Mitochondria‐Modulating Liposomes Reverse Radio‐Resistance for Colorectal Cancer

Abstract Complete remission of colorectal cancer (CRC) is still unachievable in the majority of patients by common fractionated radiotherapy, leaving risks of tumor metastasis and recurrence. Herein, clinical CRC samples demonstrated a difference in the phosphorylation of translation initiation factor eIF2α (p‐eIF2α) and the activating transcription factor 4 (ATF4), whose increased expression by initial X‐ray irradiation led to the resistance to subsequent radiotherapy. The underlying mechanism is studied in radio‐resistant CT26 cells, revealing that the incomplete mitochondrial outer membrane permeabilization (iMOMP) triggered by X‐ray irradiation is key for the elevated expression of p‐eIF2α and ATF4, and therefore radio‐resistance. This finding guided to discover that metformin and 2‐DG are synergistic in reversing radio resistance by inhibiting p‐eIF2α and ATF4. Liposomes loaded with metformin and 2‐DG (M/D‐Lipo) are thus prepared for enhancing fractionated radiotherapy of CRC, which achieved satisfactory therapeutic efficacy in both local and metastatic CRC tumors by reversing radio‐resistance and preventing T lymphocyte exhaustion.

Specimens were also scored according to the staining intensity of cancer cells as follows: 0, negative; 1, light yellow; 2, dark yellow; 3, brown.The IHC staining score was calculated by multiplying the proportion of positive cancer cells by the staining intensity of cancer cells.The staining results were evaluated by two independent pathologists who had at least 5 years working experience.All samples were obtained with approval from the Institutional Ethics Committee of the First Affiliated Hospital of Soochow University (authorisation number 2021333).

Characterization of the prepared DDS
The diameter and zeta potential of the liposomes were measured by dynamic laser scattering (DLS, DynaPro Plate Reader II, Wyatt) after dilution with sterile 1 × PBS.For visual characterization of the DDS, the liposomes were loaded onto formvar-coated nickel grids (Electron Microscopy Sciences) and negatively stained using 2% phosphotungstic acid solution (Sigma).The morphology of the liposomes was then imaged at 120 kV on an HT7700 TEM (Hitachi) in conbinaiton with a digital micrograph camera and software suite (Gatan).

Testing the drug loading capacity of DDS
Since the mixing ratio of drug to liposomal material was 1:1, we tested the drug loading as represented by metformin hydrochloride.The DDS was treated with Triton X-100 to disintegrate the phospholipid bilayers and release the encapsulated drugs.High Performance Liquid Chromatography (HPLC) was then applied to determine the concentration of the released metformin hydrochloride.To test the concentration of metformin hydrochloride in the DDS, metformin hydrochloride (0.165 mg in 1 mL solution) was utilized as the internal standard solution, while metformin hydrochloride solutions with different concentrations were utilized as the reference.The broken liposomes were purified for HPLC analysis.

In vitro experiments
Cellular proliferation assays were performed using the CCK8 Kit (Beyotime Biotechnology) in accordance with instructions provided by the manufacturer.For cell viability measurement, cells were cultured into 96well plates at least triplicate and then subjected to drug treatment as indicated.Western blot was used to detect the proteins after electrophoresis.p-eIF2α Rabbit pAb (Catalog No. R22946), eIF2α Rabbit mAb (Catalog No.

In vitro transwell migration and invasion
Cell migration and invasion experiments were performed using 24-well plates with 8 μm-polycarbonate filter inserts (#3422, Corning).CT26 cells were seeded at densities of 2 × 10 5 cells/200 μL and 1 × 10 5 cells/200 μL per well, respectively, in serum-free RPMI 1640.All cells were either uncoated or Matrigel-coated and incubated in chambers containing 600 μL of RPMI 1640 with 10% foetal serum as a chemoattractant.The cells were imaged, and their migration and invasion were captured using a microscope (Nikon, Eclipse Ti-S).
All experiments were performed thrice independently.

Plate colony formation assay
CT26 cells with different treatments were independently seeded in six-well plates at densities of 5000 cells per well and incubated at 37 °C.The medium was RPMI 1640 containing 10% foetal bovine serum, which was changed every other day.After 10 days, colony-forming cells were immersed in 4% paraformaldehyde for 20-30 min, stained with crystal violet for 2 h, and then rinsed three times with PBS to remove the excess crystal violet.Finally, images were captured using a microscope, and the number of colony-forming units was counted.

