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Cell, Tumor, and Stem Cell Biology |
1 Gruss Lipper Center for Biophotonics and Departments of 2 Anatomy and Structural Biology and 3 Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York
Requests for reprints: Jeffrey Wyckoff, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-3348; Fax: 718-430-8996; E-mail: jwyckoff{at}aecom.yu.edu.
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We have developed animal models that allow direct examination, by intravital multiphoton imaging, of the behavior of green fluorescent protein (GFP)-expressing carcinoma cells and stromal cells in live nondissected primary mammary tumors in vivo and the correlation of their behavior with metastatic potential (7–9). Intravital imaging of orthotopic rat mammary tumors has shown that increased carcinoma cell polarity and locomotion toward blood vessels correlates with increased blood burden of tumor cells and metastasis (7, 9). Further, using an in vivo invasion assay, involving the direct collection of invasive tumor cells into microneedles placed into the primary tumor, and multiphoton imaging, we have described a paracrine interaction between carcinoma cells and macrophages that functions in mammary tumors that is responsible for the migration of invasive tumor cells. This paracrine loop has been seen both in the orthotopic rat model and in transgenic mice with mammary tumors derived from the mammary epithelial–restricted expression of the polyoma middle T antigen (10). The paracrine loop signaling between macrophages and tumor cells is essential to the ability of tumor cells to invade within the primary tumor, involves epidermal growth factor (EGF) and colony-stimulating factor (CSF)-1 and their corresponding receptors (10), and is sufficient for invasion of collagen gels reconstituted with purified tumor cells and macrophages (11). This paracrine loop has been hypothesized to account for the involvement of macrophages in metastasis by mediating the migration of tumor cells to blood vessels, giving rise to increased intravasation (6, 10). However, the direct interaction of the invasive and migratory tumor cells with blood vessels has not been observed directly in live animals, and the involvement of macrophages, if any, has not been measured. In this study, we use multiphoton imaging of tumor cells during intravasation to determine if macrophages interact with tumor cells at the sites of intravasation in vivo.
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Materials and Methods |
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Multiphoton imaging of macrophage populations and tumor cell migration. Multiphoton microscopy was done as described previously (9, 18). Animals were anesthetized with isoflurane, and a small incision was made in the skin to expose the tumor site without wounding the tumor or penetrating the peritoneum. The anesthetic was maintained while imaging. The tumor was imaged using a Bio-Rad (Carl Zeiss Micro Imaging Inc., Thornwood, NY) Radiance 2000 multiphoton microscope with an inverted Olympus (Olympus America Inc., Center Valley, PA) IX70 connected to a Spectra Physics Tsunami Ti:Sapphire laser. The animal was placed within a heated chamber maintained at 30°C so as to assure that both the core body temperature and imaging field temperature were maintained during imaging within the physiologic range of 34.3°C to 36.5°C. All images were collected using non-descanned detectors. The objectives used were a 20x Plan Apo 0.7NA (air) and a 60x LUMPlan/IR 0.9NA (water). The filters used were 480/30 nm and 515/30 nm (Chroma Technology Corp., Rockingham, VT). Primary tumors (CAG-CAT-EGFP/MMTV-PyMT, Tie2-GFP/MMTV-PyMT, MMTV-PyMT/c-fms-GFP, or MMTV-PyMT/lys-GFPKi generated) were placed on an inverted microscope and imaged at 880 nm for GFP fluorescence. CAG-CAT-EGFP/MMTV-PyMT–generated primary tumors were imaged as above, after an injection into the tail vein of 200 µL of 20 mg/mL 70,000-kDa Texas red-dextran in Dulbecco's PBS, to load macrophages by phagocytosis 2 h before imaging.
The motility of individual tumor cells was observed as described previously (9, 18, 19). In particular, cell motility was observed by time-lapse imaging over 30 min at 1-min intervals, where a z-series was collected for each frame. Then, z-series reconstructions were made using ImageJ software to delineate single tumor cells. The distance traveled and the velocity of motility were then calculated as µm/min as described previously (7).
