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. 2024 Jul 9;35(7):1064–1074. doi: 10.1021/acs.bioconjchem.4c00266

AUNP-12 Near-Infrared Fluorescence Probes across NIR-I to NIR-II Enable In Vivo Detection of PD-1/PD-L1 Axis in the Tumor Microenvironment

Xinyu Zhang †, Ping Wang ‡, Guangyuan Shi §, Chu Tang ∥,*, Huadan Xue †,*
PMCID: PMC11261610  PMID: 38980173

Abstract

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The innovative PD-1/PD-L1 pathway strategy is gaining significant traction in cancer therapeutics. However, fluctuating response rates of 20–40% to PD-1/PD-L1 inhibitors, coupled with the risk of hyperprogression after immunotherapy, underscore the need for accurate patient selection and the identification of more beneficiaries. Molecular imaging, specifically near-infrared (NIR) fluorescence imaging, is a valuable alternative for real-time, noninvasive visualization of dynamic PD-L1 expression in vivo. This research introduces AUNP-12, a novel PD-L1-targeting peptide antagonist conjugated with Cy5.5 and CH1055 for first (NIR-I) and second near-infrared (NIR-II) imaging. These probes have proven to be effective in mapping PD-L1 expression across various mouse tumor models, offering insights into tumor-immune interactions. This study highlights the potential of AUNP-12-Cy5.5 and AUNP-12-CH1055 for guiding clinical immunotherapy through precise patient stratification and dynamic monitoring, supporting the shift toward molecular imaging for personalized cancer care.

Introduction

The pursuit of effective cancer treatments has led to the innovative strategy of focusing on the PD-1/PD-L1 pathway within the tumor microenvironment.1−3 By disrupting the capability of cancer cells to avoid the immune system, this strategy achieves long-term therapeutic benefits.4 It is at the forefront of oncological research, representing a significant leap in cancer treatment.5 Nevertheless, this promising avenue has limitations. Approximately 20–40% of solid tumor patients who have not been screened show a positive response to PD-1/PD-L1 inhibitors.6−8 Although hopeful, this rate underscores the difficulty in managing patients with variable responses to these therapies.9,10 Additionally, some cases exhibit hyperprogression, where tumors grow faster after immunotherapy.11 These challenges necessitate refining patient selection. Effective patient screening and expanding the beneficiary population are crucial challenges. Consequently, there is emerging evidence that the level of PD-L1 expression is a crucial indicator. The response to PD-1/PD-L1 inhibition treatment can be effectively predicted by its presence.12,13 Recognizing its utility, FDA authorized the PD-L1 expression in tumors as a criterion to select therapies.14−16 The main method for testing PD-L1 expression in clinical settings is through biopsy and immunohistochemistry.17,18 However, this method faces challenges including limited sensitivity,19 insufficient specificity,20 the invasive nature of tissue sampling,21 and the incapacity to encompass the spatial and temporal diversity of PD-L1 levels of tumors.22,23 These limitations lead to inconsistent results, affecting patient selection and treatment efficacy. Given these challenges, innovating in PD-1/PD-L1 expression monitoring is imperative.24 We aim to develop a noninvasive, sensitive, and specific technique to examine PD-1/PD-L1 levels in tumors. This method would revolutionize cancer therapy management by enabling real-time PD-L1 monitoring and treatment adjustment. Enabling the dynamic and precise utilization of PD-1/PD-L1 inhibitors can greatly improve therapeutic outcomes, making personalized cancer treatment more attainable.

In this context, molecular imaging,25−27 particularly near-infrared (NIR) fluorescence imaging,28−31 has emerged as a superior alternative. This innovative approach offers noninvasive, real-time visualization of PD-L1 dynamics across entire tumor landscapes.32−34 NIR imaging, especially the second (NIR-II) window, allows for greater tissue penetration and less autofluorescence, resulting in improved detection of PD-L1 expression with increased accuracy and sensitivity.32 NIR fluorescence imaging advances have demonstrated the potential of molecular imaging to overcome conventional PD-L1 detection limitations, facilitating improved patient selection and personalized therapy management.35,36 Zhong et al. created a targeted NIR-IIb probe for PD-L1 detection by conjugating PD-L1 antibodies, enabling real-time, noninvasive tracking of PD-L1 levels and the abundance of CD3+CD8+ T cells within the tumor microenvironment in mice with colorectal cancer.33 This approach facilitates the assessment of immunotherapy response rates, providing a theoretical foundation for personalization and optimization of tumor immunotherapy. However, while this antibody-based molecular imaging approach provides valuable insights, it faces limitations such as slower clearance rates, less effective tissue penetration, and the potential for immunogenicity-related adverse events (irAEs). These challenges underscore the necessity for the development of alternative imaging strategies such as smaller peptides, aptamers, or engineered antibody fragments, which may offer improved tissue penetration, reduced immunogenicity, and faster clearance from the body.

