Fibrogenic fibroblast-selective near-infrared phototherapy to control scarring

Rationale: Fibroblasts, the predominant cell type responsible for tissue fibrosis, are heterogeneous, and the targeting of unique fibrogenic population of fibroblasts is highly expected. Very recently, elevated glycolysis is demonstrated to play a pivotal role in the determination of fibrogenic phenotype of fibroblasts. However, it is lack of specific strategies for targeting and elimination of such fibrogenic populations. In this study, a novel strategy to use the a near-infrared (NIR) dye IR-780 for the targeting and elimination of a fibrogenic population of glycolytic fibroblasts to control the cutaneous scarring is developed. Methods: The identification and cell properties test of fibrogenic fibroblasts with IR-780 were conducted by using fluorescence activated cell sorting, transplantation experiments, in vivo imaging, RNA sequencing in human cell experiments and mouse and rat wound models. The uptake of IR-780 in fibroblasts mediated by HIF-1α/SLCO2A1 and the metabolic properties of IR-780H fibroblasts were investigated using RNA interference or signaling inhibitors. The fibrogenic fibroblast-selective near-infrared phototherapy of IR-780 were evaluated in human cell experiments and mouse wound models. Results: IR-780 is demonstrated to recognize a unique glycolytic fibroblast lineage, which is responsible for the bulk of connective tissue deposition during cutaneous wound healing and cancer stroma formation. Further results identified that SLCO2A1 is involved in the preferential uptake of IR-780 in fibrogenic fibroblasts, which is regulated by HIF-1α. Moreover, with intrinsic dual phototherapeutic activities, IR-780 significantly diminishes cutaneous scarring through the targeted ablation of the fibrogenic population by photothermal and photodynamic effects. Conclusion: This work provides a unique strategy for the targeted control of tissue scarring by fibrogenic fibroblast-selective near-infrared phototherapy. It is proposed that IR-780 based theranostic methodology holds promise for translational medicine aimed at regulation of fibrogenic behavior.


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Rotenone. To test the glycolysis stress, cells were sequentially treated with 10 mM Glucose, 1 μM Oligo and 50 mM 2-DG. Real-time ECAR and OCR were recorded according to the manufacturer's manual.  or control fibroblasts were seeded in a 24-well plates and cultured for 48 hours. The culture medium and cells were collected respectively. Lactate in the medium and cell lysates were detected using a lactate assay kit (Nanjing Jiancheng engineering # A019-2) according to the manufacturer's manual. Western blot Total proteins from fibroblasts were extracted using cell lysis buffer (Beyotime) supplemented with proteinase inhibitor cocktail (Roche). After vortexing and centrifugation at 12000 ×g for 10 min at 4°C, the supernatant was collected and protein was quantitated using a BCA protein assay kit (Beyotime). Equal amounts of protein for each sample were subjected to 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked in blocking buffer (Beyotime) for 1 hour before application of primary antibodies 4°C overnight. HRP-conjugated goat anti-mouse or anti-rabbit secondary antibody (1:1,000; Beyotime) was applied for 2 h at RT. Blots were visualized using chemiluminescence (Bio-Rad) Immunofluorescence and immunohistochemistry IR-780 labeled wound tissue cryosections (10 μm) for immunofluorescence were stained as follows. Slides were washed thrice for 10 min at RT in PBS and then permeabilized with 0.1% Triton X100

