Preclinical Study of CDH3-Targeted Zr/Lu Theranostics in Triple-Negative Breast Cancer.

1
Introduction
According to the 2022
global cancer statistics, breast cancer ranks
second in incidence among all malignancies worldwide, with 2.3 million
new cases (accounting for 11.6% of all cancers) and 666,000 deaths
annually (mortality rate, 6.9%), ranking fourth in cancer-related
mortality. The 5-year survival rate for
breast cancer patients remains below 40%. Based on molecular subtyping, breast cancer is classified into three
major subtypes: hormone receptor–positive (HR), HER2-positive,
and
triple-negative breast cancer (TNBC), the latter accounting
for
approximately 15%–20% of all cases. TNBC is defined by the absence of estrogen receptor (ER), progesterone
receptor (PR), and human epidermal growth factor receptor 2 (HER2)
expression, and represents the most aggressive
breast cancer subtype, with the poorest prognosis, the greatest therapeutic
challenges, and the highest recurrence and mortality rates,
,
making it an exceptionally challenging therapeutic target. Studies have shown that TNBC patients have significantly
shorter survival across all stages compared with non-TNBC patients, primarily due to its high malignant potential
and the lack of effective targeted therapies. Notably, although TNBC is highly heterogeneous, it exhibits greater
sensitivity to targeted therapies than other subtypes. At present, the scarcity of effective targeted
agents remains a major barrier to therapeutic progress. Therefore,
developing novel therapeutic strategies and subtype-specific interventions
for TNBC holds significant clinical and societal importance for improving
survival and prognosis in this patient population.
Targeted
radionuclide therapy (TRT) is a precision cancer treatment
strategy based on the conjugation of radioactive isotopes with targeting
molecules, allowing selective delivery of radiation to tumor cells
while sparing healthy tissues. The therapeutic response is highly
dependent on the radiation absorption dose within the tumor tissue. Currently, two FDA-approved TRT agents, [177Lu]­Lu-PSMA-617 (for prostate cancer) and [177Lu]­Lu-DOTATATE (for neuroendocrine tumors), have demonstrated clinical
efficacy.
,
Clinical studies have confirmed that 177Lu-labeled PSMA-targeted agents (Lu-PSMA I&T and Lu-PSMA-617)
exhibit both excellent safety and significant efficacy in patients
with metastatic castration-resistant prostate cancer (mCRPC). Furthermore, [177Lu]­Lu-DOTATATE has
significantly extended progression-free survival (PFS) and improved
the objective response rate (ORR) with favorable safety profiles. For diagnostic purposes, 89Zr (with
a half-life of 78.41 h) is particularly suited for detecting lesions
with low antigen expression due to its long half-life and high sensitivity
in PET imaging.
,
Therapeutically, 177Lu induces DNA damage and exhibits a ″crossfire effect″
through β-radiation (average energy of 0.497 MeV) to exert its
antitumor effects.

P-cadherin (CDH3),
a member of the cadherin superfamily, mediates tumor cell adhesion, proliferation,
and invasion. It is specifically overexpressed
in basal-like breast cancer and is significantly associated with BRCA1
mutations and poor prognosis.
,
Clinical studies have
demonstrated its prominent overexpression in various malignancies,
including breast, pancreatic, head and neck, lung, gastric, endometrial,
and colorectal cancers, while its expression is limited in normal
tissues.
,
Meta-analysis confirms that CDH3 overexpression
is linked to poor prognosis across breast cancer subtypes (Luminal,
HER2+, TNBC), positioning it as an ideal
target for TRT.
,
Preclinical studies have shown
that 90Y-labeled anti-CDH3 monoclonal antibody (mAb-6)
significantly inhibits the growth of CDH3-positive tumors, including
lung and colorectal cancers. Moreover,
CDH3-targeted antibodies or peptides conjugated with radionuclides
have demonstrated significant therapeutic efficacy in the treatment
of various malignancies, including neuroendocrine tumors and prostate
cancer.
,
However, to date, there have been no reported
studies on CDH3-targeted radionuclide therapy in TNBC. This study
constructs a CDH3-targeted radionuclide probe, 89Zr/177Lu-11E10C11, to explore the potential of CDH3 as an integrated
diagnostic and therapeutic target in TNBC, providing a novel strategy
for precision treatment.

