Targeting CAIX with [64Cu]XYIMSR-06 small molecular radio-tracer

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Targeting CAIX with [64Cu]XYIMSR-06 small molecular radio-tracer enables noninvasive PET imaging of malignant glioma in U87 MG tumor cell xenograft mice Xianteng Yang, Hua Zhu, Xing Yang, Nan Li, Haifeng Huang, Teli Liu, Xiaoyi Guo, Xiaoxia Xu, Lei Xia, Chaoyong Deng, Xiaobin Tian, and Zhi Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01210 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 26, 2019

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Molecular Pharmaceutics

1

Targeting CAIX with [64Cu]XYIMSR-06 small molecular radio-tracer enables noninvasive

2

PET imaging of malignant glioma in U87 MG tumor cell xenograft mice

3 4 5

Xianteng Yang*1,2, Hua Zhu*3, Xing Yang4, Nan Li3, Haifeng Huang1,2, Teli Liu3, Xiaoyi Guo3, Xiaoxia Xu3, Lei Xia3, Chaoyong Deng1, Xiaobin Tian5, Zhi Yang3.

6

1. Medical college of Guizhou University, Guiyang, Guizhou, 550025, China;

7

2. Department of Orthopaedics, People’s Hospital of Guizhou Province, Guiyang, Guizhou,

8

550002, China;

9

3. Key Laboratory of Carcinogenesis and Translational Research (Ministry of

10

Education/Beijing), Department of Nuclear Medicine, Peking University Cancer Hospital &

11

Institute, Beijing 100142, China;

12

4. Department of Nuclear Medicine, Peking University First Hospital, Beijing 100034, China;

13

5. Guizhou Medical University, Guiyang, Guizhou, 550004, China.

14

*These authors contributed equally to this work and are co-first authors.

15

Corresponding author: Chaoyong Deng, E-mail: [email protected]; Xiao-bin Tian, E-mail:

16

[email protected]; Zhi Yang, E-mail: [email protected]

17 18

ABSTRACT: Carbonic anhydrase IX (CAIX) plays an important role in glioma cell proliferation,

19

invasion, metastasis and resistance to radiotherapy and chemotherapy. An effective and noninvasive

20

PET molecular imaging agent targeting CAIX would help its diagnosis and treatment but is not

21

currently

22

[64Cu]XYIMSR-06, was reported to have significantly improved properties for targeting clear cell renal

available.

Recently,

a

low-molecular-weight

(LMW)

1

ACS Paragon Plus Environment

CAIX

targeting

agent,

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

cell carcinoma (ccRCC). We are encouraged to investigate the feasibility of adapting this agent for the

2

diagnosis and treatment of CAIX-overexpressing malignant glioma. In vitro cell uptake and binding

3

affinity assays were used to verify the binding capacity of [64Cu]XYIMSR-06 to U87 MG tumor cells

4

in which CAIX overexpression was confirmed. The U87 MG tumor-bearing mouse (in situ and

5

subcutaneous) model was built, and mice were injected with the radiotracer and/or coinjected with

6

acetazolamide (0.2 g/kg) as a blocking agent for noninvasive micro-PET imaging. Micro-PET imaging

7

was performed at 2, 4 and 8 h postinjection. ROI (region of interest)-based semiquantification was

8

performed in an orthotopic glioma tumor model. Biodistribution throughout each organ was

9

performed at 2, 4, 4 h block, 8 and 24 h postinjection. Hematoxylin and eosin (HE) staining and

10

immunofluorescence or immunohistochemistry (IF/IHC) staining were implemented postimaging to

11

assess the expression of CAIX in tumor organs. In vitro, [64Cu]XYIMSR-06 exhibits greater uptake

12

in glioma cells (high CAIX expression) than in HCT116 cells (low CAIX expression). The binding

13

affinity of [64Cu]XYIMSR-06 to U87 MG cell lines reaches up to 4.22 nM. Both orthotopic and

14

subcutaneous tumors were clearly visualized at 2-8 h postinjection. Biodistribution studies

15

demonstrated a maximum tumor uptake of 3.13% ID/g at 4 h postinjection, and the tumor to brain

16

ratio (T/Brain) was 6.51 at 8 h postinjection. The ROI-based T/Brain values were 7.03 and 5.46 at 2

17

h and 8 h postinjection, respectively. Histopathological analysis confirmed the overexpression of

18

CAIX in gliomas, and the area of CAIX-positive IF staining is extremely consistent with the

19

morphology on micro-PET imaging. In this study, [64Cu]XYIMSR-06 demonstrated specific

20

accumulation in CAIX-expressing U87 MG glioma tumors, indicating that the radiotracer has the

21

potential for noninvasively monitoring and guiding personalized treatment of malignant glioma and

22

other tumors overexpressing CAIX.

2


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1 2

Keywords: Carbonic anhydrase IX (CAIX), positron emission tomography imaging (PET), glioma, [64Cu]XYIMSR-06.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

3



1

Introduction

2

Glioma, especially glioblastoma multiforme (GBM), is one of the most frequent primary

3

malignancies of the central nervous system in adults and is the most aggressive subtype of brain tumor,

4

with poor prognosis 1. A combined treatment strategy including radical resection of the tumor,

5

followed by radiotherapy and chemotherapy, is considered optimal in GBM management 2. Over the

6

past few decades, despite major improvement in glioma treatment, the overall survival rate of GBM

7

patients has remained poor 3. GBM cannot be completely removed even through radical surgery

8

because of its invasive growth; almost all patients with GBM die from local recurrence near the

9

resection margin within 1-2 years after diagnosis 4, 5. Since the malignant behavior of GBM lesions can

10

be attributed to their intrinsic genetic abnormalities 6, it would be really helpful to detect and quantify

11

these specific tumor biomarkers noninvasively. Molecular imaging with positron emission tomography

12

(PET) can noninvasively measure the metabolism and molecular processes of tumors in vivo with high

13

sensitivity. PET plays a significant role in the diagnosis, prognosis, and treatment of gliomas and

14

predominantly detects gliomas by detecting their metabolic alterations. To develop new effective

15

treatment strategies for this deadly disease, tumor-specific biomarkers for noninvasive PET molecular

16

imaging have recently become the target of intense research.

