Living Bacteria‐Based Immuno‐Photodynamic Therapy: Metabolic Labeling of Clostridium butyricum for Eradicating Malignant Melanoma

Abstract Due to the complexity, aggressiveness, and heterogeneity of malignant melanoma, it is difficult to eradicate the whole tumor through conventional treatment. Herein, a strategy of metabolic engineering labeled anaerobic oncolytic bacteria (Clostridium butyricum) is demonstrated to achieve the ablation of melanoma. In this system, the metabolic substrate of C. butyricum d‐alanine (d‐Ala) is first conjugated with a photosensitizer (TPApy) showing aggregation‐induced emission (AIE). The yielded metabolic substrate of d‐Ala‐TPAPy can be metabolically incorporated into bacterial peptidoglycan to form engineered C. Butyricum. Once the engineered C. butyricum is injected into melanoma, the bacteria can only proliferate in an anaerobic zone, stimulate the tumor immune microenvironment, and ablate the tumor hypoxia region. Following that, the relatively rich oxygen content in the peripheral area can induce the death of C. butyricum. The photosensitizer (PS) on the bacteria can subsequently exert a photodynamic effect in the oxygen‐rich region and further remove the melanoma residue under light irradiation. Prominent in vivo melanoma ablation results revealed that the engineering oncolytic bacteria can provide a promising regime for solid tumor eradication.


•OH Detection in Aqueous Solution
The •OH generation potency was evaluated by using HPF as an indicator. The stock solution of HPF (5×10 −3 M in N, N-dimethylformamide) was diluted to 5×10 −6 M in the sample solution of TPApy in PBS buffer. The fluorescence signal of HPF was monitored at different time intervals in a range of 500-620 nm with the excitation wavelength at 490 nm after the solution was irradiated by light irradiation (100 mW/cm 2 ). The fluorescence intensity at 515 nm was recorded to indicate the generation rate of •OH.

•O 2− Detection in Aqueous Solution
The •O 2− generation measurements were performed using DHR123 and HKSOX-1 as indicators. The stock solution of DHR123 and HKSOX-1 were diluted to working concentration according to the protocol. And then, being mixed with TPApy in PBS buffer. The fluorescence signal of DHR123 and HKSOX-1 was monitored at different time intervals after the solution was irradiated by light irradiation (100 mW/cm 2 ).

Preparation of the D-Ala-TPApy-labeled Clostridium butyricum
The Clostridium butyricum cells were routinely grown at 37 °C and 220 rpm in LB medium. After overnight culture, the bacteria were collected and the optical density at 600 nm (OD600) was adjusted to 1.0 in PBS. The bacterial suspension was added into D-Ala-TPApy (0 ~ 50) µM solutions with the same volume, mixed and co-cultured at 37 °C for 30 min. The mixtures were centrifuged at 6000 rpm for 4 min and the supernatants were discarded, washed twice and the D-Ala-TPApy-labeled bacteria were obtained.

Characterization of peptidoglycan fragment from Clostridium butyricum
Overnight pre-cultured bacterial cells were added into a new LB medium and the bacteria were incubated on a shaker until reaching the log phase. Then, 5 mL of the bacteria were obtained by centrifugation and re-suspended in 5 mL of LB medium containing 50 μM D-Ala-TPApy and the mixture was incubated for 2 h. After that, the labeled bacteria were obtained by centrifugation at 5,000 g for 10 min and they were quickly cooled on the ice for 15 min and subsequently re-suspended in 4 mL water.
The cells were washed once with 5 mL of PBS. The cell pellets were re-suspended in

In vitro ROS detection by ABDA:
The ROS was detected using ABDA as the ROS indicator. 5 μL of ABDA stock solution was added into 1 mL of sample solution (10 μM), and the mixtures were S8 irradiated by white light (400 -800 nm) at a power density of 30 mW cm -2 . The absorbance of ABDA was monitored at different exposure times at 378 nm to obtain the decay rate of the photosensitizing process.

