Cells and cell culture

Two human HNSCC cell lines, CAL-27 (tongue, RRID:CVCL_1107, DSMZ, Braunschweig, Germany) and UD-SCC-5 (laryngeal, RRID:CVCL_L548, University of Düsseldorf, Germany) were cultured in high-glucose Dulbecco´s Eagle´s Minimum Essential Medium (DMEM) supplemented with 10% v/v heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium-pyruvate and antibiotics (10,000 IU/ml penicillin and 10 mg/ml streptomycin), at 37 °C in 5% v/v CO2. Cells were passaged regularly twice a week. For the experiment the cells were used in the exponential growth phase and their viability was tested by trypan blue exclusion. All experiments were performed with mycoplasma-free cells. The cell lines have been authenticated using STR profiling within the last three years. All cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Antibodies and peptides

The FITC-conjugated murine cmHsp70.1 monoclonal antibody (mAb, IgG1, multimmune GmbH, Munich, Germany) and an Hsp70-derived 14-mer peptide TKDNNLLGRFELSG (TPP) that has previously been demonstrated to bind to mHsp70 [16] were used. For flow cytometry and fluorescence microscopy studies, the TPP peptide was conjugated with FITC purchased from EMC Microcollections GmbH (Tübingen, Germany) and for the identification of tumor in freshly resected tissue of HNSCC patients the TPP peptide was conjugated to the near-infrared imaging probe IRDye800CW (Cambridge Research Biochemicals, Billingham, UK). Cetuximab (Erbitux®, RRID:AB_2459632, Merck, Darmstadt, Germany) which targets the epidermal growth factor receptor (EGFR), conjugated to IRDye680RD (IRDye680RD-NHS ester, LI-COR Biosciences, Lincoln, NE, USA), was used as a comparator for the identification of tumor tissue in resected tissue from patients with HNSCC.

Fluorescence microscopy

Cells (10,000 cells/well) were cultured in 8-well chamber slides. After washing with phosphate-buffered saline (PBS), unlabelled cmHsp70.1 mAb was added as a blocking agent to reduce background staining. Following another washing step, the cells were left for 5 min at room temperature to allow viable cells to internalize the cmHsp70.1 mAb-labelled epitopes and re-express free epitopes. Subsequently, FITC-conjugated cmHsp70.1 mAb (100 µg/ml) or an IgG1 isotype-matched control antibody (mouse IgG1-FITC, Cat# 340755, RRID:AB_400127, BD Biosciences, Franklin Lakes, NJ, USA) were added. Cells were then incubated for either 8 min at 4 °C for cell surface staining of Hsp70 or 60 min at 37 °C for internalization. After washing three more times, cells were fixed with PBS containing 0.5% w/v paraformaldehyde (PFA) and embedded in 4’,6-diamidine-2-phenylindole (DAPI) solution (Vector Laboratories, Burlingame, CA, USA). The slides were imaged on the Zeiss Axio Observer Zl microscope (Carl Zeiss AG, Oberkochen, Germany). Images were acquired in brightfield, FITC and DAPI channels. Multicolor images were created by overlay.

Flow cytometry

To determine the mHsp70 status, cells (0.2 × 106) were incubated with FITC-labelled cmHsp70.1 mAb (2 µg/ml) or TPP (2 µg/ml) for 30 min at 4 °C in the dark. After washing with flow cytometry buffer (PBS containing 10% v/v heat inactivated FBS) cells were analyzed using a FACSCalibur™ flow cytometer (BD Biosciences). Flow cytometry was also performed with single-cell suspensions obtained from small tissue samples taken from the tumor area by an experienced pathologist. After mechanical disintegration of the tissue, which was performed as described previously [10], the cells were incubated with cmHsp70.1-FITC mAb and CD45-APC mAb (mouse IgG1, Cat# 555485, RRID:AB_398600, BD Biosciences). CD45 staining was used to distinguish peripheral blood leukocytes (major population) from CD45-negative, non-hematopoietic tumor cells.

In each flow cytometry analysis, propidium iodine (PI) was added prior to flow cytometric analysis in order to enable the exclusion of non-viable cells from the acquisition and analysis. An IgG1-FITC isotype-matched control antibody was used to evaluate nonspecific binding. The percentage of cells stained with the control antibody was subtracted from the percentage of mHsp70 positively stained cells.

