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1 Molecular Imaging Center, Mallinckrodt Institute of Radiology, and 2 Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri; and 3 Department of Chemistry, Washington University, St. Louis, Missouri
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ABSTRACT |
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INTRODUCTION |
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Recent studies suggest that the chemokine receptor CXCR4 is an important regulator of breast cancer metastases. Analogous to directed homing of leukocytes, gradients of the chemokine CXCL12, the chemotactic ligand for CXCR4, are proposed to attract tumor cells and regulate proliferation and invasion at specific metastatic sites (3, 4, 5) . High levels of CXCL12 are produced in sites commonly affected by breast cancer metastases, and expression of the cognate receptor CXCR4 is higher in premalignant breast epithelium and breast cancer cells compared with normal breast epithelium (3 , 6 , 7) . In severe combined immunodeficient mouse models of breast cancer, a polypeptide antagonist to CXCR4 significantly reduced growth of intravenously injected cancer cells in lung (8) , and experimental and spontaneous breast cancer metastases both were reduced with a neutralizing antibody to CXCR4 (3) . CXCR4 also appears to regulate metastases of other cancer types, as evidenced by a requirement for CXCR4 in the initial proliferation of colon cancer cells in the lung after the intravenous injection of tumor cells (9) .
Expression of CXCR4 appears to be regulated by the tumor environment in vivo. Amounts of CXCR4 at the cell surface increased in breast cancer cells that were recovered and expanded from mammary fat pad xenografts in nude mice compared with parental cells in culture (10) . Additional increases in CXCR4 expression were detected in tumor cells derived from spontaneous lung metastases. After left ventricular injection of breast cancer cells in nude mice, CXCR4 was among a small number of molecules that promoted bone metastases (11) . Overexpression of CXCR4 alone in breast cancer cells also produced small increases in experimentally produced bone metastases. Although these data further support a function of CXCR4 in metastatic breast cancer, the studies may be limited by the use of immunocompromised mouse models. This is an important consideration because CXCR4 regulates normal lymphocyte trafficking, and CXCR4 signaling in the tumor microenvironment recruits plasmacytoid dendritic cells that suppress T-cell–mediated antitumor immunity (12) . Therefore, immunocompromised mice may not appropriately reproduce interactions between normal host tissues and cancer cells that regulate breast cancer progression in vivo (7) .
In addition to functions in metastatic cancer, studies with other cell types indicate that CXCR4 may affect proliferation and survival in the primary cellular environment. Administration of neutralizing antibodies to CXCR4 decreased proliferation and increased apoptosis in pancreatic ductal cells from diabetes-prone nonobese diabetic (NOD) mice (13) . Pharmacological inhibition of CXCR4 produced similar effects on tumor cell growth and survival in orthotopic xenografts of brain tumors into nude mice (14) . Although these data show that CXCR4 affects cell proliferation in normal tissue environments, it is unknown whether CXCR4 functions in the growth and progression of primary breast cancers.
We used noninvasive molecular imaging techniques to investigate CXCR4 in an immunocompetent mouse model of primary and metastatic breast cancer. When orthotopically transplanted to mammary fat pad, 4T1 breast cancer cells spontaneously metastasize in BALB/c mice, thereby reproducing many features of human breast cancer (15) . Using stable RNA interference (RNAi) to reduce expression of CXCR4 in 4T1 cells, we show that CXCR4 is necessary for the growth and metastasis of orthotopically implanted tumor cells. The initial growth of experimentally produced lung metastases also is delayed by RNAi of CXCR4 or pharmacological inhibition with the specific CXCR4 antagonist AMD3100 (16) . These data support the clinical use of CXCR4 antagonists in therapy of primary and metastatic breast cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To construct a lentiviral vector that expresses both firefly luciferase (FL) and enhanced green fluorescent protein (EGFP), we removed the human EF-1
promoter from pBudCE4.1 (Invitrogen, Carlsbad, CA) with NheI and BglII and ligated it to the corresponding sites in pGL3 Basic (Promega, Madison, WI). EF-1 and FL were excised with NheI and XbaI and blunt end ligated to the PacI site of the lentiviral vector FUGW (ref. 17
; gift of David Baltimore, California Institute of Technology) to produce FUGW-FL. FUGW-FL produces constitutive expression of FL and EGFP from EF-1 and ubiquitin promoters, respectively.
