correction notice magnetoferritin nanoparticles …...correction notice magnetoferritin...
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CORRECTION NOTICE
Magnetoferritin nanoparticles for targeting and
visualizing tumour tissues Kelong Fan, Changqian Cao, Yongxin Pan, Di Lu, Dongling Yang, Jing Feng, Lina Song, Minmin Liang and Xiyun Yan
Nature Nanotechnology 7, 459–464 (2012)
In the Supplementary Information, one of the authors was not mentioned in the author list: Lina Song has now been added. In the section ‘Preparation and characterization of M-HFn particles’ the column used for size-exclusion chromatography was incorrect: it should have been ‘Sepharose 6B’. The synthesis procedure for M-HFn nanoparticles was incorrect: it should have read ‘HFn protein shells were used as a reaction template to synthesize iron oxide nanoparticles according to the method reported by Cao et al.2 with some modification. The solution of 50 ml 100 mM NaCl with HFn (1 mg ml−1) was added to the reaction vessel, synthesized at 65 °C and pH 8.5. Fe(II) (25 mM (NH4)2Fe(SO4)2•6H2O) and stoichiometric equivalents (1:3 H2O2:Fe2+) of freshly prepared H2O2 (8.33 mM) were added. Fe(II) was added in a rate of 100 Fe/(protein min) using a dosing device (800 Dosino) connected with 842 Titrando. After theoretical 5000 Fe/ protein cage were added to the reaction vessel, the reaction was continued for another 5 min. Finally, 200 μl of 300 mM sodium citrate was added to chelate any free iron. The synthesized magnetite-containing HFn (M-HFn) nanoparticles were centrifuged and purified through size exclusion chromatography to remove the aggregated nanoparticles. The concentration of M-HFn nanoparticles was assumed to be the same as that of HFn protein and was determined using a BCA protein assay kit (Pierce). Purified M-HFn nanoparticles were obtained with a yield of about 75%.’ Reference 2 was incorrect and should have read Cao, C. Q. et al. Magnetic characterization of noninteracting, randomly oriented, nanometer-scale ferrimagnetic particles. J. Geophys. Res. 115, B07103 (2010). These errors have been corrected in this file 27 November 2012.
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SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2012.209
NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1
Magnetoferritin nanoparticles for targeting and visualizing tumour
tissues
Kelong Fan1, Changqian Cao2, Yongxin Pan2, Di Lu1, Dongling Yang1, Jing Feng1,
Lina Song1, Minmin Liang1 * & Xiyun Yan1 *
*Corresponding author.
Minmin Liang, PhD. Email : [email protected] Tel: +86 10 6488 8583; Fax: +86 10
6488 8584.
Xiyun Yan, MD. Email: [email protected] Tel: +86 10 6488 8583; Fax: +86 10 6488
8584.
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Cell lines, tissues and animal models
Cell lines HT29, K562, U937, HeLa, A375, SKOV3, PC-3, Jurkat, SW1990 and
MDA-MB-231 were obtained from the American Type Culture Collection (ATCC).
SMMC-7721, U251 and MX-1 were from the cell bank of the Committee on Type
Culture Collection of the Chinese Academy of Sciences (CTCC, Shanghai, China).
A375, PC-3, SW1990 and MDA-MB-231 cells were cultured in DMEM medium
containing 10% fetal calf serum. HT29, K562, SMMC-7721, U937, HeLa, SKOV3,
U251, Jurkat and MX-1 cells were cultured in RPMI-1640 medium containing 10%
fetal calf serum. Clinical tumour and normal tissues were obtained from the tissue
bank of the Beijing Tumor Hospital (Beijing) and Aomei Biotechnology Co. (Xi’an,
China). Female BALB/c nude mice were obtained from the Animal Center of the
Chinese Academy of Medical Sciences (Beijing). All animal studies were performed
with the approval of the Chinese Academy of Sciences Institutional Animal Care and
Use Committee. Mice were each injected subcutaneously in one thigh with 0.1 mL of
suspension containing 106 HT29, SKOV3, MX-1 or SMMC-7721 cells. When
tumours reached 0.4-0.6 cm in diameter, they were excised and fixed in 10% buffered
formalin for 24 h before embedding in paraffin. 5 µm paraffin-embedded tumor
xenograft sections were cut and used for subsequent histological staining.
