multiphoton laser scanning microscopy  (Olympus)

Journal: bioRxiv

Article Title: Antimetastatic Sulfonate-Functionalized Mesoporous Silica Nanoparticles Enhance Irinotecan Stability and Delivery for Colorectal Cancer Treatment

doi: 10.1101/2025.11.25.690433

Figure Lengend Snippet: Mechanistic evaluation of (SO 3 - )-MSN-PEG/TA on metastasis, focal adhesion turnover, angiogenesis, and tumor targeting. HCT-116 cells were treated with 200 μg/mL of (SO 3 - )-MSN-PEG/TA for 24 hours. (a) Western blot analysis of p-FAK expression levels. (b) Quantitative analysis of p-FAK protein expression relative to the control group (***p < 0.001, n = 3). (c) Immunofluorescence imaging of paxillin (green) and nuclei (blue). Scale bar: 20 μm. (d) Quantitative comparison of the number of FA per cell between control and treated groups (***p < 0.001, n = 5). (e) Schematic representation of the chicken embryo CAM assay. HCT-116 cells were seeded onto the CAM, and (SO 3 - )-MSN-PEG/TA (1 mg/egg) was administered intravenously on embryonic day 13. (f) Representative photographs of CAM vasculature on day 15. (g) Statistical analysis of vascular density performed using ImageJ software (***p < 0.001, n =5). (h) Schematic illustration of the in vivo tumor-targeting study. HCT-116 tumor-bearing mice were intravenously injected with RITC-labeled (SO3⁻)-MSN-PEG/TA (200 mg/kg) and analyzed by multiphoton microscopy. (i) Visualization of the EPR effect. Blood vessels were labeled with FITC–dextran (green), and RITC-labeled (SO3⁻)-MSN-PEG/TA (red) signals were observed 24 hours post-injection. Scale bar = 100 μm. (j) Schematic of the orthotopic colorectal cancer metastasis model. Luciferase-expressing HCT-116 tumor tissue (2 x 10⁶ cells) was implanted into the cecum wall of NOD-SCID mice to establish an orthotopic colorectal cancer model. Mice were intravenously treated with (SO 3 - )-MSN-PEG/TA (equivalent to NPs dose corresponding to 40 mg/kg of IRI@(SO 3 - )-MSN-PEG/TA). (k) Monitoring of orthotopic tumor growth by IVIS imaging from day 3 to day 31. (l) Ex vivo imaging of metastatic lesions: Post-mortem imaging of mice revealed extensive metastasis, with tumor sites identified in organs such as the liver, spleen, kidneys, stomach, and cecum (indicated by cyan arrows).

Article Snippet: Human colorectal cancer HCT-116 cells (5 × 106) were subcutaneously implanted into the left flank of NOD-SCID mice to establish a heterotopic xenograft model. Mice were intravenously injected with RITC-conjugated (SO 3 −)-MSN-PEG/TA (200 mg/kg), and tumor regions were imaged 24 hours post-injection using multiphoton laser scanning microscopy (LSM; Olympus FVMPE-RS) equipped with a tunable infrared laser (700–1080 nm).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Imaging, Comparison, Chick Chorioallantoic Membrane Assay, Software, In Vivo, Injection, Labeling, Microscopy, Luciferase, Ex Vivo