Determination of PD-L1 Expression in Circulating Tumor Cells of Hypopharyngeal and Laryngeal Cancers and Correlation with Tissue Detection

doi:10.21203/rs.3.rs-4266528/v1

Download PDF

Research Article

Determination of PD-L1 Expression in Circulating Tumor Cells of Hypopharyngeal and Laryngeal Cancers and Correlation with Tissue Detection

https://doi.org/10.21203/rs.3.rs-4266528/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

PD-L1 plays a pivotal role as an immunoregulatory checkpoint within the immune system, exerting a critical influence on the internal functioning and survival mechanisms of cancer cells. Our study aimed to elucidate the clinical significance of PD-L1 expression in circulating tumor cells (CTCs) derived from individuals afflicted with Hypopharyngeal and Laryngeal Cancers (HLC), as well as its potential implications for clinical practice. CTC were identified using an immunomagnetic positive sorting methodology. Simultaneous detection was conducted on the CTC levels among PD-L1-positive patients, aiming to ascertain the dynamic relationship between real-time CTC fluctuations and the clinicopathological indices of the patients. The constructed immunolipid magnetic nano-beads demonstrated pronounced efficacy in capturing CTCs, and the lipid nanoparticles exhibited noteworthy capture efficiency coupled with minimal cytotoxic effects. This investigation encompassed a cohort exceeding 30 individuals, wherein PD-L1 expression analysis was executed to delineate CTC variations within PD-L1-positive patients, thereby yielding auxiliary insights for clinical diagnosis. Subsequent assessment of PD-L1 expression consistency between CTCs and tissue specimens revealed a substantial agreement surpassing 70%. Furthermore, inhibition of PD-L1 yielded a significant elevation in the cytokine TNF-α levels, accompanied by a concomitant reduction in IL-10 levels. The CTC sorting system devised in this investigation boasts attributes of remarkable specificity and sensitivity. By virtue of PD-L1 expression analysis, it holds the potential to offer instructive implications for tailoring individualized treatments in clinical scenarios.

hypopharyngeal and laryngeal cancers

PD-L1

CTCs

Immunotherapy

Head and neck squamous cell cancers (HNSCCs) stand as the sixth most prevalent tumor worldwide, mainly including oral cancer, orohypopharyngeal and laryngeal cancer, laryngeal carcinoma, and hypopharyngeal carcinoma[1]. The majority of patients initially present with localized lesions, which frequently progress to advanced stages. In recent times, researchers have come to recognize that tumor heterogeneity significantly contributes to the variations and challenges encountered in tumor immunotherapy [2-3]. Notably, instances such as tumor recurrence or distant metastasis caused by lymph node infiltration often result in unfavorable prognoses, as evidenced by a less than 50% 5-year survival rate within this patient cohort[4]. Hence, it is imperative to urgently address the formidable issue of elevated tumor heterogeneity, comprehensively fathom the intricacies of tumorigenesis, and advance clinical practices rooted in gleaned tumor cell insights.

Circulating tumor cells (CTCs) are tumor cells that have acquired the capability to infiltrate the bloodstream originating from primary tumors or metastatic sites, thus facilitating dissemination to distant organs through hematogenous routes[5-7]. CTCs offer a real-time portrayal of tumor genotypes and manifest substantial genetic heterogeneity within patients. Consequently, the scrutiny of individual CTCs holds promise for elucidating the intricacies of tumor heterogeneity and delineating the dynamic, persistent genomic mutations characterizing the trajectory of tumorigenesis[8]. The principal challenge encountered in conventional CTC isolation endeavors pertains to the exceedingly low abundance of CTCs in bodily fluids (e.g., blood) [9]. The immunomagnetic nanoparticle-based isolation and enrichment system implemented in the present study allow for precise analysis of minuscule cell populations within bodily fluids. This approach demonstrates commendable reproducibility and specificity, potentially enabling the early detection of issues preceding traditional imaging modalities like computed tomography (CT) scans [10-11]. Furthermore, it boasts superior consistency and specificity when juxtaposed with alternative blood-based assessments, such as serum tumor markers. This proposed technique has already found application in diagnosing and prognostically evaluating metastatic breast cancer, colorectal cancer, lung cancer, and prostate cancer[12-15].

