Mechanisms by which tumor cells metastasize to distant organs still remain enigmatic. Immune cells have been assumed to be the root of metastasis by their fusing with tumor cells. This fusion theory, although interpreting tumor metastasis analogically and intriguingly, is arguable to date. We show in this study an alternative explanation by immune cell–derived microparticles (MPs). Upon stimulation by PMA or tumor cell–derived supernatants, immune cells released membrane-based MPs, which were taken up by H22 tumor cells, leading to tumor cell migration in vitro and metastasis in vivo. The underlying molecular basis was involved in integrin αMβ2 (CD11b/CD18), which could be effectively relayed from stimulated innate immune cells to MPs, then to tumor cells. Blocking either CD11b or CD18 led to significant decreases in MP-mediated tumor cell metastasis. This MP-mediated transfer of immune phenotype to tumor cells might also occur in vivo. These findings suggest that tumor cells may usurp innate immune cell phenotypes via MP pathway for their metastasis, providing new insight into tumor metastatic mechanism.

Crosstalk between tumor cells and their surrounding immune microenvironment is critical for tumor metastasis. Despite the killing effect of immune cells such as CTLs and NK cells, tumor cells actually are capable of devising various strategies to hijack immune cells, especially innate immune cells such as immature myeloid cells, macrophages, and mast cells for their metastasis (13). These strategies may be generally summarized as follows: the release of proangiogenic factors by activated immune cells, such as vascular endothelial growth factor, thus favoring tumor metastasis (4, 5); the release of proteases, cytokines, and growth factors by tumor-infiltrating leukocytes to potentiate the migration and invasion of malignant cells (6, 7); and premolding a favorable metastatic microenvironment by myeloid precursor cells for the homing and survival of the foreign tumor cells (8, 9). However, how and whether other mechanisms through which immune cells contribute to tumor metastasis still remain largely unexplored.

Migration is a fundamental feature of immune cells, by which immune cells interact with and extravasate endothelium to inflammatory sites (10). Notably, tumor cells also interact with and extravasate endothelial cells so to colonize in foreign tissues. According to these similarities, it is possible for tumor cells to usurp immune element(s) to cross blood vessels for their metastasis. Integrins have been well demonstrated as pivotal adhesion receptor molecules to mediate immune cell migration (11, 12). These immune-related integrins typically include LFA-1 (αLβ2 integrin or CD11a/CD18) and Mac-1 (αMβ2 integrin or CD11b/CD18). However, neither LFA-1 nor Mac-1 is usually expressed by tumor cells. Notwithstanding, this discordance may be reconciled by the old cell fusion theory, that is, the join of cancer cell and immune cell confers malignant cell with an immune phenotype to migrate through the bloodstream (1316). Alternatively, recently studies might provide a further interpretation by immune cells releasing microparticles (MPs) and tumor cells taking them up (17, 18).

Cells are capable of generating various vesicular vesicles with different sizes. In response to various stimuli, cells may change their cytoskeletal structure and result in plasma membranes encapsulating cytosolic elements that are released into the extracellular space. These specialized subcellular vesicles are called MPs with 100–1000 nm in diameter (19, 20). MPs not only contain messenger molecules, enzymes, RNAs, and even DNA, but also are capable of transferring these bioactive molecules from one cell to another (21, 22). Thus, MPs functionally appear to act as vectors to deliver molecular messages between cells, implying that tumor cells might obtain immune phenotypes through MP pathways. The present study shows that the immune adhesion molecule αMβ2 integrin is transferred from innate immune cell to hepatocarcinoma tumor cells via MP pathways. As a result, tumor cells acquire a transient immune phenotype, subsequently metastasize, and colonize in distant organs.

Murine hepatocarcinoma cell line H22 and murine B16 melanoma tumor cell line were obtained from China Center for Type Culture Collection (Wuhan, China) and cultured according to the guidelines. Female BALB/c mice, 6–8 wk old, were purchased from Center of Medical Experimental Animals of Hubei Province (Wuhan, China) for studies approved by the Animal Care and Use Committee of Tongji Medical College.

