Download Anti-VEGF Reduces Drug Delivery and Therapeutic

Supplemental Methods
VEGF-Ablation Therapy Reduces Drug Delivery and Therapeutic Response
in ECM-dense tumors
Florian Röhrig1,2,3#, Sandra Vorlová2#, Helene Hoffmann1,2,3#, Martin Wartenberg4, Freddy
E. Escorcia5, Sabrina Keller1, Michel Tenspolde1, Isabel Weigand1, Sabine Gätzner2, Katia
Manova6, Olaf Penack7, David A. Scheinberg5, Andreas Rosenwald4, Süleyman Ergün1, Zvi
Granot8 and Erik Henke1,2,3*
1
Institute of Anatomy and Cell Biology, Universität Würzburg, Würzburg, Germany
2
Institute for Clinical Biochemistry and Pathobiochemistry, Universitätsklinikum Würzburg,
Würzburg, Germany
3
Graduate School of Life Science, Universität Würzburg, Würzburg, Germany
4
Institute for Pathology, Universität Würzburg, and Comprehensive Cancer Center Mainfranken
(CCCMF), Germany
5
Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New
York, NY, USA
6
Molecular Cytology Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
7
Medizinische Klinik mit Schwerpunkt Hämatologie, Onkologie und Tumorimmunologie
Universitätsklinikum Charité, Berlin, Germany
8
Department of Developmental Biology and Cancer Research, Institute for Medical Research IsraelCanada and Hebrew University-Hadassah Medical School, Jerusalem, Israel
# These authors contributed equally to this article
* To whom correspondence should be addressed:
Erik Henke, PhD
Institute for Anatomy and Cell Biology, Universität Würzburg. Koellikerstrasse 6
97070 Würzburg, Germany
Email: [email protected]
Tel: +49-(0)931-3183270, Fax: +49-(0)931-329363
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
IHC and IF staining of tumor sections
H&E, picrosirus red, Mason-Goldner trichrome, IHC and IF staining was performed using
standard techniques on formalin fixed paraffin embedded sections. For PSR and MGTC
staining Kits from Carl Roth (Karlsruhe, Germany) were used according to the supplier’s
instructions. Antigen retrieval and staining was performed by an automated tissue stainer
(Ventana Medical Systems, Tucson, AZ). Tissues for quantitative evaluation were processed in
parallel. For quantification whole tissue sections were imaged on a Zeiss Mirax System (40x
objective, Carl Zeiss Microimaging, Germany). The whole virtual slide was used for
quantification using the ImageJ software package (rsbweb.nih.gov/ij/). Double immuno
fluorescence sections stained for NG2 and PanEC were also imaged on the Zeiss Mirax system.
Inspection of acquired images revealed that NG2 staining was observed nearly exclusively (>
95 % of stained area) in perivascular locations. Therefore, the stained area for both channels
(NG2/PanEC) was quantified and the ratio was used as a indicator for pericyte affiliation. In
addition higher power images of representative fields were taken for illustration.
Quantification of PSR staining was performed using ImageJ. RGB images were split in the
three color channels. The green channel was used for quantification of the relative area that
displayed a signal above a certain, constant threshold.
Antibodies used for IHC/IF: PanEC-Antigen (Meca32, Biolegend, 120501), p53 (Vector Labs,
VP-P956), NG2 (Millipore AB5320) Cleaved Caspase-3 (Cell Signaling, #9661), Hif1
(Novus NB100), CD31 (BD Biosciences 550274).
Hoechst distribution, lectin vessel staining and 3D image evaluation
To monitor intra-tumoral distribution of drugs, 50 µL of Hoechst 33342 (Sigma, 20 mg/mL in
0.9% NaCl) and 100 µL of Alexa 488 or Alexa 647-labeled Isolectin GS-B4 (Life
Technologies, Darmstadt, Germany. 500 µg/mL in 0.9% NaCl) were injected i.v. into tumor
2
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
bearing mice 20 min before sacrificing the animal. Tumors were removed and flash frozen in
OCT (Sakura Finetek Torrance, CA).
For Hoechst 33342 tumor distribution 10 µm sections were cut on a cryotome and mounted on
glass slides. The whole tissue sections were imaged on a Zeiss Mirax System (40x objective,
Carl Zeiss Microimaging, Germany) using the blue fluorescence channel. The whole virtual
slide was used for quantification using the ImageJ software package (rsbweb.nih.gov/ij/) by
evaluating the area fraction that showed a signal in the blue channel above a certain, constant
threshold.