Cell apoptosis analysis
To analyze the fraction of apoptotic cells, CT26 cells with different treatments were evaluated using the annexin V-APC/7-AAD apoptosis detection kit (KA3808, Abnoya).Briefly, each sample containing 1 × 10 5 cells was washed twice with cold phosphate-buffered saline (PBS) and then suspended cells in 100 μL 1 × binding buffer.Then, 5 μL of 7-AAD and 5 μL of APC annexin V were added to each sample.The cells were incubated in the dark for 15 min at room temperature.Approximately 10,000 cells/sample were analyzed by flow cytometry (Becton, Dickinson and Company, FACS Canto II).
γH2AX immunofluorescence assay γH2AX, as a critical marker of DNA double-strand breaks, was assessed in CT26 cells.In this process, cells were first cultured in a 6-well plate with a seeding density of 1×10 6 cells per well.After 24 h, the cells underwent a washing step with PBS and were subsequently exposed to 8Gy of irradiation.Following an additional 12 h of incubation, the cells were immunostained using a specific γH2AX antibody.The confocal images of cells were performed using an Olympus FV1200 microscope to analyze the formation and distribution of γH2AX foci in the cells.

Tumor model
CT26 cells cancer cells (2 × 10 6 ) in 50 μL PBS were injected into the back of BALB/c mouse to establish the subcutaneous tumor model.BALB/c mouse were intravenously injected with Luciferase-CT26 cells (2 × 10 5 cells per mouse) suspended in 0.2 μL of PBS to establish the CRC in situ tumors model.All mice were randomly divided into groups for treatment experiments.The tumor size was calculated as: length × width 2 × 0.5.The tumor-bearing mice were euthanized and sacrificed once the volume of tumor reached 1000 mm 3 .

In vivo biodistribution
The biodistribution of liposome nanoparticles in mice was monitored using an IVIS imaging system (PerkinElmer) at 2, 8, 12, 24 and 48 h after systemic administration ([DIR-labeled DDS] = 100 µg/mice).The tumors and tissues were collected from mice 48 h after i.v.injection and then placed on solid black paper for fluorescence imaging.The fluorescence intensity was measured using an IVIS imaging system (PerkinElmer) and quantified using Living Image software (PerkinElmer).The bilateral CT26 tumor-bearing mice were sacrificed 48 h after systemic administration of DIR-labeled M/D-Lipo to analyze their biodistribution behavior.The fluorescence images of mice, organs and tumors were photographed by IVIS.

Combination therapy and related mechanisms
The treatment plan was conducted when the average tumor size reached 50 mm 3 .Liposomes (M/D-Lipo ([M] = 2 mg/mouse) were intravenously injected into mice.Mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (400 mg/kg) and then mechanically immobilized in a fixture for X-ray irradiation (radiation dose rate: 113 cGy/min, RS-2000 Pro irradiator), which exposed the tumor or lower abdomen to the radiation field while shielding the rest of the body from radiation exposure.Ten days after local tumor treatment, mice were sacrificed to collect the tumors for immunofluorescence analysis of PD-L1.Five days after the treatments, the immune cells in the distant tumors and lymph nodes were analyzed by immunofluorescence slices and flow cytometry after staining with antibodies.After one week of different treatments, tumors were collected and stained with H&E for histopathological analysis.

Statistical analysis
The data are expressed as the mean plus standard deviation unless otherwise stated.T tests were used for comparisons.All statistical analyses were performed using Excel 2016 and GraphPad Prism 8 for Windows (GraphPad Software).The threshold for statistical significance was P < 0.05.
310073), ATF4 Rabbit mAb(Catalog No. 381426) and β-Tubulin mouse mAb (Catalog No. A12289) were purchased from zenbio Technology Co., Ltd (chengdu, China).Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology) was used for detection of apoptosis.The lactic acid concentration in cell culture medium was detected by Lactic Acid assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Figure S1 .
Figure S1.The clonogenic ability of CT26 cells after conventional fractionated radiotherapy was stronger than that after single fraction radiotherapy.

Figure S3 .
Figure S3.(a) The relative cell viability of CT26 cells incubated with free metformin and free 2-DG at different concentrations for 24 h.Error bars represent mean ± s.d.(n = 6).(b) The relative cell viability of CT26 cells incubated with M/D-Lipo at different concentrations for 24 h.Error bars represent mean ± s.d.(n = 6).

Figure S4 .
Figure S4.(a) Zeta potential of Lipo, M/D-Lipo, measured by DLS.Error bars represent mean ± s.d.(n = 3).(b) The fluorescence content of DiD-labeled liposomes was determined by flow cytometry compared with that of DiD-unlabeled liposomes.Error bars represent mean ± s.d.(n = 3).

Figure S11 .
Figure S11.Schematic of orthotopic mouse colorectal construction and treatment.