Quantification of plasma mutant calreticulin for diagnosis and therapeutic response assessment of peginterferon-α in myeloproliferative neoplasms: A proof-of-concept study.

Calnexin (CNX) and calreticulin (CRT) are two essential endoplasmic reticulum chaperones involved in the folding pathway dedicated to N-glycosylated proteins. The N-glycosylated protein GP64, located in the budded virus envelope of Bombyx mori nucleopolyhedrovirus (BmNPV), plays a key role in viral entry. However, the roles of CNX and CRT in GP64 processing and maturation, as well as in BmNPV infection, remain incompletely understood. In this study, we identified the two genes BmCNX and BmCRT from a B. mori transcriptome database. Spatiotemporal expression profiles showed that BmCNX and BmCRT displayed similar patterns, with expression detected in all tissues and elevated levels in the gonad, trachea, and malpighian tubule, as well as during the egg and larval stages. Following BmNPV infection, the expression of BmCNX and BmCRT was significantly upregulated in both the midgut and BmN cells. Overexpression of BmCNX and BmCRT enhanced BmNPV proliferation and GP64 expression, while knockdown of either gene suppressed both viral proliferation and GP64 expression. Furthermore, GP64 was shown to interact with BmCNX and BmCRT, respectively. These results suggest that BmNPV infection requires both BmCNX and BmCRT to facilitate GP64 expression and promote viral proliferation. This study lays the foundation for further investigation into the roles of endoplasmic reticulum chaperones in response to BmNPV infection.

Oral squamous cell carcinoma (OSCC) overexpresses CD47, enabling immune evasion via a "don't eat me" signal to macrophages. Although CD47 blockade shows promise, its efficacy is limited due to a lack of "eat me" signal and contact between macrophages and tumor cells. This study aimed to evaluate the synergistic anti-tumor effect of combining photothermal therapy (PTT) with CD47 blockade in OSCC and elucidate the underlying mechanisms. In vitro phagocytosis was assessed by flow cytometry. In vivo anti-tumor efficacy was measured by tumor growth inhibition. Mechanistic studies included detection of immunogenic cell death (ICD) markers (ATP, HMGB1, calreticulin (CRT)), confocal microscopy for CRT-macrophage co-localization, analysis of ECM component expression, and immunofluorescence for macrophage infiltration. The combination of PTT and CD47 blockade significantly enhanced macrophage phagocytosis in vitro and strongly inhibited tumor growth in vivo. PTT induced ICD, as evidenced by the release of ATP and HMGB1, and the exposure of CRT on the cell membrane. Confocal microscopy confirmed co-localization of CRT-expressing tumor cells with macrophages. Furthermore, PTT down-regulated ECM components at transcriptional and protein levels, which correlated with increased macrophage infiltration into tumors. PTT synergizes with CD47 blockade primarily by inducing CRT-dependent pro-phagocytic signaling to provide the "eat me" signal. In parallel, PTT downregulates ECM components, enabling the essential "come near me" process that facilitates macrophage infiltration into tumors. This dual approach significantly improves the macrophage based anti-tumor efficacy of CD47 blockade.