In recent decades, the field of tumor immunotherapy has undergone rapid advancement. Prominent immunotherapeutic options available in clinical settings include immune vaccine, immune checkpoint inhibitor (ICI) therapy, adoptive immune cell therapy, cytokine therapy, etc.[16]. A widely recognized tumor biomarker, PD-L1, is a transmembrane glycoprotein consisting of 290 amino acids and belongs to the B7-CD28 superfamily [17]. When PD-L1 is highly expressed ontumor cell surfaces, it can suppress immune responses and facilitate immune evasion [18-19]. Thus tumor immune escape is the key breakthrough of immunotherapy. For instance, Ghebeh et al. reported substantial PD-L1 expression in breast cancer patients' tumor cells, establishing a positive correlation between PD-L1 levels, tumor size and clinicopathological stage [20]. Furthermore, a prior study also highlighted PD-L1 overexpression in tumor cells of chronic lymphocytic leukemia, certain types of T/B-cell lymphoma, gastric cancer, esophageal cancer, renal cell carcinoma, breast cancer, and bladder cancer. This overexpression is linked to heightened Treg activity, compromised anti-tumor T cell functionality, and unfavorable prognoses[21]. In light of these insights, our study was designed to ascertain the significance of PD-L1 in gauging the propensity for recurrence and metastasis in HLC.

CTCs possess the ability to disseminate from primary tumor sites, acquire more invasive and metastatic traits through epithelial-mesenchymal transition (EMT)[22]. Building upon the foundation of the single anti-EpCAM antibody modified magnetic beads employed within the CellSearch system[23-24], our experiment introduced two additional antibodies, Vimentin and EGFR, to modify the CTC-sorted magnetic beads individually. In instances where clinical samples demonstrate PD-L1 positivity, EpCAM, Vimentin, and EGFR markers would be collectively utilized for nuanced subtyping, followed by an exploration of correlation with clinicopathological features.

Collection of clinical samples

This study was approved by the Ethics Committee of Fujian Medical University Union Hospital (2021KJT069) and carried out in strict adherence to the principles outlined in the Declaration of Helsinki (as revised in 2013). Comprehensive written informed consents was procured from all participating patients. In total, 34 samples from patients with histologically proven HLC were included by peripheral blood specimens in this study at Fujian Medical University Union Hospital from 2020 to 2022.

Inclusion criteria were: 1) patients aged ≥18 years old, 2) patients who received a confirmed diagnosis of HLC through pathological examination (including histology or cytology) according to AJCC Seventh Edition Staging Guidelines, 3) patients exhibiting least one discernible lesion that adheres to the criteria outlined in the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1), 4) patients attaining an ECOG PS score ranging from 0-1point, 5) patients displaying normal functionality of main organs, while exhibiting essentially regular results in routine blood test, blood biochemistry and coagulation function assessments.

Exclusion criteria were: 1) patients with tumors other than HLCs, 2) patients with severe anemia or organ dysfunction, 3) patients with active infection, 4) patients with the previous history of malignant tumors; and 5) patients with uncontrolled diabetes (the risk of surgical complications might be increased owing to the aggravation in the original poor tissue condition after chemotherapy).

A volume of 7.5 ml of peripheral blood was procured from each patient to facilitate subsequent CTC enumeration and analysis. Concurrently, pertinent clinicopathological data were meticulously documented. All participating patients underwent a comprehensive process of providing written informed consent.

Cells and materials

The HLC cell lines employed in this study comprised HNE1 (NO. BNCC340008), FaDu (NO. BNCC338343) and Hep-2 (NO. BNCC245666) (BNCC, https://www.bncc.org.cn/). The essential material encompassed DMEM culture medium, fetal bovine serum (FBS) and trypsin (Gibco). EpCAM, Vimentin and EGFR monoclonal antibodies were purchased from Abcam. CD45-PE, EpCAM antibody derivatives and magnetite nanoparticles were purchased from Lieyuan (Shanghai) Biomedical Technology Co., Ltd. Distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) were provided by Avanti Co., Ltd. DAPI staining solution, cholesterol (Chol), dichloromethane and other common reagents were purchased from Sigma company.

Preparation of antibody modified magnetic nanoparticles

Immunomagnetic beads were synthesized using a reverse evaporation technique as follows: Initially, 5 mg each of DOPC Chol were weighed and added into two 50 mL flasks with three necks. Subsequently, 1.0 mL Fe₃O₄-HMN solution was measured and dissolved into 3.0 mL of CH₂Cl₂ after the removal of ethanol. This Fe₃O₄-HMN/CH₂Cl₂ mixture was then transferred into the above flasks with three necks. Under an ice bath condition, the round-bottom flask underwent ultrasonic emulsification for 6 minutes using an ultrasonic instrument with a probe. Next, 2 mg of EpCAM-GHDC, Vimentin-GHDC, and EGFR-GHDC were collected and dissolved into 6 mL of double distilled water (ddH₂O), and this solution was gradually introduced into flasks. The mixture underwent further ultrasonic procession. Residual CH₂Cl₂ was eliminated using a rotary evaporator. The resulting solution was subjectetd to magnetic separation and washed three times, leading to the production of immunomagnetic microspheres.

