2.1 Hyperthermia and ultrasound triggered gene expression of URB
We first constructed the prokaryotic expression plasmid harboring temperature-actuated therapeutic circuit which carries murine IFN-γ gene or mCherry fluorescent reporter under the pR-pL tandem promoter (Fig. 2a). Then the recombinant plasmid was introduced into E. coli MG1655 (a non-pathogenic bacterium) to produce ultrasound responsive bacteria (URB). Incubation of the engineered bacteria harboring mCherry gene at 37°C did not produce any fluorescence signals, showing the suppressed expression of mCherry fluorescent gene at 37°C. In contrast, strong red fluorescent signals could be observed at 6 h after the bacterium incubation temperature rose to 45°C, revealing that TcI repression was relieved to initiate the mCherry gene expression (Fig. 2b). Prolonging the incubation time at 45°C from 5 min to 25 min increased the fluorescent signal intensities, which confirmed the controllability of thermal logic circuits of URB (Fig. 2c). To develop a noninvasive remote control of bacterial gene circuits by focused ultrasound, we optimized a series of acoustic parameters including acoustic energies, irradiation duration time at on/off state. Results showed that the parameters at 4.93 MPa acoustic pressure, 3 s ON and 5 s OFF irradiation time could keep the irradiated bacterium solution at 45°C constant temperature (Fig. 2d). After the ultrasound irradiation, URB could successfully express mCherry fluorescent protein and the fluorescence signal intensity increased gradually along with time (Fig. 2e). To elucidate the remote controllability of bacterial gene expression, we inserted a tube of bacteria into an agar phantom with or without acoustic exposure for 30 min at the optimal ultrasound parameters. Figure 2f showed the fluorescence signals only appeared in the sample received with ultrasound irradiation, but not in these bacteria that did not receive with ultrasound exposure. In addition to controlling the temperature of the bacterial solution in vitro, the temperature of the local tissue of mice could also be increased to 45°C by adjusting the appropriate ultrasonic parameters (Figure S1). In vivo animal experiment further revealed that the liver of mouse administrated intravenously with URB could emit the strong fluorescence signals after ultrasound irradiation at the above optimized acoustic parameters. By contrast, the livers from the non-irradiated control mice or the incubator-heating mice at 45°C did just emit weak background fluorescence signals (Fig. 2g-h). These results showed that ultrasound can function as a remote tool to regulate the gene expression of bacteria when they harbor some temperature-based gene control elements in vitro and in vivo. Previous report synthesized a thermosensitive therapeutic bacterium and triggered the in-situ expression of TNF-α of bacteria by near-infrared light irradiation21. However, it is difficult to act on deep tumor tissues in live animals because of weak penetration of near-infrared light. Compared to near-infrared light, ultrasound could irradiate the deeper tissue such as liver, which may be more suitable for the induction of bacteria located in the deep tumor.
2.2 In vitro treatment experiment of URB-mediated tumor immunotherapy
Next, we engineered one therapeutic URB by displacing the fluorescent mCherry gene with murine IFN-γ gene in the thermal logic circuits. Agarose gel electrophoresis confirmed the presence of IFN-γ gene fragment in the plasmid (Figure S2). SDS-PAGE gel electrophoresis analysis revealed that IFN-γ proteins appeared only in the lysate of bacteria incubated at 45°C for 30 min, but not in the lysate of bacteria incubated at 37°C, confirming the production of therapeutic IFN-γ after thermal treatment (Fig. 3a). Moreover, the therapeutic IFN-γ protein expressed from URB was water-soluble (Figure S3). Prolonging the incubation and ultrasound irradiation time at 45°C from 20 min to 80 min increased the expression level of IFN-γ proteins, showing an excellent time-dependent correlation (Fig. 3b-c). These results showed the designed therapeutic logic circuit can be strictly controlled and tuned by heating and ultrasound stimulation and their exposure duration, which is important for many therapeutic factors to execute their anti-tumor effects. To test whether the IFN-γ proteins could be secreted from their host bacteria to kill tumor cells, the calcium AM/PI staining assay was used to 4T1 breast cancer cells which were incubated with different concentrations of IFN-γ proteins from 50–150 pg/ml in the bacterial supernatant. Obviously, almost all the cells showed green fluorescence in the blank group, showing no dead cells. With the increase of IFN-γ proteins, the proportion of living cells (green fluorescence) decreased gradually, while the proportion of dead cells (red fluorescence) increased gradually (Fig. 3d). Quantitative analysis by CCK-8 kits revealed that the cell viability was 33.13 ± 16.28 %, 38.93 ± 4.72 %, 47.02 ± 72.62 %, 63.09 ± 4.14 % or 68.50 ± 3.11 % for 150 pg/ml, 125 pg/ml, 100 pg/ml, 75 pg/ml or 50 pg/ml IFN-γ proteins in the bacterial supernatant, respectively. It was significantly lower than these cells incubated with the equivalent supernatant from non-induced bacteria (Fig. 3e).
