1. Current Situation of Cancer Therapy

1.1 Cancer Incidence and Mortality Rates

The global incidence of cancer continues to rise. According to the World Health Organization (WHO), approximately 19.29 million new cancer cases were reported worldwide in 2020. Some of the most common types of cancer include breast, lung, colorectal and prostate cancer. Cancer remains one of the leading causes of death globally, with an estimation of 9.96 million deaths attributed to the disease in 2020. Fatal cancers such as lung, colorectal, liver, stomach, and breast cancer are among the most common causes of cancer-related mortality.

1.2 Current Cancer Treatments

The primary treatments for cancer include surgery, radiation therapy and chemotherapy, each playing a crucial role depending on the type and stage of cancer.

Surgery is a common method for treating many types of cancer, aiming at removing the tumour and some surrounding tissue to control or eliminate the disease. This method is most effective for cancers detected early, before they have spread to other parts of the body.

Radiation therapy uses high-energy rays, such as X-rays, to kill cancer cells or inhibit their growth. It is often employed for cancers that cannot be surgically removed or for shrinking tumours before surgery. Radiation is effective in targeting cancers in localized or fixed areas particularly.

Chemotherapy involves the use of drugs to kill cancer cells or stop them from dividing. Unlike surgery or radiation, chemotherapy can treat cancer that has metastasized because the drugs travel through the bloodstream, affecting cancer cells throughout the body. The types and combinations of chemotherapy drugs are selected based on the type of cancer, its progression, and the individual patient's condition.

1.3 The Role of Chemotherapy

Chemotherapy is a systemic treatment in which drugs circulate through the bloodstream, reaching various parts of the body. This makes it effective for treating cancers that have spread to other organs. Its systemic nature is crucial in managing advanced or metastatic cancers, where localized treatments like surgery or radiation may not be enough.

In situations where patients are unable to undergo surgery or radiation therapy due to health complications or age, chemotherapy offers a valuable alternative. For instance, in elderly patients or those with significant health risks, surgery or radiation may be too dangerous, whereas chemotherapy can still offer treatment options with lower physical risk. Chemotherapy plays a crucial role in treating malignant tumours, as many such tumours are sensitive to chemotherapy drugs. These drugs can effectively shrink tumour size, slow disease progression and, in certain cases, lead to a complete cure. Overall, chemotherapy remains a cornerstone of cancer therapy due to its broad range of applications, especially in systemic treatment or cases where other therapies are not feasible.

2. Tumour Chemotherapy Resistance: A Common Phenomenon

2.1 Incidence of Chemotherapy Resistance

The incidence of chemotherapy resistance varies based on cancer type, individual patient differences, and the specific treatment regimen employed. On average, between 50% to 70% of cancer patients develop some form of resistance during chemotherapy. Certain cancers, such as breast cancer, non-small cell lung cancer, and colorectal cancer, tend to have higher rates of resistance, making treatment more challenging in these cases. Chemotherapy resistance can develop gradually during treatment, with its incidence increasing over time. Factors such as a patient's genomic background, overall health, and the presence of pre-existing mutations in cancer cells can significantly affect the rate and extent of resistance development[1].

Tumours are composed of cancer cells with varying molecular characteristics, which may make them sensitive to different treatments or lead to the development of drug resistance. During treatment, drugs may eliminate a significant portion of the cancer cells, but a subset of these cells often survives. These surviving cells, known as drug-resistant cells, possess adaptations that allow them to evade the effects of the treatment. Over time, these drug-resistant cells multiply, resulting in the regrowth of the tumour and the formation of drug resistance, making subsequent treatments less effective.

2.2 Mechanisms of Chemotherapy Resistance in Cancer

Chemotherapy resistance in cancer is a multifaceted process involving several mechanisms at different levels:

① Drug Efflux Pumps: Cancer cells can upregulate drug efflux pumps, such as P-glycoprotein(P-gp), which actively transport chemotherapy drugs out of the cell, thereby lowering the their effective concentration.

② Drug Metabolism: Cancer cells may enhance the activity of drug-metabolizing enzymes that break down chemotherapy agents more rapidly. This accelerated drug degradation decreased the amount of active drug available in the body, weakening the treatment's impact.