17

Carbonic anhydrase IX (CAIX), a transmembrane enzyme, catalyzes the reversible reaction of

18

water and carbon dioxide to form bicarbonate and protons. It maintains acid-base balance inside and

19

outside the tumor cells. The overexpression of CAIX has been confirmed in many types of solid

20

malignancies, such as gliomas, ependymomas, and lung, ovary, breast, and renal cancers, among

21

others7, 8. It has been proven to play crucial roles in tumor cell proliferation, invasion, metastasis and

22

resistance to radiotherapy and chemotherapy

9-11.

Furthermore, CAIX expression is generally

4


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12, 13

considered to be associated with hypoxia in tumors

2

CAIX is an independent prognostic factor for poor outcomes

3

shown to enhance therapeutic efficacy in glioblastoma 17,

4

all normal tissues, with the exception of the gastrointestinal tract, gallbladder and pancreatic ducts 19, 20,

5

it could be considered an ideal specific biomarker on the surface of tumor cells for targeted therapy and

6

noninvasive glioma imaging

7

probes to screen patients for personalized treatment is of significant importance.

8 9

15-17, 21.

and is upregulated by HIF1α

14.

1

18.

15, 16,

For GBM,

and inhibition of CAIX has been

Since CAIX expression is nearly absent in

The development of effective CAIX-targeting PET molecular

Up to now, a number of radiotracers have been developed for CAIX imaging, including monoclonal antibodies (mAbs), low-molecular-weight (LMW) inhibitors and affibodies

22.

However,

10

limitations still exist for the current agents, especially LMW inhibitors, due to their low tumor uptake

11

and poor tumor-to-background ratio. The only LMW agent targeting CAIX that has entered clinical

12

trials for PET imaging, [18F]VM4-037, failed to achieve tumor imaging, including in a glioma model

13

23.

14

Recently, a series of dual-motif targeting LMW CAIX ligands ([111In]XYIMSR-01 25

and [64Cu]XYIMSR-06

25),

initially discovered by library screening

26,

24,

15

[18F]XYIMSR-04

16

reported and demonstrated excellent selectivity for CAIX in vitro and in vivo with the potential to

17

image both metastatic and localized clear cell renal cell carcinoma (ccRCC). Among these radiotracers,

18

[64Cu]XYIMSR-06 demonstrated a particularly clear image of the ccRCC tumor in a mouse model. The

19

improved molecular targeting property and the high CAIX expression in ccRCC both contributed to the

20

success of [64Cu]XYIMSR-06. Due to the common loss of the von Hippel-Lindau (VHL) tumor

21

suppressor gene, CAIX expression can be upregulated up to hundreds of times in ccRCC 27. However,

22

for other hypoxia-related tumors, the relatively low receptor expression somewhat limits the ligand

5


has been


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1

development progress with unsatisfactory maximum uptake and a lower tumor-to-background ratio in

2

gliomas

3

been investigated. Our study will evaluate its potential to guide the clinical diagnosis and treatment for

4

gliomas and other tumors that overexpress CAIX.

23.

We presume that [64Cu]XYIMSR-06 can help to solve these problems, but it has not yet

5 6 7

Materials and methods General materials and radio-synthesis of [64Cu]XYIMSR-06. XYIMSR-06 was synthesized 25.

8

and characterized as described earlier in the literature

Details about general materials and

9

radio-synthesis of [64Cu]XYIMSR-06 are described in Supporting Information.

10

Radiochemical stability. The stability of [64Cu]XYIMSR-06 in vitro was measured by

11

radio-HPLC or radio-TLC after incubation in PBS (pH = 7.0) and 5% human serum albumin (HSA) at

12

37.0°C for different times (2 h, 4 h, 8 h, 24 h and 48 h).

13

Cell lines and in vitro cell studies. The U87 MG and HCT116 cells were purchased from China

14

Infrastructure of Cell Line Resources and cultured in a humidified incubator at 37°C with 5% CO2 in

15

RPMI 1640 GlutaMAX™ media supplemented with 1% (v/v) penicillin−streptomycin and 10% (v/v)

16

heat-inactivated FBS. The expression of CAIX in U87 MG cells was measured using Western blot

17

(WB) analysis, and corresponding details are described in Supporting Information. The uptake value

18

and affinity of [64Cu]XYIMSR-06 in U87 MG cells were measured in cell uptake experiments. As a

19

negative control, HCT116 cells with lower CAIX expression were selected for simultaneous

20

measurement.

21

In vitro cell uptake. One day before the study, U87 MG and HCT116 cells were seeded in

22

24-well plates at an average of 1×105 cells per well and incubated overnight in 500 μL of RPMI 1640

6


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1

GlutaMAX™ medium. Prior to the study, the cells were washed three times with 1 mL of cold PBS,

2

after aspirating the culture solution in each well, the cells were reincubated with 500 μL of serum-free

3

medium for 2 hours at 37 °C. Then, 4 wells in every dish were divided into 1 group. To assess the

4

specific targeting ability of [64Cu]XYIMSR-06 for CAIX, two groups of the U87 MG cells (n = 4) were

5

incubated with 2 μg XYIMSR-06 for an additional 1 hour. A total of 37 kBq of [64Cu]XYIMSR-06 in

6

PBS of 20 μL was added to each well, and the cells were cultured at 37 °C with 5% CO2. After

7

coincubation with the radiotracer for 10, 30, 60 and 120 minutes, each group’s cell culture media was

8

sequentially aspirated, and the cells were rinsed three times with 1 mL of cold PBS and lysed with 500

9

μL of 0.5 M NaOH for 3 min. Cell lysates in each well were then collected and counted by a gamma

10

counter.

