughout the stroma away from clearly defined epithelial ‘nests’. Microscopic metastases were observed in H&E-stained tissue sections of the liver, lung and spleen. Positivity (one or more lesions in a tissue) was confirmed using CK19 and YFP immunohistochemistry. This data has been presented as a contingency table (Fig. 2e) and represented as the number of positive tissues out of the number of tissues scored. The ‘Any’ metastasis score is the number of mice positive for a secondary lesion found anywhere throughout the body out of the total number of mice scored. Tissues were fixed in 10% formalin overnight, dehydrated, and embedded in paraffin and 5-μm-thick sections were then processed for analyses. Immunohistochemical analysis was performed as described33. Heat-mediated antigen retrieval in 1 mM EDTA + 0.05% Tween20 (pH 8.0) for one hour (pressure cooker) was performed for Snail and Twist, 10 mM citrate buffer, pH 6.0, was used for one hour (microwave) for Ki67 or 10 min for all other antibodies. Primary antibodies are as follows: αSMA (M0851, DAKO, 1:400 or ab5694, Abcam, 1:400), cleaved caspase-3 (9661, Cell Signaling, 1:200), CD3 (A0452, DAKO, 1:200), CD31 (Dia310M, DiaNova, 1:10), CK8 (TROMA-1, Developmental Studies Hybridoma Bank, 1:50), CK19 (ab52625, Abcam, 1:100), Cnt3 (HPA023311, Sigma-Aldrich, 1:400), ENT1 (LS-B3385, LifeSpan Bio., 1:100), E-cadherin (3195S, Cell Signaling, 1:400), ENT2 (ab48595, Abcam, 1:200), Ki67 (RM-9106, Thermo Scientific, 1:400), Slug (9585, Cell Signaling, 1:200), Snail (ab180714, Abcam, 1:100), Sox4 (ab86809, Abcam, 1:200), Twist (ab50581, Abcam, 1:100), YFP (ab13970, Abcam, 1:1000), Zeb1 (NBP1-05987, Novus, 1:500), and Zeb2 (NBP1-82991, Novus, 1:100). Sections for pimonidazole adduct (HPI Inc., 1:50) or αSMA immunohistochemistry staining were blocked with M.O.M. kit (Vector Laboratories, West Grove, PA) and developed by DAB according to the manufacturer’s recommendations. Alternatively, for immunofluorescence, sections were dual-labelled using secondary antibodies conjugated to Alexa Fluor 488 or 594 or tyramide signal amplification (TSA, PerkinElmer) conjugated to FITC. Lineage-traced (YFP-positive) EMT analysis was performed on 8-μm-thick O.C.T. medium (TissueTek)-embedded frozen sections. Sections were stained for αSMA (ab5694, Abcam, 1:400) followed by Alexa Fluor 680 conjugated secondary antibody. Bright-field imagery was obtained on a Leica DM1000 light microscope or the Perkin Elmer 3DHistotech Slide Scanner. Fluorescence imagery was obtained on a Zeiss Axio Imager.M2 or the Perkin Elmer Vectra Multispectral imaging platform. The images were quantified for per cent positive area using NIH ImageJ analysis software (αSMA, Pimonidazole, Slug, and CD31), per cent positive cells using InForm analysis software (Ki67 and CD3), or scored for intensity either positive or negative (αSMA/CK8 dual staining, αSMA, CK19, YFP, Zeb1, Zeb2, Sox4, E-cadherin and cleaved caspase-3) or on a scale of 1–3 (E-cadherin) or 1–4 (ENT1, ENT2 and Cnt3). In situ hybridization (ISH) was performed on frozen tumour sections as previously described34. In brief, 10-μm-thick sections were hybridized with antisense probes to Twist1 and Snai1 overnight at 65 °C. After hybridization, sections were washed and incubated with AP-conjugated sheep anti-DIG antibody (1:2,000; Roche) for 90 min at room temperature. After three washes, sections were incubated in BM Purple (Roche) until positive staining was seen. Digoxigenin-labelled in situ riboprobes were generated with an in vitro transcription method (Promega and Roche) using a PCR template. The following primers were used to generate the template PCR product. Twist1, forward, 5′-CGGCCAGGTACATCGACTTC-3′; reverse, 5′-TAATACGACTCACTATAGGGAGATTTAAAAGTGTGCCCCACGC-3′; Snai1, forward, 5′-CAACCGTGCTTTTGCTGAC-3′; reverse, 5′-TAATACGACTCACTATAGGGAGACCTTTAAAATGTAAACATCTTTCTCC-3′. Total RNA was isolated from tumours of KPC control, KPC;TwistcKO and KPC;SnailcKO mice (n = 3 in each group) by TRIzol (15596026, Life Technologies) and submitted to the Microarray Core Facility at MD Anderson Cancer Center. Gene expression analysis was performed using MouseWG-6 v2.0 Gene Expression BeadChip (Illumina). The Limma package from R Bioconductor35 was used for quantile normalization of expression arrays and to analyse differentially expressed genes between cKO and control sample groups. Gene expression microarray data have been deposited in GEO (Accession number GSE66981). Genes upregulated in cells acquiring an EMT program were expected to be downregulated in the TwistcKO and SnailcKO tumours compared to control tumours. Blood (200 μl) was collected from KPC;LSL-YFP and KPC;TwistcKO;LSL-YFP (ROSA-LSL-YFP lineage tracing of cancer cells) mice and incubated with 10 ml of ACK lysis buffer (A1049201, Gibco) at room temperature to lyse red blood cells. Cell pellets were resuspended in 2% FBS containing PBS and analysed for the number of YFP+ cells by flow cytometry (BD LSRFortessa X-20 Cell Analyzer). The data was expressed as the percentage of YFP+ cells from gated cells, with 100,000 cells analysed at the time of acquisition. Whole blood cell pellets were also assayed for the expression of KrasG12D transcripts, using quantitative real-time PCR analyses (described below). Derivation of primary PDAC cell lines were performed as previously described36. Fresh tumours were minced with sterile razor blades, digested with dispase II (17105041, Gibco, 4 mg ml−1)/collagenase IV (17104019, Gibco, 4 mg ml−1)/RPMI for 1 h at 37 °C, filtered by a 70 μm cell strainer, resuspended in RPMI/20%FBS and then seeded on collagen I-coated plates (087747, Fisher Scientific). Cells were maintained in RPMI medium with 20% FBS and 1% penicillin, streptomycin and amphotericin B (PSA) antibiotic mixture. Cancer cells were further purified by FACS based on YFP or E-cadherin expression (anti-E-cadherin antibody, 50-3249-82, eBioscience, 1:100). The sorted cells, using BD FACSAriaTM II sorter (South Campus Flow Cytometry Core Lab of MD Anderson Cancer Center) were subsequently expanded in vitro. All studies were performed on cells cultivated less than 30 passages. As these are primary cell lines, no further authentication methods were applicable and no mycoplasma tests were performed. MTT assay was performed to detect cell proliferation and viability by using Thiazolyl Blue Tetrazolium Bromide (MTT, M2128, Sigma) following the manufacturer’s recommendations with an incubation of two hours at 37 °C. For the drug treatment studies, a cell line derived from each of the KPC, KPC;SnailcKO and KPC;TwistcKO mice was treated with 20 μM gemcitabine (G-4177, LC Laboratories) or 100 μM erlotinib (5083S, NEB) for 48 h. The relative cell viability was detected using MTT assay with a cell line derived from each of the KPC, KPC;SnailcKO and KPC;TwistcKO mice. n is defined as the number of biological replicates of a single cell line. Control conditions included 1% DMSO vehicle for erlotinib. The relative absorbance was normalized and control (time 0 h or vehicle-treated) arbitrarily set to 1 or 100% for absorbance or drug survival, respectively. RNA was extracted from whole blood cell pellets following ACK lysis using the PicoPure Extraction kit as directed (KIT0214, Arcturus), or from cultured primary pancreatic adenocarcinoma cells using TRIzol (15596026, Life Technologies). cDNA was synthetized using TaqMan Reverse Transcription Reagents (N8080234, Applied Biosystems) or High Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems). Primers for KrasG12D recombination are: KrasG12D, forward, 5′-ACTTGTGGTGGTTGGAGCAGC-3′; reverse, 5′-TAGGGTCATACTCATCCACAA-3′. 1/ΔC values are presented to show KrasG12D expression in indicated experimental groups, statistical analyses were performed on ΔC . Primer sequences for EMT-related genes are listed in Supplementary Table 1, GAPDH was used as an internal control. The data are presented as the relative fold change and statistical analyses were performed on ΔC . Tumour sphere assays were performed as previously described33. Two million cultured primary tumour cells were plated in a low-adherence 100-mm dish (FB0875713, Fisherbrand) with 1% FBS, Dulbecco’s modified Eagle’s medium, and penicillin/streptomycin/amphotericin. Cells were incubated for 7 days and formed spheres were counted at 100× magnification. Three, two and three cell lines were