Results

Section1

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Section2

• 爬片
• 流氏T抗原
• 流式黏附
• 微流控

爬片
A series of experiments were done to determine whether the cell surface display system(CSDS) correctly transports the antibody Ts to the outer membrane of the bacterium, as well as whether the antibody Ts are effective to build a cell-bacteria junction. We used cell-climbing cover glasses as the solid basement on which the cell-bacteria adhesion experiment was carried out. These cover glasses were carefully observed via the fluorescent microscope after a period of incubation, and in what pattern the bacteria adheres to the cell could be determined.
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流式T抗原
Certificate the expression of HT29 surface T antigen A specified antibody against T antigen was applied to detect the antigen on HT29’s surface.We used FACS to analysis the antigen-antibody reaction. The antibody PNA
流式粘附
Quantitative Analysis of the Validity of Antibody Ts Next, we were able to quantitatively determine whether the cell surface display system(CSDS) transports the antibody Ts to the outer membrane of the bacterium correctly, and how the antibody works. the FACS analysis was applied to the HT29 cell suspension incubated with antibody-expressing BL21. In this case, BL21 with antibody displayed on the outer membrane should attach to the HT29 cell which correspondingly have the T antigens on its surface. The reaction system was treated with 7AAD by which the dead cells can be differentiated from living ones in a low dye concentration after the co-incubation1. And the BL21 expressed EGFP in order to be detected.
To identify the positive signal, that is, the bacteria adhered to the HT29 cell, from the cell-bacteria suspension, we decided to screen out the free BL21 before we exam the fluorescence intensity. The signal that shows a similar scale of cells as well as strong fluorescence intensity is inferred as the positive signal after free bacteria are excluded(Fig.X) . In order to draw a statistically meaningful conclusion, each group had a four-times parallel repeat. As shown, positive rate in INP-EGFP(BL21 with INP and EGFP expression) was substantially higher than that of EGFP(BL21 with only EGFP expression) group(Fig.X+1).

We further investigated the efficiency of three antibody Ts. It showed that the INP antibody had a better performance with the positive rate of 24.1%, compared with Ompa-1( %) and Ompa-2(%)

Animal experiment

Overall workflow of animal experiment

Based on solid validations in vitro, we conducted animal experiments to further confirm the in vivo performance of engineered E.coli in mice colon.

Characterization of AOM/DSS-induced CRC in mice

The azoxymethane (AOM) and dextran sodium sulfate (DSS) induced colitis murine model is commonly used to study carcinogenesis and of colorectal cancer [Ref01-02], and to test the specificity and sensitivity of diagnostic tools, such as metagenome analysis based CRC risk screening [Ref03]. C57BL/6 mice were divided into groups of three to four per time point and injected with 1mg/kg AOM, followed by 2-2.5% (w/v) DSS in drinking water for 7 days, with 14 days for recovery, and repeat for 3 cycles in modeling group (Fig.7A). The endpoints of 50 days, 60 days and 70 days were chosen for morphological observation and animal experiments. Clinical symptoms were observed during the progress of carcinogenesis, such as the loss of body weight, watery diarrhea and fecal blood, consistent with the previous report. Body weight loss was observed from day 9 in C57BL/6 mice (Fig.7B), with a decrease of 6±0.2% weight loss per day in the first DSS administration cycle, and turned to mild variation in the next two cycles, which was consistent with previous studies (). Multiple polypoid masses, and occult bleeding of colon could be observed in dissected CRC modeling mice, compared to control (Fig.7C). For gross examination, 2-3 polyps around 2cm diameters could be observed from day 50 mice (Fig.7D). Correspondingly, signs of surface epithelial regeneration, moderate infiltration of inflammatory cells to the mucosa, unusual distribution of adenocarcinomatous glands pronounced the clinical adenocarcinoma status of mice (Fig.7E) moderately differentiated.

