Enhancing antitumor immunity via ROS-ERS and pyroptosis-induced immunogenic cell death in multiple myeloma

Abstract

Background

Few studies have focused on the development of multiple myeloma (MM)-specific immunotherapies. Tumor immunogenic cell death (ICD), triggered by damage-associated molecular patterns, may enhance MM-specific antitumor activity, offering a potential treatment strategy.

Methods

This study confirms that combining reactive oxygen species (ROS)-endoplasmic reticulum stress (ERS) and pyroptosis-inducers (ROS-ERS inducer 1 (REI) and Quillaja saponaria fraction 21 (QS-21), respectively) activates specific anti-MM immunity. MM cell lines were treated with REI and QS-21 alone or in combination and cytotoxicity and apoptosis were examined. ICD markers were identified, including calreticulin, ATP, heat shock protein 70, and high mobility group box 1. Additionally, changes in mitochondrial damage, endoplasmic reticulum stress, pyroptosis markers, and immune markers of dendritic cell (DC) maturation and T-cell activation were assessed both in vitro and in vivo.

Results

ROS-ERS combined with pyroptosis significantly induces MM cell apoptosis and enhances ICD marker activation. The combination treatment induces severe mitochondrial damage and endoplasmic reticulum stress, further promoting pyroptosis and MM-specific T-cell activation. In vivo, the combination treatment reduces tumor growth and improves DC and T-cell activation.

Conclusions

Thus, ROS-ERS inducers and pyroptosis inducers together significantly enhance the immunogenic response against MM, providing a promising strategy for MM treatment by activating powerful specific T-cell antitumor immunity.

Correspondence to Dr Rong Fu; [email protected] ; Dr Liu Zhaoyun; [email protected]

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Immunogenic cell death (ICD) and pyroptosis can stimulate antitumor immunity, but their individual effects are limited in overcoming the immunosuppressive microenvironment of multiple myeloma (MM).

WHAT THIS STUDY ADDS

• This study demonstrates that combining reactive oxygen species-endoplasmic reticulum stress-mediated ICD with Quillaja saponaria fraction 21-induced pyroptosis synergistically enhances MM cell immunogenicity, promotes dendritic cell maturation, and amplifies T-cell-specific antitumor responses.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Our findings provide a basis for novel combination immunotherapy strategies in MM by co-targeting ICD and pyroptosis to improve immune response and therapeutic efficacy.

Background

Multiple myeloma (MM) is a plasma cell malignancy characterized by the abnormal proliferation of plasma cells that produce excessive amounts of monoclonal immunoglobulin (M protein), leading to its accumulation and subsequent organ damage.¹ The pathogenesis of MM is complex, and immune dysregulation is one of the important pathogenic mechanisms of MM.² In patients with MM, T cells exhibit characteristics of exhaustion, leading to limited functionality. Exhausted T cells cannot recognize and attack MM cells effectively, weakening the patient’s immune response.³ Several currently used therapies include anti-CD38 antibodies,⁴ proteasome inhibitors such as bortezomib and carfilzomib,^5–7 and chimeric antigen receptor T cells,⁸ which have significantly improved the survival of patients with MM. However, almost all patients with MM will relapse or fail to respond to the current treatments.⁹ Because of immunosuppression in MM,¹⁰ it is particularly important to find treatment methods that can stimulate immunogenic responses in tumors and improve patient prognosis.

The most important method for enhancing the specific immunogenicity of tumor cells is immunogenic cell death (ICD). According to the Nomenclature Committee on Cell Death, ICD is defined as “a regulated cell death that is sufficient to activate an adaptive immune response in immunocompetent syngeneic hosts”.¹¹ Owing to the enhanced antigenicity of tumor cells and their ability to generate adjuvant signals, this complex mechanism can trigger a tumor-targeted immune response.¹² The main ICD markers include cell-surface translocation of calreticulin (CALR)¹³ and extracellular secretion of ATP. This event, along with exposure to other damage-associated molecular patterns (DAMPs) in the tumor microenvironment (TME), stimulates the antitumor immune response by inducing immature dendritic cells (DCs) to mature, thereby promoting tumor antigen processing, which activates T-cell-mediated killing and phagocytosis. Moreover, ICD induction establishes lasting protective anticancer immunity, and increased immune cell infiltration transforms an immunosuppressive TME into an immunogenic one, enhancing the tumor’s response to immunotherapy.¹⁴

In addition to ICD, pyroptosis is another newly defined mode of immunogenic programmed cell death characterized by the rapid rupture and swelling of cell membranes, contributing to adaptive immune responses.¹⁵ During pyroptosis, the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activates caspase-1 to induce gasdermin D (GSDMD)-dependent pyroptosis and promote interleukin (IL)-1β and IL-18 release. GSDM family members, including GSDMD, are cleaved by cysteine proteases to yield the active N-terminal domain—a pore-forming region that penetrates the cell membrane, creating nanoscale pores that release tumor-associated antigens and DAMPs while enhancing cytokine entry into tumor cell cytoplasm.¹⁶ Compared with ICD, the cleavage mechanism of pyroptosis has a more pronounced impact on the host’s immune system.¹⁷

While both ICD and pyroptosis play critical roles in triggering immune responses against tumors, the use of either approach alone has limited efficacy in transforming the immunosuppressive MM microenvironment. ICD primarily enhances antigen presentation and releases DAMPs. Nonetheless, the highly immunosuppressive MM microenvironment often dampens immune responses. In contrast, although pyroptosis inducers boost inflammatory responses and recruit immune cells, they may lack adequate antigen-presenting capacity on their own. Therefore, a combination strategy that integrates ICD, pyroptosis, and additional immune-modulating mechanisms is necessary to achieve a more robust antitumor immune response.

Recent evidence indicates that reactive oxygen species (ROS) and endoplasmic reticulum stress (ERS) are pivotal in ICD and pyroptosis. ROS accumulation promotes ICD via ERS induction, resulting in CALR exposure and ATP release. We hypothesize that combining ROS-ERS with pyroptosis inducers synergistically enhances tumor immunogenicity, yielding a stronger, sustained anti-MM immune response.

Here, we underscore the close relationship between ICD and pyroptosis and advocate using ROS-ERS with pyroptosis inducers for MM treatment. Our findings show that combination therapy potentially enhances tumor immunogenicity by activating T-cell-specific antitumor responses, thereby optimizing therapeutic strategies.

