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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.


PubMed | Nationwide Childrens Hospital, Ohio State University, Northwestern University, University of Ferrara and 11 more.
Type: | Journal: Clinical cancer research : an official journal of the American Association for Cancer Research | Year: 2016

Purpose The oncogenic miR-155 is upregulated in many human cancers and its expression is increased in more aggressive and therapy resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors. Experimental Design We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia and acute lymphoblastic leukemia. Results We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo. We show that anti-miR-155-DOPC can be considered non-toxic in vivo. We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism, and demonstrate that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer. Conclusions Our findings support the existence of a miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death.


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.


In Special Recognition of the work of The Stand Up To Cancer-Lustgarten Foundation Pancreatic Dream Team and in honor of Pancreatic Cancer Awareness Month SEATTLE, Nov. 11, 2016 (GLOBE NEWSWIRE) -- NanoString Technologies, Inc. (NASDAQ:NSTG), a provider of life science tools for translational research and molecular diagnostic products, today announced a new myeloid gene expression collaboration to expand the company’s immuno-oncology portfolio. The Company, in conjunction with Lisa Coussens, Ph.D., Professor & Chair, Developmental & Cancer Biology Department, OHSU Knight Cancer Institute, Portland, Oregon, is developing two new myeloid focused research panels for the study of the innate immune response to cancer.  An early version of the Myeloid Innate Immunity Panel will be made available to Dr. Coussens and her collaborators, as well as the Stand Up To Cancer – Lustgarten Foundation Pancreatic Dream Team members in an exclusive, advance offering during the month of November in conjunction with Pancreatic Cancer Awareness Month, after which the panels will be available to all researchers. “I am thrilled to be partnering with NanoString to create these novel myeloid-focused panels,” said Coussens. “We anticipate that through these efforts, we will enable a more complete understanding of the local interplay between myeloid immune components and neoplastic cells in tumors.” Myeloid cells play a key role in modulating activities fundamental to cancer development and are known to have both tumor promoting and anti-tumor functions. As myeloid cells are affected by and can have an impact on many types of cancer therapy, they are broadly applicable within immuno-oncology research. A heightened awareness of the importance of the mechanisms of immunotherapy resistance has brought the myeloid immune response into focus as a key modulator of the adaptive immune response. NanoString is currently working with Coussens on her efforts in understanding recruitment of myeloid cells into neoplastic tissue, and the subsequent regulation exerted by those myeloid cells on neoplastic cells and other cells within dynamic tumor microenvironments. The Myeloid Innate Immunity panel includes approximately 700 genes representing all major categories of myeloid cells, enabling quantitative evaluation of heterogeneous myeloid cell populations based on recruitment, differentiation, maturation status, and functional activities.  The panels are optimized to work across a range of sample types including fresh frozen tissues, formalin-fixed paraffin-embedded (FFPE) samples, peripheral blood mononuclear cells and cell lysates. “It has been a pleasure to collaborate with Dr. Coussens and we are excited to share this work with the broader community of cancer researchers. The Myeloid panel is a collection of genes that encompass the many characteristics of the innate immune response that will help advance cancer research with additional applications in infectious disease as well,” said Joseph Beechem, Ph.D., senior vice president of R&D at NanoString. “These myeloid panels are highly complementary to NanoString’s 770 gene PanCancer Immune Profiling Panel, which is by-design, more T-Cell focused. The myeloid panel will provide an orthogonal view of the regulation of the immune response.” Dr. Coussens is chair of the Department of Cell, Developmental & Cancer Biology at OHSU. Her research is focused on revealing the role that immune cells play in regulating solid tumor development. Coussens is a principal investigator on the Stand Up To Cancer – Lustgarten Foundation Pancreatic Cancer Convergence Dream Team in which her work is focused on clinical evaluation of immune-based therapies in pancreatic cancer.  