Stocks of FUGW-FL were prepared by transient transfection of 293T cells and were concentrated as described previously (17) . 4T1 cells were transduced at a multiplicity of infection (MOI) of 2 in the presence of 8 µg/mL polybrene (Sigma, St. Louis, MO). Transduced 4T1 cells were identified by expression of green fluorescent protein (GFP), and 4T1-GFP-FL cells were sorted by flow cytometry for in vitro and in vivo experiments.
For RNAi of CXCR4, we expressed short hairpin RNA molecules targeted to sites beginning at nucleotides 5 and 101 in the mouse mRNA. The following oligonucleotides (synthesized by Integrated DNA Technologies, Coralville, IA) were used: (a) position 5, 5'-GATCCCGCCGATCGTGTGAGTATATTTCAAGAGAATATACTCACACTGATCGGTTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAACCGATCAGTGTGAGTATATTCTCTTGAAATATACTCACACTGATCGGCGG-3'; and (b) position 101, 5'-GATCCCAACGTCCATTTCAATAGGATTCAAGAGATCCTATTGAAATGGACGTTTTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAAAACGTCCATTTCAATAGGATCTCTTGAATCCTATTGAAATGGACGTTGG-3'. Oligonucleotides were annealed and ligated to pSilencer 2.1-U6 hygromycin (Ambion, Austin, TX) according to the manufacturer’s directions. This vector contains a human U6 promoter to express short hairpin RNA and a hygromycin selection marker.
4T1-GFP-FL cells were transfected with short hairpin RNA plasmids targeted to either position 5 or 101 with Fugene 6 (Roche, Indianapolis, IN). Stable transfectants were isolated by culture in 250 µg/mL hygromycin (Invitrogen), and transfected RNAi 5 and RNAi 101 cells were passaged in hygromycin approximately every 2 weeks.
Immunoblotting.
80 µg whole cell lysate as determined by BCA analysis (Pierce, Rockford, IL) were loaded per lane for Western blot analysis. CXCR4 and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) proteins were detected as described previously (18)
, with primary rabbit polyclonal antibodies (Abcam, Cambridge, MA, and Santa Cruz Biotechnology, Santa Cruz, CA, respectively) and a goat antirabbit secondary antibody coupled to horseradish peroxidase (Amersham, Piscataway, NJ).
Immunohistochemistry and Quantification of Tumor Blood Vessels.
Resected primary tumors were snap-frozen in Tissue Tek O.C.T. compound (Sankura Finetek, Torrance, CA) and were sectioned at 10-µm intervals. Sections were stained for CD31 antigen with a primary rabbit polyclonal antibody (Santa Cruz Biotechnology) with standard immunohistochemistry techniques. CD31 was detected with a Vectastain ABC kit for peroxidase staining (Vector Laboratories, Burlingame, CA), and sections were counterstained with hematoxylin. Tumor vascularity was quantified by two blinded reviewers (M.S., K.L.) who counted numbers of CD31-stained vessels in 10 randomly selected x40 microscopic fields.
Calcium Assay.
Relative changes in intracellular calcium were determined with the cell permeable indicator dye X-rhod-1 (Molecular Probes, Eugene, OR). Approximately 2 x 105 cells were plated in 35-mm glass bottom dishes that were coated with 0.2% gelatin. One day later, cells were washed three times with Modified Earle’s Balanced Salt Solution (MEBSS; ref. 19
) containing 10% fetal bovine serum and were incubated for 30 minutes in MEBSS containing 1 µmol/L X-rhod-1 and 0.02% Pluronic-F127 (Molecular Probes). Cells again were washed three times with MEBSS and then were incubated for 10 minutes before assay.
Calcium assays were done on a LSM 5 PASCAL system for confocal microscopy coupled to an Axiovert 200 microscope (Zeiss, Thornwood, NY). Images were obtained every 20 seconds with a 40X water immersion objective, with argon 488 and HeNe 543 laser lines for GFP and X-rhod-1, respectively. After collecting baseline images for 2 minutes, cells were treated with 200 ng/mL mouse CXCL12- (Peprotech, Rocky Hill, NJ) or 20 µmol/L ionomycin (Sigma). In some experiments, cells were treated sequentially with CXCL12- and ionomycin. Increases in X-rhod-1 fluorescence, which correspond to increased intracellular calcium, were quantified with Zeiss LSM software to define regions-of-interest (ROI) of 10 areas within a field. Data for X-rhod-1 fluorescence were normalized to GFP constitutively expressed by transduced 4T1 cells.