Characterization of T antigen in CRC tissue

Recently, molecular mechanism laying behind the binding behavior of specific bacteria to CRC, such as Fusobacterium nucleatum was demonstrated [Ref04]. In which Thomsen-Friedenreich (TF) antigen (Galβ1, 3GalNacα-O-Ser/Thr) was characterized as typical features expressed on colorectal adenocarcinoma[Ref05-06], giving us the clue to design corresponding structure that could bind to this antigen. We firstly confirmed the expression of TF antigen on colorectums of CRC modeling mice. Fluorescein isothiocyanate (FITC)-labeled peanut agglutin (PNA), a Gal-GalNAc [Gal-β(1-3) GalNAc] lectin was specifically bound to TF antigen. We assessed the level of TF in tissue by applying this label onto colonic mice colorectum and control mice colorectum, followed with flow cytometry analysis and fluorescence microscope observation. FITC fluorescence signals were significantly higher in adenocarcinomas compared with control tissue, with PNA-FITC positive ratio of 29.3%±6.2% in CRC and 1.03%±0.36% in control, p=0.045 (Fig.8B and D). During the progression of colorectal carcinogenesis, both of day 50 and day 70 dissected CRC mice exhibited an increase of TF antigen in colorectal tissue (P<0.05) (Fig.8C). Interestingly, we observed a trend of decrease in Day 70 mice, suggesting a higher possibility of detecting early colorectal carcinoma through peptide-TF antigen binding.

Specific binding of tPep expressed E.coli to tumor tissue

To confirm that engineered E.coli attachment to CRC is peptide-TF antigen binding mediated, we applied ice nucleation protein (INP) expressed EGFP E.coli and EGFP E.coli to CRC tissue and normal mice (control) tissue, co-cultivated for 30 min at 37℃, and embedded those tissues in frozen medium, made to frozen slice, and sealed for microscopy observation (Fig.9A). Obviously, on-tumor sites of CRC tissue, which exhibited abnormal hyperplasia in H&E stain slice and higher signal of T antigen (Fig.8F), bound higher amount of INP-tPep and EGFP co-expressed E.coli (47%±3%), compared to off-tumor site (11.5%±1.5%), p<0.05 (Fig.9 B and C). Besides, an increase of INP-tPep and EGFP co-expressed E.coli was observed in the apical colon, when comparing CRC tissue with the control, illustrating the binding specificity of T peptide to T antigen. Notably, certain signals (highlighted in yellow triangles) could be observed in the basolateral side of colon, which were recognized as background because we applied E.coli onto the intact colon which prevented the in-depth invasion of bacteria to colon (Fig.9E). Therefore, we chose the apical side of the colon as the region of interest (ROI), to calculate the number of attached E.coli (Fig.9D). The overall number of E.coli was higher in INP-tPep and EGFP co-expressed E.coli treated CRC compared to that treated control, with significantly difference (p<0.0001). Besides, the number of E.coli was higher in INP-tPep and EGFP co-expressed E.coli treated CRC compared to EGFP expressed E.coli treated CRC (p<0.001). Moreover, the amount of INP-tPep E.coli in CRC tissue ranges from 2-8×10⁷, exceeding minimal cell concentration (10⁷) for ultrasonic detection as reported by Bourdeau et. al..(Ref)

section4

Ultrasonic imaging of bound engineered E.coli in colon

Before observing the gas vesicle signal in colon, we firstly built and testified the robustness of our detection system. We positioned the mice colon in 0.2% agarose gel, filled with corresponding bacteria that solidified in the 0.2% agarose, to facilitate the non-invasive ultrasonic observation of the organ. We collected the ultrasonic video and summed each frame in one figure, calculated the gray value of the image based on representative ROI. Signals of gas vesicle ARG expressed E.coli could be observed in mice colon both in transverse view (18.73±5.87) and longitudinal view (40.24±8.50), compared to nonengineered ones, p<0.05 (8.09±5.81 and 7.62±1.72 respectively) (Fig.10A, B and C). We then applied the INP-tPep and ARG co-expressed E.coli into the colorectal lumen of CRC and control mice to evaluate the ultrasonic imaging of specific binding. We injected the INP-tPep and ARG co-expressed E.coli into the lumen, co-cultivated for 30min in 37℃, and washed the unbinding bacteria with PBS for 3 times. To optimize the collection of ultrasonic signal, we set the colon vertically (Fig.11E). The corresponding results were showed in Fig.11A-B. The overall signal of gas vesicle expressed by attached E.coli showed no significant difference between CRC colon and the control, due to the high background of noise. To remove the background signal of the lumen, we chose a scanning line and collapsed the gas vesicle with 21MHz for 5min (Fig.11C), and compared the decreased signal between CRC and control. Fortunately, a significant decrease of gray values, which stood for the signal intense of gas vesicle, could be observed in CRC colon, whereas that decrease was mild in control colon (Fig.11D).

Section5

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