Intra‐Operative Definition of Glioma Infiltrative Margins by Visualizing Immunosuppressive Tumor‐Associated Macrophages

Abstract Accurate delineation of glioma infiltrative margins remains a challenge due to the low density of cancer cells in these regions. Here, a hierarchical imaging strategy to define glioma margins by locating the immunosuppressive tumor‐associated macrophages (TAMs) is proposed. A pH ratiometric fluorescent probe CP2‐M that targets immunosuppressive TAMs by binding to mannose receptor (CD206) is developed, and it subsequently senses the acidic phagosomal lumen, resulting in a remarkable fluorescence enhancement. With assistance of CP2‐M, glioma xenografts in mouse models with a tumor‐to‐background ratio exceeding 3.0 for up to 6 h are successfully visualized. Furthermore, by intra‐operatively mapping the pH distribution of exposed tissue after craniotomy, the glioma allograft in rat models is precisely excised. The overall survival of rat models significantly surpasses that achieved using clinically employed fluorescent probes. This work presents a novel strategy for locating glioma margins, thereby improving surgical outcomes for tumors with infiltrative characteristics.

All pH measurements were carried out with a MP220 pH meter (Mettler Toledo, Switzerland).

General synthesis procedure and the chemical characterization of CP2-M (Scheme 1.):
Synthesis of compound C1.C1 was prepared following the previous published procedures. [29]nthesis of compound C2.A mixture of 2,3,3-trimethylbenzoindolenine (10.0 g, 62.8 mmol) and methyl 3-bromopropanoate (11.8 g, 70.9 mmol) in anhydrous acetonitrile (80 mL) was heated to reflux for 12 h.After cooling to room temperature, the resulting solid was washed with diethyl ether until colorless, and dried in vacuo for 24 h to give compound C2 (13.7 g, 66.9%).C2 was directly used in the next step without purification. 1

Synthesis of compound CP2-M.
The compounds CP2 (100 mg, 0.13 mmol), NHS (76 mg, 0.66 mmol), DCC (136 mg, 0.66 mmol) were dissolved in 20 mL of acetonitrile and reacted at 25 °C for 0.5 h.PEG2k-mannose (500 mg, 0.25 mmol) was dissolved in aqueous solution (20 mL) at pH 8, and then added dropwise to the above reaction solution in batches for further reaction.The reaction was kept in dark for 2 h, and the product was further purified via vacuum distillation, dialysis, lyophilization and silica gel column chromatography (CH2Cl2:CH3OH= 10:1 to 3:1, V:V) to obtain pure CP2-M of 162 mg with a yield of 46.7%. 1  Synthesis of compound C5.Compound C3 (0.5 g, 0.71 mmol) was dissolved in methanol (30 mL), followed by addition of aqueous potassium hydroxide (74 mM, 20 mL) to the above solution at 0 °C and stirred at room temperature for 4 h.The methanol was removed and the saturated aqueous ammonium chloride solution was added for adjusting to pH 7, extracted with CH2Cl2 (3×).The solvent was removed under reduced pressure, and the obtained compound C5 were used directly in the next step without purification.

Synthesis of compound CB-M.
The compounds C5 (100 mg, 0.15 mmol), NHS (84.6 mg, 0.74 1640), trypsin, streptomycin and penicillin were purchased from Shanghai Ruiyuan Biomedical Technology Co., Ltd.Cell Counting Kit-8 (CCK-8) was purchased from Dalian Meilun Biotechnology Co.,Ltd.LysoTracker Green DND-26 and MitoTracker Green FM were purchased from Yeasen Biotechnology (Shanghai) Co., Ltd, China.BCA Protein Assay Kit, SDS-PAGE Gel Preparation Kit, QuickBlock™ Primary Antibody Dilution Buffer and QuickBlock™ Secondary Antibody Dilution Buffer for Western Blot were purchased from Absin Bioscience Inc., China.CD206 protein for surface plasmon resonance was purchased from Shanghai Universal Biotech Co., Ltd., China.Immunol Staining Fix Solution and Permeabilization Buffer with Triton X-100 were purchased from Dakewe Biotech Co., Ltd, China.QuickBlock™ Blocking Buffer for immunohistochemistry and antifade polyvinylpyrrolidone mounting medium were purchased from Beyotime Biotechnology Co.,Ltd, China.
H NMR spectra was recorded on a 400 MHz (Varian, USA) NMR spectrometer, and 13 C NMR spectra was recorded on a 600 MHz (Bruker, USA) NMR spectrometer.Electrophoresis apparatus trophoresis (BIO-RAD, USA) were used in Western Blotting studies.Flow cytometric studies were analyzed on BD (USA) FACSAria II flow cytometer.Zeiss LSM 710 META confocal laser scanning microscope (Carl Zeiss), ZEN 2012 software and ImageJ software were used in Confocal fluorescence imaging studies.Absorption and emission spectra were collected by UV-2550

Figure S1 .
Figure S1.Survival curves of glioma patients as a function of TAM percentages in CGGA database.

Figure S2 .
Figure S2.Representative western blotting analysis shows that CD206 is highly expressed in M2-Mφ compared to macrophages with other phenotypes.

Figure S3 .
Figure S3.The spectras of the control probe CP2, CP2-P, and CB-M.Absorption (A, E, I) and fluorescence spectra (B-C, F-G, J-K) of CP2, CP2-P, and CB-M (2.0 μM) as a function of pH.CP2, CP2-P, and CB-M was excited at 740 nm and 670 nm, respectively.(D, H) Plotting fluorescence intensity ratio (I740/I670 nm) of CP2 and CP2-P as a function of pH.Inset: the linear relationship between I740/I670 ratio and pH value.

Figure S4 .
Figure S4.The pH-dependent fluorescence images of the probe CP2-M.Fluorescence imaging change of CP2-M (3.0 μM) after treated with different pH buffer solution.CP2-M was excited at 740 nm and 670 nm, respectively.

Figure
Figure S7.CP2-M shows photophysical stability in aqueous solutions.Time course of the fluorescence intensity ratios (I740 nm/I 670 nm) of CP2-M (5.0 μM) in PBS buffer solutions with pH 4.0 and pH 7.4.

Figure
Figure S9.CP2-M shows minimized cytotoxicity.Viabilities of M2-TAMs after treatment of CP2-M for 12 h, 24 h, and 48 h with concentrations ranged from 6.25 to 50.0 μM.Cell viabilities were determined by CCK-8 assay.Data are presented as the mean ± s.d. for three replicates.

Figure
Figure S11.CP2-M respond to pH changes in vitro.(A)Confocal microscopic images of M2-Mφ (25.0 μM) treated with media with different pH values.Scale bar: 30 µm.(B)the average signal intensity in 633 nm channel at each of the following pH values: 7.4, 6.5, and 5.5.

Figure S13 .
Figure S13.Representative fluorescence (CP2-M) imaging of brain sections from healthy mice and glioma-bearing mice allograft at 4 hours post administration of CP2-M via the tail vein (5.0 μmol/kg/rat).The yellow solid lines indicate the tumor margins, scale bar: 100 μm.

Figure S15 .
Figure S15.Image-guided surgery by using clinically approved fluorescence probes including 5-ALA and ICG in live rat models bearing C6 GBM allografts.

Figure S16 .
Figure S16.Body weight of rat models after different treatments (n = 5 rats/group).