The peptide antagonist AUNP-12 represents a pioneering advancement as the inaugural competitive antagonist targeting PD-L1, meticulously engineered to exhibit high affinity by leveraging the exogenous domain of human PD-1.37 Research to date has illuminated AUNP-12′s remarkable efficacy in immune activation and its potent antitumor capabilities, alongside a reduced incidence of irAEs.38,39 Notably, AUNP-12 demonstrates an enhanced ability to bind to PD-L1, ensuring preferential uptake by cells exhibiting elevated PD-L1 expression levels.37 This specificity not only underscores AUNP-12′s potential in precision oncology but also paves the way for the development of novel probes.40

Accordingly, our team developed AUNP-12 molecular imaging probes for NIR-I and NIR-II imaging. We assessed these probes for PD-L1 monitoring in mouse tumor models expressing different PD-L1 levels and immune responses. Preliminary results indicated that the AUNP-12 fluorescence imaging probes distinctly mapped the fluctuating levels of PD-L1 expression, providing important information about the temporal and spatial dynamics of tumor-immune interactions. Notably, NIR-II imaging has proven particularly effective in delivering clearer and more accurate visualizations of the PD-L1 distribution within deep tissue environments.

The significance of this work lies in developing a method for selecting patients with immune responses. By offering a deep understanding of PD-L1 expression patterns in vivo, our AUNP-12 imaging probes can dynamically monitor the expressing levels of PD-L1 and facilitate the real-time evaluation of immunotherapy responses. Furthermore, this research offers a hopeful tool for the stratification of patients who can get more benefits from therapies contingent upon the blockade of PD-1/PD-L1. This advancement results in the further development of individualized treatment plans, optimizing therapeutic effectiveness while minimizing the potential for adverse effects. Ultimately, the development and application of AUNP-12 molecular imaging probes stand for significantly benefiting clinical outcomes in cancer immunotherapy, marking a pivotal step toward the realization of truly personalized oncological care.

Results and Discussion

Synthesis and Characterization of AUNP-12-Cy5.5 and AUNP-12-CH1055

We designed and synthesized the NIR-I probe, AUNP-12-Cy5.5, and the NIR-II probe, AUNP-12-CH1055. The absorbance spectra and fluorescence intensities of AUNP-12-Cy5.5 and AUNP-12-CH1055 were studied. The absorption spectrum peak of AUNP-12-Cy5.5 appeared at 680 nm, and its fluorescence emission spectrum revealed a peak at 698 nm (Figure 1A). For AUNP-12-CH1055, the peaks of its absorption and emission were observed at 748 and 1000 nm (Figure 1B). These results demonstrated their potential as fluorescence imaging probes. Additionally, dispersion stability, critical for the in vivo simulation, was assessed. AUNP-12-Cy5.5 was well dispersed in ordinary salt solution and serum-containing Dulbecco’s modified Eagle medium (DMEM) without any precipitation and exhibited a ζ-potential of about −1 mV (Figure 1C). The fluorescence intensity of AUNP-12-Cy5.5 was positively correlated with the probe concentration (Figure 1D). Furthermore, the fluorescence intensity of AUNP-12-CH1055 reached its peak when a 1000 nm LP filter was employed, as depicted in Figure 1E. Additionally, the strength of this effect grew steadily as the levels of the probes increased, as demonstrated in Figure 1F. We also assessed the fluorescence intensity of our probes after incubation in mouse serum for various durations to simulate in vivo conditions and evaluate the stability of the fluorescent molecular probes in vivo (Figure S1). These findings suggest the potential utility of these molecular probes in future imaging studies.

Figure 1.