In vitro Measurements of Luciferase Activity.
To quantify FL activity, 3.5 x 104 4T1-GFP-FL, RNAi 5, and RNAi 101 cells were plated in 24-well plates. One day later, cells were lysed in reporter lysis buffer (Promega), and bioluminescence from FL was measured on a luminometer (Turner Designs Luminometer Model TD-20/20). FL activity was normalized to milligrams of total cell protein assayed by BCA (Pierce). Parallel assays showed that 15,500 cells contained 0.1 mg of total cell protein.
In vitro Cell Proliferation.
One x 104 4T1-GFP-FL, RNAi 5, or RNAi 101 cells per well were seeded in triplicate 6-well plates in 2 mL of growth medium on day 0. Cells were trypsinized, resuspended in a total volume of 1 mL of medium, and counted with a hemacytometer at intervals shown on figure legends.
Animal Studies.
Animal handling and procedures were approved by the Washington University School of Medicine Animal Studies Committee. Implantation of 7 x 103 4T1-GFP-FL, RNAi 5, or RNAi 101 cells into the right inguinal mammary gland of 6-to-10-week-old female BALB/c mice (Taconic, Germantown, NY), and surgical resection of the primary tumor 21 days later, were done as described previously (20)
. The volume of primary tumors was quantified as the product of caliper measurements in three dimensions. Bioluminescence imaging was performed on all of the mice twice a week after tumor implantation, and selected animals were imaged approximately once a week by magnetic resonance imaging (MRI).
To directly produce lung metastases, mice were given injections of 1 x 106 4T1-GFP-FL, RNAi 5, or RNAi 101 cells by tail vein. Bioluminescence imaging was performed on all of the animals at the times indicated in figure legends. For experiments with the CXCR4 antagonist AMD3100 (Sigma), mice were given injections of 1.25 mg/kg AMD3100 or PBS vehicle subcutaneously twice a day beginning 3 hours after tail vein injection of 4T1 cells.
Animals were sacrificed when moribund, and sites of metastases were determined by visual inspection. In selected mice, tumor foci were localized by ex vivo luciferase assay or formation of 6-thioguanine-resistant colonies.
Bioluminescence Imaging.
Mice were intraperitoneally given injections of 150 µg/g D-luciferin in PBS, and bioluminescence imaging with a charge-coupled device (CCD) camera (IVIS, Xenogen, Alameda, CA) was initiated 10 minutes after injection (21)
. Bioluminescence images were obtained with a 15-cm field of view (FOV), binning (resolution) factor of 8, 1/f stop, and open filter. Imaging times were 1 to 60 seconds, depending on the amount of luciferase activity. Bioluminescence from ROI was defined manually, and data were expressed as photon flux (photons/s/cm2/steradian). Background photon flux was defined from a ROI drawn over a mouse that was not given an injectio0n of luciferin.
Magnetic Resonance Imaging.
MR images of mice were collected in an Oxford Instruments (Oxford, United Kingdom) 4.7 tesla, 40-cm bore magnet. The magnet is equipped with Magnex Scientific (Oxford, United Kingdom) actively shielded, high-performance (10 cm inner diameter, 60 G/cm, 100 µs rise-time) gradient coils and is interfaced with a Varian NMR Systems (Palo Alto, CA) INOVA console. Data were collected with a Stark Contrast (Erlangen, Germany) 2.5 cm birdcage RF coil. Tumor formation and development in lung and liver were monitored with multislice, coronal, respiratory-gated spin echo MR images as described previously (22)
. Spin-echo imaging settings were TR = 3 seconds, TE = 20 milliseconds, 5.0 cm FOV, slice thickness = 0.5 mm, 4 scans. During the imaging experiments, the respiration rates for all mice were regular and 
2 s-1, enabling image acquisition during postexpiratory periods.
