A pre-screening strategy to assess resected tumor margins by imaging cytoplasmic viscosity and hypoxia

Abstract

To assure complete tumor removal, frozen section analysis is the most common procedure for intraoperative pathological assessment of resected tumor margins. However, during one operation, multiple biopsies may be sent for examination, but only few of them are made into cryosections because of the complex preparation protocols and time-consuming pathological analysis, which potentially increases the risk of overlooking tumor involvement. Here, we propose a fluorescence-based pre-screening strategy that allows high-throughput, convenient, and fast gross assessment of resected tumor margins. A dual-activatable cationic fluorescent molecular rotor was developed to specifically illuminate live tumor cells' cytoplasm by emitting two different fluorescence signals in response to elevations in hypoxia-induced nitroreductase (a biochemical marker) and cytoplasmic viscosity (a biophysical marker), two characteristics of cancer cells. The ability of the fluorescent molecular rotor in detecting tumor cells was evaluated in mouse and human specimens of multiple tissues by comparing with hematoxylin and eosin staining. Importantly, the fluorescent molecular rotor achieved 100 % specificity in discriminating lung and liver cancers from normal tissue, allowing pre-screening of the tumor-free surgical margins and promoting clinical decision. Altogether, this type of fluorescent molecular rotor and the proposed strategy may serve as a new option to facilitate intraoperative assessment of resected tumor margins.

Keywords: biochemistry; chemical biology; cytoplasmic viscosity; dual-activatable; fluorescent molecular rotor; human; hypoxia; pre-screening strategy.

Figure 1.. Fluorescence emission and absorption profiles of IBS440.

(A) Fluorescence spectra of IBS440 (10 μM) in different ratios of Ethanol/Glycerol mixtures. (B) The linear response between the fluorescence intensity at 610 nm (lgI(Intensity)) of the probe IBS440 (10 μM) and the viscosity (lgη(Viscosity)) in the Ethanol/Glycerol solvent. (C) The fluorescence response of IBS440 (10 μM) to nitroreductase at the varied concentrations in reaction buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA, pH 8.0). The spectra were recorded upon treatment of IBS440 (10 μM) with nitroreductase (0–9.0 μg/mL) in the presence of NADPH (500 μM). (D) A linear correlation between the concentration of nitroreductase and the fluorescence intensity of the reaction mixture. (E) Fluorescence intensity of IBS440 (10 μM) at 610 nm in various solvents of methanol (MeOH), N, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile (CH₃CN), ethanol (EtOH), ethyl acetate (EtOAc), phosphate buffer saline (PBS), H₂O, tetrahydrofuran (THF), glycerol. (F) Fluorescence responses of IBS440 (10 μM) in the presence of NADPH (500 μM) to various species. λ_ex = 400 nm. λ_em = 520 nm. (G) Effects of pH on the response of IBS440 in solvents with different viscosity (ethanol and 50 % glycerol in ethanol). The fluorescence intensity at 610 nm was plotted against different pH values. λ_ex = 500 nm. (H) The emission intensity (at 520 nm) of IBS224 and IBS440 at different pH Tris-HCl buffer, containing 20 % DMSO as a cosolvent. Error bars represent standard deviation of three repeated experiments.

Figure 1—figure supplement 1.. Confirmation and immunoblotting analysis of nitroreductase.

(A) SDS-PAGE (12%) confirmation and (B) Immunoblotting analysis of different concentration of Recombinant nitroreductase. M: Prestained Protein Molecular Weight Marker. (A) 1–6: 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL, 250 μg/mL, 300 μg/mL of NTR. (B) 1–3: 0.2 μg/mL, 0.8 μg/mL, 1.6 μg/mL.

Figure 1—figure supplement 2.. Absorption spectra of the probe IBS440 (10 μM), IBS440 (10 μM)+ NADPH (500 μM)+ nitroreductase (9 μg/mL), IBS224 (10 μM) in reaction buffer, containing 20 % DMSO as a co-solvent.

Figure 1—figure supplement 3.. Photostability test of IBS440 (10 μM) in glycerol for 3600 s.

λ_ex = 500 nm, λ_em = 610 nm.

Figure 2.. Fluorescence localization of IBS440 in live cells and ex vivo tumor tissues.

