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.
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:
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.
Breast tumor microenvironment is composed of tumor cells, tumor-related cells, blood vessels and a series of extracellular matrix fibers. Tumor-infiltrating lymphocytes (Tils) in the microenvironment can directly or indirectly influence other components in the microenvironment, thus promoting the occurrence and development of tumors. Multiphoton microscopy (MPM) is based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). And it does not require the use of exogenous probes or staining of tissue. In this study, large-size images with subcellular resolution of the breast tumor tissue was performed using MPM. The results showed that the MPM could clearly distinguish intraepithelial Tils (iTils) and stromal compartments Tils (sTils) by comparing the signal strength and morphological difference. It demonstrated that MPM could be used as a means of pathological diagnosis and in clinical application.
Macrophages and collagen fibers are important components of the tumor microenvironment. Macrophages would secrete extracellular matrix degrading enzymes to degrade collagen, which is conducive to the formation of local infiltration and distant metastasis of tumor cells. During tumor progression, macrophages are actively recruited into tumors where they alter the tumor microenvironment to accelerate tumor progression. A high density of these tumor-associated macrophages may correlates with poor prognosis. In this work, multiphoton microscopy (MPM) using two-photon excited fluorescence combined with second harmonic generation imaging was used to monitor the changes in collagen fibers around macrophages. The experimental results show that this microscope has the ability to directly monitor the collagen changes induced by the invasion of macrophages in the absence of labels. Moreover, collagen content around macrophages in the matrix can be quantitatively calculated by image processing, and quantitative results show that the collagen content in the tumor microenvironment will significantly reduce with the appearance of macrophages. Therefore, MPM has the potential to be used as a new auxiliary tool for pathologists to quickly and effectively evaluate collagen changes in breast tumor microenvironment.
Fibroblasts are important players in proliferation, invasion, migration and metastasis of cancer cells. Until now, histological methods such as hematoxylin and eosin (H and E) staining are still a gold standard to assess fibroblasts in traditional histopathology. Multiphoton microscopy (MPM) has become a key technology for biological tissues imaging without staining at the cellular level. In this study, MPM based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was used to identify the fibroblasts in breast cancer tissue. Normal fibroblasts with spindle shape and abnormal fibroblasts with stellate shape could be clearly obtained by MPM.
Perineural invasion (PNI) is the process of neoplastic invasion of nerves. The nerve sheath includes three layers, that is, the epineurium, perineurium and endoneurium from the outside to the inside. Any one of them is invaded by malignant tumor cells, or more than 33% of the circumference of the nerve should be surrounded by tumor cells, then it can be defined as perineural invasion (PNI). Perineural invasion is another way of tumor cell metastasis, which not only indicates the poor prognosis of patients but also indicates the decline of survival rate. In this study, multiphoton microscopy (MPM) which is based on the intrinsic nonlinear optical signals—two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was applied to identify the perineural invasion in breast cancer patients, including the recognition of normal nerves and tumor cells infiltrating into the nerve. Our aim is to determine the potential feasibility of using this microscopy to identify tumor invasion around the nerves. The results mean that it is feasible to detect perineural invasion with multiphoton microscopy, and to provide a new auxiliary tool for pathologists to quickly and effectively diagnose perineural invasion.
Macrophages are at the center of the invasion and play a protumoral role in the tumor microenvironment. They can facilitate angiogenesis, extracellular matrix remodeling, invasion and migration of cancer cells, while inhibit the antitumoral immune surveillance. Tumor-associated macrophages are a major component of malignant tumors, which can stimulate the progress of tumors. In many human cancers, the presence of macrophages in tumor microenvironment has been correlated with unfavorable prognosis. Therefore, visual diagnosis of macrophages in tumor microenvironment is great significance for us to comprehend the occurrence and progress of tumor. Multiphoton microscopy (MPM) with subcellular resolution based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) is very suitable for real-time detecting morphological and structural changes in biological tissues without tissue staining and exogenous probe molecule. In this study, we describe the use of label-free MPM for analyzing the intratumoral distribution and morphological changes of macrophages in breast tumor microenvironment. Our results indicated that MPM can accurately identify macrophages in breast tumor tissues. MPM images were well consistent hematoxylin and eosin (H&E) stained images. This work would provide the basis for further quantifying these changes using MPM. With the development of miniaturized imaging devices, MPM may be a promising imaging technique for clinicians to study various structural features in tumor microenvironment.
Pleomorphic invasive lobular carcinoma (pILC) is a distinct morphological variant of invasive lobular carcinoma (ILC), which is more aggressive than classic invasive lobular carcinoma (cILC) with worse overall prognosis. It is critical to distinguish between pILC and cILC for management and treatment of patients. Multiphoton microscopy (MPM),based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), produces subcellular resolution images to show detailed information on structure and cell morphology of tissue sections without staining. In this work, MPM was introduced for label-free identification of normal breast lobules, pILC and cILC, which is consistent with haematoxylin and eosin (H&E) staining. Furthermore, analysis of nuclear area is used to quantitatively reflect their cytological characteristic. These results demonstrate that, MPM has the ability to distinguish between normal breast lobules, cILC, and pILC by histopathological features, which can provide additional indicators to determine adequate treatment strategies of patients with pILC for pathologists and surgeons. In the future, MPM may be used as a diagnostic tool of lobular cancer into routine clinical use.
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