The B16 melanoma cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Invitrogen). B16 melanoma cells were retrovirally transfected with mouse CRT fragment 39–272 cDNA and a leading sequence at the N-terminal (B16-CRT) or with empty vector (B16-EGFP) using LV5-CRT-Puro lentivirus vector. Transfection efficiency was estimated by enhanced green fluorescence protein (EGFP) fluorescence intensity in the transduced cells that were propagated in medium containing 5 µg/ml Puromycin (Sigma, USA). sCRT in supernatant was detected by Western blot analysis.

Female C57BL/6 (H-2^b) mice and TLR4-knockout mice of C57BL/6 background of 6–8 weeks of age were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). CD45.2 mice on C57BL/6 background and OT-I mice (transgenic for TCR against chicken ovalbumin peptide 257–264) were generously provided by Dr. H Liu (Suzhou University, Suzhou, China). Mouse experimental procedures were performed under specific pathogen-free conditions.

Cells were seeded at 10⁵ per well into 96-well flat-bottomed plates precoated with laminin (10 µg/ml) or fibrinogen (200 µg/ml) and incubated for 30 min. Unadhered cells were washed out twice using PBS, and the remaining cells stained with 0.1% crystal violet for 5 min and dissolved by 30% acetic acid. The plates were then read in a spectrometer (Biotek, WA, USA) at 590 nm. Cell adhesion rate was calculated using unwashed control wells as 100%.

A total of 5 × 10⁶ cells were seeded into six-well plates with three duplicates and scratched with three parallel vertical lines using 10 µl pipettes. The cells were washed three times with PBS and incubated in serum-free DMEM. Cell motility was evaluated by observing, at intervals of 0, 36, and 72 h, the movement of cells into the scratch area under a microscope. The images were recorded and the area of wound gap was measured by Image-J software. Wound closure (%) = [Gap area (T₀ − T)/Gap area T₀] × 100% (where T is the treatment time and T₀ is the time when the wound was first induced).

Groups of mice were s.c. inoculated with viable B16-CRT, or B16-EGFP cells (5 × 10⁶ cells in 100 µl saline) at the left flank. Tumor growth was monitored every 3 days. Solid tumors formed at the site of injections were measured with fine digital calipers and tumor volume was calculated by the following formula: tumor volume = 0.5 × width² × length. Tumor weight was evaluated postscarification.

For tumor metastasis experiments, mice were injected with B16-EGFP or B16-CRT viable cells (1 × 10⁶ cells in 100 µl saline) via the tail vein and monitored for up to 4 weeks. At the end of the experiments, mice were sacrificed for their lungs and livers that were fixed in Bouin’s solution and nodules on the organ surface were counted.

Single cell suspensions were treated with fluorescence-labeled Abs against mouse CRT, CD11b, Gr-1, Ly6G, Ly6C, or CD11c (either alone or in different combination) on ice for 1 h, followed by washes and resuspension in fixation buffer. The stained cells were then subjected to flow cytometric analysis on Attune NxT (Life Technology). FACS data were analyzed using FlowJo software.

Cells were prepared from spleen, bone marrow, peripheral blood, and tumor tissues. Red blood cells were lysed using ACK Lysis Buffer (Beyotime, China).

Single-cell suspensions of splenotes were prepared by mincing mouse spleens on ice and filtering to remove debris and washed twice in PBS before resuspending in RPMI-1640 complete medium.

Tumor tissue was minced on ice and then digested with Collagense Type I (3 mg/ml, Gibco) and DNase I (0.2 mg/ml, Sigma) at 37°C for 2 h. At the end of incubation, preparations were filtered to remove debris and washed twice in PBS before resuspending in RPMI-1640 complete medium.

Single-cell suspensions of splenotes from B16-CRT or B16-EGFP tumor-bearing C57BL/6 mice were sequentially treated with biotinylated antimouse Ly6G⁺ (Miltenyi Biotech) and antibiotin-coated microbeads, the positively labeled cells were then sorted using LS MACS columns according to the manufacturer’s protocol. The purity of G-MDSC populations was over 90%, as determined by flow cytometry, and the viability was higher than 95% as established by exclusion with Trypan blue.

