This study found that TSP-1 exhibited better inhibitory effects on MEC cells after 72 h of treatment. At concentrations below 0.1 µmol/L, TSP-1’s inhibitory effect on the MC-3 cell line increased with concentration in a positive correlation. Conversely, at concentrations above 0.1 µmol/L, the inhibitory effect decreased, suggesting a dose-dependent effect. Notably, at 24 h with concentrations of 0.0125 µmol/ml, 0.025 µmol/ml, 0.4 µmol/ml, 0.8 µmol/ml, and 1.6 µmol/ml, TSP-1 surprisingly promoted cell proliferation, which requires further investigation. Additionally, the potential cytotoxicity of TSP-1 at concentrations beyond 0.1 µmol/L should be rigorously explored, especially in combination with other chemotherapeutic agents, to ensure its safety and efficacy in clinical applications. However, the overall trend indicates that TSP-1 primarily inhibits MC-3 cell proliferation. When compared to PTX, a known inducer of ICD, PTX exhibited a stronger effect. Combining TSP-1 with PTX showed a more significant inhibitory effect on MC-3 cells than either treatment alone, suggesting potential clinical applications for combining TSP-1 with chemotherapy to enhance tumor treatment. Flow cytometry results indicate that TSP-1 may have some cytotoxic effects on MC-3 cells, though weaker than PTX. As a naturally secreted multifunctional matrix protein, TSP-1 lacks the potent cytotoxicity of chemotherapeutic drugs like PTX, but its ability to inhibit angiogenesis, reduce tumor adhesion, and lower invasiveness makes it a promising research target [19].

TSP-1 is a multifunctional matrix protein that acts on vascular endothelial cells during tumor angiogenesis, either promoting or inhibiting the formation of new blood vessels. TSP-1 can also directly affect tumor cells, participating in processes such as cell growth, adhesion, invasion, migration, and apoptosis [20,21,22]. TSP-1 exhibits concentration-dependent effects; at different concentrations, it can either promote or inhibit tumor cell growth, likely due to its binding to different molecular receptors and activating various signaling pathways [23, 24]. Studies have shown [15] that TSP-1, through binding to the CD36 receptor, can inhibit the growth of MEC cells, reduce their adhesion ability, and downregulate MMP-9 synthesis, thus suppressing the invasive capacity of MEC cells. Additionally, TSP-1 can inhibit MEC cell migration, although CD36 or CD47 receptors do not play a major role in this process. TSP-1 mainly induces apoptosis in MEC cells by binding to the CD47 receptor, upregulating caspase-3 expression [25, 26].

ER stress-induced CRT membrane translocation is considered one of the classic core events in ICD induction [27]. PERK is an upstream signaling molecule critical for CRT translocation, and phosphorylated PERK serves as a marker of ER stress [28]. Panaretakis et al. [14] found that CRT exposure may be related to the activation of the PERK/eIF2α signaling pathway, as anthracycline chemotherapy drugs can induce high expression of PERK, eIF2α, and CRT in mouse tumor cells, while silencing PERK reduced the expression of all three proteins.

In addition to specific chemotherapy drugs, treatments like hyperthermia and photodynamic therapy can also induce CRT exposure via the PERK/eIF2α pathway [29,30,31,32]. Studies have demonstrated that the TSP-1-derived CD47 agonist (PKHB1) induces tumor cell apoptosis in chronic lymphocytic leukemia and can activate CD47 to induce ICD, triggering CRT, HSP70, HSP90 exposure, and ATP and HMGB1 release. These results suggest that TSP-1 or its derivatives may activate CD47 to induce ICD and early CRT exposure [33]. Our findings suggest that TSP-1 may mediate CRT exposure through PERK [14, 34, 35].

To further investigate whether PERK and eIF2α are involved in TSP-1-induced CRT translocation, our immunofluorescence experiments showed that when TSP-1 was applied for 72 h at a concentration of 0.1 µmol/ml, the expression of PERK, eIF2α, and CRT in MC-3 cells significantly increased. In the TSP-1 + ISRIB group, PERK, eIF2α, and CRT expression decreased compared to the TSP-1 group, suggesting that ISRIB may downregulate the expression of these proteins via the PERK/eIF2α pathway. The expression of PERK, eIF2α, and CRT in the PTX treatment group was similar to that in the TSP-1 group, suggesting that TSP-1 might activate the same PERK/eIF2α pathway as PTX to induce CRT exposure. In the TSP-1 + PTX group, PERK, eIF2α, and CRT expression levels were higher than in the PTX group, indicating that the combination of TSP-1 and PTX may more effectively activate the PERK/eIF2α pathway. The PERK/eIF2α signaling pathway and CRT translocation have been observed in multiple tumor cells undergoing ICD [14], making this pathway a classic biological event in ICD induction.