IFN-γ can exert its cytotoxicity and immunoregulatory effects by activating Janus kinase 1 (JAK1) and signal transducers and activators of transcription1 (STAT1) signaling pathways30–32. The effect of IFN-γ expressed from URB on macrophage immune activation was further examined in vitro. The RAW 264.7 macrophages were incubated with IFN-γ expressed by bacteria for about 8 h, revealing a similar effect as the traditional IFN-γ in promoting the generation of proinflammatory M1 phenotype macrophages, with about 71.6% of macrophages staining positive for CD86 and 85.4% for CD80 at 150 pg/mL bacterial IFN-γ (Fig. 3h). Also, the significantly decreased expression of M2 phenotype marker CD206 could be observed on the macrophages, confirming that IFN-γ released from the URB can induce the macrophage polarization from M2 to M1 phonotype. Meanwhile, the bacterial IFN-γ also triggered to produce higher level of NO in the macrophages in comparison with the control group (Fig. 3f). Furthermore, we detected the tumor cell killing efficacy of these activated macrophages stimulated by different concentrations of bacterial IFN-γ, finding that higher concentrations of IFN-γ were used to stimulate macrophages, stronger cytotoxicity these activated macrophages would be (Fig. 3g). Together, the above results demonstrated that the IFN-γ produced from bacteria could effectively activate macrophages.
3.3 Tumor targeting of URB
In order to detect the tumor-targeting property of these engineered bacteria, the DiR-labelled live URB or dead URB treated with 65°C for 30 min were injected intravenously into the 4T1 tumor-bearing mice and imaged by an IVIS Spectrum imaging system at different time. As shown in Fig. 4a, the live URB revealed the efficient tumor-homing ability in the 4T1 tumor, emitting strong fluorescence at 6 h after injection. Increasing fluorescence at the tumor site was observed along with time from the mice received live DiR-labelled URB administration, but not from the control mice injected with the dead URB. 48 h after administration, mice were euthanized to analyze the fluorescence signals in various organs (Fig. 4b). According to the results, It was showed that the distribution of dead URBs are mainly in liver and spleen. On the contrary, the accumulation of the live URBs was higher in the tumor site, and less in the liver than the control group. The quantitative analysis revealed a five-fold increase of relative fluorescence intensity in the tumor of live URB group compared with dead URB, which is in consistent with the results of in vivo fluorescence imaging (Fig. 4c).
