Visualization of Alterations in Tumor Microenvironment of Esophageal Squamous Cell Carcinoma Caused by Neoadjuvant Therapy Based on the Combination of Multiphoton Microscopy and Digital Pathological Imaging Analysis

The impact of neoadjuvant PD-1 blockade in combination with chemotherapy on the tumor microenvironment of esophageal cancer has not been fully understood. In this paper, multiphoton microscopy and digital pathological imaging analysis were conducted to examine the treatment-driven microenvironment changes among 51 samples with only underwent surgical resection (SG), 49 samples with good pathological reactions (GR) and 34 samples with poor pathological reactions (PR). The results showed that treatment caused the elastic fiber to remodel in the arterioles and led the vascular walls to thicken, which might be related to drug delivery but not to the therapeutic effect. After neoadjuvant therapy, the collagen fibers in the stromal regions immediately adjacent to the tumor mass were degraded, which was a good response to treatment. In the GR group but not the PR group, although neoadjuvant therapy caused significant regression of the tumor mass, some more aggressive subclones of tumor cells were screened out, characterized by rounder nuclear shape, and smaller smoothness, with looser nuclear structure and enlarged nucleoli. In addition, stromal fibrosis also can be observed in the GR group, manifested by the more deposition of collagen fibers, longer and wider collagen fibers, and more orderly arrangement of collagen fibers existing in the stromal regions at 200 μm from the tumor mass after treatment. Correlation analysis showed that the degradation of collagen fibers adjacent to the tumor mass, nuclear morphology of the residual cells, and stromal fibrosis were significantly correlated with therapeutic efficacy.