(2024) 22:342
Jin et al. BMC Medicine
https://doi.org/10.1186/s12916-024-03559-w

BMC Medicine

RESEARCH ARTICLE

Open AccessMultispectral fluorescence imaging
of EGFR and PD‑L1 for precision detectionof oral squamous cell carcinoma: a preclinicaland clinical study
Nenghao Jin1,2,3†, Yu An4†, Yu Tian1,5†, Zeyu Zhang4†, Kunshan He3,7, Chongwei Chi3,6, Wei Mu4*, Jie Tian3,4* andYang Du3,6*

Abstract
Background Early detection and treatment are effective methods for the management of oral squamous cell carcinoma (OSCC), which can be facilitated by the detection of tumor-specific OSCC biomarkers. The epidermal growth
factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) are important therapeutic targets for OSCC. Multispectral fluorescence molecular imaging (FMI) can facilitate the detection of tumor multitarget expression with highsensitivity and safety. Hence, we developed Nimotuzumab-ICG and Atezolizumab-Cy5.5 imaging probes, in combination with multispectral FMI, to sensitively and noninvasively identify EGFR and PD-L1 expression for the detection
and comprehensive treatment of OSCC.
Methods The expression of EGFR and PD-L1 was analyzed using bioinformatics data sources and specimens.
Nimotuzumab-ICG and Atezolizumab-Cy5.5 imaging probes were developed and tested on preclinical OSCC cell line
and orthotopic OSCC mouse model, fresh OSCC patients’ biopsied samples, and further clinical mouthwash trials wereconducted in OSCC patients.
Results EGFR and PD-L1 were specifically expressed in human OSCC cell lines and tumor xenografts. NimotuzumabICG and Atezolizumab-Cy5.5 imaging probes can specifically target to the tumor sites in an in situ human OSCC
mouse model with good safety. The detection sensitivity and specificity of Nimotuzumab-ICG in patients were 96.4%
and 100%, and 95.2% and 88.9% for Atezolizumab-Cy5.5.
Conclusions EGFR and PD-L1 are highly expressed in OSCC, the combination of which is important for a precise
prognosis of OSCC. EGFR and PD-L1 expression can be sensitively detected using the newly synthesized multispectral
fluorescence imaging probes Nimotuzumab-ICG and Atezolizumab-Cy5.5, which can facilitate the sensitive and specific detection of OSCC and improve treatment outcomes.
†

Nenghao Jin, Yu An, Yu Tian and Zeyu Zhang contributed equally to thiswork.
*Correspondence:
Wei Muweimu@buaa.edu.cn
Jie Tian
ji.tian@ia.ac.cn
Yang Duyang.du@ia.ac.cn
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/.

Jin et al. BMC Medicine(2024) 22:342

Page 2 of 16

Trial registration Chinese Clinical Trial Registry, ChiCTR2100045738. Registered 23 April 2021, https://​www.​chictr.​org.​
cn/​bin/​proje​ct/​edit?​pid=​125220
Keywords Oral squamous cell carcinoma (OSCC), Epidermal growth factor receptor (EGFR), Programmed deathligand 1 (PD-L1), Multispectral fluorescence molecular imaging, Imaging probe

Background
Oral malignancies account for 3–5% of all malignant
tumors. Oral squamous cell carcinoma (OSCC) is a kindof oral malignant tumor with high malignant degree andpoor prognosis. In 185 countries around the world, there
are about 476,000 new cases of OSCC and about 226,000
deaths [1]. The overall mortality rate of oral and oropharyngeal cancers has increased by 0.5% per year overthe past 10 years [2]. After comprehensive treatment, the5-year survival rate remains at 50%, and the survival rateof patients with advanced disease decreases to 27% [3,4]. The poor prognosis of patients with OSCC is partially
due to a delayed diagnosis. Early screening and timely
treatment can effectively prevent the progression of
OSCC, thereby increasing the survival rate of patients [5,6]. Currently, the diagnostic methods for OSCC mainly
involve visual inspection and tactile examination of theoral cavity, head and neck lymph nodes, and traditional
medical imaging such as computed tomography (CT)and magnetic resonance imaging (MRI) [7]. However,some OSCCs appear similar to oral ulcers or underlying oral malignant diseases (OPMDs) [8, 9], leading to
misdiagnosis. The gold standard for a definitive OSCC
diagnosis is pathological examination, which is invasive,
time-consuming, and involves complicated procedures[10–12]. Hence, there is an urgent need for new ways todetect tumors noninvasively and sensitively.
Near-infrared fluorescence (NIRF) imaging, throughthe injection or application of fluorescent imaging
probes, allows tumors to be fluorescently lightened in
the corresponding diseased areas, providing precise
guidance for discriminating healthy and tumor tissues
safely [13]. The most common NIR dyes used for in vivo
NIRF tumor imaging are indocyanine green (ICG), Cy5,
Cy5.5, Cy7 and IRDye800CW [14]. ICG is the amphiphilic small-molecule NIRF dye approved by the food anddrug administration (FDA) for the surgical treatment ofhead and neck tumors [15]. ICG was first used for NIRF
imaging in 9 patients with head and neck squamous cell
carcinoma (HNSCC) [16]. Bredell et al. detected cervicalsentinel lymph nodes in the soft tissue after peritumoral
injection of ICG in patients with oropharyngeal cancer[17]. However, free ICG and other NIR dyes are non-specific and cannot be used for tumor-targeted imaging. NIR
dyes can be coupled with tumor-specific ligands such as
antibodies, metabolic substrates, cell surface peptides,

