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Keywords:

  • metastasis;
  • hemotogenous spread;
  • prostate cancer;
  • GFP;
  • imaging

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Metastasis is primarily responsible for the morbidity and mortality of cancer. Improved therapeutic outcomes and prognosis depend on improved understanding of mechanisms regulating the establishment of early metastasis. In this study, use of green fluorescent protein (GFP)-expressing PC-3 orthotopic model of human prostate cancer and two complementary fluorescence in vivo imaging systems (Olympus OV100 and VisEn FMT) allowed for the first time real-time characterization of cancer cell–endothelium interactions during spontaneous metastatic colonization of the liver and lung in live mice. We observed that prior to the detection of extra-vascular metastases, GFP-expressing PC-3 cancer cells resided initially inside the blood vessels of the liver and the lung, where they proliferated and expressed Ki-67 and exhibited matrix metalloprotenases (MMP) activity. Thus, the intravascular cancer cells produced their own microenvironment, where they could continue to proliferate. Extravasation occurred earlier in the lung than in the liver. Our results demonstrate that the intravascular microenvironment is a critical staging area for the development of metastasis that later can invade the parenchyma. Intravascular tumor cells may represent a therapeutic target to inhibit the development of extravascular metastases. Therefore, this imageable model of intravascular metastasis may be used for evaluation of novel anti-metastatic agents.

Metastasis accounts largely for prostate cancer–related mortality and determines the prognosis of this disease. Bone, lung, and liver are the most frequent sites of prostate cancer dissemination.1 The seed and soil hypothesis of Paget states that cancer cell metastasis is non-random and involves specific interactions between the cancer cells and the host.2 For example, close interaction between cancer cells and the endothelium occurs during the intravasation and extravasation steps of tumor metastasis3, 4 and may be tumor-type and site specific. However, this interaction and its importance in promoting the survival of cancer cells and their subsequent colonization of secondary sites are poorly characterized. The lack of in vivo information about cancer cell–endothelium interactions is due partly to technical limitations in observing this process in live animals. Immunohistochemical analysis cannot capture this process in real time and in three dimensions. Therefore, in vivo imaging is necessary for the deeper understanding of this phenomenon.

The attachment of intravascular green fluorescent protein (GFP)-expressing cancer cells to the endothelium of the lung and intravascular growth of the cancer cells has been imaged. However, these imaging studies were either conducted ex vivo on resected lungs shortly after direct injection of cancer cells into the lung,5 or the interactions were imaged in a spontaneous metastasis model post-mortem.6

In our prior studies, we pioneered real-time visualization of cellular events in tumorigenesis and progression in live intact animals using fluorescent-protein-labeled cancer cells in a variety of mouse models of human cancer.7–12 We recently reported real-time visualization of intravascular trafficking and proliferation of HT1080 sarcoma cells that express GFP in the nucleus and RFP in the cytoplasm in live intact animals.10 However, in these studies, cancer cells were injected into the epigastric cranialis vein of the mouse and thus could behave differently from intravascular-trafficking cancer cells derived from spontaneous metastasis.

To gain insight on spontaneous metastasizing cancer cells interacting with endothelium at secondary sites, we employed the clinically-relevant orthotopic GFP-PC-3 human prostate cancer model. We previously reported the extensive and widespread spontaneous skeletal and systemic organ macro metastasis (100% incidence of lung metastasis and 80% incidence of liver metastasis) in this model using fluorescence in vivo GFP imaging.13 This dissemination pattern resembles human prostate cancer.1

In this report, in order to visualize, in real time, early cellular events involved in pulmonary and hepatic metastasis of prostate cancer and the behavior of prostate cancer cells that successfully arrive at the secondary site, we employed two complementary fluorescence imaging systems: the high-resolution Olympus OV100 Small Animal Imaging System (OV100) and the quantitative VisEn Fluorescence Molecular Tomography imager (FMT). Dual-channel co-imaging with the OV100 (480 nm GFP and 680 nm near infrared) enabled visualization of the metastasizing cancer cells interacting with the endothelium and matrix metalloproteinase (MMP) activity of intravascular GFP-PC-3 cells using a near infrared-labeled molecular probe MMPSense680. Further quantification of MMP activity of intravascular GFP-PC-3 cells was accomplished using the VisEn FMT imager. This approach allowed for the first time real-time characterization of viable cancer cell and endothelium interactions in the liver and the lung early during metastatic cancer cell targeting.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