analysed for KPC control, KPC;TwistcKO and KPC;SnailcKO groups, respectively, five field of views per cell line were quantified. MRI imaging was performed using a 7T small animal MR system as previously described37. To measure tumour volume, suspected regions were drawn blinded on each slice based on normalized intensities. The volume was calculated by the addition of delineated regions of interest in mm2 × 1 mm slice distance. None of the mice had a tumour burden that exceeded 1.5 cm in diameter, in accordance with institutional regulations. All mice with measurable tumours were enrolled in the study (see Extended Data Table 3). Mice were imaged twice, once at the beginning of the enrolment (day 0), and a second time 20 days (day 19) afterwards. Surviving animals were euthanized at end point (day 21) for histological characterization. Statistical analyses were performed on the mean values of biological replicates in each group using unpaired two-tailed or one-tailed t-tests (qPCR only), or one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism, as stipulated in the figure legends. χ2 analyses, using SPSS statistical software, were performed comparing control to cKO groups for metastatic or colonization frequency across multiple histological parameters in all mice and mice ≥120 days of age in Extended Data Table 1. Fisher’s exact P value was used to determine significance. Results are outlined in Extended Data Table 2. Kaplan–Meier plots were drawn for survival analysis and the log rank Mantel-Cox test was used to evaluate statistical differences, using GraphPad Prism. Data met the assumptions of each statistical test, where variance was not equal (determined by an F-test) Welch’s correction for unequal variances was applied. Error bars represent s.e.m. when multiple visual fields were averaged to produce a single value for each animal which was then averaged again to represent the mean bar for the group in each graph. P < 0.05 was considered statistically significant.


Wu S.Y.,University of Houston | Yang X.,AM Biotechnologies, LLC | Gharpure K.M.,University of Houston | Hatakeyama H.,University of Houston | And 43 more authors.
Nature Communications | Year: 2014

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2′-O-Methyl (2′-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2′-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types. © 2014 Macmillan Publishers Limited. Source


News Article
Site: http://www.nature.com/nature/current_issue/

All common chemicals were from Sigma. Pyrrolidinedithiocarbamic acid was from Santa Cruz Biotechnology. Exo-FBS exosome-depleted FBS was purchased from System Biosciences (SBI). PTEN (9188), pAkt(T308) (9275), pAkt(S473) (4060), Pan Akt (4691), and Bim (2933) antibodies were from Cell Signaling. CD9 (ab92726), Rab27a (ab55667), AMPK (ab3759), CCL2 (ab9899), MAP2 (ab11267), and pP70S6K (ab60948) antibodies were from Abcam. Tsg101 (14497-1-AP) and Rab27b (13412-1-AP) antibodies were from Proteintech. CD81 (104901) antibody was from BioLegend. E2F1 (NB600-210) and CCR2 (NBP1-48338) antibodies were from Novus. GFAP (Z0334) antibody was from DAKO. IBA1 antibody was from WAKO. Cre (969050) antibody was from Novagen. NF-κB p65 (SC-109) and CD63 (SC-15363) antibodies were from Santa Cruz. DMA (sc-202459) and CCR2 antagonist (sc-202525) were from Santa Cruz. MK2206 (S1078) was from Selleckchem. PDTC (P8765) was from Sigma-Aldrich. Human breast cancer cell lines (MDA-MB-231, HCC1954, BT474 and MDA-MB-435) and mouse cell lines (B16BL6 mouse melanoma and 4T1 mouse breast cancer) were purchased from ATCC and verified by the MD Anderson Cancer Center (MDACC) Cell Line Characterization Core Facility. All cell lines have been tested for mycoplasma contamination. Primary glia was isolated as described13. In brief, after homogenization of dissected brain from postnatal day (P)0–P2 neonatal mouse pups, all cells were seeded on poly-d-lysine coated flasks. After 7 days, flasks with primary culture were placed on an orbital shaker and shaken at 230 r.p.m. for 3 h. Warm DMEM 10:10:1 (10% of fetal bovine serum, 10% of horse serum, 1% penicillin/streptomycin) was added and flasks were shaken again at 260 r.p.m. overnight. After shaking, fresh trypsin was added into the flask and leftover cells were plated with warm DMEM 5:5:1 (5% of fetal bovine serum, 5% of horse serum, 1% penicillin/streptomycin) to establish primary astrocyte culture. More than 90% of isolated primary glial cells were GFAP+ astrocytes. Primary CAFs were isolated by digesting the mammary tumours from MMTV-neu transgenic mouse. 