Results

ROS-ERS combined with pyroptosis inducer enhances MM cell apoptosis

We investigated whether the combination of ICD and pyroptosis inducers enhanced tumor immunogenicity, along with its underlying mechanism of action. Here, we used the ICD inducer, ROS-ERS inducer 1 (REI) (online supplemental figure S1A), which is a Pt(II)-N-heterocyclic carbene complex derived from 4,5-diaryimidazole.¹⁸ Moreover, Quillaja saponaria fraction 21 (QS-21) (online supplemental figure S1B) is a pyroptosis inducer that activates the NLRP3 inflammatory body and releases the caspase-1-dependent cytokines IL-1β and IL-18.¹⁹

To evaluate the cytotoxic effects of individual drugs on MM cells, we treated RPMI-8226 or U266 multiple myeloma cell line (U266) cells with different concentrations of REI or QS-21 and used the cell counting kit-8 (CCK8) assay to measure the survival rate of MM cells. As shown in figure 1A, the inhibitory effects of REI and QS-21 on MM cell proliferation were concentration-dependent and time-dependent. The IC50 concentration was used for 48 hours of drug action for the following experiments: REI (U266 1.28 µM and RPMI-8226 1.09 µM) and QS-21 (U266 2.67 µM and RPMI-8226 10.89 µM). In the firefly luciferase assays on Luc-RPMI-8226 cells, the combination of REI and QS-21 further reduced luciferase activity, indicating that the drug combination had strong cytotoxicity; moreover, the cell vitality and survival rate were lower than those of individual drugs (figure 1B,C). Flow cytometric analysis to detect the effects of REI and QS-21 on MM cell apoptosis revealed that the combination treatment increased the apoptosis rate (figure 1D). Additionally, light microscopy showed that the combination of drugs caused morphological changes and decreased the number of MM cells (figure 1E). These results confirm the enhanced cytotoxicity of REI combined with QS-21 on MM cells.

Figure 1. The combination of immunogenic cell death and pyroptosis inducers inhibits the growth of multiple myeloma (MM) cells. (A) Cell counting kit-8 (CCK)-8 assay: MM cell lines (RPMI-8226 and U266) were treated with different concentrations of ROS-ERS inducer 1 (REI) and QS-21 for 24, 48, and 72 hours. (B, C) Live cell imaging and microplate reader detection of the killing of Luc-RPMI-8226 using the firefly luciferase method. (D) Cell apoptosis was detected by flow cytometry. (E) Changes in cell morphology were observed under a light microscope. The concentrations of REI and QS-21 used in (B-E) were the IC50 concentrations obtained from a CCK-8 assay for 48 hours, namely REI (U266 1.28 [micro]M and RPMI-8226 1.09 [micro]M) and QS-21 (U266 2.67 [micro]M and RPMI-8226 10.89 [micro]M). Error bars, mean+-SD, n=3. Statistical analysis was performed using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. R+Q denotes the combination of REI and QS-21. ERS, endoplasmic reticulum stress: PBS, phosphate-buffered saline; QS-21, Quillaja saponaria fraction 21; REI, ROS-ERS inducer 1; ROS, reactive oxygen species; U266, U266 multiple myeloma cell line.

ROS-ERS combined with pyroptosis inducer strongly induces DAMPs in MM cells

Ecto-CALR exposure is an active process in ICD and often precedes phosphatidylserine exposure and morphological signs of apoptosis.²⁰ Exposure of CALR on the cell surface is an important marker of ICD and is the phagocytic signal of macrophages.²¹ We validated the expression of the CALR protein on the MM cell lines (RPMI-8226 and U266) and found that REI combined with QS-21 induced increased CALR expression on the cell surface in whole cells (figure 2A–E) as well as the release of the highest amounts of ATP, heat shock protein 70 (HSP70), and high mobility group box 1 (HMGB1) proteins (figure 2F,G). These results demonstrate that the combination of pyroptosis and ICD inducers leads to an increase in DAMPs, which may induce a stronger immunogenic response in tumor cells (figure 2H).

Figure 2. Immunogenicity reaction index and damage-associated molecular patterns in multiple myeloma (MM) cells. (A, B, C) Flow cytometry was used to detect the expression of ecto-CALR in MM cell lines (RPMI-8226 and U266) treated with REI and QS-21. (D, E) Western blot assays were used to detect CALR protein expression in MM cell lines (RPMI-8226 and U266) treated with REI and QS-21. (F) Western blot analysis of high mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70) protein expression in the intracellular and secreted supernatants of MM cell lines (RPMI-8226 and U266) after different treatments. (G) ATP generation after drug administration in each group. (H) Schematic diagram of MM cell death induced by immunogenic death inducers. The concentrations of REI and QS-21 used in figure 2 were the IC50 concentrations obtained from the cell counting kit-8 assay for 48 hours, REI (U266 1.28 [micro]M and RPMI-8226 1.09 [micro]M) and QS-21 (U266 2.67 [micro]M and RPMI-8226 10.89 [micro]M). Error bars, mean+-SD, n=3. Statistical analysis was performed using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. R+Q denotes the combination of REI and QS-21. CALR, calreticulin; PBS, phosphate-buffered saline; QS-21, Quillaja saponaria fraction 21; REI, reactive oxygen species-endoplasmic reticulum stress inducer 1; U266, U266 multiple myeloma cell line;PRMI-8226, PRMI-8226 multiple myeloma cell line.

ROS-ERS combined with pyroptosis inducers enhances the ROS-associated ERS and induces a stronger immunogenic response of tumor cells

We used the JC-1 kit to detect mitochondrial membrane potential. The green (monomers) to red (aggregates) fluorescence ratio was higher in the REI, QS-21, and R+Q groups than in the phosphate-buffered saline (PBS) group; it was highest in the R+Q group (figure 3A,B, online supplemental figure S1C–E). We used flow cytometry and fluorescence microscopy to detect MitoTracker Green-stained mitochondria and analyzed the number of mitochondria in the REI and QS-21 single-agent and combination treatment groups. There was a significant increase in fluorescein isothiocyanate fluorescence intensity in all three treatment groups compared with the control group, indicating an increase in the number of mitochondria or structural changes under monotherapy and combination therapy (online supplemental figure S1F,G). Moreover, the ROS-ERS combined with pyroptosis inducer triggered autophagy in MM cells, with an increase in the ratio of lipidated microtubule-associated protein 1A/1B-light chain 3 (LC3-II) to normal LC3-I, a reliable marker of autophagy.²² Compared with that of the PBS group, the expression of LC3-II was significantly increased in the REI and QS-21 groups (online supplemental figure S1H), suggesting that cell stress can induce autophagy to a certain extent. The expression of LC3-II was the highest in the R+Q group (online supplemental figure S1I), indicating that the combination treatment had the strongest ability to induce autophagy. More importantly, we analyzed the expression level of the p62 protein. Because p62, as a ubiquitin autophagy receptor, is destined to be degraded in autophagosomes,^{17 23} degradation is hindered if the lysosomal function is disrupted. p62 in the REI and R+Q groups was not effectively degraded, which confirmed that the combination of ICD and pyroptosis inducers prevented autophagy via the severe destruction of autophagosomes after lysosomes. Damaged mitochondria cannot be removed promptly, which increases the total number of mitochondria in cells. Accumulated mitochondria may also exhibit enhanced mitochondrial tracker fluorescence.

