Oral Cancer Cells Release Vesicles that Cause Pain

Abstract

Oral cancer pain is attributed to the release from cancers of mediators that sensitize and activate sensory neurons. Intraplantar injection of conditioned media (CM) from human tongue cancer cell line HSC-3 or OSC-20 evokes nociceptive behavior. By contrast, CM from noncancer cell lines, DOK, and HaCaT are non-nociceptive. Pain mediators are carried by extracellular vesicles (EVs) released from cancer cells. Depletion of EVs from cancer cell line CM reverses mechanical allodynia and thermal hyperalgesia. CM from non-nociceptive cell lines become nociceptive when reconstituted with HSC-3 EVs. Two miRNAs (hsa-miR-21-5p and hsa-miR-221-3p) are identified that are present in increased abundance in EVs from HSC-3 and OSC-20 CM compared to HaCaT CM. The miRNA target genes suggest potential involvement in oral cancer pain of the toll like receptor 7 (TLR7) and 8 (TLR8) pathways, as well as signaling through interleukin 6 cytokine family signal transducer receptor (gp130, encoded by IL6ST) and colony stimulating factor receptor (G-CSFR, encoded by CSF3R), Janus kinase and signal transducer and activator of transcription 3 (JAK/STAT3). These studies confirm the recent discovery of the role of cancer EVs in pain and add to the repertoire of algesic and analgesic cancer pain mediators and pathways that contribute to oral cancer pain.

Keywords: extracellular vesicles; miR-21; miR-221; miRNA; oral cancer; oral cancer induced pain; pain.

Figure 1.

Intraplantar injection of conditioned media from oral cancer cell lines evokes nociceptive behavior in mice. The allodynic effect of samples injected into the right hind paw was measured by assaying induced mechanical nociception (a) and thermal hyperalgesia (b) at baseline (before injection) and at 1, 3, 6, 12 and 24 hours after injection. *p< 0.05, **p< 0.01, ***p<0.001, ****p<0.0001 cell line CM versus cell line baseline indicated by color, Two-way ANOVA with Dunnett’s multiple comparisons test. Cell line CM induced nociception measured in response to mechanical (c) and thermal (d) stimuli is summarized as the negative area under the curve (AUC) measured up to 12 hours after injection relative to baseline. **p< 0.01, ***p<0.001, ****p<0.0001, Brown-Forsythe and Welch ANOVA tests with Dunnett T3 multiple comparisons test.

Figure 2.

Oral cancer cells release EVs with the characteristics of exosomes. (a) Isolated EVs express exosome endocytic marker proteins TSG101 and ALIX, and tetraspanin, CD63. Calnexin (CANX, endoplasmic reticulum marker) and GM130 (cis-Golgi network marker) were not detected. (b) Representative transmission electron micrographs of EVs isolated from HaCaT, DOK, OSC-20 and HSC-3 conditioned media. (c) The EVs from DOK cells are significantly smaller than the EVs from HaCaT, OSC-20 and HSC-3 cells. Diameters of EVs (mean ± SD) measured from TEM images taken at 92,000× magnification (10–12 fields per sample). Dotted lines indicate y-axis values = 40, 80 and 120 nm. Diameters of EVs from the cell lines (mean ± SD). HaCaT: 51.31±23.06 nm, n=201; DOK: 36.53±15.49 nm, n=615; OSC-20: 52.84±25.79 nm, n=427; HSC-3: 51.68±25.18 nm, n=193 (F_(3,1432) = 62.77, ****p<0.0001, one-way ANOVA with Tukey’s multiple comparisons test). Data independently replicated.

Figure 3.

Depletion of EVs from cancer cell line CM reverses mechanical allodynia and thermal hypersensitivity. The allodynic effect of samples injected into the right hind paw was measured by assaying induced mechanical nociception (a and b) and thermal hyperalgesia (c and d) at baseline (before injection) and at 1, 3, 6, 12 and 24 hours after injection (group sizes are given in Table S5). Cell line CM induced nociception measured in response to mechanical (c) and thermal (d) stimuli is summarized as the negative area under the curve (AUC) measured up to 12 hours after injection relative to baseline. *p<0.05, **p< 0.01, ***p<0.001, ****p<0.0001, Brown-Forsythe and Welch ANOVA tests with Dunnett T3 multiple comparisons test.

Figure 4.

Total RNA isolated from EVs is mostly small RNA. Shown are electropherograms of total RNA from EVs using the Pico (a) and Small (b) Agilent Bioanalyzer assays. (c) Composition of EV miRNA cargoes differs among oral cancer cell lines HSC-3 and OSC-20 and keratinocyte line HaCaT. MicroRNAs with counts >50 in at least one sample in the NanoString nCounter assay were clustered by Euclidean distance and Ward linkage. Shown are cell lines in columns and miRNAs in rows. (d) Both miR-21-5p and miR-221-3p are more abundant in total RNA isolated from cancer cell line EVs than from HaCaT EVs as measured by qRT-PCR. Expression levels determined as the efficiency-corrected target quantity (N₀) were measured in equal amounts of cDNA reverse transcribed from total RNA (3 ng) from three to four independent isolates of EVs. Plotted are the grand means and 95% confidence intervals. Expression levels of miR-21-5p for HaCaT: mean=0.009, n=3; OSC-20: mean=0.04, n=4; HSC-3: mean=0.09, n=3 (F_(2,7) = 34.24, *p<0.05, **p<0.01, ***p<0.001, nested one-way ANOVA with Tukey’s multiple comparisons test). Expression levels of miR-221–3p for HaCaT: mean=0.005, n=3; OSC-20: mean=0.01, n=4; HSC-3: mean=0.02, n=3 (F_(2,7) = 96.66, **p<0.01, ****p<0.0001, nested one-way ANOVA with Tukey’s multiple comparisons test). Data replicated in two independent cDNA preparations.

Figure 5.

Multiple pathways contribute to oral cancer pain. Oral cancer cells release soluble mediators and EVs into the cancer microenvironment that activate and sensitize nociceptors. Cancer pain mediators interact with membrane bound receptors to activate downstream signaling pathways that increase expression or potentiate TRPV1 and TRPA1. Neuronal interactions may occur via ligand-receptor interactions with pain mediators that are soluble or carried on EVs (e.g., proteases activating Par₂, IL6/gp130 signaling) and by internalization of EV cargoes following fusion, phagocytosis or endocytosis of the EVs, resulting in transfer of proteins and miRNAs that modify neuronal gene expression (e.g., miR-221, let-7d-5p) or act as ligands (e.g., miR-21/TLR7 signaling). Pain mediators include, in addition to those discussed in the text, endothelin (ET-1, encoded by EDN1, receptor ETAR, encoded by endothelin receptor type A, EDNRA), acids (H⁺), artemin (encoded by ARTN, receptors GFRα3/RET, encoded by GDNF family receptor alpha 3 and ret proto-oncogene, GFRA3 and RET), nerve growth factor (NGF, receptor TrkA, encoded by neurotrophic receptor tyrosine kinase 1, NTRK1), adenosine triphosphate (ATP, receptors P2X2R and P2X3R, encoded by purinergic receptors P2X 2 and 3, P2RX2 and P2RX3) and tumor necrosis factor alpha (TNFα, encoded by TNF, receptor TNFR2 encoded by TNF receptor superfamily member 1B TNFRSF1B).