Uptake and distribution of contrast agent within tumors was monitored with T1-weighted gradient-echo experiments. An intraperitoneal catheter was inserted into each mouse before its being placed into the birdcage coil. Multislice, gradient echo images were collected with TR = 60 ms; TE = 1.6 ms, 2.5-cm FOV, slice thickness = 1 cm; 8 scans. These images required approximately 1 minute to acquire. After the collection of two precontrast images, 150 µL of Omniscan contrast agent (gadodiamide; Amersham Health, Piscataway, NJ), diluted 1:10 in saline, were injected intraperitoneally and additional images were collected for a total of 25 minutes. Tumors were manually segmented, and the average intensity within the tumor was measured as a function of time with Varian’s Image Browser software (Varian, Palo Alto, CA). Data for contrast agent uptake were plotted as tumor intensity versus time.
Detection of 4T1 Tumor Cells by 6-Thioguanine Resistance.
Clonogenic 4T1 tumor cells in whole blood, liver, lung, spleen, brain, lymph nodes, or other tumor sites were detected in selected mice by inherent resistance of 4T1 cells to 60 µmol/L 6-thioguanine (Sigma) as described previously (20)
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Statistics.
Data are reported as mean values ± SEM and compared with Student’s t test. Values 
0.05 were considered significant.
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RESULTS |
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. To enable noninvasive bioluminescence imaging of primary and metastatic 4T1 cells, we transduced cells with a lentivirus that coexpresses GFP and FL. A population of transduced cells (4T1-GFP-FL) was identified by GFP expression and sorted by flow cytometry (Fig. 1B)
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. We selected two different cell lines, one each from cells that expressed RNAi constructs to positions 5 or 101, respectively. By densitometry, levels of CXCR4 in each cell line were reduced to 20% of those present in 4T1-GFP-FL cells. Stably transfected cells, designated RNAi 5 and RNAi 101, respectively, also were sorted by flow cytometry for GFP to use in subsequent experiments. Luciferase activity in cell lysates was equivalent for 4T1-GFP-FL and RNAi 101 cells, whereas RNAi 5 cells had lower luciferase activity than the other two cell types (Fig. 1C)
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CXCL12 binding to CXCR4 produces transient increases in intracellular calcium. To determine whether reduced expression levels of CXCR4 functionally inactivated signaling, we analyzed levels of intracellular calcium in response to treatment with CXCL12. In parental 4T1-GFP-FL cells, incubation with 200 ng/mL CXCL12 produced the expected rapid increase in intracellular calcium (24)
, as quantified with the calcium-sensitive dye X-rhod-1 (Fig. 2A)
. By comparison, no change in intracellular calcium was detected in RNAi 5 and RNAi 101 cells treated with the same concentration of CXCL12. As a positive control, we treated cells with 20 µmol/L ionomycin, a calcium ionophore. Incubation with ionomycin increased intracellular calcium in all cells, although the relative change was slightly lower in RNAi 5 cells compared with parental 4T1-GFP-FL and RNAi 101 cells. Potentially, this difference may be due to clonal variations or off-target effects of RNAi 5. Overall, these data show that knockdown of CXCR4 with two different RNAi constructs inhibits response to CXCL12.
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. Proliferation of 4T1-GFP-FL, RNAi 5, and RNAi 101 cells did not differ significantly (P > 0.6), demonstrating that expression levels of CXCR4 did not affect cell growth under normal culture conditions.
Reduced Expression of CXCR4 Inhibits Growth of Orthotopic 4T1 Tumors.
To investigate whether reduced CXCR4 expression and signaling affected progression of primary and metastatic breast cancer, we injected 7 x 103 4T1-GFP-FL, RNAi 5, or RNAi 101 cells into inguinal mammary fat pads of BALB/c mice. We performed RNAi bioluminescence imaging (BLI) studies of each mouse twice a week and used the well-established correlation between in vivo bioluminescence and tumor size at a given site as one measure of primary tumor growth (26)
. Over a period of 17 days, photon flux from 4T1-GFP-FL cells at the primary tumor site increased more rapidly than did either RNAi 5 or RNAi 101 cells (Fig. 3A and B)
. Differences between RNAi cell lines and parental 4T1-GFP-FL were significant (P < 0.05 or P < 0.005, respectively) at all time points except day 14 for RNAi 101 cells. Because 4T1-GFP-FL and RNAi 101 cells have comparable luciferase activities, these data demonstrate that knockdown of CXCR4 substantially inhibited the growth of primary tumors. RNAi 5 cells have lower amounts of luciferase activity than do 4T1-GFP-FL and RNAi 101 cells in vitro, accounting for the decreased sensitivity for detecting these cells with BLI. Compared with 4T1-GFP-FL cells, bioluminescence from RNAi 5 tumors is decreased to a greater extent than in vitro differences in luciferase activity, and growth rates of RNAi 5 tumors also were relatively slower. Therefore, these data indicate that the inhibition of CXCR4 also reduced growth of RNAi 5 tumors.