Figure 3. Interaction between mitochondrial damage, reactive oxygen species (ROS), and endoplasmic reticulum stress (ERS). (A, B) Fluorescence microscopy images from a JC-1 assay. (C) Transmission electron microscopy showing mitochondrial morphology. (D) Flow cytometry results of an ROS generation assay. (E) Schematic diagram of the intracellular signaling network activated by three endoplasmic reticulum transmembrane proteins (PERK, IRE1, ATF6) of the unfolded protein response. (F) Western blot assays of ERS-related proteins. REI denotes ROS-ERS inducer 1, and R+Q indicates the combination of REI and QS-21. The concentrations of REI and QS-21 used in figure 2 were the IC50 concentrations obtained from the cell counting kit-8 assay for 48 hours: REI (U266 1.28 [micro]M and RPMI-8226 1.09 [micro]M) and QS-21 (U266 2.67 [micro]M and RPMI-8226 10.89 [micro]M). Error bars, mean+-SD, n=3. Statistical analysis was determined using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. R+Q denotes the combination of REI and QS-21. ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; PBS, phosphate-buffered saline; PERK, proline extensin-like receptor kinase; QS-21, Quillaja saponaria fraction 21; REI, ROS-ERS inducer 1; U266, U266 multiple myeloma cell line;PRMI-8226, PRMI-8226 multiple myeloma cell line.

Further, we examined the mitochondrial ultrastructural changes in the REI, QS-21 single agent, and R+Q combination groups by transmission electron microscopy (TEM). Significant changes in the internal structure of mitochondria were observed in the single-treatment and combination-treatment groups, which showed signs of damage, such as membrane rupture and cristae disappearance, further suggesting that the drug treatments induced mitochondrial disruption (figure 3C).

Damage to mitochondrial metabolism may disrupt the intracellular redox balance. Therefore, ROS production was tracked using the dichlorodiacetate fluorescein method. To confirm the specificity of the fluorescence signal, we pretreated cells with N-acetylcysteine (NAC), a well-known ROS scavenger²⁴ (online supplemental figure S1G). Pretreatment with NAC significantly reduced the fluorescence intensity in all treatment groups, confirming that the signal was explicitly caused by ROS generation. ROS production in the R+Q group was higher than that in the REI, QS-21, and PBS groups (figure 3D). The combined effects of ROS and ERS activate the DAMPs.²⁵ ERS and ROS production are important components of the intracellular pathways governing ICD.²⁶ Thus, we clarified whether the response to ERS was mediated by ROS. To investigate the role of ROS and ERS in apoptosis, we used NAC and ERS inhibitor salubrinal (Sal)²⁷ to inhibit ROS and ERS, respectively. REI reduced the survival rate of MM cells, which was partially reversed by Sal (10 µM) and NAC (1 mM) (figure 1K).

To survive, cancer cells must combat numerous tumor-associated internal and environmental stresses that disrupt the protein balance within the endoplasmic reticulum (ER), promoting the accumulation of misfolded proteins in it, which causes ERS and subsequently activates the unfolded protein response (UPR). The UPR is an intracellular signaling network primarily activated by three ER transmembrane proteins (proline extensin-like receptor kinase 1 (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6) that continuously monitor protein folding within the ER lumen and initiate countermeasures to maintain ER homeostasis^{28 29} (figure 3E). Under mild or moderate ERS, homeostatic UPR initiates transcriptional and translational changes that promote cell adaptation and survival. However, if these processes fail to resolve ERS, the terminal UPR program dominates and actively signals apoptosis.³⁰ In the REI and R+Q groups, ERS-related proteins IRE1 α, PERK, p-PERK, CHOP, XBP1s, BIP, and p-eif2a increased, while eif2a decreased. Notably, after the addition of Sal and NAC, ERS was suppressed (figure 3F). Therefore, ROS and ERS are considered critical: elevated ROS levels lead to ERS, which activates ICD.

The combination of ROS-ERS and pyroptosis inducers enhances ROS production, which, in turn, enhances ERS and induces a stronger immunogenic response in tumor cells.

ROS-ERS combined with pyroptosis inducer enhances the execution of pyroptosis

We tested the effects of different treatments on pyroptosis-related proteins, including NLRP3, caspase-1, GSDMD, IL-1 β, and IL-18, by western blotting. We observed upregulated levels of NLRP3, C-caspase-1, and GSDMD-N and increased secretion of IL-1 β and IL-18 after QS-21 and R+Q treatment (figure 4A–F). Morphological observations under a light microscope revealed extensive cell swelling and membrane blebbing in the QS-21 and R+Q groups (figure 1E), reflecting the typical characteristics of pyroptosis. These results indicated that NLRP3/caspase-1/GSDMD-N-mediated pyroptosis was successfully activated.

Figure 4. Effects of ROS-ERS and pyroptosis inducers on pyroptosis in multiple myeloma (MM) cells. (A) Western blot analysis of NLRP3, caspase-1, and gasdermin D (GSDMD) in different treatment groups. [beta]-vinculin was used as a loading control. (B, C, D, E, F) The corresponding quantifications of NLRP3, C-caspase-1, GSDMD-N, IL-1[beta], and IL-18 ratios. (G) Transmission electron microscopy images show changes in the cell membrane morphology of MM cell lines after 48 hours of treatment with REI and QS-21. Error bars, mean+-SD, n=3. Statistical analysis was determined using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. REI denotes ROS-ERS inducer 1, and R+Q denotes the combination of REI and QS-21. ERS, endoplasmic reticulum stress; IL, interleukin; NLRP3, NOD-like receptor family pyrin domain containing 3; PBS, phosphate-buffered saline; PERK, proline extensin-like receptor kinase; QS-21, Quillaja saponaria fraction 21; REI, ROS-ERS inducer 1; ROS, reactive oxygen species; U266, U266 multiple myeloma cell line;PRMI-8226, PRMI-8226 multiple myeloma cell line.

Using TEM, we observed pores on the cell membranes of the QS-21 and R+Q groups, indicating damaged cell membranes (figure 4G), which further confirmed the occurrence of pyroptosis. The pores on the cell membrane in the R+Q group were larger than those in the QS-21 group and the cells were lysed.

In vitro verification of the combined effect of ROS-ERS and pyroptosis inducers in enhancing T-cell-specific antitumor immunity

Bone marrow mononuclear cells from patients with MM (online supplemental table S1) were isolated, and CD138⁺cells were obtained via magnetic bead sorting. These cells were then cultured with REI, QS-21, or their combination for 48 hours, and CALR protein expression on CD138⁺plasma cells was analyzed using flow cytometry. Notably, the REI and QS-21 combination increased both apoptosis (figure 5A,B) and CALR expression (figure 5C,D). DAMPs released into the extracellular space or exposed on the cell membrane further interact with the immune system by binding to the corresponding receptors on innate immune cells such as monocytes, DCs, and macrophages. ICD can promote the maturation of DCs and the infiltration of cytotoxic T lymphocytes. This process can reverse the immunosuppressive TME and improve sensitivity to immunotherapy.³¹ However, the immune cells in patients with MM are ineffective at initiating a strong immune response. To further evaluate the combined effect of pyroptosis and ROS-ERS inducers on MM cells and their potential to activate ICD, we isolated bone marrow-derived mononuclear cells (BM-MNCs) from patients with newly diagnosed MM and cultured them for 7 days to induce mature DCs. Next, we used an in vitro co-culture system that included pan-T cells isolated from BM-MNCs, DCs, and U266 cells (figure 5E). PBS, REI (1.28 µM), QS-21 (2.67 µM), and R+Q (1.28 µM REI+2.67 µM QS-21) were added to the co-cultured cells for 48 hours, after which cells were collected and analyzed by flow cytometry for DC activation, T-cell function, and U266 apoptosis.

