Expression of mouse CD47 on human cancer cells profoundly increases tumor metastasis in murine models

The human prostate cancer cell line PC-3 (derived from a bone metastasis), the human colon cancer cell line HCT116 and the immortalized murine macrophage cell line RAW 264.7 were obtained from the American Type Culture Collection (Manassas, VA). The PC-3 M-LN4, a PC-3 variant, was kindly provided by Dr. Isaiah J. Fidler (M. D. Anderson Cancer Center, Houston, TX). PC-3 cells were grown in complete RPMI 1640 medium supplemented with 20 mM HEPES, 2 mM L-glutamine, 10,000 U.I./mL penicillin-streptomycin, 1 mM sodium Pyruvate, 1 % MEM non-essential amino acids (Life Technologies, Grand Island, NY), and 10 % FBS (Fetal Bovine Serum, Gemini Bio-Products, West Sacramento, CA). HCT116 cells were grown in 10 % DMEM and 1 % penicillin-streptomycin. RAW 264.7 cell were cultured in DMEM containing 10 % heat-inactivated FBS and 1 % penicillin-streptomycin. Cells were cultured at 37 °C in a 5 % CO₂ water saturated atmosphere.

The murine CD47 coding sequence (GenBank: Z25524.1) was synthesized by Integrated DNA Technologies (Coralville, IA), and cloned into the mammalian expression vector pIR-PURO. A fusion gene of GFP and luciferase, which was generated in our own lab, was also cloned into the same pIR-PURO plasmid (Fig. 1). The details of plasmid pCMVpiggyBAC have been described elsewhere [13]. It was transfected together with pIR-GFP-Luc or pIR-PURO-mCD47 (Fig. 1a) into either PC-3 or HCT116 cells, using Lipofectamine 2000 and Opti-MEM (Life Technologies, Grand Island, NY). Puromycin was added to transfected cells 48 h later at a concentration of 1 μg/mL, which was gradually increased up to 3 μg/mL for two weeks, as previously described for resistant-cell selection (Rivera et al., 2011).

The puromycin-resistant, GFP-, and CD47-expressing PC-3 cells were labeled for FACS analysis (GFP and CD47 double positive cells) with either a rat PE Rat IgG2a, κ isotype control antibody (cat # 400507, Biolegend CA) or a rat PE anti mouse CD47 antibody (Cat # 127507, Biolegend). For live sorting, a concentration of ≤1.0 μg per million cells in 100 μl volume was used. Cells were incubated for 30 min at 4 °C in the dark in a PBS solution containing 2 % FBS plus either the isotype or the mCD47 antibody. Cells then were washed twice with the same solution. FACS analysis was performed on a Beckton Dickinson FACSAria (BD Biosciences, San Jose, CA). Gates were established with unstained cells operating at low pressure (20 psi) using a 100 μm nozzle. Cell clusters and doublets were electronically gated out. Cells were routinely double-sorted using GFP and PE, and post-sort analysis typically indicated purities of > 90 % with minimal cell death (<10 %). Total viable cells were used for the following experiments.

For the in vitro phagocytosis assay, Raw 264.7 cells were first activated with 50U/ml of murine IFNγ and 10 ng/ml of LPS for 24 h. The cells were then plated into a 96-well at 1 × 10⁵ per well along with 2 × 10⁴ cancer cells. The next day, phagocytosis was verified by the luciferase assay using the Bright-Glo™ Luciferase Assay System (cat # E2610, Promega, Madison, WI) according to the manufacture’s instruction. Briefly, wells were rinsed with PBS; after that, 200 μl of a 1:1 mix of PBS plus Bright-Glo reagent were poured into each well, mixed with the cells and then luciferase activity was measured in a Victor X4 Multilabel Plate Reade spectrophotometer (Perkin Elmer, Waltham MA).

All animal husbandry and experimental procedures conducted in this study were approved by the University of Houston Institutional Animal Care and Use Committee (IACUC). Six-week-old male NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice or NSG mice, NOD.CB17-Prkdc ^scid /J or NOD.Scid mice (The Jackson Laboratory, Bar Harbor, ME), and C.B-Igh-1 ^b /IcrTac-Prkdc ^scid or CB17.Scid (Taconic, Germantown, NY) were used in this study. The stable human prostate cancer cell lines PC-3 expressing either mCD47-GFP-Luc (PC3-mCD47) or GFP-Luc (PC3-GFP-Luc) were implanted subcutaneously into the mouse right flank in a concentration of 2 × 10⁶ cells. PC3-mCD47, PC3-GFP-Luc or PC-3 M-LN4 were implanted orthotopically in a concentration of 2 × 10⁴ cells.