Freshly isolated MDSCs from B16-CRT- or B16-EGFP-bearing mice were incubated for 72 h together with Dye eFluorR 670 (eBioscience, USA)-labeled splenocytes from OT-I mice at a ratio of 1:2 in the presence of 5 µg/ml OVA257–264 peptide (SIINFEKL, Eurogentec, Belgium) in U bottom 96-well plate. Proliferation of Dye eFluorR 670⁺CD8⁺ T cells was analyzed using flow cytometry.

For in vivo MDSC depletion, C57BL/6 mice were i.p. injected with Gemcitabine (LC Laboratories, MA, USA) at 100 mg/kg on days 1, 3, 6, 9, 12, 15, 18, 21, 24, and 27 after B16 tumor inoculation. MDSC depletion efficiency was assessed by flow cytometric analysis on CD11b⁺Gr1⁺ population among the splenocytes.

For in vivo BrdU incorporation assay, C57BL/6 mice (n = 6)-bearing B16 solid tumors for 18 days were i.p. injected with BrdU (10 mg/ml, 200 µl/mouse) and sacrificed 12 or 4 h thereafter for their bone marrow. For in vitro BrdU incorporation assay, BrdU (10 µM final concentration) was added directly to bone marrow culture 2 h prior to harvest. The cells were harvested and stained with fluorescence-labeled anti-CD11b, anti-Ly6G and anti-Ly6C, followed by intracellular staining with anti-BrdU-APC, for flow cytometric analysis.

Freshly isolated bone marrow cells (2 × 10⁷) from C57BL/6 mice (CD45.1 genotype) were injected into the tail veins of tumor-bearing C57BL/6 mice of the CD45.2 genotype. Five days later, the recipient animals were sacrificed for their bone marrow cells that were subsequently stained with fluorescence-labeled antibodies against CD45.1 and MDSC makers for flow cytometric analysis.

Myeloid-derived suppressor cell migration assays were performed in Transwell tissue culture plates, with transwell filters precoated with fibrinogen to prevent cells falling down. MDSCs freshly isolated from spleens of B16 tumor-bearing mice were placed in the chamber on top of that containing sCRT/39-272 or rEGFP. After 3 h incubation, transwell filters were fixed with 4% paraformaldehyde (PFA) in PBS at RT for 30 min, followed by wiping away the remaining cells in the top chamber and subsequent staining of cell nucli with DAPI for observation under a fluorescence microscope (Nikon A1, Japan).

B16-CRT and B16-EGFP solid tumor tissues from C57BL/6 mice were cut into 5-µm frozen sections and fixed in ice-cold acetone for 30 min. Sections were incubated sequentially with anti-Gr1 (Invitrogen) or Alexafluor 647-labeled goat anti-rat IgG (Invitrogen) secondary antibodies, respectively. The stained sections were analyzed by confocal scanning laser microscopy.

All statistical analyses were performed with GraphPad Prism Software version 4. Unpaired Student’s t-tests were used to compare differences between the groups. Error bars represent the SEM of the mean. A p value of 0.05 was considered statistically significant.

Although CRT is constitutively expressed in cells and can be released in soluble form during cell death, studies on the contribution of tumor-derived sCRT in tumor formation and development face many obstacles. In the present study, we constructed a murine B16 melanoma cell line expressing recombinant sCRT39-272 (B16-CRT), with recombinant EGFP (rEGFP) as a coexpression marker. B16 cells expressing rEGFP (B16-EGFP) alone were included as control (Figures 1A,B). There was virtually no difference between these two cell lines in terms of spontaneous proliferation, adhesion to lamnin- or fibrinogen-coated surface, and Transwell migration (Figures 1C–E), indicating that sCRT39-272 expression per se was unlikely to have direct effects on B16 malignancy. When s.c. inoculated into C57BL/6 mice (5 × 10⁶/mouse), however, the B16-CRT line appeared to be much more aggressive than the B16-EGFP control. Solid tumor formation at the sites of injection was observable 12 days postinoculation of the B16-CRT cells, 3 days earlier than that of the B16-EGFP control (Figure 1F). By day 21 after inoculation, average volume and weight of the B16-CRT solid tumors were by far more than that of the B16-EGFP controls (Figures 1G,H). It should also be noted that, when B16-CRT and B16-EGFP cells were i.v. injected into C57BL/6 mice (10⁶/mouse), there was no significant difference between the two groups in terms of size and number of solid tumor nodules on the surface of their lungs and livers 4 weeks postinoculation (Figure 1I), suggesting that the expression of sCRT39-272 did not render B16 cells more metastatic.

