Oncolytic Mineralized Bacteria May Be Used for Tumor Immunotherapy via Local Injection

Research Background

As a novel cancer treatment method, bacteria-based cancer immunotherapy has a long history dating back to the late 19th century when heated, inactivated bacteria were used to treat sarcomas. Initial trials found that these bacteria could trigger strong non-specific immune responses, recruiting a large number of killer cells to combat tumor cells. However, modern research has shown that while these types of bacteria (such as Coley’s toxins) were replaced by radiation and chemotherapy in the 20th century, their therapeutic effects were comparable to standard radiation and chemotherapy. Since the 1990s, Bacillus Calmette-Guérin (BCG), a live attenuated bacterium, was found to be the preferred intravesical immunotherapy for treating bladder cancer.

In recent years, multiple research groups and biotechnology companies have devoted substantial resources to developing new anti-tumor oncolytic bacterial strains, typically through genetic engineering methods to reduce their toxicity and endow them with additional anti-tumor capabilities. Furthermore, bacteria are also being used to develop cancer vaccines, providing new opportunities for bacteria-based cancer immunotherapies. Notably, Salmonella, Listeria, and Clostridium are commonly used bacterial types.

The immunosuppressive tumor microenvironment (TME) of solid tumors can inhibit the activity of innate immune cells and prevent adaptive immune cells from exerting their anti-tumor effects, leading to poor tumor responses to various therapies, especially immunotherapies. Studies have shown that injection of attenuated bacteria at the lesion site can convert immune-silent “cold” tumors into immune-activated “hot” tumors, thereby enhancing tumor responses to other therapies.

Source of the Paper

This paper was co-authored by Chenya Wang, Qian Chen from the Institute of Functional Nano & Soft Materials (FUNSOM) at Soochow University and Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, and Liping Zhong, Cong Hu from Guangxi Medical University. It was accepted for publication by “Nature Biomedical Engineering” on February 17, 2024. The paper mainly studies mineralized bacteria as a new type of cancer immunotherapy.

Research Process

1. Research Process and Methods

The study selected Salmonella typhimurium as the experimental strain and inactivated them using 4% paraformaldehyde, followed by growing manganese dioxide (MnO_2) nanostructures on the surface of the inactivated bacteria through mineralization.

In the specific preparation process, the researchers dispersed inactivated Salmonella typhimurium at a concentration of 7.5×10^9/ml in different concentrations of MnSO_4 solutions (2.5 mM, 5 mM, 10 mM, 20 mM) for 15 minutes, then reacted with NaOH. Scanning electron microscopy (SEM) and inductively coupled plasma mass spectrometry (ICP-MS) confirmed that MnO_2 particles uniformly covered the bacteria’s surface.

2. Immune Activation and Toxicity Testing at Cellular Level

In in vitro experiments, the MTT assay revealed that inactivated bacteria did not exhibit significant cytotoxicity to CT26 and 4T1 cells. By fluorescently labeling the bacteria and co-culturing them with dendritic cells, it was verified that mineralized bacteria had a higher cell phagocytosis efficiency compared to inactivated bacteria and free MnO_2 particles.

The study also assessed the immune-stimulating effects of mineralized bacteria on mouse bone marrow-derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells (MoDCs). The experiments showed that although free MnO_2, inactivated bacteria, and their mixtures could effectively stimulate BMDCs maturation, mineralized bacteria exhibited the highest stimulating effect, even surpassing the positive control lipopolysaccharide (LPS). In the MoDCs stimulation experiment, mineralized bacteria significantly activated MoDCs.

3. In Vivo Studies on Various Tumor Models

In in vivo studies, researchers used CT26 colon cancer model, 4T1 breast cancer model, B16F10 melanoma model, KPC pancreatic cancer model, and rabbit VX2 liver cancer model, treating them with a single intratumoral injection of mineralized bacteria. Compared with inactivated bacteria and free MnO_2, mineralized bacteria demonstrated a significantly more pronounced tumor growth inhibition effect with higher cure rates. Moreover, the study found that mineralized bacteria could induce a strong immune memory effect, preventing tumor recurrence in mice or rabbits re-challenged after tumor elimination.

4. Safety Evaluation

Toxicological studies found that mineralized bacteria did not cause significant cytokine elevation in mice, and no obvious damage was observed in major organs. Additionally, acute toxicity studies with high doses of mineralized bacteria indicated that, although abscesses occasionally formed at the injection site, the overall safety was satisfactory.

Research Conclusions

The research results indicate that mineralized bacteria achieve significant anti-tumor effects by activating multiple immune pathways and inducing strong and persistent NK cell and specific T-cell responses, along with good safety. Mineralized bacteria demonstrate great potential as the next generation of anti-tumor immunotherapy, especially in local administration treatments. Considering their unique capabilities, including converting solid tumors into immune-inflammatory hot zones and their efficient immune-stimulating effects, further clinical translation and application of mineralized bacteria is highly anticipated.

Research Highlights

1. Innovative Approach: This study significantly improved the immune activation capability of bacteria by mineralizing MnO_2 nanoparticles onto the surface of inactivated bacteria.
2. Validation Across Multiple Tumor Models: Mineralized bacteria have been proven to have significant anti-tumor effects in various in vivo tumor models.
3. Strong Immune Memory Effect: Successfully cured mice and rabbits exhibited strong immune memory upon re-challenge, completely preventing tumor recurrence.
4. Safety: Mineralized bacteria demonstrated good safety at high doses, without causing significant systemic adverse effects.

This study lays a solid foundation for mineralized bacteria as a novel, efficient, and safe anti-tumor immunotherapy, providing strong support for further clinical trials.