Characterization of immunolipid magnetic nano-beads

Zetasizer Nano-ZS 90 (Malvern Instruments Ltd., UK) was used to detect the particle size and potential of EpCAM/Vimentin/EGFR magnetic nano-beads. Atomic force microscopy (AFM) was used to observe the micro-morphology of different magnetic nano-beads (modified by EpCAM antibody/Vimentin antibody/EGFR antibody). PPMS-9 (QUANTUM DESIGN, USA) was further used to detect the hysteresis loop of magnetic nano-beads. The UV absorption peak of the magnetic nano-beads solution was scanned with a UV spectrophotometer. To validate the presence of antibodies on the surface of the magnetic nano-beads, subsequent confirmation and qualitative analysis of antibody content were performed.

Experimental analysis of cytotoxicity of immunolipid magnetic nano-beads

HLC cell lines HNE1, FaDu, and Hep-2 were rountinely cultivated in DMEM complete culture medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37℃ in a humidified condition containing 5% CO₂. The assessment of cytotoxicity for diverse magnetic nano-beads (modified with EpCAM antibody/Vimentin antibody/EGFR antibody) was performed using MTT colorimetry in vitro. Cells were seeded in a 96-well flat-bottom micro-detection plate at a density of 1×10⁴ cells/well, and incubated under completely humidified condition containing 5% CO₂ for 24 h. Subsequently, the absorbance was determined at the wavelength of 490 nm using a multimodal Microplate Reader (SynergyTM HT, BioTek, USA).

Separation and acquisition of CTCs

Peripheral blood samples were systematically collected from each patient at corresponding time points to faciliatet the retrieval of CTCs. The prepared EpCAM/Vimentin/EGFR magnetic nano-beads were used to enrich and screen the CTCs present in the peripheral blood specimens. The utilization of fluorescent nucleic acid dye (4, 6-diamidino-2-phenylindole, DAPI) enabled the identification of intact cells possessing nuclei. Subsequently, fluorescently labeled monoclonal antibodies were employed for CD45 and CK19 staining, serving to distinguish epithelial cells from leukocytes. The enumeration of CTCs were then conducted, adhering to predefined evaluation criteria, wherein CTCs had to exhibit FITC+, CK19+, DAPI+, and CD45-. The final quantification of CTCs was accomplished by using a multicolor fluorescent cell counter.

Immunohistochemical analysis of PD-L1

An appropriate amount of fixed fresh tissues were collected and rinsed with ultra-pure water. Subsequent steps involved the eliminination of fixed solution, progressive dehydration through a series of alcohol gradients, xylene treatment, and embedding in paraffin to craft paraffin-embedded tissue blocks. After slicing, these sections were subjected to a sealing step using 5% BSA at 37℃ for 30 min, flowing procedures like dewaxing, hydration, hydrogen peroxide treatment, and antigen restoration. Maintained at a temperature of 4℃, the sections underwent an overnight incubation with a PD-L1 antibody, followed by exposure to a DAB working solution. For slice preparation, hematoxylin staining and xylene dehydration were performed to obtain transparent slides. Under microscopic examination, PD-L1 expression was evident as a positive brown staining, with nuclei (DAPI) appearing blue in color.

Immunofluorescence staining of PD-L1 in CTCs

After membrane separation and centrifugation, the blood underwent centrifugation at 1,000 rpm, followed by a wash with PBS (800-1000 µL), and subsequently was transferred to designated centrifuge tubes. CTC capture and incubation were performed separately using EpCAM, Vimentin, and EGFR magnetic nano-beads. For staining, 15μL of DAPI staining solution was added to each centrifugation tube, followed by the addition of 10μL CK-FITC staining solution and 20μL of PD-L1 Alexa Fluor647 antibody. This mixture was then incubated in the dark for 30min. Following the removal of staining solution and antibodies by aspiration, slices were prepared and the dried samples were subjected to counting under a fluorescence microscope. PD-L1-positive cells exhibited red fluorescence, DAPI yielded blue fluorescence, and CK-FITC emitted green fluorescence.