Subsequently, the targeting efficiency of DiR-labelled URBs to the tumors was determined by fluorescence microscope observation. As Fig. 4d showed, the red fluorescence signal, which represent the distribution of DiR-labelled live URB, mainly appeared in the tumor tissues but not in the heart, kidney and lung, and a bit in the liver and spleen. By contrast, only a little of red fluorescence could be observed in the tumor for DiR-labelled dead URB. Interestingly, the red fluorescence of DiR-labelled live URB were largely distributed in the center of tumors which represents the hypoxic and necrotic region due to the insufficient nutrient supply. Different from the live URB, the dead URB mainly appeared in the peritumoral zone. These results illustrated that the live URBs rather than the dead URBs could be efficiently accumulated in the tumor site and penetrated the tumor hypoxic and necrotic region. It may be the chemoreceptors on the surface of E. coli sense the nutrient-rich and low-oxygen tumor microenvironment, promoting them to actively migrate in the tumor region33, 34. In order to further explore the behaviors of these engineered URB after intravenous injection, 4T1-bearing mice received URB at the dose of 1 × 107 colony-forming units (CFU) per mouse via intravenous injection and then sacrificed at 1, 2, 7, 14, and 21 days after injection. Various organs and tumor were collected, homogenized, serially diluted (10-10000 fold), and incubated on Luria-Bertani (LB) plates containing 100 µg/ml ampicillin. The data colony counts in each plate revealed that URB were gradually eliminated from heart, liver, spleen, lung and kidney (Fig. 4, e and f). However, the colony counts of tumors showed exponential growth along with the time, achieving the peak value after 7 days and then decreasing gradually (Fig. 4, e and g). There was not bacterial colony formation from any tissue homogenates when the dead URB were used for intravenous injection into these mice. Collectively, these results provided evidence for their capability of the engineered bacteria to home, penetrate and colonize in the tumors, attributing to the hypoxic, immunosuppressive, and biochemically unique tumor microenvironment35–37.
3.4 In vivo experiment of URB-mediated tumor immunotherapy
To evaluate the anti-tumor effect of URBs combined with focused ultrasound in vivo, unilateral 4T1 tumor-bearing mice model were treated following the therapeutic schedule in Fig. 5a. The mice were randomly separated into six groups and treated with saline (control), ultrasound alone (US), MG1655 E. coli harboring without the circuit (E. coli), URB harboring the circuit (URB), E. coli + ultrasound (E. coli + US) or URB + US group, respectively. 48 h after intravenous injection, Ultrasound irradiation was performed to trigger the expression of IFN-γ. The treatment procedure was repeated seven days later. As shown in Fig. 5b-d, the volume of tumors in each group was similar on the day 1. However, URB + US group exhibited the strongest tumor inhibitory effect against tumor growth and the longest postsurgical survival time. The average tumor volume of the mice treated with URB + US was less than 250 mm3, whereas that of other treated groups are more than 1000 mm3.The median survival time of mice in URB + US group increases to 57 days compared with 42 days for control group, which proved that application of URB combined with ultrasound irradiation could improve survival rate significantly. Compared with other groups, It was showed that apparent damages were observed on the tumor cells after the treatment of URB + US (Fig. 5e, upper row). The maximum degree of cell apoptosis was also found by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (Fig. 5e, middle row). Moreover, URB + US showed much less expression of Ki67 in immunofluorescence staining (Fig. 5e, bottom row), demonstrating significant reduced tumor proliferation potentials.
Since IFN-γ can induce polarization of M1 macrophages38, we further explored whether the treatment activated the intratumoral immune response. Flow cytometry for the polarization of tumor-associated macrophages (TAMs) demonstrated CD80+ macrophages greatly improved and CD206+ macrophages significantly decreased in the tumors treated with URB + US in comparison with control, only US and only URB groups, indicating that URB + US treatment could effectively promote the polarization of TAMs from M2 phenotype towards M1 phenotype (Fig. 5f). The polarization of TAMs markedly increased CD8+ T cells (Fig. 5g). Meanwhile, significantly higher IFN-γ and TNF-α levels but less IL-10 levels could be detected at 1, 3 and 5 days after URB + US treatment. Thus, our data suggested that URB combined with ultrasound could effectively activate antitumor immunity through inducing the expression of IFN-γ. Some previous studies have used IFN-γ for tumor treatment and found that the infiltration of lymphocytes such as CD4+ T cells, CD8+ T cells, and macrophages in the tumor microenvironment increased significantly39. The significantly reduced ability of TAM to produce arginase-1 and iNOS was also demonstrated after IFN-γ treatment40. All these data suggested that IFN-γ can promote M1 macrophages in the tumor microenvironment, thereby improving the efficacy of tumor immunotherapy.