and growth factors to recognize tumor cells [18–21].
In HNC, Cetuximab-800CW was evaluated by fluorescence-guided imaging (FGI) for high sensitivity to tumor
detection [22, 23]. Multispectral fluorescence imaging(FMI) mainly uses broad-spectrum light and can simultaneously detect multiple biomarkers expressed in tumors.
Chen et al. demonstrated the feasibility of detecting multiple targets in human for the first time in Barrett’s neoplasia study. Multispectral FMI can facilitate precise andearly detection of tumors [24]. Compared with targetFMI with a single tracer, multispectral FMI can improve
the sensitivity and specificity of tumor identification [25].
For localized, small-scale tumors, the extracellular matrix(ECM) may prevent the penetration of intravenously
administered macromolecules, leading to missed diagnoses of small lesions [26]. The use of micro-dose fluorescent imaging probes to spray or smear the diseased
area enables almost instant imaging [27]. For OSCC, theentire oral mucosa can be topically applied with targeted
imaging probes, and suspected cancer lesions can bedirectly lightened and correspondingly detected [28–32].
The epidermal growth factor receptor (EGFR) and programmed death-ligand 1 (PD-L1) are two key importanttherapeutic targets for OSCC. Targeted therapy, represented by EGFR antibody drugs, and immunotherapy, represented by anti-PD-1/PD-L1 antibodies, haveprovided new strategies for the treatment of HNSCC.
Cetuximab, an anti-EGFR monoclonal antibody (mAb),
was the first FDA-approved molecular-targeted drug forthe treatment of HNSCC [33] and has been approved
as a first-line treatment for relapsed and/or metastatic
HNSCC. Fluorescently labeled cetuximab can be utilized
for targeted FMI of OSCC to evaluate the targeted recognition of anti-EGFR fluorescently labeled antibodies inand around the tumor after systemic administration [34].
de Wit JG et al. [23] reported that Cetuximab-800CW
can be used for the detection of positive tumor margins
through 66 OSCC tumors and can safely locate surgicalmargins during surgery. Nimotuzumab has a lower affinity than Cetuximab, and has less effect on the recognition
and killing of normal tissue cells [35]. PD-L1 is a transmembrane receptor that, after binding to PD-L1/PD-L2,
can prevent cellular immunity. PD-L1 antibody drugs canrelieve this inhibition and enhance the cellular immune
response against tumor cells by blocking the PD-1/
PD-L1 signaling pathway [36]. The potential for imaging

Jin et al. BMC Medicine(2024) 22:342

with Atezolizumab and its ability to predict responses
to PD-L1 immunotherapy were initially assessed in ahuman study using zirconium-89-labeled Atezolizumab(anti-PD-L1) [37]. Many cancers, including OSCC, aremolecularly heterogeneous, and the detection of multiple
targets is needed for more accurate clinical diagnosis andguidance for personalized therapy.
Hence, the aims of this study is to validate EGFR and
PD-L1 as specific biomarkers for OSCC and develop
multispectral Nimotuzumab-ICG and AtezolizumabCy5.5 fluorescence imaging probes to quantify the
dynamic expression of dual-target EGFR and PD-L1 frompreclinical in vitro human OSCC cell lines, in vivo OSCC
orthotopic mouse models, to clinical OSCC patient
tumor specimens, and topical application as mouthwash
in OSCC patients. The new imaging strategies, quick andsensitive detection methods, and experimental evidence
for the sensitive detection of OSCC biomarkers provided
in this study will help to provide guidance for the early
detection of OSCC, targeted therapy and immunotherapy (Fig. 1).

Methods
Bioinformatics analysis

The data used to compare EGFR and PD-L1 expressionin HNSCC and normal tissues are available in the geneFig. 1 Study flowchart

Page 3 of 16

expression profiling interactive analysis (GEPIA) database at http://​gepia.​cancer-​pku.​cn.
Clinical model prediction based on receiver operator
characteristic (ROC) analysis

One hundred and twelve OSCC patients treated at the
Chinese PLA General Hospital between December 2018and December 2019 were retrospectively analyzed. The
inclusion criteria were patients who underwent surgery
for the first time at our hospital and were diagnosed with
OSCC based on complete clinical and follow-up records.
Exclusion criteria: (1) presence of other concomitanttumors; (2) presence of distant metastases; (3) historyof preoperative antineoplastic therapy; (4) serious postoperative complications. The follow-up period was three
years, and the patients were re-examined every threemonths. The time of recurrence, metastasis, or death
was defined as the cutoff time. The follow-up period
ended on December 1, 2022. Receiver operating characteristic (ROC) curve analysis was performed based on
EGFR and PD-L1 expression. EGFR and PD-L1 expression was evaluated by two independent pathologists in
our hospital who were blinded to the study design. EGFR
expression was detected using immunohistochemistry (IHC). The negative, weak positive, moderate positive, strong positive staining were respectively defined as

Jin et al. BMC Medicine(2024) 22:342—, + , +  + , +  +  + . PD-L1 expression was measured using
PD-L1 IHC 22C3 pharmDx (Dako 22C3, Dako, Denmark) and ranged from 0 to 99% as determined using theTumor Proportion Score (TPS).
EGFR and PD‑L1 expression analysis via IHC of human
OSCC

Analyses of EGFR and PD-L1 expression were conductedon biospecimens from the Department of Stomatology,First Medical Center, Chinese PLA General Hospital.
All biopsy specimens were diagnosed as OSCC by the
Department of Pathology. The biospecimens were thenembedded and sliced continuously. Tissue sections wereincubated with bovine albumin (BSA, GC305010; Servicebio, Wuhan, China) for 30 min and Rabbit monoclonal antibody to EGFR (RRID: AB_869579; Abcam,
Cambridge, UK) and Rabbit monoclonal antibody
to PD-L1 (RRID: AB_2687878; Abcam, Cambridge,UK) at 4 °C overnight. The slides were then exposed to
HRP peroxidase-labeled Goat Anti-Rabbit IgG (RRID:
AB_2811189; Servicebio, Wuhan, China) and incubatedfor 1 h. Subsequently, the sections were stained withhematoxylin and eosin (H&E) for analysis. The expression levels of EGFR and PD-L1 were quantified using
H-scores (0–300). Images were then captured usinga scanner (Digital Sight DS-U3, Eclipse E100, Nikon,
Japan), and H-scores were calculated by ImageJ Fiji (NIH,
Bethesda, MD, USA).
Cell culture

Normal human oral keratinocytes (HOK) (ScienCell
Research Laboratories Inc., USA) cells were cultured
in Roswell Park Memorial Institute 1640 (RPMI-1640)
medium (Thermo Fisher Scientific) containing 10% fetal
bovine serum (FBS) (Thermo Fisher Scientific Australia Pty Ltd., Australia) and 1% penicillin–streptomycin (Macgene Biotechnology, China). Human OSCC
CAL27 cells (RRID: CVCL_1107; ATCC, Manassas, VA,
USA), CAL27-Fluc cells (RRID: CVCL_1107; Cobioer
Biosciences Co., Ltd., China) and HSC3 cells ((RRID:
CVCL_1288; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)(Thermo Fisher Scientific (CHINA) Co., Ltd., China)
containing 10% FBS and 1% penicillin–streptomycin.
All cells were cultured in a Cell Culture Dish (Corning
Inc., USA). All experiments were performed using mycoplasma-free cells. All human cell lines were authenticatedusing STR profiling within the last three years.
EGFR and PD‑L1 expression analysis in different cells
via western blotting and flow cytometry