Preparation and characterization of M-HFn nanoparticles.
Recombinant human ferritin shells composed of 100% heavy-chain subunits
were produced in Escherichia coli and purified as described1. Briefly, Escherichia
coli lysate expressing HFn was sonicated on ice and then centrifuged at 10000 g for
30 min. The supernatant was heated at 70°C for 10 min to precipitate most of the
Escherichia coli proteins. After centrifugation, the supernatant was precipitated again
by ammonium sulfate (520 g/L). The precipitate was collected by centrifugation at
22,000×g, and then dissolved in PBS. After dialyzing out the ammonium sulfate,
HFn was purified by size-exclusion chromatography on a Sepharose 6B column. The
final yield of HFn was >100 mgL-1 from the bacterial lysate.
HFn protein shells were used as a reaction template to synthesize iron oxide
nanoparticles according to the method reported by Cao et al2 with some modification.
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The solution of 50 mL 100 mM NaCl with HFn (1 mg/ml) was added to the reaction
vessel, synthesized at 65°C and pH 8.5. Fe(II) (25 mM (NH4)2Fe(SO4)2•6H2O) and
stoichiometric equivalents (1:3 H2O2:Fe2+) of freshly prepared H2O2 (8.33 mM) were
added. Fe(II) was added in a rate of 100 Fe/(protein min) using a dosing device (800
Dosino) connected with 842 Titrando. After theoretical 5000 Fe/ protein cage were
added to the reaction vessel, the reaction was continued for another 5 min. Finally,
200 μL of 300 mM sodium citrate was added to chelate any free iron. The synthesized
magnetite-containing HFn (M-HFn) nanoparticles were centrifuged and purified
through size exclusion chromatography to remove the aggregated nanoparticles. The
concentration of M-HFn nanoparticles was assumed to be the same as that of HFn
protein and was determined using a BCA protein assay kit (Pierce). Purified M-HFn
nanoparticles were obtained with a yield of about 75%.
The prepared M-HFn nanoparticles and HFn protein were characterized using
TEM (Tecnai F20, Philips), cryo-TEM (FEI Titan Krios 300kV, FEI, Oregon), HFn
proteins were negatively stained with uranyl acetate for TEM observation while iron
oxide cores encapsulated in HFn proteins were unstained. For cryo-TEM observation,
M-HFn nanoparticle samples were embedded in vitreous ice using an FEI Vitrobot
Mark VI and imaged with an FEI 300-kV Titan Krios cryo-TEM equipped with a
Gatan UltraScan4000 (model 895) 16-megapixel CCD camera. M-HFn nanoparticles
were imaged with an absolute magnification micrograph of 96,000, and the dose for
each micrograph was about 20e-/Å.
A peroxdiase activity test was carried out on M-HFn nanoparticles at room
temperature. M-HFn at 0.5 µM was mixed with 500 mM H2O2 in 0.2 M sodium
acetate buffer (pH 4.5), using 0.2 mg/mL TMB (Sigma) as the substrate. Colour
reactions were recorded 30 min after addition of the substrate. The reaction buffer
used for the DAB (Sigma) substrate was 0.05 M Tris-HCl, pH 7.5.
The prepared M-HFn nanoparticles were further characterized using
size-exclusion chromatography (SEC, Amersham Pharmacia Biotech, Piscataway)
and dynamic light scattering (DLS, DynaPro Titan TC, Wyatt Technology). SEC
analysis were performed on a Superose 12 column installed on a Waters 515 solvent
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delivery system (Waters, Milford) equipped with an in-line radioactivity detector and
a Waters UV2487 dual wavelength absorbance detector. DLS analysis was performed
at room temperature. The concentration of M-HFn used was 1.5 mg/ml in PBS buffer.
As shown in Figure S1a, the M-HFn nanoparticles were found to be monodisperse in
solution with a outer diameter of 12~16 nm. In addition, HFn and M-HFn had
identical SEC elution times (Figure S1b,c). These results indicate that the
mineralization process does not significantly perturb the overall protein cage
architecture of HFn and that the iron oxide core is sequestered within the protein
shell.
