Head and Neck Squamous Cell Carcinoma (HNSCC)-Derived Extracellular Vesicles (EVs) Contribute to Varied Chemokine Expression Profiles in M0 THP-1 Macrophages (MΦ) Aadi Gadekar1, Christopher J Papayannakos1, James A DeVoti1, Mohd Israr1, Fung Lam1, Bettie M Steinberg1, Vincent Bonagura1 1Institute of Molecular Medicine, The Feinstein Institutes for Medical Research at Northwell
BACKGROUND
• 900k new cases of HNSCC annually • Therapy-resistance growing issue •
RESULTS
1. Median EV Diameter Varied in HNSCC Cell Lines
Tumor-associated macrophages (TAMs) • Aid tumor growth • Shape tumor microenvironment (TME) with cytokines and chemokines
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Extracellular Vesicles (EVs), like exosomes, are membrane-bound nanoparticles • Role in tumor-macrophage crosstalk
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Significant differences in EV characteristics observed across HNSCC cell lines
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Significant differences observed in EV secretion rate across HNSCC cell lines
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CM derived from all HNSCC cell lines induced all markers • SCC25 & SCC154 showed highest induction • Mirrors findings from primary monocytes stimulated with CM
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Cytokines influence immune responses, chemokines are chemotactic for immunocytes • IL-6 = inflammation + angiogenesis • CXCL5 = recruits neutrophils • CXCL10 = recruits T-cells •
2. EV Diameters Distributions Were Mostly Similar Across HNSCC Cell Lines
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CONCLUSION
• n=6, *** p <0.001, *p<0.05.
3. EV Secretion Varied Across Cell Lines
Representative distribution from NTA measurements. ***p<0.001
4. SCC25 + SCC154, CM & EVs, Induce IL-6 •
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HPV+ and HPV- EVs induced IL-6 and CXCL10 expression • Limited induction of CXCL5 Induction of CXCL10 by SCC25 EVs suggests MΦ mediated T-cell recruitment
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Future Work
OBJECTIVES
1.
2.
Investigate the physical characteristics and EV secretion rate across HNSCC cell lines. Investigate M0 macrophage response to EVs from different HNSCC cell lines. METHODS
1. THP-1 cells differentiated into MΦ with 100 ng/mL of PMA.
n=6, ***p<0.001, **p<0.01.
5. Robust Induction by CM, Mild EV Induction
IL-6 upregulation observed by SCC25 and SCC154. IL-6 creates pro-tumor TME with angiogenesis and recruitment of myeloid-derived stem cells.
6. SCC25 CM & EVs Induce CXCL10
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Optimize PMA-differentiation of THP-1 cells
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Conduct visualization studies to verify EV uptake by MΦ
3.
Conduct experiments with in primary human monocytes and in vivo models Implications
1. EV-mediated MΦ expression of chemokines and cytokines contributes to immunosuppressive TME
2. Conditioned media (CM) by HNSCC cell lines for 48 hours.
2. New potential therapeutic window for boosting treatment efficacy
3. EVs isolated from CM with ultracentrifugation.
3. Warrants further study on HNSCC-derived exosomal cargo
4. EVs characterized by nanoparticle tracking analysis (NTA). 5. MΦ stimulated with 10 or 100 EVs per cell for 24 hours. 6. qPCR measured gene expression changes normalized to GAPDH.
EVs induced CXCL5 less than CM. CXCL5-mediated neutrophil invasion in the TME shown to have poorer outcomes.
EV-mediated CXCL10 upregulation is similar to the upregulation seen by CM. CXCL10-mediated T-cell recruitment boosts anti-tumor immunity.
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