ELISA detection of cytokines

Peripheral blood mononuclear clear cells (PBMCs) were isolated by employing gradient density centrifugation (Sigma-Aldrich) to spearte lymphocytes. These PBMCs were inoculated onto 96-well plates at a cell density of 2×10⁵ cells/well. To stimulate T cell activation, anti-CD3 (10g/L) and anti-CD28 (2g/L) were introduced, and this activation phase wa sustained for 48 hours. For siPD-L1-FaDu cells, siRNA pre-treatment faciliatedt their generation. These treated cells were subsequently subjected to a 12-hour reaction. After this, purified activated T cells were co-cultured with FaDu cells by a ratio of 10:1 for 48 h. The co-cultured supernatant was collected for the detection of TNF-α and IL-10 expression using ELISA.

Statistical analysis

The data batained from each group within our study underwent analysis using SPSS 19.0 statistical software. Measurement data was presented as mean ± standard deviation (±s), with a predetermined significance level of α=0.05 (bilateral), and the rank-sum test was used for statistical analysis. Further calculations were performed to determine diagnostic metrics such as sensitivity, specificity, effecacy, positive predictive value, and negative predictive value. A significance level of P<0.05 indicated a statistically significant difference.

Flow chart for the preparation of magnetic nano-beads, sorting and identification of CTCs

The initial step in the process involves the preparation of LMB (Anti-EpCAM, Anti-Vimentin, and Anti-EGFR) for CTC enrichment and capture on the platform. Figure 1 illustrated the comprehensive LMB preparation process. Subsequently, the enriched and captured CTCs underwent analysis, including immunofluorescence staining by using the aforementioned antibodies. Simultaneously, the assessment of PD-L1 expression in the serum was conducted, and an immunohistochemical analysis of PD-L1 in tissue samples was employed to facilitate a consistency analysis.

Material science evaluation of LMB

The particle sizes of the three immunomagnetic spheres in aqueous solution are similar, and the particle size distribution is uniform. EpCAM-LMB had an average size of 113.1±7.6 nm with a PDI of 0.262 (Figure 2A). Vimentin-LMB exhibited an average size of 125.8±5.3 nm with a PDI of 0.275 (Figure 2B). EGFR-LMB displayed an average size of 129.4±8.7 nm with a PDI of 0.192 (Figure 2C). Consistent with previous studies, the three immunomagnetic spheres have higher potential in aqueous solution. EpCAM-LMB had a charge of +19.7±4.26 mV (Figure 2D); Vimentin-LMB displayed a charge of +31.5±3.87 mV (Figure 2E); and EGFR-LMB exhibited a charge of +25.4±3.93 mV (Figure 2F). The ultraviolet test (Figure 2G) showcased broad absorption peaks around 280 nm for all three LMBs. Concurrently, the FT-IR spectrum test (Figure 2H) depicted the emergence of new peaks about 2840-2930cm^-1. The observation results of EpCAM-LMB, Vimentin-LMB, and EGFR-LMB under AFM are shown in Figure 2 I-K, respectively.

Analysis of the captured CTCs

The clinical data of the patients whose peripheral blood samples were collected underwent comprehensive statistical analysis, with detailed insights presented in Table 1. The results indicated that the clinical samples with CTCs greater than or equal to 5 were much more than those with CTCs count less than 5. The acquired peripheral blood samples were analyzed by using the established magnetic nano-beads, followed by the sorting and identification of tumor cells extracted from clinical samples of HLC. To elaborate, the divided blood samples were sorted and captured using EpCAM-LMB, Vimentin-LMB, EGFR-LMB and total number of CTCs respectively. According to our statistics, males exhibited a noteworthy prevalence over females. More specifically, the results of CTC counts showcased consistent male predominance regardless of separate target or combined counting method employed. CTC counts of Male patients were higher than female patients (Figure 3A).

Taking 60 years old as a demarcation point, analysis unveiled that patients aged above 60 years manifested higher EpCAM-LMB capture rates and total CTCs capture rates, whereas Vimentin-LMB capture and EGFR-LMB capture displayed the reverse trend (Figure 3B). Evaluation based on tumor stage highlighted elevated total CTCs capture rates among advanced stage patients as compared to those at an early stage (Figure 3C). In addition, a notable disparity was observed, wherein total CTC capture was significantly greater in patients with moderately and highly differentiated tumors in contrast to those with poorly differentiated tumors (Figure 3D). Moreover, intriguingly, vimentin and EGFR exhibited specific capture potential for distinct patient subsets, a finding of profound significance for clinical research. Supplementary Figure 1 provides representative fluorescence results of CTCs captured using magnetic beads modified by the three antibodies, presenting characteristics like DAPI+, CK8/18/19+, and CD45-.