Figure S1. (a) DLS analysis of M-HFn nanoparticles. M-HFn nanoparticles have
an outer diameter of 12~16 nm (i.e. 6~8 nm in radius). (b) SEC of HFn protein and (c)
M-HFn nanoparticles by in-line UV detection at 280 nm (protein) and 410 nm (iron
oxide core).
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Peroxidase activity of M-HFn, natural holoferritin and apoferritin
A comparison of the peroxidase activity of M-HFn, natural holoferritin and
apoferritin was performed on a PVDF membrane. Briefly, 4 µg of M-HFn, natural
holoferritin and apoferritin was respectively mixed with 1 µl of TMB substrate (10
mg/mL), 1 µl of 30% H2O2 and 5 µl of sodium acetate buffer (pH 4.5) and the mixture
was transferred to a PVDF membrane. The image was taken 30 min after transfer.
Mineral cores within ferritin exhibited peroxidase activity, catalyzing the oxidation of
substrate TMB and giving a coloured dot on the membrane. The strong staining
intensity demonstrates the high peroxidase activity of mineral cores. As shown in
Figure S2, M-HFn shows higher peroxidase activity than natural holoferritin due to
their differences in mineral phase composition. The apoferritin control without a
mineral core exhibited no peroxidase activity.
Figure S2. Direct comparison of peroxidase activity between magnetoferritin,
natural holoferritin and apoferritin. Horse spleen holoferritin from Sigma was used
here as natural holoferritin.
Saturation binding assay
The binding affinity of HFn to TfR1 was measured using a saturation binding
assay. A total of 5×105 SMMC-7721 cells were incubated with increasing amounts
of FITC-conjugated HFn at concentrations ranging from 0 to 400 nM for 1 h at 4°C.
The total binding of FITC-conjugated HFn was calculated after cells were washed
(Figure S3a, black line). Nonspecific binding was determined as the binding of
FITC-conjugated HFn to non-TfR1 sites. In order to measure nonspecific binding, the
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same reaction mixtures were prepared with the addition of an excess of (40 µM)
unconjugated HFn. Nonspecific binding of FITC-conjugated HFn was obtained after
washing with cold PBS (Figure S3a, red line), and the specific binding of
FITC-conjugated HFn was calculated by subtracting nonspecific binding from total
binding (Figure S3b). The dissociation constant (Kd) was calculated using GraphPad
Prism 4.0. As shown in Figure S3, the saturation binding curve clearly shows that the
binding of HFn to SMMC-7721 cancer cells is saturable and that the binding can be
significantly inhibited by adding an excess dose of unconjugated HFn. This confirms
again that HFn binding is specific. Scatchard analysis demonstrated that HFn binds to
SMMC-7721 cells with a high affinity (Kd of 50 nM).
Figure S3. Saturation binding curve for FITC-HFn binding to TfR1 on
SMMC-7721 cells. (a) Total (black line) and non-specific binding (red line). Excess
unconjugated HFn was used to determine nonspecific binding. Each data point
represents the average value from triplicate wells. (b) Specific binding was obtained
after subtraction of non-specific binding from total binding. Binding of FITC-HFn to
SMMC-7721 cells is saturable. Binding is inhibited by excess unconjugated HFn. The
Kd of HFn was 50 nM.
Reactivity of HFn to human cancer cells
The binding activity of HFn protein shells to cancer cells was confirmed using
human hepatocellular carcinoma, colon carcinoma, breast adenocarcinoma, melanoma,
erythroleukemia, cervical carcinoma, ovarian carcinoma, prostate carcinoma,
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glioblastoma, histiocytic lymphoma and T-cell leukemia cell lines by flow cytometric
analysis. As illustrated in Figure S4, HFn strongly bound to these cancer cell lines,
demonstrating the ability of HFn to universally recognize cancer cells.
Figure S4. Flow cytometric analysis of HFn reactivity with human cancer cells.
The ten cancer cell lines examined were glioblastoma (U251), ovarian carcinoma
(SKOV-3), histiocytic lymphoma (U937), pancreatic cancer (SW1990), cervical
carcinoma (HeLa), prostate carcinoma (PC-3), breast adenocarcinoma
(MDA-MB-231), melanoma (A375), erythroleukemia (K562), and T-cell leukemia
(Jurkat).