11

In vitro cell binding assay. The conﬂuent cells were separated and resuspended 24 hours before

12

the binding assay, and aliquots of 5x104 cells were transferred to each well in the 48-well plate. Then,

13

different concentrations of [64Cu]XYIMSR-06 (0.037, 0.185, 0.37, 1.85, 3.7, 18.5, 37, 185 KBq) in 20

14

μL PBS were added to each well and coincubated to test the specific binding ability of the radiotracer

15

to the cells. After 2 hours of incubation at 37℃ with 5% CO2, each well was washed three times with 1

16

mL of cold PBS, trypsinized with 0.5 M NaOH buffer for 3 min, and then the NaOH solution was

17

collected for analysis by gamma counter. Each procedure in the experiment was repeated four times.

18

Mouse models. All animal experiments were conducted in accordance with protocols approved by

19

the Peking University Cancer Hospital Animal Care and Use Committee. Six-week-old BALB/c nude

20

mice were obtained from Beijing Huafukang (HFK) Bioscience Co. Ltd. (Beijing, China) and were

21

subcutaneously injected into the lateral right flank with 1x106 U87 MG cells. Tumor growth was

22

monitored, and mice were subjected to PET imaging when the volume of the tumor increased to 300

7



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1

mm3. For orthotopic glioma, U87 MG cells in 3 μl PBS (1×105 cells) were injected into the right

2

cerebral hemisphere of the mouse according to previously reported methods

3

injection, the tumor-bearing mice implanted in situ were used for research.

28.

Eight days after the

4

Micro-PET imaging. Mice bearing U87 MG xenografts were injected with [64Cu]XYIMSR-06

5

(0.2 ml, 18.5 MBq) via the tail vein, placed in the prone position and fixed on a micro-PET scanner

6

after anesthesia with 3% isoflurane. As a comparison, acetazolamide, a general carbonic anhydrase

7

inhibitor, was used for intraperitoneal injection of three subcutaneous tumor-bearing mice at a dose of

8

0.2 g/kg 8 h before the injection of the radiotracer. Whole-body, 1-bed PET imaging was performed on

9

a Super Argus PET scanner (Sedecal, Spain) at the scheduled time points (2 h, 4 h, 8 h and 24 h)

10

postinjection of [64Cu]XYIMSR-06, and mice were maintained under 1.5% isoﬂurane. The parameters

11

for obtaining PET images were 80 mm diameter Transaxial FOV, 900–1200 s PET acquisition time,

12

and OSEM 3D reconstruction iterative algorithms with random and attenuation corrections.

13

Afterwards, in the PET images of orthotopic gliomas, ROIs were sketched to estimate the SUV

14

(standard uptake value) of the radiotracer in each organ.

15

Quantitative biodistribution studies. Twenty BALB/c mice carrying subcutaneous U87 MG

16

gliomas with an approximate diameter of 0.8 cm were randomly divided into five groups. To verify the

17

specificity of the [64Cu]XYIMSR-06 to target CAIX, one of the mouse groups was intraperitoneally

18

injected with a dose of acetazolamide (0.2 g/kg) 8 h in advance. Each mouse received an intravenous

19

injection of [64Cu]XYIMSR-06 (0.2 ml, 1.11 MBq) via the tail vein. At specific times postinjection (2

20

h, 4 h, 4 h block, 8 h and 24 h), tumor-bearing mice were sacrificed by cervical dislocation, and organ

21

or tissue samples, including blood, heart, lung, brain, liver, spleen, kidney, stomach, small intestine,

22

large intestine, bone, muscle and tumor, were collected, weighed, and measured for radioactivity with a

8


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1

γ-counter. The percentage of injected dose per gram (%ID/g) of each tissue was calculated by

2

comparison with one percent of the original standard dose. Mean ± standard deviation (SD) were used

3

to describe the data.

4

Pathological analysis. Hematoxylin and eosin (HE), immunohistochemistry (IHC) and

5

immunofluorescence (IF) staining were performed to confirm the expression and distribution

6

characteristics of CAIX in glioma tumor tissues. Details are described in the Supporting Information.

7

Statistical analysis. Quantitative data was presented as the mean ± SD and was analyzed by

8

one-way analysis of variance to determine significant differences between the two sets of data.

9

Statistical analysis was performed using SPSS 21.0 (IBM, Armonk, NY, USA). A value of P﹤0.05 was

10

considered statistically significant.

11 12

RESULTS

13

Radio-synthesis and quality control. The structures of XYIMSR-06 and [64Cu]XYIMSR-06 are

14

descriped in Figure 1A. [64Cu]XYIMSR-06 was successfully obtained by radiolabeling. The average

15

radiochemical yield was 87.06 ± 4.05% (n=5). After purification, the radio-chemical purification yield

16

was over 99%, and the specific activity was 21.3-35.1 GBq/μmol (n=5). As shown in Supporting

17

Information Figure S1, free

18

relevant quality control indicators are shown in Table 1.

19

64Cu

was not found in the final product by radio-HPLC. Clinically

Table 1. Production and quality control of [64Cu]XYIMSR-06. Parameter

QC Specification

QC Result

Appearance

Clear, colorless

Pass

Volume

1.0-2.0 mL

1.0-1.5 mL

9



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pH

5.0-8.0

7.40

Radio-TLC

> 95%

> 96%

Radio-HPLC

> 95%

> 99%

Ethanol

< 5%

0

Endotoxins

< 15 EU/mL

pass

Sterility

Sterile

Pass

Specific Activity

13.5-100 GBq/µmol

21.3-35.1 GBq/µmol

1 2

Stability of [64Cu]XYIMSR-06 in vitro. The in vitro stability of [64Cu]XYIMSR-06 in PBS and

3

5% HSA was evaluated by radio-HPLC and radio-TLC. After 24 hours of incubation in both solutions

4

at 37℃, no significant dissociation or decomposition of the product was observed. After another 24

5

hours of incubation, [64Cu]XYIMSR-06 in PBS decreased slightly from 99.6% at 24 hours to 97.5% at

6

48 hours (Supporting Information Figure S2). These results indicate that [64Cu]XYIMSR-06 has

7

reliable stability for further clinical application.

