Human tissues and cells

Human protocols and informed consent were approved by University Institutional Review Boards. These include an initial clinical study to examine PDPN expression on OSCC cells in oral lesion resections (Rutgers #CR00006596 and Rowan #Pro2012001544) and a Phase 1 clinical trial (USFDA IND #118210 and clinicaltrials.gov #NCT04188665) to study the effects of MASL on OSCC cell morphology, PDPN expression, growth, and motility in oral lesion biopsies and resections (Rutgers #CR00024686 and Rowan #Pro20140000009). Patients diagnosed with OSCC were randomized 1:1 by coin toss and treated with placebo or oral lozenges containing 100 mg of essentially pure MASL prepared and compounded at the University of California GMP facility (2315 Stockton Boulevard, Sacramento, CA 95817) in a manner consistent with best care practice. This 100 mg MASL dose was based on the concentration of M. amurensis lectins found in traditional concoctions used to treat cancer and inflammation (Hellmig et al. 2023). Cells obtained from surgical biopsies and resections were cultured from minced tissue washed in PBS, digested with trypsin (Hyclone SH30042.01 or Gibco 25200-056), and incubated in DMEM (Hyclone SH30021) supplemented with 25 mM HEPES (Hyclone SH30237), 1000/100 IU/ml penicillin/streptomycin (Corning 30-002-CI), 2.5 ug/ml amphotericin b (HyClone SV30078.01), and 10% FBS (Seradigm 1400–500) at 37 °C in 5% CO2 and 100% humidity as described (Yin et al. 2024). Cell morphology and pan-cytokeratin expression were evaluated to confirm the malignant identity of these cells. Cells obtained from 11 out of the 19 patients (~ 58%) were successfully adapted to culture while 8 (~ 42%) failed to grow or succumbed to contamination.

Immunohistochemistry

Surgical specimens were fixed in 10% formalin in PBS, paraffin embedded, sectioned (4 μm), and processed for hematoxylin/eosin (H&E) staining and immunohistochemistry with antibodies specific for PDPN (D2-40 Agilent M361901-2), CD3 (2GV6 Roche/Ventana 790–4341), E-cadherin (26 Roche/Ventana 790–4497), and N-cadherin (SP90 Fisher PIMA516324) as described (Ochoa-Alvarez et al. 2015; Retzbach et al. 2018; Yin et al. 2024). Tissue morphology and PDPN expression was graded on a scale of 1 to 5, with five displaying the highest level of cellular dysplasia and PDPN expression, by two board certified pathologists in an independent and blind manner. Numbers of CD3 positive T cells were counted in 200 × 200 micron areas central to OSCC tissue to quantitate patient immune cell infiltration into tumors. Cells were visualized by phase contrast and bright field microscopy with a Zeiss Axiovert 5 microscope equipped with ZeissAxioCam Mrc cameras and Zen software as previously described (Nicoletto et al. 2024; Yin et al. 2024).

Western blot analysis

Western blotting was performed as previously described (Nicoletto et al. 2024; Sheehan et al. 2022; Yin et al. 2024). Briefly, cells were grown to confluence and lysed with 2% SDS, 10% glycerol, 10 mM EDTA, 50 nM DTT, 50 mM NaF, 1 mM Na3VO4, and 1 mM PMSF in 62.5 mM Tris pH 6.8. Protein concentration was measured by Coomassie (Fisher 1856209) with BSA standards (Fisher 23210). Equal amounts of protein (10ug/lane) were resolved by SDS-PAGE, transferred to Immobilon membranes (Millipore IPVH00010), and incubated with primary antiserum specific for PDPN (Agilent Dako M3619), pan-cytokeratin (Cell Signaling 4068 S), and GAPDH (Cell Signaling 2118 S). Primary antiserum was probed with appropriate secondary anti-rabbit (Cell signaling 7074 S) or anti-mouse (Invitrogen 31430) IgG antibodies and detected by chemiluminescence (Fisher 32106 or Fisher 34095). Signal intensity was quantitated by Image J (NIH version 1.54f). Transferred gels and membranes were stained with Coomassie dye and India ink, respectively, to verify equal loading and transfer.

Cell viability and migration assays

Effects of reagents on OSCC cell growth and motility were performed as described (Yin et al. 2024). Briefly, cells were grown to confluence on 6 well tissue culture cluster plates (Falcon 353224). For migration assays, cell monolayers were scratched and visualized at 0 and 24 h after treatment with 0 nM, 770 nM, 1540 nM, and 3080 nM MASL. Images were taken at 0 and 24 h after treatment, and migration was quantitated as the distance cells traveled from the edge to the center of the wound. Sister plates treated with MASL for 24 h were incubated with alamarBlue (BioRad #BUF012A) for 2 h and assayed (ex/em: 570/600 nm) to assess cell viability. Cell morphology was also examined on cells cultured on chambered culture slides (Falcon 354104) and stained with H&E (Abcam ab2458801). Cells were visualized by phase contrast microscopy with a Zeiss Axiovert 5 microscope equipped with ZeissAxioCam Mrc cameras and Zen software as previously described (Nicoletto et al. 2024; Yin et al. 2024). All assays were performed at least 3 times with similar results.

Cell surface PDPN expression analysis by flow cytometry

OSCC cells were grown to confluence, trypsinized, suspended in media (2 × 105 cells/ml), incubated with 1 ug/ml PE labeled anti-PDPN mAb clone NC-08 (Biolegend 337003) (Suzuki et al. 2022) or PE labeled isotype mAb control (Biolegend 402304) for one hour at 4 °C, washed with PBS, and analyzed with a flow cytometer (FACSLyric, BD Biosciences, San Jose, CA, USA) with FlowJo software (BD Biosciences). PDPN expression in each cell line was compared as relative fluorescent intensity (RFI) calculated as the median fluorescent intensity (MFI) of antibody staining divided by the MFI of isotype control.

IR700 antibody conjugation and NIR-PIT analysis

IR700 antibody conjugation and NIR-PIT analysis were performed as described (Furusawa et al. 2022; Kato et al. 2023). Briefly, 1 mg (6.8 nmol) NZ-1 mAb was incubated with 66.8 µg (34.2 nmol) IR700 NHS ester (LI-COR Biosciences, Lincoln, NE, USA) in phosphate buffer (pH 8.5) at room temperature for one hour. Conjugated antibodies were purified by Sephadex G25 column chromatography (PD-10; Cytiva, Piscataway, NJ, USA) and concentrations were determined by absorption at 689 nm using UV-Vis (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). Effects of IR700 conjugated antibodies on OSCC cell viability after near-infrared exposure were evaluated by flow cytometry. Briefly, OSCC cells were seeded on 24 well plates (2 × 105 cells/well) and incubated overnight to form confluent monolayers. IR700 labeled antibody was then added to each well to a final concentration of 4 ug/ml and incubated for 1 h at 37 °C. NIR light (150 mW/cm2) was applied to achieve 0, 5, 10, 20, or 50 Joules (J) before cells were immediately trypsinized, collected, stained with Fixable Viability Dye eFluor 506 (ThermoFisher Scientific 65–0866), and analyzed by flow cytometry (FACSLyric, BD Biosciences, San Jose, CA, USA) with FlowJo software (BD Biosciences).

Statistical analysis

Statistical analysis was performed with Prism software (GraphPad Prism version 10) as described in text and figure legends.