Aim: To evaluate the feasibility of using multiphoton microscopy (MPM) to assess a tumor regression grading (TRG) system.
Methods: Fresh specimens from seven patients with colorectal carcinoma undergoing neoadjuvant radiochemotherapy at the Fujian Medical University Union Hospital were obtained immediately after proctectomy. Specimens were serially sectioned (10 µm thickness) and used for MPM or stained with hematoxylin and eosin for comparison. Sections were imaged by MPM using 810 nm excitation, and images were collected in two wavelength channels corresponding to second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) signals. The ratio of these signal intensities was used to distinguish fibrosis from normal mucosal and serosal tissues.
Results: TRG of specimens assessed by MPM were in complete agreement with histologic grading performed by a consulting pathologist. SHG and TPEF images clearly revealed collagen fibers and fragmented elastic fibers in the muscularis propria specimens following neoadjuvant radiochemotherapy. Additionally, blood vessel hyperplasia was observed as thickening and fibrosis of the intima and media, which was accompanied by minimal inflammatory cell infiltration. Furthermore, the SHG/TPEF ratio in stromal fibrosis (4.15 ± 0.58) was significantly higher than those in the normal submucosal (2.31 ± 0.52) and serosal (1.47 ± 0.10) tissues (P < 0.001 for both). Analysis of emission spectra from cancerous tumor cells revealed two peaks corresponding to nicotinamide adenine dinucleotide hydrogen and flavin adenine dinucleotide signals; the ratio of these values was 1.19 ± 0.02, which is close to a normal metabolic state.
Conclusion: MPM can be used to perform real-time diagnosis of tumor response after neoadjuvant treatment, and can be applied to evaluate TRG.
Keywords: Multiphoton microscopy; Neoadjuvant treatment; Second-harmonic generation; Tumor regression grading; Two-photon excited fluorescence.