2
Materials and Methods
2.1
Cell Lines and Cell Culture
This
study utilized three human TNBC cell lines, authenticated by STR profiling:
HCC1806, BT-549, and MDA-MB-231, all purchased from Wuhan PnnoScience Life Technology Co., Ltd. HCC1806 and BT-549 cells
were cultured in RPMI-1640 medium, while MDA-MB-231 cells were cultured
in DMEM, both supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin,
and incubated at 37 °C with 5% CO2.
,
Cells were passaged for no more than 6 months, and collected for
further experiments when cell density reached 75–90%.

2.2
Establishment of Animal Models
The
experiment used 6–8 week-old female BALB/c-nu nude mice (Sichuan
Paixiwei Biotech Co., Ltd.), which were housed in an SPF barrier system
(22 ± 2 °C, 50 ± 5% humidity, 12-h light/dark cycle)
for acclimatization. HCC1806 cells in the logarithmic growth phase
were mixed with Matrigel at a 1:1 ratio (total volume 100 μL),
and 1 × 107 cells per mouse were subcutaneously injected
into the right axillary flank of the mice to establish the HCC1806-CDH3
xenograft tumor model. Mice were monitored daily for health status,
and tumor volume changes were measured.The animal research protocol
and all animal experiments were approved by the Animal Ethics and
Welfare Committee of Mianyang Central Hospital (Ethical approval number:
S20250310–02).

2.3
Flow Cytometry Analysis of In Vitro Cell Lines
Cells (5 × 104 per well) were seeded in a 96-well
U-bottom plate, followed by centrifugation at 4 °C, 1300 rpm
for 5 min. The cells were washed twice with prechilled FACS buffer
(95% PBS + 5% FBS). The cells were incubated on ice with 25 μg/mL
of monoclonal antibody for 60 min. After washing, fluorescent secondary
antibody was added and incubated in the dark for 30 min. Following
incubation and washing, the cells were resuspended in 200 μL
of buffer. A total of 3,000 events were acquired using a FACSCalibur
flow cytometer, and the data were analyzed with FlowJo v10.8 software.

2.4
Synthesis of DFO-11E10C11
A 50 kDa
ultrafiltration membrane (13,000 rpm, 10 min, 4 °C) was used
to remove solvents and small molecular impurities from the CDH3 monoclonal
antibody solution, followed by three washes with 0.15 M carbonate
buffer (pH 9.5). The purified antibody was then mixed with DFO at
a 10:1 molar ratio (DFO dissolved in 20 nmol/uL DMSO) and incubated
at 37 °C with shaking at 70 rpm for 1 h. The reaction mixture
was subsequently washed three times with 0.5 M sodium acetate buffer
(pH 5.5) using ultrafiltration, and the final product was collected
for further use.

2.5
Synthesis of DOTA-11E10C11
The CDH3
monoclonal antibody solution was subjected to ultrafiltration using
a 50 kDa membrane (13,000 rpm, 10 min, 4 °C) to remove solvents
and small molecular impurities, followed by three washes with prechilled
PBS (pH 7.4). The purified antibody was then mixed with DOTA at a
10:1 molar ratio (20 nmol/μL DMSO solution) and incubated at
37 °C with shaking at 70 rpm for 1 h. Subsequently, an equimolar
amount of tetrachloroethylene peroxide reductant was added to continue
the reaction for another hour. The final product was washed three
times with 0.5 M sodium acetate buffer (pH 5.5) using ultrafiltration
and collected for further use.