231-xenograft CAFs were isolated by digesting the mammary tumours from MDA-MB-231 xenograft. For the mixed co-culture experiments, tumour cells were mixed with an equal number of freshly isolated primary glia, CAFs or NIH3T3 fibroblast cells in six-well plate (1:3 ratio). Co-cultures were maintained for 2–5 days before magnetic-bead-based separation. For the trans-well co-culture experiments, tumour cells were seeded in the bottom well and freshly isolated primary glia, CAFs or NIH3T3 cells were seeded on the upper insert (1:3 ratio). Co-cultures were maintained for 2–5 days for the further experiments. Lentiviral-based packaging vectors (Addgene), pLKO.1 PTEN-targeting shRNAs and all siRNAs (Sigma), Human Cytokine Antibody Array 3 (Ray biotech), and lentiviral-based vector pTRIPZ-PTEN and pTRIPZ-CCL2 shRNAs (MDACC shRNA and ORFome Core, from Open Biosystems) were purchased. The human PTEN-targeting shRNA sequences in the lentiviral constructs were: 5′-CCGGAGGCGCTATGTGTATTATTATCTCGAGATAATAATACACATAGCGCCTTTTTT-3′ (targeting coding sequence); 5′-CCGGCCACAAATGAAGGGATATAAACTCGAGTTTATATCCCTTCATTTGTGGTTTTT-3′ (targeting 3′-UTR). The human PTEN-targeting siRNA sequences used were: 5′-GGUGUAAUGAUAUGUGCAU-3′ and 5′-GUUAAAGAAUCAUCUGGAU-3′. The human CCL2-targeting siRNA sequences used were: 5′-CAGCAAGUGUCCCAAAGAA-3′ and 5′-CCGAAGACUUGAACACUCA-3′. The mouse Rab27a-targeting siRNA sequences used were: 5′-CGAUUGAGAUGCUCCUGGA-3′ and 5′-GUCAUUUAGGGAUCCAAGA-3′. Mouse pLKO shRNA (shRab27a: TRCN0000381753; shRab27b: TRCN0000100429) were purchased from Sigma. For lentiviral production, lentiviral expression vector was co-transfected with the third-generation lentivirus packing vectors into 293T cells using Lipo293 DNA in vitro Transfection Reagent (SignaGen). Then, 48–72 h after transfection, cancer cell lines were stably infected with viral particles. Transient transfection with siRNA was performed using pepMute siRNA transfection reagent (SignaGen). For in vivo intracranial virus injection, lentivirus was collected from 15 cm plates 48 h after transfection of packaging vectors. After passing a 0.45 μm filter, all viruses were centrifuged at 25,000 r.p.m (111,000g) for 90 min at 4 °C. Viral pellet was suspended in PBS (~200-fold concentrated). The final virus titre (~1 × 109 UT ml−1) was confirmed by limiting dilution. Cell isolation was performed based on the magnetic bead-based cell sorting protocol according to manufacturer’s recommendation (Miltenyi Biotec Inc.). After preparation of a single-cell suspension, tumour cells (HCC1954 or BT474) were stained with primary EpCAM-FITC antibody (130-098-113) (50 μl per 107 total cells) and incubated for 30 min in the dark at 4 °C. After washing, the cell pellet was re-suspended and anti-FITC microbeads (50 μl per 107 total cells) were added before loading onto the magnetic column of a MACS separator. The column was washed twice and removed from the separator. The magnetically captured cells were flushed out immediately by firmly applying the plunger. The isolated and labelled cells were analysed on a Gallios flow cytometer (Beckman Coulter). For EpCAM-negative MDA-MB-231 tumour cells, FACS sorting (ARIAII, Becton Dickinson) was used to isolate green fluorescent protein (GFP)+ tumour cells from glia or CAFs. Isolation of primary glia was achieved by homogenization of dissected brain from P0–P2 mouse pups. After 7 days, trypsin was added and cells were collected. After centrifugation and re-suspension of cell pellet to a single-cell suspension, cells were incubated with CD11b+ microbeads (Miltenyl Biotec) (50 μl per 107 total cells) for 30 min at 4 °C. The cells were washed with buffer and CD11b+ cells were isolated by MACS Column. CD11b+ cells were analysed by flow cytometry and immunofluorescence staining. Western