Tumors implanted subcutaneously were let grow for up to 3 or 4 weeks or until they reached approximately 1500 mm³ and then excised. Tumor growth was monitored every 3 days by measuring two perpendicular tumor diameters with a caliper, and their volume was calculated by the formula ½ (Length × Width²). For the orthotopic model, each mouse received intraprostatic injections of PC3-mCD47, PC3-GFP-Luc or PC-3 M-LN4. Bioluminescent imaging was done weekly for a month to quantitate the luciferase signal from PC3-mCD47 and PC3-GFP-Luc cells as described in more detail in the following section. Mice were sacrificed after that, lungs and livers were harvested and metastatic lesions on these organs were counted after H&E staining.

For histological staining, organ tissues including lungs and livers were collected and fixed in 10 % formalin. Serial 5-μm cross-sections of pulmonary and hepatic metastases from mice implanted with either PC3-mCD47 or PC3-GFP-Luc cells were prepared and H&E stained for examination with light microscopy. Five fields of each single representative section were examined for each organ from each of the five mice in each group using an Olympus BX51 microscope, a camera Olympus DP73, and its associated software, Olympus cellSens® 1.9 (Olympus Imaging America Inc., Center Valley, PA).

The coding sequence for murine CD47 was synthesized according to GenBank (Z25524.1), and cloned into the transposon vector pIR-PURO [14]. To facilitate in vitro and in vivo characterization, a fusion gene of GFP luciferase (GFP-Luc) was included in all the transposon vectors, with the control vector containing GFP-Luc only. The detailed composition of these transposon vectors is depicted in Fig. 1a. To ensure that the established cells overexpress the transgenes, pIR-mCD47-GFP-Luc or pIR-GFP-Luc was co-transfected with pCMVpiggyBAC [13]. The piggyBac transposase expressed from pCMVpiggyBAC would allow the transgenes contained in the transposon donor plasmids to be efficiently integrated into the chromosomes of PC-3 cells at multiple copy numbers, ensuring a high level of transgene expression [13, 15]. The cells were initially selected by puromycin treatment followed by flow cytometry sorting as described [14]. Expression of mCD47 was confirmed by flow cytometry (Fig. 1b). The newly established cell lines are designated PC3-mCD47 and PC3-GFP-Luc (the control cell line), respectively.

We further characterized the PC3-mCD47 cells by comparing them with PC3-GFP-Luc control cells for their sensitivity to the phagocytic effect of activated macrophages. To determine the generality of the impact from mCD47 overexpression, we included in this experiment another tumor cell that was similarly transduced with mCD47 but is from different tissue origin. HCT116 is a human colon cancer cell line and it was transduced with the same transposon constructs to establish HCT116-mCD47 and HCT116-GFP-Luc. The tumor cells were mixed with activated macrophages and, as all the tumor cells express luciferase, their viability was conveniently determined by measuring intracellular luciferase activity after the cells were extensively washed. The results show that the viability of both PC3-mCD47 and HCT116-mCD47 was significantly higher than that of their respective control cells (Fig. 2). These results show that overexpression of mCD47 in human cells can negatively impact the phagocytic effect of murine macrophages when they encounter each other.

To compare tumor growth properties, both PC3-GFP-Luc and PC3-mCD47 cells were implanted subcutaneously to the right flank of NSG mice. Local tumor growth was measured every 3 days. The size of PC3-mCD47 tumor was only marginally bigger than that of PC3-GFP-Luc in NSG mice (Fig. 3a). The local tumors were then surgically removed to allow the monitoring of tumor cell metastasis to the sentinel lymph node and distant organs by IVIS optical imaging. The image signal (radiance in p/s unit) became detectable at week 3 in mice implanted with PC3-mCD47 cells, mainly in the body area that covers the draining lymph node and some distant organs including lung and liver (Fig. 3b). The radiance then got increasingly stronger, and by week 4, it reached to an extremely high level while the radiance in mice planted with PC3-GFP-Luc was only barely detectable. The experiment was terminated at this point, and the draining lymph node, lung and liver were collected. The sentinel lymph nodes in mice implanted with PC3-mCD47 cells were vastly enlarged. Their average size is 4 times larger than that in mice implanted with PC3-GFP-Luc cells (Fig. 3c). Microscopic examination of the sections prepared from the collected lung and liver revealed significantly more and larger metastatic lesions in mice implanted with PC3-mCD47 cells than in the PC3-GFP-Luc group (Fig. 3d and e). These data demonstrate that, although overexpression of mCD47 in PC-3 cells only marginally enhances the establishment of local tumors, it significantly potentates the spontaneous metastasis of tumor cells to the sentinel lymph node and distant organs including lung and liver.