Figure 5. In vitro cell co-culture system construction and dendritic cell (DC) activation. (A, B) Flow cytometry was used to detect the apoptosis of CD138 + cells in the bone marrow of patients with multiple myeloma after REI and QS-21 treatment. (C, D) Flow cytometry was used to detect the expression of ecto-calreticulin (CALR) in bone marrow CD138 + cells of patients with MM after REI and QS-21 treatment. (E) Schematic of an in vitro co-culture system for immunogenic cell death (ICD) induction. MM cells were subjected to different treatments for ICD induction. Subsequently, DCs and pan-T cells from patients with MM were added to the MM co-culture system. Different cell types were quantitatively analyzed via flow cytometry after 48 hours of co-culture. (F) The proportion of DCs after the in vitro induction of DC, as measured by flow cytometry. (G) Quantification of data shown in figure 3C . Error bars, mean+-SD, n=10. Statistical analysis was determined using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. REI denotes reactive oxygen species-endoplasmic reticulum stress inducer 1 and R+Q denotes the combination of REI and QS-21. MHC, major histocompatibility complex; MM, multiple myeloma; PBS, phosphate-buffered saline; QS-21, Quillaja saponaria fraction 21; REI, reactive oxygen species-endoplasmic reticulum stress inducer 1; U266, U266 multiple myeloma cell line;PRMI-8226, PRMI-8226 multiple myeloma cell line.

As shown in figure 5F,G, both the REI and QS-21 groups showed increased expression of the DC activation markers CD86, CD80, CD70, and major histocompatibility complex II (MHC-II). The R+Q combination group demonstrated a significant increase in DC activation markers, suggesting that the combination of REI and QS-21 further promoted DC maturation and activation, which is an important part of ICD induction.

During ICD, mature DCs stimulate T cells by interacting with CD28 and T-cell receptor (TCR). Activated T lymphocytes play a crucial role in tumor cell cytokine release and ICD induction. We further analyzed the activation of T lymphocytes, represented by CD3⁺ and CD3⁺CD8 ⁺T cells. The percentages of CD3⁺CD27⁺CD28⁺ and CD3⁺CD8⁺CD27⁺CD28⁺ T cells (figure 6A,B) increased in both the REI and QS-21 groups and were the highest in the R+Q combined group with statistical significance. Interestingly, the percentages of senescent CD57⁺CD3⁺T lymphocytes and CD3⁺CD8⁺CD57⁺T decreased in the monotherapy and combination groups. Additionally, compared with the PBS, REI, and QS-21 groups, the effector memory (CCR7⁻CD45RA⁻CD8⁺) subset of CD8⁺T lymphocytes in the combined R+Q group tended to increase, and the number of naïve T cells decreased. Thus, naïve T cells may transform into effector memory T cells, suggesting that combination therapy with REI and QS-21 may exert antitumor immune effects.

Figure 6. T-cell activation and multiple myeloma (MM) cell apoptosis in an in vitro co-culture system. (A, B) Population and distributions of CD3 + and CD3 + CD8 + T cells in different groups. (C) Apoptosis of MM cells in each group after co-culture in vitro. (D) Transwell co-culture model diagram. (E) MM apoptosis caused by anti-MM T cells. (F) ELISA for cytokines in the co-culture supernatants. Error bars, mean+-SD, n=10. Statistical analysis was determined using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. REI denotes reactive oxygen species-endoplasmic reticulum stress inducer 1 and R+Q denotes the combination of REI and QS-21. DC, dendritic cell; IFN, interferon; IL, interleukin; REI, reactive oxygen species-endoplasmic reticulum stress inducer 1; TEM, transmission electron microscopy; TNF, tumor necrosis factor.

We analyzed CD3⁺ T cells and CD8⁺ T cells for activation markers (CD69, PD-1, CD25, 4-1BB, and CD107a) using flow cytometry. No significant changes in CD69, PD-1, or CD25 were observed among treatment groups; however, 4-1BB and CD107a expression was significantly increased in the R+Q group compared with the PBS, REI, and QS-21 groups (online supplemental figure S2A,B).

We performed cell viability assays (CCK8 and apoptosis assays) on REI and/or QS-21-treated DCs, T cells, and MM cells. The viability of immune cells (including DCs and T cells) remained above 80% in all treatment groups, indicating minimal cytotoxic effects of treatment on immune cells (online supplemental figure S2G–I). Furthermore, we assessed the apoptosis of U266 cells after co-culture. Apoptosis was increased in the R+Q group compared with that in the individual agent groups; as shown in figure 6C, the mean apoptosis rate was significantly higher in the R+Q group (74.05±5.21, p<0.01) than in the monotherapy (REI: 50.52±6.10, QS-21: 54.50±8.76) and control (28.05±1.97) groups, confirming the enhanced antitumor effect in vitro.

We performed an additional Transwell co-culture experiment to verify the enhanced function of T-cell activation and resistance to MM cells. Two groups were established: one untreated and one treated with combined R+Q. DC and T cells were added to the upper chamber, while U266 cells were placed in the lower chamber to induce T-cell activation and antigen specificity. After 24 hours, the T-cell-DC inserts were transferred to fresh U266 cells in a drug-free environment, and apoptosis was measured after an additional 24 hours. The apoptosis rate of U266 cells was approximately 10–13% in the untreated group but increased to 21–24% in the R+Q-treated group (figure 6D,E). This finding indicates that T cells exposed to R+Q treatment can acquire cytotoxic potential against MM cells even without continuous drug exposure.

We measured tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), IL-10, and IL-6 levels in the supernatant of the co-culture system for each treatment group (figure 6F). TNF-α and IFN-γ levels were significantly higher in the R+Q group than in the monotherapy or control groups, indicating enhanced T-cell activation. Conversely, IL-10 and IL-6 levels tended to decrease in the R+Q group, indicating a reduced immunosuppressive environment. Therefore, the combination treatment of REI and QS-21 not only enhanced the activation markers of DC and T cells but also significantly improved their functional output and promoted a robust antitumor immune response.

Thus, these in vitro results suggest that a combination of pyroptosis and ROS-ERS inducers can induce DC maturation and activate T cells in the context of DAMPS, restoring depleted immunity and triggering an antitumor immune response. Moreover, this process counteracts T-cell aging and promotes antitumor immune effects.