GFP Gene Transduction of PC-3 Cells

The pLEIN vector (Clontech Laboratories, Inc.) expressing enhanced GFP was transfected to the PT67 packaging cell line (Clontech Laboratories, Inc.). PC-3 cells were infected with the viral supernatants collected from PT67 cells as previously described.7

Surgical Orthotopic Implantation (SOI)

The SOI of GFP-PC-3 tumors in the prostate gland of 6-week-old BALB/c nu/nu male mice was performed as previously described.13 Two tumor fragments (1 mm3) were implanted by SOI in the capsule of the dorsolateral lobe of the prostate in each of five nude mice.

Imaging Tumor Metastasis in Live Mice

The Olympus OV100 Small Animal Imaging System (Olympus Corp., Tokyo, Japan) was used for imaging tumor metastasis in live mice. To prepare the animals for an imaging experiment, mice were anesthetized and an arc-shaped incision was made in the abdominal or chest skin folds to prepare a reversible abdominal or chest skin flap, respectively.14 Intravascular and extravascular metastasis images were acquired in real time in 8-bit format with the OV100. The OV-100 imaging system used in our studies is equipped with a highly sensitive Hamamatsu Electron Multiplying (EM) CCD camera. The length of time to acquire high-resolution planer images or stack images of a single field was in the millisecond range such that the motion from breathing or the heartbeat had no effect on image acquisition. For conducting real-time visualization and quantification of MMP activity, The MMPsense680 near infrared probe was purchased from VisEn Medical and tail-vein injected at a dosage of 2 nM/mouse 22 hrs prior to imaging. Eight bit images were acquired through the 680 channels of both the OV100 and the VisEn FMT to detect and to visualize MMPsense680 uptake. GFP and MMPsense680 Z-stacks of liver were obtained over 750 μm at 10 μm steps and imaging analysis was performed by compiling the Z-stack into a 3-dimensional projection image using ImageJ (NIH). Planner images were also converted into RGB color Tiff images using ImageJ, imported into Photoshop. Contrast and brightness adjustment were applied to the whole image when necessary. The FMT system was used for measuring lung and liver MMPsense680 uptake at 22 h post-tail-vein injection of the probe. The FMT software program contains algorithms that can quantify picomolar concentrations of the activated probe in the metastatic lung and liver.

H&E Staining and Immunohistochemistry

Dissected livers and lungs were fixed in 10% buffered formalin, embedded in paraffin. Five-μm-thick sections were cut and processed with either H&E staining or for immunohistochemical analysis of cytokeratin (DAKO, M3515) and Ki-67 (DAKO, M7240) staining. The Envision + anti-mouse system was used for antigen-antibody detection. The slides were briefly immersed in hematoxylin for counterstaining and evaluated under light microscopy.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Intravascular cancer cells at the site of metastasis have been frequently observed in clinical cases.15–17 However, the majority of intravascular cancer cells were found to be nonviable upon histological examination.18, 19 While there is increasing experimental evidence which suggests that intravascular cancer cells interact directly with the endothelium and their initial proliferation may take place in the blood vessels prior to their extravasation,5, 6, 10, 20–22 animal models have not been developed to demonstrate this phenomena in real time in vivo. We recently reported intravascular proliferation of HT1080 GFP/RFP dual-color-labeled cells injected into the epigastric cranialis vein of nude mice10 indicating that intravascular cancer cells maintained their proliferative potential. However, this model did not represent spontaneous metastasis.

In the present report, using an orthotopic spontaneous metastasis model of human prostate cancer, we demonstrate for the first time, in real-time, in live animals that intravascular trafficking PC-3-GFP cancer cells in the liver and lung were actively proliferating and possessed MMP protease activities.

In vivo imaging of intravascular PC-3 cancer cells in the liver and lung

To investigate, in live mice, intravascular behavior of trafficking tumor cells in the lung and liver spontaneously produced from orthotopic PC-3-GFP tumors, the OV100 imaging system that affords rapid and high-resolution visualization capability was employed. The non-luminous liver and lung blood vessels appeared as sharply defined dark networks against tissue auto-fluorescence. No blood vessel-enhancing contrast agent was required for imaging experiments. This method allows long-term visual monitoring of spatial-temporal relationships between cancer cells and endothelium.