Given that tumor cell-derived sCRT39-272 was unable to directly promote B16 cell proliferation (Figure 1C), its malignancy enhancing effect has to be explained by alternative mechanisms such as proangiogenesis or immunosuppression. Contradictory reports on the antiangiogenisis roles of the CRT N domain have been documented (6, 16). In our hands, recombinant sCRT39-272 was unable to exert any measurable suppression or promotion effect on the growth of blood vessel endothelial cells in vivo (data not shown). Consistent with our notion that sCRT39-272 might be able to facilitate the differentiation/generation of MDSCs through CD14/TLR4 signaling, the number of Gr1⁺ cells in tissue sections of B16-CRT solid tumors (harvested from C57BL/6 mice 21 days postinoculation) was substantially higher than that of the B16-EGFP group (Figure 2A), which is confirmed by flow cytometric analysis for CD11b⁺Gr-1⁺ cells among the disseminated cells from the solid tumor tissues (Figure 2B).

The increased presence of CD11b⁺Gr1⁺ MDSCs in B16-CRT solid tumors could be the results of selective chemotaxis and/or de novo expansion. The MDSC chemotaxis potential of sCRT39-272 was confirmed by Transwell assays in which sCRT39-272 was significantly more effective than rEGFP in inducing MDSC migration (Figure 2C). Additionally, sCRT39-272 was also able to block their apoptosis in vitro (Figure 2D). These results indicate that sCRT/39-272 has multiple impacts on the survival and chemotaxis of MDSCs.

CD11b⁺ immature myeloid cells (IMCs), derived from hematopoietic stem cells in the bone marrow, are the source of common myeloid progenitor cells that differentiate into DCs (CD11b⁺CD11c⁺) or macrophages (CD11b⁺F4/80⁺) upon appropriate stimulation in peripheral organs (17). In the case of tumor-bearing individuals, however, IMCs accumulate in tumor microenvironment where they stop differentiation and functionally behave as MDSCs (17), which is accompanied by increased IMC percentage in the bone marrow as well as periphery (18). Based on our above shown results, we reasoned that tumor-derived sCRT39-272 might further enhance IMC/MDSC generation/proliferation in tumor-bearing animals. To assess this possibility, BrdU was i.p. injected into C57BL/6 mice-bearing B16-CRT or B16-EGFP solid tumors, 12 h later BrdU-positive cells among bone marrow CD11b⁺Ly6G⁺Ly6C^low G-MDSCs and CD11b⁺Ly6G⁻Ly6C^high M-MDSCs were numerated by flow cytometry. As shown in Figure 3A, in the G-MDSC population of bone marrow cells from B16-CRT-bearing mice, BrdU-positive cells accounted for 28.7%, significantly higher than that (15.2%) from the B16-EGFP controls, indicating accelerated proliferation of these T cell suppressing cells in the presence of sCRT-producing tumors. However, there was no significant difference in the proportion of BrdU-positive cells among bone marrow M-MDSC subset from mice-bearing B16-CRT or B16-EGFP tumors (66.2 vs. 67.8%). The selective expansion of G-MDSCs by rCRT was further confirmed by in vitro experiments, in which bone marrow cells were treated in GM-CSF-conditioned medium in the presence of sCRT/39-272 (Figure 3B).

Increased proliferation of G-MDSCs in the bone marrow of tumor-bearing mice was also reflected in the periphery. Compared with the B16-EGFP-bearing animals, B16-CRT-bearing mice showed a significant increase of G-MDSCs among splenocytes (13 vs. 7%) and WBCs (23 vs. 16%) (Figure 3C). Percentages of M-MDSCs among splenocytes and WBCs remained relatively low in both groups and difference between them was not statistically significant.

To exclude the possibility that the more significant generation of G-MDSCs in B16-CRT-bearing mice was purely due to the larger size of the solid tumors, C57BL/6 mice (CD45.2)-bearing B16-EGFP or B16-CRT tumors (inoculated 17 or 10 days earlier, respectively) of approximately the same size were adoptively transferred with CD45.1⁺ bone marrow cells (freshly isolated from CD45.1 transgenic mice), 5 days later MDSC percentage among CD45.1⁺ cells in the periphery of the recipient mice were monitored. Figure 3D shows that proportion of G-MDSCs in the CD45.1⁺ population in the spleen, blood, and solid tumors was significantly higher in B16-CRT-bearing mice than that in B16-EGFP controls. By contrast, percentage of M-MDSCs among the CD45.1⁺ population in the blood was significantly lower in B16-sCRT-bearing mice than that in B16-EGFP controls (7 vs. 19%), while there was no significant difference in the spleen and solid tumors. These results together suggest that the development of sCRT-secreting solid tumors is associated with more significantly increased proliferation/generation and output of G-MDSCs in vivo.