2.6
Determination of the Grafting Ratio
A 5 mL aliquot of arsenazo III solution was mixed with 5 mL of standard
lead solution and reacted for 10 min. Then, 640 μL of the reaction
mixture was aliquoted into 1.5 mL centrifuge tubes. To each tube,
40 μL of NaCl solution (1 M) was added, followed by the addition
of 120 μL, 116 μL, 110 μL, 100 μL, 90 μL,
and 80 μL of NH4OAc buffer (0.15 M), and correspondingly
0 μL, 4 μL, 10 μL, 20 μL, 30 μL, and
40 μL of standard Mal-DOTA solution (0.4 nmol/μL). All
samples were thoroughly mixed and incubated at 37 °C in the dark
for 20 min. A standard curve was constructed by plotting the absorbance
at 656 nm against the concentration of Mal-DOTA. Under identical conditions,
11E10C11-DOTA was introduced into the reaction system, and the absorbance
was measured. The molar ratio of DOTA to antibody was determined based
on the standard curve.

2.7
Synthesis of [89Zr]­Zr-DFO-11E10C11
A solution of 89Zr­(IV) oxalate (China Academy of Engineering
Physics, Institute of Nuclear Physics and Chemistry) was adjusted
to pH 7.0 using 0.5 M Na2CO3 and equilibrated
at room temperature for 3 min. CDH3-DFO conjugate was then added at
a 1:2 molar ratio, and the volume was adjusted with HEPES buffer (0.1
M, pH 7.0). The reaction mixture was incubated at 37 °C in a
metal bath for 90 min, with shaking for 10 s every 10 min. The labeling
efficiency was assessed by thin-layer chromatography (TLC). A 2 μL
aliquot of the reaction mixture was applied to a 10 × 1.5 cm
silica gel fiber paper, and upward development was performed using
0.5 M sodium citrate buffer (pH 5.5) as the mobile phase.

2.8
Synthesis of [177Lu]­Lu-DOTA-11E10C11
CDH3-DOTA was mixed with 177LuCl3 (China
Academy of Engineering Physics, Institute of Nuclear Physics and Chemistry)
at a 1:2 molar ratio and incubated at 42 °C in a metal bath for
1 h, with shaking for 10 s every 10 min. The labeling efficiency was
determined by TLC after the reaction. The reaction mixture was analyzed
for purity by high-performance liquid chromatography (HPLC).

2.9
In Vitro Stability
To assess the
in vitro stability of [177Lu]­Lu-DOTA-11E10C11, 5 μL
of the labeled product was mixed with 20 μL of either physiological
saline (0.9% NaCl), PBS (pH 7.4), or RPMI-1640 medium containing 10%
FBS. The labeling efficiency was measured at time points of 4, 24,
48, 72, 96, 144, and 192 h using TLC at room temperature. This approach
systematically evaluated the effect of different media on the stability
of the labeled complex.

2.10
Saturation Binding and Block Assays
CDH3-positive cells in the logarithmic growth phase (5 × 104 cells) were preincubated with 12 gradient 2-fold serial dilutions
of unlabeled monoclonal antibody (50 times the initial concentration)
for 30 min (shaken at 200 rpm). Subsequently, 5 μCi of [177Lu]­Lu-DOTA-11E10C11 was added, and incubation was continued
for 1 h at room temperature. A specificity control group containing
only the labeled antibody was also included. 11E10C11, with an initial
concentration of 50 times its half-maximal effective concentration
(EC5
0), was subjected to 12-step serial 2-fold
dilutions and then added to the cell culture system. After incubation
at 200 rpm for 30 min, an equimolar concentration of [177Lu]­Lu-DOTA-11E10C11 was introduced, followed by an additional 1-h
reaction period to perform a competitive binding blockade assay. All
samples were washed three times with FACS buffer (4 °C, 2800
rpm, 4 min) and subsequently analyzed using a gamma counter to determine
both bound and unbound radioactivity, thereby enabling evaluation
of the targeting binding properties of the radiolabeled antibody.