Bortezomib-induced ICD and its effects on DC maturation and T-cell activation

To assess whether R+Q-induced DAMP release and immune activation differ from general cytotoxic effects, we used bortezomib (BTZ) as a control. We measured intracellular and extracellular HSP70 and HMGB1 by western blot after BTZ at 0, 2.5, 5, and 10 nM, and CALR levels by flow cytometry. BTZ-treated MM cells showed increased surface CALR compared with the PBS control (online supplemental figure S3A,B), yet CALR exposure was significantly lower than in the R+Q group. Similarly, HMGB1 and HSP70 secretion increased in BTZ-treated cells versus PBS (online supplemental figure S3C), indicating that while BTZ induces DAMP release, its extent is less pronounced than that of R+Q.

To assess the immune response, cells were divided into four groups: control (untreated U266 cells), U266+BTZ (5 nM), DC+T cells + U266, and DC+T cells + U266 + BTZ (5 nM). We evaluated tumor cell apoptosis, DC activation, and T-cell function. Although BTZ induced significant U266 cell death (online supplemental figure S3D), the release of DC activation markers (CD70/CD40/CD80/CD86/MHC-II) was lower than with R+Q (online supplemental figure S3E). Moreover, T-cell activation (CD57/CD27CD28/TEM/naïve T/4-1BB/CD107a/PD-1/CD69/CD25) was less pronounced in the BTZ group (online supplemental figure S3F), suggesting that, despite BTZ-induced DAMPs release, the R+Q combination elicits greater DAMPs release along with increased DC and T-cell activation and function, thereby enhancing immunogenic effects.

Taken together, these findings imply that while BTZ can induce ICD and modest immune activation, REI and QS-21 treatment more effectively enhance DAMPs release, DC maturation, and T-cell activation, culminating in a stronger antitumor immune response.

ROS-ERS combined with pyroptosis inducer was verified to enhance antitumor immunity in vivo

Encouraged by the synergistic tumor-killing mechanism of ICD and pyroptosis in vitro, we evaluated the combined effects of these compounds in vivo. We first established an NOG (NOD-Cg-Prkdc^scidIL2rg^tm1sug/JicCrl) mouse model lacking an immune system. This model allowed us to evaluate the immune responses of patients with MM and the antitumor performance of human MM cell lines. A mouse MM model³² was established by subcutaneous injection of U266 cells into NOG mice. Mononuclear cells from the peripheral blood of patients with MM were injected intravenously into NOG mice carrying U266 tumors to create a humanized immune system (figure 7A). MM mice were randomly divided into four groups, with four mice in each group: PBS group, REI group, QS-21 group and R+Q group. REI (5 mg/kg) was intraperitoneally injected on days 0, 2, 4, 6, 8, and 10, and QS-21 (0.33 mg/kg) was subcutaneously injected weekly. The PBS group was injected intraperitoneally with an equal volume of PBS, as shown in figure 7B. Tumor growth was rapid in the PBS group, with a tumor volume of approximately 1,200 mm³. REI and QS-21 alone moderately inhibited MM tumor growth, whereas the antitumor efficiency was improved and the tumor volume was reduced in the R+Q group. On the 26th day, one mouse from each group was selected for positron emission tomography-CT (PET-CT) scanning, and the standardized uptake value (SUV) of the subcutaneous tumor was measured. The higher the number of tumor cells, the more vigorous the glucose metabolism and the higher the SUV. In the REI, QS-21, and R+Q groups, the SUV decreased, with the R+Q group showing the lowest SUV (figure 7C). The mice were euthanized, the tumors were removed and the tumor volume of each group was observed (figure 7D). The tumor weight of the R+Q group was the smallest (figure 7E). The mice in all groups showed good biocompatibility (figure 7F).

Figure 7. In vivo experimental model of immunogenic cell death (ICD) and pyroptosis-mediated effects in a multiple myeloma (MM) xenograft model. (A) In vivo therapeutic schedule. (B) Tumor growth curves in different groups of mice. (C) Positron emission tomography-CT imaging of different groups of mice after treatments. (D) U266 tumor photo of different groups of mice after treatments. (E) The tumor weight in different groups of mice. (F) Body weight changes of MM tumor-bearing mice in different groups over 26 d. Error bars, mean+-SD, n=4. Statistical analysis was determined using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. REI denotes reactive oxygen species-endoplasmic reticulum stress inducer 1 and R+Q denotes the combination of REI and QS-21. PBS, phosphate-buffered saline; QS-21, Quillaja saponaria fraction 21; REI, reactive oxygen species-endoplasmic reticulum stress inducer 1; SUV, standardized uptake value.

In addition, to further elucidate the mechanism of the antitumor effect of ICD and pyroptosis therapy and the role of ICD, we immuno-stained with corresponding antibodies and analyzed key antitumor immune cells, such as DCs and T cells, in mouse subcutaneous tumors using flow cytometry. The percentage of mature DC markers, including CD80, CD70, CD40, MHC-II, and CD86, was higher in the R+Q combination group than in the other three groups (figure 8A, online supplemental figure S4A). Increased expression of DC activity markers helps activate DCs to bind to receptors on immature T cells, thereby activating T cells.

Figure 8. Anti-multiple myeloma (MM) effect and tumor immune status in the MM xenograft model. (A) Quantification of the surface expression of functional molecules on murine tumor DCs measured via flow cytometry. (B, C, D) Quantification of the distribution of different CD3 + and CD8 + T cells in murine tumors in these groups. (E, F, G) Apoptosis of myeloma cells (CD45-) in murine tumors was detected via flow cytometry. (H) Serum cytokine levels of tumor necrosis factor-[alpha] and interferon-[gamma] in mice post treatment. Error bars, mean+-SD, n=4. Statistical analysis was determined using a one-way analysis of variance. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. REI denotes reactive oxygen species-endoplasmic reticulum stress inducer 1 and R+Q denotes the combination of REI and QS-21. DC, dendritic cell; IFN, interferon; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; QS-21, Quillaja saponaria fraction 21; REI, reactive oxygen species-endoplasmic reticulum stress inducer 1; TEM, transmission electron microscopy; TNF, tumor necrosis factor.

We then analyzed the T-cell markers in the tumor tissue. Senescent T cells exhibited abnormal phenotypes, such as CD57 upregulation,^{33 34} and the numbers of CD3⁺CD57⁺ and CD3⁺CD8⁺CD57⁺T cells in the R+Q group were significantly lower than those in the PBS, REI and QS-21 groups (figure 8B–D, online supplemental figure 4B,C). Blood cell analysis and liver and kidney function tests in the different groups of mice showed normal biochemical indices (online supplemental figure S4D). This suggests that the combination treatment of REI and QS-21 can reverse T-cell aging in vivo. Currently, T-cell senescence is a major obstacle to T-cell therapy, indicating that MM therapy has great potential. In addition, we observed an increasing trend in CD3⁺CD27⁺CD28⁺T cells and a significant increase in the number of CD3⁺CD8⁺CD27⁺CD28⁺T cells, suggesting that the combination of REI and QS-21 induces T-cell activation and potentially generates antitumor immunity. In addition, we found increased levels of IFN-γ and TNF-α in the lymphocytes of the last three groups after treatment (figure 8H), indicating ICD activation in NOG mice.