During hematogenous metastasis, the cancer cell can attach to the endothelium or be trapped in a vessel followed by extravasation into the tissue parenchyma.23 In our studies, using the zoom function (1.6x–16x) of the OV100 imaging system, extensive intravascular cancer cells were observed in the liver through an abdominal skin-flap in all mice examined [Figure 1a (zoom 3x), Figure 2a (zoom 16x),]. Indeed, at day 27 after orthotopic transplantation of PC-3-GFP cells, almost all of the micro-metastatic foci detected in the liver were within the blood vessels. While some formed non-continuous “segments” in both the small and large vessels, others appeared to grow along the inner wall of the blood vessels (Fig. 1a, arrows), either filling the vessel lumens or wrapping around the vessels (Fig. 1a, arrowheads). We obtained high resolution 750 μm Z-stack images with the 1.6x zoom of OV100 and image acquisition for each frame required only milliseconds. Reconstruction of the Z-stacks also revealed the extensive presence of intravascular GFP-PC-3 cancer cells in the liver (Supporting Information Video #1). Despite an intense search for extravasated PC-3-GFP cells, we found no tumor foci outside the blood vessels, suggesting at early stages of hepatic metastasis, the metastasizing cancer cells are highly dependent on the host endothelial microenvironment prior to the establishment of their own extravascular microenvironment. It is also likely that intravascular cancer cells, even after extravasation, remain closely associated with the endothelium on the outer surface of the blood vessel. This observation is consistent with a previous report that early tumor growth occurred within the vasculature in the liver in the presinusoidal portal vein branches.24, 25

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Figure 1. In vivo imaging of intravascular cancer cells in the liver and lung. (a): Extensive intravascular cancer cells were visualized in the liver with the OV100 Small-Animal Imaging System using the zoom function (a—3x and b—16x magnification). Metastasized cancer cells clustered in the vessels and blocked blood flow (a, arrows). Individual fluorescent cancer cells can often be distinguished. Some fluorescent cancer cells seem to be “wrapped around” the vessel outer wall (a, arrowheads). Intravascular cancer cells in the lung were less extensive than in the liver and fewer clusters were found in the lung vessels. (b): 7.6x magnification; (c): 16x magnification. Arrows: intravascular cancer cell clusters. Arrowheads: extravascular cancer cell clusters. Scale bars: 500 μm in (a); 100 μm in (b) and (c). Four tumor-bearing mice were imaged. Images presented here are representitave of the average observations of all animals. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Figure 2. In vivo imaging of MMP activity of metastasized cancer cells in the liver. (a): Extensive intravascular cancer cells were visualized in the liver with the OV100 Small-Animal Imaging System using the zoom function (16x magnification). (bd): In vivo imaging of MMP activity of metastasized cancer cells. MMPSense680 can be activated by MMP-2, -3, -9, -13 released by cancer cells and become fluorescent. The probe was given 22 hours before imaging. The livers of the tumor-bearing mice were imaged at the same field through the GFP channel (a) and 680 channel at 16x magnification (b). Cancer cells in the vessels shown in a were MMP positive (b). (c), Merge of (a) and (b). (d), Merged images of GFP and 680 channels of the OV100 acquired at 1.6x magnification. Scale bars: 100 μm in (a), (b), and (c); 500 μm E. Four tumor-bearing mice were imaged. Images presented here are representative of observations of all animals. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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To compare the behavior of spontaneous metastasis of prostate cancer cells at other secondary sites, the lungs of GFP-PC-3 tumor-bearing live mice were also imaged with the OV100 via a skin flap exposing the entire front chest region. In contrast to liver metastasis, much less extensive metastasis was found in the lung of all tumor-bearing mice examined. Most of the metastases were observed in the lung capillaries and small vessels, and “segment-like” metastases were shorter than those found in the liver (Fig. 1b and 1c, arrows). These data suggested that intravascular proliferation of metastasizing cancer cells in the lung was less pronounced than in the liver. A few extravascular metastatic foci were also observed (Fig. 1b, arrow head) indicating that extravasation from the vessels occurred earlier in the lung compared with that at the liver. The question whether intravascular proliferation leads to extravascular invasion, or whether they are two independent events remains to be answered.

Intravascular PC-3-GFP cancer cells possess MMP protease activity.