PD-L1+ CTC analysis

Subsequent to the selection of samples exhibiting positive PD-L1 expression, a more refined determination of the CTC count was undertaken. The outcomes illuminated that out of the HNSCC patient pool, 18 individuals showcased positive PD-L1 expression (Figure 4A). This comprehensive examination encompassed the quantification of PD-L1+CTCs through diverse modalities: Epithelial (EpCAM-PD-L1+), interstitial (Vimentin-PD-L1+), and EGFR-PD-L1+. Notably, the findings affirmed that a greater number of PD-L1+CTCs were captured using EpCAM-LMB. Meanwhile, Vimentin-LMB exhibited superior capture of PD-L1+CTCs in comparison to EpCAM-LMB; whereas the number of PD-L1+CTCs captured by EGFR-LMB was the least. Intriguingly, among the samples, eight displayed the capture of CTCs featuring EGFR-PD-L1+. In addition, Figure 4B provides representative fluorescence outcomes showcasing PD-L1+CTCs captured via magnetic beads modified with the three distinct antibodies separately.

Comparison of CTC and tissue-derived PD-L1

In this study, 17 cases of laryngeal carcinoma, 2 case of hypopharyngeal carcinoma and 1 case of a neck tumor were encompassed. The evaluation of PD-L1 were used to seek to establish both consistency and disparity between tissue samples and CTC samples. Supplementary Figure 2A&2B provides representative images showcasing negative and positive PD-L1 expression in tissue. Among the laryngeal carcinoma samples, 14 displayed positive PD-L1 expression in both tissue and CTC samples. Two cases exhibited PD-L1 positivity exclusively in CTC samples, without corresponding detection in tissue samples. One case showcased negative PD-L1 expression in both tissue and CTC samples, thus lacking any positive expression detection. For hypopharyngeal carcinoma, one case had no detectable PD-L1 expression in both tissue and CTC samples. Another hypopharyngeal carcinoma case demonstrated PD-L1 positivity exclusively in CTC samples, with no corresponding detection in tissue samples. Similarly, for the neck tumor case, PD-L1 was positively expressed in the CTC sample, while being undetected in the tissue sample. Drawing from these results, the PD-L1 positive detection rate consistency exceeded 70% between tissue samples and CTC samples (Table 2).

To delve into the gene mutation profile of HLC patients, NGS was conducted and analyzed on leukocyte, tumor cells, and CTCs from patients. Filtering criteria encompassed the elimination of Non-exonic region mutation sites, adapter, non-monoclonal fragments (multiple barcode), and synonymous mutations (SNV). Specifically, non-synonymous mutations were utilized for subsequent analyses. According to these criteria, the mutational landscape of four primitive tumor lesions was obtained (Supplementary Figure 3). Employing multiple linear regression models, the analysis involved a subset of 96 mutational contexts in four HLC patients. This profile was compared with 30 mutational signatures within the COSMIC database (Figure 5). Isolation of CTCs from four patients was accomplished using CTC isolation kits, and Whole Exome Sequencing (WES) was conducted to compare them with primitive tumors. The ensuing results, presented in Supplementary Figure 4, revealed a substantial presence of somatic mutations. While certain mutations were identical between primitive tumors and CTCs, it was notable that somatic mutations within CTCs significantly outnumbered those observed in the primitive tumor.

Correlation between PD-L1+ CTC and clinicopathological indicators

In PD-L1-positive samples, the examination of PD-L1 expression levels across different stages is presented in Figure 6A. The findings elucidate that in stage IV, EpCAM PD-L1+ exhibits the highest CTC count, wherein the trend for Vimentin PD-L1+ mirrors that of EpCAM PD-L1+. In contrast, EGFR+ PD-L1+ showcases minimal variation in CTC trends across stages I, II, and IV (Figure 6B). Furthermore, among PD-L1-positive patients, the assessment of PD-L1 expression levels corresponding to different degrees of differentiation is illustrated in Figure 6C. The outcomes reveal that among well-differentiated samples, EpCAM PD-L1+ yields the highest CTC count. Notably, for Vimentin PD-L1+ and EGFR+ PD-L1+, CTC counts exhibit minimal fluctuations across poorly differentiated, moderately differentiated, and well-differentiated stages (Figure 6D).