In an investigation of the cytotoxicity of MM tumor cells in each treatment group and evaluation of apoptosis in tumor cells (CD45⁻) in mouse models, we observed increased apoptosis of tumor cells in the REI, QS-21, and R+Q groups and a significant increase in the proportion of apoptotic cells in the R+Q group, with statistically significant differences between the groups (figure 8E–G).

Discussion

Immunotherapies for MM, including CD38 monoclonal antibodies and chimeric antigen receptor-T cells, show promising therapeutic effects. However, studies on MM cell-specific immunotherapy remain limited. Therefore, we reviewed the MMRF-coMMpass database and found that combined CALR and NLRP3 expression in MM cells correlated with patient prognosis (online supplemental figure S5A–C). Our study demonstrates that combining ROS-ERS and pyroptosis inducers synergistically enhances antitumor immune responses in MM therapy, producing a potent immunostimulatory effect that improves antigen presentation, immune cell recruitment, and T-cell activation.

ICD releases DAMPs from dead tumor cells and activates tumor-specific immune responses, thereby stimulating the long-term efficacy of anticancer drugs by directly killing tumor cells and stimulating antitumor immunity. DAMPs include cell surface exposure of CALR and the extracellular release of ATP, HSP70, and HMGB1.³⁵ During pyroptosis, endogenous GSDME and GSDMD are cleaved by the caspase family, including caspase-1 and caspase-3, releasing active GSDMD-N and GSDME-N termini that form membrane pores.³⁶ Induction of ICD and pyroptosis in MM can significantly enhance the immune response against tumors. ICD triggers the release of DAMPs, which stimulate an immune response, whereas pyroptosis, a form of programmed cell death, releases inflammatory cytokines that further amplify this effect. Together, these processes lead to the activation of specific T cells and other immune cells, creating a more robust and targeted immune response against MM, thereby improving the effectiveness of immunotherapy.

To confirm our results, we systematically evaluated the cytotoxicity of ROS-ERS inducer REI combined with pyroptosis inducer QS-21 on MM cells and its mechanism against tumor immunity. R+Q significantly enhanced MM cell cytotoxicity, reduced luciferase activity, increased apoptosis, and induced morphological changes. R+Q strongly triggered the release of DAMPs—CALR, ATP, HSP70, and HMGB1—with increased CALR exposure at the cell surface serving as a key immunogenic signal that attracts macrophages and initiates an immune response.^{37 38}

The combination treatment induced significant mitochondrial dysfunction, confirmed by decreased mitochondrial membrane potential, increased mitochondrial number, and ultrastructural damage observed via TEM after R+Q treatment. Mitochondrial damage disrupted redox balance, leading to increased ROS generation. Simultaneously, the combination triggered autophagy, indicated by an increased LC3-II/LC3-I ratio. Nevertheless, the p62 protein was not efficiently degraded in the REI and R+Q groups, suggesting that combination therapy disrupted autophagosome-lysosome fusion and prevented clearance of damaged mitochondria.³⁹ Consequently, this accumulation further increased ROS production, central to enhancing immunogenic responses.⁴⁰ Combination therapy significantly elevated ROS levels, thereby inducing ERS. Sustained ERS ultimately triggered apoptosis and DAMP release, enhancing MM cell immunogenicity. Inhibition of ROS and ERS with NAC (ROS scavenger) and Sal (ERS inhibitor) partially reversed REI’s cytotoxic effects, confirming the critical role of the ROS-ERS axis in ICD.

The combination treatment effectively activated the cellular pyroptosis pathway. Specifically, it upregulated NLRP3, activated caspase-1 (C-caspase-1), and GSDMD-N; increased IL-1β and IL-18 secretion; and induced cellular membrane changes with pore formation observed by TEM. Compared with the QS-21 group, larger membrane pores in the R+Q group suggest that the combination promoted more extensive pyroptosis, facilitating the release of tumor-associated antigens and DAMPs and further enhancing the immune response.

Further, pyroptosis leads to the release of intracellular HMGB1 and ATP.⁴¹ ATP released by dying cells activates purinergic P2X7 receptors on DCs, subsequently activating the NLRP3-ASC inflammasome and promoting IL-1β secretion. IL-1β, along with antigen presentation, enhances CD8⁺ T-cell activation, contributing to IFN-γ production and the development of adaptive immune responses against cancer cells.⁴² All of these molecules can be used as DAMPs to stimulate further inflammatory responses.

Mechanistically, combining ICD and pyroptosis induced significant mitochondrial damage and ERS, while exposure to ICD markers markedly inhibited autophagy and enhanced pyroptosis. Activation of the focal death pathway NLRP3/GSDMD enlarged the tumor cell membrane,⁴³ increased permeability, and activated and matured DCs and T cells; moreover, cytokines more readily entered cells, thereby amplifying the immunogenic death response of MM cells and improving therapeutic efficacy.

DC and T-cell activation was confirmed in in vitro experiments as well. The maturation of DCs involves the upregulation of co-stimulatory molecules such as CD80, CD86, CD70, MHC-II, and CD40. Interaction between CD70 on DCs and CD27 on T cells plays a synergistic stimulatory role in the interaction between DCs and T cells,^{44 45} promoting the proliferation and survival of T cells. The binding of CD80/CD86 to CD28 provides a positive co-stimulatory signal that promotes T-cell proliferation and cytokine secretion.⁴⁶ MHC-II is responsible for antigen processing and presentation to CD4⁺ T cells. The interaction between CD40 on DCs and CD40L on T cells is crucial for the complete activation of DCs. It can enhance the antigen presentation ability of DCs and increase their cytokine secretion, thereby promoting T-cell proliferation and differentiation. Mature DCs stimulate T cells by interacting with CD28 and TCR. CD57 is present in CD3⁺ and CD8⁺T cells that become senescent after differentiation.³³ The expression of CD57 in lymphocytes indicates their inability to proliferate or participate in immune responses.^{47 48} Therefore, the combination of REI and QS-21 may reverse T-cell aging, enabling them to actively participate in the immune response, kill tumor cells, and improve the dysfunctional MM microenvironment. 4-1BB as a co-stimulatory domain, for chimeric antigen receptor T cells, improves T-cell proliferation and survival as well as reduces T-cell exhaustion.^{49 50} CD107a (LAMP-1) may be a marker for degranulation of NK and activated CD8⁺ T cells.⁵¹ We observed a significant increase in 4-1BB and CD107a expression in the R+Q group. This indicates increased T-cell activation, degranulation and cytotoxic activity.