Macrophage pixel intensity was determined by capturing z-series of CAG-CAT-EGFP/MMTV-PyMT–generated tumors labeled with Texas red-dextran, with a 20x Plan Apo 0.7NA (air) objective, in which the viewing field is 512 x 512 µm and reconstructing in ImageJ. For the 60x LUMPlan/IR 0.9NA objective, a field of view is 128 x 128 µm. Pixel intensity was measured by drawing equal size boxes on a 90° view of the z-stack and averaging the pixels for each slice. Motility of tumor cells was scored as net tumor cell centroid translocation of >20 µm over 30 min of time-lapse imaging. A tumor cell was scored as moving near a macrophage if the motility occurred within 20 µm of a macrophage. Intravasation was scored as number of events per 128 x 128 x 100 = 1.6 x 106 µm3 volume of tumor. All measurements were double blind scored.
Counts of perivascular macrophage clusters were done by injecting 200 µL of 20 mg/mL Texas red-dextran in Dulbecco's PBS into the tail vein of MMTV-PyMT and Csf1op/Csf1op/PyMT mice 2 h before imaging followed by a second injection of FITC-dextran, at the same concentration into the carotid artery to insure vessel labeling, immediately before imaging to label blood vessels. Mice were not under anesthesia for the tail vein injections but were for carotid artery injections done just before imaging. After multiphoton imaging of FITC-dextran–labeled vessels, the number of macrophages per 100 µm of vessel length in each field was counted using a double blind counter. One hundred vessels from each type of mouse were counted. Perivascular macrophage clusters were also observed in Tie2-GFP/MMTV-PyMT mice using similar methods.
Total numbers of macrophages were counted in whole tumor tissue using Texas red-dextran labeling and the counting method described by us previously (20).
Measuring the number of carcinoma cells in circulating blood. To measure the number of viable carcinoma cells in circulating blood from MMTV-PyMT and Csf1op/Csf1op/PyMT mice, animals were anesthetized with isoflurane and blood was drawn from the right ventricle of the heart using a heparin-coated 25-gauge needle and a 1-cm3 syringe. For these experiments, +/op used as "wild-type" control animals (12) were littermates of the Csf1op/Csf1op/PyMT mice. Blood was placed in a tissue culture dish containing DMEM with 10% fetal bovine serum (FBS) and incubated overnight. The next day, the dishes were rinsed with PBS and the DMEM/10% FBS was replaced. Colonies were counted after 7 days. Each colony was scored as one tumor cell from the blood. To test the effect of PD153035, 80 mg/kg PD153035 in 8% DMSO was injected i.p. (21) into MMTV-PyMT mice 1 h before blood collection. One hour after injection was chosen to have a circulating concentration of PD153035 high enough to optimally block tyrosine phosphorylation. It was shown previously that phosphorylation occurs after 3 h (21). As a control, 8% DMSO was injected into identical MMTV-PyMT mice. To test the effect of a monoclonal anti-mouse CSF-1 receptor function blocking antibody (courtesy of Dr. S. Nishikawa, Kyoto University Medical School, Kyoto, Japan; ref. 22), 2.5 µg in 150 µL PBS were injected i.p. 4 h before blood collection. As a control, the same concentration of nonimmune IgG was injected 4 h before blood collection. Experiments were done 4 h after injection because it was determined that 4 h is required for adequate equilibration of the antibody in the extracellular space and to provide a sufficient dose of circulating antibody to neutralize the CSF-1 receptor for a 2-day period (23).
Cell collection. Cell collection using the in vivo invasion assay was carried out as described previously (24, 25). For testing the chemotactic response of dextran-loaded macrophages, Texas red-dextran was injected via tail vein 2 h before cell collection in both CAG-CAT-EGFP/MMTV-PyMT and MMTV-PyMT/c-fms-GFP mice. To test the efficacy of the monoclonal anti-mouse CSF-1 receptor function blocking antibody (22), 2.5 µg in 150 µL PBS were injected i.p. 4 h before cell collection.
