Pyroptosis Remodeling Tumor Microenvironment to Enhance Pancreatic Cancer Immunotherapy Driven by Membrane Anchoring Photosensitizer

Abstract Immunotherapy, the most promising strategy of cancer treatment, has achieved promising outcomes, but its clinical efficacy in pancreatic cancer is limited mainly due to the complicated tumor immunosuppressive microenvironment. As a highly inflammatory form of immunogenic cell death (ICD), pyroptosis provides a great opportunity to alleviate immunosuppression and promote systemic immune responses in solid tumors. Herein, membrane‐targeted photosensitizer TBD‐3C with aggregation‐induced emission (AIE) feature to trigger pyroptosis‐aroused cancer immunotherapy via photodynamic therapy (PDT) is applied. The results reveal that pyroptotic cells induced by TBD‐3C could stimulate M1‐polarization of macrophages, cause maturation of dendritic cells (DCs), and activation of CD8+ cytotoxic T‐lymphocytes (CTLs). Pyroptosis‐aroused immunological responses could convert immunosuppressive “cold” tumor microenvironment (TME) to immunogenic “hot” TME, which not only inhibits primary pancreatic cancer growth but also attacks the distant tumor. This work establishes a platform with high biocompatibility for light‐controlled antitumor immunity and solid tumor immunotherapy aroused by cell pyroptosis.


Instruments
The main instruments used in this project were as follows:

Western Blot Analysis
To detect the FL-GSDMD protein after the treatment of TBD-3C (10 μM, 40 mW/cm 2 ) and detect the PRAP protein after TBD-3C (10 μM, 40 mW/cm 2 ) treatment or STS stimulation (2 μM for 4 h), the KPC and Panc02 cells were harvested after the corresponding treatment.
Protein concentration was qualified by BCA assay. Then equal amount of protein was resolved in 10% SDS-PAGE, transferred to polyvinylidence fluoride (PVDF) membranes, blocked with 5% milk for 1 hour at room temperature and then incubated with primary antibodies at 4°C overnight. After washing with PBS three times, secondary antibody was incubated with the membrane. The results were detected and photographed by ChemiScope.

Isolation and differentiation of BMDM and BMDC
BMDM and BMDC were isolated from C57BL/6 mice. Briefly, the mice were sacrified and disinfected with 70% alcohol, the hip bones without skin and muscles were harvested and the 3 epiphyses of the bones were cut, and then the marrow were flushed into a 15 mL centrifuge tube using a 1 mL syringe. The resulting marrow were lysed with red blood cell lysis buffer and filtered through nylon mesh filters (70 μm), and centrifuged at 600×g and 4°C for 5 minutes. The obtained cells were diluted at a concentration of 1 × 10 6 cells/mL, and M-CSF was added at a final concentration of 20 ng/mL to differentiate BMDM or 10 ng/mL GM-CSF and 5 ng/mL IL-4 were added to differentiate BMDC. 10 mL of each type of cells were severally seeded onto cell culture-treated petri dish and incubated at 37°C with 5% CO2 for 3 days. Replaced the medium and incubated for another 3 days. M1 and M2 macrophages were obtained after adding LPS (0.5 g/mL) and IL-4 (20 ng/mL) separately for 48 h. The induced BMDMs were incubated with CD11b and F4/80 antibodies for 30 minutes. The expression of CD11b and F4/80 were measured using flow cytometer to confirm their phenotype. The BMDMs were incubated with CD86 and CD206 antibodies to show the differential expressions in the LPS-induced M1-like and IL-4-induced M2-like macrophages by flow cytometry to confirm their phenotype. M1-like and M2-like macrophages were defined as CD86 hi CD206 lo and CD86 lo CD206 hi , respectively. The BMDCs were incubated with CD11c and MHC II antibodies to confirm their phenotype. The BMDMs and BMDCs were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin.

Isolation of T lymphocytes
T lymphocytes were obtained from the spleen of C57BL/6 mice. Spleen was mechanically dissociated into small pieces using a grind rod. The digested tissues were milled into single cells using a 70-μm cell strainer and washed with PBS for 3 times. Red blood cell lysis buffer was used to remove red blood cells. Then the CD8 + T cell were purified using a mice CD8 + T cell isolation kit (Miltenyibiotec, 130-096-495) according the manufacturer's instructions.
The CD8 + T cells were further stained using CD8-PE and CD3-FITC antibodies to confirm their phenotype. The isolated CD8 + T cells were labeled with 4 μmol/L CFSE for 10 min at 37°C to obtain the CFSE-labeled CD8 + T cells and were mixed with the Non-CD8 + T cells in the column of the isolation kit for further use.

In vivo Biocompatibility assay
At the end of the TBD-3C PDT treatment in vivo, the peripheral blood (1mL per mouse) was collected under anesthesia for blood biochemistry detection. The biochemistry indicators including creatine kinase (CK), creatinine (CREA), uric acid, urea were the liver and kidney 4 function index. The liver and kidney function tests were performed to investigate the biocompatibility of the TBD-3C PDT treatment.
And the major organs (hearts, livers, spleens, lungs and kidneys) of the mice were collected to evaluate the toxicity of the TBD-3C PDT treatment by H&E staining. For the H&E assay, the organs were fixed in 10% neutral buffered formalin for 24 h. After paraffin embedding, they were cut into 4 μm-thick sections and deparaffinized after being baked at 68°C for 90 minutes. The sections were dehydrated in graded ethanol and immersed in hematoxylin staining solution for 5 minutes. The color of the sections changed from blue to red 2 seconds after adding 50 uL 1% hydrochloric acid ethanol. The sections were then immersed in eosin staining solution for 5 minutes and washed with ddH2O. Finally, the representative images were captured by ImageScope software.