8

In vitro cell experiments. Using β-actin as an internal standard, the expression of CAIX in cells

9

was measured by Western blot. The relative expression level of CAIX in U87 MG cells is 1.32 ± 0.16

10

and in HCT116 cells is 0.28 ± 0.01 (t=11.60, p=0.01), which indicates the overexpression of CAIX in

11

U87 MG cells (Figure 1 B-C). The cellar uptake rate of [64Cu]XYIMSR-06 in U87 MG cells (‰ in 1×

12

105 cells) was significantly higher than that in HCT116 cells, and the difference was statistically

13

significant (3.73 ± 0.39 vs 0.45 ± 0.08, t =16.60, p = 0.00, at 1 h and 3.68 ± 0.27 vs 0.69 ± 0.15, t

14

=19.40, p = 0.00, at 2 h). After blocking with the precursor XYIMSR-06, the value of uptake was

15

significantly reduced at 1 and 2 h of coincubation (3.73 ± 0.39 vs 0.88 ± 0.14, t = 13.83, p = 0.00, at 1 h

10


Page 11 of 36

1

and 3.68 ± 0.27 vs 0.91 ± 0.21, t = 16.31, p = 0.00, at 2 h) (Figure 1 D). These results suggest that

2

[64Cu]XYIMSR-06 has a specific targeting ability for CAIX-overexpressing tumor cells. Based on the

3

cell binding affinity determination, the KD value reached 4.22 nM (Figure 1 E).

4

Relative expression of CAIX

c1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

5

D

6

***

U87 MG

HCT116

Tumor cell type

Cellular uptake rate (‰)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

U87 MG HCT116 U87 MG-Block

***

10min

*** ***

*** ***

***

30min

1h

2h

Time

11



1 2

Figure 1. (A) Synthetic scheme of [64Cu]XYIMSR-06. (B, C) Relative CAIX expression in U87

3

MG and HCT116 cancer cells. The expression of CAIX in U87 MG was significantly higher than that

4

in HCT116. (D) The radioactivity uptake of [64Cu]XYIMSR-06 in U87 MG and HCT116 cells (1×105).

5

The radioactivity uptake of [64Cu]XYIMSR-06 in U87 MG cells was significantly higher than that in

6

HCT116 cells and was drastically reduced after blocking, and the difference was statistically

7

significant. ***P < 0.001. (E) The affinity of [64Cu]XYIMSR-06 in U87 MG cells. (F) IHC staining of

8

U87 MG tumor. (F1) 0.4x magnification of the original micrographs. (F2) (40x magnification of the

9

original micrographs) shows that the overexpression of CAIX is mainly located on the surface of the

10 11

cell membrane.

Micro-PET imaging. To observe the accumulation of radiotracer in glioma tumors in vivo,

12

we performed micro-PET imaging with

64Cu-labeled

XYIMSR-06. Representative, decay-corrected

13

PET images obtained at different time points (2 h, 4 h, 8 h and 24 h) after injection of the tracer in U87

14

MG tumor-bearing mice are shown in Figures 2-3. At 2 h, the tumor can be clearly seen with additional

15

background signal in the lungs, gastrointestinal tract, kidneys and urine. With the quick clearance of

16

the probe from the nontumor organs, relatively selective tumor images becomes quite clear at 4 h, and

17

good contrast images in tumors were obtained at 8 h (Figure 2 A, Figure 3 A-B). However, due to the

18

decay and excretion of the nuclide, legible PET images could not be acquired at 24 h. In the PET

12


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1

imaging of orthotopic gliomas, the SUV values were 0.24 ± 0.03 and 0.21 ± 0.01 at 2 and 8 h (Figure 3

2

C), and the tumor to brain and tumor to muscle ratios were, respectively, 7.03 ± 0.80 and 5.71 ± 0.75 at

3

2 h and 5.46 ± 0.48 and 5.36 ± 0.27 at 8 h. Throughout the entire imaging period, the tumor uptake of

4

the radio-tracer is significantly higher than the uptake of the brain tissue (Figure 2 A, Figure 3 A-B),

5

which is indicative of the potential of [64Cu]XYIMSR-06 for clinical applications. After blocking with

6

a carbonic anhydrase inhibitor, acetazolamide, the PET image of the tumor becomes blurred (Figure 2

7

C). Comparing the ratio of tumor to background after blocking vs no blocking, tumor to muscle from

8

biodistribution (Bio-T/M), tumor to blood from biodistribution (Bio-T/Blood), tumor to brain from

9

biodistribution (Bio-T/Brain), tumor to muscle from the ROIs in PET imaging (ROI-T/M) and tumor to

10

brain from the ROIs in PET imaging (ROI-T/Brain), all ratios are drastically reduced after blocking,

11

and the differences were statistically significant (Figure 2 D).

12

Biodistribution studies. The biodistribution results of [64Cu]XYIMSR-06 in subcutaneous

13

U87 MG glioma tumors are described below (Figure 2 B), and the specific values from the

14

biodistribution are described in Supporting Information Table S1. At 2 h postinjection, the retention

15

of [64Cu]XYIMSR-06 in the blood was only 0.72% ID/g, suggesting rapid blood clearance that is

16

consistent with the characteristics of LMW probes. The radioactivity uptake value in the tumor reached

17

3.13 ± 0.2 and 3.09 ± 0.47% ID/g at 4 and 8 h after injection, respectively. After blocking with

18

acetazolamide, the uptake value in the tumor for the radioactive ligand is drastically reduced, to 0.70 ±

19

0.08% ID/g, at 4 h after injection. The uptake in the gastrointestinal tract overexpressing CAIX was

20

also significantly reduced and was accompanied by the decline in the uptake in other organs, such as

21

blood, lung, kidney, after blocking. At 8 h, the tumor to blood, tumor to brain and tumor to muscle

22

ratios were 9.33 ± 2.18, 6.51 ± 2.50 and 4.36 ± 1.25, respectively. Of all organs, the kidney had the

13



1

highest uptake rate, followed by the stomach, lungs or intestines, demonstrating that

2

[64Cu]XYIMSR-06 is mainly excreted from the urinary system. Within 24 h, slight radiotracer uptake

3

in the liver was observed, indicating that the probe is partially metabolized in the liver.