Isolating cells from primary tumors and fluorescence-activated cell sorting. To determine if the Texas red-dextran–labeled cells were macrophages, tumors were collected from MMTV-PyMT and MMTV-PyMT/c-fms-GFP mice and dual-label fluorescence-activated cell sorting (FACS) sorted in three different ways. To isolate the macrophages from a primary tumor, a small piece of tumor was minced and filtered twice through a nylon filter to obtain a single-cell suspension and washed with PBS. First, to determine macrophages by using macrophage markers, the cells were incubated with F4/80 primary antibody for 15 min at 37°C (26) followed by R-phycoerythrin–labeled anti-rat secondary antibody (PharMingen, San Diego, CA) 1:100 for 15 min at 37°C. FACS was done based on their GFP (c-fms-GFP) expression and coexpression of F4/80 expression in macrophages using a FACSVantage cell sorter (Becton Dickinson, San Jose, CA). For Texas red-dextran–labeled cells, mice were preinjected as described above 2 h before cell sorting. Cells from the MMTV-PyMT tumor were isolated as above and then incubated with F4/80 primary antibody for 15 min at 37°C followed by FITC-labeled anti-rat secondary antibody (1:100 for 15 min at 37°C). FACS was done based on Texas red fluorescence and coexpression of F4/80 as detected by the FITC-labeled antibody. Third, cells from a MMTV-PyMT/c-fms-GFP mouse, preinjected with Texas red-dextran, were FACS sorted based on Texas red fluorescence and on their GFP (c-fms-GFP) expression. The percentage of macrophages identified with F4/80 or c-fms-GFP that costained with Texas red-dextran was calculated as a gated population with (total cells – cells only labeled with Texas red-dextran) / total cells x 100 = 97%. To determine the degree of colocalization of the Texas red-dextran–loaded macrophages with those labeled with the F4/80 antibody, a Pearson's correlation coefficient was calculated, where each element X was a pixel value from one probe and Y was the corresponding pixel of the other probe at the same spatial location and N was the number of perimeter pixels in each annulus:
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Real-time PCR. Quantitative reverse transcription-PCR (QRT-PCR) analysis was done to determine the change of expression of EGF in BAC1.2F5 cells cultured with or without 200 µg/mL Texas red-dextran (Invitrogen/Molecular Probes, Carlsbad, CA) for 2 h followed with or without 36 ng/mL CSF-1 (a gift from Chiron Corp., Emeryville, CA) for 4 h. PCR was done using the ABI 7900 (Applied Biosystems, Foster City, CA) with sequence-specific primer pairs for EGF as described before (11).
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To determine if macrophages in the stroma of mammary tumors after dextran loading were still active in paracrine-mediated invasion (24), the in vivo invasion assay was used to collect invading tumor cells and macrophages from mice with MMTV-PyMT/c-fms-GFP tumors 30 min after i.v. injection of Texas red-dextran. Under these conditions, 100% of the invading macrophages accompanying tumor cells were loaded with Texas red-dextran and collected in the same numbers as from control tumors in animals not preinjected with Texas red-dextran. This indicates that the EGF/CSF-1 paracrine loop remains functional after phagocytosis of Texas red-dextran by macrophages and that macrophages throughout the volume of the primary tumor were Texas red-dextran labeled. This result was consistent with the ability of macrophages to produce EGF after CSF-1 stimulation even when dextran loaded (Fig. 1D).
