Fig 1.
Specific binding of M2pep to and internalization into in vitro differentiated M2 macrophages.
(A) M1 and M2 macrophages were incubated with M2pep or scM2pep (20 μM) for 30 min and analyzed by flow cytometry. (M2pep: n = 6; scM2pep: n = 4). (B) Representative immunofluorescence images of M1 and M2 macrophages incubated with 200 μM M2pep for 30 min (blue–Hoechst 33342; red–M2pep; green–phalloidin; white–F4/80; Scale 10 μm). The percentage of M2pep positive macrophages was quantified by counting the PE-CF594 positive cells in the M1 or M2 macrophage population (M1/M2 n = 10). Values are means ± SEM; *P<0.05. Statistical analysis was performed with the Wilcoxon-matched pair test.
Fig 2.
Gating strategy used in the FACS analyses to determine binding of M2pep to TAMs/liver macrophages in tumor and non-parenchymal liver cell suspensions.
(A) Representative flow cytometry gating scheme. Liver macrophages or TAMs within extracted cell suspensions were identified by staining of CD45, F4/80 and CD11b. Discrimination between M1 and M2 marcophages was performed by their expression of CD206 and CD86, respectively. The Alexa 633 fluorescence intensity (AF633) was analyzed for all macrophages and for M1 an M2 macrophages in particular. Non-parenchymal liver cell as well as tumor cell suspensions were treated with 20 μM M2pep or scM2pep for 30 min on ice, followed by analysis for the Alexa 633 fluorescence intensity. (B) Representative histograms of the activation status of liver macrophages or TAMs within extracted cell suspensions. Shown are the flow cytometry results of the tumor cell suspension of the TGFα/c-myc mouse model.
Fig 3.
M2pep binding to TAMs in HCC cell suspensions.
Tumor cell suspensions were treated with 20 μM M2pep or scM2pep for 30 min on ice, followed by analysis for the Alexa 633 fluorescence intensity. (A) and (B) Comparison of the median Alexa 633 fluorescence intensity of M2pep in M2 and M1 macrophages in tumor cell suspensions from (A) TGFα/c-myc mice (n = 4) and (B) HepG2 Xenograft nude mice (n = 5). (C) and (D) Comparison of the median Alexa 633 fluorescence intensity between tumor cell suspensions of (C) TGFα/c-myc mice (n = 4) and (D) HepG2 Xenograft nude mice (n = 5) treated with M2pep or scM2pep. Values are means ± SEM; *P<0.05. Statistical analysis was performed with Students t-test; n. s., not significant.
Fig 4.
M2pep binding to macrophages in non-parenchymal liver cell suspensions.
Suspended non-parenchymal liver cells obtained from (A) TGFα/c-myc mice (n = 4) and (B) HepG2 Xenograft nude mice (n = 5) were treated with 20 μM of Alexa 633-labeled M2pep or scM2pep for 30 min on ice. For the identification of the macrophages the cells were stained and analyzed by FACS analysis as described in Fig 2. Values are means ± SEM; *P<0.05. Statistical analysis was performed using Students t-test.
Fig 5.
M2pep shows highly selective binding to TAMs in the dissociated 4T1 tumors that strongly exceeded that in the two dissociated HCCs.
(A) Non-parenchymal liver cell and tumor cell suspensions of 4T1-bearing Balb/c mice were treated with 20 μM M2pep for 30 min on ice. Peptide binding was quantified based on the Alexa 633 fluorescence intensity by flow cytometry as described in Fig 2 (n = 3). (B) and (C) Non-parenchymal liver and tumor cells were prepared simultaneously from HCC-bearing (B) TGFα/c-myc mice (n = 4) or (C) HepG2 Xenograft nude mice (n = 5). The suspended cells were incubated with 20 μM M2pep for 30 min on ice. Subsequently, the cell suspensions were analyzed for association with M2pep by flow cytometry as described in Fig 2. Values are means ± SEM; *P<0.05. Statistical analysis was performed with Students t-test; n. s., not significant.