4

B

2h 4h 4h-Block 8h 24h

35 30 25 ID/g%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20 15 10 5

***

0

5

art liver leen lung ney one ach tine tine scle mor rain lood b om tes tes b he b tu kid sp mu st ll in e in m larg a s X Axis Title

6 7

Figure 2: Micro-PET images and biodistribution of tumors. (A) Micro-PET images of

8

[64Cu]XYIMSR-06 in U87 MG xenograft nude mice within the upper flank. Representative whole PET

9

images were obtained at 2, 4 and 8 h after intravenous injection of 18.5 MBq (500 μCi) of

14


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1

[64Cu]XYIMSR-06. White arrows indicate tumors. (B) Biodistribution histogram of [64Cu]XYIMSR-06

2

in vivo. Biodistribution values were measured in %ID/g in glioma tumor (U87 MG)-bearing BALB/c

3

nude mice at 2 h, 4 h, 4 h block, 8 h and 24 h after the intravenous application of [64Cu]XYIMSR-06.

4

After blocking with the carbonic anhydrase inhibitor acetazolamide, the tumor uptake value was

5

significantly reduced, and the difference was statistically significant. ***P < 0.001. (C): Systemic PET

6

images obtained at 4 h after injection of radiotracer; arrows indicate tumor. (C1) The tumor can be

7

clearly observed. (C2) After blocking with carbonic anhydrase inhibitor in advance, the image of the

8

tumor becomes blurred. (D) Comparison of the tumor to background tissue ratios after blocking with

9

acetazolamide. The ratios T/M, T/Blood and T/Brain from both biodistribution and ROIs were

10

significantly reduced, and the difference was statistically significant for all. **P < 0.01, ***P < 0.001.

11

12

15



C 1.0

2h 8h

0.8 SUV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.6 0.4 0.2 0.0 y ch ne ma kid sto

er liv

e tin es int

g lun

le art sc he mu

in bra

or tum

1 2

Figure 3: Micro-PET images of orthotopic U87 MG xenografts after injection of

3

[64Cu]XYIMSR-06. (A, B) Figure clearly shows the intracranial tumor of the mouse at 2 and 8 hours

4

after the injection, where the white arrows indicate the tumor. (C) shows the SUV value of organs in

5

orthotopic gliomas based on semiquantification of ROIs. (D) shows the appearance of an orthotopic

6

glioma and its immunohistochemical staining. (D1) The tumor can be seen in the right cerebral

7

hemisphere, with tumor indicated by the red circle. (D2) shows CAIX immunohistochemical staining

8

of orthotopic gliomas, and tumor tissue can be clearly distinguished, as indicated by the red circle. (D3)

9

shows no CAIX expression in normal brain tissue, and (D4) shows CAIX overexpression in the tumor

10

tissue (40x magnification of original micrographs).

11

Pathological analysis. Images of tumor HE staining and the distribution pattern of CAIX

12

antibody staining in U87 MG tumor is shown in Figure 4 and Figure 1F. (A) Significant necrotic

13

regions appear in the center of the tumor (A1), and the diffuse area of the tumor cells is located at the

14

edge of the tumor (A2). (B) The area of CAIX-positive IF staining is mainly distributed between the

15

necrotic and tumor cell diffusion areas, which is consistent with the uptake distribution at the center of

16

the coronal plane in the tumor PET imaging (C). This suggests that the [64Cu]XYIMSR-06 is

17

specifically targeting the region of CAIX expression. Comparing the IHC-stained images between

16


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1

CAIX and hypoxyprobe-1, the positive staining areas of the two images were extremely similar,

2

suggesting that CAIX is mainly expressed in the area of tumor hypoxia (Figure 4E VS 4F). On the

3

magnified microscopic image of IF staining and the IHC section, overexpression of CAIX was

4

observed to be mainly at the surface of the cell membrane (Figure 1F, Figure 4D and Figure 4F2).

5

However, the area of hypoxyprobe-positive staining is mainly distributed in the nucleus and cytoplasm

6

(Figure 4E2).

7

8

17



1 2

Figure 4: Image of tumor HE, IHC and IF staining. (A) shows tumor HE staining. (B) shows IF

3

staining, and (C) shows a PET image of the centralized region of the coronal plane of the tumor, with a

4

red square indicating the tumor. From the HE-stained images, areas of tumor necrosis and cell

5

proliferation can be distinguished (A), with (A1) showing the regions of necrosis and (A2) showing

6

regions of diffuse tumor cells. (B) On the IF image, the area of CAIX-positive staining is located

7

around the necrosis of tumor cells. (B, C) Through the center of the coronal plane of the tumor, the

8

morphology of the PET images and the distribution of CAIX IF staining are extremely similar. (D)

9

Microscopic distribution of CAIX antibodies in tumor tissues. (D1) DAPI staining image of the nucleus

10

in U87 MG tumor. (D2) CAIX IF staining of tumors. (D3) Merged images of DAPI and CAIX staining.

11

(E) IHC-staining image of hypoxyprobe-1 in U87 MG tumor. (F) IHC-staining image of CAIX in U87

12

MG tumor. It shows that the overexpression of CAIX is mainly located on the surface of the cell

13

membrane (Figure 4 D, F2).

14

F2: 40 x magnification of the original micrographs.

B, E1, F1: 0.4 x magnification of the original micrographs; A1-2, D, E2,

15 16

DISCUSSION

18


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1 2

In this study, we evaluated the potential clinical application of [64Cu]XYIMSR-06 as an LMW PET imaging probe for the noninvasive screening of CAIX expression in gliomas.