For Hoechst 33342 tissue penetration and 3D-vessel evaluation, tissue was cut on a cryotom to
200 µm slices and mounted on glass slides. Z-stacks were acquired by confocal imaging in the
blue and near infrared channel (Leica SP4/2, 20x objective). Tissue penetration was measured
as the maximal distance from the vessel surface (by Alexa 647 staining) that Hoechst 33342
staining was present using ImageJ. For this purpose the acquired z-stacks were evaluated at the
same tissue depth for isolated, longitudinal cut blood vessels. The maximal distance of Hoechst
33342 staining was measured perpendicular to both sides of each blood vessel, the arithmetic
mean of the two values was used. Each blood vessel was evaluated at several positions. At least
10 vessels per stack, and four stacks per biological sample were evaluated.
3D vessel-evaluation was done using the ImageJ software package (rsbweb.nih.gov/ij/) or its
Fiji distribution (http://fiji.sc/wiki/index.php/Fiji) with additional plugins: Skeletonize 3D
(http://imagejdocu.tudor.lu/doku.php?id=plugin:morphology:skeletonize3d:start) (1), Tubeness
(http://www.longair.net/edinburgh/imagej/tubeness/). For vessel ramification analysis binary
stack images were converted with the skeletonize plugin and evaluated for branching points.
Vessel surface area was evaluated with the tubeness plugin.
3
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
Biodistribution of doxorubicin
For biodistribution studies a bolus of 100 µg doxorubicin or doxil was given on specified days
to doxorubicin naïve animals. Mice were sacrificed 2h (doxorubicin) or 24h (Doxil) post
injection when doxorubicin could be expected to be cleared from the blood stream (2). Tissue
samples were flash frozen and stored at – 80 C until extraction. The method described by
Laginha et al. was used with slight modifications (3). In brief: Tissue samples were
homogenized by sonification in 9 parts (v/w) water. 200 µL homogenate were combined with
50 µL 10% Triton X-100 (v/v) and 750 µL 0.75 N HCl in 2-propanol. The mixture was
vortexed briefly and extracted for 12h at -20 C. Samples were again vortexed at r.t. and
cleared by centrifugation (20 min, 4°C, 20,000 x g). Fluorescence was read (Ex.: 470 nm, Em.:
590 nm) in a microplate reader and corrected against extracts from tissue samples of nontreated animals. A standard curve was established by adding defined amounts of
doxorubicin/doxil to homogenates of non-treated tissue samples prior to extraction.
Biodistribution of 3H-paclitaxel
1 µCi of 3H-paclitaxel (Moravec, Brea, CA) were injected into tumor bearing mice at the
specified days. Animals were euthanized 2 h later and major organs were harvested. Specific
amounts of the organs (up to 200 mg) were dissolved in 2 mL SoluEne-350 (PerkinElmer,
Waltham, MA) at 55 °C. To decolorize 200 µL 30 % H2O2 were added in aliquots and the
samples again incubated for 60 min at 55 °C. After addition of 10 mL Hionic-Fluor Cocktail
(PerkinElmer) the samples were read on a LS 6000 -scintillation counter (Beckman, Fullerton,
CA).
Cell toxicity studies
Cells were plated (1500 cells in 100 µL DMEM/well) in 96 well MTP and left to adhere
overnight. After 24 h 100µL of DMEM containing twice the indicated concentration of
cytotoxic chemotherapeutics were added to each well without prior removal of medium. Each
4
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
concentration was tested in a 6-fold replicate. Cells were incubated with the therapeutics for 72
h before media were removed. Relative remaining cell numbers were determined using a
fluorescence based cell quantification kit (CyQuant, Life Technologies, Darmstadt, Germany).
EC50-values were determined by using a non-linear regression based model using the Prism
software (Prism 5, GraphPad, LaJolla, CA)
ECM Extraction
Extracellular matrix proteins were extracted from tumor tissue using a modified protocol from
Kleinman et al. (4).
In brief, tumors (size 300 – 500 mm3) were excised, weighted, snap frozen and stored at -80 °C
until further work-up. The tumors were homogenized in 2 mL/g WW high salt extraction buffer
(HSEB, 3.4 M NaCl, 50 mM Tris HCl, 4 mM EDTA, pH 7.4) on ice with a tissue homogenizer
(UltraTurax, IKA, Staufen, Germany). Non-soluble material, including ECM proteins, was
pelleted by ultracentrifugation (100.000 x g, 4 °C, 30 min). This HSEB extraction was repeated
once, supernatants were collected for western analysis. The pellet was washed with water and
PBS, and finally re-suspended in PBS.