Figure 1

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Characterization of AUNP-12-Cy5.5 and AUNP-12-CH1055. (A, B) Absorbance spectra and fluorescence intensities of AUNP-12-Cy5.5 and AUNP-12-CH1055. (C) ζ-Potential of AUNP-12-Cy5.5 in different solvents. (D) NIR-I fluorescence intensities of AUNP-12-Cy5.5 with different concentrations. (E) Fluorescence intensities under different long-pass filters of AUNP-12-CH1055. (F) NIR-II fluorescence intensity of AUNP-12-CH1055 at different concentrations.

Expression Characteristics of PD-L1 and Targeting of AUNP-12-Cy5.5 In Vitro

To demonstrate that AUNP-12-Cy5.5 is a specific molecular imaging probe capable of monitoring PD-L1 levels in tumors, we initially verified PD-L1 expression in several tumor cells, including 4T1, CT26, B16F10, MDA-MB-231, and MCF-7. These results showed that CT26, B16F10, and MDA-MB-231 cell lines exhibited high levels of PD-L1 expression, whereas 4T1 and MCF-7 cell lines showed comparatively lower expression of PD-L1 (Figure 2A,B). We further validated the targeted ability of AUNP-12-Cy5.5 against CT26 and B16F10 in vitro. Among them, the targeting binding rate of CT26 or B16F10 was the highest in the AUNP-12-Cy5.5 group. After that, the AUNP-12 block group, aPD-L1 block group, and phosphate-buffered saline (PBS) group were ranked in order. This indicated that AUNP-12-Cy5.5 had high specificity in binding to tumor cells with high PD-L1 expression (Figures 2C and S2). To precisely identify the binding site of AUNP-12-Cy5.5 on the cell, we performed additional cellular-level experiments. Through immunofluorescence staining and confocal fluorescence microscopy, we observed that AUNP-12-Cy5.5 primarily binds to PD-L1 on the cell membrane surface (Figure S3). Peptide probes have shown superior penetration into tumor tissues compared with antibody probes, which was confirmed through three-dimensional (3D) tumor spheroid experiments. After coincubating with CT26 cell-derived 3D tumor spheroids for 3 h, AUNP-12-Cy5.5 exhibited significantly stronger fluorescence signals within the tumor spheroids than aPD-L1-Cy5.5 (Figure S4).

Figure 2.

Figure 2

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Expression characteristics of PD-L1 and targeting of AUNP-12-Cy5.5 in vitro. (A, B) PD-L1 showed varying expression levels across several cancer cell lines: higher in B16F10, CT26, and MDA-MB-231 and lower in 4T1 and MCF-7. (C) Flow cytometry indicated that AUNP-12-Cy5.5 more effectively targeted CT26 cells (high PD-L1 expression) in the AUNP-12-Cy5.5 group.

Biological Distribution of AUNP-12-Cy5.5 In Vivo in Different Tumor Models

To verify the tumor targeting ability of AUNP-12-Cy5.5 in different tumor models, we constructed subcutaneous mouse tumor models of 4T1 and CT26, respectively. The results showed that in the 4T1 subcutaneous mouse tumor models with low levels of PD-L1, there was no notable discrepancy in the degree of probe targeting on the tumors between the aPD-L1 block group and AUNP-12 block group (Figures 3A–C and S5A). However, in the CT26 groups with high expression of PD-L1, the probe group had greater targeting at the tumor site (Figures 3D–F and S5B). The results indicated that AUNP-12-Cy5.5 could specifically image the PD-L1 expression in various tumors. After NIR-I imaging, the tumors were removed for tissue clearing according to the benzyl benzoate (BABB) protocol.41 Subsequently, we employed the advanced capabilities of three-dimensional light sheet fluorescence microscopy (3D LSFM) to further investigate tumor tissues ex vivo. The transition from living subjects to dissected tissues allowed us to maintain the integrity of the tumor environment while offering a more detailed view of PD-L1 distribution. We observed differential expression patterns of PD-L1, revealing significantly higher levels in CT26 tumors compared to 4T1 tumors within the 3D microenvironment of the tumor models. On the contrary, the aPD-L1 and AUNP-12 block groups showed weaker fluorescence signals. This distinction in PD-L1 expression between the two mouse tumor models suggested differences in immunological landscapes and immune response abilities. The high-resolution three-dimensional imaging provided by 3D LSFM allowed for precise quantification and spatial mapping of PD-L1 expression, offering improved comprehension of the tumor microenvironment (Figure 3G).

