Expression analysis of the IL-2 protein in vitro
To allow expression of the IL-2 protein in a prokaryotic system, the IL-2 gene was cloned into three different inducible expression vectors (Supplementary Fig. 1 A, 1 B and 1 C). The differential proteins expressed by the three recombinant plasmids in E. coli BL21 (DE3) were verified by Western blotting and mass spectrometry, which proved that the target protein IL-2 was successfully expressed (Supplementary Fig.2 and Table S1). Coomassie Brilliant Blue staining showed that the recombinant protein showed soluble expression under the action of SUMO and IF2 tags (Fig. 1 A). Engineered bacteria need to continuously secrete recombinant proteins inside a tumour, induce immune cell activation, attack tumour cells, and inhibit tumour growth. Therefore, the IL-2 protein can be continuously expressed in hypoxic tumours under the oxygen-dependent promoter of the haemoglobin gene (vhb) of Vitreoscilla and the pelB leader sequence (Fig. 1B and Supplementary Fig. 3). Engineered bacteria were cultured overnight in LB medium, and the recombinant protein was successfully expressed in engineered EcN bacteria, as verified by SDS-PAGE analysis (Fig. 1 C). Western blot analysis indicated that the IL-2 protein was presented in both the cell lysate and medium supernatant of EcN (hIL-2) (Fig. 1 D). To reduce the effect of tags on IL-2 protein activity, a SUMO fusion system with a relatively low molecular weight was selected for subsequent experimental research.
SUMO-IL-2 protein can promote the proliferation of PBMCs
BL21 (pSmartI-IL 2) bacteria were cultured, and IL-2 protein was collected by Ni-NTA Sefinose (TM) Resin Kit. The IL-2 protein at different concentrations was cocultured with peripheral blood mononuclear cells (PBMCs), and PBS was added to the control group. PBMCs were cocultured with the recombinant protein for 24 h, and then Cell Counting Kit-8 was added to detect the cell survival rate. The results showed that cell proliferation was obviously promoted after the addition of SUMO-IL-2 (Sumo is the solubilizing label on pSmartI), and the proliferation rate of cells also increased as the SUMO-IL-2 protein concentration increased (Fig. 2 A). Additionally, the cell culture medium was centrifuged after the incubation, and the culture supernatant was collected. The concentrations of IFN-γ and TGF-β in the supernatant of the culture medium were detected by enzyme-linked immunosorbent assay (ELISA). The results showed that compared with that in the supernatant of the control group, the concentration of IFN-γ in the supernatant of the experimental group was significantly increased (Fig. 2 B), while the concentration of TGF-β was significantly decreased (Fig. 2 C).
EcN specifically colonizes tumour regions in tumour-bearing mice
An IVIS can accurately observe the real-time location of bacteria in animals without causing damage to the animals[16, 17]. After intraperitoneal injection of 5×106 CFU/100 µL EcN(Lux) into tumour-bearing mice in the experimental group and injection of sterile PBS into tumour-bearing mice in the control group, bacterial colonization in the mice was observed by an IVIS. The results showed that the tumour-bearing mice exhibited a significant fluorescence signal in the tumour area for 5 days after the bacteria were injected, and the fluorescence signal was still observed on the 7th day after injection (Fig. 3 A). The control group did not exhibit a detectable signal. After the mice were euthanized on the 7th day, the tumour, liver, kidneys and spleen of the mice were obtained. IVIS analysis showed that 5 days after EcN(Lux) was intraperitoneally injected into tumour-bearing mice, a strong fluorescence signal was detected in the tumour tissues of the mice, and no fluorescence signal was detected in other organs (Fig. 3 B). These results showed that EcN has excellent targeting to the tumours in CT26 tumour-bearing mice. Bacteria can quickly accumulate in the tumour area and grow and reproduce in the tumour area after intraperitoneal injection into mice, while bacteria in other parts of mice can be removed by the local immune response quickly.
Antitumour effect of EcN(hIL-2)
To validate the successful expression of the IL-2 molecule carried by EcN in tumour areas, we performed immunohistochemistry on samples from each group of mouse tumours. Mice were euthanized on the 7th day after the third administration, and the tumour tissue was removed, fixed in 4% paraformaldehyde, and then embedded in paraffin. These results showed that a yellow-grey signal appeared in the tumour tissue sections of the EcN (hIL-2) experimental group, while those of the other three groups did not show a positive signal (Fig. 4 A). These results indicate that IL-2 is successfully expressed in the tumour region. The antibody used in immunohistochemistry is Anti-His Tag Rabbit Polyclonal Antibody.