To investigate the relative locations of tumor cells and macrophages to blood vessels, multiphoton microscopy of CAG-CAT-EGFP/MMTV-PyMT mice (mice with GFP-expressing tumor cells) was done as described previously (10, 14). Macrophages were identified by Texas red-dextran loading. Blood vessels were unequivocally identified using (a) i.v. injected fluorescent dextrans to label the blood space immediately before imaging (Fig. 1C), (b) light scattering from erythrocytes in blood vessels in the absence of dextrans, and (c) multiphoton imaging of mice with MMTV-PyMT/Tie2-GFP tumors in which endothelial cells express GFP from the tie2 promoter (Fig. 1A; Supplementary Fig. S1; ref. 17). Multiphoton microscopy of mice with MMTV-PyMT/Tie2-GFP tumors in which endothelial cells express GFP from the tie2 promoter, and of macrophages labeled by their ability to phagocytose i.v. injected Texas red-dextran (28), shows that the perivascular macrophages are located adluminal within the stroma of the tumor and not luminal within the blood space of the vessel (Fig. 1A; Supplementary Fig. S1).
Interactions between macrophages, tumor cells, and blood vessels leading to intravasation in mammary tumors. Using the methods to unequivocally label all macrophages in living tumors described above, multiphoton microscopy showed that macrophages are present in large numbers at the margin of the tumor and then decreasingly throughout the stroma as one imaged deeper inside the tumor (Fig. 2A ). In the center of the tumor where most of the blood vessels are located, many macrophages were found in association with blood vessels. The same distribution of macrophages was seen in histologic sections of primary tumors from identical mice, although the tumor volume was collapsed due to dehydration (Fig. 2B). The density of macrophages per unit volume of tissue decreased from the tumor margin to the deeper regions of the tumor where many perivascular macrophages were seen (Fig. 2C, bottom). This decrease in macrophage density was not due to loss of fluorescence detection efficiency because tumors with GFP-expressing tumor cells showed excellent fluorescence intensity to the same depth of imaging as that used for macrophages (Fig. 2C). Perivascular macrophages were best imaged by multiphoton microscopy and not by conventional confocal microscopy in undissected tumors because most perivascular macrophages reside deep within the tumor beyond the depth of imaging of pinhole confocal microscopy. This same distribution pattern of macrophages was seen in tumors using all three methods described above for visualizing macrophages (Lys-GFPKi, c-fms-GFP, and fluorescent dextran phagocytosis), consistent with the results described above that all three methods detected macrophages equally well. Furthermore, the density of tumor-associated macrophages seen with Texas red-dextran labeling was the same as observed using anti-F4/80 immunohistochemistry (20).
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To determine if perivascular macrophages are the sites of intravasation of tumor cells in PyMT tumors, mice with GFP-expressing tumor cells were i.v. injected with Texas red-dextran to label macrophages and then time-lapse imaged to observe if the intravasation of GFP-expressing tumor cells is either near or distant from the perivascular macrophages. A 1.6 x 106–µm3 volume of tumor containing a blood vessel and at least one macrophage was selected at random and time-lapse imaged for 30 min to score intravasation. Time-lapse imaging showed that tumor cells migrate toward the vessel-associated macrophages and become closely associated with the vessel surface (Fig. 3C; Supplementary Movie 1). In addition, tumor cells were seen to intravasate only where perivascular macrophages were present (Supplementary Fig. S3 and Fig. 4A; Supplementary Movies 2 and 3). The intravasation events scored live by time-lapse imaging (18 events in 96 volumes in 19 animals) always occurred within 20 µm of a macrophage (Supplementary Movie 4 shows the intravasation and disappearance of tumor cells as they are swept away in the blood flow). z-series analysis of the site of intravasation confirmed the presence of a macrophage above or below the plane of imaging even if not seen in the same plane of focus as the tumor cell, indicating that in all cases a macrophage was within one cell diameter of the intravasating tumor cell.
