Abstract
Background: We report here imaging of the behavior of αv integrin linked to green fluorescent protein (GFP) in human osteosarcoma cells colonizing the lung of nude mice. Materials and Methods: 143B osteosarcoma cells expressing αv integrin–GFP were generated by transfection of an αv integrin–GFP fusion-gene vector pCMV–AC– ITGAV–GFP. In order to generate experimental lung metastases, 143B osteosarcoma cells (1×106), stably expressing αv integrin–GFP, were injected intravenously via the tail vein. The osteosarcoma cells were transplanted orthotopically in the tibia of nude mice in order to generate spontaneous metastases. Lungs were harvested and imaged by confocal microscopy within 1 hour. Results: In the experimental lung-metastasis model, extravasating and deformed osteosarcoma cells expressing αv integrin–GFP were observed. Pseudopodia of the osteosarcoma cells contained small puncta of αv integrin–GFP. In early-stage spontaneous lung metastasis, tumor emboli were observed in pulmonary vessels. At high magnification, small αv integrin–GFP puncta were observed in the tumor embolus. In late-stage spontaneous metastasis, tumor emboli were also observed in pulmonary vessels. Invading cancer cells with strong expression of αv integrin–GFP were observed at the margin of the tumor emboli. Conclusion: The results of this study demonstrate that molecular dynamics of αv integrin–GFP can be imaged in lung metastasis, which will allow further understanding of the role of αv integrin in this process. The results also suggest a general concept for imaging molecular behavior in vivo.
- αv Integrin
- green fluorescent protein
- osteosarcoma
- real-time imaging
- confocal microscopy
- nude mice
- lung metastasis
The αv integrin subfamily consists of at least five members including αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8 (1) and have been implicated in tumor progression (2-5), including osteosarcoma (6). In a previous study, we used a powerful subcellular in vivo imaging model to demonstrate how an anti-integrin antibody inhibits seeding to and growth of osteosarcoma cells on the lung (7). 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 these double-labeled osteosarcoma cells, single cancer-cell seeding in the lung after i.v. injection of osteosarcoma cells was imaged in real time (7). 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. AIIB2 also significantly inhibited spontaneous lung metastasis from 143B-GFP-RFP tumors growing in the tibia 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 (7).
We then began to develop imaging of molecular dynamics in vivo. For this purpose, we used 143B osteosarcoma cells expressing an αv integrin–GFP fusion gene in order to visualize the molecular dynamics of αv integrin in osteosarcoma cells interacting with RFP-expressing blood vessels using color-coded imaging (8).
We previously developed subcellular in vivo imaging (9-34). In the present report, we demonstrate subcellular imaging of αv integrin behavior in osteosarcoma cells during lung metastasis. In this study, we imaged the molecular dynamics of αv integrin–GFP in osteosarcoma cells (34) during experimental and spontaneous lung metastasis in nude mice.
Materials and Methods
Cells. The 143B human osteosarcoma cell line was maintained with RPMI-1640 medium (Irvine Scientific, Santa Ana, CA, USA) containing 10% fetal bovine serum (FBS) (Omega Scientific, San Diego, CA, USA) and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO2.
Establishment of human osteosarcoma cells expressing αv integrin–GFP. The pCMV6-AC-ITGAV-GFP vector, containing αv integrin linked to GFP, was obtained from OriGene Technologies (Rockville, MD, USA). 143B cells were transformed to express αv integrin–GFP as follows: At 80% confluency, cultures were transfected with pCMV-AC-ITGAV-GFP using Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. After transfection, stable cells were selected with G418 (800 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA), starting at 24 h after transfection. Stable colonies were selected and maintained in RPMI-1640 medium containing 10% FBS and 500 μg/ml G418 (8, 35).
Experimental and spontaneous lung metastasis models in nude mice. Nude (nu/nu) mice were bred and housed in a barrier facility (AntiCancer Inc., San Diego, CA, USA). To image experimental lung metastases of nude mice, 143B cells (1×106) expressing αv integrin–GFP were injected into the tail vein of 4- to 5-week-old nude mice. Twenty-four hours after injection, nude mice were euthanized with a ketamine mixture (10 μl ketamine HCl, 7.6 μl xylazine, 2.4 μl acepromazine maleate, and 10 μl water) (Butler-Schein, Dublin, OH, USA). Lungs were harvested from each mouse at necropsy and were placed on a glass slide with a cover. To image spontaneous lung metastases in nude mice, 143B cells (1×106) expressing αv integrin–GFP, were transplanted into the left tibia of 4- to 5-week-old nude mice as previously described (36). Mice were euthanized 4 and 8 weeks after implantation, and lungs were harvested and observed. All animal studies were conducted in accordance with the principals and procedures outlined in the NIH Guide for the Care and Use of Laboratory Animals under assurance number A3873-1.
Imaging. Imaging was performed with an FV 1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan) with a XLUMPLFL 20× (0.95 NA) water-immersion objective (37). GFP was excited at 488 nm. Collagen fibers were imaged in reflectance mode with excitation at 488 nm, and scattered (reflected) photons were collected at the same wavelength. Images were produced with FV10-ASW Fluoview software (Olympus) and ImageJ (NIH, Bethesda, MD, USA) and were not modified beyond the standard adjustment of intensity levels.