HOK, CAL27, CAL27-Fluc and HSC3 cells were cultured and collected. Total protein was extracted fromPage 4 of 16

cells using precooled Pierce RIPA Buffer (Thermo Fisher
Scientific, USA) and Halt Protease Inhibitor Cocktail(Thermo Fisher Scientific, USA), and western blotting
was conducted utilizing Rabbit monoclonal antibody
to EGFR (RRID: AB_869579) and PD-L1 monoclonal
antibody (RRID: AB_2756526; Proteintech Group, Inc.,USA), polyclonal rabbit β-actin antibody (Cell Signaling
Technology, USA), and secondary antibody (1:10,000,
Jackson ImmunoResearch, USA) were used for westernblot. All four cell types (1 × ­106) were incubated with
FITC labeled Anti-EGFR antibody (RRID:AB_298005)
and APC Anti-PD-L1 antibody (ab206967, Abcam, Cambridge, UK). After 30 min incubation, the cells were suspended in Phosphate-Buffered Saline (PBS) (Thermo
Fisher Scientific Co., Ltd., China) and analyzed using
flow cytometry (FACSCanto™ II; BD Biosciences, USA).
FlowJo (version10.8.1) software was used for data analysis. The experiment was repeated three times, and theaverage of the results was taken.
Human OSCC orthotopic mouse modelAll animal experiments were approved according to
the guidelines of the Institutional Animal Care and Use
Committee (Permit No: IA21-2203–24) of the Instituteof Automation, Chinese Academy of Sciences. PBS (50μL) containing 5 × ­106 HSC3 or CAL27-Fluc cells were
injected into the tongues of 6-week-old male BALB/cnu/nu mice anesthetized with isoflurane (RWD Life Science, China). The feeding method is to give soft food orsoftened food with feed water. HSC3 and CAL27-Fluctumor xenografts were harvested 7–10 days after inoculation; H&E staining was performed. Primary anti-EGFR
antibody (RRID: AB_869579) and anti-PD-L1 antibody(RRID: AB_2687878) were used for immunohistochemical and immunofluorescence staining to analyze theexpression of EGFR and PD-L1 in human OSCC tumor
xenograft mouse models.
Synthesis of Nimotuzumab‑ICG and Atezolizumab‑Cy5.5

ICG-NHS-ester (330 nM, dissolved in 300 μL DMSO,
Sigma-Aldrich) was added to humanized monoclonal
EGFR antibody (5 mg, 33 nM, Nimotuzumab, Biotech
Pharma, China) (dye/antibody ratio was 1:20) in conjugation buffer (5 mL, 0.1 M PBS), and the solution was
reacted in the dark at 25 °C with continuous oscillation
overnight. The mixture was concentrated to 0.8 mL using
a centrifugal filter unit (3 kDa MWCO, Amicon) andpurified three times via centrifugation at 8,500 rpm for
15 min. Similarly, Cy5.5-NHS-ester (330 nM, dissolved in
300 μL 0.1 M PBS, New Research Bioscience, China) was
added to humanized monoclonal anti-PD-L1 antibody(Atezolizumab, Roche Registration GmbH, Switzerland)in conjugation buffer (5 mL, 0.1 M PBS), and the solution

Jin et al. BMC Medicine(2024) 22:342

was reacted in the dark at 25 °C with continuous oscillation overnight. The mixture was concentrated and purified thrice using a centrifugal filter unit (3 kDa MWCO,
Amicon). The conjugation efficiency was calculated.
In vitro cell viability experiments and spectroscopic
analysis

Logarithmic growth-phase HOK, CAL27-Fluc, and
HSC3 cells were seeded (5 × ­103/well) in 96-well culture
plates (Corning Inc., USA) overnight. NimotuzumabICG, Atezolizumab-Cy5.5 and the mixture were added
into the culture plates at different concentrations (2.5, 5,
10, 20, 40, 80, 160, and 320 μg/mL), respectively. After
24 h incubation, cells were washed thrice with PBS andcultured in 10% CCK-8 medium (CA1210; Solarbio Science and Technology Co., Ltd., China) for 30 min; theabsorbance at 450 nm was determined using a spectrophotometric microplate reader (Synergy HT; BioTek,
USA).
The spectra of excitation light and absorption light ofNimotuzumab-ICG and Atezolizumab-Cy5.5 were tested
by fluorescence spectrophotometer (F-7000, Hitachi,
Japan) (The scanning speed is 1200 nm/min, the spectral
bandwidth is 10 nm, and the sampling interval is 0.2 nm)
and near-infrared spectrophotometer (UV-3600 Plus,
Shimadzu, Japan), respectively.
In Vivo and Ex vivo fluorescence molecular imaging (FMI)and safety evaluation

CAL27-Fluc OSCC tumor-bearing mice were injectedwith Nimotuzumab-ICG (0.2 mg) and AtezolizumabCy5.5 (0.2 mg), and their mixture via the caudal vein.
The block group (normal mice) was intraperitoneallyinjected with Nimotuzumab (0.2 mg) and Atezolizumab(0.2 mg) 1 h before the intravenous injection of the imaging probes. FMI images were captured at different time
points (pre-, 2, 6, 12, and 48 h) using an IVIS® Spectrum/
CT (Caliper Life Sciences, USA), and the tumor to background ratio (TBR) was calculated using the region ofinterest (ROI) measurement tool with Fiji software.
After 24 h, the mice were sacrificed, and the serum
were collected for detection of liver function indices,
including alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) using an automatic blood biochemical auto analyzer (7600; Hitachi, Tokyo, Japan).
The tongues, hearts, livers, spleens and kidneys werecollected for ex vivo FMI by IVIS® SpectrumCT. H&E
staining was performed on organs after ex vivo imaging.
Resected fresh CAL27-Fluc tumor-bearing mouse tongue
tumor specimens were analyzed using immunofluorescence (IF) staining for EGFR and PD-L1 expression.

