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Ultrasound-Triggered Cuproptosis: A Novel Strategy for Targeted Cancer Therapy
Inducing precise and efficient cancer cell death while stimulating an immune response remains a critical focus in oncology. A recent study published in Advanced Materials introduces a novel approach: ultrasound-triggered nanoparticles (RC NPs) that, for the first time, integrate cuproptosis with sonodynamic therapy (SDT). This innovative platform offers a spatiotemporally controlled and immunoactivating strategy for treating pancreatic and other aggressive cancers.
01 Ultrasound-triggered nanoparticles (RC NPs)
Recently, Chinese scientists developed ultrasound-triggered nanoparticles (RC NPs) at Advanced Materials. Developed by the research team, RC NPs act like "Trojan horse-like strategy" implanted in cancer cells, awaiting the signal to eliminate cancer cells sequentially.
RC NPs are self-assembled from a degradable sonosensitive polymer (Poly RA) and a polyphenol-structured polymer capable of loading metal ions (Poly MPN).
Tips:
Sonosensitive Polymer (Poly RA): Ruthenium complex (Ruthenium Complexes, RBB) Sonosensitizer (Em=534 nm). Under ultrasound irradiation, RBB can generate reactive oxygen species (ROS), which act like “nano-scissors” to destroy cancer cell membranes.
Copper Ion Reservoir (Poly MPN): This is a metal-polyphenol network structure with a thioketal bond "insurance" ROS sensitivity and metal binding affinity, effectively locking Cu2+. Ultrasound and ROS trigger thioketal bond cleavage, releasing copper ions that disrupt mitochondrial function and induce cuproptosis.
RC NPs can kill cancer cells by generating ROS and inducing cuproptosis. The platform has unique advantages, such as deeper tissue penetration, excellent tissue selectivity, and higher safety, and has broad application prospects. RC NPs are also targeted at pancreatic cancer, the "king of cancers", and have shown amazing therapeutic effects in a variety of animal models[1].
Figure 1. RC NPs are biodegradable nanocarriers self-assembled from amphiphilic polymers Poly RA and Poly MPN[1]. A: Schematic diagram of the composition of RC NPs. Poly RA is a biodegradable polymer containing RBB molecules and thioketal bonds. On the one hand, this structure gives Poly RA the ability to generate ROS (Reactive Oxygen Species) On the other hand, ROS generation disrupts the thioketal bonds, leading to the degradation of Poly MPN. Poly MPN is an amphiphilic polymer synthesized by a similar method that has ROS-sensitive properties and can bind to metals. BD: Appearance and stability of RC NPs. RC NPs enter cells to promote cuproptosis, and TEM imaging analysis shows that they are uniformly distributed spherical structures (B, C). In PBS with or without 10% fetal bovine serum (FBS), the particle size of RC NPs did not change significantly within 14 days, showing considerable stability (D).
02 RC NPs have a dual killing effect on cancer cells
RC NPs can be rapidly degraded under ultrasound irradiation (~5 min), releasing Cu2+ locally in the tumor and ROS (Using DPBF as a specific probe for singlet oxygen (1O2 ),~83% of DPBF was oxidatively degraded after 2.5 min of ultrasonic treatment, confirming ROS generation by RC NPs). Along with the aggregation of lipoylated proteins and the depletion of iron-sulfur cluster proteins, RC NPs further cause mitochondrial damage and metabolic pathway disruption, and finally activate cuproptosis[1].
RC NPs also synchronously trigger immunogenic cell death (ICD), activating systemic anti-cancer immune responses, including damage-associated molecular patterns (DAMPs) Furthermore, RC NPs activated systemic T cell immunity and remodeled the immunosuppressive microenvironment, enhancing effector T cells (CD8+ T cells) infiltration, activating natural killer cells, and inhibiting regulatory T cells (Treg)[1].