3.5 In vivo experiment of URB-mediated tumor immunotherapy
Since the antitumor immunity of URB could be effectively activate by ultrasound in vitro, we next wander whether the immune response triggered by URB combined with ultrasound can inhibit the growth of distant tumors. Bilateral 4T1 tumor-bearing mice model were established to explore the treatment-elicited abscopal (left) therapeutic effect by treating the primary (right) tumor. As depicted in Fig. 6a, 4T1 cancer cells were inoculated at day − 10 (right) and day − 7 (left), respectively. A week later when the right flank tumors were about 100 mm3 (day 0), the engineered URB at 107 CFU were systemic administrated by tail vein and followed by ultrasound irradiation at two days later (day 2). As expected, significant antitumor effects were observed in the primary tumors received with ultrasound irradiation and URB administration (Fig. 6b-c). Interestingly, the distant tumors from URB + US group were also greatly inhibited in comparison with control, US, E. coli, URB, E. coli + US groups (Fig. 6b and d). The survival rate of the mice treated with URB + US was greatly improved, with more than 80% of them still alive on 60th day post the tumor inoculation (Fig. 6e). Notably, only very few metastatic foci were observed in the lungs of mice treated with URB + US (Fig. 6e–g), suggesting effective inhibition of lung metastasis. In addition, we also demonstrated that systemic administration of URB did not cause severe side effects to mice by body weight monitoring (Figure S4). All of these data indicated URB + US resulted in a robust anti-tumor immune effect to protect mice from distant tumors and metastasis.
To understand the mechanism of the antitumor systemic effects triggered by URB plus ultrasound, immune cells in the spleen were assessed on the 7th day after the first treatment by bilaterally 4T1 cancer model. The percentage of M1 phenotype macrophages (CD80+) and M2 phenotype macrophages (CD206+) in spleens was evaluated. The results showed that the proportion of CD80+ macrophages in the URB + US group was 18.9 ± 3.38%, with a significantly increase compared with saline control group (8.78 ± 2.66%). In contrast, the proportion of CD206+ macrophages in spleen were significantly decreased to 19.5 ± 3.02% (Fig. 6i). Specifically, the percentage of CD8+ T cell in the URB + US group occupied 34.7 ± 4.49% with an increase by nearly 3.7 times in comparison with the control group (Fig. 6j). There was a statistically increase in the percentage of CD4+ T cell in the spleen treated with URB + US group (Figure S7 a), while the proportion of DC and NK cell was slightly increased in the combined treatment (Figure S7 b-c). The antitumor systemic immune response elicited by URB + US was further verified by the cytokine detection. It has been found that URB + US significantly promoted the IFN-γ and TNF-α level in blood (Fig. 6k-l). These results indicated that the URB + US combined therapy could trigger the systematic antitumor immunity to inhibit the distant tumor growth and metastasis41–43.
In order to further explore the biosafety of this therapeutic strategy, the changes of mice weight and major organs histopathologic features was evaluated. It is found that no abnormal weight changes of mice were found in the different treatment (Figure S5), demonstrating the good biosafety of the URB-mediated immunotherapy. In addition, cell damage was not observed in the H༆E staining slices of various organs of different group mice (Figure S8). The blood biochemistry and blood routine were recorded that all the indexes of the URB + US group were not significantly different from those of the other group (Table S1-S2). Because URB had less accumulation in normal tissues such as organs and blood. With the elimination of the immune system, URB has the short half-life in major organs. Previous studies used a similar bacteria strain to our study, E. coli MG1655, and confirmed that the strain was safe and did not affect the homeostasis of the body21. Together, the results demonstrated that the novel targeted therapeutic strategy integrating URB with ultrasound holds great potential in cancer immunotherapy.