③ Enhanced DNA repair: Some chemotherapy drugs cause DNA damage to kill cancer cells. However, resistant cancer cells may increase their DNA repair capabilities, allowing them to quickly fix the chemotherapy-induced damage, thus reducing the drug's cytotoxic effects.

④ Evasion of Cell Death: Chemotherapy often induces cancer cell death by triggering apoptosis. Resistant cancer cells can evade this process by upregulating anti-apoptotic proteins or downregulating pro-apoptotic proteins, which prevent the programmed cell death that chemotherapy aims to induce.

⑤ Tumour Heterogeneity: The heterogeneity within a tumour means that different cells may respond differently to chemotherapy. Some cancer cells might inherently possess or acquire resistance, enabling them to survive treatment and proliferate, further driving the development of resistance[2].

2.3 Consequences of Chemotherapy Resistance

The development of chemotherapy resistance has significant consequences for both patient health and economic well-being:

Health Impact: Chemotherapy resistance reduces the effectiveness of treatment, allowing cancer to progress and worsening patient outcomes. This may lead to decreased survival rates and a lower quality of life. Patients may also be forced to undergo more aggressive and toxic treatments, which can further damage their health.

Economic Impact: The cost of treating resistant cancer escalates, as patients often require expensive alternative medications or additional supportive therapies. Resistance can also result in longer hospital stays, more frequent treatments, and overall increased medical expenses, intensifying the financial strain on patients and healthcare systems.

3. Unspecific Cytotoxic Effects of Chemotherapy

Chemotherapy's non-specific cytotoxic effects refer to the damage caused to normal cells alongside cancer cells. Because many chemotherapy drugs lack high selectivity, they primarily target rapidly dividing cells—a characteristic shared by both cancer cells and some normal cells, such as those in the bone marrow, gastrointestinal tract, and hair follicles.

Bone Marrow Suppression: Chemotherapy often suppresses bone marrow activity, reducing the production of blood cells, which leads to complications such as Leukopenia (low white blood cell count), Anemia (low red blood cell count), Thrombocytopenia (low platelet count).

Gastrointestinal Damage: Chemotherapy affects the gastrointestinal lining, resulting in symptoms like nausea, vomiting, diarrhea, constipation, and oral ulcers.

Hair Loss: Due to the rapid division of hair follicle cells, chemotherapy commonly causes hair loss. Skin and nails may also be affected, causing dryness, itching, pigmentation changes, or brittleness.

Reproductive System Impact: Chemotherapy can affect fertility, leading to reduced sperm count or quality in men, and menstrual irregularities, amenorrhea, or even infertility in women.

Neurotoxicity: Certain chemotherapy drugs cause neurological side effects, such as numbness, tingling, or muscle weakness, particularly in the extremities (hands and feet).

Given these wide-ranging non-specific effects, patients often experience multiple side effects. Doctors counteract these with supportive therapies, dose adjustments, or by switching to less toxic treatment regimens to improve patient quality of life during chemotherapy.

4. Inspiration

As previously mentioned, there are mainly two problems with traditional chemotherapy. First, chemotherapy resistance often occurs during treatment, increasing the risk of recurrence. Second, chemotherapeutic agents are less selective, often leading to serious side effects during treatment. Therefore, once the chemotherapy resistance can be reduced and the selectivity of chemotherapeutic agents can be increased, there's a possibility of significantly enhancing the effects of chemotherapy and improving the overall prognosis and quality of life of patients. Motivated by the desire to address these issues, we consulted with specialists who have been working on the research of malignant tumours. During the conversation, Jin Duo, the General Manager of Jilin Qizhong Biological Co., Ltd. showed us a report on the small-nucleic acid drug industry ‘Small nucleic acid drug industry report -自古雄才多磨难，小核酸迎新纪元’, which told us that using RNA interference technology in the treatment of tumour has ushered in a new era of development.

Taking this as an opportunity, we determined to utilize RNA interference (RNAi), a promising technology, to down-regulate genes responsible for chemotherapy resistance, thereby increasing the sensitivity of tumour cells to chemotherapeutic agents[3]. However, challenges remain in improving the targeting ability and biosafety of small nucleic acid drugs used in the treatment of solid tumours.