Antibody and transferrin blocking studies
Antibody blocking study was performed on SMMC-7721 cells. Briefly, 0.3 µM
of FITC-HFn was added to the wells in the presence or absence of a 10-fold molar
excess of anti-TfR1 mAbs. After incubation for 1 h on ice, the cells were washed
three times in cold PBS and then collected. Cell-bound fluorescence was measured by
flow cytometry. In addition, the incubated cells were also examined under a confocal
laser scanning microscope. Results are shown in Figure S5. Anti-TfR1 mAb
completely blocks the binding of HFn to SMMC-7721 cancer cells as determined by
both flow cytometry and confocal studies, further confirming that TfR1 is the binding
receptor of HFn and mediates its specific binding to cancer cells.
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Figure S5. (a) Fluorescence staining of SMMC-7721 human liver cancer cells
using FITC-conjugated HFn in the presence (right) or absence (left) of a 10-fold
molar excess of anti-TfR1 mAbs. (Scale bar = 50 µm). (b) Flow cytometry analysis of
the binding of FITC-conjugated HFn to SMMC-7721 cancer cells in the presence or
absence of a 10-fold molar excess of anti-TfR1 mAbs. (n = 3, bars represent means ±
SD)
Competitive binding by transferrin was tested using SMMC-7721 cells. Briefly,
5X105 cells were incubated for 1 h on ice with 0.3 µM FITC-HFn and transferrin at
concentrations ranging from 0 to 40 µM. After washing three times in cold PBS, the
fluorescence intensity of cells was measured by flow cytometry. Figure S6 shows that
transferrin competes with HFn for binding to TfR1. However, transferrin at a 100-fold
molar excess only inhibits HFn binding by 50%.
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0 10 20 30 40100
120
140
160
180
200
220
Fluo
resc
ence
Inte
nsity
Transferrin(µM)
Figure S6. Transferrin competes with HFn for binding to TfR1. Binding of
FITC-HFn to SMMC-7721 cells is only partially inhibited by transferrin.
Fluorescence and mineral core-peroxidase staining analysis
We assessed the co-localization of fluorescence staining and mineral
core-peroxidase staining by incubating HT-29 cancer xenograft tumours with
FITC-conjugated M-HFn. Briefly, two sequential sections from an HT-29 cancer
xenograft were first stained with FITC-labeled M-HFn. One section was examined
after M-HFn-peroxidase staining by light microscopy while the other was examined
by fluorescence microscopy. By comparing these sequential sections we can see that
the fluorescence staining co-localized with mineral-peroxidase staining in tumour
cells (Fig. S7).
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Figure S7. Fluorescence staining co-localized with mineral core-peroxidase
staining in tumour cells on the sequential sections. Two sequential sections from an
HT-29 cancer xenograft were stained both with FITC-labeled M-HFn. One section
was examined after M-HFn-peroxidase staining by light microscopy while the other
was examined by fluorescence microscopy. (Scale bar = 50 µm )
HFn staining of human tissue arrays
To study the potential correlation of HFn binding with the grade and growth
pattern of tumours, tissue arrays including hepatocellular carcinoma, lung squamous
cell carcinoma, cervical squamous cell carcinoma, prostate adenocarcinoma, ovarian
serous papillary carcinoma, and colonic adenocarcinoma (about 20 cases/type), and
their corresponding normal and lesion tissues (about 5 cases/type) were incubated
with FITC-labeled HFn (1 µM) at 4°C overnight. The stained tissues were imaged
under a confocal laser scanning microscope (Olympus). As shown in Figure S8-13,
HFn based fluorescence staining positively correlated with differentiation, grades and
growth patterns of hepatocellular carcinoma, lung squamous cell carcinoma, cervical
squamous cell carcinoma, prostate adenocarcinoma, ovarian serous papillary
carcinoma and colonic adenocarcinoma. The corresponding normal tissues, necrotic
tumours, chronic inflammatory tissues and hyperplastic tissues showed negative
staining.