Results and Discussion
143B cells expressing αv integrin–GFP. 143B αv integrin–GFP cells have strikingly bright GFP fluorescence in the cytoplasm in vitro (Figure 1A). Fibronectin is an extracellular ligand binding to αv integrin (37). When 143B αv integrin–GFP cells were seeded on a fibronectin-coated dish (BD Pharmingen, San Diego, CA, USA), punctate expression of αv integrin–GFP was observed interacting with fibronectin coated on the culture dish (Figure 1B).
Imaging 143B αv integrin–GFP experimental lung metastases in nude mice. To understand the molecular dynamics of αv integrin–GFP in osteosarcoma experimental lung metastasis, 143B αv integrin–GFP cells were injected into the tail vein of nude mice. Twenty-four hours after injection, lungs were harvested and observed by confocal microscopy. A single cancer cell with small αv integrin–GFP puncta was observed on the surface of the lung (Figure 2A). Extravasating and deformed osteosarcoma cells expressing αv integrin–GFP were also observed (Figure 2B). There were multiple pseudopodia of osteosarcoma cells with small αv integrin–GFP puncta (Figure 2C). These results suggested that the behavior of αv integrin is associated with cancer-cell adaptation to the pulmonary microenvironment at an early stage of lung metastasis.
Imaging of early-stage 143B αv integrin–GFP spontaneous lung metastases. In order to image the molecular dynamics of αv integrin–GFP in osteosarcoma cells during spontaneous lung metastasis, we transplanted osteosarcoma cells into the tibia of nude mice to generate a primary bone tumor, which subsequently spontaneously generated lung metastases. Four weeks after implantation, lungs were harvested and observed. Tumor emboli were observed in pulmonary vessels. The expression of αv integrin–GFP was scattered in the emboli (Figure 3A). With higher magnification, small αv integrin–GFP puncta were also observed in the tumor embolus (Figure 3B). Osteosarcoma cells adjacent to lung tissue had puncta strongly expressing αv integrin–GFP (Figure 3C). Metastatic cells around a small vessel, strongly expressed diffuse αv integrin–GFP (Figure 3D).
Imaging of late-stage spontaneous lung metastases of 143B αv integrin–GFP cells. In order to image the behavior of αv integrin–GFP in osteosarcoma cells in late-stage spontaneous lung metastases, lungs were harvested 8 weeks after orthotopic implantation of 143B cells into the tibia and examined. Tumor emboli were observed in pulmonary vessels (Figure 4A). Invading cancer cells with strong expression of αv integrin–GFP were observed at the margin of tumor emboli (Figure 4A). Spontaneously invading osteosarcoma cells had extensive αv integrin–GFP puncta (Figure 4B). With higher magnification, αv integrin–GFP puncta were readily imaged at the subcellular level (Figure 4C) and pseudopodia with small αv integrin puncta were observed (Figure 4D).
143B cells expressing αv integrin–green fluorescent protein (GFP). A: 143B cells stably expressing αv integrin–GFP in vitro. Bar: 100 μm. B: Punctate expression of αv integrin–GFP was observed at the bottom of the cells binding to fibronectin coated on the plastic cell-culture dish. Bar: 50 μm. FV1000 confocal microscopy.
Imaging of αv integrin–green fluorescent protein (GFP) molecular dynamics in experimental lung metastases of 143B osteosarcoma cells in nude mice. A: Small αv integrin–GFP puncta in the cytoplasm of a 143B cell. B: Extravasating deformed 143B cells have αv integrin–GFP puncta. C: Multiple pseudopodia of 143B cells with small αv integrin–GFP puncta. Bar: 20 μm. FV1000 confocal microscopy.
The in vivo molecular imaging technology and mouse models of osteosarcoma metastasis described in the present report will be a very valuable tool for investigating the molecular dynamics of αv integrin and other proteins in lung metastasis of osteosarcoma (7, 19, 38). The results also suggest a general concept for imaging molecular behavior in vivo.
Imaging of αv integrin–green fluorescent protein (GFP) molecular dynamics in 143B spontaneous lung metastases in nude mice (early stage). A: Tumor embolus in a large vessel in the lung. Diffuse expression of αv integrin–GFP in the 143B osteosarcoma cells in the tumor embolus. Bar: 100 μm. B: Small αv integrin–GFP puncta observed at higher magnification (×40). Bar: 50 μm. C: Osteosarcoma cells adjacent to lung tissue with puncta strongly expressing αv integrin–GFP (arrow). Bar: 50 μm. D: Cells strongly expressing αv integrin–GFP surrounded a smaller vessel (V). Bar: 50 μm. FV1000 confocal microscopy. T: Tumor. V: Vessel.
Imaging of αv integrin–green fluorescent protein (GFP) molecular dynamics in spontaneous lung metastases of 143B cells in nude mice (late stage). A: Tumor embolus in pulmonary vessel. Invading cancer cells with strong expression of αv integrin–GFP at the margin of the embolus (arrows). Bar: 50 μm. B: Invading cancer cells with more αv integrin–GFP puncta. Bar: 50 μm. C: αv integrin–GFP puncta higher magnification (×60). Bar: 20 μm. D: Pseudopodium of 143B cells with small αv integrin–GFP puncta. Bar: 30 μm. FV1000 confocal microscopy.
Footnotes
This article is freely accessible online.
Conflicts of Interest
No potential conflicts of interest were disclosed.
Dedication
This paper is dedicated to the memory of A. R. Moossa, M.D., and Sun Lee, M.D.
- Received May 2, 2016.
- Revision received June 5, 2016.
- Accepted June 7, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
