In this study, the team focused on the clinical challenge of poor prognosis in the treatment of highly malignant tumors. Centering on the treatment concept of combining “reducing tumor burden” with “systemic immunity,” the team proposed a new treatment strategy that combines Wknife ablation technology with immunomodulation, leveraging the synergistic advantages of immediate tumor ablation and inhibition of metastatic tumors. They carried out interdisciplinary collaborative cooperation and scientific research, studied the host immune response and the regulation of the tumor microenvironment induced by pulsed electric field tumor ablation, and proposed a new tumor treatment strategy of “Wknife” combined with nano-immune drugs to potently activate systemic antitumor immune responses.

Since 2000, the team of Professor Yao Chenguo at Chongqing University has been dedicated to research on tumor cell electroporation, and the related results were first published in an international academic journal in 2004. Since then, Professor Yao Chenguo’s team has continuously developed and improved product technology and formed unique independent intellectual property rights, and is currently internationally recognized as a pioneer and leader in electroporation tissue ablation technology. Building on the team’s earlier research and starting from the perspective of pulse parameter optimization, they proposed a new pulse form combining nanosecond pulses and microsecond pulses, and developed the first micro-nanosecond synergistic electric pulse tumor ablation system in China and abroad — the Wknife.

The original text is as follows:

The incidence of malignant tumors continues to rise, and the mortality rate remains high, making the treatment situation severe and seriously threatening the life and health of people worldwide. China has listed diagnostic and treatment technologies for major diseases such as malignant tumors as a key development direction and a priority area in urgent need of breakthroughs. With the continuous deepening of comprehensive, multi-scale research on molecules, genes, cells, tissues, and organisms, analyzing complex tumor characteristics and proposing advanced and effective treatment technologies urgently require the intersection and integration of multiple disciplines such as medicine, biology, and engineering. Among these, medicine-engineering integration is the key to addressing the actual needs of clinical application and breaking through medicine’s “bottleneck” technologies; by applying engineering technology to the medical field, more advanced and more precise diagnostic and treatment devices can be developed. At present, tumor ablation technology developed from pulsed electric field irreversible electroporation has become a new type of physical treatment modality. With its outstanding advantages such as unique non-thermal effects, minimal invasiveness, and selectivity, it has shown broad medical prospects in the treatment of highly malignant tumors located in special sites adjacent to blood vessels or important organs that are unresectable or that have failed radiotherapy and chemotherapy.

Since 2000, the team of Professor Yao Chenguo at Chongqing University has been dedicated to research on tumor cell electroporation, and the related results were first published in an international academic journal in 2004. Since then, Professor Yao Chenguo’s team has continuously developed and improved product technology and formed unique independent intellectual property rights, and is currently internationally recognized as a pioneer and leader in electroporation tissue ablation technology. Building on the team’s earlier research and starting from the perspective of pulse parameter optimization, they proposed a new pulse form combining nanosecond pulses and microsecond pulses and developed the first micro-nanosecond synergistic electric pulse tumor ablation system in China and abroad — the Wknife. It has passed the Class III medical device registration testing of the National Medical Products Administration (NMPA), successfully carried out the first clinical trials for pancreatic cancer and liver cancer, and has new applications in areas such as ablation of benign prostatic hyperplasia and thyroid lesions. In order to break through the clinical difficulties faced by pulsed electric field tumor ablation technology and to continue expanding its clinical applications, interdisciplinary collaborative cooperation and scientific research were carried out to study the host immune response and the regulation of the tumor microenvironment induced by pulsed electric field tumor ablation, and a new tumor treatment strategy of “Wknife” combined with nano-immune drugs was proposed to potently activate systemic antitumor immune responses. The related work was published in Advanced Healthcare Materials under the title “Optimal Irreversible Electroporation Combined with Nano-Enabled Immunomodulatory to Boost Systemic Antitumor Immunity.”

In this study, the authors proposed a combination treatment strategy that integrates irreversible electroporation with nano-immunomodulation to enhance systemic immune responses and prevent secondary tumor growth (Figure 1A). First, through a finite element analysis model, in vitro cell killing, and in vivo tumor ablation experiments, the optimal electric field parameters for inducing damage-associated molecular patterns (DAMPs) were determined. Meanwhile, the authors used FDA-approved PEG-PLGA to encapsulate the small-molecule TLR7 receptor agonist R837 (imiquimod). To inhibit or block the PD1/PDL1 pathway, the surface of the nanoparticles was further coated with a layer of genetically engineered cell membrane overexpressing PD1 protein. At the same time, the pulsed electric field can also remodel the tumor microenvironment, promoting the penetration of the PPR@CM-PD1 nano-drug and immune cells. The authors verified the efficacy of this combined treatment from two aspects: eradication of the primary tumor and inhibition of distant tumors. These findings highlight the great potential of the new combination treatment regimen for cancer immunotherapy and pave the way for future clinical translation.