Figure 3.

Figure 3

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In vivo NIR-I imaging and biodistribution of AUNP-12-Cy5.5 for the subcutaneous mouse tumor models. (A) Fluorescence images of the 4T1 subcutaneous mouse tumor models in vivo at different time points (n = 3). (B) Ex vivo fluorescence images of the 4T1 subcutaneous mouse tumor models. (C) Quantitative analysis of TBR over 24 h. (D) Fluorescence images of the CT26 subcutaneous mouse tumor models in vivo at different time points (n = 3). (E) Ex vivo fluorescence images of the CT26 subcutaneous mouse tumor models. (F) Quantitative analysis of TBR over 24 h. (G) 3D LSFM images of tumors in the 4T1 and CT26 subcutaneous mouse tumor models. Scale bars: 200 μm. TBR: tumor-to-background ratio. The data were presented as mean ± standard error of the mean (SEM) *p < 0.05, **p < 0.01, ***p < 0.001.

AUNP-12-Cy5.5 Probe is Able to Monitor the Heterogeneous Expression of PD-L1 in Tumors

To determine if AUNP-12-Cy5.5 is capable of monitoring PD-L1 expression in various cancers, both breast cancer cell lines, MCF-7 (right) and MDA-MB-231 (left), with varying PD-L1 expression levels, were chosen and implanted into the mammary glands of both sides of mice (Figure 4A). This approach allowed monitoring of the heterogeneity of PD-L1 across distinct tumor regions within a single individual. These results showed that AUNP-12-Cy5.5 had a better targeting ability in MDA-MB-231 tumors with higher levels of PD-L1 expression, in contrast to MCF-7 tumors with lower levels of PD-L1 expression (Figure 4B–D). The same verification was obtained from ex vivo imaging results (Figures 4E and S5C).

Figure 4.

Figure 4

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In vivo NIR-I imaging and biodistribution of AUNP-12-Cy5.5 in the MDA-MB-231 tumor (left) and MCF-7 tumor (right). (A) In vivo fluorescence images of bilateral mammary gland mouse tumor models at different time points (n = 3). (B–D) Quantitative analysis of TBR in MDA-MB-231 and MCF-7 tumors over 24 h. (E) Ex vivo fluorescence images of bilateral mammary gland mouse tumor models. TBR: tumor-to-background ratio. The data were presented as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001.

AUNP-12-CH1055 Probe in the NIR-II Region Shows Higher Imaging Sensitivity and Can Detect Tumors with Low PD-L1 Expressing

The research revealed that AUNP-12-Cy5.5 successfully identified tumor tissues with high levels of PD-L1, yet exhibited limited sensitivity in detecting tumors expressing low levels of PD-L1. NIR-II offers superior imaging resolution, increased tissue penetration depth, and reduced tissue autofluorescence compared with NIR-I fluorescence imaging. Therefore, we designed fluorescence imaging probe AUNP-12-CH1055, which could be applied to NIR-II imaging. We verified the detection effect of the probe in the 4T1 mouse tumor model exhibiting low levels of PD-L1 expressing. As indicated by the findings, AUNP-12-CH1055 demonstrated superior tumor detection in cases of low PD-L1 levels when compared to AUNP-12-Cy5.5 (Figures 5A–C and S5D).

Figure 5.

Figure 5

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In vivo NIR-II imaging and biodistribution of AUNP-12-CH1055 in the 4T1 subcutaneous mouse tumor models. (A) Fluorescence images of the 4T1 subcutaneous mouse tumor models in vivo at different time points (n = 3). (B) Quantitative analysis of mean fluorescence intensity over 48 h. The fluorescence intensity of tumors in AUNP-12-CH1055 groups was higher than that in the two block groups after injection of AUNP-12-CH1055. (C) Ex vivo fluorescence images of tumors and major organs. The data were presented as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001.

Safety Evaluation of Probes

We evaluated the biosafety of imaging probes obtained 7 days post injection into the CT26 subcutaneous mouse tumor models, respectively. Among them, aspartate transaminase (AST) and alanine transaminase (ALT) are markers of liver function, while BUN is a marker of kidney function. The values of these parameters fell within the normal ranges (Figure 6A–C). The hematoxylin and eosin (H&E) staining of major organs such as heart, liver, spleen, lung, and kidney showed normal. All of these findings showed that the constructed imaging probes were safe in vivo (Figure 6D).

