Comparison of primary tumor sections from MMTV-PyMT or MTLn3-ErbB3-

Transcription

1 Supplemental Data Comparison of primary tumor sections from MMTV-PyMT or MTLn3-ErbB3- GFP tumors in mice either injected with control or clodronate-containing liposomes and stained for macrophages using an anti-mouse F4/80 antibody, showed no decrease in the number of tumor-associated macrophages (TAMs) after clodronate liposome treatment. Since macrophages themselves are involved in clearance of dead or dying cells, inhibition of macrophage function over a 48 hour period might occur without a clear drop in the number of macrophages. Therefore we measured macrophage function directly within the primary tumor by detection of fluorescent 70 kd dextran macropinocytosis (1). We used multiphoton intravital imaging or wide-field fluorescence microscopy of dissected organs to detect the uptake of Texas Red or tetramethylrhodamine dextran as described previously (1), as well as FACS analysis to distinguish between macrophages and neutrophils. To evaluate the cells taking up liposomes, control (empty) liposomes were labeled using 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA). The labeled liposomes were injected iv into MMTV- PyMT/c-fms-GFP mice (2). 48hrs post injection the animals were placed under isoflurane anesthesia and a skin flap surgery was done to expose the tumor so as to image it using multiphoton microscopy (1). Uptake of liposomes was seen by the colocalization of the GFP labeled cells (green) and the DiA emission (red) (Supplemental Fig. 1A). Facs analysis of PyMT and MTLn3-ErbB3 tumors using rat APC anti-mouse Gr-1 and rat PE-Cy5 anti-mouse F4/80 antibodies to label neutrophils and macrophages respectively, showed that 95% of the cells that took

2 up the DiA-labeled liposomes were F4/80 positive while 5% were Gr-1 positive. Thus the majority of cells taking up liposomes are macrophages. To test the effect of clodronate-containing liposomes on macrophage function, either control or clodronate-containing liposomes were injected iv in both MMTV- PyMT/c-fms-GFP and MTLn3-ErbB3-GFP models 48hrs prior to imaging. 2 hours prior to imaging, 200μL of 10mg/ml Texas Red or tetramethylrhodamine dextran (70KDa) was injected via tail vein. Functionality of the macrophages was determined by the ability to take up dextran in both the tumor and the spleen. Images were quantitated using ImageJ. Intravital imaging of primary tumors from both the MMTV- PyMT/c-fms-GFP and MTLn3-ErbB3-GFP models (Supplemental Fig 1.B-E) showed a dramatic decrease in dextran uptake by stromal cells in the clodronate liposome treated tumors compared to the control liposome treated tumors. There was no visible difference in the number of GFP-labeled cells in the MMTV- PyMT/c-fms-GFP tumors treated with either control or clodronate-containing liposomes, consistent with our immunohistochemistry results indicating no decrease in F4/80 staining cells. For the PyMT model, the fraction of GFPlabeled cells containing dextran was determined and was significantly reduced: the ratio of red(dextran)/green(gfp cells) pixel intensity in control liposome treated tumors was 1.16+/-0.37 compared to 0.34+/-0.18 for clodronate treated tumors (two-tailed T test p<.002, mean and standard deviation of 6 measurements from 2 control liposome tumors and 6 measurements from 2 clodronate liposome tumors), showing that treatment with clodronate reduces macrophage function compared to treatment with control liposomes. For the MTLn3-ErbB3-GFP model, the amount of dextran uptake in stromal cells inside the tumor was determined by quantitating the fraction of pixels inside the tumor that were