PMA was purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse CD11b and CD18 blocking Abs were purchased from BioLegend (San Diego, CA). PE-conjugated anti-mouse CD11b and CD3 Abs; allophycocyanin-conjugated anti-mouse MHC-II; and FITC-conjugated anti-mouse CD18, F4/80, Gr-1, and CD19 Abs were purchased from eBioscience (San Diego, CA).

Mouse splenic cells were cultured in the presence or absence of 50 ng/ml PMA for 12 h. Cell viability was determined at the end of the incubation period by trypan blue staining. Viability was found to be >97% in all experiments. The supernatants from activated or resting cells were harvested and centrifuged for 10 min at 2,500 × g and then for 2 min at 14,000 × g to get rid of cells and cellular debris. Then the supernatant was further centrifuged for 60 min at 20,000 × g to pellet MPs. Isolated MPs were suspended with PBS that was prefiltered through 0.1-μm filter and passed through 1-μm filter to further exclude background noise or nonspecific events for the following experiments. In this study, 3 × 105 MPs were yielded from 5 × 106 immune cells. A total of 6 × 106 MPs mixed with 3 × 105 tumor cells (a ratio of 20:1) was used in the following experiments.

MPs were passed through 1-μm filter and fixed at room temperature for 60 min with 4% paraformaldehyde in 0.01 M PBS. After washing with PBS, the preparations were postfixed in 1% OsO4 (Taab) for 30 min. After rinsing with distilled water, the pellets were dehydrated in graded ethanol, including block staining with 1% uranylacetate in 50% ethanol for 30 min, and embedded in Taab. After overnight polymerization at 60°C and sectioning for electron microscope, the ultrathin sections were analyzed with a JEM1010 electron microscope (JEOL).

Isolated MPs were labeled with a red fluorescent cell linker (PKH26; Sigma-Aldrich), according to the manufacturer’s protocol. Then labeled MPs were coincubated with CFSE-stained H22 cells at 37°C for 20 h. H22 cells taking up MPs were washed three times and observed under a two-photon fluorescent microscope (LSM 710 and ConfoCor 3 systems; Carl Zeiss).

Isolated MPs were labeled with a green-fluorescent cell linker (PKH67; Sigma-Aldrich) and coincubated with H22 cells at 37°C for 20 h. Then cells were incubated with Lyso-, ER-, or Golgi-Tracker Red (Beyotime), respectively, according to the manufacturer’s protocol, and visualized by two-photon confocal microscope.

Isolated MPs were suspended in 250 μl PBS with 3 μm nonfluorescent beads (LB-30; Sigma-Aldrich). After mixing, beads were run to control for MP size and count the number of MPs by a flow cytometer (BD LSR II flow cytometer; BD Biosciences). The forward and side scatters were set at logarithmic gain. If 10,000 counts of LB30 were collected, the number of MP can be calculated with the following formula: n = 10,000 × (MP percentage/LB30 percentage).

Fluorescent dye-conjugated anti-mouse CD3, CD11b, CD18, CD19, Gr-1, F4/80, and MHC-II Abs were added at the appropriate dilution to prewashed MPs or H22 cells, respectively. MPs or H22 cells were kept on ice for 30 min, resuspended in 300 μl PBS, and then analyzed by flow cytometry, using CellQuest software (BD Biosciences).