(A) The cells were treated with IBS440 (5 μM) for 20 min and then stained with Hoechst 33,342 (1x) and Mito Tracker Green (10 μM) for 10 min. Blue channel (λ_ex = 405 nm, λ_em = 430–480 nm). Green channel (λ_ex = 488 nm, λ_em = 500–550 nm). Red channel (λ_ex = 488 nm, λ_em = 580–630 nm). Scale bar: 25 μm. (B) Visualization in 3D reconstruction (i) and the max merge image of 3D structure (ii). Scale bar: 100 μm.

Figure 2—figure supplement 1.. Cell viability test (%) in three cell lines (FaDu cell, MHCC97H cell and A549 cell) incubated with IBS440 or compound IBS224 (5–50 μM) for different incubation time.

The error bar is the mean standard deviation of six separate measurements.

Figure 2—figure supplement 2.. Fluorescence imaging of cancerous cells (MHCC97H, FaDu and A549 cells) and noncancerous cells (RAW264.7 and A7r5 cells).

They were incubated with a commercial lipophilic mitochondrial dye Mito-Tracker Green (1 μM) for 20 min. Blue channel (λ_ex = 405 nm, λ_em = 430–480 nm). Green channel (λ_ex = 488 nm, λ_em = 500–550 nm). Scale bar: 25 μm.

Figure 2—figure supplement 3.. Fluorescence imaging of hepatocellular tumor and normal tissues.

They were incubated with a commercial lipophilic mitochondrial dye Mito-Tracker Green (1 μM) for 20 min. Blue channel (λ_ex = 405 nm, λ_em = 430–480 nm). Green channel (λ_ex = 488 nm, λ_em = 500–550 nm).

Figure 2—figure supplement 4.. HIF-1α expression level in three different cell lines under normoxia (20 % O₂) and hypoxia (2 % O₂) conditions for 12 hr or 24 hr by immunoblotting.

β-actin were served as loading control.

Figure 2—figure supplement 5.. Fluorescence imaging of hypoxia in live cells.

(A) Cells were cultured under normoxic (20 % O₂) for 12 hr and then treated with IBS440. (B) Cells were cultured under hypoxic (2 % O₂) conditions for 12 hr and then treated with IBS440. Blue channel (λ_ex = 405 nm, λ_em = 430–480 nm). Green channel (λ_ex = 488 nm, λ_em = 500–550 nm). Scale bar: 25 μm.

Figure 2—figure supplement 6.. Fluorescence imaging of viscosity in cancer cells (MHCC97H, FaDu and A549 cells) and normal cells (RAW264.7 and A7r5 cells) treated with IBS440 (5 μM) for 20 min.

Blue channel (λ_ex = 405 nm, λ_em = 430–480 nm). Red channel (λ_ex = 488 nm, λ_em = 580–630 nm). Scale bar: 25 μm.

Figure 2—figure supplement 7.. Visualization of the normal tissues of mice (A).

heart, B. liver, C. spleen, D. lung, E. kidney, F muscle, G. fat, H. brain and MHCC97H tumor tissue (I) in confocal fluorescence 3D reconstruction. All tissues were incubated with IBS440 (10 μM) for 20 min. Blue channel (λ_ex = 405 nm, λ_em = 430–480 nm). Red channel (λ_ex = 488 nm, λ_em = 580–630 nm). Scale bar: 50 μm.

Figure 3.. In vivo and ex vivo fluorescence imaging of mouse tumors.

(A) In vivo fluorescence imaging of the tumor-bearing mice after stained with IBS440. (B) Ex vivo fluorescence imaging of the major organs (heart, liver, spleen, lung, and kidney) and tumor tissues of tumor-bearing mice. (C) Hematoxylin and eosin microscopic imaging of the resected tumor tissues. Scale bar: 100 μm.

Figure 3—figure supplement 1.. Hematoxylin and eosin staining of the major organ of the tumor-bearing mice.

Figure 4.. Photograph imaging, fluorescence imaging, and hematoxylin and eosin microscopic imaging of nine pairs of cancerous/noncancerous tissues derived from nine individual hepatocellular cancer patients.

They were incubated with IBS440 (10 μM) for 20 min and taken imaging. Scale bar: 100 μm.

Figure 4—figure supplement 1.. The scatter plot represented the maximal gray-scales signal intensities of nine pairs of liver tissue samples in nitroreductase and viscosity detection channels.

The threshold (160.3,164.3) was computed by K-means clustering algorithm.

Figure 5.. Photograph imaging, fluorescence imaging, and hematoxylin and eosin microscopic imaging of five pairs of cancerous/noncancerous tissues derived from five individual lung cancer patients.

They were incubated with IBS440 (10 μM) for 20 min and taken imaging. Scale bar: 100 μm.