3 red. The red pixels did not overlap with the green (GFP labeled) pixels in the MTLn3- ErbB3-GFP tumors, consistent with a stromal cell such as a macrophage taking up the label. The average uptake was again significantly reduced in the clodronate treated animals (fraction of pixels in control liposome treated tumors:1.5%+/-1.6% compared to 0.1%+/-0.07% for clodronate treated tumors, two-tailed T test p<.04, mean and standard deviation of 9 measurements from 3 control liposome tumors and 6 measurements from 2 clodronate liposome tumors). FACS analysis of the MTLn3-ErbB3-GFP tumors treated with either clodronate or control liposomes using anti-gr-1 and anti-f4/80 antibodies to label neutrophils and macrophages, respectively, corroborated the imaging data. It showed that the number of macrophages did not change upon clodronate treatment, but there was a decrease in the number of macrophages that had taken up tetramethylrhodamine dextran in the clodronate treated tumors (2-fold down). Imaging of the spleens from mice either treated with control or clodronatecontaining liposomes (Supplemental Fig 1.F-G), also showed a very dramatic impairment of macrophage function upon clodronate-liposome treatment as measured by Texas Red dextran uptake. FACS analysis of these spleens using an anti-f4/80 antibody to label macrophages showed a marked decrease in the percentage of macrophages present in clodronate liposome treated mice vs control liposome treated mice (2-3 fold reduction). Furthermore there were fewer macrophages labeled with dextran present in clodronate liposome treated spleens vs control liposome treated spleens (5 fold reduction). These results agree with previous literature showing that spleens of clodronate liposome treated mice have fewer macrophages compared to control liposome treated mice (3).

4 Based on the above data, we conclude that the primary target of clodronate liposome treatment in our tumor models is the macrophage. Supporting this conclusion, we find that 95% of the cells that have taken up dextran are macrophages. This is consistent with our prior in vivo invasion studies in which the major stromal cell coinvading with tumor cells is the macrophage (1, 4). Treatment with clodronate significantly reduces an important macrophage function: macropinocytosis as measured by dextran uptake. We think that direct neutrophil contributions to our measurements in the in vivo invasion assay are unlikely to be significant for the following reasons. Only 5% of the cells taking up liposomes are Gr-1 positive. In our in vivo invasion measurements there are few if any neutrophils that invade into the needles with the tumor cells and macrophages. The MTLn3-ErbB3-GFP tumors showed significantly more Gr-1 staining cells than the PyMT tumors, and treatment of MTLn3-ErbB3-GFP tumors with clodronate liposomes reduced the number of Gr-1 staining cells to the levels present in PyMT tumors treated with control liposomes, indicating that neutrophil density is not closely correlated with in vivo invasion measurements. Finally, clodronate liposome treatment has been reported to have limited effects on neutrophil function (3). Supplemental Methods: Labeling of control liposomes with 4-(4-(dihexadecylamino)styryl)-Nmethylpyridinium iodide (DiA). A previously published procedure (5) for labeling with DiI was used for labeling with DiA, in order to simultaneously image GFP and DiA in the multiphoton microscope. DiA solution was prepared by adding 1 ml of 100% ethanol to 2.5 mg of DiA crystals (Molecular Probes catalog number D3883) and sonicating in a

5 warm waterbath at 35 khz until the crystals were completely dissolved. 10 µl of this solution was added per 1ml of control liposomes and shaken thoroughly, followed by a 10 minute incubation in the dark at room temp. Unbound DiA was washed away with sterile PBS by centrifugation for 10 min at 20,000 x G. Liposomes were resuspended to the original volume in sterile PBS and either used right away for injections or stored in the dark at 4 C. Intravital imaging of DiA labeled liposomes and Texas Red dextran uptake in primary tumors and spleens weeks female MMTV- PyMT/c-fms-GFP mice (2) and MTLn3-ErbB3-GFP orthotopically injected SCID mice (4 week-old tumors) were injected iv with either DiA labeled liposomes, unlabeled control liposomes or clodronatecontaining liposomes (100μL liposome solution per 10g animal weight). To image DiAlabeled liposome uptake, 48hrs post liposome injection the MMTV- PyMT/c-fms-GFP mouse was placed under isoflurane anesthesia and a skin flap surgery was done to expose the tumor (1). The animal was placed on an Olympus IX 70 inverted microscope and the GFP-labeled stromal cells were imaged using a Radiance 2000 MP multiphoton at an excitation of 870nm and a 20X 0.95NA water objective (1). Uptake of liposomes was seen by the colocalization of the GFP-labeled cells and the DiA emission (Supplementary Fig. 1A). The primary tumor cells did not take up the label. To visualize the effect of control and clodronate-containing liposomes on macropinocytosis, 48 hours post liposome injection, animals were injected iv with 200µL 10mg/ml Texas Red dextran (70Kda, Molecular Probes D1830) for 2 hours. Imaging was done as described above. Macropinocytosis was determined by the ability of cells to take up dextran in both the tumor and the spleen. Quantitation of MMTV- PyMT/c-fms-GFP images was done using

