Yasunori Tome

High efficacy of tumor-targeting Salmonella typhimurium A1-R on a doxorubicin- and dactolisib-resistant follicular dendritic-cell sarcoma in a patient-derived orthotopic xenograft PDOX nude mouse model

Follicular dendritic-cell sarcoma (FDCS) is a rare and recalcitrant disease. In the present study, a patient-derived orthotopic xenograft (PDOX) mouse model of FDCS was established in the biceps muscle of nude mice. The FDCS PDOX was resistant to both doxorubicin (DOX) and NVP-BEZ235, dactolisib (BEZ) an experimental agent which is a dual pan-phosphoinositide 3-kinase-mammalian target of rapamycin inhibitor. However, in contrast to DOX and BEZ, the FDCS PDOX was sensitive to the tumor-targeting bacterial strain, Salmonella typhimurium A1-R (S. typhimurium A1-R). The combination of S. typhimurium A1-R and either DOX or BEZ did not increase the antitumor efficacy of S. typhimurium A1-R, indicating that DOX and BEZ were not active in this PDOX model. The efficacy of S. typhimurium A1-R in this recalcitrant FDCS gives strong impetus to move bacterial therapy to clinical trials for this disease. The findings of the present study are of particular importance since it demonstrates that S. typhimurium A1-R is effective in a PDOX model of FDCS established from a patient who failed DOX therapy.

The role of hair follicle nestin-expressing stem cells during whisker sensory-nerve growth in long-term 3D culture.

We have previously reported that nestin‐expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND‐GFP mice), and were placed in 3D histoculture supported by Gelfoam®. β‐III tubulin‐positive fibers, consisting of ND‐GFP‐expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F‐actin. These findings indicate that β‐III tubulin‐positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND‐GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin‐expressing stem cells in the hair follicle is for growth of the follicle sensory nerve. J. Cell. Biochem. 114: 1674–1684, 2013.

Spatial–temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness

The phase of the cell cycle can determine whether a cancer cell can respond to a given drug. We report here on the results of monitoring of real-time cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI) before, during, and after chemotherapy. In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor are in G0/G1 and 70% in S/G2/M. In contrast, approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 µm) tumor blood vessels of an established tumor are in G0/G1. Longitudinal real-time imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, had little effect on quiescent cancer cells, which are the vast majority of an established tumor. Moreover, resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Our results suggest why most drugs currently in clinical use, which target cancer cells in S/G2/M, are mostly ineffective on solid tumors. The results also suggest that drugs that target quiescent cancer cells are urgently needed.

In Vivo Gene Transfer Between Interacting Human Osteosarcoma Cell Lines Is Associated With Acquisition of Enhanced Metastatic Potential

We report here in vivo gene transfer between cancer cells is associated with acquisition of high metastatic behavior. The 143B‐GFP cell line with high metastatic potential and the MNNG/HOS‐RFP cell line with low metastatic potential, both derived from the TE85 human osteosarcoma cell line, were either co‐transplanted or transplanted alone in the tibia in nude mice. Upon mixed transplantation of the two differently labeled sublines, resulting metastatic colonies are single colored either red or green, thereby demonstrating their clonality and enabling facile color‐coded quantification. When MNNG/HOS‐RFP and 143B‐GFP were co‐transplanted in the tibia, the number of lung metastases of MNNG/HOS‐RFP increased eight‐fold compared to MNNG/HOS‐RFP transplanted alone (P < 0.01). In contrast, no enhancement of MNNG/HOS‐RFP metastases occurred when MNNG/HOS‐RFP and 143B‐GFP were transplanted separately in the right and left tibiae, respectively. This result suggests that the presence of 143B‐GFP increased the metastatic potential of MNNG/HOS‐RFP within the mixed tumor. We observed transfer of the Ki‐ras gene from 143B‐GFP to MNNG/HOS‐RFP after they were co‐implanted suggesting the Ki‐ras played a role in increasing the metastatic potential of MNNG/HOS‐RFP in the presence of 143B‐GFP. These data suggest the possible role of in vivo gene transfer in enhancing the metastatic potential of cancer cells. The data also further demonstrated the power of color‐coded imaging to visualize cancer‐cell/cancer‐cell interactions in vivo. J. Cell. Biochem. 108: 362–367, 2009.