Figure 5—figure supplement 1.. The scatter plot represented the maximal gray-scales signal intensities of five pairs of lung tissue samples in nitroreductase and viscosity detection channels.

The threshold (164.4,165.8) was computed by K-means clustering algorithm.

Figure 5—figure supplement 2.. Photograph imaging, fluorescence imaging, and hematoxylin and eosin microscopic imaging of five pairs of cancerous/noncancerous tissues derived from five individual oral cancer patients.

They were incubated with IBS440 (10 μM) for 20 min and taken imaging. Scale bar: 100 μm.

Figure 5—figure supplement 3.. Photograph imaging, fluorescence imaging, and hematoxylin and eosin microscopic imaging of six pairs of cancerous/noncancerous tissues derived from six individual renal cancer patients.

They were incubated with IBS440 (10 μM) for 20 min and taken imaging. Scale bar: 100 μm.

Figure 5—figure supplement 4.. The scatter plot represented the maximal gray-scales signal intensities of five pairs of oral tissue samples in nitroreductase and viscosity detection channels.

The threshold (164.7,165.3) was computed by K-means clustering algorithm.

Figure 5—figure supplement 5.. The scatter plot represented the maximal gray-scales signal intensities of six pairs of renal tissue samples in nitroreductase and viscosity detection channels.

The threshold (163.5, 167.1) was computed by K-means clustering algorithm.

Figure 6.. Fluorescence pre-screening of 35 pieces of resection margin tissues derived from one hepatocellular cancer patient.

(A) Photograph imaging and fluorescence imaging of resected tissues. (B) Clustering analysis using K-means algorithm on the maximal gray-scales signal intensities of each tissue sample in nitroreductase and viscosity detection channels. (C) Hematoxylin and eosin microscopic imaging of the representative liver samples. Scale bar: 50 μm. (D) The correlation analysis of liver tissue samples between fluorescence signals and hematoxylin and eosin staining results. Pre-screening: (++): definitely positive; (+): suspiciously positive; (-): negative. Paraffin H&E Positive: P; Paraffin H&E Negative: N.

Figure 6—figure supplement 1.. Hematoxylin and eosin staining results of 35 pieces of resected margin tissues derived from one hepatocellular cancer patient.

Figure 7.. Fluorescence pre-screening of 16 pieces of resection margin tissues derived from one lung cancer patient.

(A) Photograph imaging and fluorescence imaging of resected tissues. (B) Clustering analysis using K-means algorithm on the maximal gray-scales signal intensities of each tissue sample in nitroreductase and viscosity detection channels. (C) Hematoxylin and eosin microscopic imaging of the representative lung samples. Scale bar: 50 μm. (D) The correlation analysis of lung tissue samples between fluorescence signals and hematoxylin and eosin staining results. Pre-screening: (++): definitely positive; (+): suspiciously positive; (-): negative. Paraffin H&E Positive: P; Paraffin H&E Negative: N.

Figure 7—figure supplement 1.. Hematoxylin and eosin staining results of 16 pieces of resected margin tissues derived from one lung cancer patient.

Figure 8.. Schematic presentation of intraoperative pathological assessment.

(A) The current strategy used during a tumor operation. Based on surgeon’s gross examination, multiple resected tissues are prioritized for frozen-section analysis. Occasionally, an extended resection is indicated with positive feedback to avoid unnecessary re-operations. In principle, frozen section analysis takes minimally 30 min to 1 hr; while in practice, such procedure requires more time to complete due to a large number of samples received in overloaded pathological laboratories. (B) Fluorescence-based pre-screening strategy described in this paper. It allows the pre-screening of tumor involvement with high sensitivity by incubating multiple resected ex vivo tissues with chemical fluorescent agent for minutes and then imaged. (i) Negative result is rapidly conveyed to the operation room to close the case with 20 min. (ii) Positive ones are prioritized for further frozen-section double-checks and reviews.

Scheme 1.. Synthesis of IBS224 and IBS440.

Appendix 1—figure 1.. ¹³C-NMR (DMSO-d6) spectrum of IBS440.

Appendix 1—figure 2.. ¹H-NMR (DMSO-d6) spectrum of IBS440.

Appendix 1—figure 3.. ¹³C-NMR (DMSO-d6) spectrum of IBS224.

Appendix 1—figure 4.. ¹H-NMR (CDCl₃) spectrum of IBS224.

Appendix 1—figure 5.. MS spectrum of IBS224.

Appendix 1—figure 6.. MS spectrum of IBS440.