Internalization kinetics of cmHsp70.1 antibody and TPP

The targeting capacity of FITC-labelled cmHsp70.1 mAb and TPP was assessed by flow cytometry. For this, 200,000 cells were transferred to 1.5 ml tubes and washed with PBS. Each aliquot was incubated with the antibody or peptide at 4 °C or 37 °C for varying periods of time (0, 1, 2, 5, 10, 15, 20, 30, 60 to 120 min). After the indicated time points, the cells were transferred to tubes containing ice-cold PBS to stop internalization. After washing, cells were resuspended in flow cytometry buffer and analyzed as described above. Only viable (PI-) negative cells were gated and analyzed. The IgG1-FITC isotype-matched control antibody was included in all analyses.

Patients

This study was approved by the Ethics Committee of the Klinikum rechts der Isar, Technical University Munich (TUM) School of Medicine and Health. All patients provided informed written consent 24 h before start of the study. A total of 11 patients with local surgical resection of HNSCC were included into the study. For the analysis of the tracer in lymph nodes one patient with Merkel cell carcinoma and one patient with adenoid cystic carcinoma who underwent primary tumor excision and neck dissection were included into the study. Patients were treated between April 2021 and Juni 2024 in the Department of Otorhinolaryngology at Klinikum rechts der Isar. Patients with varying tumor localizations and TNM-stages were included into the study.

Ex vivo imaging

Immediately after excision, the tissue was covered for 5 min with a blocking solution (5% w/v milk powder and 0.5% v/v Triton-X-100 in PBS, all purchased from Sigma-Aldrich). For a direct comparison, the whole specimen was firstly sprayed with Cetuximab-IRDye680 (100 µg/ml) and then with TPP-IRDye800 (100 µg/ml). The concentration of 100 µg/ml for TPP was based on previous data indicating that this concentration provides an optimal TBR in vivo [14]. With respect to Cetuximab also a concentration of 100 µg/ml was chosen which is in line with a previous study with a topical application [20]. In vitro experiments displayed no overlap in the fluorescence signals with different wavelengths after a sequential application of Cetuximab-IRDye680 followed by TPP-IRDye800 on the same specimen. Specimens were incubated with both tracers for 5 min at room temperature (RT), followed by an intensive rinsing of the sample with PBS and 1% v/v Triton-X-100 before imaging. The IRDye600-dye was then excited with a 670 nm CW diode laser, and the emitted signal was passed through a 780/10 nm filter before being imaged with a back-illuminated EM CCD camera. IRDye800 was excited with a 780 nm CW diode laser, and the emitted signal was passed through an 800 nm long-pass filter. The camera captured an area of 2 × 2 cm. Images of larger specimen were stitched together using the ImageJ software (https://imagej.net/ij) [21]. In addition, lymph nodes with and without histologically proven metastases were imaged using TPP-IRDye800.

TBRs were determined by dividing the mean fluorescence value of cancerous regions of interest (ROIs) by the mean signal intensities of non-cancerous ROIs. The ROIs were selected randomly on the epithelial side of each resected specimen, maintaining a small distance from the electrosurgical cutting borders. For comparison of TPP and Cetuximab, identical ROIs on the same specimen were chosen. All fluorescence image analyses were performed using ImageJ software. Frozen sections of the tumors were obtained from pathologist as part of their intraoperative analysis. The glass slides with the frozen tissue were sprayed with TPP-IRDye680, and wide-field NIR macroscopic images were taken, as previously described. For microscopic imaging, the slides were embedded in DAPI solution and imaged using the 680-nm channel, as outlined in the fluorescence microscopy section.

Evaluation of diagnostic accuracy

A three-step evaluation was performed for 12 selected ROIs. Initially, all ROIs were categorized using a binary system based on histopathology (1: tumor, 0: tumor-free) and validated by an experienced pathologist. Subsequently, the fluorescence intensity of either Hsp70- or EGFR-based imaging was quantified for each region, and a signal-to-background ratio was calculated by dividing each signal by the mean fluorescence value of the verified tumor-free ROIs within the same specimen. This facilitated a more effective intertumoral comparison.