At both the protein and gene levels, this study confirms that TSP-1 induces CRT translocation in MEC cells by activating the PERK/eIF2α pathway. WB results showed that after 72 h of TSP-1 treatment, PERK, eIF2α, and CRT expression in MC-3 cells increased. The expression levels in the PTX and TSP-1 groups were similar, but the combination treatment showed the best results. Adding ISRIB significantly reduced the expression of PERK, eIF2α, and CRT. qPCR experiments further confirmed the relationship between TSP-1, PERK, eIF2α, and CRT, consistent with the Western blot results. This suggests that TSP-1 may promote CRT translocation via the PERK/eIF2α pathway.

The process of CRT membrane translocation is quite complex, regulated by various factors such as reactive oxygen species (ROS) and the activation of caspases [14, 36]. TSP-1 does not act directly on the endoplasmic reticulum (ER); rather, it indirectly induces ER stress by inhibiting the expression of the transcription regulator BRD4, which affects the normal transcription and translation of other important proteins. As a result, TSP-1-induced ICD resembles anthracycline-induced ICD and falls under Type I ICD. This type of ICD is regulated by the caspase family, and inhibiting caspase activation can block CRT membrane translocation triggered by Type I ICD inducers [37, 38]. Compared to Type II ICD induced by photodynamic therapy and radiation, TSP-1-induced CRT exposure is more gradual, whereas photodynamic therapy can upregulate CRT expression on the surface of human bladder cancer T24 cells within 30 min [36].

The ICD-inducing capacity of TSP-1 presents multifaceted opportunities for clinical translation. Mechanistically, its synergy with conventional chemotherapy agents such as paclitaxel (PTX) could be strategically leveraged—while PTX exerts direct cytotoxic effects, TSP-1 may amplify immunogenic signaling through PERK/eIF2α-mediated calreticulin exposure, potentially creating a therapeutic loop that enhances both tumor elimination and antigen-specific immune activation. This combinatorial approach could be particularly advantageous in tumors exhibiting chemotherapy resistance, where dual-pathway targeting might overcome treatment refractoriness. Furthermore, the immunomodulatory properties of TSP-1 in remodeling the tumor microenvironment provide a rational basis for integration with immune checkpoint inhibitors. By promoting dendritic cell maturation through DAMPs release, TSP-1-induced ICD may convert “cold” tumors into immunologically “hot” niches, thereby potentiating PD-1/PD-L1 blockade efficacy. Notably, TSP-1’s dual functionality in simultaneously suppressing angiogenesis via CD36 receptor binding and inducing pro-immunogenic apoptosis through CD47 interactions suggests its unique value in highly vascularized malignancies like MEC. However, critical challenges persist in optimizing dose scheduling to balance its concentration-dependent biphasic effects, as our in vitro data revealed paradoxical proliferation promotion at supraphysiological concentrations (> 0.1 μmol/L). Future therapeutic designs must also address potential off-target effects on normal vasculature and develop biomarker-driven strategies to identify patients most likely to benefit from TSP-1-based regimens.

Although the study provides insights into the role of TSP-1 in inducing ICD and CRT membrane translocation, several key questions remain unanswered. First, the exact mechanism by which TSP-1 influences CRT translocation through the PERK/eIF2α signaling pathway is not fully elucidated. While the study suggests that TSP-1 indirectly causes ER stress by inhibiting BRD4, the potential involvement of reactive oxygen species (ROS) and the extent to which caspase activation regulates CRT exposure require further investigation. Additionally, the comparison between TSP-1-induced Type I ICD and the more rapid Type II ICD triggered by photodynamic therapy raises questions about the timing and efficacy of TSP-1 as an ICD inducer. Our study primarily focused on the short-term effects of TSP-1 on cell proliferation and apoptosis, with a treatment duration of 72 h. Future studies should investigate the chronic effects of TSP-1 on tumor progression, recurrence, and resistance mechanisms. The clinical relevance of our findings requires validation in animal models and, ultimately, clinical trials. Investigating the efficacy of TSP-1 in combination with established immunotherapies will be crucial for establishing its therapeutic potential. Lastly, the broader implications of TSP-1’s effects on other DAMPs in MC-3 cells remain unclear, warranting future research to explore these signaling pathways in greater detail.