 by heartbeat and respiratory movement. CLSM imaging was performed using the FV1000 confocal laser microscope (Olympus Corp, Tokyo, Japan) with two-laser diodes (473 nm and 559 nm). A 4 × (0.20 numerical aperture immersion) objective lens and 20 × (0.95 numerical aperture immersion) objective lens (Olympus) were used. 800 × 800 pixels and 1.0 µm z steps were scanned, which took 1-2 s per section. Scanning and image acquisition were controlled by Fluoview software (Olympus). (C) The schematic diagram shows the method of longitudinal intravital CLSM imaging of FUCCI-expressing gastric cancer cells growing in the liver using an abdominal skin-flap window. The abdominal skin-flap window method enables ten laparotomies during 150 days.

Skin-Window System
The stabilization of a target organ is one of the most indispensable steps for single-cell live imaging with a confocal laser microscope (Olympus Corp, Tokyo, Japan). Cross-sections are needed for single-cell imaging. Chittajallu et al. [42] demonstrated that a dorsal skin-fold-chamber enabled visualization of drug response of FUCCI-expressing HT1080 soft-tissue sarcoma. Ritsma et al. [43,44] also demonstrated that an abdominal window using a coverslip enabled visualization of the biological behavior of Colo26 mouse colon cancer cells in the liver of a mouse. Both window methods are useful and convenient for intravital single-cell imaging. However, a coverslip window limits the area for imaging [45], and the glass pressed on the cancer cells may affect their behavior in vivo.

Minimal Organ-Stabilization System Using Styrofoam and Pins
Therefore, Yano et al. [46] developed a convenient, minimally-invasive organ stabilization system using a styrofoam board, tape, pins, and a cover glass over the liver of a mouse ( Figure 1B). This system enabled laser-scanning microscopy imaging of cancer cells in the liver of the live mouse without vibration caused by heartbeat and respiratory movement of a mouse under anesthesia. This system also enabled tracing the same location and the same cancer cells at the single-cell level in a live mouse, even with repeat laparotomies ( Figure 1C).

Cell-Cycle Distribution within a Tumor
Monitoring cell cycle dynamics during tumor growth is very important for improving our understanding of cancer. Yano et al. [46] demonstrated intravital real-time monitoring of orthotopic FUCCI-expressing tumors in the liver of live mice during tumor growth ( Figure 2A). Nascent tumors (7 days after inoculation) consisted of a majority of proliferating cancer cells ( Figure  Chittajallu et al. [42] clearly visualized G 1, late G 1 /early S, S/G 2, and mitosis in vivo combining a nuclear-morphology reporter (histone H2B-CFP) and the FUCCI system. Chittajallu et al. [42] could image for only two weeks, since tumor growth was limited to the depth of the chamber.  [46] with the permission of Taylor and Francis).

Established Tumors Consist of a Vast Majority of Quiescent Cancer Cells
Solid tumors are well known to be heterogeneous, which makes it difficult to understand cancer biology [47,48]. Our abdominal skin-flap method enabled reconstruction of three-dimensional images ( Figure 3A) [46]. Yano et al. [46] showed that a nascent tumor (7 days after inoculation) consisted of cells that were mostly (90%) in S/G2/M ( Figure 3B,E). In contrast, a medium-sized established tumor (21 days after inoculation) had regions of both G2/M cells (65 to 30%) and G0/G1

Established Tumors Consist of a Vast Majority of Quiescent Cancer Cells
Solid tumors are well known to be heterogeneous, which makes it difficult to understand cancer biology [47,48]. Our abdominal skin-flap method enabled reconstruction of three-dimensional images ( Figure 3A) [46]. Yano et al. [46] showed that a nascent tumor (7 days after inoculation) consisted of

Intravital Orthotopic FUCCI Imaging Reveals the Relationship between Cell Cycle Phase of Cancer Cells and the Juxtaposition of Tumor Blood Vessels
It is also important to investigate the relationship between cancer cells and tumor blood vessels [49]. Kienast et al. [50] demonstrated intravital single-cell imaging of multistep-brain metastasis of cancer cells using a combination of a multiphoton laser microscope and a cranial window. Kienast et al. [50] showed that cancer cells are initially arrested at a blood vessel branch, when they extravasted, and then grew at the perivascular position with angiogenesis. To investigate the cell-cycle position of cancer cells near and far from vessels, transgenic mice with nestin-promoter driving GFP (nestindriven GFP [ND-GFP]) were used to label nascent blood vessels with GFP [24,25] (Figure 4A,B). Yano

