(G-270) Tracking lipid transfer between pancreatic ductal carcinoma cells and cancer-associated fibroblast cells with two-photon fluorescence and stimulated Raman microscopy

Introduction:: Pancreatic ductal carcinoma (PDAC) is the most common type of malignant pancreatic cancer. Exploring the metabolism of PDAC with its microenvironment may help diagnose and treat this cancer. The goal of this study is to use stimulated Raman scattering microscopy (SRS) to determine lipid transformation between PDAC and cancer-associated fibroblasts (CAF) cells, using deuterium-labeled oleic acid (OA-d34) as a specific marker to track and quantify this process.


Materials and Methods:: Cells used in the research are PDAC Kras-control and CAF C7-TA-PSC. The regular culture medium is DMEM supplied with high glucose, glutamine, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. The nutrient-depleted medium is DMEM without glucose and glutamine. The CAF cells were first treated with 200 µM OA-d34 for 36 h before being co-cultured with PDAC cells in a nutrition-depleted medium. To track the lipid transfer between PDAC and CAF cells, SRS microscopy was performed to detect a carbon-deuterium band (C-D) at 6, 12, 18, and 24 h.




Results, Conclusions, and Discussions:: We treated CAF cells with 200 µM deuterium oleic acid for 36 h. We observed a significant increase in intracellular lipid droplets (LDs) in the CAF cells. To mimic the PDAC tumor microenvironment, we co-cultured PDAC with the OA-treated CAF cells at a ratio of 1:10. SRS imaging of the C-D bond at 2112 cm^-1 was used to track the quantity and dynamics of lipid droplet transfer between these cells. This C-D Raman peak is free from the cellular background, enabling more accurate LD segmentation and quantification. The CAF and PDAC cells were labeled with GFP and mCherry, respectively. Two-photon fluorescence imaging was employed to identify the cell types. Our integrated imaging system was able to acquire SRS and the two-channel, two-photon fluorescence imaging simultaneously, simplifying colocalization and segmentation analysis. After co-culture for 6 h, we observed the accumulation of C-D labeled LDs in PDAC cells (Fig. 1a). At different time points of 12, 18, and 24 h, we found increasing C-D labeled LDs in PDAC cells. Accordingly, we observed decreased LDs in the CAF cells (Fig. 1b). These experimental observations indicate that lipids were actively transferred from the CAF to the PDAC cells under nutrient-depleted conditions, revealing the supporting role of CAF to cancer cells in the tumor microenvironment. In this study, we will use live-cell imaging to apprehend the dynamics of LDs and the metabolic crosstalk between the two cell types to unveil how lipids are transferred from CAF to PDAC cells. Conclusion: The lipid nutrition transfer from CAF to PDAC cells was directly observed with SRS imaging of deuterium labeling. Our results revealed that lipids are a significant nutrition support to PDAC cells. This work may provide new clues to developing anti-cancer strategies by suppressing the supporting role of CAF cells to cancer cells regarding nutrient supplies.



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