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Figure S8. HFn staining of a lung tissue array. Tissues were incubated with
FITC-labeled HFn. (a) Grade 1 lung squamous cell carcinoma showing weak staining
intensity with 10-20% positive cells. (b) Grade 2 lung squamous cell carcinoma
showing medium staining intensity with 20-50% positive cells. (c) Grade 3 lung
squamous cell carcinoma showing strong staining intensity with >50% positive cells.
(d) Necrotic carcinoma tissue showing negative staining. (e) Normal lung showing
negative staining. (f) Normal lung with congestion showing slight staining. The
tumours were graded according to Gleason’s grading system. Scale bar = 100 µm
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Figure S9. HFn staining of a liver tissue array. Tissues were incubated with
FITC-labeled HFn. (a) Grade 1 hepatocellular carcinoma tissue showing medium
staining intensity with 50-80% positive cells. (b) Grade 2 hepatocellular carcinoma
showing strong staining intensity with >80% positive cells, (c) Grade 3 hepatocellular
carcinoma tissue showing strong staining intensity with >80% positive cells. (d)
Normal liver showing negative staining. (e) Liver tissue of hepatitis showing negative
staining. (f) Cirrhotic tissues showing negative staining. The tumours were graded
according to Gleason’s grading system. Scale bar = 100 µm
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Figure S10 HFn staining of a cervical tissue array. Tissues were incubated with
FITC-labeled HFn. (a) Grade 1 cervical squamous cell carcinoma showing weak
staining intensity with 20-50% positive cells. (b) Grade 2 cervical squamous cell
carcinoma showing medium staining intensity with 20-50% positive cells. (c) Grade 3
cervical squamous cell carcinoma showing strong staining intensity with >50%
positive cells. (d) Cancer adjacent normal cervical tissue showing negative staining. (e)
Cervical chronic inflammatory tissue showing negative staining. (f) Normal cervical
tissue showing negative staining. The tumours were graded according to Gleason’s
grading system. Scale bar = 100 µm
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Figure S11 HFn staining of a prostate tissue array. Tissues were incubated with
FITC-labeled HFn. (a) Grade 1 prostate adenocarcinoma showing weak staining
intensity with 10-20% positive cells. (b) Grade 2 prostate adenocarcinoma showing
medium staining intensity with 20%-50% positive cells. (c) Grade 3 prostate
adenocarcinoma showing medium staining intensity with 50-80% positive cells. (d)
Grade 4 prostate adenocarcinoma showing strong staining intensity with >80%
positive cells. (e) Prostatic hyperplasia showing negative staining. (f) Prostate smooth
muscle showing negative staining. (g) Cancer adjacent normal prostate tissue showing
negative staining. (h) Grade 1 prostatic intraepithelial neoplasia showing slight
staining only in prostate duct epithelium. The tumours were graded according to
Gleason’s grading system. Scale bar = 100 µm
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Figure S12. HFn staining of an ovary tissue array. Tissues were incubated with
FITC-labeled HFn. (a) Grade 1 ovarian serous papillary carcinoma showing medium
staining intensity with 50-80% positive cells. (b) Grade 2 ovarian serous papillary
carcinoma showing strong staining intensity with >80% positive cells. (c) Grade 3
ovary serous papillary carcinoma showing strong staining intensity with >80%
positive cells. (d) Cancer adjacent normal ovary showing negative staining. The
tumours were graded according to Gleason’s grading system. Scale bar = 100 µm
Figure S13. HFn staining of a colon tissue array. Tissues were incubated with
FITC-labeled HFn. (a) Grade 1 colonic adenocarcinoma showing weak staining
intensity with 10-20% positive cells. (b) Grade 2 colonic adenocarcinoma showing
strong staining intensity with 20-50% positive cells. (c) Grade 3 colonic
adenocarcinoma showing strong staining intensity with >50% positive cells. (d)
Normal colon showing negative staining. The tumours were graded according to
Gleason’s grading system. Scale bar = 100 µm
M-HFn-based peroxidase-like reaction mechanism
To understand the mechanism of the M-HFn-based peroxidase-like reaction, we
detected the formation of •OH during the reaction by electron spin resonance
(ESR),using 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping reagent,
a method well-known to effectively determine •OH. Briefly, 100 µL reaction mixtures
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were taken after 10 min reactions at room temperature and mixed with 20 µl of 200
mM DMPO to form a DMPO-OH adduct. The amount of hydroxyl radical was
determined from ESR signals using a Bruker model ESP 300E spectrometer. ESR
measurements were carried out at room temperature under the following conditions:
microwave power 15.89 mW, modulation amplitude 3.081 G, scan range of 100.00 G,
modulation frequency 100.00 kHz, and center field 3485.00 G. As shown in Figure
S14 (a, b), •OH was produced during the peroxidase-like reaction in the presence of
both M-HFn nanoparticles and H2O2. With the addition of the •OH scavenger, ethanol,
the formed •OH disappeared (Figure S14 c) and the peroxidase activity of M-HFn
nanoparticles decreased to 20% of the original activity (Figure S14 d,e), indicating
that the •OH formed during the peroxidase-like reaction is responsible for the
catalytic oxidation of peroxidase substrate to give the colored precipitate at the site of
its target.