To evaluate the antitumour efficacy of EcN (IL-2), we subcutaneously injected CT26 colon cancer cells into the right axillary area of BALB/c mice. The resultant xenograft tumour model was used to study the antitumour effect of EcN (hIL-2). When the tumours in the mice grew to approximately 60 mm3, the mice were randomly divided into 4 groups (n = 5, 6, or 7), and the groups were treated respectively by intraperitoneal injection of sterile PBS, EcN, EcN (28a) or EcN (hIL-2). Body weight and tumour volume were measured every two days during the observation period until the animals were sacrificed. The results of the experiment showed that the tumour growth in the EcN (hIL-2) group was significantly inhibited (Fig. 4 B), while the xenograft tumour growth in the other three groups have no difference. The final tumour weight of the EcN (hIL-2) group was also significantly lower than that of the other 3 groups (Fig. 4 C). The tumour volume in the PBS group reached 4.93 ±1.35 cm3, but the tumour volume in the EcN (hIL-2) group was significantly smaller, reaching a volume of 2.32 ± 1.43 mm3. Tumour volume was calculated according to the formula (Table 1). Tumour growth in the EcN (hIL-2) group was inhibited approximately 53.91% compared to that in the PBS group.
Tumour histomorphology and safety monitoring of E. coli Nissle 1917
Haematoxylin-eosin staining (H&E staining) is one of the commonly used staining methods for paraffin sections. Haematoxylin dyeing solutions are alkaline, mainly causing chromatin in the nucleus and nucleic acid in the cytoplasm to be stained purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and the extracellular matrix red. Our experimental results showed that the tumour staining results in the PBS group showed normal tumour cell morphology, and no necrotic areas were observed. However, infiltrating inflammatory cells were observed in the EcN experimental group, and the cell morphology was irregular, the phenomenon that inflammatory cells gather in the inflammatory focus. (Fig. 5 A).
Additionally, we also performed H&E staining of liver, kidney and spleen tissues from the PBS group and EcN experimental group. The results showed that there was no significant change in histopathological morphology in the liver, kidneys or spleen between the two groups, indicating that EcN had no obvious side effects on the liver, kidneys or spleen in mice (Fig. 5 A). During the experiment, to assess the systemic effects of EcN on the whole body after intraperitoneal injection, we measured mouse body weight every two days. At the end of the experiment, the liver, kidneys and spleen of the mice in each group were excised and weighed, and there were no differences in weight among the four groups (Fig. 5 B). Although the mice exhibited a slight decrease in body weight during treatment, they all approached the same weight by the end of the experiment (Fig. 5 C). All of the above results demonstrate that the toxicity of intraperitoneal administration of EcN to mice is negligible.
Examining the tumour microenvironment
We explored the mechanism underlying the antitumour immune activity of EcN (hIL-2) in a CT26 colon cancer tumour model. We investigated the immune cell profile in the tumour microenvironment and the changes in cytokines in the blood using the CT26 tumour model. We selected specific antibodies to be combined with antigens on the surface of T lymphocytes, neutrophils and M1 macrophages, and compared the changes in the content of these three cells in the four groups of tumors by immunohistochemistry. Immunohistochemical results showed that the levels of tumour-infiltrating T lymphocytes and neutrophils were increased in the EcN (hIL-2)-treated group compared with the other three groups (Fig. 6 A, B). The results also showed an increase in the level of M1 macrophages in the total macrophage population in the tumour microenvironment after treatment with EcN (hIL-2) (Fig. 6 C). We believe that the yellow signals in the other three groups are the original T lymphocytes, neutrophils and M1 macrophages in the mouse tumor tissues, while the EcN (hIL-2) treatment group has a large area of yellow signals, and the positive signal can be seen more clearly from the 400× partial enlarged image. This can further explain the increase in the number of T lymphocytes, neutrophils and M1 macrophages in tumor-bearing mice after EcN (hIL-2) treatment. Next, we examined the levels of two immune factors in the blood in four groups of mice. The data showed that the IFN-γ level was significantly increased and the TGF-β level was decreased in the blood of mice in the EcN (hIL-2)-treated group compared with that of mice in the other experimental groups (Fig. 6 D, E). Collectively, these data indicate that EcN (hIL-2) treatment improves the immune microenvironment of tumour-bearing mice to some extent, leading to improved survival outcomes after EcN (hIL-2) treatment.