For urea extraction the HSEB non-soluble pellet was re-suspended in 1.8 mL/g (starting
material) of an urea extraction buffer (UEB, 2M urea, 150 mM NaCl, 50 mM Tris HCl, 4 mM
EDTA, pH7.4) briefly homogenized and extracted overnight at 4 °C. Still non-soluble material
was again pelleted by ultracentrifugation (26.000 x g, 1h 4 °C). The UEB supernatants were
dialyzed against a low salt buffer (150 mM NaCl, 50 mM Tris HCl, 4 mM EDTA, pH7.4) for
48 h at 4 °C with two buffer changes. Protein content was determined with the BCA Assay Kit.
To all extraction buffers Complete proteinase inhibitor cocktail (Roche Diagnostics,
Mannheim, Germany) was added.
5
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
Lysyl oxidase activity assay
A modified form of the fluorometric microwell assay described by Fogelgren was used (5). In
short: In a black 96-well microtiterplate was added to 120 µL of an freshly prepared assay
solution consisting of 75 µL 2x PBS, 15 µL N-Acetyl-Resorufin (100 µM, Ampliflu-Red,
Sigma-Aldrich), 15 µL 2,5-diaminopentane (100 mM, cadaverine a substrate of all five lysyl
oxidases (5-7)) and 15 µL horseradish-peroxidase (5U/mL, Sigma-Aldrich). For each sample
the assay solution was prepared in two triplicate rows. To one triplicate 30 µL water, to the
other 30µL APN (3.5 mM in water) was added. Finally 50 µL of cell culture supernatant or
protein containing lysate was added and the fluorescence of released resorufin was recorded
continuously over 3h in a fluorescence plate reader (PerkinElmer, Wallac II; Ex: 530nm, Em:
570nm). Slope of the fluorescence signal in the linear range was calculated for both APNinhibited and non-inhibited samples; the difference was used as a measure of lysyl oxidase
activity.
Note: There a conflicting reports whether APN inhibits LOXL2. While some authors
demonstrated inhibition of LOXL2 (7-9), at least one group reported that LOXL2 is not
inhibited by APN(10). Our own results using recombinant expression of hLOXL2 also
indicate that LOXL2 is inhibited by APN.
Transwell ECM drug penetration assay
The membranes of transwell inserts (24 well MWD format, 33 mm2 membrane area, Costar,
Cölbe, Germany) were coated with 3 µg/mm2 of the respective ECM extract or protein by
adding the protein suspension in 50 µL of buffer and letting the membranes air dry overnight.
ECM was reconstituted by adding 150 µL of PBS to the upper chamber of the transwell and
incubation for 1 h. 850 µL of 20 µg/mL doxorubicin in PBS were added to the lower
compartment. The plate was read continuously for 6 h in a fluorescence plate reader
(PerkinElmer, Wallac II; Ex: 530nm, Em: 570nm).
6
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
For LOX modification assays, 140 µg (10 µg/mm2) of matrigel were mixed with 10 µg purified
recombinant hmLOX or hLOXL2 in 50 µL PBS (+/- 500 µM BAPN), the suspension was
applied to transwell inserts (96 well MWD format, 14 mm2 membrane area, Costar, Cölbe,
Germany) and incubated for 6 h at 37 °C. Afterwards the suspension was dried over night and
subjected to the assay described above (100 µL PBS in upper chamber, 300 µL 20 µg/mL
doxorubicin in PBS in lower chamber).
Recombinant expression and purification of hLOX and hLOXL2
The cDNA encoding both enzymes was amplified from cDNA generated from RNA isolated
from HUVEC. To amplify the active, mature hmLOX (AAs 169 to 417, missing the signal and
propeptide
sequences)
the
primers
5´-
TGCAGGAATTCGCCACCATG
GACGACCCTTACAACCCCTAC AAG – 3’ (italics: EcoRI site, underlined: start codon) and
5´-AGCTCTCGAGGCTAGCCTAGTGGTGATGGTGATGATGACCTCCATACGGTG
AAATTGTGCAGCCTGAGGCAT-3´ (italics: XhoI site, bold: NheI site, underlined: 6xHistag) were used. The resulting DNA was cloned in the expression vector pGEX-4T1 (GE
Healthcare Europe, Freiburg, Germany). The pGEX system was used for heterologous
overexpression of hmLOX in E. coli after IPTG induction. The active enzyme was purified by
from the media supernatant was purified via a Co2+ affinity column (Thermo Fisher, Rockford,
IL) with elution using 250 mM imidazol at pH 7.2. The eluted enzymes were subjected to
dialysis versus PBS and used for enzymatic modification of ECM (matrigel).