PD-L1 analysis at cell line level

The analysis of PD-L1 expression was carried out in laryngeal carcinoma cell lines HNE1, FaDu, and Hep-2. As illustrated in Figure 7A, discernible PD-L1 expression was observed in the laryngeal carcinoma cells, displaying varying levels of expression. In this context, the control group consisted of K562 cells. To delve deeper, a co-culture model involving FaDu cells and activated T cells was established to validate the impact of PD-L1 on cytokine expression, particularly TNF-α and IL-10. ELISA results unequivocally demonstrated that upon co-culturing T cells with PD-L1 inhibited FaDu cells, a notable increase in TNF-α levels was evident, whereas IL-10 levels exhibited a significant decrease (Figure 7B&7C). The preceding outcomes established that the number and the marker change of CTCs indicated the effect of therapy. As well as highlighted the need for a comprehensive understanding of the underlying causative mechanisms. An insight into this was offered by the fact that Wnt/β-catenin signaling could potentially induce apoptosis in FaDu cells post treatment with cisplatin combined with paclitaxel. Supporting this notion, the cellular viability of FaDu was markedly suppressed in the cisplatin combined with paclitaxel group in contrast to the control group (P<0.01, Supplementary Figure 5A). Furthermore, the application of cisplatin combined with paclitaxel led to significant reductions in the levels of EBV and N-cadherin in FaDu cells when compared to the control group (P<0.01, Supplementary Figure 5B&5C).

In this study, the successful construction of the immunolipid magnetic beads (EpCAM-LMB, Vimentin-LMB, and EGFR-LMB) facilitated the effective sorting and identification of CTCs derived from HLCs through the utilization of immunomagnetic positive sorting. Meanwhile, the system demonstrated its capability to measure CTC concentrations in PD-L1-positive patients. The efficiency of the detection system was affirmed to surpass 80% in terms of sorting CTCs from HLCs. In parallel, the assessment of consistency between CTCs and tissue-derived PD-L1 was carried out, revealing a remarkable consistency rate surpassing 70%. Furthermore, the study explored the impact of PD-L1 inhibition on cytokine levels in laryngeal carcinoma cell lines, confirming a significant increase in TNF-α levels and a concurrent reduction in IL-10 levels post PD-L1 inhibition.

The predictive value of PD-L1 expression in primary tissues is notably limited, underscored by the weak correlation observed between PD-L1 expression in primary tumors and its manifestation in distant metastatic sites [25]. This discrepancy suggests that primary tumors might not serve as adequate surrogates for gauging PD-L1 expression in metastatic sites. A critical insight emerges from the fact that CTCs encapsulate a wealth of clinical molecular information, making them a potential remedy for the aforementioned limitation [26-27]. Moreover, the analysis of PD-L1 expression derived from CTCs holds the potential to augment the availability of clinical samples[28-30]. In this study, the EpCAM single antibody, as utilized by CellSearch, was employed to modify magnetic beads. Subsequently, the construction of magnetic beads was extended through the incorporation of both Vimentin and EGFR antibodies. This concerted effort aimed to capture CTCs within patients diagnosed with HLCs, thereby facilitating the comprehensive characterization of PD-L1 expression[31-32]. Furthermore, immunohistochemical analysis was conducted to detect the presence of PD-L1 protein within tumor tissues. This evaluation holds pivotal significance as it informs clinical treatment decisions, guiding the identification of anti-PD-1/PD-L1 antibody therapies with the highest possibility of effectively targeting tumors. A distinctive aspect of this study lies in its pioneering nature, representing the first comprehensive exploration of this avenue. Notably, PD-L1 manifests within tumor cells of several significant cancer types and can even be detected within immune cells. Its expression spans multiple components such as the cytoplasm or cell membrane, showcasing considerable variation in expression levels at an individual cellular level [33-34]. In this context, the assessment of PD-L1 expression within CTCs emerges as a pivotal supplement to traditional tissue-based PD-L1 analysis. This endeavor bears the potential to furnish a foundation for refining clinical protocols and decision-making processes in oncological practice.

Building upon the foundation of the CellSearch system, our research embarked on CTC detection employing a novel approach utilizing immunomagnetic positive sorting facilitated by EpCAM\ EGFR\Vimentin immunolipid magnetic nano-beads. This innovation led to the development of a high-precision CTC separation and detection system specifically tailored for patients afflicted with HLCs. At the same time, our study also ventured into the realm of PD-L1 analysis, a domain scarcely explored in existing literature on laryngeal carcinoma. Nonetheless, it is important to acknowledge certain limitations in our study. Presently, our research stands as a single-center study, a fact that inevitably constrains the size of our sample cohort. As a consequence, certain correlation trends might not appear as pronounced as desired. Recognizing this limitation, our subsequent research endeavors are strategically designed to transcend this shortcoming by encompassing a larger sample size. This approach aims to provide more robust theoretical underpinnings and a comprehensive data foundation for future clinical investigations grounded in this system. In summation, the present study not only successfully forged a cutting-edge system for separation and identification of CTCs from the peripheral blood of laryngeal carcinoma patients but also ventured into exploring the intricate relationship between CTC counts and PD-L1 positivity. In doing so, our work holds promise in offering a valuable clinical reference for the unfolding landscape of research into head and neck tumors.