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). Differences in tumor volume between control and RNAi cells were proportionately greater than were differences in luciferase activity measured by BLI. This disparity likely is due to the different conditions measured with each technique. Only viable cells produce luciferase activity (BLI), whereas necrotic and viable tissue both contribute to tumor volume. Before surgical resection (day 21), many of the control 4T1-GFP-FL tumors developed areas of ulceration. Areas of ulceration are included in 4T1-GFP-FL tumor volumes but do not produce bioluminescence. By comparison, RNAi tumors did not have ulceration, so a smaller measured volume of tumor cells will produce relatively greater bioluminescence because all of the cells are viable. Overall, the combination of BLI data and tumor volumes demonstrate that knockdown of CXCR4 substantially limits the growth of orthotopic primary 4T1 breast cancer cells.
One possible mechanism for more rapid growth of 4T1-GFP-FL tumors is enhanced angiogenesis. CXCL12 signaling through CXCR4 stimulates the production of vascular endothelial growth factor (VEGF; ref. 27
), and VEGF also has been demonstrated to up-regulate CXCR4 in breast cancer cells (4)
. Knockdown of CXCR4 potentially could disrupt this autocrine-positive feedback loop in RNAi tumors. To noninvasively investigate steady-state perfusion and permeability of primary tumors, we used MRI with the blood-pool contrast agent gadodiamide to study four mice each with 4T1-GFP-FL and with RNAi 101 tumors. After intraperitoneal injection of gadodiamide, sequential images of primary tumors were obtained over 25 minutes, and changes in signal intensity within tumors were normalized to baseline images without contrast (Fig. 4A and B)
. Time-dependent increases in signal were comparable in both tumor types, indicating that steady-state delivery and retention of the vascular contrast agent was not affected by CXCR4 levels in 4T1-GFP-FL and RNAi 101 cells.
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. Intratumoral vessels did not differ between 4T1-GFP-FL and RNAi 101 tumors, which confirmed MRI data for tumor perfusion. By comparison, RNAi specimens had a small but significant (P < 0.05) reduction in blood vessels, which could contribute to the decreased growth of RNAi 5 primary tumors. Because angiogenesis was not affected by both RNAi constructs, it is likely that decreased numbers of blood vessels in RNAi 5 tumors are due to clonal variations rather than to effects of CXCR4 inhibition on angiogenesis.
Knockdown of CXCR4 Prevents Macroscopic Metastases of 4T1 Cells.
4T1 breast cancer cells spontaneously form metastases, independent of primary tumor resection (28)
. Surgical resection of primary tumors reproduces clinical treatment of patients with breast cancer. In our model system, the removal of large primary tumors improves the sensitivity of BLI for detecting metastases because high amounts of luciferase activity in these tumors potentially could obscure small metastatic tumors. We resected primary tumors in all eight of the mice into which 4T1-GFP-FL cells were implanted, and we resected the four largest tumors each in mice into which RNAi 5 and RNAi 101 cells, respectively, were implanted. In the remaining mice that received RNAi cells, primary tumors were <3 mm in diameter and, therefore, were not removed.
We used BLI and MRI to detect and monitor breast cancer metastases in the mice described above that had received implants of 4T1-GFP-FL, RNAi 5, and RNAi 101 cells. All eight mice with 4T1-GFP-FL tumors developed tumor metastases with lung as the predominant organ (Fig. 5)
. These mice all died as a result of metastatic breast cancer within 56 days of tumor implantation. Sites of macroscopic metastases identified with noninvasive imaging were confirmed in selected mice by the ex vivo imaging of luciferase activity or the isolation of 4T1 cells from tissues based on 6-thioguanine resistance (data not shown). In all of the mice, macroscopic metastases were confirmed by dissection and visual inspection at autopsy (Fig. 5C)
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RNA Interference or Pharmacological Inhibition of CXCR4 Delays Initial Growth of Experimental Lung Metastases.