Page 5 of 16

Probe fluorescence imaging of fresh human specimens

First, 10 fresh OSCC specimens were pre-tested todetermine the optimal incubation concentration of theprobe, and then a formal fluorescence imaging experiment on 20 OSCC specimens was followed.. A total of
30 resected fresh human OSCC specimens were rinsed
in 5% goat serum (SL038, Solarbio Science and Technology Co., Ltd., China) to remove debris and mixed
and incubated with Nimotuzumab-ICG and Atezolizumab-Cy5.5 at 20 °C for 3 min. Subsequently, thespecimens were washed with PBS 3 times. A homemade multispectral fluorescence imaging system (KeyLab of Molecular Imaging, CAS) was used to perform
imaging experiments on the specimens. The device canachieve optical resolution of 50 μm, bandwidth of 5 nm,
imaging field of 12.5 × 12.5 ­cm2, 50 fps time resolution(adjustable according to actual conditions), real-time
imaging of white light image, near-infrared dual imaging channels of 665 nm and 785 nm simultaneously.
After imaging experiments, the specimens were fixed
in 4% paraformaldehyde (P1110; Macgene Biotechnology) for subsequent H&E, EGFR, and PD-L1 IHC and
IF staining.
Fluorescence imaging of OSCC in human patients

This study was approved by the Ethics Committee of theChinese PLA General Hospital (No. S2021-097–01) andregistered in the Chinese Clinical Trial Registry (Registration Number: ChiCTR2100045738). All humanpatients signed informed consent, and the patients gargled 5 mL of Nimotuzumab-ICG (0.4 mg/mL) and Atezolizumab-ICG (0.4 mg/mL) for approximately 30 s.
Next, the patients gargled ultrapure water for 30 s threetimes, and the interval between Nimotuzumab-ICG and
Atezolizumab-ICG imaging was 1 h. White-light andfluorescence images were obtained using a commercialized Digital Precision Medicine imaging device H2800(DPM, Beijing, China). After surgical treatment, fresh
specimens were obtained for the probe-spraying experiment, H&E staining, EGFR, PD-L1 IHC staining, and IF
staining. EGFR and PD-L1 expression were defined as—, + , +  + , +  +  + for negative, weak positive, medium
positive, and strong positive expression.
Statistical analysis

The optimal cutoff values for the expression of EGFR and
PD-L1 were determined from the ROC curves. Statisticalanalyses were performed using SPSS (version 21.0; IBM,
Armonk, NY, USA) and GraphPad Prism software (version 9; GraphPad Software Inc., San Diego, CA, USA).
Differences between paired data were tested using the

Jin et al. BMC Medicine(2024) 22:342

Wilcoxon test. Statistical significance was set as *P < 0.05,** P < 0.01, ***P < 0.001, **** P < 0.0001.

Results
Validation of EGFR and PD‑L1 as specific OSCC biomarkers

According to the GEPIA dataset, the expression levelsof EGFR and PD-L1 were significantly higher in HNSCC
tumors than para tumor tissues (Fig. 2A). The EGFR and
PD-L1 gene expression profiles across all HNSCC tumor
samples (n = 519) and paired normal tissues (n = 44) wereexpressed as log2[transcripts per million (TPM) + 1]].
Patients with OSCC had high EGFR and PD-L1 expression. The expression of EGFR and PD-L1 is associatedwith the prognosis of patients with HNSCC. The correlation between EGFR expression and disease-free survival (DFS) and overall survival (OS) in 493 patients with
HNSCC, and PD-L1 expression, DFS, and OS in 466
patients with HNSCC were analyzed using the GEPIA
dataset. EGFR overexpression was significantly associated with OS (*P = 0.045), whereas high expressionof PD-L1 was associated with shorter DFS (*P = 0.01)(Fig. 2B).
The statistical data for OSCC patients (n = 112) are
shown in Supplementary Table 1. The areas under the
curve corresponding to high expression of EGFR and
PD-L1 were 0.791 and 0.641, respectively, but the corresponding curve area for the combined prediction is 0.836(95% Confidence Interval (CI): 0.744 to 0.928), suggestingthat the combination of EGFR and PD-L1 dual-targets isimportant for the precise prognosis of OSCC (Fig. 2C).
To confirm that EGFR and PD-L1 are specific biomarkers for OSCC, biospecimen slides from patients with
OSCC (n = 59) were immunohistochemically stained with
anti-EGFR and anti-PD-L1 antibodies. The demographics
of patients with OSCC (n = 59) are shown in Additional
file 1: Table S1. EGFR and PD-L1 expression levels weredistinctly higher in OSCC tumors than in para tumor
tissues (Fig. 2D). We clearly distinguished tumor tissues
from normal tissues by immunohistochemical staining for EGFR and PD-L1. The EGFR H-Score and PD-L1
H-Score in OSCC and para tumor tissues were 141.2 vs.
67.19 and 116.2 vs. 69.66, respectively (**** P < 0.0001)(Fig. 2E).
Detection of EGFR and PD‑L1 expression in in vitro OSCC
cells and in vivo orthotopic OSCC tumor model

The expression of EGFR and PD-L1 was relatively higher
in CAL27, CAL27-Fluc, and HSC3 cells than in HOK
cells, used as control (Fig. 3A). EGFR and PD-L1 protein
expression was significantly higher in CAL27 (~ 4.75fold, **** P < 0.0001, ~ 1.19-fold, * P < 0.05), CAL27-Fluc(~ 2.36-fold, *** P < 0.001, ~ 1.85-fold, **** P < 0.0001),
and HSC3 (~ 2.65-fold, **** P < 0.0001, ~ 2.34-fold, ****

Page 6 of 16

P < 0.0001) cells than in HOK cells (Fig. 3B). IHC staining showed that EGFR and PD-L1 were both highly
expressed in the tumor area in both CAL27-Fluc andHSC3 mouse models (Fig. 3C). EGFR and PD-L1 double
IF staining also confirmed their high expression in the
CAL27-Fluc mouse tumor xenografts (Fig. 3D). In general, our experiments confirmed that EGFR and PD-L1
were expressed at relatively higher levels in OSCC tumor
tissues than in normal tissues.
Characterization of Nimotuzumab‑ICG
and Atezolizumab‑Cy5.5 imaging probes

