Real-Time Determination of the Cell-Cycle Position of Individual Cells within Live Tumors Using FUCCI Cell-Cycle Imaging

Most cytotoxic agents have limited efficacy for solid cancers. Cell-cycle phase analysis at the single-cell level in solid tumors has shown that the majority of cancer cells in tumors is not cycling and is therefore resistant to cytotoxic chemotherapy. Intravital cell-cycle imaging within tumors demonstrated the cell-cycle position and distribution of cancer cells within a tumor, and cell-cycle dynamics during chemotherapy. Understanding cell-cycle dynamics within tumors should provide important insights into novel treatment strategies.

Our laboratory has developed in vivo intravital imaging techniques of tumors growing orthotopically in the brain, liver, other organs of mice, and circulating cancer cells in blood vessels and lymphatic ducts during metastasis [1,2,13,17,20,21]. In vivo intravital imaging also demonstrated the difference in behavior of cancer cells at the orthotopic and subcutaneous sites in real-time [31][32][33][34][35].
In this review, we focus on intravital imaging with the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging of individual cells in tumors. Intravital FUCCI-imaging provides a new paradigm of cell-cycle-based treatment of solid cancers.
In this review, we focus on intravital imaging with the fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging of individual cells in tumors. Intravital FUCCI-imaging provides a new paradigm of cell-cycle-based treatment of solid cancers.

FUCCI (Fluorescence Ubiquitination Cell Cycle Indicator) Repoters
Sakaue-Sawano et al. [36] have reported that the cell-cycle phase in viable cells can be identified using FUCCI. Red nuclei in FUCCI-expressing cells (FUCCI-red) indicate the quiescent G0/G1 phase [37], green nuclei in FUCCI-expressing cells (FUCCI-green) indicate the proliferating late-S/G2/M phase, and yellow nuclei in FUCCI-expressing cells (FUCCI-yellow) indicate the early S phase ( Figure  1A). Intravital FUCCI imaging enabled visualization of cell-cycle dynamics of individual cancer cells within tumors. Moreover, FUCCI imaging also visualized cell-cycle dynamics within tumors during chemotherapy. Sakaue-Sawano et al. also demonstrated that the FUCCI 2 that emits red (mCherry) and green (mVenous) fluorescence and provides better color contrast than original FUCCI [38]. Bajar et al. modified the original FUCCI in order to visualize four cell-cycle phases [39]. Furthermore, Sakaue-Sawano et al. [40] recently developed two new FUCCI; FUCCI (CA); and FUCCI (SCA). FUCCI (CA) produced a sharp triple-color distinct separation of G1, S, and G2, while FUCCI (SCA) permitted a two-color readout of G1 and S/G2 phases. Oki et al. [41] modified FUCCI, which enabled the distinction between G0 and G1. A bright fluorescence signal is the most important for intravital imaging in vivo. Moreover, these fluorescent proteins should not be easily bleached. The original FUCCI for intravital real-time in vivo imaging of cancer cells at the single cell level met these criteria. The schematic diagram shows the method of longitudinal intravital confocal laser scanning microscopy (CLSM) imaging of FUCCI-expressing gastric-cancer cells growing in the liver using a skin-flap window. All animal procedures were performed under anesthesia using s.c. administration of a ketamine mixture (10 μl ketamine HCl, 7.6 μl xylazine, 2.4 μL acepromazine maleate, and 10 μL PBS). FUCCI-expressing MKN45 cells were harvested by brief trypsinization. Single-cell suspensions were prepared at a final concentration of 2 × 10 5 cells/5 μl Matrigel (BD). After laparotomy, FUCCI-expressing cancer cells were subserosally injected directly into the left lobe of the liver using a 31 gauge needle. After cancer-cell implantation, the abdominal wall of the mice was closed with 6-0 sutures. Mice were anesthetized as described above and placed on a custom-designed (A) FUCCI-expressing MKN45 gastric cancer cells in G 0 /G 1 , S, or G 2 /M phases are red, yellow, or green, respectively. (B) The schematic diagram shows the method of longitudinal intravital confocal laser scanning microscopy (CLSM) imaging of FUCCI-expressing gastric-cancer cells growing in the liver using a skin-flap window. All animal procedures were performed under anesthesia using s.c. administration of a ketamine mixture (10 µl ketamine HCl, 7.6 µl xylazine, 2.4 µL acepromazine maleate, and 10 µL PBS). FUCCI-expressing MKN45 cells were harvested by brief trypsinization. Single-cell suspensions were prepared at a final concentration of 2 × 10 5 cells/5 µl Matrigel (BD). After laparotomy, FUCCI-expressing cancer cells were subserosally injected directly into the left lobe of the liver using a 31 gauge needle. After cancer-cell implantation, the abdominal wall of the mice was closed with 6-0 sutures. Mice were anesthetized as described above and placed on a custom-designed imaging box. The liver was exteriorized and placed on a Styrofoam box, and a cover glass was gently placed on the liver, which inhibited vibration caused 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-irradiation promotes the migration of FUCCI-red cancer cells while reducing the FUCCI-green proliferative cancer cells. These results suggested that FUCCI-red quiescent cancer cells are resistant to irradiation and play a key role in metastasis. Furthermore, Bouchard et al. showed that pre-irradiation of mammary gland promotes lung metastasis [71]. Onozato et al. [72] demonstrated using FUCCI real-time imaging of tumor spheroids that FUCCI-green proliferative cancer cells located at the outside of the spheroids are sensitive to irradiation; in contrast, FUCCI-red cancer cells located in the center of the spheroids are dormant 40 days after irradiation, and then survive for more than two months, indicating that they are radioresistant.