Abstract Prostate cancer remains the most commonly diagnosed malignancy in men in the United States and the second leading cause of cancer-related mortality. Identifying new molecular mechanisms that influence tumor–immune interactions is essential for improving therapeutic outcomes. Calreticulin (CRT), an endoplasmic reticulum (ER) chaperone, can translocate to the cell surface where it functions as a damage-associated molecular pattern (DAMP) during immunogenic cell death (ICD), serving as an “eat-me” signal that links the stressed tumor cell to innate immune recognition and subsequent adaptive T-cell activation. Although CRT expression in prostate epithelial cells is known to be androgen-regulated, the mechanism governing its surface localization is poorly understood. Post-translationally modified forms of CRT have been detected at the cell surface, yet its potential acetylation-dependent trafficking has not been examined, despite proteome-wide studies identifying numerous acetylated proteins in prostate cancer. Here, we investigated the role of CRT acetylation in its surface translocation in prostate cancer models. Using 7,8-diacetoxy-4-methylcoumarin (DAMC) as an exogenous acetyl donor, we evaluated surface and intracellular expression of wild-type and lysine-mutant (K206R/K207R/K209R) GFP-tagged CRT in LNCaP cells for later comparison with primary prostate epithelial cells. Cells were treated with DAMC (100 µM) in combination with trichostatin A (50 nM) and thapsigargin (100 nM) for 24 h. Immunofluorescence analysis demonstrated increased surface CRT in treated cells compared with controls, with a positive correlation between surface CRT and lysine acetylation signals. Surface protein isolation and immunoblotting confirmed CRT at the plasma membrane, although acetyl-lysine signal was diminished, suggesting that acetylation facilitates CRT trafficking but may be reversed or transferred upon membrane localization. Experiments with mutant CRT isoforms and analysis assessing whether DAMC-induced CRT acetylation is accompanied by other hallmarks of ICD, including extracellular ATP and HMGB1 release are ongoing to determine whether acetylation enhances the full immunogenic signature of dying prostate cancer cells. These findings support a model in which CRT acetylation enhances its surface exposure and immunogenic function, providing a novel mechanism by which post-translational modification can modulate tumor immunogenicity. Elucidating this pathway may reveal new strategies to exploit the prostate tumor microenvironment, enhance immune recognition of prostate cancer cells, and inform combination approaches with immunotherapy or radiation. Citation Format: Courtney Thomas, Janiyah Vogle, Kandace Rankin, Emmanuel Frimpong, Kayla Glover. Targeting calreticulin acetylation to enhance tumor immunogenicity in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr A063.

Proton beam therapy is widely regarded for its cost-effectiveness, precision, and protection of normal tissues. Emerging evidence shows that conventional radiotherapy can inhibit primary tumors and promote immunogenicity of distant tumors, possibly via damage-associated molecular patterns (DAMPs) like calreticulin (CRT) and high mobility group box 1 (HMGB1) released during immunogenic cell death (ICD). However, the role of proton beams in inducing DAMPs and enhancing immunogenicity remains unclear. This study aimed to investigate the effects of proton beam-induced DAMPs on the colonization of distal tumors. In this study, in vitro cell irradiation experiments were conducted to identify the optimal proton beam dose for enhancing DAMPs expression in mouse colon carcinoma Colon-26 cells. Based on the optimal proton dose determined in vitro, a tumor-bearing mouse model was employed to evaluate its efficacy in inhibiting distal tumor colonization. To explore the mechanisms behind the anti-tumor effects, shRNA targeting DAMPs-related immunogenic molecules was applied to assess the immune response. In vitro findings indicated high-dose proton irradiation markedly induces HMGB1 release yet exerts no significant effect on CRT membrane exposure. Following high-dose proton beam irradiation, tumor cells transfected with shRNA exhibited a significant reduction in CRT and HMGB1 expression compared with the con-shRNA-irradiated control group. In vivo experiments demonstrated that HMGB1 knockdown reduced distal-tumor rejection by 60%, whereas CRT knockdown reduced it by only 20%, indicating that HMGB1 release may dominate proton-induced ICD. Our research results indicated that high-dose proton irradiation trigger the rejection of distal tumor colonization through a signaling pathway that depends on HMGB1. This research advanced our understanding of proton beam therapy immunological mechanisms and offered insights for improving tumor treatment outcomes.

