Biophotonics discovery最新文献

Significance: Radioresistance remains a significant problem for head and neck squamous cell carcinoma (HNSCC) patients. To mitigate this, the cellular and molecular pathways used by radioresistant HNSCC that drive recurrence must be studied.

Aim: We aim to demonstrate optical imaging strategies to provide flow cytometry-like single-cell level analysis of hypoxia-inducible factor 1-alpha (HIF-1α)-mediated metabolic changes in the radioresistant and radiosensitive HNSCC cells but in a more efficient, cost-effective, and non-destructive manner. Through both optical imaging and flow cytometry studies, we will reveal the role of radiation-induced HIF-1α overexpression and the following metabolic changes in the radioresistance development for HNSCC.

Approach: We optimized the use of two metabolic probes: 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) (to report glucose uptake) and Tetramethylrhodamine ethyl ester (TMRE) (to report mitochondrial membrane potential) with both a standard fluorescence microscope and a flow cytometry device, to report the changes in metabolism between radioresistant (rSCC-61) and radiosensitive (SCC-61) HNSCC cell lines under radiation stresses with or without HIF-1α inhibition.

Results: We found that the matched HNSCC cell lines had different baseline metabolic phenotypes, and their metabolism responded differently to radiation stress along with significantly enhanced HIF-1α expressions in the rSCC-61 cells. HIF-1α inhibition during the radiation treatment modulates the metabolic changes and radio-sensitizes the rSCC-61 cells. Through these studies, we demonstrated that a standard fluorescence microscope along with proper image processing methods can provide flow cytometry-like single-cell level analysis of HIF-1α-mediated metabolic changes in the radioresistant and radiosensitive HNSCC cells.

Conclusions: Our reported optical imaging strategies may enable one to study the role of metabolism reprogramming in cancer therapeutic resistance development at the single-cell level in a more efficient, cost-effective, and non-destructive manner. Our understanding of radiation resistance mechanisms using our imaging methods will offer opportunities to design targeted radiotherapy for improved treatment outcomes for HNSCC patients.

Significance: Heart disease is the leading cause of death in the United States, yet research is limited by the inability to culture primary cardiac cells. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) are a promising solution for drug screening and disease modeling.

Aim: Induced pluripotent stem cell-derived CM (iPSC-CM) differentiation and maturation studies typically use heterogeneous substrates for growth and destructive verification methods. Reproducible, tunable substrates and touch-free monitoring are needed to identify ideal conditions to produce homogenous, functional CMs.

Approach: We generated synthetic polyethylene glycol-based hydrogels for iPSC-CM differentiation and maturation. Peptide concentrations, combinations, and gel stiffness were tuned independently. Label-free optical redox imaging (ORI) was performed on a widefield microscope in a 96-well screen of gel formulations. We performed live-cell imaging throughout differentiation and early to late maturation to identify key metabolic shifts.

Results: Label-free ORI confirmed the expected metabolic shifts toward oxidative phosphorylation throughout the differentiation and maturation processes of iPSC-CMs on synthetic hydrogels. Furthermore, ORI distinguished high and low differentiation efficiency cell batches in the cardiac progenitor stage.

Conclusions: We established a workflow for medium throughput screening of synthetic hydrogel conditions with the ability to perform repeated live-cell measurements and confirm expected metabolic shifts. These methods have implications for reproducible iPSC-CM generation in biomanufacturing.

Significance: Increased collagen linearization and deposition during tumorigenesis can impede immune cell infiltration and lead to tumor metastasis. Although melanoma is well studied in immunotherapy research, studies that quantify collagen changes during melanoma progression and treatment are lacking.

Aim: We aim to image in vivo collagen in preclinical melanoma models during immunotherapy and quantify the collagen phenotype in treated and control mice.

Approach: Second-harmonic generation imaging of collagen was performed in mouse melanoma tumors in vivo over a treatment time course. Animals were treated with a curative radiation and immunotherapy combination. Collagen morphology was quantified over time at an image and single-fiber level using CurveAlign and CT-FIRE software.

Results: In immunotherapy-treated mice, collagen was reorganized toward a healthy phenotype, including shorter, wider, curlier collagen fibers, with modestly higher collagen density. Temporally, collagen fiber straightness and length changed late in treatment (days 9 and 12), while width and density changed early (day 6) compared with control mice. Single-fiber collagen features calculated in CT-FIRE were the most sensitive to the changes among treatment groups compared with bulk collagen features.

Conclusions: Quantitative second-harmonic generation imaging can provide insight into collagen dynamics in vivo during immunotherapy, with key implications in improving immunotherapy response in melanoma and other cancers.