The combination of REI and QS-21 induced more intense release of DAMPs, more pronounced DC maturation, and more potent T-cell activation than BTZ, a drug commonly used in MM therapy.⁵² Although BTZ induced ICD to some extent, the levels of CALR exposure, HMGB1 and HSP70 secretion, DC activation, and T-cell activation were significantly lower in the BTZ group compared with the R+Q group. This suggests that the combination therapy has a unique and more effective immunomodulatory effect.

In in vivo models, the combination treatment significantly inhibited tumor growth, increased the activation markers of DCs and T cells, reduced senescent T cells, and enhanced antitumor immune responses.

The combination of ROS-ERS inducers and cellular pyroptosis inducers offers a promising new approach to MM therapy with the potential to overcome current therapeutic limitations. However, our study focused on ICD induced by the combination of REI and QS21 and its immune-activating effects; the interactions among cell death pathways remain to be investigated. Future studies will block distinct T-cell death pathways using specific inhibitors to clarify their respective contributions and reveal how different death modes synergistically enhance immunogenicity. This will optimize ICD induction strategies and provide new ideas for MM immunotherapy.

In conclusion, our study provides a foundation for developing novel immunotherapeutic strategies against MM. By integrating ROS-ERS and pyroptosis induction, we demonstrated a synergistic approach that enhanced MM cell immunogenicity, activated antitumor immune responses and improved in vitro and in vivo antitumor efficacy. Further research may yield more effective and personalized therapies for patients with MM.

Conclusion

Overall, this work represents a promising strategy. ROS-ERS combined with pyroptosis can significantly enhance the immunogenic response of MM, providing a promising strategy for the treatment of MM by activating powerful specific T-cell antitumor immunity.

Experimental section/methods

Cell lines

The MM cell line RPMI-8226 was purchased from the American Type Culture Collection and then cultured in Iscove’s Modified Dulbecco’s Medium (Gibco) with 20% fetal bovine serum (Gibco) and with 5% CO₂ at 37°C. The MM cell line U266 was obtained from the American Type Culture Collection and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin/streptomycin at 37°C in a 5% CO₂ humidified incubator.

Cell viability assay

CCK-8 from GlpBio Technology (Montclair, California, USA) was used to determine cell viability. A total of 10,000 cells per well were seeded into 96-well plates. The medium was removed after 24, 48, and 72 hours of incubation and replaced with a medium containing different concentrations of REI or QS-21. CCK-8 solution (10 µL) in fresh medium (100 µL) was added to each well and incubated at 37°C for 2–3 hours according to the manufacturer’s instructions. The optical density was measured using a microplate reader (BioTek, Winooski, Vermont, USA).

Cell apoptosis analysis

After the cells were washed with PBS, the media and washings were collected and pelleted by centrifugation (3 min, 800 g). The cells were stained with annexin V and propidium iodide according to the manufacturer’s instructions (BD556547, BD Biosciences). After incubation in the dark for 15 min at ambient temperature, each sample was diluted with annexin V binding buffer (200 µL) and subjected to fluorescence-activated cell sorting analysis using the BD system. Finally, the data were analyzed using FlowJo V.10.23,58 software.

Firefly luciferase assay

RPMI-8226, an MM human cell line, expressing the luciferase gene (Luc) was constructed by genetic engineering. To determine the killing efficiency after adding the Luc luminous substrate during the killing experiment, the number of living cells was determined using the strength of the fluorescence signal emitted. After 48 hours of drug treatment, 2% Triton X⁃100 (Sigma) solution (50 µL) was added to each well, mixed, and 50 µL from each well was removed and added to a white 96-well plate for detection. Next, the substrate solution (50 µL of 300 µg/mL D⁃Luciferin aqueous solution and 2 mg/mL ATP aqueous solution in a 3∶1 volume ratio) was added to each well, mixed, and analyzed after 5 min. The detection instrument can be a multifunctional enzyme marker and living imager

Detection of cell surface calreticulin (CRT)

Myeloma cells were collected in 1 mL cold PBS and the cell suspension was centrifuged (1,200 rpm for 5 min) at 4°C and the supernatant was discarded. Anti-calreticulin (CRT) antibody conjugated to phycoerythrin (Ab209577; Abcam, Cambridge, UK) was added to the cell pellet (5 µg/mL) at room temperature for 30 min in the dark. Next, the cells were washed using 1 mL cold PBS, centrifuged (1,200 rpm for 5 min at 4°C) again to discard the supernatant, and finally suspended in 200 µL PBS before subjecting to flow cytometry test.

Study participants

This study enrolled 22 patients with newly diagnosed MM (median age, 65 years; range, 47–80 years; online supplemental table S1) between April 2024 and April 2025 and the samples were obtained from the Hematology Department, Tianjin Medical University General Hospital, China.

DC and pan-T lymphocyte extraction

Induction of DCs and determination of activation

Human bone marrow was used to isolate BM-MNCs, which were cultured overnight in RPMI-1640 solution with 10% fetal bovine serum. The supernatant was removed and IL-4 (50 ng/mL) (I4269, Merck, Darmstadt, Germany) and granulocyte-macrophage colony-stimulating factor (500 ng/mL) (GP20353-0.005, Glpbio, California, USA) were added. Then, the hanging half-medium of cells and an equal amount of culture medium were added every 48 hours. On day 7, the cells were collected and co-cultured with the treated U266 cells for 24 hours. Flow cytometry was used to analyze the lineage of HLA-DR⁺ cells as DCs.^{53 54}

Collection of naïve T cells and determination of activation: isolated BM-MNCs were handled with a magnetic bead sorting technique using the Pan-T Cell Isolation KIT II (130-045-201, Miltenyi, Germany) to separate pan-T cells. The cells were then co-cultured with treated U266 cells and DCs for 24 hours, and DuraClone IM T Cell Subsets were used to detect the activation and function of T cells.

Detection of DC surface functional molecules

After harvesting the co-cultured cells, single-cell suspensions of treated mouse tumor tissue precipitate were washed with 1 mL cold PBS and centrifuged (1,000 rpm for 5 min) at 4°C. Next, the DCs were incubated with CD80/phycoerythrin (5 µL), CD86/allophycocyanin (5 µL), MHC-II/peridinin-chlorophyll-protein (5 µL), CD40/allophycocyanin-A750 (5 µL), lineage/FITC (20 µL), and HLA-DR/PB450 (10 µL) for 20 min at room temperature in the dark. The cells were then washed two times with PBS and finally suspended in 200 µL PBS to be tested. The aforementioned antibodies for flow cytometry were purchased from BioLegend (San Diego, California, USA).

