Dual-color fluorescence imaging distinguishes tumor cells from induced host angiogenic vessels and stromal cells

Materials and Methods

GFP Transgenic Animals. Transgenic C57/B6-GFP mice (10) were obtained from the Research Institute for Microbial Diseases (Osaka University, Osaka). The C57/B6-GFP mice expressed the Aequorea victoria GFP under the control of the chicken β-actin promoter and cytomegalovirus enhancer. All of the tissues from this transgenic line, with the exception of erythrocytes and hair, fluoresced green under excitation light. The GFP gene, regulated as described above, was crossed into nude mice on the C57/B6 background. Both immunocompetent and nude GFP transgenic mice were used in the present study.

Expression Vectors. The pLNCX₂ vector was purchased from Clontech. The pLNCX₂ vector contains the neomycin resistance gene for antibiotic selection in eukaryotic cells. The RFP (DsRed2, Clontech) was inserted in the pLNCX₂ vector at the EglII and NotI sites.

RFP Vector Production. For retroviral transduction, PT67, an NIH 3T3-derived packaging cell line expressing the 10 Al viral envelope, was purchased from Clontech. PT67 cells were cultured in DMEM (Irvine Scientific) supplemented with 10% heat-inactivated FBS (Gemini Biological Products, Calabasas, CA). For vector production, packaging cells (PT67), at 70% confluence, were incubated with a precipitated mixture of DOTAP reagent (Boehringer Mannheim) and saturating amounts of pLNCX₂-DsRed2 plasmid for 18 h. Fresh medium was replenished at this time. The cells were examined by fluorescence microscopy 48 h posttransfection. For selection of brightly fluorescing cells producing high-titer retroviral supernatants, the RFP-expressing packaging cells were cultured in the presence of 500-2,000 μg/ml G418 increased in a stepwise manner (Life Technologies, Grand Island, NY) for 7 days.

RFP Gene Transduction of Tumor Cell Lines. For RFP gene transduction, 20% conf luent rodent B16F0 melanoma cells, MMT060562 mammary tumor cells, Dunning prostrate carcinoma cells, as well as human PC-3 prostate carcinoma cells and HCT-116 colon cancer cells were incubated with a 1:1 precipitated mixture of retroviral supernatants of PT67 cells and RPMI 1640 or other culture media (GIBCO) containing 10% FBS (Gemini Biological Products) for 72 h. Fresh medium was replenished at this time. Tumor cells were harvested with trypsin/EDTA and subcultured at a ratio of 1:15 into selective medium, which contained 50 μg/ml G418. To select brightly fluorescent cells, the level of G418 was increased to 800 μg/ml in a stepwise manner. Clones expressing RFP were isolated with cloning cylinders (Bel-Art Products) by trypsin/EDTA and were amplified and transferred by conventional culture methods in the absence of selective agent.

RFP-Expressing Cutaneous Melanoma Model. Six-week-old male C57/B6-GFP mice were injected s.c. with 10⁶ RFP-expressing mouse B16F0 melanoma cells. Cells were first harvested by trypsinization and washed three times with cold serum-containing medium and then kept on ice. Cells were inoculated by intradermal injection of the dorsal skin of the animal in a total volume of 50 μl within 40 min of harvesting.

RFP-Expressing Orthotopic Breast Cancer Model. Six-week-old female C57/B6-GFP mice were injected orthotopically with 10⁶ RFP-expressing mouse MMT060562 mammary tumor cells. Cells were first harvested by trypsinization and washed three times with cold serum-containing medium and then kept on ice. Cells were injected in the mammary fat pads of the animal in a total volume of 50 μl within 40 min of harvesting.

RFP-Expressing Orthotopic Prostate Cancer Models. Six-week-old male C57/B6-GFP immunocompetent mice were injected orthotopically with a single dose of 5 × 10⁵ RFP-expressing rat Dunning prostate cancer cells. Similarly, C57/B6 GFP nude mice were injected with 10⁶ PC-3-RFP human prostate cancer cells. Cells were first harvested by trypsinization and washed three times with cold serum-containing medium and then kept on ice. The cells were injected in a total volume of 30 μm within 40 min of harvesting. The bladder and prostate were exposed after a lower midline abdominal incision. The incision in the abdominal wall was closed with a 6-0 surgical suture in one layer. The animals were kept under isoflurane anesthesia during surgery. All procedures of the operation described above were performed with a ×7 magnification stereomicroscope.

RFP-Expressing Orthotopic Colon Cancer Model. Six-week-old transgenic female GFP nude mice were used as the host for HCT-116-RFP human colon cancer cells. HCT-116-RFP cells were first harvested by trypsinization and washed three times with cold serum-free medium and then resuspended with serum-free RPMI medium 1640. The cells were injected within 40 min of harvesting. After proper exposure of the colon through a lower left abdominal incision, 10⁶ HCT-116-RFP cells in 50 μl were injected under the serosa of the descending colon by using a 25-μl syringe (Hamilton). The incision in the abdominal wall was closed with a 6-0 surgical suture in one layer. The animals were kept under ketamine anesthesia during surgery.