Data Availability

All data generated and analyzed during this study are included in this published article.

Funding

This study was supported by Natural Science Foundation of Fujian Province (Grant numbers: 2021J01784), Fujian provincial health technology project [Grant numbers: 2021GGA017] and Double high-level medical institution construction fund of Fujian Province（2021-76).

Conflict of interest disclosure

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval statement

The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Ethics Committee of Fujian Medical University Union Hospital (2021KJT069).

Patient consent statement

Written informed consents have been obtained from all patients.

CHAI R C, LAMBIE D, VERMA M, et al. Current trends in the etiology and diagnosis of HPV-related head and neck cancers [J]. 2015, 4(4): 596–607.
Pramudita R Prasetyanti, Jan Paul Medema Intra-tumor heterogeneity from a cancer stem cell perspective 2017;16(1):41.
Vitor Manuel Leitão de Sousa, Lina Carvalho Heterogeneity in Lung Cancer 2018;85(1–2):96–107.
CHEN W, ZHENG R, BAADE P D, et al. Cancer statistics in China, 2015 [J]. 2016, 66(2): 115–32.
PANTEL K, BRAKENHOFF R H J N R C. Dissecting the metastatic cascade [J]. 2004, 4(6): 448–56.
PANTEL K, SPEICHER M J O. The biology of circulating tumor cells [J]. 2016, 35(10): 1216–24.
WOO D, YU M J T R. Circulating tumor cells as “liquid biopsies” to understand cancer metastasis [J]. 2018, 201: 128–35.
HABLI Z, ALCHAMAA W, SAAB R, et al. Circulating tumor cell detection technologies and clinical utility: Challenges and opportunities [J]. 2020, 12(7): 1930.
Sandhya Sharma 1, Rachel Zhuang 2, Marisa Long 2 et all Mirjana Pavlovic Circulating tumor cell isolation, culture, and downstream molecular analysis 2018 Jul-Aug;36(4):1063–1078.
Hoyoung Park 1, Mintai P Hwang, Kwan Hyi Lee Immunomagnetic [10] nanoparticle-based assays for detection of biomarkers 2013;8:4543–52.
Leila Hatami Giklou Jajan, Seyed Nezamedin Hosseini, Mohsen et all Abolhassani Progress in affinity ligand-functionalized bacterial magnetosome nanoparticles for bio-immunomagnetic separation of HBsAg protein 2022;17(7):e0267206.
BIDARD F-C, PEETERS D J, FEHM T, et al. Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data [J]. 2014, 15(4): 406–14.
DE BONO J S, SCHER H I, MONTGOMERY R B, et al. Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer [J]. 2008, 14(19): 6302–9.
KREBS M G, SLOANE R, PRIEST L, et al. Evaluation and prognostic significance of circulating tumor cells in patients with non–small-cell lung cancer [J]. 2011, 29(12): 1556–63.
COHEN S J, PUNT C, IANNOTTI N, et al. Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer [J]. 2008.
André T, Shiu KK, Kim TW et al. KEYNOTE-177 Investigators. Pembrolizumab in Microsatellite-Instability-High Advanced Colorectal Cancer. N Engl J Med. 2020; 383(23): 2207–2218.
Markus Eckstein, Alessia Cimadamore, Arndt Hartmann et all PD-L1 assessment in urothelial carcinoma: a practical approach 2019;7(22):690.
Jossie Rotman 1 2, Leontine A S den Otter 1, Maaike C G Bleeker et all PD-L1 and PD-L2 Expression in Cervical Cancer: Regulation and Biomarker Potential 2020;11:596825.
Edjah K Nduom, Jun Wei, Nasser K Yaghi, Neal Huang et all PD-L1 expression and prognostic impact in glioblastoma 2016;18(2):195–205.
Ghebeh H,Mohammed S, A1-Omair A et al. The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors [J] Neoplasia, 2006; 8(3):190–198.
Nakanishi J,Wada Y, Matsumoto K et al. Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers. [J] Cancer Immunol lmmu-nother, 2007;56(8):1173–1182.
ZOU D, CUI D J C B, MEDICINE. Advances in isolation and detection of circulating tumor cells based on microfluidics [J]. 2018, 15(4): 335.
SWENNENHUIS J F, VAN DALUM G, ZEUNE L L, et al. Improving the CellSearch® system [J]. 2016, 16(12): 1291–305.
WANG L, BALASUBRAMANIAN P, CHEN A P, et al. Promise and limits of the CellSearch platform for evaluating pharmacodynamics in circulating tumor cells; proceedings of the Seminars in oncology, F, 2016 [C]. Elsevier.
Robert Brody, Yiduo Zhang, Marc Ballas et all PD-L1 expression in advanced NSCLC: Insights into risk stratification and treatment selection from a systematic literature review 2017;112:200–215.
C Luchini, F Bibeau, M J L Ligtenberg et all ESMO recommendations on microsatellite instability testing for immunotherapy in cancer, and its relationship with PD-1/PD-L1 expression and tumour mutational burden: a systematic review-based approach 2019;30(8):1232–1243.
Michal Šmahel PD-1/PD-L1 Blockade Therapy for Tumors with Downregulated MHC Class I Expression 2017;18(6):1331.
Mazel M, Jacot W, Pantel K, Bartkowiak K, Topart D, Cayrefourcq L, Rossille D, Maudelonde T, Fest T, Alix-Panabieres C. Frequent expression of PD-L1 on circulating breast cancer cells. Mol Oncol. 2015;9:1773–82.
Jilaveanu LB, Shuch B, Zito CR, Parisi F, Barr M, Kluger Y, Chen L, Kluger HM. PD-L1 expression in clear cell renal cell carcinoma: an analysis of nephrectomy and sites of metastases. J Cancer. 2014; 5: 166–72.
Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, Tibbe AG, Uhr JW, Terstappen LW. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004;10:6897–904.
Warkiani ME, Guan G, Luan KB, Lee WC, Bhagat AA, Chaudhuri PK, Tan DS, Lim WT, Lee SC, Chen PC, et al. Slanted spiral microfluidics for the ultra-fast, label-free isolation of circulating tumor cells. Lab Chip. 2014;14:128–37.
Khoo BL, Warkiani ME, Tan DS, Bhagat AA, Irwin D, Lau DP, Lim AS, Lim KH, Krisna SS, Lim WT, et al. Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells. PLoS One. 2014;9:e99409.
George A Crabill 1, Theresa S Pritchard 1, Tracee L McMillerMechanisms regulating PD-L1 expression on tumor and immune cells2019;7(1):305.
Shunli Peng 1, Rong Wang 1, Xiaojuan Zhang EGFR-TKI resistance promotes immune escape in lung cancer via increased PD-L1 expression2019;18(1):165.

