Cyanine Phototruncation Enables Spatiotemporal Cell Labeling

Abstract

Photoconvertible tracking strategies assess the dynamic migration of cell populations. Here we develop phototruncation-assisted cell tracking (PACT) and apply it to evaluate the migration of immune cells into tumor-draining lymphatics. This method is enabled by a recently discovered cyanine photoconversion reaction that leads to the two-carbon truncation and consequent blue-shift of these commonly used probes. By examining substituent effects on the heptamethine cyanine chromophore, we find that introduction of a single methoxy group increases the yield of the phototruncation reaction in neutral buffer by almost 8-fold. When converted to a membrane-bound cell-tracking variant, this probe can be applied in a series of in vitro and in vivo experiments. These include quantitative, time-dependent measurements of the migration of immune cells from tumors to tumor-draining lymph nodes. Unlike previously reported cellular photoconversion approaches, this method does not require genetic engineering and uses near-infrared (NIR) wavelengths. Overall, PACT provides a straightforward approach to label cell populations with spatiotemporal control.

Figure 1.

a) Cyanine phototruncation reaction. b) Depiction of the application of (i) spatially controlled PACT to (ii) cellular migration from tumor to TDLN.

Figure 2.

a) Absorbance curves of 3 (R₁ = OMe, R₂ = H) before and after irradiation with 730 nm LED (500 mW/cm² for 1 h (50 μM, pH 7.4 PBS). b) Samples of 3 and HITCl (50 μM, pH 7.4 PBS) were irradiated with a 730 nm LED (50 mW/cm²) for up to 1 h and monitored by UV-vis absorbance designated time intervals. c) Cellular photoconversion data. MC38 cells were incubated with PBS containing 3’-OMe-DiR (20 μM) or DiR (20 μM) for 30 min at 37 °C. The cells were then washed with PBS containing 1% FBS twice and then exposed to NIR light at 10 and 100 J/cm² (780 nm, 150 mW/cm²). Photoconversion was analyzed by flow cytometry using Cy7 and Cy5 filter sets with identical gating (Figure S4)

Figure 3.

a) Fluorescence imaging of photoconversion in MC38 tumors (implanted in the right lower limb). After intra-tumoral injection of 3’-OMe-DiR or DiR, fluorescence images were acquired with Cy5 and Cy7 filter settings just before and after NIR light exposure (in yellow circle). b) Fluorescence microscopy of frozen sections of MC38 tumors. After intra-tumoral injection of 3’-OMe-DiR or DiR, the tumor was exposed to NIR light. Frozen sections of the tumor were prepared and observed with a fluorescence microscope. Cy5 and Cy7 fluorescence are shown in magenta and green, respectively. Representative pictures are shown (images; ×200; scale bar, 100 μm). c-d) Flow-cytometric analysis of photoconversion in MC38 tumors. After intra-tumoral injection of 3’-OMe-DiR or DiR, the tumor was exposed to NIR light, followed by the preparation of a single-cell suspension of the tumor. Representative dot plots (c) and comparison of photoconversion rates between 3’-OMe-DiR and DiR (D; n = 3; unpaired t test; **, P < 0.01, ***, P < 0.001) were shown for T cells, macrophages, dendritic cells, and tumor cells. Photoconversion rates were calculated as ((photoconverted cell number)/(sum of non-photoconverted and photoconverted cell numbers)) × 100. e-f) Tracking of photoconverted intra-tumoral immune cells migrated into tumor-draining lymph nodes in MC38 cells. Tumors were exposed to NIR light after intra-tumoral injection of 3’-OMe-DiR or DiR (see methods section). Tumor-draining lymph nodes were harvested 1 h and 24 h after NIR light exposure. e) Representative dot plots of migrated macrophages and dendritic cells with 3’-OMe-DiR 24 h after NIR light exposure. f) Comparison of rates of migrated cells among the mice with 3’-OMe-DiR and DiR staining 1 h and 24 h after NIR light exposure (F; n = 3; one-way ANOVA followed by Tukey’s test; ***, P < 0.001, ****, P < 0.0001) were shown.