Background/Objectives: To improve the response rate of immune checkpoint inhibitors (ICIs), inducing immunogenic cell death (ICD) is a promising approach. Photothermal therapy (PTT) induces immunogenic cell death and activates anti-tumor immunity. While there are various ICD inducers, the difference in ICD induction by various modalities is poorly understood. In this study, we found previously unrecognized advantages of PTT compared to anti-cancer drugs and showed the usefulness of PTT as an anti-cancer drug-free approach to be combined with immunotherapy. Methods: Gold nanorods were synthesized as photothermal agents and added to culture medium or locally administered to tumor tissues. Mitoxantrone (MIT), an ICD inducer, and cisplatin (CDDP), a non-ICD inducer, were compared with PTT. To assess the induction of ICD, the subcellular localization and amounts of high mobility group box 1 (HMGB1) and calreticulin (CRT) were observed using immunofluorescent staining. FM3A tumor-bearing mice were treated with PTT or anti-cancer drugs, and cell death and DAMPs localization in tumor tissues were analyzed. Also, the supra-additive effect of PTT on ICI was observed. Tumor-infiltrating CD8+ T cells were examined to evaluate the immune status in tumor tissues. Results: In vivo assays showed that PTT induces HMGB1 release and increased expression of CRT on the cell membrane. Moreover, PTT showed a supra-additive effect in terms of therapeutic effect and anti-tumor activation when combined with an immune checkpoint inhibitor. Conclusions: In this study, we demonstrated that PTT induced ICD-related signaling and improved the response rate of ICI, which means PTT is a promising combination therapy with ICI.

Calreticulin (CALR) mutations are prevalent in 20%-30% of patients with BCR::ABL1-negative myeloproliferative neoplasms (MPN). Mutant calreticulin (mutCALR), presented by the thrombopoietin receptor (MPL, also known as TPOR or CD110) on the surface of the disease-initiating MPN progenitors, represents an ideal target for curative immunotherapies including monoclonal antibodies, bispecific T cell engaging antibodies (TCE), and CAR-T cell therapies. Despite that two clinical TCE candidates have advanced into phase 1 trials in recent 2 years, depletion of mutCALR+ hematopoietic stem cells and normalization of hematopoiesis remained absent in preclinical evaluation. Here, we developed a bispecific T cell engager DX1-2C11 that specifically and efficiently eradicates mutCALR-expressing cells via recruiting polyclonal T cells. DX1-2C11 depleted Ba/F3 cells expressing mutCALR, as well as primary murine myeloid cells in a dose-dependent manner invitro. In CALRdel52 transgenic mice, a single dose of DX1-2C11 activated CD4+ and CD8+ T cells in the peripheral blood, spleen and bone marrow within 24 h. Furthermore, a single dose of DX1-2C11 reduced platelet counts in the periphery and decreased mutant stem/progenitor cell populations in the spleen and bone marrow by Day 7 posttreatment. Notably, the reduction of mutant burden was durably maintained in secondary recipient mice. In the disseminated NSG model, DX1-2C11 delivered immediate tumor burden reduction and significantly prolonged the overall survival of mice compared to the control group. Taken together, these data suggest that bispecific T cell engaging antibody targeting mutCALR represents a curative strategy that efficiently eliminates mutant MPN stem cells invivo.

Design, engineering, and functional evaluation of nanobody-based anti-CD73 for immunogenic cell death induction in chemoresistant colorectal Cancer cell line.

BackgroundAge, gender, and mutation type are key risk factors for myeloproliferative neoplasms (MPNs). Africa remains under-represented in global cancer statistics due to limited population-based genomic data.ObjectiveTo determine the frequency and demographic associations of common MPN-related genetic abnormalities in the South African population.MethodsA retrospective cross-sectional analysis of cytogenetic results for Janus kinase-2 p.V617F (JAK-2 p.V617F), Janus kinase-2 exon 12 (JAK-2 exon 12), calreticulin (CALR), myeloproliferative leukaemia virus oncogene (MPL), and breakpoint cluster region-Abelson kinase 1 (BCR::ABL1) was conducted from 01 January 2018 to 31 May 2023. Data were retrieved from the National Health Laboratory Service and analysed for associations with age and gender using Fisher’s Exact Test or Pearson’s Chi-Square Test (p < 0.05).ResultsA total of 8934 patient records were analysed; 58% were male patients and 42% female patients, with a mean age of 50 ± 17 years. Among sequence variant changes, 18.2% of MPN cases were positive for BCR::ABL1, 8.5% for JAK-2 p.V617F, 0.5% for CALR, 0.04% for MPL, and none for JAK-2 exon 12. BCR::ABL1 showed equal sex distribution, while JAK-2 p.V617F increased with age and showed slight female predominance (p = 0.002). CALR and MPL frequencies were too low for meaningful association testing.ConclusionBCR::ABL1 was the most frequent abnormality, especially in younger age groups, whereas JAK-2 p.V617F was linked to increasing age and female predominance.What this study addsMPN genetic testing in South Africa predominantly targeted male patients (ratio 1.4:1). BCR::ABL1 was the most common abnormality, particularly in individuals aged 18 to 49 years, while JAK-2 p.V617F showed a slight female predominance (1:1.2).