Key Mechanism 1: Controllable release of copper ions and copper-induced death
First, after cancer cells internalized RC NPs, the intracellular ROS level and copper ion concentration increased significantly. Western blotting (Western blot) results showed that the expression levels of iron-sulfur cluster proteins FDX1, ACO2, and SDHB were significantly downregulated, directly inhibiting the activity of the key enzyme in tricarboxylic acid cycle (TCA cycle), leading to energy metabolism collapse. Subsequently, it was observed that DLAT, a key marker of cuproptosis, formed prominent aggregated plaques in the cytoplasm (Enhanced red fluorescence signal) in confocal laser scanning microscopy (CLSM) assay, confirming abnormal aggregation of lipoylated proteins. In addition, it was shown that mitochondria in tumor cells treated with RC NPs/US showed reduced cristae structure in biological transmission electron microscopy (Bio-TEM) assay, which also increased membrane density, and overall atrophy, indicating that mitochondrial function was severely impaired.
Figure 2. Intracellular anticancer properties of RC NPs[1]. A-C: RC-NPs can effectively internalize cancer cells, increase intracellular ROS levels and copper ion concentrations, and inhibit cell viability. In Miapaca-2 cells treated with RC NPs/US, ROS generation was 4.0 and 3.4 times higher than that after RC NPs and MPN NPs/US treatment, respectively (A); and the Cu content was 9.1-fold higher in cells treated with CuCl₂ than in those treated with RC NPs/US (B); thus, the survival rate of Miapaca-2 cells treated with 20 μM RC NPs/US was only 28%, with an IC50 of 8.7 μM (C). D-E: RC-NPs induce cuproptosis by inducing the aggregation of lipoylated proteins and the reduction of iron-sulfur cluster proteins. RC NPs/US treatment downregulated the expression levels of iron-sulfur cluster proteins (such as POLD1, ACO2, SDHB, LIAS and FDX1) (D); and DLAT significantly aggregated in the cytoplasm, marking the occurrence of cuproptosis (E). F-G: RC NPs target cancer cell mitochondria and inhibit mitochondrial membrane potential. In pancreatic cancer cell Miapaca-2 treated with RC NPs/US, the mitochondrial morphology was mostly atrophic, mitochondrial cristae were diminished or absent, and the mitochondrial membrane density increased (F); at the same time, the mitochondrial membrane potential of the cells changed, and JC-1 could not aggregate in the mitochondrial matrix, showing strong green fluorescence and weak red fluorescence (G).
The study found that RC NPs triggered the release of DAMPs, a characteristic molecule of ICD. DAMPs mainly include calreticulin (CRT) externalization of calreticulin (CRT), ATP release, and HMGB1 translocation. CLSM and flow cytometry (FCM) quantitative results showed that the fluorescence intensity and expression of CRT on the membrane surface of Miapaca-2 cells treated with RC NPs/US were higher than those in the control group, indicating the initiation of ICD. ELISA detected an increase in the concentration of HMGB1 in the culture supernatant of Miapaca-2 cells, and FCM showed an increase in ATP release. The two synergistically promoted antigen presenting cells (APCs) recruitment and activation.
Moreover, RC NPs simultaneously mediated the systemic activation of immune effector cells and promoted dendritic cells (DC) Maturation and polarization reprogramming of macrophages. FCM analysis demonstrated that bone marrow-derived dendritic cells (BMDCs) were reprogrammed after co-culture with tumor cells treated with RC NPs/US. The maturation of BMDCs was significantly enhanced, as indicated by increased expression of the co-stimulatory molecule CD80+. In addition, the expression of the M2 macrophage marker CD206+ was markedly reduced, suggesting a phenotypic shift from M2 to M1 polarization. These results indicate that RC NPs/US treatment reversed the immunosuppressive tumor microenvironment into a proinflammatory state.