2.11
Immunoreactive Fraction Assay
HCC1806
cells were aliquoted into 0.5 mL centrifuge tubes at an initial density
of 800,000 cells/200 μL and subjected to 2-fold serial dilution
to generate 12 concentration gradients. Each tube received a cold
antibody at a starting concentration of 50× EC5
0, followed by identical 2-fold serial dilution. After gentle shaking incubation for 30 min to
establish 100% blocking controls, the tubes were centrifuged (3000
rpm, 5 min) and the supernatant was discarded. An equal volume (200
μL) of [177Lu]­Lu-DOTA-11E10C11 was added to each
tube, followed by incubation on an orbital shaker (100 rpm) for 1
h. The tubes were subsequently centrifuged, washed twice with PBS,
and the cell-associated radioactivity (cpm) was measured. A scatter
plot was constructed with the number of cells per tube on the x-axis and the ratio of total cpm to (measured cpm minus
cpm of the 100% blocked tube) on the y-axis. The
data were fitted to a linear equation y = ax + b (R2 ≥
0.99), and the immunoreactive fraction was calculated as 100% ×
(1/b).

2.12
Internalization Assay
HCC1806 cells
(2 × 104 cells per well) were seeded in a 12-well
plate and cultured overnight. Subsequently, 0.2 μCi of [177Lu]­Lu-DOTA-11E10C11 was added to each well and incubated
for 0, 4, 24, and 48 h. At each time point, the culture supernatant
and PBS wash were collected as extracellular fractions. Surface-bound
components were collected after 10 min of treatment with 0.1 M glycine-HCl
(pH 2.7), while the internalized fractions were obtained by lysis
with 1% SDS for 10 min. The radioactive activity of each fraction
was measured using a gamma counter (decay-corrected). The internalization
rate was calculated as [internalized fraction/(surface + internalized
fraction)] × 100%. The experiment was performed in triplicate.

2.13
Positron Emission Tomography/Computed Tomography
(PET/CT) Imaging
Tumor-bearing BALB/c-nu mice were intravenously
injected with 3.7 MBq of [89Zr]­Zr-DOTA-11E10C11 (100 μL).
PET/CT imaging was performed at seven time points postinjection (4,
24, 48, 72, 96, 144, and 192 h) under 2.5% isoflurane anesthesia.
Quantitative analysis was conducted using AMIDE software: standardized
uptake values (SUV) were automatically calculated based on decay-corrected
administered activity and body weight. The SUVmean for each organ
and tissue was determined using spherical regions of interest (ROIs)
with diameters of 2–3 mm, and time-activity curves were generated
to analyze the dynamic distribution characteristics of the tracer.

2.14
Biodistribution
Tumor-bearing BALB/c-nu
mice were intravenously injected with 0.74 MBq of [177Lu]­Lu-DOTA-11E10C11
(100 μL). Mice were sacrificed at seven time points postinjection
(4, 24, 48, 72, 96, 144, and 192 h) for tissue collection. After weighing
the major organ tissues, radioactive activity was measured using a
gamma counter. The data were decay-corrected based on the physical
half-life of 177Lu (6.65 days), and the in vivo distribution
of [177Lu]­Lu-DOTA-11E10C11 was quantitatively analyzed
as the percentage of injected dose per gram of tissue (%ID/g).

2.15
Radionuclide Therapy
HCC1806 tumor-bearing
mice (tumor volume 50 ± 10 mm3) were randomly assigned
to three groups (n = 5 per group): experimental group (3.7 MBq [177Lu]­Lu-DOTA-11E10C11), antibody control group (100 μg
11E10C11), and negative control group (equivalent volume of physiological
saline), all of which were administered via tail vein injection (100
μL). After 30 days of treatment, the mice were sacrificed, and
blood samples were collected for complete blood count analysis. Tumors
and major organs were fixed in 4% paraformaldehyde, followed by H&E
staining and tumor tissue immunofluorescence analysis to systematically
assess the therapeutic efficacy and safety.

2.16
Growth and Survival Analysis
During
the 30-day treatment period, mouse body weight was monitored every
3 days using an electronic balance, and tumor dimensions (long axis,
L; short axis, W) were measured with calipers. Tumor volume was calculated
using the formula (W2 × L × 0.5). Mice were observed daily for signs of distress,
and euthanasia was performed if any of the following criteria were
met: tumor diameter ≥ 2.0 cm, weight loss >20%, or signs
of
severe distress (including anorexia, reduced activity, or ulceration). Survival status of mice was monitored through
day 55, and survival curves were generated to assess therapeutic efficacy.

