Head and Neck Cancer N-glycome traits are cell line and HPV-status dependent

Announcement date

2023/07/05

Responsible:

Daniel Kolarich . Associate Professor, Griffith University

Description

Glycosylation is the most common post-translational modification of proteins, and glycosylation changes at cell surfaces are frequently associated with malignant epithelia. Many head and neck squamous cell carcinoma (HNSCC), especially those originating in the lymphoid mucosa of the oropharynx, are driven by so-called high-risk genotypes of Human Papillomavirus (HPV). Five-year survival remains poor, averaging around 50% globally: this is partly related to late diagnosis. Specific protein glycosylation signatures on malignant keratinocytes have promise as diagnostic, and prognostic biomarkers and as therapeutic targets. Nevertheless, HNSCC specific glycome is to date largely unknown. Six established HNSCC cell lines were used to capture the qualitative and semi-quantitative N-glycome using porous graphitized carbon LC−ESI-MS/MS. Oligomannose type N-glycans were the predominant features in all HNSCC cell lines analysed (57.5-70%). The levels of sialylated N-glycans showed considerable cell line dependent differences ranging from 24-35%. Importantly, α2-6 linked sialylated N-glycans were dominant across most HNSCC cell lines except in SCC-9 cells where similar levels of α2-6 and α2-3 sialylated N-glycans were observed. Furthermore we found that HPV-positive cell lines contained higher levels of phosphorylated oligomannose N-glycans, which hint towards an upregulation of lysosomal pathways. Almost all fucose type N-glycans carried core-fucose residues with just minor levels (<4%) of Lewis-type fucosylation identified. We also observed paucimannose type N-glycans (2-5.5%), though in low levels. Finally, we identified oligomannose N-glycans carrying core-fucose residues and confirmed their structure by tandem MS. This first systematic mapping of the HNSCC N-glycome revealed diverse and specific glycosylation features in HNSCC cell lines, paving the way for further studies aimed at assessing their possible diagnostic relevance.

Sample preparation

---

1. Sample Origin

General information:
PGC-nanoLC-ESI-MS/MS glycomics

1.1 Biologically derived material

Biologically derived material - Recombinantly produced material

Cell type:
epithelial cell

Growth/harvest conditions for recombinantly produced material:
All of the HNSCC cell lines were STR profiled and authenticated. This gave us the confidence that our cell lines are correctly identified, and not cross contaminated with other cells. In addition, we have also performed mycoplasma testing using Lonza's MycoAlert® Mycoplasma Detection Assays and were confirmed to be of mycoplasma free. Six HNSCC cell lines, including two HPV positive, were purchased from ATCC (American Type Culture Collection) or a generous gift (Supplementary Table 1). Cell culture was performed according to ATCC guidelines. Cells were cultured under standard conditions in humidified incubators at 37°C, 20% O2, 5% CO2. Briefly, 0.6 – 0.7 million cells were seeded into T75 mL flasks and incubated with medium consisting of RPMI-1640, 10% foetal bovine serum and penicillin/streptomycin, of which 2A3 cells were incubated with media consisting of hGlucose DMEM-10%FBS+2nM glutamine +G418 200ug/mL. Cells were harvested (by scraping) using lysis buffer (Ripa buffer) once cell density reached 85-90% confluency and the protein concentration was quantified using BCA kit, Thermo Fisher Scientific.

Biologically derived material - Biological origin of Material

Origin (biological fluid, tissue, etc):
N/A

Species:
Homo sapiens (Human)

Treatments and/or storage conditions:
N/A

Glycoprotein:
N/A

Biologically derived material - Purchased from commercial manufacturer

Vendor and applicable item information:
N/A

1.2 Chemically derived material

Synthesis steps or specify where the equivalent reaction protocol is available:
N/A

Description of starting material:
N/A

2. Sample Processing

2.1 Sample Processing - Isolation

Enzymatic treatments

Enzymes used for oligosaccharide removal or modification of starting material:
PNGase F

Describe vendor or expression and purification procedure:
New England Biolabs

Sample material treated in-solution or immobilized? State also temperature, duration, volume, enzyme concentration:
N/A

Chemical treatments

Define the technique for oligosaccharide release or other chemical modifications:
N/A

Reaction conditions (temperature, duration, volume and chemical concentrations):
N/A

2.2 Sample Processing - Modification

Enzymatic modifications

Describe any treatments made to the isolated material:
N/A

Enzyme concentration, supplier, biological source, incubation time and temperature:
N/A

If novel glycosidase was used, provide information indicating the origin (i.e. species) of the enzyme:
N/A

Chemical modifications

Describe any treatments made to the isolated material:
N/A

Explain the type of modification employed:
N/A

Source of materials, description of kits used, reaction conditions and detailed workflow:
N/A

2.3 Sample Processing - Purification

Purification steps:
C18 CARBON CLEAN UP

3. Defined Sample

Sample name:
N-linked oligosaccharides

Liquid chromatography

---

N/A

MS

---

1. General features

(a) Global descriptors

Instrument manufacturer:
Bruker Daltonik

Instrument model:
6340 Ion Trap LC/MS

Customizations:
N/A

Ion mode:
Negative

(b) Control and analysis software

Software name:
DataAnalysis

Version:
N/A

Upgrades not reflected in version number:
N/A

Switching criteria (tandem only):
N/A

Isolation width (global, or by MS level):
N/A

Location of ‘parameters’ file:
N/A

2. Ion sources

(a) Electrospray Ionisation (ESI)

Supply type (static, or fed):
CAPTIVE SPRAY

Interface name:
N/A

Catalog number, vendor, and any modifications made to the standard specification:
N/A

Sprayer name:
CID

Sprayer type, coating, manufacturer, model and catalog number (where available):
N/A

Relevant voltages where appropriate (tip, cone, acceleration):
N/A

Degree of prompt fragmentation evaluated:
N/A

Whether in-source dissociation performed:
N/A

Other parameters if discriminant for the experiment (such as nebulizing gas and pressure):
N/A

(b) MALDI

Plate composition (or type):
N/A

Matrix composition (if applicable):
N/A

Deposition technique:
N/A

Relevant voltages where appropriate:
N/A

Degree of prompt fragmentation evaluated:
N/A

PSD (or LID/ISD) summary, if performed:
N/A

Operation with or without delayed extraction:
N/A

Laser type (e.g., nitrogen) and wavelength (nm):
N/A

Other laser related parameters, if discriminating for the experiment:
N/A

3. Ion transfer optics

Hardware options:
N/A

(a) Post-source componentry - Collision cell

Collision-Induced Dissociation (CID)

Gas composition:
nitrogen drying gas

Gas pressure:
12 psi

Collision energy CID/function:
N/A

Electron Transfer Dissociation (ETD)

Reagent gas:
N/A

Pressure:
N/A

Reaction time:
N/A

Number of reagent atoms:
N/A

Electron Capture Dissociation (ECD)

Emitter type:
CID

Voltage:
N/A

Current:
N/A

(b) Post-source componentry - TOF drift tube

Reflectron status (on, off, none):
N/A

(c) Post-source componentry - Ion trap

Final MS stage achieved:
N/A

(d) Post-source componentry - Ion mobility

Gas:
N/A

Pressure:
N/A

Instrument-specific parameters:
N/A

(e) Post-source componentry - FT-ICR

Peak selection:
N/A

Pulse:
N/A

Width:
N/A

Voltage:
N/A

Decay time:
N/A

IR:
N/A

Other parameters:
N/A

(f) Post-source componentry - Detectors

Detector type:
N/A