Figure 1. Schematic illustration of the synergistic efficacy of the “Wknife” and PPR@CM-PD1, and single-cell simulations under different pulsed electric fields.

Simulation studies showed (see Figure 1) that microsecond pulses and nanosecond pulses act on cells at different sites. Microsecond pulsed electric fields (μPEF) mainly trigger electroporation responses of the cell membrane, whereas nanosecond pulsed electric fields (nPEF) mainly affect intracellular organelles. This targeted effect can be attributed to the high-frequency components in nPEF, whose frequency is lower than or close to the charging time constant of the cell membrane. This causes electro-osmosis of the cell membrane, thereby affecting organelles within the cell, such as the nucleus. Therefore, micro-nanosecond synergistic pulsed electric fields (nμPEF) can not only simultaneously target both the inner and outer cell membranes, but also enhance the electroporation effect on both membranes, thereby exerting a stronger effect on cells. Through cell-killing experiments, we found that the killing effect of synergistic pulses on tumor cells was significantly higher than the sum of the effects of nanosecond pulses and microsecond pulses alone. This provided a strong basis for further in vivo experiments and helped determine appropriate parameter settings. We further investigated the effect of immunogenic cell death under conditions where the three pulses produced the same killing rate on cells. Compared with microsecond pulses alone, under synergistic pulses the proportion of CRT-positive cells on the cell membrane increased by about 20%, the proportion of HMGB1-negative decreased by about 5%, and the amount of ATP release increased by about one-fold. This indicates that synergistic pulses can more fully release tumor-associated antigens, promote the maturation of dendritic cells, and thereby trigger a stronger cellular immune response (see Figure 2).

Figure 2. In Vitro Cell-Killing Effect of Pulsed Electric Fields

Further in vivo tumor ablation experiments showed that all three pulses could induce cell necrosis and effectively inhibit tumor cell proliferation. Tumors treated with synergistic pulses released more tumor-associated antigens and DMAPs (death-associated molecular patterns), thereby stimulating the maturation of dendritic cells in lymph nodes, which increased by about 10%. In addition, synergistic pulses also promoted the proliferation and differentiation of T cells in the spleen, with the number of helper T cells increasing by about 6% and the number of cytotoxic T cells increasing by about 3-fold. This process also stimulated the release of intratumoral cytokines and activated the immune response of the mice (see Figure 3). The application of micro-nanosecond synergistic pulses has been shown to more effectively stimulate the body’s immune response. This may be because synergistic pulses can act in a targeted manner on multiple sites of tumor cells, including the outer membrane and internal organelles, thereby maximizing the exposure of immunogenic substances.

Figure 3. Systemic Immune Response Triggered by Pulsed Electric Fields

At the same time, the effect of pulsed electric field ablation on primary tumors and its impact on distant tumors were evaluated. All three pulses could immediately destroy tumor cells, thereby safely and effectively ablating the primary tumor. Compared with only 2 mice in the nanosecond pulse group and the microsecond pulse group whose tumors completely disappeared, the synergistic pulse group was more effective in controlling the primary tumor, with a cure rate of 50%. In addition, pulsed electric fields can also alter the tumor microenvironment by promoting increased vascular permeability, alleviating hypoxia, and softening the dense stroma. Unfortunately, none of the three pulses appeared to effectively inhibit the growth of distant tumors; compared with the control group, no significant difference was observed in distant tumor growth (Figure 4). These findings demonstrate the ability of pulsed electric fields to rapidly and effectively eliminate primary tumors. However, the immune effects of pulsed electric fields alone are not sufficient to eliminate all residual tumor cells and inhibit the development of distant tumors. These results highlight the importance of combining ablation therapy with immunotherapy to enhance systemic immune responses.

Figure 4. In Vivo Antitumor Effect of Pulsed Electric Fields

Considering the superior characteristics of nanomaterials as modular platforms for targeting and delivering multiple drugs, the research team developed a genetically engineered nanovesicle, named PPR@CM-PD1, to serve as an immunomodulator to enhance the “in situ vaccine” vaccination response triggered by the pulsed electric field. This nanovesicle was prepared using a nanoprecipitation method, using PEG-PLGA polymer to encapsulate R837, while coating the outer layer of the nanoparticle with a cell membrane highly expressing PD1 protein. The synthesized PPR@CM-PD1 nanoparticles have stable performance. After the pulsed electric field modulates the tumor stroma, these nanoparticles can more easily penetrate the tumor tissue and thus exert their effects more effectively (see Figure 5).

