Tumor microenvironment study is of huge significance in terms of cancer invasiveness observation and characterization. Traditional fluorescence microscope imaging techniques, while highlighting specific features of tumor tissue by labeling, like nucleus, lose many other molecular information that can indicate some biological structures found in tumor microenvironments. Our label-free multi-modal multi-photon imaging system, aiming for the intrinsic optical response of tumor tissue, manage to visualize various kinds of biological features in tumor microenvironments, which are either absent or covered in traditional labeled fluorescent imaging.
Macroscopic events in the tumor microenvironment identified by multicontrast imaging. Each image contains 380 × 380 pixels with 0.5-mm pixel size. (A) Natural angiogenesis (An). (B) Lymphangiogenesis (LA) near a blood vessel (BV), which can be differentiated from the LA by the presence of c(3)R2850 contrast. Some native cells (red arrows) and non-native cells (blue arrows) are also marked. (C) Degraded basement membrane (DBM) from lymphatic vessels or mammary ducts/lobules. Some nonnative cells (blue arrows) are also marked. (D) Collagen production and fibroblast activation demonstrated by paralleled formation of fibroblasts, collagen fibers, and angiogenic vessels (area marked by broken magenta line). A fibrosis feature of dense collagen is also identified. (E) Non-native cell recruitment. Tumor cells (red arrows), nontumor native stromal cells (magenta arrows), and non-native cells (blue arrows) can be discriminated against each other according to c(3)THG contrast and c(3)THG-AF(2)–covisible tumor-associated extracellular vesicles.
Large-area of co-localized single-modality multiphoton images revealing vital signatures of local tumor invasion in a week 8 tumor. Tumor cells are visible by 2PAF, 3PAF, and R3050 (top of the images), and form a clear boundary separate from the stroma below. Collective tumor cell invasion (CTCI) is reflected by a protrusion of tumor cells or cell debris visible by R3050/2PAF/3PAF, angiogenesis (An) by a protrusion of 3PAF-visible vesicles, tumor-associated collagen structure-3 (TACS-3) by a protrusion of collagen fibers, and lymphangiogenesis (LA) by a protrusion of elastin fibers. The four protrusions infiltrate from the tumor side into the stroma in parallel fashion so that local tumor invasion is promoted.
Furthermore, our label-free imaging systems preserve living tissue structures that would lose vitality or even be washed out during staining and rinsing routine of traditional fluorescent imaging. Especially, tumor-associated extracellular vesicles (TEV) are visualized ex vivo for human breast tumor tissue and in vivo for rat mammary tumor tissue using our imaging setup, which are important indicators to cancer invasiveness. Using bench-top imaging system, we are able to observe the distribution and motion of TEV by real-time imaging, and also quantify the concentration of them in order to correlate with pathological results.
AF(3) image of unperturbed mammary tumor from a carcinogen-injected rat. Several lipid vesicles and adipocytes from the χ(3) R2850 image also appear. One marked area reveals several FAD-rich vesicles. Disorganized fluorescent vesicles in region of dense collagen and region near angiogenesis are co-visible in the χ(3) THG image. The reason why most fluorescent vesicles are distributed in tubular formations rather than the disorganized formations of the marked areas (yellow rectangles) can be explained by the composite χ(3) R2850/AF(3) image analysis.
Tu H, Liu Y, Marjanovic M, Chaney EJ, You S, Zhao Y, Boppart SA. Concurrence of extracellular vesicle enrichment and metabolic switch visualized label-free in the tumor microenvironment. Scieince Advances, 3(1): e1600675, 2017.
Tu H, Liu Y, Turchinovich D, Marjanovic M, Lyngsø JK, Lægsgaard J, Chaney EJ, Zhao Y, You S, Wilson WL, Xu B, Dantus M, Boppart SA. Stain-free histopathology by programmable supercontinuum pulses. Nature Photonics, 10: 534-540, 2016.
|You S, Tu H, Zhao Y, Liu Y, Chaney EJ, Marjanovic M, Boppart SA, Raman spectroscopic analysis reveals abnormal fatty acid composition in tumor micro- and macroenvironments in human breast and rat mammary cancer. Scientific Reports, 6: 32922, 2016.|
However, since TEVs are highly vital. It is still unknown how their vitality and biological properties would vary with time after incision. Therefore, apart from the powerful bench-top imaging system, a portable label-free multi-modal multi-photon imaging system is built and used to collect images in operating room right after tissue excision, which is believed to preserve the TEV vitality to the maximum extent. The photograph shows this portable imaging system.
This new portable system has has been used to collect many interesting images and also effectively visualize TEVs.
Preliminary results show a clear difference between breast tumor tissue and healthy breast reduction tissue in terms of extracelluar vesicle concentration. We believe more intraoperative data from operating room can further help find valid correlation with pathological results.