In addition, RC NPs mediated systemic T cell immune activation and immunosuppressive microenvironment remodeling, enhancing T cell infiltration and immune surveillance. RC NPs/US significantly increased the proportion and infiltration rate of CD8+ T cells in the Panc 02 subcutaneous tumor model, and the proportion of CD8+ T cells increased by 2.2 times compared with the control group. It also increased the proportion of NK cells in tumor tissues to nearly 3-fold that of the PBS group (5.24%); and increased by nearly 3-times; and significantly lowered the proportion of Treg cells in the tumor than that in the PBS group (7.9%).
Figure 3. RC NPs can induce tumor-specific immune responses[1]. A-D: RC NPs effectively activate DAMPs and activate immune responses. RC NPs induce CRT of Miapaca-2 cells to migrate to the cell membrane, exposing it on the cell surface and increasing the release of HMGB1 in the cancer cell nucleus (A). RC NPs treatment increases the flow cytometry curve area (B) and FCM analysis index of CART of Miapaca-2 cells (C), and the amount of ATP produced is 1.3 times that of cells treated with RC NPs (D). E-H: RC NPs/US induces DC maturation, and mature DC cells (CD80+ CD86+ ) increased in number (E-F); NPs/US and induced macrophage polarization from M2 to M1 (H). I-K: RC NPs/US activated immune responses in tumors and lymph nodes. RC NPs/US enhanced T cell immunity and increased CD80+ CD86+ The cells increased the proportion of Treg cells (I), activated and enhanced the anti-tumor response of NK cells (J), and reduced the proportion of Treg cells in the tumor tissue of mice (K).
03 RC NPs change the fate of pancreatic cancer mice
RC NPs may alter the fate of pancreatic cancer-bearing mice through their "dual-killing" effect, activated by ultrasound-induced cuproptosis and immune activation. In the C57BL/6 mouse model of Panc 02 subcutaneous pancreatic cancer, RC NPs accumulated at the tumor site following tail vein administration. Under the guidance of fluorescence imaging, ultrasound treatment was performed at the locked position. RC NPs induced cuproptosis in tumors, exerting a significant and superior tumor growth inhibition rate (TGI) than Gemcitabine (GEM), achieving a tumor inhibition rate of 90% [1].
Figure 4. Antitumor effect of RC NPs combined with SDT in the subcutaneous pancreatic cancer model[1]. A-C: In vivo imaging and antitumor properties of RC NPs/US. Biodistribution and ex vivo imaging after intravenous injection of Cy7.5-RC NPs at different time points, the fluorescence signal reached a peak at 24 h (A). Mice were injected with PBS, Gemcitabine (GEM), MPN NPs, MPN NPs/US, RC NPs, and RC NPs/US via the tail vein, followed by ultrasound treatment. The tumor size in the RC NPs/US-treated group was significantly smaller than that in the other treatment groups (B), showing a significant tumor treatment effect, with a tumor inhibition rate of approximately 90% (C). D: H&E and Ki67 staining results of tumor sections showed that tumor cell proliferation was significantly inhibited in mice treated with RC NPs/US, and extensive nuclear shrinkage, fragmentation, and loss were observed in tumor tissues.
Orthotopic pancreatic cancer model in mice (Pancreatic injection of Panc 02-Luciferase cells) and clinical patient-derived xenografts (PDX) In the model, RC NPs/US achieved a significant therapeutic breakthrough[1].
In an orthotopic mouse pancreatic cancer model, the tumor growth rate of the RC NPs/US group was significantly lower than that of the control group; immune analysis showed that the ratio of mature dendritic cells in the tumor (CD80+ CD86+) increased to 41.2%, and the rate of CD8+ T cell infiltration increased to 25.1%, forming a strong anti-tumor immune response, allowing the immune system to remember the characteristics of cancer cells and prevent recurrence. RC NPs/US achieved a highly efficient and low-toxic therapeutic effect in the orthotopic tumor model.
In the mouse PDX model, conventional treatment (PBS, GEM) resulted in the death of all mice within 40 days, while 80% of the mice in the RC NPs/US group survived for more than 60 days, and some individuals even achieved complete tumor regression. At the end of the 30-day treatment course, the tumor volume in the RC NPs/US group was only 1.2 times the initial volume, while the tumor volume in the control group increased by more than 15 times. RC NPs/US can effectively inhibit tumor growth and improve survival rate in PDX models that highly simulate clinical tumor microenvironment (SR).