3

First, further study of a potential probe is generally considered worth while if the expression level

4

of the targeted receptors on the cancer cells is more than three times higher than in normal cells29.

5

CAIX has been confirmed to be overexpressed in gliomas but absent in normal brain tissue7, 8, and

6

CAIX has been confirmed to be upregulated by hypoxia-inducible factor 1α (HIF-1α), which is mainly

7

localized around the necrotic tissue of tumors30. Moreover, an ideal target receptor is usually expressed

8

on the surface of cancer cells rather than in the cytoplasm or in the nucleus. Since ligands that target

9

intracellular receptors and carry imaging contrast or cytotoxic payload must be nonspecifically

10

permeable or passively diffuse across plasma membranes, they will noticeably also enter healthy cells,

11

causing unnecessary damage to healthy cells29. We performed Western blot and immunohistochemical

12

analysis of U87 MG glioma cells and specimens using anti-CAIX antibodies. Overexpression of CAIX

13

in gliomas was observed from the results of both WB (the relative expression level of CAIX in U87

14

MG is 4.7 times higher than in HCT116) and IHC. In the IF-image staining of CAIX, high expression

15

of CAIX was primarily located around the necrotic area of tumor tissue. Comparing with the

16

IHC-stained images of hypoxyprobe-1, CAIX is mainly expressed in the area of tumor hypoxia rather

17

than the entire tumor cell. These findings suggested that CAIX is associated with tumor hypoxia. On

18

the microscopic images, CAIX was spotted to be expressed on the surface of the cancer cell membrane.

19

These direct findings were consistent with previous research results9-11,

20

an ideal, specific target in gliomas and indicating that molecular probes targeting CAIX for the

21

diagnosis and treatment of glioma deserve further development.

22

15,

demonstrating CAIX to be

Second, the application potential of a molecular imaging probe critically relies on the ability of the

19



1

ligands to strongly bind to receptors and selectively localize at the site of disease. An ideal imaging

2

probe should boast optimal affinity, specificity, suitable size and physical characteristics for its

3

application29.

4

In our research, the labeling and purification process of [64Cu]XYIMSR-06 took only 15 minutes,

5

and the average radiochemical yield reached 87%, suggesting a more convenient labeling synthesis

6

with higher yield. After purification, more than 99% of the radiochemical products were collected, with

7

a specific activity of 21.3-35.1 GBq/μmol. There was no free 64Cu in the final product. After 24 hours

8

of incubation, superior in vitro stability of [64Cu]XYIMSR-06 was observed in 5% HSA and PBS at

9

37.0℃. In addition, the radionuclide 64Cu-labeled XYIMSR-06 product has been validated in previous

10

studies25. These findings show that [64Cu]XYIMSR-06 can meet quality control standards for

11

long-range delivery or clinical applications.

12

In the cell uptake experiment, the uptake of [64Cu]XYIMSR-06 in U87 MG cells, which has

13

higher CAIX expression, was significantly higher than in HCT116 cells, which has lower CAIX

14

expression, and could be blocked by excessive XYIMSR-06. After blocking, the uptake drastically

15

decreased from 3.73 to 0.88 at 1 h and from 3.68 to 0.91 at 2 h. In the biodistribution study, the uptake

16

value of [64Cu]XYIMSR-06 in the tumor sharply decreased after blocking with a carbonic anhydrase

17

inhibitor, acetazolamide. At the same time, the uptake of the probe in the gastrointestinal tract, which

18

has higher expression of CAIX, was also significantly blocked and was accompanied by a decrease in

19

the uptake of blood, lung, kidney and other organs. It is suggested that acetazolamide mainly blocks

20

tissues with high expression of CAIX. The decrease in uptake in other organs may be due to the slight

21

diuretic effect of acetazolamide, which is consistent with the drug metabolism characteristics of

22

acetazolamide31. Subsequent micro-PET image studies have also shown that tumor images from

20


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1

blocked mice become blurred compared to unblocked images. These findings suggest that

2

[64Cu]XYIMSR-06 can specifically bind to CAIX in U87 MG glioma. The KD value of

3

[64Cu]XYIMSR-06 binding CAIX is 4.22 nM, indicating that [64Cu]XYIMSR-06 has a higher affinity

4

for CAIX.

5

Biodistribution studies were performed in nude mice bearing U87 MG gliomas, and results

6

demonstrated moderate tracer uptake in tumors. Radiotracer uptake within the tumor reached a

7

maximum of 3.13% ID/g at 4 h postinjection, then slowly cleared from the tumor, down to 0.81% ID/g

8

by 24 h after injection. Although the [64Cu]XYIMSR-06 uptake value in the tumor is 3.09% ID/g at 8 h,

9

the ratio of tumor to blood, tumor to brain and tumor to muscle is 9.33, 6.51 and 4.36, respectively,

10

indicating a better background ratio between tumor and normal brain region. At the same time,

11

[64Cu]XYIMSR-06 showed the highest uptake in the kidneys, indicating that it is mainly excreted

12

through the urinary system. This was followed by stomach, which has a higher uptake because CAIX

13

expression is relatively high in this organ under normal conditions8,

14

had a high radiotracer intake. The possible reason is that CAIX is overexpressed in the lungs of mouse

15

embryonic tissues

16

demonstrating the advantages of rapid excretion of low-molecular-weight agents relative to monoclonal

17

antibodies33,

18

management.

34,

32.

20, 22.

Subsequently, the lungs also

In addition, [64Cu]XYIMSR-06 was rapidly cleared from the blood,

which will reduce the exposure of patients to radiation exposure and facilitate hospital

19

In the micro-PET images, both in situ and subcutaneous tumors can be clearly observed at 2 h

20

after injection and a fairly good contrast image of the tumor was obtained at 8 h. After injection of

21

acetazolamide, a recognized carbonic anhydrase inhibitor, the image of the tumor becomes unclear.