Because RNAi of CXCR4 prevented macroscopic metastases of 4T1 cells from the mammary fat pad, we could not assess the effects of CXCR4 on tumor growth in other organs. We injected 1 x 106 4T1-GFP-FL, RNAi 5, or RNAi 101 cells by tail vein to directly introduce cancer cells to lung, an organ with high levels of CXCL12 (3)
and the most common site of metastases from orthotopically placed 4T1-GFP-FL tumors. Mice were imaged over 7 days after the injection, and data for luciferase activity in lung were normalized to initial bioluminescence at 3 hours to account for differences in injection of tumor cells and lower luciferase activity in RNAi 5 cells (29)
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ROI analysis of lungs showed an immediate decrease in bioluminescence between 3 and 16 hours for all cells, with luciferase activity at 16 hours being approximately 20% of that at the initial 3-hour time point (Fig. 6)
. Decreases in cell number were comparable for both cell lines, indicating that the initial inefficiency of the metastatic process in lung was not affected by levels of CXCR4 (30
, 31)
. As measured by ROI analysis of BLI images, normalized photon flux from 4T1-GFP-FL cells progressively increased through 1 week, reaching levels more that 11-fold greater than the initial lung value at 3 hours. By comparison, normalized luciferase activity from RNAi 5 and 101 cells in lung was significantly lower than that from 4T1-GFP-FL cells at all times after 24 hours, indicating that proliferation and/or survival of RNAi cells was inhibited significantly (P < 0.05). These early differences in growth or survival of breast cancer cells in lung could be mediated by the mitogenic and/or antiapoptotic effects of CXCR4, respectively (14
, 25
, 32)
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As an alternative to RNAi, we treated mice with AMD3100, a specific inhibitor of CXCR4 (16)
that has been shown recently to inhibit growth of intracranial brain tumor xenografts in mice (14)
. Mice were given injections via tail vein with 1 x 106 4T1-GFP-FL cells and were imaged with BLI 3 hours later to establish a baseline for luciferase activity. Animals then were treated twice a day with 1.25 mg/kg AMD3100 or PBS, injected subcutaneously (14)
. Similar to data with RNAi 101 cells, the inhibition of CXCR4 with AMD3100 significantly decreased numbers of 4T1-GFP-FL cells in lung after 24 hours, as determined by BLI (P < 0.05; Fig. 6B
). The lag in cell proliferation and/or survival produced by AMD3100 was less than that observed in RNAi cells, likely because intermittent dosing of AMD3100 was less effective than RNAi in blocking CXCR4 signaling. After 7 days, cells in mice treated with AMD3100 had overcome the initial delay in proliferation, showing normalized bioluminescence that did not differ from that of 4T1-GFP-FL cells in mice receiving PBS vehicle control. Similar to results with RNAi cells, AMD3100 treatment of mice with 4T1-GFP-FL cells did not prolong survival relative to vehicle control. These data with RNAi and pharmacological blockade of CXCR4 indicate that this chemokine receptor substantially enhances the initial growth of breast cancer cells in lung, although interrupting the CXCR4 signaling alone is not sufficient to prolong overall survival.
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DISCUSSION |
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B–dependent fashion (10)
, again suggesting that CXCR4 signaling is an important determinant of metastatic breast cancer. Furthermore, humans with invasive ductal breast carcinoma show a significant correlation between relative expression levels of CXCR4 and the extent of lymph node metastases (36)
. Whereas previous studies suggest that CXCR4 regulates breast cancer metastases, our current data extend these observations by demonstrating that CXCR4 has substantial functions in both primary and metastatic breast cancer in immunocompetent mice. We used stable RNAi directed against two different sites in CXCR4 mRNA to knockdown expression of CXCR4 and CXCL12-mediated signal transduction in murine 4T1 cells. After orthotopic implantation into mammary fat pads, parental 4T1-GFP-FL cells with endogenous expression of CXCR4 grew more rapidly than RNAi knockdown cells. In addition to delayed growth of RNAi cells in primary tumors, some of these tumors regressed after approximately 2 weeks. These data suggest that CXCR4 is necessary for proliferation and survival of breast cancer cells in the primary tumor site. This result contrasts with cell growth under standard culture conditions, in which levels of CXCR4 did not affect proliferation rates. These data indicate that CXCR4 is essential for cell growth under conditions in which survival and growth factors are limiting, such as the in vivo tumor microenvironment. Similar positive effects of CXCR4 on cell growth have been reported for an ovarian cancer cell line cultured under suboptimal conditions in vitro (37) .