The entire hLOXL2 CDS including the signal peptide was amplified from HUVEC cDNA. In
the process a 6xHis-tag was C-terminal fused to the CDS. The amplified DNA was cloned into
the lentiviral vector pLVX-puro (Clontech, Mountain View, CA). Lentiviral particles were
generated in HEK 293 cells by co-transfection with the pCMV-dR8.9 and pCMV-VSV-G(11)
(both plasmids were obtained from Addgene, Cambridge, MA) using a standard CaCl 2-based
transfection method. Supernatant was used to transfect HEK 293 cells. Stable cells selected
with puromycin (3.5 µg/mL). Active enzyme from the media supernatant was purified via a
7
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
Co2+ affinity column (Thermo Fisher, Rockford, IL) with elution using a stepwise pH gradient
(pH 10.5 – pH 6). The eluted enzymes were subjected to dialysis versus PBS and used for
enzymatic modification of ECM (Matrigel).
Collagen Crosslinking Analysis
ECM from APN treated and control tumors was obtained by high salt extraction of cellular
components. The insoluble ECM was re-suspended in water and used to coat glass slides
(angiogenesis µ-slides, Ibidi, Martinsried, Germany) at µg/well. Interferences reflection
Images were acquired as z-stacks (30 slides, z-distance: 1.0 µm) on a Nikon A1 microscope in
reflection mode using am 60x oil immersion objective and an 647 nm laser following a
published protocol(12). Identifiable collagen fibers in optical fields were manually counted.
RNA-isolation
RNA was isolated from 2-3 10 µm sections of archival FFPE tumor samples (app. 10 mg tissue
per sample) using the Agencourt FormaPure Kit (BeckmanCoulter, Krefeld, Germany)
following the manufacturer’s instructions. In a limited number of cases RNA yield was
insufficient (below 400 ng) and amplified using the SensationPlus FFPE Amplification kit
(Affymetrix) using 50 ng of RNA.
RNA was isolated from cells tumors using the RNeasy Kit (Qiagen, Hilden, Germany)
according to the manufacture’s recommendations.
RNA was isolated from fresh tumor samples using the Trizol reagent (Life Technologies,
Darmstadt, Germany) according to the manufactures recommendation.
mRNA-Quantification
mRNA-expression levels were quantified using the GeXP-System (BeckmanCoulter, Krefeld,
Germany). Protocols for reverse transcription, amplification, labeling, gel electrophoresis and
quantification were used as recommended by the manufacturer. RNA-levels were normalized to
levels of housekeeping genes -2-microglobulin (B2M) and ribosomal protein S29
8
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
(RPS29)(13) in murine, and -2-microglobulin (B2M) in human samples respectively. Gene
specific primer sequences are shown in Table 1. Analysis was done with three technical
replicates per biological sample. Mean values of technical replicates was used for statistical
analysis.
Table 1: Gene specific primer sequences for mRNA quantification using the GeXPsystem. The Sequences contain the necessary tag sequences. Primers were combined to several
multiplex sets, that each included the respective primers for B2M and RPS29.