Nimotuzumab and Atezolizumab were reacted with ICGNHS and Cy5.5-NHS to yield Nimotuzumab-ICG and
Atezolizumab-Cy5.5, respectively (Additional file 1: Fig.
S1). The conjugation efficiency was 93.42% and 92.02%,
respectively. Nimotuzumab-ICG had an excitation wavelength of 808 nm and absorption peak at 790 nm, whichwere partially offset from those of free ICG (Excitation:
815 nm), indicating that the dye was conjugated to Nimotuzumab. The spectral characterization of AtezolizumabCy5.5 also showed a shift from the excitation wavelengthof free Cy5.5 (Excitation: 691 nm), indicating that thedye was linked to Atezolizumab, with excitation andabsorption peak of Atezolizumab-Cy5.5 were 670 nm and679 nm, respectively (Additional file 1: Fig. S2). The cell
viability of different human OSCC cells was not influenced when the concentrations of Nimotuzumab-ICG
and Atezolizumab-Cy5.5, were lower than 320 μg/mL.
These results suggest that Nimotuzumab-ICG and Atezolizumab-Cy5.5 were relatively biocompatible and safe
for further in vivo study (Additional file 1: Fig. S3).Thein vivo toxicity of Nimotuzumab-ICG and AtezolizumabCy5.5 was evaluated in healthy male BALB/c-nu/nu mice;
The mice were euthanized 24 h post-injection of PBS,
Nimotuzumab-ICG, Atezolizumab-Cy5.5, and Nimotuzumab-ICG and Atezolizumab-Cy5.5 injection (4 mg/kg).
Serum assessment showed that the ALT and AST levels
were within the normal range in all four groups (Additional file 1: Fig. S4). In addition, no obvious histopathological changes were observed in the major organs (heart,
liver, kidney, or spleen) (Additional File 1: Fig. S5). Nimotuzumab-ICG and Atezolizumab-Cy5.5 were further
tested for in vivo transoral toxicity and proved to be safe
(Additional file 1: Table S2). These results demonstrated
that Nimotuzumab-ICG and Atezolizumab-Cy5.5 were
well-developed and safe.
The in vivo biodistribution and tumor targetingof Nimotuzumab‑ICG and Atezolizumab‑Cy5.5

An orthotopic human CAL27-Fluc OSCC tumor mousemodel was established and bioluminescence imaging (BLI) was performed to locate the OSCC tumor

Jin et al. BMC Medicine(2024) 22:342

Page 7 of 16

Fig. 2 Expression and analysis of EGFR and PD-L1 in HNSCC in GEPIA database and OSCC patients. A Statistical analysis of EGFR and PD-L1
expression in HNSCC tumors and normal tissues obtained from the GEPIA database. Log2(TPM + 1) was used for the log-scale. HNSCC, Headand Neck Squamous Cell Carcinomas. B The correlation of EGFR and PD-L1 expression related to disease free survival (DFS) and overall survival(OS). C Receiver operating characteristic analysis of EGFR and PD-L1. FPR, False Positive Rate; TPR, True Positive Rate; AUC, Area Under Curve. D
Representative EGFR and PD-L1 IHC and H&E histology obtained from OSCC biospecimens. Scale bar of overview, 2 mm. Scale bar of tumor
and para tumor, 100 μm. (E) IHC H-scores of EGFR and PD-L1 expression in IHC samples (n = 59 patients). **** p < 0.0001

Jin et al. BMC Medicine(2024) 22:342

Page 8 of 16

Fig. 3 Detection of EGFR and PD-L1 Expression in in vitro OSCC cells and in vivo orthotopic OSCC tumor model. A FACS analysis of HOK, CAL27,
CAL27-fLUC, and HSC3 cells for detection of EGFR and PD-L1 expression. B Western blotting analysis of EGFR and PD-L1 expression in HOK,CAL27, CAL27-fLUC, and HSC3 cell lines. *P < 0.05, ***P < 0.001, ****P < 0.0001. C H&E, EGFR and PD-L1 IHC staining of OSCC tumor xenograft. Scalebar, 200 μm. D White light, H&E, immunofluorescence staining (IF) obtained from CAL27-fLUC tumor xenografts. H&E Scale bar, 1,000 μm. Both
PD-L1 and EGFR are over-expressed in the cell membrane and stained in green and red, respectively; the nucleus is stained in blue. Scale bar, 500
and 20 μm

xenograft (Additional file 1: Fig. S6). CAL27-Flucoral tumor-bearing mice were divided into different groups: Nimotuzumab-ICG, Atezolizumab-Cy5.5,

Nimotuzumab-ICG + Atezolizumab-Cy5.5, and corresponding blocking groups. The fluorescence signals of
ICG and Cy5.5 were detected at 785 nm and 665 nm,

Jin et al. BMC Medicine(2024) 22:342

respectively (Fig. 4A). The fluorescence signal in the
Nimotuzumab-ICG group was detected at 2 h post-injection, peaked at 12 h post-injection, gradually decreased,
and remained detected 48 h post-injection. The fluorescence signal of the Atezolizumab-Cy5.5 group was
detected at 2 h post-injection and peaked at 12 h postinjection. After the simultaneous injection of Nimotuzumab-ICG and Atezolizumab-Cy5.5, both fluorescencesignals were detected at 2 h and peaked at 12 h postinjection. The FMI TBR of the target group was higher
than that of the corresponding block group at the corresponding time points. The difference in the TBR was
more significant at 12 h than other time points. (Fig. 4B).
After 48 h of in vivo observation, the tumors and majororgans were dissected for ex vivo FMI. The fluorescencesignal of the tumor region in the targeted group was
higher than that in the blocking group. The fluorescencesignals of Nimotuzumab-ICG and Atezolizumab-Cy5.5
were stronger in the liver and certain signals in the kidney, indicating that the imaging probes were mainly
metabolized through the liver and some through kidney.
Animal study suggested that Nimotuzumab-ICG and
Atezolizumab-Cy5.5 dual-targeting imaging probes possessed good OSCC tumor-targeting capability and safety,
making them suitable for further clinical application andtranslation.
Identification of EGFR and PD‑L1 expression on OSCC
patients’ fresh biopsied samples using Nimotuzumab‑ICG
and Atezolizumab‑Cy5.5 imaging probes

For the identification of multitarget expression, a homemade multispectral fluorescence imaging system wasdeveloped and used to conduct imaging experiments
on patient specimens, and white light and multispectral
fluorescence images were obtained. Figure 5A shows
the working model diagram and prototype of the multispectral fluorescence imaging system. Fresh OSCC
specimens from 10 patients were topically treated with
Nimotuzumab-ICG and Atezolizumab-Cy5.5 with different concentrations. The optimal incubation concentration of Nimotuzumab-ICG and Atezolizumab-Cy5.5
was found to be 0.4 mg/mL (Additional file 1: Fig. S7)
for 3 min (Additional file 1: Fig. S8). The obvious fluorescence signals of Nimotuzumab-ICG and AtezolizumabCy5.5 from the OSCC tumor region could be specifically