Our results are consistent with a recent study of human breast cancer that also demonstrates the importance of the primary tumor microenvironment in CXCL12-CXCR4 signaling (7) . Expression of CXCR4 was increased in invasive ductal carcinoma specimens compared with ductal carcinoma in situ and normal breast epithelium, and CXCL12 was overexpressed by normal breast stromal cells adjacent to malignant cells. The authors [Allinen et al. (7) ] proposed that CXCL12 in the tumor microenvironment functioned to promote breast cancer proliferation, migration, and invasion. RNAi against CXCR4 in breast cancer cells could interrupt this paracrine signaling pathway, potentially accounting for the impaired growth and survival of primary tumors observed in our study.
All of the mice into which parental 4T1 cells were implanted developed palpable tumors within 3 weeks and, subsequently, had spontaneous breast cancer metastases that most commonly affected lung. All mice into which parental 4T1 cells were implanted died within 60 days. Animals with RNAi cells survived through 90 days without evidence of metastases, likely because of the limited growth of primary tumors. The inhibition of primary breast tumor growth and spontaneous metastases were comparable with both of the stable RNAi molecules, providing strong evidence that effects were specific to CXCR4 and not due to variations in cell lines or off-target results of RNAi. Overall, these data show that the in vivo proliferation of 4T1 cells in the primary tumor site and the development of macroscopic metastases are both highly dependent on expression levels of CXCR4.
CXCL12-CXCR4 activates numerous signaling pathways in breast cancer cells in vitro (38) , so that there are many potential mechanisms through which CXCR4 could affect both primary and metastatic breast cancer. Because expression of CXCR4 and VEGF can provide a positive feedback loop and because angiogenesis is essential for tumor growth and metastases (reviewed in ref. 39 ), we initially investigated whether primary tumor vasculature contributed to in vivo differences between parental and RNAi 4T1 cells. MRI and immunohistochemistry showed comparable accumulation of an intravascular contrast agent and numbers of tumor blood vessels, respectively, in parental and RNAi 101 tumors, whereas RNAi 5 tumors had somewhat diminished vascularity. Because effects on vascularity were not concordant between RNAi 5 and 101 tumors, differences likely were due to clonal variations and were not specifically caused by blocking CXCR4 signaling in breast cancer cells. Further investigation is needed to dissect how other downstream effectors of CXCR4, such as AKT and extracellular signal-regulated kinases 1 and 2 (Erk 1 and 2), regulate primary breast cancer proliferation, survival, and invasion.
To investigate the effects of CXCR4 on breast cancer growth in a metastatic site, we produced experimental lung metastases with parental and RNAi cells and used noninvasive BLI to measure temporal changes in cell number. After intravenous injection, all of the cells had comparable decreases in bioluminescence, indicating that reduced expression of CXCR4 did not affect initial metastatic inefficiency. Compared with parental 4T1 cells, growth of RNAi 5 and 101 lung metastases was delayed substantially, showing that reduced CXCR4 correlated with a marked reduction in cell proliferation and/or survival. A similar function for CXCR4 signaling in growth of experimental colon cancer metastases in lung has been observed recently (9) . Using an "intrakine" to decrease plasma membrane expression of CXCR4, Zeelenberg et al. showed that CXCR4-deficient cells survived in lungs but did not proliferate, whereas numbers of control cells proliferated rapidly after up-regulating expression of CXCR4 (9) . The delay in cell proliferation likely is due to increased apoptosis and/or delayed cell cycle entry in cells with reduced CXCR4. These effects may be mediated by reduced activation of AKT or Erk 1 and 2, pathways that have been correlated with CXCR4–mediated cell proliferation and survival in vivo (14) . Dissecting how specific CXCR4-regulated signaling pathways contribute to multiple steps in the progression of primary and metastatic breast cancer will further our understanding of CXCR4 in breast cancer, and it is an important area of future research.