Table 1

The clinical data of patients
Characteristic	n	EpCAM+	Vimentin+	EGFR+	CTC < 5	CTC ≥ 5	P
Age							0.650
≥ 60	10	10	9	9	1	9
<60	12	11	11	11	2	10
Gender							0.010**
Male	21	21	20	20	2	19
Female	1	0	0	0	1	0
T stage							0.891
T1/T2	6	6	6	6	1	5
T3/T4	14	13	12	12	2	12
Differentiation degree							0.240
Poorly Differentiated	9	9	9	9	0	9
Moderately differentiated	9	8	8	8	2	7
High differentiated	3	3	2	2	1	2
Tumor Location							0.860
Left	4	4	4	4	0	4
Right	8	8	8	8	1	7
Bilateral	7	7	6	6	1	6
Other	1	1	1	1	0	1
Type							0.378
Pharyngeal Cancer	7	7	7	7	0	7
Laryngocarcinoma	8	7	6	6	2	6
Other	6	6	6	6	1	5

Table 2

Comparison of CTC and tissue-derived PD-L1
No.	1		2		3		4		5		6		7		8		9		10		11		12		13		14		15		16		17		18		19		20
PD-L1	-	-	-	-	+	+	+	+	+	+	+	-	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	+	-	+	+	+	-	+	-	+	+
PD-L1	0		0		1		1		1		1	0	1		1		1		1		1		1		1		1		1		1	0	1		1	0	1	0	1
Type	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T	B	T

Note: “0”: PD-L1 negative, “1” PD-L1 positive, B: Blood; T: Tissue

No competing interests reported.

SupplementaryFigure.zip

Download PDF

Version 1

posted

You are reading this latest preprint version

Determination of PD-L1 Expression in Circulating Tumor Cells of Hypopharyngeal and Laryngeal Cancers and Correlation with Tissue Detection

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Results

Discussion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1