Figure 5. Preparation and Characterization of PPR@CM-PD1 Nanovesicles

Combining pulsed electric fields with immune drugs can achieve efficient “cold-to-hot” tumor conversion, improve the efficacy of immunotherapy, effectively eliminate tumor cells, and inhibit recurrence and metastasis. When pulsed electric fields and PPR@CM-PD1 were used simultaneously for combination treatment, the primary tumor almost completely disappeared by day 12, the growth of distant tumors was significantly reduced by about 70%, and the median survival of the mice was significantly prolonged, from 14 days to 30 days (Figure 6). These findings indicate that treatment with pulsed electric fields combined with PPR@CM-PD1 potently eliminated the primary tumor and effectively inhibited the growth of distant tumors, representing a promising tumor treatment modality.

Figure 6. In Vivo Antitumor Effect of Synergistic Pulses Combined with PPR@CM-PD1

In addition to directly killing tumor cells, pulsed electric fields can induce immunogenic cell death and release DAMPs, and lead to the release of tumor-associated antigens (TTA) hidden within cells, thereby activating and maturing dendritic cells in lymph nodes. These mature dendritic cells can effectively process antigens and activate naive T cells, thereby initiating T cell-mediated immune responses. At the same time, synergistic pulses loosened the tumor tissue, alleviated the immunosuppressive tumor microenvironment, and promoted the infiltration of immune cells, thereby eliminating residual cells and completely clearing the primary tumor. In addition, the nano-immune drug more easily accumulates in tumor tissue; the immune adjuvant R837 promoted the maturation of dendritic cells, and the PD1 protein strengthened the adaptive immune system through PD1/PDL1 checkpoint blockade. Because T cells were activated, the spleen produced more memory T cells, which effectively targeted distant tumors (Figure 7). Therefore, synergistic pulses altered the tumor microenvironment, leading to increased accumulation of PPR@CM-PD1 within the primary tumor, producing a more effective systemic antitumor immune response.

Figure 7. Systemic Immune Response of Synergistic Pulse Combined with PPR@CM-PD1 Treatment

[Article Conclusions and Discussion, Inspiration and Outlook]

In summary, the authors proposed a new tumor treatment strategy that combines ablation therapy with nano-immunomodulation, leveraging the synergistic advantages of immediate tumor ablation and inhibition of metastatic tumors. The authors used micro-nanosecond synergistic pulsed electric fields (Wknife) for primary tumor ablation, showing advantages such as multi-site action on cells, higher cell-killing efficiency, and initiation of immune responses. At the same time, the authors further developed a genetically engineered nanoparticle containing the immune adjuvant imiquimod (R837) and PD1 protein (PPR@CM-PD1), which acts on the innate immune system through the TLR7 agonist and on the adaptive immune system through PD1/PDL1 checkpoint blockade. Local treatment with synergistic pulses, combined with systemic administration of PPR@CM-PD1, can elicit an effective systemic antitumor immune response, thereby inhibiting both the primary tumor and distant tumors. Given that the pulsed electric field and the components of PPR@CM-PD1 have received FDA approval or are of biological origin, we anticipate that this innovative approach is expected to achieve clinical translation.

Professor Yao Chenguo of the School of Electrical Engineering, Chongqing University; Researcher Wu Ming of the Key Laboratory of Mengchao Hepatobiliary Hospital of Fujian Medical University; and Xue Yanan of the School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, are the co-corresponding authors of this paper. Dr. Peng Wencheng, doctoral student Cao Yanbing, and master’s student Zhang Yuting are the co-first authors of this paper. This study was funded by projects including the National Natural Science Foundation of China (52237010 and 62175031), the Fujian Provincial Science and Technology Program (2022J011282 and 2021J011285), the Chongqing Natural Science Foundation (cstc2020jcyj-msxmX0932), the Fuzhou Science and Technology Program (2022-Y-007), the Fuzhou Health Commission Science and Technology Project (2022-S-rc1), and the Fuzhou “14th Five-Year Plan” Key Clinical Specialty (20220203).

Original article link:

https://onlinelibrary.wiley.com/doi/10.1002/adhm.202302549