She has received numerous awards, including: the V Foundation Scholar Award, the AACR-Women in Cancer Research Charlotte Friend Memorial Lectureship, and the 2015 recipient of the 13th Rosalind E. Franklin Award from the National Cancer Institute. This is the latest in a series of research partnerships NanoString has with global leaders in immuno-oncology. NanoString and Coussens will be presenting independently at the upcoming Society for Immunotherapy of Cancer (SITC) conference taking place Wednesday, November 9 through Sunday, November 13 at the Gaylord National Hotel & Convention Center in National Harbor, Maryland. Results from NanoString’s previously announced collaborations with Merck and MD Anderson Cancer Center will also be presented this week at AMP and SITC. - Title: Beyond PD-L1 IHC:  A Gene Expression Based Test in development for anti-PD-1 response on the nCounter® Dx Analysis System - Speaker: Dr. Matthew Marton, Director of Genomics and Companion Diagnostics, Merck - Date/time: Wednesday, November 9th, 8 AM - 9 AM. - Title: The increasing clinical relevance of predictive biomarkers in cancer immunotherapy: can we afford to move forward without them? - Speakers: Alessandra Cesano, Alex Rueben (MDACC) & Jared Lunceford (Merck). - Date/time: Saturday, November 12th, 12:00 PM – 1:00 PM. About the OHSU Knight Cancer Institute: The Knight Cancer Institute at Oregon Health & Science University is a pioneer in the field of precision cancer medicine. The institute's director, Brian Druker, M.D., helped prove it was possible to shut down just the cells that enable cancer to grow. This breakthrough has made once-fatal forms of the disease manageable and transformed how cancer is treated. The OHSU Knight Cancer Institute is the only National Cancer Institute-designated Cancer Center between Sacramento and Seattle – an honor earned only by the nation's top cancer centers. It is headquarters for one of the National Cancer Institute's largest research collaboratives, SWOG, in addition to offering the latest treatments and technologies as well as hundreds of research studies and clinical trials. For additional information on the OHSU Knight Cancer Institute visit www.ohsu.edu/xd/health/services/cancer or follow us on Facebook and Twitter. About the Stand Up To Cancer Initiative Stand Up To Cancer (SU2C) raises funds to accelerate the pace of research to get new therapies to patients quickly and save lives now. SU2C, a program of the Entertainment Industry Foundation (EIF), a 501(c)(3) charitable organization, was established in 2008 by film and media leaders who utilize the industry’s resources to engage the public in supporting a new, collaborative model of cancer research, and to increase awareness about cancer prevention as well as progress being made in the fight against the disease. As SU2C’s scientific partner, the American Association for Cancer Research (AACR) and a Scientific Advisory Committee led by Nobel Laureate Phillip A. Sharp, PhD, conduct rigorous, competitive review processes to identify the best research proposals to recommend for funding, oversee grants administration, and provide expert review of research progress. Current members of the SU2C Council of Founders and Advisors (CFA) include Katie Couric, Sherry Lansing, Lisa Paulsen, Rusty Robertson, Sue Schwartz, Pamela Oas Williams, Ellen Ziffren, and Kathleen Lobb. The late Laura Ziskin was also a co-founder.  Sung Poblete, Ph.D., R.N., has served as SU2C’s president since 2011. For more information on Stand Up To Cancer, visit www.standup2cancer.org. About the Lustgarten Foundation The Lustgarten Foundation is the largest private foundation dedicated to funding pancreatic cancer research. The Foundation supports research to find a cure for pancreatic cancer, facilitates dialogue within the medical and scientific community, and educates the public about the disease through awareness campaigns and fundraising events. Since its inception, the Foundation has directed more than $125 million to research and assembled the best scientific minds with the hope that one day, a cure can be found. Thanks to private funding, 100 percent of every dollar donated to the Foundation goes directly to pancreatic cancer research. For additional information, please visit www.lustgarten.org. About NanoString Technologies, Inc. NanoString Technologies provides life science tools for translational research and molecular diagnostic products. The company's nCounter Analysis System has been employed in life sciences research since it was first introduced in 2008 and has been cited in more than 1,300 peer-reviewed publications. The nCounter Analysis System offers a cost-effective way to easily profile the expression of hundreds of genes, proteins, miRNAs, or copy number variations, simultaneously with high sensitivity and precision, facilitating a wide variety of basic research and translational medicine applications, including biomarker discovery and validation. The company's technology is also being used in diagnostics. The Prosigna® Breast Cancer Prognostic Gene Signature Assay together with the nCounter Dx Analysis System is FDA 510(k) cleared for use as a prognostic indicator for distant recurrence of breast cancer. In addition, the company is collaborating with multiple biopharmaceutical companies in the development of companion diagnostic tests for various cancer therapies, helping to realize the promise of precision oncology. For more information, please visit www.nanostring.com. The NanoString Technologies logo, NanoString, NanoString Technologies, nCounter, 3D Biology, and Prosigna are registered trademarks of NanoString Technologies, Inc.