Based on these results, we propose the following mechanism for the
M-HFn-based peroxidase-like reaction. With the addition of H2O2 and peroxidase
substrate into the M-HFn reaction solution, diffusing H2O2 enters the ferritin cavity
through its hydrophilic channels and interacts with the iron oxide core of M-HFn to
generate •OH on the surface of the iron core. The generated •OH then oxidizes the
peroxidase substrate (e.g., DAB) diffused nearby to form an insoluble colored
precipitate at the site of M-HFn, which is targeted to cancer cells. The colored
precipitates are only formed at the site of M-HFn since •OH radicals are highly
reactive and short-lived, and can only oxidize nearby substrates to give colored
precipitates. The clear boundary between tumour and normal tissues on M-HFn
stained tissue slides (Figure S15) also proves that the colored precipitates are
generated right at the site of M-HFn-targeted cancer cells, and the oxidized colored
precipitates do not diffuse away from their targets.
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Figure S14 ESR spectra of DMPO-OH adducts in the M-HFn-based peroxidase-like
reaction. (a) H2O2/40 mM DMPO; (b) H2O2/0.5 µM M-HFn nanoparticles/40 mM
DMPO; (c) H2O2 /M-HFn nanoparticles/40 mM DMPO/•OH scavenger ethanol; The
reaction was initiated by adding 500 mM H2O2. All the mixtures were in 200 mM
acetate buffer (pH = 4.5). (d) Peroxidase activity of M-HFn nanoparticles with (right
tube) or without the •OH scavenger ethanol. M-HFn catalyzed the oxidation of
peroxidase substrate TMB in the presence of H2O2 to give a colored product. (e)
Absorbance at 652 nm of the TMB reaction solution catalytically oxidized by M-HFn
with or without the •OH scavenger ethanol.
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Figure S15 The M-HFn-stained tumour area is clearly demarcated from normal
tissues. A HT-29 colon cancer xenograft was stained by M-HFn and visualized by
DAB color development. The clear boundary between tumour and normal tissues on
M-HFn stained tissue slides demonstrates that the colored precipitates are generated
right at the site of M-HFn-targeted cancer cells, and that the oxidized colored
precipitates do not diffuse away from their targets. (Scale bar = 50 µm)
Comparison of immunohistochemical and M-HFn approaches
Sequential liver tissue sections containing tumours were stained respectively by
M-HFn nanoparticles and anti-TfR1 Abs. Paraffin-embedded tissues of two
hepatocellular carcinoma cases identified by a pathologist were deparaffinized in
xylene and then hydrated progressively in an ethanol gradient. After quenching
endogenous peroxidase activity, the tissue sections were blocked with goat serum, and
then incubated with M-HFn or polyclonal rabbit anti-TfR1 antibody, respectively. The
stained sections were analyzed under a microscope and the results were assessed by
two independent pathologists. M-HFn-stained tissues showed positive staining of
tumour cells and negative staining of normal liver cells (Figure S16 a, c). Anti-TfR1
antibody-based immunohistochemical staining could not distinguish between tumours
and normal liver tissues. (Figure S16 b, d).