As a potential therapeutic model, we used the specific CXCR4 antagonist AMD3100 (16) to treat mice with experimentally produced 4T1-GFP-FL lung metastases. Inhibition of CXCR4 with AMD3100 delayed the initial growth of 4T1-GFP-FL cells in lung compared with the growth in mice that received vehicle control. The early temporal lag in growth was similar to that produced by stable RNAi of CXCR4, although effects of AMD3100 were quantitatively lower and less sustained. This difference likely is due to the persistent inhibition of CXCR4 expression with RNAi compared with the variable antagonism of CXCR4 produced by the rapid decrease in plasma levels of the compound after intermittent subcutaneous dosing (40) .
Neither RNAi nor AMD3100 prolonged the survival of mice with experimental lung metastases compared with parental cells treated with vehicle alone. Incomplete effects of CXCR4 inhibition in breast cancer have been reported previously with a peptide antagonist of CXCR4, which produced only a small reduction in experimental lung metastases (35) . Potentially, this could be due to the selection of variant cells that are not dependent on CXCR4. Alternatively, CXCR4 may be needed only for the initial growth of lung metastases. These data imply that combination therapy with a CXCR4 inhibitor and other chemotherapeutic drugs may be needed for potential clinical benefits in breast cancer patients.
In summary, we have shown that reduced expression of CXCR4 limits orthotopic growth of breast cancer cells in vivo and prevents development of macroscopically detectable metastases from primary tumors. These results extend potential therapeutic applications of CXCR4 inhibitors to the treatment of both primary and metastatic breast cancer. Our model system also demonstrates the advantages of noninvasive imaging techniques to monitor the progression of breast cancer in the same cohort of mice. As new antagonists of CXCR4 become available, in vivo imaging techniques will be important tools for determining the therapeutic efficacy of candidate inhibitors and for analyzing how CXCR4 regulates the development and progression of primary and metastatic breast cancer.
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FOOTNOTES |
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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.
Note: M. Smith, K. Luker, and G. Luker are currently in the Department of Radiology, University of Michigan Medical School, Ann Arbor, Michigan.
Requests for reprints: Gary D. Luker, Department of Radiology, University of Michigan Medical School, 1150 West Medical Center Drive, Room 9303 MSRB III, Ann Arbor, MI 48109-0648. Phone: (734)-763-5849; Fax: (734)-647-2563; E-mail: gluker{at}umich.edu
Received 5/26/04. Revised 9/15/04. Accepted 9/ 3/04.
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Z. Wang, L. Zhang, A. Qiao, K. Watson, J. Zhang, and G.-H. Fan Activation of CXCR4 Triggers Ubiquitination and Down-regulation of Major Histocompatibility Complex Class I (MHC-I) on Epithelioid Carcinoma HeLa Cells J. Biol. Chem., February 15, 2008; 283(7): 3951 - 3959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pattarozzi, M. Gatti, F. Barbieri, R. Wurth, C. Porcile, G. Lunardi, A. Ratto, R. Favoni, A. Bajetto, A. Ferrari, et al. 17 -Estradiol Promotes Breast Cancer Cell Proliferation-Inducing Stromal Cell-Derived Factor-1-Mediated Epidermal Growth Factor Receptor Transactivation: Reversal by Gefitinib Pretreatment Mol. Pharmacol., January 1, 2008; 73(1): 191 - 202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Miao, K. E. Luker, B. C. Summers, R. Berahovich, M. S. Bhojani, A. Rehemtulla, C. G. Kleer, J. J. Essner, A. Nasevicius, G. D. Luker, et al. CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature PNAS, October 2, 2007; 104(40): 15735 - 15740. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, A. Mora, H. Shim, A. Stecenko, K. L. Brigham, and M. Rojas Role of the SDF-1/CXCR4 Axis in the Pathogenesis of Lung Injury and Fibrosis Am. J. Respir. Cell Mol. Biol., September 1, 2007; 37(3): 291 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Hirbe, J. Rubin, O. Uluckan, E. A. Morgan, M. C. Eagleton, J. L. Prior, D. Piwnica-Worms, and K. N. Weilbaecher Disruption of CXCR4 enhances osteoclastogenesis and tumor growth in bone PNAS, August 28, 2007; 104(35): 14062 - 14067. [Abstract] [Full Text] [PDF]
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|
|
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B. Schade, S. H.L. Lam, D. Cernea, V. Sanguin-Gendreau, |
, statistically significant difference (P < 0.05). In C, tumor sizes in three dimensions were measured by calipers 17 days after implantation of cancer cells; data are presented as mean ± SEM for tumor volume (n = 8 for each cell type).