22

Consistent with the biodistribution, significant uptake of radiotracer was observed in the kidneys,

21



1

stomach and lungs. After comparative analysis of the distribution characteristics of IF staining with the

2

imaging area of tumors, we found that the staining patterns are extraordinarily consistent, suggesting

3

that the radiotracer mainly accumulates in the regions of CAIX overexpression in tumor tissues.

4

Compared with previous studies

5

lower quality of imaging. In previous research, the expression of CAIX has been confirmed to be

6

upregulated by HIF1α 14, and VHL tumor suppressor gene inhibits HIF1α expression. In ccRCC, due to

7

the common loss of VHL, the expression of HIF1α is significantly increased, resulting in the

8

overexpression of CAIX

9

hypoxia-associated tumors. In this study, radiotracer uptake in gliomas is much less than in ccRCC 25,

10

resulting in worse images and significant differences in data. But T/Brain ratio in orthotopic glioma

11

PET imaging were 7.03 and 5.46 at 2 and 8 h, respectively, demonstrating that the probe has an

12

excellent signal-to-noise ratio in gliomas. [64Cu]XYIMSR-06 shows superior imaging performance in

13

glioma imaging compared with [(18)F]VM4-03723, which is a low-molecular-weight reagent approved

14

for clinical trials. These findings indicate the specific targeting ability of [64Cu]XYIMSR-06 for CAIX

15

and its potential to guide clinical applications.

27.

25,

[64Cu]XYIMSR-06 has lower uptake in gliomas and a slightly

Therefore, CAIX is much more highly expressed in ccRCC than other

16

In addition, the blood-brain barrier (BBB) is a focus of attention when developing imaging and

17

therapeutic drugs for in brain tumors. In the research of glioma models, tumor cells migrate, proliferate

18

and eventually wrap blood vessels along the cerebral vasculature35. The implanted tumor cells insert

19

between endothelial cells and perivascular astrocytes, interrupting cell-cell interactions and increasing

20

vascular permeability, leading to BBB rupture36,

21

patients, it was found that the blood volume of the tumor was relatively increased, and the contrast

22

agent was extravasated, which also indicated that BBB decomposition is a characteristic of GBM38.

37.

In the imaging examination results from GBM

22


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1

Previous studies have shown that IgG with a molecular weight of approximately 150 kDa can leak

2

from cerebral blood vessels into tumor tissues of orthotopic glioma39. In our research, the orthotopic

3

glioma transplanted into the mouse brain can be clearly observed by micro-PET after injection of the

4

radiotracer. Therefore, [64Cu]XYIMSR-06, a low-molecular-weight reagent with a molecular weight of

5

approximately 1.5 kDa, can reach the tumor tissue through the BBB partially destroyed by the tumor

6

cells. These findings suggest that [64Cu]XYIMSR-06 has potential for clinical applications.

7

In conclusion, high-purity [64Cu]XYIMSR-06 is achievable through easy synthesis and labeling. It

8

shows a higher affinity for specific binding to glioma cells and tumor tissues overexpressing CAIX.

9

The tumor can be clearly observed using a micro-PET device 2 to 8 hours after injection. Our research

10

indicates that [64Cu]XYIMSR-06 can be used to screen for gliomas overexpressing CAIX and other

11

tumors overexpressing carbonic anhydrase and has the potential for clinical application for guiding

12

personalized treatment.

13 14

Supporting Information

15

General materials, radio-synthesis of [64Cu]XYIMSR-06, in vitro cell studies, pathological

16

analysis, radio-synthesis and quality control, radiochemical stability and the detailed values of the

17

biodistribution are described in the supporting information.

18 19 20

Notes All authors declare no conflict of interest.

21 22

Acknowledgement

23



1

The current research was financially supported by Beijing Nova Program (Z171100001117020),

2

Beijing Excellent Talents Funding (2017000021223ZK33), Beijing Municipal Administration of

3

Hospitals-Yangfan Project (ZYLX201816). Beijing Natural Science Foundation, Jing-Jin-Ji special

4

projects for basic research cooperation (H2018206600), National Natural Science Foundation of China

5

(81571705, 81671733, 81871386, 81560356, and 81871387). Major State Research Development

6

Program of China (2016YFC0100402). The science and Technology Foundation of Guizhou (Grant

7

nos. [2015] 3044 and [2015] 2089). Science and Technology Foundation of Health and Family

8

Planning Commission of Guizhou Province (gzwjkj2018-1-040).

9 10 11

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Farin, A.; Suzuki, S. O.; Weiker, M.; Goldman, J. E.; Bruce, J. N.; Canoll, P. Transplanted

Zagzag, D.; Amirnovin, R.; Greco, M. A.; Yee, H.; Holash, J.; Wiegand, S. J.; Zabski, S.;

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1

837-849.

2

37.

3

Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells. Nat

4

Commun. 2014, 5, 4196.

5

38.

6

PET imaging for assessment of treatment response in patients with gliomas. Lancet Neurol. 2010, 9, (9),

7

906-920.

8

39.

9

I.; Matsumura, Y. Molecular imaging using an anti-human tissue factor monoclonal antibody in an

10

Watkins, S.; Robel, S.; Kimbrough, I. F.; Robert, S. M.; Ellis-Davies, G.; Sontheimer, H.

Dhermain, F. G.; Hau, P.; Lanfermann, H.; Jacobs, A. H.; van den Bent, M. J. Advanced MRI and

Takashima, H.; Tsuji, A. B.; Saga, T.; Yasunaga, M.; Koga, Y.; Kuroda, J. I.; Yano, S.; Kuratsu, J.

orthotopic glioma xenograft model. Sci Rep. 2017, 7, (1), 12341.