blotting was done as previously described. In brief, cells were lysed in lysis buffer (20 mM Tris, pH 7.0, 1% Triton X-100, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA and protease inhibitor cocktail). Proteins were separated by SDS–PAGE and transferred onto a nitrocellulose membrane. After membranes were blocked with 5% milk for 30 min, they were probed with various primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature, and visualized with enhanced chemiluminescence reagent (Thermo Scientific). In brief, total RNA was isolated using miRNeasy Mini Kit (Qiagen) and then reverse transcribed using reverse transcriptase kits (iScript cDNA synthesis Kit, Bio-rad). SYBR-based qRT–PCR was performed using pre-designed primers (Life Technologies). miRNA assay was conducted using Taqman miRNA assay kit (Life Technologies). For quantification of gene expression, real-time PCR was conducted using Kapa Probe Fast Universal qPCR, and SYBR Fast Universal qPCR Master Mix (Kapa Biosystems) on a StepOnePlus real-time PCR system (Applied Biosystems). The relative expression of mRNAs was quantified by 2−ΔΔCt with logarithm transformation. Primers used in qRT–PCR analyses are: mouse Ccl2: forward, 5′-GTTGGCTCAGCCAGATGCA-3′; reverse: 5′-AGCCTACTCATTGGGATCATCTTG-3′. Mouse Actb: forward: 5′-AGTGTGACGTTGACATCCGT3′; reverse: 5′-TGCTAGGAGCCAGAGCAGTA-3′. Mouse Pten: forward: 5′-AACTTGCAATCCTCAGTTTG-3′; reverse: 5′-CTACTTTGATATCACCACACAC-3′. Mouse Ccr2 primer: Cat: 4351372 ID: Mm04207877_m1 (Life technologies) Synthetic miRNAs were purchased from Sigma and labelled with Cy3 by Silencer siRNA labelling kit (Life Technologies). In brief, miRNAs were incubated with labelling reagent for 1 h at 37 °C in the dark, and then labelled miRNAs were precipitated by ethanol. Labelled miRNAs (100 pmoles) were transfected into astrocytes or CAFs in a 10-cm plate. After 48 h, astrocytes and CAFs containing Cy3-miRNAs were co-cultured with tumour cells (at 5:1 ratio). Genomic DNA was isolated by PreLink genomic DNA mini Kit (Invitrogen), bisulfite conversion was performed by EpiTect Bisulphite Kit and followed by EpiTect methylation-specific PCR (Qiagen). Primers for PTEN CpG island are 5′-TGTAAAACGACGGCCAGTTTGTTATTATTTTTAGGGTTGGGAA-3′ and 5′-CAGGAAACAGCTATGACCCTAAACCTACTTCTCCTCAACAACC-3′. Luciferase reporter assays were done as previously described27. The wild-type PTEN promoter driven pGL3-luciferase reporter was a gift from A. Yung. The pGL3-PTEN reporter and a control Renilla luciferase vector were co-transfected into tumour cells by Lipofectamine 2000 (Life Technologies). After 48 h, tumour cells were co-cultured with astrocytes or CAFs. Another 48 h later, luciferase activities were measured by Dual-Luciferase Report Assay Kit (Promega) on Luminometer 20/20 (Turner Biosystems). The PTEN 3′-UTRs with various miRNA binding-site mutations were generated by standard PCR-mediated mutagenesis method and inserted downstream of luciferase reporter gene in pGL3 vector. The activities of the luciferase reporter with the wild-type and mutated PTEN 3′-UTRs were assayed as described above. Astrocytes or CAFs were cultured for 48–72 h and exosomes were collected from their culture media after sequential ultracentrifugation as described previously. In brief, cells were collected, centrifuged at 300g for 10 min, and the supernatants were collected for centrifugation at 2,000g for 10 min, 10,000g for 30 min. The pellet was washed once with PBS and purified by centrifugation at 100,000g for 70 min. The final pellet containing exosomes was re-suspended in PBS and used for (1) transmission electron microscopy by fixing exosomes with 2% glutaraldehyde in 0.1 M phosophate buffer, pH 7.4; (2) measure of total exosome protein content using BCA Protein Assay normalized by equal number of primary astrocytes and CAF cells; (3) western blotting of exosome marker protein CD63, CD81 and Tsg101; and (4) qRT–PCR by extracting miRNAs with miRNeasy Mini Kit (Qiagen). Fixed samples were placed on 100-mesh carbon-coated, formvar-coated nickel grids treated with poly-l-lysine for about 30 min. After washing the samples on several drops of PBS, samples were incubated on drops of buffered 1% gluteraldehyde for 5 min, and then washed several times on drops of distilled water. Afterwards, samples were negatively stained on drops of millipore-filtered aqueous 4% uranyl acetate for 5 min. Stain was blotted dry from the grids with filter paper and samples were allowed to dry. Samples were then examined in a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 Kv. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp.). For exosome detection, 100 μl exosomes isolated from 10-ml conditioned media of astrocytes or CAFs were incubated with 10 μl of aldehyde/sulfate latex beads (4 μm diameter, Life Technologies) for 15 min at 4 °C. After 15 min, PBS was added to make sample volume up to 400 μl, which was incubated overnight at 4 °C under gentle agitation. Exosome-coated beads were washed twice in FACS washing buffer (1% BSA and 0.1% NaN in PBS), and re-suspended in 400 μl FACS washing buffer, stained with 4 μg of phycoerythrin (PE)-conjugated anti-mouse CD63 antibody (BioLegend) or mouse IgG (Santa Cruz Biotechnology) for 3 h at 4 °C under gentle agitation and analysed on a FACS Canto II flow cytometer. Samples were gated on bead singlets based on FCS and SSC characteristics (4 μm diameter). For Annexin V apoptosis assay, after 24 h doxorubicin (2 μM) treatment, the cells were collected, labelled by APC-Annexin V antibody (Biolegend) and analysed on a FACS Canto II flow cytometer. CD11b+ and BV2 cells were stained with CCR2 antibody (Novus) at 4 °C overnight; they were then washed and stained with Alexa Fluor 488 anti-rabbit IgG (Life Technologies) at room temperature for 1 h. The cells were then analysed on a FACS Canto II flow cytometer. All animal experiments and terminal endpoints were carried out in accordance with approved protocols from the Institutional Animal Care and Use Committee of the MDACC. Animal numbers of each group were calculated by power analysis and animals are grouped randomly for each experiment. No blinding of experiment groups was conducted. MFP tumours were established by injection of 5 × 106 tumour cells in 100 μl of PBS:Matrigel mixture (1:1 ratio) orthotopically into the MFP of 8-week-old Swiss nude mice as done previously28. Brain metastasis tumours were established by ICA injection of tumour cells (250,000 cells in 0.1 ml HBSS for MDA-MB-231, HCC1954, MDA-MB-435, 4T1 and B16BL6, and 500,000 cells in 0.1 ml HBSS for BT474.m1 into the right common carotid artery as done previously29). Mice (6–8 weeks) were randomly grouped into designated groups. Female mice are used for breast cancer experiments, both female and male are used for melanoma experiments. Since the brain metastasis model does not result in visible tumour burdens in living animal, the endpoints of in vivo metastasis experiments are based on the presence of clinical signs of brain metastasis, including but not limited to, primary central nervous system disturbances, weight loss, and behavioural abnormalities. Animals are culled after showing the above signs or 1–2 weeks after surgery based on specific experimental designs. Brain metastasis lesions are enumerated as experimental readout. Brain metastases were counted as micromets and macromets. The definition of micromets and macromets are based on a comprehensive mouse and human comparison study previously published30. In brief, ten haematoxylin and eosin (H&E)-stained serial sagittal sections (300 μm per section) through the left hemisphere of the brain were analysed for the presence of metastatic lesions. We counted micrometastases (that is, those ≤ 50 μm in diameter) to a maximum of 300 micrometastases per section, and every large metastasis (that is, those > 50 μm in diameter) in each section. Brain-seeking cells from overt metastases and whole brains were dissected and disaggregated in DMEM/F-12 medium using Tenbroeck homogenizer briefly. Dissociated cell mixtures were plated on tissue culture dish. Two weeks later, tumours cells recovered from brain tissue were collected and expanded as brain-seeking sublines (Br.1). For the astrocyte miR-19 knockout mouse model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected with Ad5-GFAP-Cre virus (Iowa University, Gene Transfer Vector Core) 2 μl (MOI ~108 U μl−1) per point, total four points at the right hemisphere (n = 9). Control group (n = 7) was injected with the same dose Ad5-RSV-βGLuc (Ad-βGLuc) at the right hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week after virus injection, mice were intracarotidly injected with 2 × 105 B16BL6 tumour cells. After two weeks, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, and IHC staining were evaluated. Only parenchymal lesions, which are in close proximity of adenovirus injection, were included in our evaluation. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For the intracranial tumour model, Mirc1tm1.1Tyj/J mice (Jax lab) (6–8 weeks) were intracranially injected as described above. Seven mice were used in the experiment. One week later, these mice were intracranially injected with 2.5 × 105 B16BL6 tumour cells at both sides where adenoviruses were injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above. For the Rab27a/b knockdown mouse model, seven C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total three points at the right hemisphere; concentrated control lentivirus containing pLKO.1 scramble were injected at the left hemisphere. All intracranial injections were performed by an implantable guide-screw system. One week later, mice were intracranially injected with 5 × 104 B16BL6 tumour cells at both sides where they had been infected. After one week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation, histological phenotypes of H&E-stained sections, IHC staining were evaluated. When performing metastases size quantification, only parenchymal lesions that were in close proximity to the adenovirus injection sites were included in the analyses. Tumour size was calculated as (longest diameter) × (shortest diameter)2/2. For exosome rescue experiments, eight C57BL6 mice (Jax lab) (6–8 weeks) were intracranially injected with concentrated lentivirus containing shRab27a and shRab27b (ratio 1:2) 2 μl per point, total 3 points at both hemispheres. One week later, these mice were intracranially injected with 5 × 104 B16BL6 tumour cells with 10 μg exosome isolated from astrocyte media at the right sides where they had been injected with lentivirus; 5 × 104 B16BL6 tumour cells with vehicle were injected at the left sides where lentivirus had been injected. After another week, whole brains were dissected and fixed in 4% formaldehyde, and embedded in paraffin. Tumour formation and phenotype were analysed as above. For in vivo extravasation assay, equal numbers of cells labelled with GFP-control shRNA and RFP-PTEN shRNA (Open Biosystems) were mixed and ICA injected. After cardiac perfusion, brains were collected and sectioned through coronal plan on a vibrotome (Leica) into 50-μm slices. Fluorescent cells were then counted. For inducible PTEN expression in vivo, mice were given doxycycline (10 μg kg−1) every other day. To quantify brain metastasis incidence and tumour size, brains were excised for imaging and histological examination at the end of experiments. Ten serial sagittal sections every 300 μm throughout the brain were analysed by at least two pathologists who were blinded to animal groups in all above analyses. Reverse-phase protein array of PTEN-overexpressing cells was performed in the MDACC Functional Proteomics core facility. In brief, cellular proteins were denatured by 1% SDS, serial diluted and spotted on nitrocellulose-coated slides. Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. The signal obtained was amplified using a Dako Cytomation-catalysed system and visualized by DAB colorimetric reaction. The slides were analysed using customized Microvigene software (VigeneTech Inc.). Each dilution curve was fitted with a logistic model (‘Super curve fitting’ developed at the MDACC) and normalized by median polish. Differential intensity of normalized log values of each antibody between RFP (control) and PTEN-overexpressed cells were compared in GenePattern (http://genepattern.broadinstitute.org). Antibodies with differential expression (P < 0.2) were selected for clustering and heat-map analysis. The data clustering was performed using GenePattern. Two studies in separate