2.17
Hematoxylin and Eosin (H&E) Staining
Tissue samples were fixed in 4% paraformaldehyde (PFA, pH 7.4)
for 24 h, followed by dehydration through a graded ethanol series
and clarification with xylene. The samples were then embedded in paraffin
and sectioned at a thickness of 4 μm. After deparaffinization
and rehydration, the sections were stained with hematoxylin and eosin,
followed by differentiation with hydrochloric acid ethanol and bluing
with running water, before being dehydrated and mounted. Whole-slide
images of stained tissues were acquired using a Nanozoomer S360 digital
scanner (Hamamatsu Photonics). Microscopic examination revealed typical
staining characteristics: cell nuclei appeared blue-purple, while
the cytoplasm and stromal components displayed a red gradient.

2.18
Immunofluorescence
Tissue sections
were fixed in 4% PFA (pH 7.4) and permeabilized with 0.1% Triton X-100
to enhance antibody penetration. Nonspecific binding sites were blocked
with 3–5% bovine serum albumin (BSA). The sections were incubated
overnight at 4 °C with primary antibodies (53BP1, γH2AX,
BRCA1/2, all purchased from HUABIO). After washing with PBS, the sections
were incubated with fluorescent secondary antibodies at room temperature
for 1 h. The nuclei were counterstained with DAPI and mounted. Following
image acquisition by fluorescence microscopy, the fluorescence results
were scanned using a 3DHistech Pannoramic MIDI digital slide scanner,
and fluorescence intensity was quantified using ImageJ software.

2.19
Statistical Analysis
All experimental
data were analyzed using GraphPad Prism 10.4.1 software. Results are
presented as mean ± standard deviation (Mean ± SD). Intergroup
comparisons were performed using unpaired two-tailed t test or one-way analysis of variance (ANOVA). Survival analysis
was conducted using the Kaplan–Meier method with Log-rank test.
A p-value of <0.05 was considered statistically significant.

3
Results
3.1
Expression of CDH3 in Triple-Negative Breast
Cancer
Flow cytometry analysis revealed significant differences
in CDH3 expression levels across the three human TNBC cell lines (Figure
A-C). MDA-MB-231
cells exhibited no CDH3 expression, BT-549 cells displayed moderate
expression, while HCC1806 cells showed markedly high CDH3 expression.
Flow cytometry results also demonstrated that the binding capacity
of the 11E10C11 antibody to target cells remained unchanged after
conjugation with DOTA (Figure
A).

3.2
Radiospecific Cell Binding and Internalization
Radiative cell binding assays of TNBC cell lines with high CDH3
expression (HCC1806) demonstrated a clear dose-dependent increase
in cellular uptake of [177Lu]­Lu-DOTA-11E10C11. Competitive
binding assays confirmed that preincubation with a 50-fold excess
of unlabeled 11E10C11 monoclonal antibody effectively inhibited radiolabeled
antibody uptake. The saturation binding of [177Lu]­Lu-DOTA-11E10C11
to HCC1806 cells yielded a Bmax of 534.9 ± 32.4 nmol and a dissociation
constant (K
d) of 8.49 ± 1.91 nmol/L
(Figure
E). For BT-549
cells, Bmax was 90.91 ± 9.6 nmol, and K
d was 9.85 ± 2.52 nmol/L (Figure D.1). The cell blockade assay results demonstrated that the radioactive
uptake of [177Lu]­Lu-DOTA-11E10C11 in the HCC1806 binding
group was 0.84% ± 0.03% of the added dose, while that in the
blockade group was 0.1% ± 0.05% (Figure
F). The immunoreactive fraction of 177Lu-DOTA-11E10C11 in HCC1806 cells was determined to be 82.3% (Figure
G). Internalization
studies in the CDH3-high expressing HCC1806 cells showed progressive
internalization of [177Lu]­Lu-DOTA-11E10C11 over time. The
internalization rates at 0, 4, 24, and 48 h were 0.54 ± 0.49%,
2.95 ± 1.33%, 6.13 ± 0.66%, and 8.52 ± 1.43%, respectively,
with statistically significant differences between time points (P < 0.05, Figure
D).