In Special Recognition of the work of The Stand Up To Cancer-Lustgarten Foundation Pancreatic Dream Team and in honor of Pancreatic Cancer Awareness Month SEATTLE, Nov. 10, 2016 (GLOBE NEWSWIRE) -- NanoString Technologies, Inc. (NASDAQ:NSTG), a provider of life science tools for translational research and molecular diagnostic products, today announced a new myeloid gene expression collaboration to expand the company’s immuno-oncology portfolio. The Company, in conjunction with Lisa Coussens, Ph.D., Professor & Chair, Developmental & Cancer Biology Department, OHSU Knight Cancer Institute, Portland, Oregon, is developing two new myeloid focused research panels for the study of the innate immune response to cancer.  An early version of the Myeloid Innate Immunity Panel will be made available to Dr. Coussens and her collaborators, as well as the Stand Up To Cancer – Lustgarten Foundation Pancreatic Dream Team members in an exclusive, advance offering during the month of November in conjunction with Pancreatic Cancer Awareness Month, after which the panels will be available to all researchers. “I am thrilled to be partnering with NanoString to create these novel myeloid-focused panels,” said Coussens. “We anticipate that through these efforts, we will enable a more complete understanding of the local interplay between myeloid immune components and neoplastic cells in tumors.” Myeloid cells play a key role in modulating activities fundamental to cancer development and are known to have both tumor promoting and anti-tumor functions. As myeloid cells are affected by and can have an impact on many types of cancer therapy, they are broadly applicable within immuno-oncology research. A heightened awareness of the importance of the mechanisms of immunotherapy resistance has brought the myeloid immune response into focus as a key modulator of the adaptive immune response. NanoString is currently working with Coussens on her efforts in understanding recruitment of myeloid cells into neoplastic tissue, and the subsequent regulation exerted by those myeloid cells on neoplastic cells and other cells within dynamic tumor microenvironments. The Myeloid Innate Immunity panel includes approximately 700 genes representing all major categories of myeloid cells, enabling quantitative evaluation of heterogeneous myeloid cell populations based on recruitment, differentiation, maturation status, and functional activities.  The panels are optimized to work across a range of sample types including fresh frozen tissues, formalin-fixed paraffin-embedded (FFPE) samples, peripheral blood mononuclear cells and cell lysates. “It has been a pleasure to collaborate with Dr. Coussens and we are excited to share this work with the broader community of cancer researchers. The Myeloid panel is a collection of genes that encompass the many characteristics of the innate immune response that will help advance cancer research with obvious applications in infectious disease as well,” said Joseph Beecham, Ph.D., senior vice president of R&D at NanoString. “These myeloid panels are highly complementary to NanoString’s 770 gene PanCancer Immune Profiling Panel, layering a unique dimension of gene expression information that will provide insights into the modulation activities of the innate immune response.” Dr. Coussens is chair of the Department of Cell, Developmental & Cancer Biology at OHSU. Her research is focused on revealing the role that immune cells play in regulating solid tumor development. Coussens is a principal investigator on the Stand Up To Cancer – Lustgarten Foundation Pancreatic Cancer Convergence Dream Team in which her work is focused on clinical evaluation of immune-based therapies in pancreatic cancer.  