Finally, five experienced otolaryngologists independently assessed the unstained ROIs visually using a five-point Likert scale: (1) indicating a definite absence of tumor; (2) likely absence; (3) indicating uncertainty; (4) suggesting probable presence; and (5) definite presence. The otolaryngologists are practicing in a certified University Hospital Center with a special focus on head and neck tumor surgery which assesses and treats a large number of tumor patients every year and are hence well trained. The surgeons’ evaluations were based on a white light image of the tumors, mirroring their intraoperative judgments. The inter-rater agreement was assessed and a receiver operator curve (ROC) was calculated using the conventional histologic assessment as gold standard.

Histological assessment

Histological evaluation was performed on Hematoxylin and Eosin (H&E) stained slides. For the Hsp70 and EGFR immunohistochemistry staining, deparaffinized and rehydrated FFPE sections (4 µm) of the patient tumors were used and heated by microwaving in target retrieval buffer (pH 6) to unmask antibody epitopes. Nonspecific binding was blocked by incubating with a protein blocking solution (10% v/v rabbit serum in PBS and 1% w/v BSA) for 30 min. After each step, the sections were washed in PBS. An overnight incubation at 4 °C with the cmHsp70.1 mAb (dilution, 1:750 in PBS and 1% w/v BSA) or anti-EGFR mouse mAb (dilution, 1:100 in PBS and 1% w/v BSA, Cat# sc-373746, Santa Cruz Biotechnology, USA) was followed by another incubation with HRP-labelled rabbit anti-mouse secondary reagent (Cat# P0260, RRID:AB_2636929, Dako-Agilent, Santa Clara, CA, USA) for 2 h. Thereafter, a 3,3-diaminobenzidine (DAB + ) chromogen reaction, which was limited to exactly 5 min for all staining procedures was performed. Cell nuclei were counterstained with Hematoxylin. In a final step, all sections were embedded in Eukitt® (Sigma-Aldrich) mounting medium. A reference section with a defined staining intensity was always examined as an internal control. Snapshot images of the stained sections were acquired using the Aperio Slide Scanner (Leica Biosystems, USA). Unless otherwise stated, the staining reagents were purchased by Agilent DAKO, USA. The expression levels of Hsp70 and EGFR in the tumor area were analyzed. To evaluate the staining intensity, tumor sections were categorized as very weak (value 0), weak (value 1), moderate (value 2), strong (value 3), and very strong (value 4). Adjacent normal mucosa was used as a control. For quantitative evaluation, the percentage of fields showing positively stained cells was considered. The values ranged from 0: < 1% positive stained tumor cells; 1: 1–25% positive tumor cells; 2: 25–50%; 3: 50–75% and 4: > 75%. The final expression level was then calculated by combining the qualitative and quantitative scores. Two independent investigators performed the assessment. All scores were blinded to the grading and the fluorescence intensities of each tumor sample.

Clinical imaging system

The KARL STORZ IMAGE1 S™ Rubina® (KARL STORZ, Tuttlingen, Germany), a technology developed for NIR/ICG imaging, was evaluated for the detection of TPP-IRDye800. For this study the HOPKINS® NIR/ICG telescope (0°, 10 mm, 20 cm) was used in white light and NIR mode. Color videos and fluorescence images are displayed in real time. Via the IMAGE 1 S™ software menu control, different NIR/ICG visualization modes can be activated (overlay, intensity map, monochromatic). For comparison of the preclinical and clinical imaging systems, the exact same ROIs were selected and analyzed using ImageJ.

Statistical analysis

Statistical analysis of the cell culture experiments as well as the comparison of TBR between TPP and Cetuximab was performed using Student´s t-test. Data are presented by mean ± SD. P-values ≤ 0.05 were considered significantly different between the compared groups. ROC analysis was used to evaluate the diagnostic performance of the fluorescence spray and the tumor assessment by surgeons compared to histopathology as gold standard. The differences between the calculated areas under the curve (AUC) were compared using the DeLong test. Analyses were performed using SigmaPlot software (Systat Software Inc, USA). The inter-rater agreement was assessed using a weighted κ coefficient for multiple raters with quadratic weights using the statistical software R [22] and its package irrCAC [23].