Atg4B and Cathepsin B-Triggered in Situ Luciferin Formation for Precise Cancer Autophagy Bioluminescence Imaging

Autophagy plays a crucial role in tumorigenesis and progression, but current approaches to visualize it in vivo show limited precision due to their single-analyte-responsive mode. Hence, by simultaneously employing dual autophagy enzymes Atg4B and cathepsin B to trigger the in situ formation of luciferin, we herein propose a strategy for precise autophagy bioluminescence imaging. An Atg4B-responsive peptide Ac-Thr-Phe-Gly-d-Cys (TFGC) and a cathepsin B-activatable compound Ac-Lys-Gly-Arg-Arg-CBT (KGRR-CBT) were rationally designed. During tumor autophagy, these two compounds were uptaken by cancer cells and cleaved by their corresponding enzymes to yield d-cysteine and 2-cyano-6-aminobenzothiazole, respectively, which underwent a CBT-Cys click reaction to yield d-aminoluciferin, turning the bioluminescence “on”. The responsiveness of these two compounds toward the two enzymes was tested in vitro, and the ability to turn bioluminescence “on” was validated in living cancer cells and in vivo. We anticipate that our precise autophagy imaging strategy could be further applied for the diagnosis of autophagy-related diseases in the near future.


General methods
All the starting materials were obtained from Aladdin Chemistry Co., Ltd.(China), GL Biochem Co., Ltd.(Shanghai), Sigma-Aldrich (Shanghai, China).Commercially available reagents were used without further purification, unless noted otherwise.All other chemicals were analytical reagent grade or better.Deionized (DI) water (18.2MΩ•cm) was purified by a Milli-Q system (Millipore) and used throughout the experiments.Atg4B was purchased from Abnova (Taiwan, China).Cathepsin B (CTSB) was obtained from Bio-Techne (USA).
High performance liquid chromatography (HPLC) analyses were performed on an Agilent 1260 HPLC system equipped with a G1322A pump and in-line diode array ultraviolet (UV) detector using an Agilent Zorbax 300SB-C18 RP column with CH3CN (0.1% of trifluoroacetic acid (TFA)) and water (0.1% of TFA) as the eluent.The spectra of electrospray ionization-mass spectrometry (ESI-MS) were obtained on a Finnigan LCQ Advantage ion trap mass spectrometer (ThermoFisher Corporation) that was equipped with a standard ESI source.
Fluorescence imaging was conducted on a fluorescence microscope (Ti2-U, Nikon, Japan).
Bioluminescence images of cells and animals were captured by a PerkinElmer animal imaging system.

Expression of firefly luciferase (fLuc)
The expression and purification protocol of fLuc was according to the literature method. 1 In detail, BL 21 cells were transformed with the appropriate plasmids (pET-6xHis/Luciferase) and cultured in lysogeny broth (LB) medium supplemented with 100 μg/mL ampicillin at 37 °C until their OD600 reached 0.6-0.8.Afterwards, the BL 21 cells were induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16 °C for 20 h.The bacterial cells were then precipitated (4500 rpm, 10 min) and re-suspended in binding/wash buffer (Sangon Biotech, China).After disrupting the bacterial cells via sonication on ice.The His-tagged enzymes were purified using Ni-NTA-agarose (Sangon Biotech, China) equilibrated in TBS buffer (50 mM Tris, 150 mM NaCl, pH 8.0) supplemented with 10 mM imidazole.After the enzyme supernatant was loaded in the Ni-NTA column, rotated for 30 min, and the column was washed with binding/wash buffer for 3 times.Then, the enzyme was eluted out by elution buffer (Sangon Biotech, China) for 3 times.The protein collected from elution after dialysis was used without further purification and the enzyme concentration was determined by BCA protein assay kit (KeyGen Biotech, China).

Cytotoxicity studies
For the cell viability assay, fLuc-transfected MDA-MB-231 cells were seeded in 96-well plates (5 × 10 3 per well), and cultured with cell culture medium for 24 h.Afterwards, the cell culture medium was removed and cells were treated with different concentrations of compounds of interests.The cell viabilities were further detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide (MTT) assay.In detail, 10 μL MTT solution (5 mg/mL) was added into each well and the plates were incubated in the incubator for 4 h.Afterwards, 150 μL dimethyl sulfoxide (DMSO) was used to replace the reaction solution in each well.After measuring the absorbance of each well at 570 nm with multimode microplate reader (Spectra Max iD3, BD, USA), the cell viability percentage relative to the untreated cells was calculated.
The MTT assay was performed in sextuplet, and the average values of the six measurements were taken.