To determine if intravasation efficiency is related to the density of perivascular macrophages, Csf1op/Csf1op/PyMT mice defective in CSF-1 production were used. These mice show tumor growth like wild-type and +/op animals but slower tumor progression and decreased invasion and metastasis consistent with the requirement for CSF-1 signaling for invasion and metastasis and the observed lower densities of tumor-associated macrophages in Csf1op/Csf1op/PyMT mammary tumors (20, 31). Therefore, we investigated the relative number of perivascular macrophages in +/op MMTV-PyMT and Csf1op/Csf1op/PyMT tumors in animals from the same litter and found that the +/op/PyMT tumors had more than six times as many perivascular macrophages per 100 µm length of blood vessel (Fig. 4B). To determine if intravasation efficiency is correlated with the number of perivascular macrophages, the number of viable tumor cells circulating in the blood was determined by drawing blood from the right ventricle of the heart directly downstream from the mammary tumor, thereby avoiding intervening capillary beds that might trap tumor cells. The results show that +/op/PyMT mice with 6-fold more perivascular macrophages had 16-fold more viable tumor cells per mL of blood than Csf1op/Csf1op/PyMT mice with few perivascular macrophages (Fig. 4B and C, columns a and b). To compensate for differences in vascular density between the +/op/PyMT and Csf1op/Csf1op/PyMT mice, the numbers of cells collected from the blood were normalized to the vessel density of primary tumors from wild-type and Csf1op/Csf1op mice (Fig. 4D; ref. 20). The number of tumor cells in the blood of Csf1op/Csf1op animals remained significantly reduced compared with that in +/op animals when the data were normalized to vessel density (Fig. 4D). These results indicate that it is the number of perivascular macrophages and not the number of blood vessels that correlates with intravasation.
In previous studies using an in vivo invasion assay, a paracrine loop was identified between carcinoma cells and macrophages, where tumor cell EGF receptors and macrophage CSF-1 receptors are signaled by macrophage-produced EGF and tumor cell–produced CSF-1, respectively, leading to chemotaxis-mediated comigration and invasion of both cell types (10). To test the hypothesis that this paracrine signaling loop, involving EGF and CSF-1 receptors, is involved during the intravasation of carcinoma cells at perivascular macrophage clusters, EGF receptor activity was inhibited by i.p. injection of 80 mg/kg PD153035, an erbB1 and erbB2 receptor tyrosine kinase inhibitor. This concentration of PD153035 correlates to 15 µmol/L in the blood plasma after 1 h (21). One hour after injection of PD153035, the number of tumor cells exiting the primary tumor in the blood dropped by 5-fold in MMTV-PyMT mice treated with PD153035 compared with controls mock injected with buffer (Fig. 4C, columns c and d).
To determine if the CSF-1 receptor activity is also essential for intravasation, we injected i.p. 2.5 mg/mL of monoclonal antibody that is function blocking for CSF-1 receptor activity (22). Four hours after i.p. injection, the time of peak inhibition of receptor activity (23), the number of tumor cells found in the blood was 6-fold less than MMTV-PYMT mice injected with nonimmune IgG (Fig. 4C, columns e and f).
To confirm that the antibody blocked the paracrine loop described previously (10), the in vivo invasion assay was done 4 h after i.p. injection into 18-week-old PyMT x c-fms-GFP tumor-bearing mice. The number of collected cells per needle (tumor cells plus macrophages) from needles containing 25 nmol/L EGF was 
1,000 total cells on average (75% tumor cells and 25% macrophages) from mice with control antibodies but reduced to 127 ± 48 total cells (3% macrophages) from mice injected with CSF-1 receptor function blocking antibody. These numbers are very similar to what was seen when the same antibody was placed in the needle during the invasion assay (10), showing the inhibition of CSF-1-receptor activity in the tumor following i.p. injection of the function blocking antibody as used in the current study.
In total, these experiments show that intravasation follows the same pattern of dependence on macrophages (Fig. 4B), and EGF and CFS-1 signaling (Fig. 4C and D), as does the paracrine loop-dependent collection of invasive tumor cells in the in vivo invasion assay (10). These results are consistent with previous work showing a correlation between the accumulation of polarized (chemotactic) tumor cells around blood vessels, intravasation and metastasis (7), and the loss of metastatic potential in the absence of tumor-associated macrophages (12).