T lymphocytes are the main targets for MDSC suppression. To assess whether sCRT-generated MDSCs were functionally different from their conventional counterparts, MDSCs isolated from mice-bearing B16-CRT and B16-EGFP tumors were compared for ability to inhibit T cell proliferation in vitro, no significant difference was observed (Figure 3E).

Next, we asked whether B16-CRT tumors would be more powerful than B16-EGFP tumors in blocking MDSC differentiation into DCs or macrophages in vivo, which could be reflected by percentage changes in DCs and/or macrophages in the periphery. Figure 4A shows that percentage of DCs (but not macrophages) among WBCs of B16-CRT-bearing mice was significantly lower than that of B16-EGFP-bearing animals (5.5 vs. 12.5%). Decreased DC percentage in the spleen and tumor tissues was also observed in B16-CRT-bearing mice albeit not statistically significant. To further assess whether sCRT could directly suppress differentiation of MDSCs toward DCs, murine splenic G-MDSCs derived from tumor-bearing mice were incubated with GM-CSF for 5 days in the presence of recombinant sCRT39-272 or rEGFP followed by flow cytometric analysis for CD11b⁺CD11c⁺ DCs. Apparently sCRT39-272, but not rEGFP, substantially inhibited DC differentiation in vitro (Figure 4B).

S100A8 and S100A9 are proinflammatory proteins essential for functional maturation and chemotactic migration of MDSCs (19, 20). Interestingly, S100A8/9 mRNA expression level, quantitated by Q-PCR, was significantly higher in murine splenic G-MDSCs under stimulation with sCRT39-272 compared with rEGFP. This difference was partially (S100A8), or completely (S100A9), diminished when murine splenic G-MDSCs from TLR4-knockout mice were employed (Figure 4C), indicative of the involvement of TLR4 in sCRT-assisted G-MDSC generation which is in line with our previous observation that sCRT39-272 activates myeloid cells through interaction with CD14 and TLR4 (10). The same phenomenon was also observed on protein levels as evidenced by Western blotting assays on splenic MDSCs of tumor-bearing mice treated with sCRT39-272 or rEGFP (Figure 4D). Moreover, neutralizing antibodies against S100A8/9 effectively reversed sCRT39-272-mediated suppression of DC differentiation from G-MDSC as well as sCRT39-272 inducing G-MDSC migration in vitro (Figures 4E,F). Note that G-MDSCs from TLR4-knockout mice were poor responders to sCRT-induced differentiation toward DCs and migration in vitro (Figures 4G,H). Thus, sCRT39-272 could directly interact with MDSCs derived from tumor-bearing animals through TLR4, leading to production of S100A8/9, which in turn blocks MDSC differentiation. This at least partially explains the increased number of G-MDSCs in B16-CRT tumor-bearing mice.

Data accumulated thus far indicate that the malignancy-enhancing effect of sCRT/39-272 is related to TLR4⁺ MDSCs in vivo. Indeed, when TLR4-knockout mice were employed as recipients to B16-CRT and B16-EGFP tumors, no significant differences in tumor size, G-MDSC percentage in the blood and spleen, and infiltrating MDSCs in solid tumor tissues were observed between the two groups (Figures 5A,B). Taking this further, gemcitabine was utilized to selectively deplete MDSCs in tumor-bearing C57BL/6 mice (21). The depletion efficacy was confirmed by significant reduction of CD11b⁺Gr1⁺ MDSCs in tumor-bearing mice given gemcitabine injections (Figure 5C). In the MDSC-depleted animals, no significant difference was observed between the B16-CRT and B16-EGFP groups in terms of solid tumor growth (volume and weight) up to 27 days after inoculation (Figures 5D,E), strongly supporting our hypothesis that MDSCs are essentially important for the enhanced malignancy of B16-CRT tumor cells. It is also of interest to note that gemcitabine treatment reversed the suppression of CD4⁺ and CD8⁺ T cell proportions in B16-CRT-bearing animals in these experiments (Figure 5C).