3.3
Synthesis and Radiolabeling of 89Zr/177Lu-11E10C11
We successfully synthesized
the radiolabeled complex of 11E10C11 by conjugating the DFO chelator
with 89Zr for PET imaging ([89Zr]­Zr-DFO-11E10C11),
and by conjugating the DOTA chelator with 177Lu for targeted
radionuclide therapy ([177Lu]­Lu-DOTA-11E10C11). The DOTA
to 11E10C11 ratios were approximately 7.69 (Figure A.1). HPLC analysis confirmed the radiochemical purity (RCP)
exceeding 99% for 11E10C11, 11E10C11-DOTA, and [177Lu]­Lu-DOTA-11E10C11
(Figure A.2–4). TLC analysis demonstrated
labeling yields >99% for both radiolabeled conjugates (Figure B.1–2). Stability tests indicated
that both radionuclide-labeled antibodies maintained over 99% labeling
efficiency after 8 days of incubation at room temperature in physiological
saline (NS), phosphate-buffered saline (PBS), and RPMI 1640 medium
containing 10% fetal bovine serum (Figure
A-B,Figure C.1–12), demonstrating excellent in vitro stability.

3.4
[89Zr]­Zr-DFO-11E10C11 PET/CT Imaging
PET/CT imaging of mice showed that 24 h postinjection, [89Zr]­Zr-DFO-11E10C11 preferentially accumulated at the tumor site.
At 48 h postinjection, clear tumor imaging was observed. Between 72
and 192 h postinjection, the radioactive uptake in the tumor steadily
increased and maintained high levels of radioactivity (Figure
C). From 4 to 192 h postinjection,
nontarget organs (heart, liver, spleen, lungs) exhibited decreasing
uptake over time, with [89Zr]­Zr-DFO-11E10C11 showing significantly
longer retention in the tumor compared to nontarget organs. Further
regional analysis and quantification of radioactivity in the tumor,
heart, liver, spleen, lungs, muscle, and bone were performed (Figure
D). The maximum tumor
uptake of [89Zr]­Zr-DFO-11E10C11 in tumor-bearing mice occurred
at 72 h, with a mean SUV of 2.11 ± 0.31. After 72 h, tumor uptake
gradually decreased over time, with a mean SUV of 1.4 ± 0.14
at 196 h. Radioactive uptake in nontarget organs, including the heart,
liver, spleen, and lungs, decreased over time, while muscle and bone
uptake initially slightly increased before gradually decreasing.

3.5
Biodistribution of [177Lu]­Lu-DOTA-11E10C11
The biodistribution study demonstrated significant tumor targeting
of [177Lu]­Lu-DOTA-11E10C11 in mice (Figure
E). Tumor uptake of [177Lu]­Lu-DOTA-11E10C11
showed a time-dependent increase, reaching a peak at 96 h (39.34 ±
5.9%ID/g). Pharmacokinetic analysis of blood clearance revealed a
peak blood uptake at 4 h (26.78 ± 1.77%ID/g), which gradually
decreased over time and was cleared from the bloodstream by 192 h
(blood uptake at 192 h: 2.1 ± 0.38%ID/g). Lung uptake peaked
at 4 h (9.19 ± 1.29%ID/g), which was higher than other nontarget
tissues, but gradually decreased over time, with a value of 1.42 ±
0.13%ID/g at 192 h. Between 4 and 192 h postinjection, the tumor-to-blood
uptake ratio ranged from 0.33 ± 0.11 to 4.24 ± 0.16%ID/g,
the tumor-to-lung ratio from 0.91 ± 0.38 to 6.32 ± 0.17%ID/g,
and the tumor-to-liver ratio from 0.90 ± 0.49 to 8.98 ±
2.20%ID/g (Figure
F–H).