Ultraprecision Therapy for Type 1 vs Type 2 CALR+ MPN by Dual Epitope Targeting that Restores Ruxolitinib Sensitivity.

Calreticulin attenuates intervertebral disc degeneration by suppressing NLRP3 inflammasome activation through PINK1/Parkin-mediated mitophagy.

Multi-bioactive poly(amino acid)-metal-organic framework nanocomposite for reinforced cascading photodynamic immunotherapy of cancer.

TNFSF10 drives hyperactive immune responses via NLRP3 inflammasome and endoplasmic reticulum stress in autoimmune and inflammatory diseases.

Platelets display immunophenotypic alterations and dysregulated transcriptomic signature in Philadelphia-negative myeloproliferative neoplasms.

Background: A significant challenge in oncology is the development of therapies that selectively eliminate cancer cells while engaging the immune system for durable protection. Oncogene-driven transformation has been shown to rewire cellular metabolism and organelle function, creating unique dependencies. Lysosomes, traditionally considered mere degradative centers, are emerging as critical regulators of cell survival and death. We hypothesized that oncogenic transformation, particularly by drivers like Her2, creates a lysosomal "Achilles' heel," priming these organelles for permeabilization and offering a novel therapeutic target. Methods: Using the MCF-10A-neoT model of oncogene-driven transformation, we first characterized lysosomal alterations. We then conducted a high-throughput screen for compounds inducing Lysosomal Membrane Permeabilization (LMP), focusing on Reactive Oxygen Species (ROS) inducers. Lead compounds were evaluated for their ability to trigger established markers of Immunogenic Cell Death (ICD), including surface calreticulin (CRT), ATP release, and HMGB1 release. Selectivity and efficacy were further validated in co-culture models and immunocompetent mouse models. Results: Oncogene-transformed cells exhibited significantly enlarged lysosomes, altered subcellular distribution, and increased cathepsin B/L activity compared to their non-transformed counterparts. Our screen identified several ROS-generating compounds that selectively induced LMP in cancer cells. Treatment with these lead compounds resulted in the robust exposure of CRT, and the extracellular release of ATP and HMGB1, confirming the induction of ICD. In co-culture, the compounds demonstrated a clear therapeutic window, preferentially killing cancer cells. In vivo studies in immunocompetent mice confirmed tumor regression and the establishment of a protective anti-tumor immune memory. Conclusion: Our findings validate that targeted LMP induction is a viable strategy for selectively killing oncogene-addicted cancer cells. Moreover, by triggering ICD, this approach not only directly eliminates tumor cells but also stimulates a systemic immune response, addressing the critical need for therapies that provide long-term anti-cancer immunity.

Bladder cancer (BC) remains a prevalent urothelial malignancy characterized by high recurrence and mortality rates, severely compromising patients' quality of life. Current intravesical chemotherapies, although locally administered, are limited by rapid renal excretion and poor tumor accumulation, which undermines treatment efficacy. Moreover, these conventional agents often cause immunosuppression, further diminishing therapeutic outcomes. To address these challenges, we developed MC/Pep, an innovative amphiphilic peptide-based nanoplatform that self-assembles into spherical nanoparticles capable of codelivering mitoxantrone (MT) and cinnamaldehyde (CA). A key innovative feature of this system is its matrix metalloproteinase 2 (MMP2)-responsive structural transformation, which triggers morphological rearrangement into highly aggregated nanostructures within the tumor microenvironment, enabling enhanced targeted accumulation and retention. In addition to improving drug delivery, MC/Pep induced endoplasmic reticulum oxidative stress-mediated immunogenic cell death (ICD) through the triggering of reactive oxygen species (ROS) generation via intracellular redox reactions. MC/Pep also promoted the production of mitochondria-derived ROS by inducing changes in mitochondrial membrane permeability, thereby synergistically enhancing the ICD effect. This system induced the ectopic displacement of calreticulin (CRT) and the exocytosis of high-mobility group protein B1 (HMGB1) and facilitated DC maturation and T-cell activation in vivo, thereby eliciting an antitumor immune response. Owing to its promising pharmacokinetic properties, tumor-targeting ability, and ability to enhance both immunomodulation and drug accumulation, MC/Pep represents a novel and clinically promising nanotherapeutic strategy for BC, offering a viable path toward translation in immunotherapy-enhanced chemotherapy.