She has received numerous awards, including: the V Foundation Scholar Award, the AACR-Women in Cancer Research Charlotte Friend Memorial Lectureship, and the 2015 recipient of the 13th Rosalind E. Franklin Award from the National Cancer Institute. This is the latest in a series of research partnerships NanoString has with global leaders in immuno-oncology. NanoString and Coussens will be presenting independently at the upcoming Society for Immunotherapy of Cancer (SITC) conference taking place Wednesday, November 9 through Sunday, November 13 at the Gaylord National Hotel & Convention Center in National Harbor, Maryland. Results from NanoString’s previously announced collaborations with Merck and MD Anderson Cancer Center will also be presented this week at AMP and SITC. - Title: Beyond PD-L1 IHC:  A Gene Expression Based Test in development for anti-PD-1 response on the nCounter® Dx Analysis System - Speaker: Dr. Matthew Marton, Director of Genomics and Companion Diagnostics, Merck - Date/time: Wednesday, November 9th, 8 AM - 9 AM. - Title: The increasing clinical relevance of predictive biomarkers in cancer immunotherapy: can we afford to move forward without them? - Speakers: Alessandra Cesano, Alex Rueben (MDACC) & Jared Lunceford (Merck). - Date/time: Saturday, November 12th, 12:00 PM – 1:00 PM. About the OHSU Knight Cancer Institute: The Knight Cancer Institute at Oregon Health & Science University is a pioneer in the field of precision cancer medicine. The institute's director, Brian Druker, M.D., helped prove it was possible to shut down just the cells that enable cancer to grow. This breakthrough has made once-fatal forms of the disease manageable and transformed how cancer is treated. The OHSU Knight Cancer Institute is the only National Cancer Institute-designated Cancer Center between Sacramento and Seattle – an honor earned only by the nation's top cancer centers. It is headquarters for one of the National Cancer Institute's largest research collaboratives, SWOG, in addition to offering the latest treatments and technologies as well as hundreds of research studies and clinical trials. For additional information on the OHSU Knight Cancer Institute visit www.ohsu.edu/xd/health/services/cancer or follow us on Facebook and Twitter. About the Stand Up To Cancer Initiative Stand Up To Cancer (SU2C) raises funds to accelerate the pace of research to get new therapies to patients quickly and save lives now. SU2C, a program of the Entertainment Industry Foundation (EIF), a 501(c)(3) charitable organization, was established in 2008 by film and media leaders who utilize the industry’s resources to engage the public in supporting a new, collaborative model of cancer research, and to increase awareness about cancer prevention as well as progress being made in the fight against the disease. As SU2C’s scientific partner, the American Association for Cancer Research (AACR) and a Scientific Advisory Committee led by Nobel Laureate Phillip A. Sharp, PhD, conduct rigorous, competitive review processes to identify the best research proposals to recommend for funding, oversee grants administration, and provide expert review of research progress. Current members of the SU2C Council of Founders and Advisors (CFA) include Katie Couric, Sherry Lansing, Lisa Paulsen, Rusty Robertson, Sue Schwartz, Pamela Oas Williams, Ellen Ziffren, and Kathleen Lobb. The late Laura Ziskin was also a co-founder.  Sung Poblete, Ph.D., R.N., has served as SU2C’s president since 2011. For more information on Stand Up To Cancer, visit www.standup2cancer.org. About NanoString Technologies, Inc. NanoString Technologies provides life science tools for translational research and molecular diagnostic products. The company's nCounter Analysis System has been employed in life sciences research since it was first introduced in 2008 and has been cited in more than 1,300 peer-reviewed publications. The nCounter Analysis System offers a cost-effective way to easily profile the expression of hundreds of genes, proteins, miRNAs, or copy number variations, simultaneously with high sensitivity and precision, facilitating a wide variety of basic research and translational medicine applications, including biomarker discovery and validation. The company's technology is also being used in diagnostics. The Prosigna® Breast Cancer Prognostic Gene Signature Assay together with the nCounter Dx Analysis System is FDA 510(k) cleared for use as a prognostic indicator for distant recurrence of breast cancer. In addition, the company is collaborating with multiple biopharmaceutical companies in the development of companion diagnostic tests for various cancer therapies, helping to realize the promise of precision oncology. For more information, please visit www.nanostring.com. The NanoString Technologies logo, NanoString, NanoString Technologies, nCounter, 3D Biology, and Prosigna are registered trademarks of NanoString Technologies, Inc.