Cell culture
The melanoma cell line B16F10 was cultured in Dulbecco's Modified Eagle Medium (DMEM). The culture medium contains 10% fetal bovine serum (FBS) and antibiotics (50 units mL -1 and 50 units mL -1 streptomycin) at 37°C under a humidified atmosphere containing 5% CO 2 for 24 h and then replaced the fresh medium.

Cell uptake study of D-Ala-TPApy-labeled Clostridium butyricum
The cellular uptake of D-Ala-TPApy-labeled Clostridium butyricum in B16F10 cell lines was evaluated. B16F10 were plated on 8-well plates at a density of 5×10 4 /well and grown in DMEM supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., USA) and 10 μg/mL gentamicin (Thermo Fisher Scientific Inc, USA) for 20 h at 37 °C at a humidified 5% CO 2 incubator. The medium was replaced with 300 µL of fresh medium and 100 µL of D-Ala-TPApy-labeled Clostridium butyricum at bacteria/cell ratio of 20 were added to each well. After 2 h incubation, the medium was discarded. After 2 hours, the medium was replaced by media

Experiment protocols involving animals were authorized by the Animal Ethics
Committee of the School of Medicine, Fudan University. C57 BL/6 male mice (4 weeks of age) were purchased from the Chinese Academy of Science (Shanghai). The male mice were injected subcutaneously with 100 µL of cell suspension containing 5×10 5 B16F10 cells. The tumor volume was allowed to grow to about 100 mm 3 before in vivo experiment.

In vivo pharmacodynamic study
The melanoma-bearing C57 BL/6 mice were randomly divided into six groups (n = 8).
Mice in various groups were one-time intra-tumoral injection different agents with blue light irradiation (100 mW/cm 2 ) for 5 min or without light illumination for 9 days.
The length and width of the tumor and the bodyweight of mice were measured before every injection during the therapy. The tumor volume was calculated using the S10 formula V (mm 3 ) = 1/2×length (mm)× width (mm) 2 After 9 days' treatment, some of the mice were sacrificed and tumors were separated, weighed, and photographed. Additionally, the tumors were cut into small pieces, fixed in 40% formalin, and embedded in the paraffin. Then, the tissues were sectioned for histopathological analysis with H&E staining, immunohistochemical analysis, and

S12
The male C57 BL/6 mice harboring B16F10 tumor model were used to evaluate for the influence of CD4 + , CD8 + T cells, and cytokines. Briefly, after various treatments, five mice per group were sacrificed and the tumors, spleens, and blood were harvested for detection. The tissues were digested with collagenase IV (175 U/mL), hyaluronidase (100 U/mL), and DNase ( For cytokine quantification, the harvested spleen cells were suspended in 5 mL of the DMEM culture medium supplemented with 10 % FBS for 24 h. The supernatants were collected and cytokines were quantified using mouse IL-4, IL-6, IL-12, and IFN-γ ELISA kits (Life technologies, USA).

Statistics study
Quantitative data were presented as mean ± standard error of the mean (SEM).
ANOVA analysis was used for multiple comparisons, and Student's t-test was used for two-group comparisons. Sample size (n) for in vitro and in vivo studies was 3 and 7, respectively. The differences in each group were analyzed by the Kaplan-Meier method, and the P-value was determined by the log-rank test. All statistical analyses were carried out using GraphPad Prism 5.0. (P-value: *P < 0.05, **P < 0.01, ***P < 0.001). S14 piperidine, N 2 ; d, CuBr, TEA, DMSO, Ar. Figure S1. 1 H NMR spectrum of compound 1 (CDCl 3 ). S15 Figure S2. 1 H NMR spectrum of compound 2 (CD 3 OD).         The scale bar is 50 μm. Figure S18. Microscopic images of H&E-stained sections of the major organs (heart, liver, spleen, lung, kidney) after tumor-bearing nude mice being treated with different formulations. The scale bar is 50 μm.