Detection of T-cell surface functional molecules

After harvesting, co-culture system cells, single-cell suspensions of treated mouse tumor tissue precipitate were resuspended in PBS and washed (as mentioned above). The cells were then incubated with the DURA Clone IM T Cell Subsets kit (H1017246, Beckman Coulter, California, USA) for 20 min at room temperature and protected from light. Subsequently, myeloma cells were resuspended in PBS and washed two times. Finally, cells were resuspended in 200 µL PBS to be analyzed.^{55 56}

Western blot analysis

RPMI-8226 or U266 cells were treated with PBS, REI, or QS-21 for 48 hours. Cell extracts were prepared by washing the cells in cold PBS, followed by resuspension in radioimmunoprecipitation assay lysis buffer (#P0013B, Beyotime Biotechnology, Shanghai, China). The samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the calculated protein concentrations at 20 mA for 120 min. The transfer membrane was operated at 100 V for 90 min in an ice-bath environment. After transfer, the membrane was blocked with 5% skim milk for 1 hour, washed thrice with Tris-buffered saline with Tween 20 (TBST) for 5 min each. Next, the various specific primary antibodies (HSP70, HMGB1, CALR, CHOP, GRP78, XBP1s, eIF2α, IRE1α, PERK, p-PERK, LC3, P62, p-eIF2α, NLRP3, caspase-1, GSDMD, IL18, and IL-1β) were diluted (1:1,000) in 5% bovine serum albumin in TBST containing 1% NaN3 and incubated at 4°C overnight. Next, after washing, the membrane was incubated with the secondary antibody for 1 hour at room temperature with constant shaking. The membrane was washed thrice with TBST and finally incubated with enhanced chemiluminescence (#36208ES76, Yeasen). A gel imager (C500, Azure BioSytems, Dublin, California, USA) was used to image and analyze the bands.

Transwell assay

U266 cells (2×10⁵) were added to the bottom wells with full medium (500 µL), while DCs (1×10⁵) and T cells (3×10⁵) were put into the upper wells (8 µm membrane) together. After drug treatment and incubation at 37°C for 24 hours, DC and T cells were removed from the upper surface and added to the bottom wells of new unstimulated U266 cells, which were incubated at 37°C for 24 hours to detect apoptosis of U266 cells.

Animal model

The animal experiments were conducted in accordance with the guidelines of the Tianjin Experimental Animal Use and Care Committee, and the full experiment protocol was approved by the Animal Ethics Committee of Tianjin Medical University General Hospital. The laboratory was accredited by the Tianjin Science and Technology Commission under the accreditation number IRB2023-DWFL-328.NOG (NOD-Cg-Prkdc^scidIL2rgt^m1sug/JicCrl) mice (6-week-old, male) were provided by the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China).

A mouse model was established as previously described.^{57 58} Each mouse was injected with 10⁷ U266 cells (–14 days), and after 14 days, when the myeloma tissue size grew to approximately 100 mm³, the MM mice were randomly divided into four groups (four mice in each group subjected to the following treatments: PBS, REI, QS-21 and R+Q). Approximately 2×10⁷ lymphocytes derived from patients with MM (day 0) were input into mice from the tail vein, and then REI (5 mg/kg) was intraperitoneally injected at 0, 2, 4, 6, 8, and 10 days, and QS-21 (0.33 mg/kg) was subcutaneously injected once a week. PBS group was intraperitoneally injected with equal volume of PBS. On day 26, one mouse (among the four in each group) was removed from each group and subjected to PET-CT; additionally, 500 L of peripheral blood was obtained and centrifuged (1,500 rpm, 5 min). The liver and renal function and cytokine levels in MM mice were detected in the 300 µL supernatant; 500 µL of blood cells was used for routine blood and immune cell subgroup analyses. Unlike in other mouse models, the immune system of the NOG mice was imported into human immune cells after neoplasia, thus reflecting the effects observed in humans.

Mice PET-CT

¹⁸F-fluorodeoxyglucose positron emission tomography and CT scanning (Inliview-3000B, NOVEL MEDICAL, China) were performed to identify and quantify the growth of tumors in the xenograft model. Tumor activity was evaluated via maximal SUV (SUVmax).

ATP assay

ATP Assay Kit (S0026; Beyotime, Shanghai, China) was used to detect the ATP level.⁵⁹ First, myeloma cells were gathered and washed three times with PBS, followed by lysis using a lysis solution. Simultaneously, a gradient level of ATP standards was prepared with the fresh lysate; to each assay plate, 100 µL samples and the diluted gradient ATP standards were added, followed by 100 µL working solution from the kit and the absorbance values were measured using a microplate reader at 28°C for 5 min. Finally, the ATP assay was performed according to protein quantification.

Measurement of mitochondrial membrane potential (MMP)

The MitoProbe JC-1 Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to determine the mitochondrial membrane potential according to the manufacturer’s protocol. Briefly, after treatment, the cells were stained with JC-1 at 37°C for 20 min, washed two times with buffer, observed under a fluorescence microscope, and subjected to fluorescence-activated cell sorting

ROS assay

An ROS Assay Kit (S0033M, Beyotime Biotechnology, Shanghai, China) was used to measure ROS levels. Cells were inoculated in 12-well plates at 2×10⁶ cells per well and pre-incubated in a 37°C incubator with 5% CO₂ for 12 hours or 24 hours under the intervention. After incubation with the fluorescent probe 2,7-dichlorofluorescein diacetate,⁶⁰ ROS levels were detected using flow cytometry or fluorescence microscopy.

MitoTracker labeling

MitoTracker Green (Beyotime) was used to determine the mitochondrial number.⁶¹ After treatment, cells were incubated with the fluorescent probe for 30 min at 37°C in the dark. The cells were then washed two times and fresh medium was added. Observations were made using a fluorescence microscope, immediately photographed, and analyzed using flow cytometry.

Statistical analysis

Data are presented as the mean±SD and analyzed using Prism V.9.0 (GraphPad Software, La Jolla, California, USA). Statistical significance was determined using a one-way analysis of variance with a post hoc Dunnett’s test. Statistical significance was set at p<0.05.

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information.

Ethics statements

Patient consent for publication

Not applicable.

Ethics approval

The clinical study protocol was approved by the Ethics Committee of the Tianjin Medical University General Hospital (Ethics No. IRB2023-KY-251).

Footnote

LZ and HW contributed equally.

Contributors LZ and RF designed the study. HW and LZ performed experimental analysis and wrote the manuscript. CY, XZ, LH, JS and KD collected and arranged the data and helped in data analysis. RF, LZ and HW revised the manuscript and analyzed the data to ensure its accuracy. LZ and HW made equal contribution to this study. All authors read and approved the final manuscript. RF is responsible for the overall content as the guarantor.

Funding This work was supported by the Key research and development projects of the Ministry of Science and Technology (grant no. 2024YFC2510500), the National Natural Science Foundation Project (grant no. 82270142, 81900131), the Tianjin Municipal Natural Science Foundation (grant no. 24ZGSSSS00050), the Tianjin Science and Technology Planning Project (grant no. 24ZXGZSY00090, 24ZXGQSY00020), Tianjin Municipal Health Commission Project (grant no. TJWJ2023XK003), China Postdoctoral Science Foundation (2023M742624), Tianjin Municipal Natural Science Foundation (grant no. 23JCQNJC00870).

Competing interests None declared.

Provenance and peer review Not commissioned; externally peer reviewed.

Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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