3.6
Therapeutic Efficacy of [177Lu]­Lu-DOTA-anti-CDH3
At day 45-day postinjection, the tumor volume of the [177Lu]­Lu-DOTA-11E10C11 treatment group was 137.5 ± 5.41 mm3, significantly lower than that of the other groups (NS group:
596.2 ± 28.82; 11E10C11 group: 351.2 ± 20.83). The [177Lu]­Lu-DOTA-11E10C11 treatment group exhibited the strongest
tumor suppression, with a tumor growth inhibition (TGI) rate of 74.94%,
while the 11E10C11 monoclonal antibody group showed a modest tumor
suppression effect, with a TGI rate of 42.68%. Statistical analysis
revealed that tumor suppression in both the [177Lu]­Lu-DOTA-11E10C11
group and the 11E10C11 group was significantly superior to that in
the control group (P < 0.0001). Moreover, a significant
difference in tumor volume changes was observed between the [177Lu]­Lu-DOTA-11E10C11 group and the 11E10C11 group (P < 0.0001) (Figure
A,B). The [177Lu]­Lu-DOTA-11E10C11 treatment
group demonstrated 100% survival rate at 55 days, whereas decreased
survival rates were observed in both the normal saline (NS) control
group and the 11E10C11 monoclonal antibody group (Figure
C). All groups of tumor-bearing
mice showed a gradual increase in body weight, with no significant
intergroup differences (Figure
D).

3.7
Toxicity Assessment of [177Lu]­Lu-DOTA-anti-CDH3
After 30 days of treatment, hematological analysis of the NS, 11E10C11,
and [177Lu]­Lu-DOTA-11E10C11 groups showed that red blood
cell, white blood cell, hemoglobin, and platelet counts remained within
normal ranges, with no significant differences between the groups
(Figure
A). Hematoxylin
and eosin staining revealed no significant differences in cell morphology
and structure across the major organs of the NS, 11E10C11 monoclonal
antibody, and [177Lu]­Lu-DOTA-11E10C11 treatment groups,
with no notable pathological changes observed in any of the major
organs (Figure
B).
Immunofluorescence staining showed that the 53BP1 fluorescence intensity
in the [177Lu]­Lu-DOTA-11E10C11 group was higher than that
in the NS and 11E10C11 groups (P < 0.01; Figure
A). The γH2AX
fluorescence intensity in the [177Lu]­Lu-DOTA-11E10C11 and
11E10C11 groups was higher than that in the NS group (P < 0.0001; Figure
B). The BRCA1/2 fluorescence intensity in the 11E10C11 group was
higher than that in the [177Lu]­Lu-DOTA-11E10C11 group (P < 0.05; Figure
C). The differences among the groups were statistically significant.