3‐Dimensional Tissue Is Formed From Cancer Cells In Vitro on Gelfoam®, But Not on MatrigelTM

Cell and tissue culture can be performed on different substrates such as on plastic, in Matrigel™, and on Gelfoam®, a sponge matrix. Each of these substrates consists of a very different surface, ranging from hard and inflexible, a gel, and a sponge‐matrix, respectively. Folkman and Moscona found that cell shape was tightly coupled to DNA synthesis and cell growth. Therefore, the flexibility of a substrate is important for cells to maintain their optimal shape. Human osteosarcoma cells, stably expressing a fusion protein of αv integrin and green fluorescent protein (GFP), grew as a simple monolayer without any structure formation on the surface of a plastic dish. When the osteosarcoma cells were cultured within Matrigel™, the cancer cells formed colonies but no other structures. When the cancer cells were seeded on Gelfoam®, the cells formed three‐dimensional tissue‐like structures. The behavior of 143B osteosarcoma cells on Gelfoam® in culture is remarkably different from those of these cells in monolayer culture or in Matrigel™. Tissue‐like structures were observed only in Gelfoam® culture. The data in this report suggest a flexible structural substrate such as Gelfoam® provides a more in vivo‐like culture condition than monolayer culture or MatrigelTM and that MatrigelTM does not result in actual three‐dimensional culture. J. Cell. Biochem. 115: 1362–1367, 2014.

Imaging UVC‐induced DNA damage response in models of minimal cancer

We have previously demonstrated that the ultraviolet (UV) light is effective against a variety of cancer cells in vivo as well as in vitro. In the present report, we imaged the DNA damage repair response of minimal cancer after UVC irradiation. DNA‐damage repair response to UV irradiation was imaged on tumors growing in 3D culture and in superficial tumors grown in vivo. UV‐induced DNA damage repair was imaged with GFP fused to the DNA damage response (DDR)‐related chromatin‐binding protein 53BP1 in MiaPaCa‐2 human pancreatic cancer cells. Three‐dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1‐GFP nuclear foci to be observed within 1 h after UVC irradiation, indicating the onset of DNA damage repair response. A clonogenic assay showed that UVC inhibited MiaPaCa‐2 cell proliferation in a dose‐dependent manner, while UVA and UVB showed little effect on cell proliferation. Induction of UV‐induced 53BP1‐GFP focus formation was limited up to a depth of 40 µm in 3D‐culture of MiaPaCa‐2 cells. The MiaPaCa‐2 cells irradiated by UVC light in a skin‐flap mouse model had a significant decrease of tumor growth compared to untreated controls. Our results also demonstrate that 53BP1‐GFP is an imageable marker of UV‐induced DNA damage repair response of minimal cancer and that UVC is a useful tool for the treatment of residual cancer since UVC can kill superficial cancer cells without damage to deep tissue. J. Cell. Biochem. 114: 2493–2499, 2013.

Dynamic color‐coded fluorescence imaging of the cell‐cycle phase, mitosis, and apoptosis demonstrates how caffeine modulates cisplatinum efficacy

Caffeine enhances the effect of certain anticancer drugs, but the mechanism of modulation is poorly understood. In this study, modulation of cisplatinum efficacy induced by caffeine was visualized at the subcellular level by real‐time fluorescent‐protein imaging. Mitotic and apoptotic changes were observed by imaging 143B human osteosarcoma dual‐color cells, in which GFP is expressed in the nucleus and RFP is expressed in the cytoplasm. Modulation of the cell cycle was imaged using time‐lapse imaging of HeLa cells expressing a fluorescent ubiquitination‐based cell cycle indicator (FUCCI) in the nucleus. Clonogenic assays showed that caffeine increased the inhibition by cisplatinum on cell proliferation. Subcellular imaging demonstrated that cisplatinum decreased mitosis and induced apoptosis in 143B cells. The combination of cisplatinum and caffeine enhanced mitosis and subsequently increased apoptosis. Time‐lapse imaging showed that cisplatinum strongly induced cell‐cycle arrest in the S/G2 phase in HeLa‐FUCCI cells. Caffeine overcame the cell‐cycle arrest induced by cisplatinum, thereby increasing its efficacy, since cisplatinum is ineffective against quiescent cells. The data in this report indicate that caffeine modulates the cell cycle in cancer cells, thereby enhancing efficacy of cell‐cycle‐dependent anticancer drugs such as cisplatinum. J. Cell. Biochem. 114: 2454–2460, 2013.

Labeling the Stroma of a Patient-Derived Orthotopic Xenograft (PDOX) Mouse Model of Undifferentiated Pleomorphic Soft-Tissue Sarcoma With Red Fluorescent Protein for Rapid Non-Invasive Imaging for Drug Screening.