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Figure S16 A side-by-side comparison of standard antibody-based
immunohistochemical and M-HFn-based approaches in two hepatocellular
carcinoma cases identified by pathologists. Sequential liver tissue sections
containing tumours were stained respectively by M-HFn nanoparticles and anti-TfR1
antibody. (a, c) M-HFn-stained tissues showed positive staining of tumour cells and
negative staining of normal liver cells. (b, d) Anti-TfR1 antibody-based
immunohistochemical staining could not distinguish tumours from normal liver
tissues. Scale bar = 100 µm
We further did a literature search to establish the range of sensitivities and
specificities of different antibodies-based detections to make valid comparsions with
our M-HFn-based approach. The search results are shown as following in
Supplemental Table 1. Of the 56 different antibodies widely used in the detection of
the 15 main cancer biomarkers for 7 different types of cancer reported in the literature,
only the best ones (AMACR antibody P504S: sensitivity of 80%~95%; specificity:
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79%~100%; and PSA antibody ER-PR8: sensitivity of 82%~94.4%, specificity of
100%) had a similar tumour detection sensitivity and specificity with our M-HFn.
However, they can only detect one type of cancer. When M-HFn nanoparticles were
used to screen 474 clinical specimens from patients with nine types of cancer, it had a
total sensitivity of 98% and a total specificity of 95% (Table 1 in manuscript),
indicating that M-HFn are an excellent reagent for cancer screening.
Table S1 Sensitivity and specificity of various antibody-based
immunohistochemistry and immunofluorescence detection methods. The
antibodies listed below represent most of the antibody population reported in the
literature or available commercially for cancer biomarker detection.
Tumor Biomarker Antibody (Source) Sensitivity Specificity References
Hepatocellular carcinoma
CEA 11-7 (DAKO) 46.88~79% 52.44~97% [3],[4],[5] AFP α-AFP mAb (DAKO) 17~61.5% 97.00% [5],[6] TfR OKT9, 5E9, RBC4, B3/25, Tu15 97.06% 86.67% [7]
Lung carcinoma
TTF-1 SPT24 (Novocastra), 8G7G3/1 (DAKO) 65~87% 92%~100% [8],[9]
p53 DO-7 (Novocastra), PAb240 (Oncogene Science)
46~79.2% 83.7~100% [10],[11],[12]
CK-7 M7018 (DAKO) 75.00% 77% [13]
Colonic adenocarcinoma
CEA 11-7 (DAKO) 50~59.4% 53.23% [3],[14]
p53 PAb240 (Oncogene Science), α-p53 mAb (DAKO), DO-7 (Novocastra)
49~80% 91.63% [15],[16],[17]
Ki-67 α-Ki-67 mAb (CHANGDAO), α-Ki-67 mAb (DAKO)
21.6%, 87.5%
100%, 86.67%
[18],[19]
Cervical squamous cell carcinoma
p53 BP53-12 (BioGenex), DO-7 (Novocastra Laboratories), α-p53 mAb (DAKO)
17.1~85.7% 87~100% [20],[21],[22], [23],[24]
Ras α-Ras mAb RP35, α-Ras mAb Y13 259 38.7~100% 50~90% [25],[26]
C-erbB-2 α-C-erbB-2 pAb, NCL-CBll (Novocastra laboratories)
12.1~38.7% 83~100% [27],[28],[29],[30]
Prostate adenocarcinoma
EPCA α-EPCA pAb 84~94% 85% [31],[32] AMACR P504S (Corixa) 80~95% 79~100% [33],[34],[35]
PSA ER-PR8 (DAKO), A0562 (DAKO)
82~94.4%, 100%
100%, 68%
[36],[37],[38]
Ovarian serous papillary carcinoma
ER 6F-11 (Novocastra Laboratories) 63~86.36% 95~97.7% [39],[40],[41] WT1 6F-H2(DAKO) 82~86% 95~97% [39],[41]
p53 DO-7 (Novocastra Laboratories), PAb1801 (Cambridge Research Biochemicals)
55~73.7% 61.54~100% [39],[42],[43], [44],[45]
Breast Her-2 28 different antibodies including TAB250 6~82% 92~100% [46], [47]
© 2012 Macmillan Publishers Limited. All rights reserved.
carcinoma (Berlex biosciences), 2H11 (Genetech), and 3E8 (Genetech)
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