Intravital Orthotopic FUCCI Imaging Reveals the Relationship between Cell Cycle Phase of Cancer Cells and the Juxtaposition of Tumor Blood Vessels
It is also important to investigate the relationship between cancer cells and tumor blood vessels [49]. Kienast et al. [50] demonstrated intravital single-cell imaging of multistep-brain metastasis of cancer cells using a combination of a multiphoton laser microscope and a cranial window. Kienast et al. [50] showed that cancer cells are initially arrested at a blood vessel branch, when they extravasted, and then grew at the perivascular position with angiogenesis. To investigate the cell-cycle position of cancer cells near and far from vessels, transgenic mice with nestin-promoter driving GFP (nestin-driven GFP [ND-GFP]) were used to label nascent blood vessels with GFP [24,25] (Figure 4A,B). Yano et al. [46,51] also reported that proliferating cancer cells exist only near tumor vessels or the tumor surface; in contrast, cancer cells far from vessels or in the center of tumors are quiescent ( Figure 4C,D).
Cells 2018, 7, x FOR PEER REVIEW 6 of 14 et al. [46,51] also reported that proliferating cancer cells exist only near tumor vessels or the tumor surface; in contrast, cancer cells far from vessels or in the center of tumors are quiescent ( Figure 4C,D).

Intravital Orthotopic FUCCI Imaging Reveals that Quiescent Cancer Cells are Resistant to Conventional Chemotherapy
Resistance to conventional chemotherapy is an important clinical problem that results in tumor recurrence and poor prognosis in cancer patients. Most currently-used anticancer agents are effective only on cycling cancer cells [52] and have no effect on quiescent/dormant cancer cells [53][54][55][56][57][58]. To overcome chemoresistance, intravital imaging was performed [59][60][61][62][63]. The cell-cycle phase determines the cancer-cells response to chemotherapy [64][65][66][67][68][69][70]. Yano et al. [46] demonstrated by FUCCI imaging that currently-used cytotoxic anticancer agents are ineffective for solid tumors, since they comprise mostly non-cycling, quiescent cancer cells ( Figure 5). Chittajallu et al. [42] confirmed our results using intravital single-cell level FUCCI imaging in nude mice. Intravital FUCCI imaging determined that the cell cycle phase distribution of cancer cells in vivo differed from 2D in vitro cell culture.

Intravital Orthotopic FUCCI Imaging Reveals that Quiescent Cancer Cells are Resistant to Conventional Chemotherapy
Resistance to conventional chemotherapy is an important clinical problem that results in tumor recurrence and poor prognosis in cancer patients. Most currently-used anticancer agents are effective only on cycling cancer cells [52] and have no effect on quiescent/dormant cancer cells [53][54][55][56][57][58]. To overcome chemoresistance, intravital imaging was performed [59][60][61][62][63]. The cell-cycle phase determines the cancer-cells response to chemotherapy [64][65][66][67][68][69][70]. Yano et al. [46] demonstrated by FUCCI imaging that currently-used cytotoxic anticancer agents are ineffective for solid tumors, since they comprise mostly non-cycling, quiescent cancer cells ( Figure 5). Chittajallu et al. [42] confirmed our results using intravital single-cell level FUCCI imaging in nude mice. Intravital FUCCI imaging determined that the cell cycle phase distribution of cancer cells in vivo differed from 2D in vitro cell culture.

Intravital Orthotopic FUCCI Imaging Unveils the Adverse Effect of Irradiation Therapy
Radiotherapy is an important part of breast cancer treatment. Radiation therapy is well known to kill cancer cells, sometimes eradicate tumors, and then reduce the recurrence rate and prolong overall survival. However, radiation is recognized to induce chronic inflammation, which increases the risk of developing several types of cancer, including breast cancer. Bouchard et al. [71] reported that pre-irradiation of the mammary gland of nude mice promoted the migration of D2A1 FUCCI-expressing breast cancer cells using intravital imaging. Intravital FUCCI imaging showed that pre