Despite the recent advances in traditional anti-cancer methods such as surgery, chemotherapy and radiotherapy, the mortality of cancer patients has been increasing year by year. Living bacteria therapy shows a promising therapeutic potential against cancer. When using bacteria to treat cancer, the oxygen-rich environment in normal tissue and the clearance by the immune system leads to a rapid decline in the number of bacteria in the healthy tissue, promoting complete elimination of the bacteria and avoiding potential toxicity to the host [12]. At the same time, complex physiological functions (including migration, chemotaxis, biocompatibility, etc.) enable the bacteria to specifically target and colonize in the anoxic or necrotic area within the tumor tissue, enabling the efficient delivery of therapeutic agents [13, 14]. Effective bacterial anti-cancer therapy models activate the immune cells and mount a tumor specific immune response with minima side effects, despite the immunosuppressive TME, [13, 15]. In addition, the ease of genetic manipulation of the bacteria enables the reprogramming of the bacteria, which could synthesize and secrete antineoplastic drugs, change their metabolic pathways, and secrete therapeutic toxin proteins and anti-cancer bacterial derivatives [16–18]. Albumin has a good biocompatibility and biodegradability, and can form complexes with insoluble or exogenous substances and assist in material transport. Because albumin binding proteins (such as SPARC, gp60) are highly expressed in many tumors, albumin is often loaded into nanoparticles as a drug delivery system targeting tumors, and BSA is used as a connecting agent on the surface of living bacteria. On the one hand, albumin based drug delivery systems provide sufficient amino acids and energy for fast-growing cancer cells, and on the other hand, it also enables the accurate delivery of targeted therapeutic agents to the tumor [11, 19–22]. Based on the above advantages, albumin modification promotes the escape of the nanoparticles from the complex human immune system, vascular system and TME, and unfavorable conditions such as low pH, high interstitial fluid pressure and cytoplasmic matrix released by the TME [23, 24]. We modified the surface of E. coli with BSA, and show the feasibility of this approach. By co-culturing with tumor cells, we confirmed that E. coli modified by BSA had an improved ability to target and adhere to tumor cells.
In biotinylated E. coli, due to the difference in primary amine density, surface charge and total surface area between E. coli, there may be an impact on the coupling efficiency [25, 26]. This may lead to only a small amount of E. coli to be coupled to biotin, resulting in a partial difference in the fluorescence signal produced by E. coli when biotinylated. Escherichia coli biotinylation was verified by streptavidin with green fluorescent. When the denaturing and non-reducing reaction of streptavidin coupled BSA was carried out, the purity of BSA was insufficient, which led to the residual shadow above the main band. Due to pH, protein isoelectric point and other potential reasons, streptavidin tetramer (M = 58KD) could not be completely dissociated, so the streptavidin tetramer and polymer appear in the same Fig. 2 Nevertheless, the molecular weight of the mixture of streptavidin coupled with BSA showed that BSA with the molecular weight of 66 KD was successfully coupled with the dissociated streptavidin monomer, and the number of BSA molecules bound to a streptavidin monomer was the largest. In addition, Styo9-PI, as a working reagent of LIVE/DEAD Bacterial Viability Kit, is a powerful nucleic acid dye that can penetrate the cell membrane and stain the nucleus. After E. coli-BSA was co-incubated with T24 tumor cells for 1 hour, some of the T24 tumor cells were also stained green, but this did not affect the conclusions of the experiment.
When immunofluorescence signal from streptavidin was positive, the surface protein modification of living bacteria had been completed. Consistent with this, Mostaghaci et al. reported in 2017 that the synthetic particles modified by biotin-streptavidin binding on the surface of bacteria could be more effectively attached to urinary tract epithelial cells and gastrointestinal epithelial cells because of their affinity to mannose molecules expressed on the cell surface [27]. Vargason and Anselmo et al. achieved the same results in 2020 when adhesin BSA molecules were modified on the surfaces of Lactobacillus casei (LC), E. coli (EC) and Bacillus coagulans (BC). It was also proved that before and after modification, the growth capacity and activity of bacteria remained unchanged, but their adhesion to gastrointestinal tract and resistance to the colonization of pathogenic bacteria were enhanced [25, 26]. What is more gratifying is that when we co-incubated E. coli modified BSA with TCCSUP bladder cancer cells with a higher expression of SPARC, we saw that a large number of E. coli were swallowed by TCCSUP cells after 12 hours.(Supplementary video 1 and 2)These data not only provide a feasible support for the developement of BSA protein modification strategy on the surface of living bacteria, but also reveal the anticancer potential of such a strategy. In particular, some solid tumors with special anatomical location can be administered by intratumoral injection, perfusion, oral administration and so on. Furthermore, our novel strategy could prevent bacteria-protein conjugates from being recognized and cleared by human liver, kidney, lysosome and complex immune system when transported to the tumor site [10, 28].
The complexity of living bacteria as organisms determines the difficulty and risk of turning them into anti-tumor weapons. However, complexity is precisely the most fascinating part of living bacteria. Different species of bacteria have different biological, physical and chemical characteristics. Bacteria in tumors can have complex effects on tumor progression or inhibition by affecting different immune pathways, resulting in diverse anti-tumor effects after their surface modification [29–33]. This allows scientists to fine-tune the functions of different strains to achieve anti-tumor activity that other treatments cannot achieve [34]. Fortunately, most of the easily modifiable engineered strains contain primary amine groups, which provides essential conditions for surface BSA modification and anticancer application of engineered living bacteria. At present, a variety of bacterial therapies have been successfully implemented in humans and have entered phase I/II clinical trials [35–38]. In these clinical trials, bacteria were mostly used as anticancer agents carrying cytotoxic proteins, cytokines, angiogenesis inhibitors, antigens and antibody. Despite the complex pathophysiological characteristics of tumor and the development of bacterial therapy, traditional anticancer methods such as surgery, radiotherapy and chemotherapy are still the current first-line anticancer choice. Although the bacterial vector has achieved gratifying results in the experimental model, there are still some challenges in assessing the safety, effectiveness and accuracy of these agents, and further research is needed to evaluate its efficacy and toxicity in the treatment of cancer in the clinic. The combination of bacterial therapy and conventional therapy has shown better therapeutic efficacy and disease prognosis, and provides a better treatment for patients with cancer pedigree. In the future, many traditional single treatments will be replaced by multidisciplinary and multidirectional combination therapy. The method of protein modification on the surface of bacteria in the treatment of cancer may help to create a new model of cancer treatment, however, further research is warranted to enable the clinical translation of this strategy for cancer therapy.