11 12

Table 1. Production and quality control of [64Cu]XYIMSR-06. Parameter

QC Specification

QC Result

Appearance

Clear, colorless

Pass

Volume

1.0-2.0 mL

1.0-1.5 mL

pH

5.0-8.0

7.40

Radio-TLC

> 95%

> 96%

Radio-HPLC

> 95%

> 99%

Ethanol

< 5%

0

Endotoxins

< 15 EU/mL

pass

Sterility

Sterile

Pass

Specific Activity

13.5-100 GBq/µmol

21.3-35.1 GBq/µmol

29



1 2

3

Relative expression of CAIX

c1.6 1.2 1.0 0.8 0.6 0.4 0.2 0.0

U87 MG

HCT116

Tumor cell type

D

5

***

1.4

4

Cellular uptake rate (‰)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

U87 MG HCT116 U87 MG-Block

***

10min

*** ***

*** ***

***

30min

1h

2h

Time

30


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1 2

Figure 1. (A) Synthetic scheme of [64Cu]XYIMSR-06. (B, C) Relative CAIX expression in U87

3

MG and HCT116 cancer cells. The expression of CAIX in U87 MG was significantly higher than that

4

in HCT116. (D) The radioactivity uptake of [64Cu]XYIMSR-06 in U87 MG and HCT116 cells (1×105).

5

The radioactivity uptake of [64Cu]XYIMSR-06 in U87 MG cells was significantly higher than that in

6

HCT116 cells and was drastically reduced after blocking, and the difference was statistically

7

significant. ***P < 0.001. (E) The affinity of [64Cu]XYIMSR-06 in U87 MG cells. (F) IHC staining of

8

U87 MG tumor. (F1) 0.4x magnification of the original micrographs. (F2) (40x magnification of the

9

original micrographs) shows that the overexpression of CAIX is mainly located on the surface of the

10

cell membrane.

11

12

31



B

2h 4h 4h-Block 8h 24h

35 30 25 ID/g%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20 15 10 5

***

0

1

art liver leen lung ney one ach tine tine scle mor rain lood b om tes tes b he b tu kid sp mu st ll in e in g m r la sa X Axis Title

2 3

Figure 2: Micro-PET images and biodistribution of tumors. (A) Micro-PET images of

4

[64Cu]XYIMSR-06 in U87 MG xenograft nude mice within the upper flank. Representative whole PET

5

images were obtained at 2, 4 and 8 h after intravenous injection of 18.5 MBq (500 μCi) of

6

[64Cu]XYIMSR-06. White arrows indicate tumors. (B) Biodistribution histogram of [64Cu]XYIMSR-06

7

in vivo. Biodistribution values were measured in %ID/g in glioma tumor (U87 MG)-bearing BALB/c

8

nude mice at 2 h, 4 h, 4 h block, 8 h and 24 h after the intravenous application of [64Cu]XYIMSR-06.

9

After blocking with the carbonic anhydrase inhibitor acetazolamide, the tumor uptake value was

10

significantly reduced, and the difference was statistically significant. ***P < 0.001. (C): Systemic PET

11

images obtained at 4 h after injection of radiotracer; arrows indicate tumor. (C1) The tumor can be

12

clearly observed. (C2) After blocking with carbonic anhydrase inhibitor in advance, the image of the

32


Page 32 of 36

Page 33 of 36

1

tumor becomes blurred. (D) Comparison of the tumor to background tissue ratios after blocking with

2

acetazolamide. The ratios T/M, T/Blood and T/Brain from both biodistribution and ROIs were

3

significantly reduced, and the difference was statistically significant for all. **P < 0.01, ***P < 0.001.

4

5

6

C 1.0

2h 8h

0.8 SUV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.6 0.4 0.2 0.0 y ch ne ma kid sto

er liv

e tin es int

g lun

le art sc he mu

in bra

or tum

7

33



1

Figure 3: Micro-PET images of orthotopic U87 MG xenografts after injection of

2

[64Cu]XYIMSR-06. (A, B) Figure clearly shows the intracranial tumor of the mouse at 2 and 8 hours

3

after the injection, where the white arrows indicate the tumor. (C) shows the SUV value of organs in

4

orthotopic gliomas based on semiquantification of ROIs. (D) shows the appearance of an orthotopic

5

glioma and its immunohistochemical staining. (D1) The tumor can be seen in the right cerebral

6

hemisphere, with tumor indicated by the red circle. (D2) shows CAIX immunohistochemical staining

7

of orthotopic gliomas, and tumor tissue can be clearly distinguished, as indicated by the red circle. (D3)

8

shows no CAIX expression in normal brain tissue, and (D4) shows CAIX overexpression in the tumor

9

tissue (40x magnification of original micrographs).

10

11

12

34


Page 34 of 36

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1 2

Figure 4: Image of tumor HE, IHC and IF staining. (A) shows tumor HE staining. (B) shows IF

3

staining, and (C) shows a PET image of the centralized region of the coronal plane of the tumor, with a

4

red square indicating the tumor. From the HE-stained images, areas of tumor necrosis and cell

5

proliferation can be distinguished (A), with (A1) showing the regions of necrosis and (A2) showing

6

regions of diffuse tumor cells. (B) On the IF image, the area of CAIX-positive staining is located

7

around the necrosis of tumor cells. (B, C) Through the center of the coronal plane of the tumor, the

8

morphology of the PET images and the distribution of CAIX IF staining are extremely similar. (D)

9

Microscopic distribution of CAIX antibodies in tumor tissues. (D1) DAPI staining image of the nucleus

10

in U87 MG tumor. (D2) CAIX IF staining of tumors. (D3) Merged images of DAPI and CAIX staining.

11

(E) IHC-staining image of hypoxyprobe-1 in U87 MG tumor. (F) IHC-staining image of CAIX in U87

12

MG tumor. It shows that the overexpression of CAIX is mainly located on the surface of the cell

13

membrane (Figure 4 D, F2).

14

F2: 40 x magnification of the original micrographs.

B, E1, F1: 0.4 x magnification of the original micrographs; A1-2, D, E2,

35



Graphic abstract

Headline: A small molecule probe targeting CAIX, [64Cu]XYIMSR-06, was successfully evaluated for its potential as a PET imaging agent for stratified diagnosis and personalized treatment in glioma.


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Targeting CAIX with [64Cu]XYIMSR-06 small molecular radio-tracer

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