We started with the tumour microenvironment to find ways of improving targeting ability. In the center of the tumour lies a hypoxic region, with an oxygen concentration of less than 0.5%, which is apparently different from normal organs (oxygen concentration of 1-5%). The partially anaerobic attenuated Salmonella(Salmonella spp)which is characterized by intracellular survival and invasiveness, especially in solid tumour tissues has a dominant colonization and aggregation effect. Therefore, we propose to use attenuated Salmonella as a delivery system for siRNAs targeting different tumour-resistance genes to reduce tumour resistance to chemotherapeutic agents. In addition, to further improve the targeting ability and safety of attenuated Salmonella to tumour tissues, we propose to modify the genome of attenuated Salmonella and the corresponding plasmid expressing siRNAs.

Nanotechnology has been widely used in cancer therapy. We can gain effective treatment responses and reduce harmful side effects by using chemotherapeutic agents loaded in nanoparticles (NPs). However, the delivery efficiency and targeting ability of NPs are limited.

According to the literature, only 0.7% of the administered dose of nanoparticles is ultimately delivered to solid tumours[4]. Hence it still remains a great challenge to improve the accumulation of nanomaterials in tumour sites. From that we considered whether combining drug-loaded NPs with engineered attenuated Salmonella might take the advantage of high selectivity of attenuated Salmonella towards tumours to increase the delivery efficiency of NPs, thus providing the therapeutic effects of chemotherapeutic agents.

JLU-NBBMS 2024 designed a synergistic treatment strategy combining engineered bacteria delivering RNAi and NPs loaded chemotherapeutic agents, aiming for reducing the resistance of chemotherapeutic agents and improving the selectivity and efficiency of chemotherapeutic agents to bring out the potent treatment effects while reducing the side effects of chemotherapy, and ultimately result in the improvements of outcomes.

The delayed lysis attenuated Salmonella owned by The Laboratory of Molecular Biology, School of Basic Medical Sciences, Jilin University acts as the base and is underwent modular design by the concept of synthetic biology. Our therapeutic system not only meets the current research goals, but also has a high degree of flexibility to further enhance its functionality or adapt to new therapeutic needs by modifying or optimizing different modules.

1. Targeting module

We use the Lpp-OmpA structure to show the RGD peptide on the outer membrane of Salmonella, to increase its specific targeting ability toward tumours.

Extension

We may replace the RGD peptide with other tumour-targeting peptides or antibody fragments to further enhance the tumour-targeting ability of the therapeutic system or to accommodate the therapeutic needs of different types of tumours. For example, the RGD peptide can be replaced by a single chain antibody fragment (scFv) targeting HER2 for targeting HER2-positive tumour cells in breast cancer or, like BNUZH-CHINA 2023, to enable Salmonella to express carcinoembryonic antigen (CEA)- specific scFvs.

2. Gene regulation module

We used engineered Salmonella-mediated trans-kingdom RNAi to suppress the expression of tumour chemoresistance genes. Briefly speaking, engineered Salmonella is able to produce shRNAs targeting chemoresistance genes via the T7 expression cassette, invade tumour cells, release the shRNAs into the cytoplasm with the help of listeriolysin O, the encoded product of the hlyA gene and ultimately activate an RNAi-associated complex to silence the relevant resistance genes. The hlyA gene expression controlled by the hypoxic promoter pnirB and the tumour-targeting ability of Salmonella can effectively restrict this process within tumour tissues, thereby enhancing the efficacy and safety of the treatment.

Extension

In fact, the functions of this module are far from gene regulation. We can use T7 expression cassette to efficiently express other gene products, for instance, Cytotoxic proteins, immunomodulatory factors or anticancer peptides, thus extending the application range of the system so that it can not only silence drug-resistant genes but also directly kill tumour cells or enhance the immune response.

3. Safety module

The delayed lysis strain we used deleted the asd and murA genes, which prevented the mutant bacteria from synthesising an intact cell wall and inserted the murA and asd genes on a corresponding expression plasmid. However, the expression of these genes was under the control of the araCPBAD promoter, which means that the murA and asd genes are expressed only in the presence of arabinose, ensuring the survival of attenuated Salmonella. This bacterial system not only serves as a screening system for successful plasmid transformation, but also allows Salmonella to be lysed and die as the concentration of arabinose gradually decreases after Salmonella enters the organism, thus further ensuring the safety of attenuated Salmonella.