6 ImageJ to obtain the pixel intensity of the GFP-labeled stromal cells (green) and dextran (red). Random fields were selected for image analysis of both control and clodronate treated mice. GFP-labeled stromal cells were circled and pixel intensity was measured in the GFP channel as well as the same area in the Texas Red dextran channel. After background subtraction for both channels, the Red/Green ratio was calculated. In the MTLn3-ErbB3-GFP xenograft model the amount of dextran uptake by stromal cells was determined by quantitating the fraction of pixels inside the tumor that were red. Preparation of tissue samples for facs analysis. The MTLn3-ErbB3-GFP xenograft model was used allowing 4 weeks for tumor growth. The animals were weighed and injected with control or clodronate liposomes (100μL liposome solution per 10g weight) via tail vein. 48 hours post injection, mice were injected with 200μL 10mg/ml tetramethyl rhodamine dextran (70Kda, Molecular Probes D1818). Primary tumors and spleens were dissected out of the mice 2 hours post dextran injection. Tissue samples were kept on ice for the entire procedure. Non-necrotic areas of the tumor and spleen were minced with razors in PBS-0.2% BSA. Cell suspensions were spun down at 1,000rpm for 5 min, supernatants discarded and samples washed with PBS-0.2%BSA by spinning at 1,000rpm for 5 min. Red blood cells were subsequently lysed on ice for 10min using 1X RBC lysis buffer, samples were spun down, washed again with PBS- 0.2%BSA and passed through a 30μm filter (Partec ) to obtain a single cell suspension. Primary tumor cell suspensions were counted with a hemacytometer and density adjusted to 1x10 7 cells per ml, 1 ml was then used for the rest of the procedure. The entire spleen samples were used for this protocol. Cell suspensions were spun down at 8,500 rpm for 15 sec, supernatants discarded and samples resuspended in 100μL PBS-

7 0.2%BSA. The Fc receptor was blocked by adding 5μL rat anti-mouse CD16/CD32 antibody (BD Pharmigen ) per sample for 15 min. Cell suspensions were washed twice with PBS-0.2%BSA and neutrophils and macrophages labeled for 45 mins using rat APC anti-mouse Gr-1 (ebioscience ) and rat PE-Cy5 anti-mouse F4/80 (ebioscience ) antibodies at 1:100. Samples were washed twice with PBS- 0.2%BSA and placed in 5ml polypropylene tubes (Fisher Sci A) to be analyzed using a Becton Dickinson FacsAria and FlowJo software. Immunohistochemistry. Tissues were fixed in buffered formalin for 24 hrs, then switched to 70% ethanol solution, and paraffin-embedded. Staining for Gr-1: paraffin sections were melted at 60 C for 45 min and deparaffinized in xylene, re-hydrated through graded alcohols to water and washed in TBS (tris buffer). Slides were pretreated with 3% hydrogen peroxide for 10 min, washed in TBS, and antigen retrieval was performed using ph mm sodium citrate buffer in a steamer for 20 min, then cooled for 30 min at RT. Slides were blocked in 5% normal donkey serum/2% BSA for 1 hour at RT before incubating with primary antibody for 1 hour at RT, rat-anti-mouse Gr- 1 (BD Biosciences #557445) at 1/50, diluted in blocking solution. Slides were then washed 4 times, 3 min each with TBS before applying biotin labeled secondary antibody (rabbit-anti-rat, DAKO) at 1/300 for 1 hour at RT. Slides were washed again and incubated for 20 min with the avidin-biotin-hrp complex as directed by DAKO. Slides were washed in TBS and DAB applied (DAKO) for 5 min before lightly counterstaining with Harris Hematoxylin (Poly Scientific s212). F4/80 Staining: the immunohistochemistry was performed as described previously (6). Briefly, paraffin sections were deparaffinized and rehydrated, then quenched with 2% hydrogen peroxide