Page 9 of 16

detected, while adjacent normal areas did not show fluorescence signals (Fig. 5B). Quantitative analysis showedthat Nimotuzumab-ICG imaging discriminated between
tumor and para tumor. Under 785 nm NIR, the TBR of
the tumor was higher than para tumor (30.05 ± 1.87
vs 20.27 ± 2.35, ** P < 0.01). In addition, under
665 nm + 785 nm NIR, the TBR of tumors was higher
than that of para tumor (27.59 ± 1.33 vs 16.81 ± 1.39, ***P < 0.001). Similarly, Atezolizumab-Cy5.5 could clearly
distinguish tumors from parathyroid tumors. Under
665 nm NIR and 665 nm + 785 nm NIR, the TBR oftumor and para tumor were (23.18 ± 2.52 vs 14.07 ± 1.51,** P < 0.01) and (20.15 ± 1.55 vs 11.32 ± 1.02, ** P < 0.01),
respectively. This observation was further confirmed
by H&E, EGFR, and PD-L1 immunohistochemical andimmunofluorescence staining of patient’s specimens. The
expression levels of EGFR and PD-L1 in OSCC tumor tissues were also observed based on the fluorescence intensity produced by excitation light irradiation at 665 nm
and 785 nm. The higher the fluorescence intensity, thehigher were the biomarker expression levels (Fig. 5C).
Therefore, we further performed multiplexed fluorescence imaging of EGFR and PD-L1 in 30 fresh OSCC
patients’ specimens. FMI results, immunohistochemical
staining, and immunofluorescence staining showed thatthe sensitivity and specificity of Nimotuzumab-ICG were
96.4% (95%CI: 79.8% ~ 99.8%) (27/28) and 100% (95%CI:
19.8% ~ 100%) (2/2), respectively. The sensitivity andspecificity of Atezolizumab-Cy5.5 were 95.2% (95%CI:
74.1% ~ 99.7%) (20/21) and 88.9% (95%CI: 50.7% ~ 99.4%)(8/9), respectively. The high imaging sensitivity and specificity of the imaging probes with high safety indicate thefeasibility of topical application in human patients with
OSCC in situ.
Fluorescence imaging of Nimotuzumab‑ICG
and Atezolizumab‑ICG in OSCC patients in situ

Nimotuzumab-ICG and Atezolizumab-ICG were topically applied as mouthwashes to 8 OSCC patients with
OSCC. The fluorescence imaging of EGFR and PD-L1
dual targets was mainly performed using the Digital Precision Medicine imaging device H2800 (DPM, Beijing,
China), which possesses an endoscopic detector that canbe applied to patients’ mouths. Quantitative analysis aftergargling with Nimotuzumab-ICG (0.4 mg/ml) showed(See figure on next page.)
Fig. 4 Biodistribution of Nimotuzumab-ICG and Atezolizumab-Cy5.5 in an orthotopic OSCC mouse model. A In vivo FMI of an OSCC orthotopic
mouse model injected with Nimotuzumab-ICG, Atezolizumab-Cy5.5, or a combination of both, respectively. The blocking group represents
the pre-administration of corresponding antibody drugs Ex vivo FMI of the tongue with OSCC tumor and major organs 48 h post-injection. B TBR
analysis of in vivo FMI signal intensity in different groups at different time points. The TBR was significantly different at 12 h postinjection. FMI,
fluorescence molecular imaging; TBR, tumor to background ratio. *P < 0.05, **P < 0.01

Jin et al. BMC Medicine(2024) 22:342

Fig. 4 (See legend on previous page.)

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Jin et al. BMC Medicine(2024) 22:342

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Fig. 5 Nimotuzumab-ICG and Atezolizumab-Cy5.5 imaging probes were used to detect fresh OSCC biopsy samples. A The working model diagramand prototype of the home-made multispectral fluorescence imaging system. B Representative NIR and NIR overlay image of Nimotuzumab-ICG
and Atezolizumab-Cy5.5 staining of fresh OSCC biospecimen from patients. Orange and white arrows correspond to the position and orientationof the cross-sectional fluorescence intensity profiles of the NIR and NIR overlay regions, respectively. Quantitative analysis showedthat the fluorescence intensity in the tumor area and the para tumor area. NIR, Near-infrared. C White light, H&E, EGFR IHC, PD-L1 IHC, and IF stainingof fresh biospecimen from OSCC patients. Scale bar, 1,000 μm and 100 μm

that EGFR expression was specifically highly expressed intumor tissues but not in normal mucosa tissues (Fig. 6A,
top, 23.9 ± 1.17 vs 3.01 ± 0.24, **** P < 0.0001). After surgical treatment, tissue specimens were subjected to ex vivo
fluorescence imaging, H&E staining, and immunohistochemical staining. Quantitative analysis showed that

the TBR in the tumor was higher than that in the paratumor (Fig. 6A, bottom, 48.02 ± 4.65 vs 19.27 ± 1.973, ***P < 0.001). Similarly, after gargling with AtezolizumabICG (0.4 mg/ml), quantitative analysis showed that the
TBR in the tumor was higher than that in the para tumor
in both in situ and ex vivo experiments. The TBR of the

Jin et al. BMC Medicine(2024) 22:342

tumor and para tumor were 23.4 ± 2.19 vs 5.34 ± 0.61, (***
P < 0.001) and 27.47 ± 2.43 vs 17.06 ± 1.52 (** P < 0.01),
respectively (Fig. 6B). The fluorescence imaging data of
8 cases of OSCC are summarized in Additional file 1:
Table S3. The ex vivo FMI, HE, and IHC data were almost
consistent with in vivo FMI observations, showing thatspecific tumor areas can be fluorescently illuminated by
the targeted imaging probes.
Interestingly, a patient had buccal mucosal SCC on theleft side and buccal mucosal lichen planus on the rightside of the mouth. After gargling with NimotuzumabICG and Atezolizumab-ICG imaging probes, fluorescence signal was observed in the left buccal mucosa,
whereas no trace of fluorescence signal was observed in
the right buccal mucosa (Fig. 6C). The data validated thespecificity of the Nimotuzumab-ICG and AtezolizumabICG probes for the detection of OSCC. These in vivo
observations were further verified via subsequent IHC
and IF experiments (Fig. 6D).

Discussion
Sensitive detection of tumor-specific biomarkers canfacilitate the early and accurate detection of OSCC andprovide guidance for precision therapy. At present, a
number of studies have demonstrated the feasibility of
OSCC fluorescence guided localization and guidanceof surgical margin resection. Early diagnosis of OSCC
was achieved through oral spray fluorescence recognition imaging [29, 30]. EGFR and PD-L1 are key targets
in OSCC. Herein, we developed Nimotuzumab-ICG
and Atezolizumab-Cy5.5 imaging probes combined withmultispectral FMI to facilitate the noninvasive detection of EGFR and PD-L1 with high sensitivity, specificity
and safety. We further translated our dual-target imaging strategy from preclinical OSCC bearing animal studies to clinical patients with OSCC, which showed great
potential for the comprehensive management of OSCC
for early detection and image-guided therapy, including
targeted therapy, surgery, and immunotherapy.