Figure 6.

Figure 6

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Assessment of the safety of two imaging probes. The mice were sacrificed after 7 days treatment, and serum and major organs were collected. Assessment of liver (A, B) and renal (C) functions in mice treated with PBS, AUNP-12-Cy5.5, or AUNP-12-CH1055. (D) H&E staining of major organs after treatment with PBS, AUNP-12-Cy5.5, or AUNP-12-CH1055. Scale bars: 50 μm.

In this research, we have successfully created two novel NIR fluorescence imaging probes, AUNP-12-Cy5.5 and AUNP-12-CH1055, specifically designed for application in the NIR-I and NIR-II windows, based on AUNP-12 polypeptides. These probes have been utilized for the preclinical in vivo molecular imaging of PD-L1 levels, including in the 4T1 and CT26 subcutaneous mouse tumor models as well as bilateral MCF-7 and MDA-MB-231 mouse tumor models. Our findings suggest these two imaging probes could specifically identify the in vivo dynamic expression of PD-L1, which was confirmed through immunohistochemistry. This approach marks a significant shift away from the traditional PD-L1 expression analysis methods, primarily reliant on static and invasive immunohistochemistry techniques, toward a dynamic, noninvasive, and real-time approach. This development is particularly crucial for screening patients who can get more benefits from immune checkpoint inhibitor (ICI) therapy and offering personalized treatment plans that align with the unique tumor characteristics and response patterns of individual patients.

Building on this foundation, we see that the application of molecular imaging, especially through our newly developed NIR fluorescence imaging probes, presents significant advantages in visualizing immune targets. Unlike traditional imaging probes, which often employ antibodies for targeting PD-L1, our approach uses peptides specifically engineered to include sequences from the extracellular PD-1-binding domain. These peptides, utilized in our AUNP-12-Cy5.5 and AUNP-12-CH1055 probes, offer distinct advantages over antibodies due to their smaller size, better tissue penetration, and faster clearance from nontargeting tissues. This results in a higher sensitivity and more specific imaging of PD-L1 biomarkers compared to antibody-based probes. Moreover, the use of peptides avoids the potential immunogenicity associated with antibodies, making repeated imaging sessions safer and more feasible for patients. This is particularly important for longitudinal studies and monitoring of the dynamic changes of PD-L1 in tumors over time.

Furthermore, because we chose both NIR-I and NIR-II for our probes, we significantly enhanced the quality of imaging. These NIR wavelengths were selected for their ability to penetrate deeper into tissues and have less autofluorescence compared to the visible spectrum, leading to substantial improvements in the signal-to-noise ratio of imaging.42 This critical enhancement enables a clearer, more precise visualization of PD-L1 distribution in tumors. The introduction of the NIR-II window, particularly with AUNP-12-CH1055 probe, offers higher sensitivity and the ability to detect proteins with low expression levels that are challenging to identify with NIR-I-based imaging. This attribute is particularly valuable as it enables the detection of deeper-seated tumors and provides a clearer and comprehensive view of the tumor, which enhances our understanding of tumor biology and immunotherapy efficacy.

In this work, we have validated the capability of AUNP-12-Cy5.5 to monitor PD-L1 expression in several mouse tumor models with various levels of PD-L1, from in vivo imaging to ex vivo 3D LSFM imaging. These validations not only demonstrate the probe’s effectiveness in accurately mapping PD-L1 distribution but also confirm its high safety. These results showed that compared to tumor tissues with low expressing levels of PD-L1, such as in the 4T1 subcutaneous mouse tumor models, AUNP-12-Cy5.5 probe had better imaging effect in the tumor tissues with high expressing levels of PD-L1, such as in the CT26 subcutaneous mouse tumor models, indicating that AUNP-12-Cy5.5 could be used in real-time, noninvasive, and quantitative monitoring of tumor tissues with high PD-L1 expression.