Relative CTSB activity measurements
1 × 10 5 fLuc-transfected MDA-MB-231 cells with or without rapamycin (RAPA) treatment were collected and washed with ice-cold PBS for 3 times.Afterwards, the cells were resuspended in 50 µL RIPA lysis buffer (Beyotime, China) and lysed on ice for 30 min.The CTSB activity of cell lysate was measured with Cathepsin B Activity Fluorometric Assay Kit (Genmed Scientifics, China).

Western blot
After RAPA treatment, fLuc-transfected MDA-MB-231 cells were washed with PBS followed by lysing with RIPA buffer (Beyotime, China) containing 1% Protease Inhibitor Cocktail Set III (Beyotime, China) on ice for 30 min.After centrifugation, the supernatants were collected and the total protein concentration of each sample was analyzed by BCA protein assay kit (KeyGen Biotech, China).The protein samples were then added with reduced protein loading buffer and diluted to the same concentration prior to the denaturation in boiling water bath for 15 min.Afterwards, the protein samples were loaded on a 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel which was subsequently transferred to a polyvinylidene difluoride (PVDF) membrane for 120 min.After incubation with NcmBlot blocking buffer (NCM Biotech, China) at room temperature for 10 min, the PVDF membrane was washed with Tris-buffered saline (TBS) containing 0.5% Tween-20 (Solarbio, China) (TBST) for 3 times followed by the incubation with a rabbit monoclonal antibody against Atg4B (abcam, UK) (the primary antibody) at 4 °C with shaking overnight.Next, the PVDF membrane was washed with TBST and then incubated with the corresponding horse radish peroxidase (HRP)-conjugated secondary antibody at room temperature for 60 min.Finally, the target proteins were detected after incubating the PVDF membrane with enhanced chemiluminescence (ECL) reagent (Tanon, China) and visualized via a chemiluminescence imaging system (Tanon 5200, China).GAPDH was set as the loading control.

Immunofluorescence staining
FLuc-transfected MDA-MB-231 cells were treated with cell culture medium or 10 μM RAPA for 4 h.Afterwards, the cells were immobilized by 0.1% glutaraldehyde for 10 min at room temperature.Followed by the permeabilization by 0.1% Triton-X 100 for 10 min, the cells were then incubated with 3% bovine serum albumin (BSA)solution for 2 h prior to the incubation
with the LC3A/B antibody (Bioss, China) in BSA solutions at 4 o C overnight.Next, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (KeyGen Biotech, China) at room temperature for 2 h.Before confocal fluorescence imaging, the cells were washed with PBS for 3 times to remove unbound antibodies.Animal model Three-week-old female BALB/c nude mice were obtained from Yangzhou University Medical Center (Yangzhou, China).All experimental procedures at the animal level were performed under the approval of the Animal Care Committee of Southeast University (No.20230410031) and in compliance with the Regulations for the Administration of Affairs Concerning Experimental Animals of China.To establish tumor-bearing mouse model, 5 × 10 7 fLuctransfected MDA-MB-231 cells (suspended in 100 µL of PBS) were subcutaneously injected into the right tight of each mouse.After 10 days, tumor models were established and the mice were randomly divided into five groups (n = 4).Each group were tumor-directly injected with PBS or 5 μmol/kg RAPA for 4 h to construct tumor autophagy-inactivated or autophagyactivated models.

Figure S2 .
Figure S2.ESI-MS spectrum of compound A.

Figure S17 .
Figure S17.ESI-MS spectrum of the HPLC peak at 10.2 min in Figure S16.

Figure S20 .
Figure S20.ESI-MS spectrum of the HPLC peak at 15.1 min in Figure S19.

Figure S24 .
Figure S24.(a) CTSB activities of fLuc-transfected MDA-MB-231 after RAPA treatment for 4 h.(b) Western blotting of Atg4B in fLuc-transfected MDA-MB-231 cells after 10 μM RAPA treatment for 4 h and corresponding quantitative analysis of band intensity of Atg4B/GAPDH.(c) Immunofluorescence staining of fLuc-transfected MDA-MB-231 cells with (or w/o) RAPA treatment using LC3A/B antibody and FITC-labeled goat anti-rabbit IgG antibody.(d) TEM images of fLuc-transfected MDA-MB-231 cells without (the top row) or with (the bottom row) 10 μM RAPA treatment for 4 h.Red arrows indicate the locations of autolysosomes.

Figure S25 .
Figure S25.HPLC traces of TFGC (a) and KGRR-CBT (b) before/after incubation in PBS or 10% FBS at 37 o C for 24 h.

Figure S29 .
Figure S29.Time course quantified total flux of the images in Figure 2.

Table S1 .
The limits of detection (LODs) of Atg4B in representative reported methods.

Table S2 .
The LODs of CTSB in representative reported methods.