Figure 5. Antitumor effect of RC NPs combined with SDT in subcutaneous pancreatic cancer model and PDX model[1]. A-D: In vivo antitumor effect of RC NPs in an orthotopic model of pancreatic cancer in mice. The model was established by injecting luciferase-expressing Panc 02 cells orthotopically into the pancreas of mice. After the model was successfully established, RC NPs were injected twice via the tail vein (A). After adding ultrasound treatment, RC NPs/US effectively inhibited tumor growth, and the average tumor weight was only 1/6 and 1/2 of that in the GEM group and RC NPs group (B); and increased the number of mature DC cells in the tumor (C) and CD8+ T cell infiltration rate (D). The body weight of mice did not change significantly during RC NPs treatment. E-G: In vivo antitumor effect of RC NPs in patient-derived xenograft (PDX) model. Small pieces of fresh tumor tissue from clinical pancreatic patients were subcutaneously transplanted into mice to establish a PDX model. The therapeutic agent was injected into the mice via the tail vein on the 1st and 3rd days, respectively (E). The results showed that RC NPs/US effectively inhibited tumor growth, and the survival rate of mice was significantly longer than that of other groups (F); and the tumor volume of mice in the RC NPs/US group was significantly smaller than that of the control group (G).
SDT+Nanoparticles can be well applied to deep solid tumors (such as liver cancer, glioblastoma) and drug-resistant tumors (Doxorubicin-resistant U87 cells). Combination therapy packages can also be added to expand the treatment scope. For example, SDT+immunotherapy, combined with PD-L1 antibody after inducing ICD, may significantly increase the infiltration rate of CD8+ T cells. Or SDT+Gene therapy, through ultrasound-targeted delivery of siRNA, silence tumor-related genes (such as MDR1), reversing chemotherapy resistance.
Technical advantages of SDT[1][2]:
Precise and controllable: by adjusting ultrasound parameters (frequency, intensity, pulse mode) can achieve precise activation of local tumors and avoid causing systemic toxicity. Avoiding systemic toxicity and "accidental damage" to healthy cells.
Deep penetration: Ultrasound, as a mechanical wave, has a significantly better penetration depth than photodynamic therapy (PDT) Light source (such as visible light/near infrared light) can reach deep tumor tissue (such as deep organs such as the liver and pancreas), and is suitable for treating solid tumors that are difficult to reach with traditional therapies.
04 Summary
This article introduces the effects of RC NPs in subcutaneous pancreatic cancer models, orthotopic models, and patient-derived xenografts (PDX). The results showed that RC NPs had a significant tumor-suppressing effect in the model. At the same time, fluorescence imaging and flow cytometry experiments also confirmed the good biosafety and immune activation ability of RC NPs. This achievement overcomes the limitations of traditional copper delivery systems (e.g., poor spatiotemporal control and systemic toxicity) and opens new avenues for combining sonodynamic therapy with novel cell death mechanisms”, providing a highly promising candidate for clinical transformation. Perhaps in the near future, non-invasive and precise nanotherapy will become the standard for anti-cancer treatment, turning more "incurable diseases" into "curable diseases!"
RC nanoparticles (Cy7.5-RC NPs) were labeled, and the biodistribution and enrichment of the nanoparticles in the tumor model were monitored by in vivo imaging system (IVIS).
ROS probe, which visualizes and semi-quantitatively analyzes the generation level of reactive oxygen species (ROS) in tumor cells through changes in green fluorescence intensity.
Detect the concentration of copper ions (Cu2+) in tumor cells. The green fluorescence intensity is positively correlated with the intracellular (Cu2+) concentration.
The changes in the red-green fluorescence ratio were used to evaluate the changes in mitochondrial membrane potential, reflecting the damage to mitochondrial function and the process of cuproptosis.