We further constructed the AUNP-12-CH1055 fluorescence imaging probe in the NIR-II region to detect tumor tissues with low PD-L1 expressing. Using the 4T1 subcutaneous mouse tumor models as an example, AUNP-12-CH1055 probe could more clearly illuminate the tumor tissue of the 4T1 experimental group of mice, which indicated that fluorescence imaging in NIR-II region had higher sensitivity, lower background signal, and better imaging depth than fluorescence imaging in NIR-I region. Moreover, we mirrored PD-L1 heterogeneity by implanting mice in bilateral mammary glands with breast cancer cell lines (MCF-7 and MDA-MB-231) that had distinct PD-L1 expression levels, reflecting the observed variability in patient tumors. The results demonstrated that our fluorescence imaging probe preferentially targeted the MDA-MB-231 tumors, which showed a higher PD-L1 expression. This selective uptake underscores the probe’s potential in distinguishing tumor tissues based on PD-L1 levels, thereby offering insights into the complexity of tumor heterogeneity. Moreover, we are committed to validating our probes against traditional methods in clinical settings to establish their accuracy and utility.

In this study, we administered a single dose of 2 mg/kg, which is lower than the therapeutic dose reported in previous literature (3 mg/kg/day for 14 days).43 This dosage achieved excellent imaging results without affecting therapeutic effects. AUNP-12, as a therapeutic peptide for immune checkpoint blockade therapy, holds potential for synergistic effects when combined with other innovative treatment strategies, such as photodynamic therapy,44 even at lower doses. This dual functionality enables AUNP-12 to be a promising theranostic probe for future integrative diagnostic and therapeutic applications. Next research will focus on exploring the synergistic therapeutic potential and optimizing the multifunctional capabilities of this probe.

In the future, our goal is to apply the developed AUNP-12-Cy5.5 and AUNP-12-CH1055 NIR fluorescence imaging probes for precise and real-time visualization of PD-L1 expressing levels in clinical settings. They can offer a novel approach for the selection of patients who are most likely to derive optimal benefits from PD-1/PD-L1 therapy, which ensures that patients receive the most appropriate and effective treatments based on their individual tumor characteristics.

Experimental Procedures

Synthesis of the Probe AUNP-12-Cy5.5

Cy5.5-NHS (3 mg) and AUNP-12 (10 mg) were combined with dimethyl sulfoxide (DMSO) (1 mL) and 20 μL of triethylamine (TEA) was added. They were agitated for 12 h, and the liquid above was taken out through freeze-drying. High performance liquid chromatography (HPLC) was utilized to purify crude materials, resulting in the isolation of the yellow solid, which was AUNP-12-Cy5.5.

Synthesis of the Probe AUNP-12-CH1055

CH1055 (3 mg) and AUNP-12 (10 mg) were mixed with DMSO (1 mL) for dissolution. They were subsequently supplemented with N,N-diisopropylethylamine (DIPEA) (0.5 mg, 3.8 μM), N-hydroxysuccinimide (NHS) (2 mg, 9 μM), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (2.4 mg, 20 μM). The reaction lasted for 24 h. Then the liquid above was taken out through freeze-drying. The purity of AUNP-12-CH1055 was assessed by HPLC.

Measurement of Fluorescence Properties of AUNP-12-Cy5.5 and AUNP-12-CH1055

A UV-2600 UV–Vis spectrophotometer was used to measure excitation and absorption spectra, while emission spectra were measured using the FLS98. AUNP-12-Cy5.5 imaging efficiency was examined in detail using different solvents. PBS and DMEM with serum were utilized as the solutions. Multiple dilutions were used to establish the concentration gradients of 0.01, 0.1, 0.5, 1, and 5 μM AUNP-12-Cy5.5 solution in PBS as the solvent to study the relevance between fluorescence intensity and concentration. The same concentration gradient of free Cy5.5 was set as above. NIR-II fluorescence imaging was conducted with 880, 1000, and 1300 nm LP filters, respectively. The excitation laser was operated at a wavelength of 808 nm. Parallel experiments were conducted AUNP-12-CH1055 and free CH1055 under identical concentration gradients to study the associations between the fluorescence intensity and concentration.