4
Discussion
This study systematically
demonstrates, for the first time, the
therapeutic value of CDH3 as a specific molecular target for TNBC.
First, CDH3 exhibits characteristic overexpression in TNBC HCC1806
cells. Second, the diagnostic probe [89Zr]­Zr-DFO-11E10C11
enables precise detection of primary or metastatic tumors. Finally,
the therapeutic agent [177Lu]­Lu-DOTA-11E10C11 demonstrates
significant tumor suppression and favorable safety profiles. This
work represents a breakthrough in CDH3-targeted theranostics for TNBC,
providing a potentially translatable therapeutic strategy to improve
clinical outcomes.
Flow cytometry confirmed the specific overexpression
of CDH3 in
TNBC, consistent with prior reports[23][24]. A CDH3-high-expressing
HCC1806 xenograft mouse model was successfully established. Significant
differences in tumor volume progression were observed between treatment
groups, with the [177Lu]­Lu-DOTA-11E10C11 group demonstrating
marked tumor growth suppression, confirming its potent antitumor efficacy.
The antitumor effect of the 11E10C11 antibody group aligned with antibody-dependent
cellular cytotoxicity (ADCC) mechanisms, indicating that 11E10C11 primarily exerts cytotoxic effects via
ADCC. Notably, this study is the first to systematically elucidate
the differential therapeutic outcomes of CDH3-targeted therapy across
treatment groups, validating CDH3 as a feasible therapeutic target
for TNBC.
We successfully developed a CDH3-targeted PET/CT imaging
probe,
[89Zr]­Zr-DFO-11E10C11, which clearly visualized primary
and metastatic tumors at 72 h postinjection while demonstrating time-dependent
accumulation characteristics, enabling noninvasive assessment of CDH3
expression. This study provides a novel molecular imaging tool with
potential clinical translation value for early diagnosis and treatment
monitoring of TNBC. However, it should be noted that [89Zr]­Zr-DFO-11E10C11 showed nonspecific accumulation in the liver and
lungs, which may be associated with circulating shed CDH3 affecting
antibody pharmacokinetics, suggesting
the need for further optimization of its pharmacokinetic properties.
We systematically evaluated the in vivo safety profile of [177Lu]­Lu-DOTA-11E10C11. Pharmacokinetic analysis revealed rapid
blood clearance, with a 92.18% clearance rate between 4 and 192 h,
significantly reducing the risk of hematologic damage and bone marrow
suppression. Hematological analysis confirmed the absence of hematotoxicity
associated with [177Lu]­Lu-DOTA-11E10C11. We hypothesize
that circulating 177Lu and [177Lu]­Lu-DOTA-11E10C11
have minimal stimulatory effects on white blood cells and weak toxicity
toward blood cells, without inducing cellular damage. Pathological
examination of major organs, including the liver and lungs, showed
no evidence of radiotoxicity.
By further investigating the antitumor
mechanism of [177Lu]­Lu-DOTA-11E10C11, we found that, compared
with the NS group and
the 11E10C11 monoclonal antibody group, γH2AX and 53BP1 signals
were significantly increased in tumor tissues after [177Lu]­Lu-DOTA-11E10C11 treatment, indicating that radionuclide therapy
effectively induced extensive DNA double-strand breaks and activated
the DNA damage response. meanwhile, BRCA1/2 fluorescence signals were
markedly decreased in the [177Lu]­Lu-DOTA-11E10C11 group,
suggesting suppression of homologous recombination repair. collectively,
the immunofluorescence results of 53BP1, γH2AX, and BRCA1/2
indicate that the radionuclide-conjugated antibody induces severe
local DNA damage in tumors while disrupting high-fidelity DNA repair
processes, thereby leading to repair imbalance, and this molecular
phenotype of high damage and low repair provides mechanistic evidence
for its pronounced antitumor efficacy and highlights its potential
advantages as a targeted radionuclide therapy strategy.
The
clinical implications of this study extend beyond TNBC treatment.
Given the characteristic overexpression of CDH3 in various malignancies
including pancreatic, gastric, and colorectal cancers, the [177Lu]­Lu-DOTA-11E10C11 therapeutic
strategy could potentially be extended to other CDH3-overexpressing
tumors. Although nonspecific accumulation of [177Lu]­Lu-DOTA-11E10C11
in normal tissues was observed, optimization of antibody structure
(e.g., molecular weight reduction, bispecific targeting design) may
significantly improve its pharmacokinetic profile. These findings
not only validate the feasibility of CDH3-targeted therapy but also
provide an innovative treatment option for advanced metastatic solid
tumors, demonstrating significant clinical translation potential.
This study has several limitations. The differences between mouse
models and human TNBC may affect clinical translatability, necessitating
further validation in humanized models. Additionally, the nonspecific
hepatic accumulation of the probe may reduce its diagnostic and therapeutic
efficacy, which could potentially be improved through antibody optimization
to enhance targeting specificity.

5
Conclusion
This study innovatively
demonstrated the potential value of CDH3
as an integrated theranostic target for TNBC. Both [89Zr]­Zr-DFO-11E10C11
(diagnostic) and [177Lu]­Lu-DOTA-11E10C11 (therapeutic)
exhibited excellent diagnostic specificity and remarkable tumor-suppressive
efficacy in TNBC mouse models, providing a novel approach with promising
translational potential for precision theranostics in TNBC.
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Supplementary Material