Due to the presence of additional IL2 receptor gamma chain deletion, the immunodeficiency in NSG mouse was significantly more severe than that of NOD.Scid mouse [16]. Consequently, some human tumor cells show an improved metastatic potential in NSG mouse [17]. Thus, it is desirable to determine if PC3-mCD47 cells also have an increased metastatic property in the less immunodeficient NOD.Scid mice. Additionally, it is also important to determine if the enhanced metastatic property of PC3-mCD47 cells could be recapitulated following orthotopic route of implantation. As such, we implanted both PC3-GFP-Luc and PC3-mCD47 cells intraprostatically into the NOD.Scid mice. We also added an additional group of animals in which the more metastatic PC-3-LN4 cells were implanted by the same orthotopic route so that the metastatic outcome could be compared among the different groups. Bioluminescent imaging was done weekly on mice implanted with PC3-mCD47 and PC3-GFPLuc cells to monitor and confirm tumor growth at the orthotopic site (data not shown). Mice were euthanized five weeks after the orthotopic tumor cell implantation and lungs and liver were harvested. Following H&E staining, the metastatic lesions were enumerated as in Fig. 3d. The results show that animals orthotopically implanted with PC3-mCD47 cells had 2.5- and 3-fold more pulmonary lesions than the mice implanted with PC3-GFP-Luc and PC-3 M-LN4 cells, respectively (Fig. 4a). The same metastatic trend was detected in the liver, with mice in the PC3-mCD47 group having 3- and 4-fold more hepatic lesions than mice in PC3-GFP-Luc and PC-3 M-LN4 group, respectively (Fig. 4b). Interestingly, our data also showed that PC-3 M-LN4 cells were not significantly more metastatic than PC3-GFP-Luc cells after orthotopic implantation, which is in line with the observation made by other groups [3]. Together, these data suggest that PC3-mCD47 cells have a superior ability to spontaneously metastasize to distant organ tissues than the control PC3-GFP-Luc cells as well as the selected metastatic PC-3 M-LN4 cells following orthotopic implantation.

Although mCD47 was found to enhance metastases of PC-3 cells to the distant organs in both NSG and NOD.Scid mice after subcutaneous or orthotopic implantation, the control PC3-GFP-Luc cells showed metastases to these organs as well albeit at a significantly lower efficiency (Figs. 3 and 4). It has been reported that CB17.Scid mouse was significantly less permissive than NOD.Scid in supporting the engraftment of human hematopoietic stem cells [18, 19]. One of the reasons for the difference on this permissiveness is that, due to the SIRPα polymorphism among different mouse strains, SIRPα in Balb/c genetic background (from which CB17.Scid is derived) has a much lower binding affinity than its counterpart in C57BL/6 (from which NOD.Scid is derived) to human CD47 [19]. As such, we also implanted both PC3-mCD47 and PC3-GFP-Luc cells subcutaneously to CB17.Scid mice to compare the local tumor growth as well as migration to the draining lymph nodes and metastasis to the distant organs. The results show that, unlike in NSG and NOD-Scid mice, the control PC3-GFP-Luc cells completely failed to migrate to the draining lymph node and to metastasize to either lung or liver (Fig. 5b and c). Expression of mCD47 moderately enhanced local tumor growth (Fig. 5a) but significantly potentiated the tumor cell migration to the draining lymph node (Fig. 5b) and distant metastasis to the lung (Fig. 5c). Liver metastasis was detected in the mice from PC3-mCD47 group, but at a low frequency. These data demonstrate that mCD47 expression could enable PC-3 tumor cells to initiate spontaneous local and distant metastasis in a mouse strain in which otherwise the tumor cells would not be able to metastasize.