RNA sequencing and quantitative real time
for 5 min at RT and blocked in blocking buffer (PBS + 5% donkey serum (Beyotime) for 1 h at RT and then incubated overnight with primary antibodies diluted in PBS in a humidified chamber. The following day, slides were washed thrice in PBS for 10 min at RT and stained with Alexa Fluor 488 or cy3-conjugated secondary antibodies (diluted in PBS ) for 30 min at 37 ℃. After three washes in PBS, slides were stained with DAPI at RT for 3min and mounted with Prolong Diamond antifade Reagent. The protocols of cell climbing slices for immunofluorescence were the same as the cryosections, except the permeation process: slides were permeabilized with 0.1% Triton X100 for 30 min at RT. The protocols of dewaxed paraffin sections (4 μm) for immunofluorescence were the same as cryosections, except the permeation process: slides were incubated in 0.1M citric acid > 95 ℃ for 20 min. Dewaxed paraffin sections (4 μm) for immunohistochemistry were washed thrice for 10 min at RT in PBS and incubated in 0.1M citric acid > 95 ℃ for 20 min. The slides were then blocked and incubated overnight with primary antibodies.
The following day, slides were washed thrice in PBS for 10 min at RT and incubated with HRP-conjugated secondary antibodies (diluted in PBS ) for 30 4 min at 37 ℃. After three washes with PBS, slides were incubated with DAB and co-stained with hematoxylin. All the sections were captured with Leica confocal microscrope or Olympus fluorescence microscope and processed with image J.

Cell transplantation
Human fibroblasts transplantation to rat wound model After dorsal skin wounding, 18 rats were randomly divided into three groups including IR-780 H group, control cell group and PBS group. Rats of IR-780 H group received 2×10 6 IR-780 H human fibroblasts in 0.2 ml PBS injected from tail vein. Rats of control cell group received 2×10 6 control human fibroblasts. Rats in PBS group were performed with 0.2 ml PBS. Wound tissues were harvested after wound healed (about 15 days after wounding). The wound scar area, scar depth, the ECM deposition and the expression of α-SMA were analyzed. 10 days latter, all skin tissues were harvested for histology. for 10 min. After thrice washes, cells were subjected to NIR light irradiation using an infrared thermal camera ( 808 nm, 0.25, 1.25, 5.01 W cm -2 ) for 5 min. 3) to study the photodynamic and photothermal mechanism of IR-780, primary fibroblasts were treated with or without NAC(5 mM, Sigma) for 24 hours and then incubated with IR-780 (0.4 μM) for 10 min. After thrice washes, cells incubated with or without ice were subjected to NIR light irradiation (1.25 W cm -2 ) for 5 min. 24 hours after different treatments, cell viability was assessed using theCCK-8 assay (as described in section "Growth curve assay")

Calcein AM/(Propidium Iodide) PI staining
Fibroblasts were seeded in the 6-wells plates (5×10 5 cells/ well) and cultured overnight. Cells were subjected to different treatments before testing the cell survival with Calcein AM/PI staining. To test the photodynamic and photothermal effect of IR-780, FACS-sorted IR-780 H or control human fibroblasts were incubated with IR-780 (0.4 μM) at 37 ℃ for 10 min. After 6 thrice washes, cells were irradiated with NIR laser light (808 nm, 1.25 W cm -2 ) for 5 min. 6 hours after different treatments, cells were stained with Calcein AM(2ug/ml)+PI(15ug/ml) in PBS for 20 min at 37℃ in the dark. After thrice washes, Fluorescent images were captured by Olympus fluorescence microscope and processed with image J.

In vitro and vivo assessment of PTT property
For in vitro detection, 2 ml IR-780 (10 μM) in PBS was filled in a 30 mm dish and irradiated with NIR laser light (808 nm, 1.25 W cm -2 ) for 5 min. The real-time temperature change was imaged by the infrared thermal camera (Ti32, Fluke, USA). For in vivo detection, mice at 2 days after wounding were received intraperitoneal injection of IR-780 (0.334 mg/kg) in 0.2 ml PBS. 24 hours latter, the real-time temperature change of mice was imaged by the infrared thermal camera (Ti32, Fluke, USA) when the whole wound tissues were exposed to the continuous NIR laser beam (808 nm, 0.75 W/cm 2 , 5 min).

In vivo therapeutic effect of IR-780
For photodynamic and photothermal therapeutic effects, mice were intraperitoneally injected with IR-780 (0.334mg/kg) at 1, 8 days post wounding. 24 hours latter, wound tissues were irradiated with NIR laser light 8 (808 nm, 1.25 W cm -2 ) for 5 min. Wound samples were collected for histology at 3, 10 days and when the wounds healed.   Cell viability testing of human IR-780 H and control fibroblasts exposed to different IR-780 concentration and irradiation power.  Scale bar, 100 µm.