Breast cancer (BC) is a significant health concern for women, with its classification into multiple stages contingent upon the dimensions of the tumor, the extent of lymph node involvement, and the presence of distant metastasis. Despite the application of uniform treatment protocols to cases of similar staging, the outcomes are subject to variability due to the inherent heterogeneity of the disease, highlighting an urgent need for further investigation. The tumor microenvironment (TME) plays a pivotal role in tumor progression and metastasis, with collagen fibers emerging as a critical component of the TME that is implicated in these processes. However, the precise interplay between collagen fibers and tumor staging remains to be elucidated. Advancements in multiphoton microscopy (MPM), which capitalizes on nonlinear optical phenomena, have yielded impressive imaging capabilities, facilitating the real-time visualization of tumor histology and the quantification of metabolic activity within tumors. Recent studies have underscored the intricate relationship between collagen fibers and the dynamics of tumor evolution.
In this study, we utilized multiphoton microscopy to image three distinct tumor-associated collagen signatures (TACS) at the invasive front of the tumor. We then used MATLAB to extract the corresponding collagen morphological features and analyzed their correlation with clinical staging. Our results revealed significant changes in the morphological features of collagen fibers in TACS across different stages of BC at the tumor invasion front. Notably, the proportionate area and number of collagen fibers were found to be inversely correlated with the clinical staging risk group of the disease. Our findings offer new perspectives for the clinical staging of BC, providing valuable insights that may enhance the predictive accuracy of disease progression and prognostic outcomes.
Ductal carcinoma in situ (DCIS) accounts for approximately 20% of all breast cancer. DCIS is a form of breast cancer that is restricted to the ducts and has not invaded surrounding breast tissue or spread to lymph nodes or other parts of the body. The grades of DCIS are classified as low, intermediate, and high, based on cytonuclear features, and high-grade DCIS has a higher risk of progressing into invasive ductal carcinoma (IDC). The collagen fibers are an important component of the tumor microenvironment (TME) in DCIS and play an important role in tumor formation and progression. Multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) can monitor the morphological changes of collagen fibers around DCIS. SHG is currently considered the gold standard for visualizing collagen fibers and has been widely employed in various cancer-related studies of collagen fibers. Our investigation employed MPM imaging of breast tissue to observe the differences in collagen fibers within three distinct grades of DCIS. Through image processing, we were able to quantify various attributes of collagen fibers enveloping DCIS lesions of varying grades. The study found that collagen fibers surrounding low-grade DCIS were denser and exhibited more sinuous shapes, whereas collagen fibers around intermediate and high-grade DCIS lesions were less dense and exhibited a more organized arrangement. The study suggests that MPM imaging is a powerful tool for investigating the microenvironment of DCIS and may provide valuable information for predicting disease progression and prognosis.
The invasion and metastasis of tumor cells are not only related to the tumor cells themselves, but also closely intertwined with other components of the tumor microenvironment. The connective cells, including fibroblasts and myofibroblasts, play a crucial role in determining the remodeling of collagen fibers in the tumor microenvironment during the process of tumor invasion, which determinant of tumor migration. In this study, we used the multi-photon imaging system to identify two different prognostic tumor-associated collagen signatures (TACS4 and TACS6). Then, the multi-photon images were used to co-locate with HE images, and open-source convolutional neural network Hover-Net was used to segment, classify and quantifythe nuclear feature. Our results showed that the connective cells are opposed to tumor cells and do not mix with tumor cells in TACS4, while in TACS6, they are scattered promiscuously with tumor cells. The spatial distribution of connected cells in TACS4 is denser than that in TACS6. These differences in spatial distribution of connected cells may potentially have different prognostic information.
Necrosis is a form of cell death which is histologically characterized by homogeneous clusters and sheets of dead cells. Although several studies have indicated that the presence of tumor necrosis in pathological specimens may provide adverse prognostic information in solid tumor, the mechanism of necrosis is still unclear. Based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG), multiphoton microscopy (MPM) is commonly used to monitor the morphological changes of biological tissues. In this study, we performed MPM imaging of the breast tissue and found that MPM can be used to rapidly classify the early and late-stage tumor necrosis in invasive breast cancer according to the changes of intracellular proteins. It demonstrated that MPM may provide a new assistant tool for pathologists to quickly and effectively classify the early and late-stage tumor necrosis.
Necrosis is a form of cell death caused by an external factor of the cell, such as hypoxia. It is usually associated with rapidly growing malignancies in the breast, colon, brain, lungs, kidney, and pancreas. Multiphoton microscopy (MPM) based on intrinsic nonlinear optical signals were used to monitor the morphological changes of biological tissues and identify tumor tissue necrosis in breast cancer patients, as well as surrounding tumor cells and collagen. In this study, we performed MPM imaging of the breast tissue and found that there were two types of necrosis in the breast tissue, namely intraluminal necrosis and interstitial necrosis. Different types of necrosis may have different effects on the prognosis. It means MPM may provide a new assistant tool for pathologists to quickly and effectively identify tumor necrosis. It is expected that rapid identification of tumor necrotic areas can provide prognostic information for early recurrence or death, thus helping to diagnose and treat cancer.
The presence of blood vessel invasion (BVI) in breast tumor microenvironment has been recognized as an unfavorable prognostic factor. Invasion of cancer cells into vessels is one of the critical steps for metastasis. Therefore, visualization of BVI is vital for comprehending the progress of tumor. Multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) can monitor morphological changes in biological tissues. In this study, we found significant differences in morphology between normal breast blood vessel and abnormal blood vessel encountered with tumor invasion using label-free MPM. Our study demonstrated that MPM has the ability to not only identify BVI in breast tumor environment but also reveal the morphological changes of breast blood vessel. By comparing with the hematoxylin and eosin (HE) stained image, it was confirmed that MPM provides a new assistant tool for pathologists to identify BVI effectively. Keywords:
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