Gene
Symbol
Fragment
Size
Forward Primer
Reverse Primer
mFn1
132
AGGTGACACTATAGAATATGTGCACGTGCC
TGGGCAAT
GTACGACTCACTATAGGGAACGGGAGGACACAGGG
CTCC
mLama5
135
AGGTGACACTATAGAATAGGTCACACTTAT
CAGCCGTGGCA
GTACGACTCACTATAGGGAGTCGTCCTGCGTGATG
CGCT
mCol3a1
126
AGGTGACACTATAGAATAAGAGGCTTTGAT
GGACGCAA
GTACGACTCACTATAGGGAAGCTCCGTTGTCTCCT
GGAA
mLamc1
142
AGGTGACACTATAGAATACGCACTGTCCGA
CTGGCACT
GTACGACTCACTATAGGGACACGGGCGGCACAGTC
TCAC
mCol2a1
147
AGGTGACACTATAGAATAAGAAGGGTCTGG
CTGGCGCT
GTACGACTCACTATAGGGAGCTCGCCTCGTTCACC
AGCA
mCol18a1
151
AGGTGACACTATAGAATACGTGGCGCTGGC
CTCTTTGT
GTACGACTCACTATAGGGAGACAGTGCACAGGGCT
CACCC
mFbn1
154
AGGTGACACTATAGAATAAGGCCCCCTGCA
GTTACGGT
GTACGACTCACTATAGGGACCTCGGCCCATGCCCA
TTCC
mLama1
188
AGGTGACACTATAGAATATGGCCTCGGTGC
TCTGGGTC
GTACGACTCACTATAGGGACGGCACGTGCTCCACG
AGTTT
mCol13a1
146
AGGTGACACTATAGAATAGACGAAGGGAGG
CCTGGAGCG
GTACGACTCACTATAGGGAGGAACCTCTGCTCCCG
GGTCG
mFbn2
172
AGGTGACACTATAGAATATCTCTGGATGCC
TCTGGGCTGA
GTACGACTCACTATAGGGAACTGGTAGTGCTGGAC
GTAGCC
mCol4a1
175
AGGTGACACTATAGAATAGCATTGGCGGCT
CTCCAGGG
GTACGACTCACTATAGGGAGGGCCGGGTACACCTT
GGTC
mLamb1
191
AGGTGACACTATAGAATATGCGCCCCTGTG
GATGGAGT
GTACGACTCACTATAGGGAGCGTTGCTGTTCCGGC
CTTC
mCol1a1
196
AGGTGACACTATAGAATATGATGGCAAAAC
CGGCCCCC
GTACGACTCACTATAGGGATCCGGGAAGGCCTCGC
TCTC
mCol5a1
201
AGGTGACACTATAGAATATGCCCACCAAGC
AGCTGTACC
GTACGACTCACTATAGGGAAGAGGAAGACAGGGGA
GCGGC
mHspg2
217
AGGTGACACTATAGAATAACGGCCTGGCAT
CGTGCAAA
GTACGACTCACTATAGGGAACCGGCAGGCACTCGG
ATCT
AGGTGACACTATAGAATAAGTGGCTGCTAC
TCGGCGCT
GTACGACTCACTATAGGGAGGCGGGTGGAACTGTG
TTACG
242
mB2m
mLOX1
225
AGGTGACACTATAGAATAGGCCACCCAGCC
ACATAGATCG
GTACGACTCACTATAGGGAAGTAGGGGTCGGGCAC
CAGG
mLOXL1
207
AGGTGACACTATAGAATACCGCGTGCTGGA
GCCACCT
GTACGACTCACTATAGGGAGCCTGCACGTAGTTAG
GGTCCG
mLOXL2
187
AGGTGACACTATAGAATATTCTTCTGGGCA
ACCAGGGCG
GTACGACTCACTATAGGGAGCTAGGCTCAGGGAAG
GCAGC
mLOXL3
178
AGGTGACACTATAGAATATCCAGCCTCTGG
AGTTGTGCC
GTACGACTCACTATAGGGAACGGAGACCCCACACT
GAAGC
mLOXL4
127
AGGTGACACTATAGAATAAGGCCCGTTAGC
GTACGACTCACTATAGGGATGGGGCCACATCATGG
9
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
mVEGFA
mCAIX
226
166
mRPS29
255
hB2M
213
hLOX
195
hLOXL1
177
hLOXL2
146
hLOXL3
167
hLOXL4
135
GCTGCTCTG
AGGTGACACTATAGAATAGGCCTCCGAAAC
CATGAACT
AGGTGACACTATAGAATAGGTGCACCTCAG
TACTGCTT
AGGTGACACTATAGAATATTCCTTTCTCCT
CGTTGGGC
TGATTTCAG
GTACGACTCACTATAGGGAGTCCACCAGGGTCTCA
ATCG
GTACGACTCACTATAGGGAATGGGACAGCAACTGT
TCGT
GTACGACTCACTATAGGGATCCATTCAAGGTCGCT
TAGTCC
AGGTGACACTATAGAATATCGGGCCGAGAT
GTCTCGCT
AGGTGACACTATAGAATAGGGCGACGACCC
TTACAACCC
AGGTGACACTATAGAATACCGCGGTCTCCC
TGACTTGG
AGGTGACACTATAGAATAACCGGCCGTGGT
GAGTTGTG
AGGTGACACTATAGAATAGCCCTTAGGGTC
CTGCTCGGC
AGGTGACACTATAGAATAAACTGGGGGCTC
ACCGAAGC
GTACGACTCACTATAGGGACAATGTCGGATGGATG
AAACCCAGA
GTACGACTCACTATAGGGATAGGGGTCGGCCACCA
GGTC
GTACGACTCACTATAGGGATCGGTGGCCTCAGGGG
CATA
GTACGACTCACTATAGGGACACCGCCTCTCAGTCG
CACC
GTACGACTCACTATAGGGACCGTGGAAGGGGACGG
AGAC
GTACGACTCACTATAGGGATGGCGTCCCCGACCAG
AACC
Human samples
RNA was isolated for expression analysis from fully anonymized archival FFPE tissue. A
general approval for use of the samples in the study was given by the Research Ethics
Committee of the Universitätsklinikum Würzburg.