Increased levels of MMP (-1, -2, -9, -10, -13) expression were found in the PC-3 cells. The MMPs have been shown to affect metastasis and angiogenesis.26 To visualize MMP activity of cancer cells at secondary sites, a near-infrared fluorescence probe, MMPSense680, was injected through the tail vein 22 hours before OV100 imaging. MMPSense680 is activated by matrix metalloproteinases (MMPs), including MMP-2, -3, -9, and -13. This probe is not fluorescent in its inactivated state and becomes highly fluorescent following protease-mediated cleavage. Twenty-two hours after probe injection, dual-channel OV100 imaging was conducted to visualize, in real-time, MMP activities of the intravascular PC-3-GFP cells metastasized to the liver using MMPSense680 (Fig. 2b and 2c at 16x zoom and Fig. 2d at 1.6x zoom). Additionally, reconstruction of a 750 μm high-resolution and dual-channel (480nm for GFP and 680nm for MMPSense680) Z-stack of images demonstrated that intravascular GFP-PC-3 cancer cells possessed MMP activities (Supporting Information Video #1).

To confirm the observations from OV100 imaging, a second imaging modality, VisEn FMT, was utilized to quantify MMP activity in the lung and liver from tumor-bearing mice. VisEn FMT enables non-invasive quantification and tomographic slicing of fluorochrome concentration and distribution within living small animals with sub-millimeter spatial resolution and picomolar sensitivity. As shown in Figure 3, VisEn FMT scanning allowed visualization and quantification of MMPSense680 uptake in the liver and lung. Four corresponding z-slices (Z3, Z9, Z15 and Z21) showed both qualitative and quantitative differences between MMPSense680 activities in non-tumor bearing control mice and that of tumor-bearing mice (Fig. 3a and 3b). Twenty-four hours after injection of 2 nmol of the MMPSense680 probe, 24 pmol total MMP activity was detected in the chest and liver area of the control mouse (Fig. 3a). In contrast, PC-3 tumor-bearing mouse produced 91 pmol of total MMP activity in this region (Fig. 3b). Consistent with OV100 observations (Fig. 1 and Fig. 2), the MMPSense680 signal from the tumor-bearing mouse was mostly from the liver, suggesting that the liver contained more viable metastasized cancer cells than the lung. Similar results were obtained with the other three PC-3-GFP tumor-bearing mice (data not shown). The low basal MMP activity detected in control mice could be from other MMP-expressing cells such as endothelial cells, macrophages, or some other inflammatory cells.

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Figure 3. MMP activity quantitatively measured by FMT. Twenty-four hours after 2 nmol MMPSense680 probe was injected, mice were scanned with the VisEn FMT in order to measure total MMP activity in the lungs and liver. The yellow boxes in the left frame of (a) and (b) indicate the scanning position, while four small images in the right frame demonstrate signals from four comparable Z-stack slices (Z63, Z9, Z15 and Z21). The color bar represents the intensity of the MMPSense680 signal ranging from 12 to 114 pmol. A non-tumor-bearing mouse gave rise to 24 pmol of MMP activity (a). In contrast, a PC-3 tumor-bearing mouse produced 91 pmol of MMP activity (b). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Our data demonstrate that the employment of dual-imaging modalities with complementary features and molecular probes measuring the biological function of the cancer cells offer improved imaging of spontaneously-trafficking intravascular cancer cells. While fluorescence imaging such as with the OV100 offers high-resolution visualization and anatomical details, tomography (VisEn FMT) yields three-dimensional imaging. The combined information from multiple imaging modalities should facilitate better understanding of mechanisms of human prostate cancer metastasis.