Tumor Tissue Sampling. Tumor tissue biopsies were processed from 3 days to 4 weeks after inoculation of tumor cells. Fresh tissue was cut into ≈1-mm³ pieces and pressed on slides for fluorescence microscopy. For analyzing tumor angiogenesis, the tissues were digested with trypsin/EDTA at 37°C for 5 min before examination. After trypsinization, tissues were put on precleaned microscope slides (Fisher Scientific) and covered with another microscope slide.

Whole-Body Imaging. Whole-body imaging (11) was performed in a fluorescent light box illuminated by fiber-optic lighting at 470 nm (Lightools Research, Encinitas, CA). Emitted fluorescence was collected through a long-pass filter GG475 (Chroma Technology, Brattleboro, VT) on a Hamamatsu C5810 three-chip cooled color CCD camera (Hamamatsu Photonics, Bridgewater, NJ). High-resolution images of 1,024/724 pixels were captured directly on an IBM PC. Images were processed for contrast and brightness and analyzed with the use of IMAGE PRO PLUS 3.1 software (Media Cybernetics, Silver Spring, MD).

Fluorescence Imaging Microscopy. An Olympus BH 2-RFCA fluorescence microscope equipped with a mercury 100-W lamp power supply was used. To visualize both GFP and RFP fluorescence at the same time, excitation light was produced through a D425/60 band pass filter, 470 DCXR dichroic mirror. Emitted fluorescence light was collected through a long pass filter GG475 (Chroma Technology). High-resolution images of 1,024/724 pixels were captured by a Hamamatsu C5810 three-chip cooled color CCR camera (Hamamatsu Photonics) (10) and directly stored on an IBM PC. Images were processed for contrast and brightness and analyzed with the use of IMAGE PRO PLUS 4.0 software (Media Cybernetics).

All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health National Research Council's Guide for the Care and Use of Laboratory Animals (available at http://oacu.od.nih.gov/regs/guide/guidex.htm) under assurance number A3873-1. Animals were kept in a barrier facility under HEPA filtration. Mice were fed with autoclaved laboratory rodent diet (Tecklad LM-485, Western Research Products, Orange, CA).

Results

Whole-Body Imaging of RFP-Expressing Colon Tumor in GFP Mouse. Whole-body imaging in a fluorescence light box visualizes the orthotopically growing RFP-expressing HCT-116 human colon cancer contrasted to the GFP-expressing nude mouse host (Fig. 1). The dual-color imaging system readily distinguishes the tumor from the host, suggesting that fluorescence microscopy of fresh tissue would visually differentiate tumor and host at the cellular level.

Visualizing Angiogenesis Onset and Development in Fresh Tumor Tissue. Dual-color images of early events in tumor angiogenesis induced by an RFP-expressing B16F10 mouse melanoma in the transgenic GFP-expressing mouse are shown in Fig. 2A. Host-derived GFP-expressing fibroblast cells (arrows) and endothelial cells (arrowheads) that form nascent blood vessels are seen clearly against the red fluorescent background of the RFP-expressing mouse melanoma. Fig. 2B shows well developed, host-derived GFP-expressing blood vessels within the RFP-expressing mouse melanoma.

Fig. 2C compares tumor vasculature in viable tumor tissue to necrotic tumor tissue in the same tumor mass. GFP-expressing tumor vasculature can be readily identified in the area where the tumor tissue maintained good viability. In the necrotic area, however, only remnants of GFP-expressing vasculature can be visualized.

The images were acquired 3 weeks after s.c. injection of B16F10-RFP melanoma cells in the GFP mouse.

Visualization of Interaction of Host Dendritic Cells and Tumor Cells in Live Tumor Tissue. Fig. 3 shows many host-derived GFP-expressing dendritic cells directly contacting B16F10-RFP melanoma cells with their dendrites.

Host Lymphocyte Infiltration Visualized in Fresh Tumor Tissue. In the case of orthotopically implanted MTT060562-RFP mouse breast cancer cells, strong immune responses could be observed by day 3 after tumor implantation. Numerous host-derived GFP-expressing lymphocytes are seen infiltrating the RFP-expressing tumor mass (Fig. 4). This apparent immune response is consistent with this tumor's eventual regression.

Visualization of Host Macrophage-Tumor Cell Interaction in Live Tumor Tissue. Fig. 5 shows host macrophages expressing GFP 35 days after orthotopic implantation of human prostate carcinoma cells in the transgenic GFP nude mouse. The host macrophages, identified by morphology, expressing GFP are observed contacting and engulfing prostate cancer cells expressing RFP (Fig. 5).

Discussion

This dual-color tumor-host interaction model system allows visualization of tumor-host interaction by whole-body imaging as well as in fresh tissue. Both the tumor and the host are uniquely identified by their fluorescence color: RFP for the tumor and GFP for the host. The model has shown the specificity of various types of host cells for the tumor. For example, we have visualized tumor cells being contacted by host dendritic cells, macrophages engulfing tumor cells, and lymphocytes attacking the tumor, which eventually regressed. The dual-color, tumor-host interaction model system allows observation of tumor-host interaction at the single-cell level in fresh tissue, affording further insights into the role of host cells in tumor growth and progression. This may be particularly important for understanding the angiogenic process. The model can be used to elucidate factors whose expression within the tumor or host cells may play a role in malignancy. The model can also be used to develop specific therapeutics that attack or support host cells that affect tumor growth and progression.