Thrombospondin-1 induces immunogenic cell death in human mucoepidermoid carcinoma MC-3 cells via the PERK/eIF2α signaling pathway: potential implications for tumor immunotherapy

Abstract

Objective: To investigate whether Thrombospondin-1 (TSP-1) induces immunogenic cell death (ICD) in human mucoepidermoid carcinoma (MC-3) cells and explore its potential to induce calreticulin (CRT) exposure via the PERK/eIF2α signaling pathway.

Methods: The MC-3 cell line was used as the research model. The CCK-8 assay was performed to determine the optimal seeding density, TSP-1 concentration, and treatment time. Annexin V/PI double staining combined with flow cytometry was used to assess apoptosis across different experimental groups (blank control, TSP-1, paclitaxel (PTX), TSP-1 + PTX). Cells were divided into groups: blank control, PTX, TSP-1, TSP-1 + ISRIB (ISRIB: Integrated Stress Response Inhibitor), and TSP-1 + PTX, and CRT expression was detected by flow cytometry. Immunofluorescence, Western blot, and qPCR were used to detect the expression of PERK (Protein Kinase R-like Endoplasmic Reticulum Kinase), eIF2α (eukaryotic Initiation Factor 2α), and CRT. All experiments were performed in triplicate, and data were analyzed using GraphPad Prism 8.0 software. Statistical significance was set at P < 0.05.

Results: At a seeding density of 2 × 10⁴/mL, MC-3 cells reached the growth plateau by day six. The optimal concentration and duration of TSP-1 treatment were 0.1 μmol/L and 72 h, respectively. Flow cytometry, immunofluorescence, Western blot, and qPCR results revealed that TSP-1 significantly induced CRT exposure in MC-3 cells (P < 0.05), accompanied by the upregulation of PERK and eIF2α expression (P < 0.05). Co-treatment with PTX further enhanced these effects, while the addition of ISRIB reduced the expression of PERK, eIF2α, and CRT (P < 0.05).

Conclusion: TSP-1 induces ICD in MC-3 cells, accompanied by CRT exposure, potentially mediated through the activation of the PERK/eIF2α signaling pathway. These findings suggest that TSP-1 may have potential as an adjunct to chemotherapy for enhancing tumor immunotherapy.

Keywords: Calreticulin; Immunogenic death; MC-3 cell line; PERK/eIF2α signaling pathway; Thrombospondin-1; Tumor immunotherapy.

Fig. 1

Optimal seeding density and growth curve of MC-3 cell line

Fig. 2

Inhibitory effect of TSP-1 protein on the MC-3 cell line

Fig. 3

Detection of apoptosis by flow cytometry. A Apoptosis rate of the MC-3 cell line after culturing with the same volume of DMEM (control); B apoptosis rate of the MC-3 cell line after induction by TSP-1; C apoptosis rate of the MC-3 cell line after culturing with PTX; D apoptosis rate of the MC-3 cell line after culturing with TSP-1 + PTX

Fig. 4

Detection of CRT positive expression rate by flow cytometry. A Selection of gated cells for CRT expression detection by flow cytometry; B CRT positive expression rate after culturing in the same volume of DMEM; C CRT positive expression rate after culturing with PTX; D CRT positive expression rate after culturing with TSP-1; E CRT positive expression rate after culturing with TSP-1 + ISRIB; F CRT positive expression rate after culturing with TSP-1 + PTX

Fig. 5

PERK immunofluorescence in MC-3 cells (×400). Cytoplasmic PERK expression shown in red fluorescence (a, d, g, j, m); DAPI staining shown in blue fluorescence under the same field (b, e, h, k, n); Merged results of PERK red fluorescence and DAPI blue fluorescence (c, f, i, l, o); Blank control group (a–c); PTX treatment group (d–f); TSP-1 treatment group (g–i); TSP-1 + ISRIB treatment group (j–l); TSP-1 + PTX treatment group (m–o); (scale bar = 50 μm)

Fig. 6

eIF2α immunofluorescence staining in MC-3 cells (×400). Cytoplasmic eIF2α expression shown in green fluorescence (a, d, g, j, m); DAPI staining shown in blue fluorescence under the same field (b, e, h, k, n); merged results of eIF2α green fluorescence and DAPI blue fluorescence (c, f, i, l, o); blank control group (a–c); PTX treatment group (d–f); TSP-1 treatment group (g–i); TSP-1 + ISRIB treatment group (j–l); TSP-1 + PTX treatment group (m–o); (scale bar = 50 μm)

Fig. 7

CRT Immunofluorescence Staining in MC-3 Cells (× 400)Cytoplasmic CRT expression shown in red fluorescence (a, d, g, j, m); DAPI staining shown in blue fluorescence under the same field (b, e, h, k, n); merged results of CRT red fluorescence and DAPI blue fluorescence (c, f, i, l, o); blank control group (a–c); PTX treatment group (d–f); TSP-1 treatment group (g–i); TSP-1 + ISRIB treatment group (j–l); TSP-1 + PTX treatment group (m–o); (scale bar = 50 μm)

Fig. 8

PERK, eIF2α, and CRT protein expression levels in different groups