Cell Line and Animal Models

All of the experiments were carried out on various cell lines, including B16F10, CT26, 4T1, MDA-MB-231, and MCF-7 cancer cell lines. They were grown in RPMI-1640 medium or DMEM with serum and penicillin-streptomycin. BALB/c mice and BALB/c nude mice were raised in a facility free of pathogens at 25 °C and offered unlimited food and water. Procedures involving animals were carried out in accordance with the humane and responsible guidelines (application number: XHDW-2022-016) in Peking Union Medical College Hospital. For the subcutaneous mouse tumor models, BALB/c mice were implanted with CT26 or 4T1 cells (106 cells per mouse, 200 μL) at the flanks of each mouse. For the bilateral mouse tumor models, BALB/c nude mice were implanted with MDA-MB-231 cells (106 per mouse, 200 μL, left mammary gland) and MCF-7 cells (106 per mouse, 200 μL, right mammary gland).

Immunoblotting for PD-L1 Expression

The cells were broken down using lysis buffer that included the protease inhibitor cocktail. The extracts from cells or tissues were denatured before being analyzed by using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then the protein was separated on SDS-PAGE gels and transferred to poly(vinylidene difluoride) (PVDF) membranes. The blots were obstructed by 5% nonfat dry milk in tris buffered saline with tween 20 (TBST). Next, they were exposed to primary antibodies. Signals were identified with horseradish peroxidase (HRP)-conjugated secondary antibodies and the enhanced chemiluminescence (ECL) detection method. Servicebio provided the primary antibody against PD-L1 (GB11339A) and the HRP-conjugated secondary antibody against rabbit (GB23303).

Assessment of Targeting Specificity In Vitro

To conduct flow cytometry, the cells were adjusted to 1 × 10∧7 cells/mL in the staining buffer. The incubation period of 30 min was conducted at room temperature with all sample mixtures containing 100 μL of the cell suspension mixed with 500 μL of fixation buffer. After fixation, the cells underwent centrifugation, and the liquid above was removed. The cell pellet was then washed by membrane permeation washing solution, followed by centrifugation and removal of the liquid above. The cells were subsequently resuspended in 150 μL of the same membrane permeation washing solution.

For blocking experiments, CT26 or B16F10 cells were divided into different groups: one group was incubated with 60 μg of aPD-L1 (no. BE0101, Bioxcell) to create the aPD-L1 block group, and another group was treated with 6 μg of AUNP-12 (HY-P1812, MedChemExpress) to form the AUNP-12 block group. After the initial blocking step, these cells along with the cells in the AUNP-12-Cy5.5 group were treated with AUNP-12-Cy5.5 probe for a duration of 2 h. The control group, labeled as the PBS group, received no treatment.

After the incubation period, all groups were washed twice with membrane permeation wash solution to remove any unbound probe, followed by one wash with a flow cytometry staining solution. Finally, analysis was conducted with a BD LSRII flow cytometer.

In Vivo NIR-I Imaging of PD-L1 in Mice with AUNP-12-Cy5.5

Each of the subcutaneous mouse tumor models and the bilateral mouse tumor models was randomly divided into 3 groups. The aPD-L1 block group was intraperitoneally injected with 200 μg of aPD-L1 both 12 and 1 h prior to imaging, while the AUNP-12 block group was intraperitoneally injected with 3 mg of AUNP-12 at the same respective time points. After the initial blocking step, all groups including the AUNP-12-Cy5.5 group were intravenously injected with AUNP-12-Cy5.5 probe. In the next step, isoflurane-anesthetized mice were injected with AUNP-12-Cy5.5 (IVIS spectrum) to perform fluorescence molecular imaging (FMI). Images were taken before injection and after 1, 2, 4, 6, 12, and 24 h. After 1 d following injection, the mice with tumors in each group were euthanized in order to collect their major organs and get the ex vivo imagings. The efficacy of targeting was assessed using the tumor-to-background ratio (TBR). It was calculated by dividing tumor fluorescence intensity by background fluorescence intensity. Data analysis was performed by using IVIS LivingImaging 4.4 software (PerkinElmer).

In Vivo NIR-II Imaging of PD-L1 in Mice with AUNP-12-CH1055

The 4T1 subcutaneous mouse tumor models were randomly separated into 3 groups. The aPD-L1 block group was intraperitoneally injected with 200 μg of aPD-L1 both 12 and 1 h prior to imaging, while the AUNP-12 block group was intraperitoneally injected with 3 mg of AUNP-12 at the same time points. After the initial blocking step, all groups, including the AUNP-12-CH1055 group, were intravenously injected with AUNP-12-CH1055 probe. Next, mice under isoflurane anesthesia were subjected to FMI following the administration of AUNP-12-CH1055 via intravenous injection. NIR-II images were captured at 2, 4, 6, 12, 24, and 48 h after the injection. Then, the mice with tumors in each group were euthanized to collect their tumors, livers, hearts, spleens, kidneys, and brains, along with their ex vivo images, followed by analysis using ImageJ software.