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.


PubMed | University of Houston, Pacific Northwest National Laboratory, MDACC, Catholic University of the Sacred Heart and 3 more.
Type: Journal Article | Journal: Cell reports | Year: 2016

Even though hyperthermia is a promising treatment for cancer, the relationship between specific temperatures and clinical benefits and predictors of sensitivity of cancer to hyperthermia is poorly understood. Ovarian and uterine tumors have diverse hyperthermia sensitivities. Integrative analyses of the specific gene signatures and the differences in response tohyperthermia between hyperthermia-sensitive and -resistant cancer cells identified CTGF as a key regulator of sensitivity. CTGF silencing sensitized resistant cells to hyperthermia. CTGF small interfering RNA (siRNA) treatment also sensitized resistant cancers to localized hyperthermia induced by copper sulfide nanoparticles and near-infrared laser in orthotopic ovarian cancer models. CTGF silencing aggravated energy stress induced by hyperthermia and enhanced apoptosis of hyperthermia-resistant cancers.


NEW YORK and MELBOURNE, Australia, Dec. 06, 2016 (GLOBE NEWSWIRE) -- Mesoblast Limited (Nasdaq:MESO) (ASX:MSB) today announced that MD Anderson Cancer Center (MDACC) in Texas and the United States National Institutes of Health (NIH) will fund a clinical trial combining Mesoblast's two synergistic proprietary technologies, Mesenchymal Precursor Cell (MPC)-based expansion and ex-vivo fucosylation of hematopoietic stem cells (HSCs) for cord blood transplantation in cancer patients. The trial will provide clinical data on whether the combination of these two technologies synergistically facilitates more rapid cord blood HSC engraftment for bone marrow transplant patients than can be achieved by either technology alone.  The number of allogeneic bone marrow transplants performed globally each year could be substantially increased beyond the current 30,000, for cancer and non-cancer indications, if safe and effective alternative sources of allogeneic HSCs are available, such as cord blood, for patients who cannot find a matched donor. Unfortunately, cord blood transplants are associated with prolonged engraftment times due to insufficient numbers and inadequate homing capacity of cord blood HSCs, adversely impacting their clinical outcomes. Combining Mesoblast's proprietary technologies using MPC-based expansion plus ex-vivo fucosylation of cord blood HSCs aims to overcome the two key limitations to using cord blood for rapid, early engraftment and bone marrow reconstitution in adult bone marrow transplant patients. This novel clinical strategy has the potential to significantly increase the number of patients who can receive unrelated donor transplants. Previously, Mesoblast conducted a Phase 2 clinical trial which demonstrated that transplantation of HSCs from MPC-expanded cord blood resulted in a reduced engraftment time, from a median of 24 days for placebo-treated cells to a median of 15 days for co-cultured cells.i Separately, another Phase 2 clinical study showed that transplantation of fucosylated, but non-expanded, cord blood HSCs also resulted in a reduced median engraftment time of 17 days.ii More recently, preclinical results from a group led by Dr Elizabeth J. Shpall, Director of the Cell Therapy Laboratory and a Professor in the Department of Stem Cell Transplantation at MDACC, where MPC-based expansion and ex vivo-fucosylation technologies were combined, showed a very rapid engraftment time of approximately seven days. “Our data suggest that combining Mesoblast’s MPC-based HSC expansion and ex vivo fucosylation technologies may be the optimal clinical strategy for rapid engraftment of cord blood transplants, potentially making cord blood transplantation a real option for many desperate patients who cannot find a suitable alternative,” said Dr Shpall. The new trial of up to 25 patients, entitled ‘Cord Blood Ex-vivo MPC Expansion Plus Fucosylation to Enhance Homing and Engraftment’, is supported by a grant from the NIH National