Intravital Orthotopic FUCCI Imaging Identifies Cell Cycle-Related Genes
Kagawa et al. [73] demonstrated with intravital multiphoton microscopy and FUCCI imaging cell cycle-associated cancer cell mobilization and invasion. S/G 2 /M-phase FUCCI-green proliferating cells, but not G 0 /G 1 -phase FUCCI-red quiescent cells, invaded surrounding tissues. Kagawa et al. also performed cDNA microarray-based comparative analyses between FUCCI-green and -red cells in culture and in vivo to elucidate the molecular mechanism of the control of cell cycle-dependent motility. Arhgap11A was identified as a cell cycle-dependent mobility-controlling molecule [73].

New Cell-Cycle-Based Approaches to Treatment of Solid Tumor: Decoy, Trap, and Shoot Therapy
Intravital FUCCI imaging demonstrated that quiescent, chemoresistant cancer cells should be targeted. Therefore, it was hypothesized that mobilizing the cancer-cell cycle from G 0 /G 1 phase to S/G 2 /M phase would make the cancer cells sensitive to DNA damage drugs or to antimitotic drugs. Yano et al. [74] demonstrated that a telomerase-specific oncolytic adenovirus, OBP-301 [75,76], decoyed and trapped formerly quiescent cancer cells in early S phase and sensitized the decoyed cancer cells to currently-used cytotoxic anticancer agents ( Figure 6). Yano et al. [77] also showed with FUCCI imaging that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G 2 ( Figure 7). Moreover, Yano et al. [78] also showed that cancer-targeting Salmonella typhinurium A1-R [79] also decoyed cancer cells from G 0 /G 1 phase to G 2 /M phase (Figure 7), and when cancer cells were subsequently treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G 2 (Figure 7) [80]. Tumors decoyed by S. typhinurium A1-R and trapped by rMETase were significantly more sensitive to conventional chemotherapy than cancer cells that were not pretreated with this strategy (Figure 7).

Conclusions
Intravital real-time monitoring of FUCCI-expressing tumors demonstrated cell-cycle dynamics of each cancer cell in a tumor in a live animal, suggesting why current cytotoxic agents are mostly ineffective. FUCCI imaging enabled us to develop a curative strategy to overcome cancer-cell quiescence in tumors.
Funding: This article was supported by grants-in-aid from the Ministry of Education, Science and Culture, Japan, No. 18K16313 to S.Y.