Histopathological examination of dissected lungs and livers

To confirm the observations derived from OV100 and VisEn FMT in vivo imaging, dissected lungs and livers from imaged tumor-bearing mice and control mice were examined by H&E and immunohistochemistry staining. In the liver, metastasized cancer cells were found within small and large vessels, either in clusters (few cells-100 cells) (Fig. 4a) or as single cells (data not shown). No extravasation was observed at this early time point (3 weeks after primary tumor inoculation) in any liver section. In the lung, small clusters (less than 10 cells) or single cancer cells were largely intravascular (Fig. 4b). Most of the intravascular cancer cells were restricted to the pulmonary micro-vessels without invasion. Extensive extravascular metastasis was observed in the hilar region (data not shown). The epithelial origin of PC-3 cancer cells was confirmed by positive staining with anti-cytokeratin AE1/AE3 antibody [Fig. 4c, i and ii]. The viability of intravascular PC-3 cells was further confirmed by positive Ki-67 immunohistochemical staining [Fig. 4c, iii and iv], consistent with their observed MMP activity (Figures 1 and 3). Further, positive Ki-67 staining indicated that intravascular PC-3-GFP cells were proliferating in these vessels. To compare the differences in intravascular growth between PC-3 cells in the liver and the lung, we measured the size of 11 and 16 tumor cell clusters identified on serial liver and lung H&E sections, respectively. The size of intravascular PC-3 tumor clusters in the liver was significantly larger (diameter 94.3 ± 28.6 μm) than that in the lung (diameter 18.3 ± 8.1 μm), p = 0.0016. Consistent with this observation, the number of tumor cells in a Ki-67-positive cluster in the liver was considerably more (19.4 ± 11.1 cells) than that of in the lung (1.4 ± 0.6 cells), p = 0.04. These results indicate that hepatic blood vessels may be a more favorable microenvironment for intravascular PC-3 cell growth prior to extravasation.

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Figure 4. Histopathological examination of metastasized cancer cells in the liver and lung. (a) Cancer cells were found in clusters in the liver vessels. Cancer cells interacted with vascular endothelial cells either directly [iii] or through a “matrix-like” structure [i, ii, iv]. H&E staining: [i-iii]; trichrome staining: [iv]. (b): Single or small cancer cell clusters were found in the small vessels of lung tissue; or sometimes a few cancer cells lined up alongside the vessels [ii, iii]. H&E staining: [i]; trichrome staining: [ii-iv]. (c): Immunohistochemistry staining showed that intravascular cancer cells in the liver were cytokeratin (AE1/AE3)-positive [i-ii], indicating their epithelial origin. Positive Ki-67 staining of intravascular PC-3 cells in the liver [iii] and lung [iv] indicates that intravascular cancer cells maintained proliferation potential. Magnification levels were as indicated. Arrow: Intravascular GFP-PC-3 cancer cells. Scale bars: 40 μm in 4A(iv), 20 μm for all others. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Moreover, in the liver vessels, while some cancer cells were found to interact with endothelial cells directly; most cancer cell clusters and single cells were surrounded by a “network-like” fibrin matrix that connected the cancer cells to the endothelium [Fig. 4a, i and iii]. It is unknown whether the fibrin matrix came from the host vessels or was secreted by the cancer cells themselves to establish a microenvironment. Additionally, trichrome staining revealed intact endothelium of the blood vessels in the liver [Fig. 4a, iv].

In summary, our study demonstrated in real time, in intact live animals, and confirmed by histology in resected tissues, that PC-3 cancer cells, after arriving at the liver and lung, can remain in the blood vessels of the secondary sites to proliferate and develop into micro-metastases. Utilizing dual-channel in vivo imaging systems and activatable MMPSense680, critical cellular functions such as the MMP activity of intravascular cancer cells can be visualized. A second imaging modality was used in the study to provide complementary quantitative and validating results. The cancer cell-endothelium interaction revealed by in vivo imaging represents a novel microenvironment and a unique therapeutic target to inhibit the subsequent development of macro-metastases. This imageable metastasis model should prove useful for evaluating the efficacy of novel anti-metastatic agents active in the intravascular microenvironment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

This work was partially supported by University of Chicago Cancer Center pilot project award (HRX) for molecular imaging and NCI grants CA103563, CA132971 and CA099258 to AntiCancer, Inc.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results and Discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_24979_sm_suppinfovideo.wmv533KSupplementary Video #1. In vivo imaging of MMP activity of metastasized cancer cells in the liver. The Olympus OV100 Small Animal Imaging System was used for imaging tumor cell and endothelium interaction in the liver in live mice through a reversible abdominal skin-flap. For conducting real-time visualization and quantification of MMP activity, The MMPsense680 near infrared probe was purchased from VisEn Medical and tail-vein injected at a dosage of 2 nM/mouse 22 hrs prior to imaging. Eight bit images were acquired through the 680 channels of the OV100 to visualize MMPsense680 uptake. GFP and MMPsense680 Z-stacks of liver were obtained over 750 μm at 10 μm steps and imaging analysis performed by compiling the Z-stack into a 3-dimensional projection image using ImageJ (NIH).

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.