Cancer Institute (NCI Grant R01 CA061508-19) and will be led by Dr Amanda Olson, Assistant Professor, Department of Stem Cell Transplantation and Dr Shpall at MDACC. If the results of the combination study are positive, Mesoblast's proprietary ex vivo-fucosylation technology may be incorporated into the company's Phase 3 program of MPC-expanded HSCs. About HSC Transplantation Treatment of Patients with Advanced Blood Cancers Many patients with advanced blood cancers, such as acute myeloid leukemia, require a stem cell transplant to repopulate bone marrow HSCs after treatment with high dose chemotherapy. Patients typically undergo transplant with blood stem cells taken from the bone marrow or peripheral blood of a donor with a matched tissue type. However, it may be difficult to find a matched donor, especially for patients who are part of a racial or ethnic minority. While transplants using cord blood-derived stem cells do not require the same degree of donor matching as blood and marrow, this approach has had limited success due to the low yield of stem cells in cord blood and their reduced ability to localize within the recipient’s bone marrow. About Ex-Vivo Fucosylation Technology Ex-vivo fucosylation is the addition of the sugar fucose to surface receptors on cells, including HSCs and mesenchymal lineage stem cells. This process modifies receptors on these cells by adding carbohydrate or sugar sequences which allows them to be recognized by and bound to their ligands present on endothelial cells lining blood vessels in inflamed tissues and in human bone marrow.  As a result, such modified cells demonstrate enhanced homing properties to bone marrow or to tissues that are inflamed. Mesoblast has exclusively licensed the ex-vivo fucosylation technology, which was developed at the Harvard Medical School by Dr Robert Sackstein, for use with HSCs and with allogeneic mesenchymal lineage cells.  This cell targeting technology resulted in engraftment of systemically infused human HSCs into mouse bone marrow at a rate ten times that of unmodified human HSCs.iii About Mesoblast Mesoblast Limited (Nasdaq:MESO) (ASX:MSB) is a global leader in developing innovative cell-based medicines. The Company has leveraged its proprietary technology platform, which is based on specialized cells known as mesenchymal lineage adult stem cells, to establish a broad portfolio of late-stage product candidates. Mesoblast’s allogeneic, ‘off-the-shelf’ cell product candidates target advanced stages of diseases with high, unmet medical needs including cardiovascular diseases, immune-mediated and inflammatory disorders, orthopedic disorders, and oncologic/hematologic conditions. Forward-Looking Statements This press release includes forward-looking statements that relate to future events or our future financial performance and involve known and unknown risks, uncertainties and other factors that may cause our actual results, levels of activity, performance or achievements to differ materially from any future results, levels of activity, performance or achievements expressed or implied by these forward-looking statements. We make such forward-looking statements pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws. Forward-looking statements should not be read as a guarantee of future performance or results, and actual results may differ from the results anticipated in these forward-looking statements, and the differences may be material and adverse. You should read this press release together with our risk factors, in our most recently filed reports with the SEC or on our website. Uncertainties and risks that may cause Mesoblast's actual results, performance or achievements to be materially different from those which may be expressed or implied by such statements, and accordingly, you should not place undue reliance on these forward-looking statements. We do not undertake any obligations to publicly update or revise any forward-looking statements, whether as a result of new information, future developments or otherwise.

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