Statistical Analysis
All statistical analysis was done using the Prism5 Software (GraphPad, LaJolla, CA).
Differences between two groups were analyzed using an unpaired, two-tailed Student’s T-test.
In parallel the samples were tested for significant variation of variance. All statistical tests were
performed between sets of individual biological replicates. Appropriate sample and cohort size
for animal studies were calculated based on previous results from previous experiments
according to standard equations (14).
10
Röhrig, Vorlova, Hoffmann et al.
Supplemental Methods
References
1.
Lee TC, Kashyap RL, Chu CN. Building Skeleton Models Via 3-D Medial Surface Axis
Thinning Algorithms. Cvgip-Graph Model Im. 1994;56(6):462-78.
2.
Harrison K, Wagner NH, Jr. Biodistribution of intravenously injected [14C]
doxorubicin and [14C] daunorubicin in mice: concise communication. J Nucl Med.
1978;19(1):84-6.
3.
Laginha KM, Verwoert S, Charrois GJ, Allen TM. Determination of doxorubicin
levels in whole tumor and tumor nuclei in murine breast cancer tumors. Clin Cancer Res.
2005;11(19 Pt 1):6944-9.
4.
Kleinman HK, McGarvey ML, Hassell JR, Star VL, Cannon FB, Laurie GW, et al.
Basement membrane complexes with biological activity. Biochemistry. 1986;25(2):312-8.
5.
Fogelgren B, Polgar N, Szauter KM, Ujfaludi Z, Laczko R, Fong KS, et al. Cellular
fibronectin binds to lysyl oxidase with high affinity and is critical for its proteolytic
activation. The Journal of biological chemistry. 2005;280(26):24690-7.
6.
Kim S, Kim Y. Variations in LOXL1 associated with exfoliation glaucoma do not
affect amine oxidase activity. Molecular vision. 2012;18:265-70.
7.
Rodriguez HM, Vaysberg M, Mikels A, McCauley S, Velayo AC, Garcia C, et al.
Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor. J
Biol Chem. 2010;285(27):20964-74.
8.
Xu L, Go EP, Finney J, Moon H, Lantz M, Rebecchi K, et al. Post-translational
modifications of recombinant human lysyl oxidase-like 2 (rhLOXL2) secreted from
Drosophila S2 cells. J Biol Chem. 2013;288(8):5357-63.
9.
Vadasz Z, Kessler O, Akiri G, Gengrinovitch S, Kagan HM, Baruch Y, et al. Abnormal
deposition of collagen around hepatocytes in Wilson's disease is associated with
hepatocyte specific expression of lysyl oxidase and lysyl oxidase like protein-2. J Hepatol.
2005;43(3):499-507.
10.
Kim YM, Kim EC, Kim Y. The human lysyl oxidase-like 2 protein functions as an
amine oxidase toward collagen and elastin. Molecular biology reports. 2011;38(1):145-9.
11.
Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS, et al. Lentivirusdelivered stable gene silencing by RNAi in primary cells. Rna. 2003;9(4):493-501.
12.
Wong CC, Gilkes DM, Zhang H, Chen J, Wei H, Chaturvedi P, et al. Hypoxia-inducible
factor 1 is a master regulator of breast cancer metastatic niche formation. Proc Natl Acad
Sci U S A. 2011;108(39):16369-74.
13.
de Jonge HJ, Fehrmann RS, de Bont ES, Hofstra RM, Gerbens F, Kamps WA, et al.
Evidence based selection of housekeeping genes. PLoS One. 2007;2(9):e898.
14.
Motulsky H. Intuitive biostatistics. New York: Oxford University Press; 1995. xx,
386 p. p.
11