Page 12 of 16

EGFR and PD-L1 are important therapeutic targets in
OSCC. EGFR, a transmembrane glycoprotein, is highly
expressed in 90% of HNSCC cases and is a strong prognostic indicator of head and neck cancer [38, 39]. Maurizi et al. found that EGFR levels are associated with therisk of recurrence and death [40]. PD-L1 is also highly
expressed on the surface of HNSCC [41]. The expression of PD-L1 in various solid tumors, such as esophageal, gastric, and thyroid cancers, is associated withpoor prognosis [42, 43]. Bioinformatics analysis fromGEPIA Datasets and immunohistochemical staining of
59 human OSCC biological specimens from the Chinese
PLA General Hospital showed that EGFR and PD-L1
were highly expressed in OSCC. The dataset obtained
based on GEPIA also verified that high EGFR expression in HNSCC was correlated with OS. Overexpression of PD-L1 in HNSCC is associated with DFS, not OS,
which is consistent with previous findings [44]. However,whether the expression of EGFR or PD-L1 in HNSCC iscorrelated with OS or DFS may be subjected to differences due to the sample numbers and follow-up time.
Based on a 3-year retrospective analysis of 112 patientswith OSCC treated at the Chinese PLA General Hospital, a ROC curve was used to predict the clinical model.
The combination analysis of EGFR and PD-L1 expression
in predicting the 3-year OS of patients with OSCC can
improve the prediction accuracy. Therefore, both EGFR
and PD-L1 can not only be used as prognostic markers
for OSCC but also as potential markers for guiding antitumor therapy.
Multiplexed FMI technology provides good opportunities for noninvasive and multitarget detection with highsensitivity and safety. A variety of malignant tumors,
including OSCC, have tumor molecular heterogeneity [45], The multispectral fluorescence probes make up
for the situation that the imaging of a single mode or therecognition of a single target makes the ability of tumor
characterization and boundary analysis relatively challenging. And multispectral is more powerful than single
spectrum in defining tumor heterogenous properties and(See figure on next page.)
Fig. 6 Fluorescence imaging of Nimotuzumab-ICG and Atezolizumab-ICG in OSCC human patients. A White light, NIR, and NIR overlay images wereobtained by oral gargling experiment of Nimotuzumab-ICG on OSCC patients in clinical application. A dotted white line was drawn from tumor
area to para tumor tissue to calculate the counts of fluoresce signal. The expression of EGFR was further imaged with Nimotuzumab-ICG in fresh
biopsied specimens of OSCC patients, and verified using IHC and H&E staining. Scale bar, 2000 μm. B White light, NIR, and NIR overlay images
were obtained by oral gargling experiment of Atezolizumab-ICG on OSCC patients for clinical application. The expression of PD-L1 was imaged
with Atezolizumab-ICG in fresh biopsied specimens of OSCC patients, and verified using IHC. Scale bar, 2000 μm. C Nimotuzumab-ICG
and Atezolizumab-ICG were used in the oral gargling experiment, and white light, NIR, and overlayed NIR images of both the left (with OSCC area)
and right (with lichen planus) buccal mucosa were obtained. The fluorescence intensity was measured, calculated, and verified via pathology. D
H&E, EGFR IHC, PD-L1 IHC, and IF staining were performed on fresh OSCC specimens on the patient’s left buccal mucosa. The yellow line indicated
the gross tumor range in fresh surgical specimens. The black line shows the tumor areas in H&E staining and IHC staining, while the white line
shows the tumor areas in IF staining. Scale bar, 2,000 μm

Jin et al. BMC Medicine(2024) 22:342

Fig. 6 (See legend on previous page.)

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Jin et al. BMC Medicine(2024) 22:342

boundary analysis [24]. And thus the detection of multitargets is most likely to improve both the sensitivity andspecificity of tumor recognition and for accurate clinicaldiagnosis and guidance of treatment. The fluorescencelabeled anti-EGFR antibody Cetuximab-IRDye800CW
has been used to locate tumors and surrounding tissues in advanced HNSCC [34]. Intravenous injection ofCetuximab-IRDye800 in patients with HNSCC achieved
high sensitivity and specificity and successfully distinguished tumors from normal tissues in FMI-guided
surgical studies [46, 47]. As a humanized monoclonal
antibody, Nimotuzumab has a high affinity and can bindto the extracellular domain of EGFR and inhibit EGF
binding [48] Nimotuzumab has been approved in several
countries for the treatment of HNSCC [49] and glioma[50] based on clinical trials of various malignancies [51].
Nimotuzumab has the advantage of low toxicity compared with Cetuximab [35]. Therefore, in this study, wedeveloped a novel EGFR targeting Nimotuzumab-ICG
fluorescence imaging probe. In OSCC tumor tissues withhigh EGFR expression, Nimotuzumab-ICG showed ahigher fluorescence imaging signal and better image contrast than images of tumors with low or no EGFR expression. Moreover, we developed an Atezolizumab-Cy5.5
fluorescence imaging probe targeting the PD-L1 molecule, which can realize noninvasive and dynamic imaging of PD-L1 expression and provide guidance for safe
immunotherapy. Targeted molecular imaging of PD-L1 isan effective method for dynamic and noninvasive evaluation of PD-L1 expression; however, the main administration method is the intravenous route [37, 52–54]. In thisstudy, we administered imaging probes through topicallyadministration [27–32], which can achieve almost instantimaging of EGFR and PD-L1 by spraying the target areawith micro-doses of fluorescent imaging probes anddetecting suspected cancer lesions. Multispectral FMI
imaging of Nimotuzumab-ICG and Atezolizumab-Cy5.5
is a new non-invasive, sensitive and specific way to identify tumors. Ultimately, we will be able to offer more precise and personalized treatments.
To further promote clinical translation, we applied ourimaging probes as mouthwashes to 8 OSCC patients.
Since the homemade multispectral fluorescence imaging
system is still in the prototype stage, it cannot be used in
the surgery room. Hence, we utilized the commercialized Digital Precision Medicine imaging device H2800,
though this system has only one channel for detecting
ICG fluorescence signal. Hence, we developed Nimotuzumab-ICG and Atezolizumab-ICG imaging probes andapplied them consecutively to OSCC patients througha mouthwash. Our imaging method is quick sensitiveand specific in detecting EGFR and PD-L1 expression

Page 14 of 16

in patients with OSCC, which was further validatedby post-surgical histology and immunohistochemistry.
Moreover, our imaging strategy can discriminate tumor
lesions from other oral diseases, such as buccal mucosa
lichen planus. For the future clinical application of homemade multispectral fluorescence imaging system can not
only provide early identification and diagnosis of OSCC,
but also provide guidance for clinical surgical boundary
recognition and anti-tumor targeted therapy and immunotherapy. In addition, it is still necessary to further optimize the imaging algorithm and autofocus algorithm,
eliminate the image distortion caused by oral haze, andcontinuously improve the image recognition ability.