Our laboratory pioneered patient‐derived orthotopic xenograft (PDOX) mouse models using surgical orthotopic implantation (SOI). PDOX models are patient‐like, in contrast to the ectopic subcutaneous‐transplant cancer models. In the present study, we demonstrate that an undifferentiated pleomorphic soft‐tissue sarcoma (UPS‐STS) PDOX model acquired bright RFP‐expressing stroma through one passage in red fluorescent protein (RFP) transgenic mice, which upon passage to non‐colored nude mice was non‐invasively imageable. A PDOX nude mouse model of UPS‐STS was established in the biceps femoris of nude mice. After the tumors grew to a diameter of 10 mm, the tumors were subsequently passaged to RFP transgenic mice, and after tumor growth were then passaged to non‐transgenic nude mice. Tumors were divided into small fragments and transplanted in the biceps femoris at each passage. The OV100 Small Animal Fluorescence Imaging System and FV1000 laser scanning confocal microscope were used to image RFP fluorescence in the UPS‐STS PDOX models. UPS‐STS PDOX tumors, previously grown in RFP transgenic nude mice for only one passage, had very bright fluorescence and after passage to non‐transgenic nude mice maintained the bright fluorescence and were non‐invasively imageable. FV1000 confocal imaging revealed diffusely distributed bright RFP stromal cells in the PDOX tumor, both in RFP transgenic mice and after passage to non‐transgenic mice. These results demonstrate a powerful method to make the PDOX UPS‐STS model brightly fluorescent for non‐invasive imaging, as well as for confocal microscopy of individual stromal cells associated with the tumor. The RFP‐labeled UPS PDOX has the potential to rapidly screen for novel effective agents for individual patients, including stroma‐targeting drugs, whereby the stromal cells are a visual target. J. Cell. Biochem. 118: 361–365, 2017.

Imaging the inhibition by anti‐β1 integrin antibody of lung seeding of single osteosarcoma cells in live mice

Integrins play a role in tumor growth and metastasis. However, the effect of integrin inhibition has not been visualized on single cancer cells in vivo. In this study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti‐integrin antibody affects seeding and growth of osteosarcoma cells on the lung. The 143B human osteosarcoma cell line, expressing red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus, was established. Such double‐labeled cells enable imaging of apoptosis and mitosis and other nuclear‐cytoplasmic dynamics. Using the double‐labeled osteosarcoma cells, single cancer‐cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged. The anti‐β1 integrin monoclonal antibody, AIIB2, greatly inhibited the seeding of cancer cells on the lung (experimental metastasis) while a control antibody had no effect. To image the efficacy of the anti‐integrin antibody on spontaneous metastasis, mice with orthotopically‐growing 143B‐RFP cells in the tibia were also treated with AIIB2 or control anti‐rat IgG1 antibody. After 3 weeks treatment, mice were sacrificed and primary tumors and lung metastases were evaluated with fluorescence imaging. AIIB2 significantly inhibited spontaneous lung metastasis but not primary tumor growth, possibly due to inhibition of lung seeding of the cancer cells as imaged in the experimental metastasis study. AIIB2 treatment also increased survival of mice with orthotopically growing 143B‐RFP.

Comparison of UVB and UVC Effects on the DNA Damage-Response Protein 53BP1 in Human Pancreatic Cancer

We have previously demonstrated that ultraviolet (UV) light is effective against a variety of cancer cells expressing fluorescent proteins in vivo as well as in vitro. In the present report, we compared the DNA damage repair (DDR) response of pancreatic cancer cells after UVB or UVC irradiation. The UV‐induced DNA damage repair was imaged with green fluorescent protein (GFP) fused to the DDR‐related chromatin‐binding protein 53BP1 in MiaPaCa‐2 human pancreatic cancer cells growing in 3D Gelfoam® histoculture and in superficial tumors grown in nude mice. 53BP1‐GFP forms foci during DNA damage repair. A clonogenic assay in 2D monolayer culture initially showed that UVC and UVB inhibited MiaPaCa‐2 cell proliferation in a dose‐dependent manner, with UVC having more efficacy. Three‐dimensional Gelfoam® histocultures and confocal imaging enabled 53BP1‐GFP foci to be observed within 1 h after UV irradiation, indicating the onset of DDR response. UVB‐induced 53BP1‐GFP focus formation was observed up to a depth of 120 µm in MiaPaCa‐2 cells on Gelfoam® compared to 80 µm for UVC. UVB‐induced 53BP1‐GFP focus formation was observed up to a depth of 80 µm in MiaPaCa‐2 cells, implanted within skin flaps in mice, at a significantly greater extent than UVC. MiaPaCa‐2 cells irradiated by UVB or UVC in the skin‐flap mouse model had a significant decrease in tumor growth compared to untreated controls with UVB having more efficacy than UVC. Our results demonstrate that UVB has greater tissue penetration than UVC because of its longer wavelength and has clinical potential for eradicating superficial cancer. J. Cell. Biochem. 115: 1724–1728, 2014.