The role of cell-cell interactions in defining the microenvironment for tumor progression and metastasis has been studied extensively (32). In this regard, macrophages have been hypothesized to play roles in both tumor rejection and increased malignancy (6, 27, 33–35). Part of the reason for this apparent paradox is the lack of direct observations about the behavior and interactions of macrophages with tumor cells in vivo that can lead to a mechanistic understanding of the macrophage contribution to malignancy. The new insight resulting from the direct observation of macrophages and their interactions with tumor cells in vivo described here is that almost all tumor cell migration and intravasation occur in association with macrophages.
Tumor-associated macrophages have been shown to form clusters in the intratumoral region, and these macrophage dense "hotspots" are correlated with angiogenesis and found to have increased levels of vascular endothelial growth factor (29, 30). However, other groups have reported that the accumulation of macrophages invading carcinomas does not correlate with microvessel density, suggesting that the association of macrophages with blood vessels may contribute to malignancy independently of angiogenesis (36). Here, we describe for the first time that perivascular macrophages of the mammary tumor are associated with tumor cell intravasation in the absence of local angiogenesis.
Previous studies have identified macrophages as a source of EGF, which is chemotactic for tumor cells in mammary tumors in vivo (10, 24) and in vitro (11, 37). The fact that macrophages are a source of EGF probably explains why tumor cells are polarized toward blood vessels in mammary tumors (9) because these vessels have perivascular macrophages as shown in this study. The involvement of chemotaxis in macrophage-associated tumor cell migration and intravasation is consistent with the gene expression patterns of the invasive population of tumor cells (the invasion signature) collected from mammary tumors as they comigrate with macrophages (38). The "invasion signature" implicates signaling pathways involved in chemotaxis of carcinoma cells (37, 39, 40), consistent with the chemotactic behavior of tumor cells adjacent to macrophages observed in vivo in this study and previously in vitro (11).
Our results show that the perivascular macrophages of mammary tumors are specifically involved in intravasation. An understanding of how the perivascular macrophage population arises, its relationship to the general population of tissue associated macrophages, and how the interaction of perivascular macrophages with endothelial cells affects vessel integrity and transendothelial migration of tumor cells will be essential in targeting intravasation selectively.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank the laboratory members and the members of the Gruss Lipper Center for Biophotonics for their contributions to the work discussed in this article.
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Footnotes |
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Received 5/18/06. Revised 12/18/06. Accepted 12/28/06.
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M. Sidani, D. Wessels, G. Mouneimne, M. Ghosh, S. Goswami, C. Sarmiento, W. Wang, S. Kuhl, M. El-Sibai, J. M. Backer, et al. Cofilin determines the migration behavior and turning frequency of metastatic cancer cells J. Cell Biol., November 19, 2007; 179(4): 777 - 791. [Abstract] [Full Text] [PDF]
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C. E. Lewis, M. De Palma, and L. Naldini Tie2-Expressing Monocytes and Tumor Angiogenesis: Regulation by Hypoxia and Angiopoietin-2 Cancer Res., September 15, 2007; 67(18): 8429 - 8432. [Abstract] [Full Text] [PDF]
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W. Wang, J. B. Wyckoff, S. Goswami, Y. Wang, M. Sidani, J. E. Segall, and J. S. Condeelis Coordinated Regulation of Pathways for Enhanced Cell Motility and Chemotaxis Is Conserved in Rat and Mouse Mammary Tumors Cancer Res., April 15, 2007; 67(8): 3505 - 3511. [Abstract] [Full Text] [PDF]
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) in the first frame of the time-lapse movie 10 µm below the plane of focus of the other panels; middle left, a different plane of focus at the same time frame as the left. Middle left, middle right, and right, same plane of focus and the movement of tumor cells into the vessel. Line, vessel wall; arrow, point of intravasation of tumor cells.