Conclusions
We developed a multispectral FMI imaging strategy
that demonstrated the feasibility of concurrently detecting multiple tumor biomarkers and the potential for the
early detection of tumors that are molecularly heterogeneous. We synthesized the multispectral fluorescenceimaging probes Nimotuzumab-ICG and AtezolizumabCy5.5 with OSCC targeted imaging performance andgood biosafety. Make up for the situation that the imaging method of a single mode or the recognition of a single target makes the ability of tumor characterization
and boundary analysis relatively Challenging. Combinedwith the homemade multispectral fluorescence imaging
system, the EGFR and PD-L1 expression can be identified and quantified from preclinical OSCC tumor-bearing mouse models to clinical OSCC patients, whichfacilitates early cancer detection and provide guidance
for anti-tumor targeted therapy, image-guided surgery,and immunotherapy of OSCC. We demonstrated a
proof-of-concept for detecting multiple targets concurrently in patients with OSCC neoplasia, which is promising for a wide range of clinical applications, including
early screening and surveillance, biopsy guidance, quick
staining of fresh biopsies, in vivo diagnosis, and intraoperative margin delineation.
Abbreviations
OSCC	Oral squamous cell carcinoma
EGFR	Epidermal growth factor receptor
PD-L1	Programmed death-ligand 1
FMI	Fluorescence molecular imaging
CT	Computed Tomography
MRI	Magnetic Resonance Imaging
NIRF	Near-infrared fluorescence
ICG	Indocyanine green
HNSCC	Head and Neck squamous cell carcinoma
ROC	Receiver operator characteristic
IHC	Immunohistochemistry
H&E	Hematoxylin and eosin
ROI	Region of interest
TBR	Tumor to background ratio

Jin et al. BMC Medicine(2024) 22:342

Page 15 of 16

Supplementary Information

Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China. 4 Key Laboratory of Big Data-Based
Precision Medicine (Beihang University), Ministry of Industry and Information Technology of the People’ S Republic of China, School of Engineering Medicine, Beihang University, Beijing 100191, China. 5 Departmentof Stomatology, Beijing Integrated Traditional Chinese and Western Medicine
Hospital, Beijing 100039, China. 6 University of Chinese Academy of Sciences,
Beijing 100080, China. 7 State Key Laboratory of Computer Science and Beijing Key Lab of Human‑Computer Interaction, Institute of Software, Chinese
Academy of Sciences, Beijing 100190, China.

The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12916-​024-​03559-w.
Additional file 1: Table S1. Demographics of OSCC patients. Table S2.
Results of the Acute Oral Toxicity Test. Table S3. Clinical data of mouthwash trials for OSCC patients. Fig. S1. Synthesis of Nimotuzumab-ICG and
Atezolizumab-Cy5.5. Fig. S2. Excitation and absorption spectra of Nimotuzumab-ICG and Atezolizumab-Cy5.5 under excitation light at 780 nm and
640 nm, respectively. Fig. S3. HOK, HSC3 and CAL27-fLUC cell lines weretreated with cell viability at different concentrations of Nimotuzumab-ICG
and Atezolizumab-Cy5.5. Fig. S4. Serum assessment of liver function markers. Fig. S5. The in vivo toxicity of Nimotuzumab-ICG and AtezolizumabCy5.5 were evaluated in healthy BALB/c-nu/nu male mice. H&E staining ofheart, liver, kidney and spleen in different groups. Scale bar, 100 μm. Fig.
S6. Bioluminescence imagingexperiments demonstrated the location of
tongue tumors in a mouse model. Fig. S7. Optimal concentration analysis
of Nimotuzumab-ICG and Atezolizumab-Cy5.5. Fig. S8. Analysis of optimum incubation time of Nimotuzumab-ICG and Atezolizumab-Cy5.5
Additional file 2. Original images of Western blotting
Acknowledgements
The authors thank Weisheng Wen, Jingqiu Bu, Lei Zhang, Peng Chen, Qing Xi,
Huawei Liu, Yipeng Ren, Lin Feng, Fei Chen, Lejun Xing, Bo Wei and Ye Tao forthe support during the fresh OSCC patients’ biospecimens collection, Rui Wu
for manuscript proofreading.
Authors’ contributions
NH J drafted the manuscript. NH J and Y T performed the experiments, collected OSCC tissue specimen. Y A developed a home-made multispectral
fluorescence imaging system. NH J, ZY Z and KS H analyzed the data. CW C
performed part of the experiments. W M, J T and Y D designed the study andrevised the manuscript. All authors read and approved the final manuscript.
Funding
This work was supported by the National Natural Science Foundation of China(82272111, 92159303, 62027901), the National Key R&D Program of China(Grant number 2023YFC3402800), and Beijing Natural Science Foundation
under Grant (L232097).
Availability of data and materials
The datasets used and analyzed during our study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Our study was approved by the Ethics Committee of Chinese PLA General
Hospital (No. S2021-097–01). All participants provided written informed
consent. The animal experiments were approved by the guidelines of theInstitutional Animal Care and Use Committee (Permit No: IA21-2203–24) at theInstitute of Automation, Chinese Academy of Sciences, and the experiments
were conducted in conformity with approved guidelines. Author confirm
that all the experiments on the use of human tissue samples confirm that all
experiment were performed in accordance with the Declaration of Helsinki’s
guidelines and regulations. The study was approved and the participants gave
their informed consent.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Medical School of Chinese PLA, Beijing 100853, China. 2 Departmentof Stomatology, The First Medical Centre, Chinese PLA General Hospital,
Beijing 100853, China. 3 CAS Key Laboratory of Molecular Imaging, Beijing Key

Received: 24 December 2023 Accepted: 13 August 2024

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