8 in a 50/50 solution of methanol and PBS for 30 minutes. They were then washed two times in water and once in PBS, and then blocked for 30 minutes in 5% normal rabbit serum and 20 minutes in 200 ug/ml human IgG FC (Cappel, #55911) before incubating with primary rat F4-80 antibody (7) at 1:100 concentration for 2 hours RT. Two washes were done with PBS followed by incubation with secondary biotinylated anti-rat IgG (5ug/ml, Vector #BA-4001) for 30 minutes at RT and washed two times with PBS. Avidin-biotin conjugate (ABC kit, Vector #PK-6100) was applied for 30 minutes, then washed two times with PBS. DAB was applied and color was checked under the microscope. Slides were then washed in PBS, then water, counterstained, dehydrated and covered by coverslip.

9 Supplemental Figure 1.

10 Supplemental Figure 1. Clodronate liposomes are taken up by TAMs, making them functionally inactive. A. Control liposomes were labeled with DiA and imaged using intravital imaging in a multiphoton microscope as described previously (1). DiA labeled liposomes were injected iv into MMTV- PyMT/c-fms-GFP mice 48 hrs prior to imaging. GFP labeled cells (green) in the tumor are shown to have taken up the DiA liposomes (red) (arrow). B-C. Unlabeled control liposomes (B) or clodronate-containing liposomes (C) were injected iv 48 hrs prior to imaging in MMTV- PyMT/c-fms-GFP mice. Two hrs prior to imaging, mice were injected iv with Texas Red dextran to visualize functionally active macrophages that take up dextran within the primary tumor. As has been shown previously (1), GFP-labeled stromal cells took up Texas Red dextran. Injection of control liposomes did not affect the ability of GFP-labeled stromal cells (green) to take up dextran (red) (arrow in B). Clodronate liposomes significantly impaired the ability of GFP-labeled stromal cells (arrow in C) to take up dextran. D-E. Unlabeled control liposomes (D) or clodronate-containing liposomes (E) were injected iv 48 hrs prior to imaging in the MTLn3-ErbB3-GFP xenograft model. Two hrs prior to imaging Texas Red dextran was injected iv. GFP labeled carcinoma cells (green), Texas Red dextran (red). A large number of stromal cells stromal cells taking up dextran (red) were present in the primary tumors of control liposome-injected mice (D) while very few were seen in the tumors of mice injected with clodronate-containing liposomes (E). F-G. Intravital imaging of the spleens from the MTLn3-ErbB3-GFP xenograft model showed a high number of cells that take up Texas Red dextran in the control liposome injected mice (red in F) while very few were seen in the clodronate liposome injected mice (G). Representative images are shown for each experiment. Scale bar for all = 25 μm.

11 PyMT SDF-1 invasion 6 5 Relative number of cells Matrigel EGF SDF1 SDF1+IRESSA Supplemental Figure 2. MMTV-PyMT tumors invasive response to SDF-1 is blocked by Iressa. In vivo invasion of MMTV-PyMT tumors to 25nM EGF or 62.5nM SDF-1 relative to buffer levels (matrigel only) in the presence or absence of 1μM Iressa. Means and standard errors of the mean are shown.

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