Pyroptosis has recently emerged as a promising therapeutic strategy owing to its unique advantages in inducing lytic cell death and eliciting immunogenic responses. Moderate induction of pyroptosis not only directly eliminates cancer cells but also activates antitumor immunity through the release of tumor-associated antigens and proinflammatory cytokines. However, most available pyroptosis inducers suffer from poor solubility, instability, and nonspecific biodistribution, which significantly hinder their translational potential. To address these challenges, we developed a pyroptosis-inducing delivery platform based on carboxybetaine zwitterionic nanogels for the efficient encapsulation of celastrol. These zwitterionic nanogels exhibit excellent resistance to protein adsorption and prolonged circulation in vivo. Upon drug release, celastrol promotes mitochondrial reactive oxygen species (ROS) accumulation, activates the caspase-3/GSDME axis, and triggers pyroptosis accompanied by immunogenic signals, including adenosine triphosphate (ATP) secretion, lactate dehydrogenase (LDH) release, and calreticulin (CRT) exposure. When combined with the anti-PD-1 checkpoint blockade, this platform further enhances dendritic cell maturation and antigen presentation, thereby amplifying pyroptosis-driven immune responses. In summary, our study demonstrates an effective strategy for inducing pyroptosis by integrating zwitterionic nanogel-mediated celastrol delivery with immune checkpoint inhibition, providing new insights and potential breakthroughs for cancer immunotherapy.

Breast cancer remains the most common malignancy worldwide and the leading cause of cancer-related mortality. Recently, extracellular vesicles (EVs) derived from adipose tissue-derived stem cells (ADSCs) have attracted increasing attention for their potential to modulate inflammatory signaling and influence tumor cell behavior. This in vitro study was designed to investigate the effects of ADSC-EVs on MCF-7 breast cancer cells. EVs were isolated from ADSC culture supernatants and applied to MCF-7 cells at concentrations ranging from 0 to 80% (v/v). Cell viability, migration, and expression of IL-6/STAT3 pathway-related genes were evaluated using MTT, scratch assays, and qRT-PCR. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test, with significance set at p < 0.05. The results showed that 20% EV treatment markedly inhibited MCF-7 cell activity, significantly reducing viability and almost completely blocking migration, with wound closure rates of 35.4% ± 3.80 at 24 h and 47.6% ± 4.2 at 48 h, compared with 48% ± 4.6 and 67% ± 4.2 in the control group, respectively. Notably, expression levels of IL-6, IL-6RST, and STAT3 were significantly downregulated (fold changes 0.155 ± 0.02 and 0.258 ± 0.012, p < 0.01), accompanied by severe disruption of the microtubule network. Immunofluorescence imaging revealed a disorganized microtubule architecture and irregular filament distribution in EV-treated cells, corresponding with decreased expression of TubA1 and CALR genes. These findings indicate that ADSC-EVs not only suppress IL-6/STAT3 inflammatory signaling but also destabilize the intracellular microtubule system, collectively contributing to the inhibition of MCF-7 breast cancer cell migration and survival. This provides an important molecular basis for developing novel EV-based therapeutic strategies in breast cancer treatment.

Targeting calreticulin (CALR) in tumors: Cellular mechanisms, structural insights and ligand development advances.

Synergistic chemoimmunotherapy using pirarubicin-loaded tumor-derived extracellular vesicles for triple-negative breast cancer (TNBC).