cohorts were conducted. The first one was a retrospective evaluation of PTEN in two cohorts. (1) Archived formalin-fixed and paraffin-embedded brain metastasis specimens (n = 131) from patients with a history of breast cancer who presented with metastasis to the brain parenchyma and had surgery at the MDACC (Supplementary Information). Tissues were collected under a protocol (LAB 02-486) approved by the Institutional Review Board (IRB) at the MDACC. (2) Archived unpaired primary breast cancer formalin-fixed and paraffin-embedded specimens (n = 139) collected under an IRB protocol (LAB 02-312) at the MDACC (Supplementary information). Formal consent was obtained from all patients. The second study was a retrospective evaluation of PTEN, CCL2 and IBA1 in the matched primary breast tumours and brain metastatic samples from 35 patients, of which there are 12 HER2-positive, 14 triple-negative and nine oestrogen-receptor-positive tumours according to clinical diagnostic criteria (Supplementary Information). Formalin-fixed, paraffin-embedded primary breast and metastatic brain tumour samples were obtained from the Pathology Department, University of Queensland Centre for Clinical Research. Tissues were collected with approval by human research ethics committees at the Royal Brisbane and Women’s Hospital (2005/022) and the University of Queensland (2005000785). For tissue microarray construction, tumour-rich regions (guided by histological review) from each case were sampled using 1-mm cores. All the archival paraffin-embedded tumour samples were coded with no patient identifiers. Standard IHC staining was performed as described previously28. In brief, after de-paraffinization and rehydration, 4 μm sections were subjected to heat-induced epitope retrieval (0.01 M citrate for PTEN). Slides were then incubated with various primary antibodies at 4 °C overnight, after blocking with 1% goat serum. Slides underwent colour development with DAB and haematoxylin counterstaining. Ten visual fields from different areas of each tumour were evaluated by two pathologists independently (blinded to experiment groups). Positive IBA1 and Ki-67 staining in mouse tumours were calculated as the percentage of positive cells per field (%) and normalized by the total cancer cell number in each field. TUNEL staining was counted as the average number of positive cells per field (10 random fields). We excluded necrotic areas in the tumours from evaluation. Immunofluorescence was performed following the standard protocol recommended by Cell Signaling. In brief, after washing with PBS twice, cells were fixed with 4% formaldehyde. Samples were blocked with 5% normal goat serum in PBS for 1 h before incubation with a primary antibody cocktail overnight at 4 °C, washed, then incubated with secondary antibodies before examination using confocal microscope. Pathologists were blinded to the group allocation during the experiment and when assessing the outcome. Publicly available GEO data sets GSE14020, GSE19184, GSE2603, GSE2034 and GSE12276 were used for bioinformatics analysis. The top 2 × 104 verified probes were subjected to analysis. Differentially expressed genes between metastases from brain and other sites (primary or other metastatic organ sites) were analysed by SAM analysis in R statistical software. The 54 commonly downregulated genes in brain metastases from GSE14020 and GSE19184 were depicted as a heat-map by Java Treeview. For staining of patient samples, we calculated the correlation by Fisher’s exact test. For survival analysis of GSE2603, the patient samples were mathematically separated into PTEN-low and -normal groups based on K-means (K = 2). Kaplan–Meier survival curves were generated by survival package in R. Multiple group IHC scores were compared by Chi-square test and Mantelhaen test in R. All quantitative experiments have been repeated using at least three independent biological repeats and are presented as mean ± s.e.m. or mean ± s.d.. Quantitative data were analysed either by one-way analysis of variance (ANOVA) (multiple groups) or t-test (two groups). P < 0.05 (two-sided) was considered statistically significant.

Discover hidden collaborations