Besides, the mutant strains deleted the msbB gene-encoded endotoxin as well, which significantly reduced the competence of endotoxin.

Extension

the expansion of this module lies in the broader suicide module concept itself. We can replace the delayed lysis strain with other strategies, such as a toxin-antitoxin system or a kill switch circuit, to create a more versatile and adaptable suicide module.

4. Drug delivery module

We have chosen to encapsulate the chemotherapy drugs in mPEG-PLGA-PLL nanoparticles and load them onto Salmonella. Methoxy polyethylene glycol (mPEG) helps the nanoparticles evade phagocytosis by the reticuloendothelial system, poly lactic-co-glycolic acid (PLGA) allows for sustained drug release through gradual degradation in the body, while the cationic polymer poly-L-lysine (PLL) provides good biocompatibility and tunability, aiding in targeted and controlled drug release.

Extension

Furthermore, PLL retains a positive charge and has a flexible, stable structure with adjustable molecular weight, allowing for the introduction of side chains and specific targeting groups to modify the polymer backbone. This modification improves the carrier's performance, achieving controlled drug release. Customizing the side chains of PLL opens possibilities for targeting different sites and providing more personalized functions.

5. Model

We aim to more effectively guide wet experiments through dry experiments by simulating the functionality of our treatment system from three aspects: tumour targeting, therapy, and safety switch.

With the desire to specifically kill tumour cells, we screened for markers applicable to tumours and found that the expression of integrin was elevated in the majority of tumours. So we looked forward to targeting tumours with RGD peptides. With the current development, there are many improved types of RGD, such as c(RGDfK) and iRGD, which are all functionally improved. However, we currently do not know the structural stability and their binding ability to integrins of common RGDs as well as modified types after loading them onto the outer membrane presentation system, so we hope to judge the stability by structural simulations and compare the binding ability of common RGD peptides and modified RGD peptides to integrins by molecular docking. In this process, we found that we could not fully judge the affinity by molecular docking alone. Therefore, we further used a molecular dynamics simulation of the binding of different types of RGD peptides to integrins to select RGD peptides. Finally, we concluded that common RGD peptides are sufficient for targeting in terms of affinity and stability.

For treatment, on the one hand, we need to screen genes of different tumours that lead to drug resistance, and on the other hand, we need to simulate the process of drug release for treatment. Firstly, for the screening of drug-resistant genes, we have screened the drug use of patients with different tumours from the TCGA and GEO database, labeled their drug usage, carried out survival analysis based on the gene expression of the patients, scored by their prognosis and screened out the drug-resistant genes with the highest degree of unfavourable prognosis as the target of RNAi silencing. For the simulation of the drug release for the therapeutic process, we want to construct a correlation model through mathematical modeling, firstly to construct a model of the efficiency of drug release from nanoparticles in the periphery of the tumour tissue; and based on different drug release efficiencies (such efficiencies may be subject to different), to construct the efficiency of the treatment of the drug after it has approached/entered the tumour cells.

For the safety switch, we want to simulate the changes in the human immune system (especially the changes in the human peripheral blood for immune changes) after the injection of Salmonella, the variables are mainly the amount or concentration of injected Salmonella, and we hope to simulate the changes in the amount of immune cells, or the changes of immune substances (cytokines, etc.) in the plasma/tissue fluids. Furthermore, we want to simulate the hypoxic promoters during the process of transcriptional regulation which focuses on the changes in gene transcription due to the binding of oxygen to regulatory proteins. For the delayed lysis system, we mainly simulate the regulation of bacterial lysis by arabinose, for the nonlinear changes in arabinose concentration with cellular metabolism after the injection. Combining these three models, we simulate the safety situation during the actual implementation of our project.

In summary, JLU-NBBMS is dedicated to implementing the modular design concept of synthetic biology to develop an innovative tumour therapeutic system by combining engineered attenuated Salmonella and nanotechnology. This system not only effectively reduces drug resistance of tumour, but also significantly improves the safety and efficacy of treatment through precise delivery of chemotherapeutic agents. We firmly believe that by continuously optimizing and expanding the functionality of each module, our research will open up new directions and possibilities for future cancer treatments.