Fixation and Clearing of Tumor Samples

Following the initial NIR-I imaging, the mice were anesthetized and euthanized. Tumors were aseptically taken out, rinsed with PBS, and then immersed in 4% formalin for 24 h in the absence of light. Next, tumors were dried using methanol with concentrations varying from 25 to 100% for a duration of 2 h, followed by immersion in pure methanol for 1 d. Next, these tumors were washed by using benzyl alcohol and benzyl benzoate (BABB).

Light Sheet Fluorescence Microscopy of Mice Tumors Ex Vivo

The cleared tumors were examined using LSFM from LaVision BioTec in Germany. The filters were adjusted to a 680 nm excitation and 710 nm emission in order to match the Cy5.5 dye for detecting PD-L1 in the samples. The increment was established at 5 μm, with a maximum span of 2 mm for scanning across the tumor. Each tumor sample was imaged for a total of 7 min with measurements taken using a 400 ms exposure time per slice. Imaris software was applied to generate three-dimensional (3D) reconstructions of the tumor images.

Ex Vivo Histology and Examination of Liver and Kidney Function

After 7 days following the injection, major organs were placed in 10% formalin and subsequently encased in paraffin. H&E staining was conducted to analyze the biotoxicity of the probes. Blood of mice was obtained and centrifuged at 2000 rpm for 0.5 h to get serum. The levels of alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) in serum were detected with a chemistry analyzer.

Statistical Analysis

Quantitative data have been presented as mean ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 10 (GraphPad software, Inc., San Diego, CA). Comparisons between means of two groups were performed using the student’s t-test. Statistical significance was established at *p < 0.05, **p < 0.01, and ***p < 0.001.

Acknowledgments

This study was financially supported by Beijing Hospital Authority Clinical Medicine Development special funding (Grant number ZLRK202333), the National Key R&D Program of China (Grant number 2023YFC3402800), the National Natural Science Foundation of China (Grant numbers 82071896, 82272111, 62027901, 92159303, and 82302338), the Shaanxi Fundamental Science Research Project for Chemistry and Biology (22JHQ088), National High Level Hospital Clinical Research Funding (2022-PUMCH-D-001), and Key Research and Development Program of Xianyang (L2023-ZDYF-SF-016).

Glossary

Abbreviations Used

3D

three-dimensional

3D LSFM

three-dimensional light sheet fluorescence microscopy

ALT

alanine transaminase

AST

aspartate transaminase

BABB

benzyl alcohol and benzyl benzoate

BUN

blood urea nitrogen

DMEM

Dulbecco’s modified Eagle medium

ECL

enhanced chemiluminescence

FBS

fetal bovine serum

FMI

fluorescence molecular imaging

H&E

hematoxylin and eosin

HPLC

high performance liquid chromatography

HRP

horseradish peroxidase

ICI

immune checkpoint inhibitor

irAEs

immunogenicity-related adverse events

NIR

near-infrared

NIR-I

the first near-infrared

NIR-II

the second neared-infrared

PBS

phosphate-buffered saline

PVDF

poly(vinylidene difluoride)

SDS-PAGE

sodium dodecyl-sulfate polyacrylamide gel electrophoresis

TBR

tumor-to-background ratio

TBST

tris buffered saline with tween 20

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.4c00266.

  • Experimental methods, analysis of the in vivo stability of AUNP-12-Cy5.5 probe, validation of the targeting specificity of AUNP-12-Cy5.5 probe in the B16F10 cell line, analysis of the localization of AUNP-12-Cy5.5 probe on CT26 cells, evaluation of the tissue penetration ability of AUNP-12-Cy5.5 probe in a 3D tumor spheroid model, and additional ex vivo signal-to-background ratio (SBR) statistics for organs and tumors (PDF)

The authors declare no competing financial interest.

Supplementary Material

bc4c00266_si_001.pdf (461.2KB, pdf)

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Supplementary Materials

bc4c00266_si_001.pdf (461.2KB, pdf)

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