A transwell system that incorporated a polycarbonate filter membrane with a diameter of 6.5 mm and pore size of 8 μm (Corning) was used to assess the migration and metastasis of H22 and B16 cells. The Matrigel (5 μg in 10 μl serum-free RPMI 1640 medium) was added to the filter to form a thin gel layer and dried in a hood overnight. Tumor cells were suspended in 200 μl medium and added to the upper chamber of the transwell insert. The lower chamber was filled with 600 μl medium containing 20% FBS. After 20 h of incubation at 37°C, B16 cells on the upper surface of the filter were removed by using a cotton swab. B16 cells that penetrated to the lower surface of the filter were fixed in methanol, and then sections were stained with hematoxylin and observed under a microscope. CFSE-prestained H22 cells that transferred to the lower chamber were directly counted using a fluorescent microscope.

H22 liver tumors were collected and weighted, and single-cell suspensions were prepared. Briefly, 100 mg tumors were cut into small pieces and incubated with collagenase, hyaluronidase, and DNase I for 1 h at 37°C and homogenized with semifrosted slides. All enzymes were purchased from Sigma-Aldrich. After lysis of RBCs, the suspensions were fractionated by centrifugation on a Ficoll (GE Healthcare, Piscataway, NJ) density gradient, and tumor-infiltrating leukocytes (TILs) were harvested from the 75∼100% Ficoll interface.

H22 tumor cells (3 × 105) were i.v. injected into BALB/c mice (n = 6 per group). After tumor formation, photos of the mice were taken. Scarified mice were used for tumor detection, and the left mice were fed for the long-term survival study. To study the blockade effects of CD18 and CD11b molecular, MP-treated H22 cells were i.v. injected to BALB/c mice, and these mice were administrated with 30 μg anti-CD18 Ab twice, 1 h before and 12 h after the tail vein injection of H22 cells.

All experiments were performed three times. Results were expressed as mean values ± SD and interpreted by repeated-measure ANOVA. The p values <0.05 were considered statistically significant. The analysis was conducted using the SPSS 11.0 software.

Given the capability of cells to release MPs upon activation (23), CFSE-labeled splenic cells were stimulated with 50 ng/ml PMA for 12 h. The released MPs were confirmed by the observed fluorescent dots under two-photon fluorescent microscope (Fig. 1A). The structure and size of MPs were further determined under electron microscope (Fig. 1B). In parallel, the flow cytometric analysis showed ∼6-fold increases in the production of MPs after PMA stimulation (Fig. 1C).

FIGURE 1.

Characterization of immune cell–derived MPs. (A) CFSE-labeled splenic cell–derived MPs were observed under two-photon confocal microscope. (B) The structure and size of MPs were observed by electron microscopy. (C) Splenic cells were cultured in the presence or absence of 50 ng/ml PMA for 12 h. The released MPs were counted by flow cytometry. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments. The number of MPs in PBS group was designed as 1. #p < 0.001 compared with PBS group. (D) MPs were stained with different fluorescent dye-conjugated Abs and analyzed by flow cytometry.

FIGURE 1.

Characterization of immune cell–derived MPs. (A) CFSE-labeled splenic cell–derived MPs were observed under two-photon confocal microscope. (B) The structure and size of MPs were observed by electron microscopy. (C) Splenic cells were cultured in the presence or absence of 50 ng/ml PMA for 12 h. The released MPs were counted by flow cytometry. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments. The number of MPs in PBS group was designed as 1. #p < 0.001 compared with PBS group. (D) MPs were stained with different fluorescent dye-conjugated Abs and analyzed by flow cytometry.

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To clarify immune phenotypes, these MPs were stained with fluorescent dye-conjugated Abs against CD3, CD19, Gr-1, F4/80, and MHC-II and analyzed by flow cytometry. MPs from the normally cultured splenic cells showed a low proportion of CD3, CD19, Gr-1, F4/80, or MHC-II expression. In contrast, upon PMA stimulation, the proportion of Gr-1+ or F4/80+ MPs, but not CD3+ or CD19+ MPs, was significantly increased (Fig. 1D), suggesting that, relative to T or B cells, myeloid cells probably are the major donors for MPs under PMA stimulation.

Next, we clarified whether immune phenotypes on the above immune MPs could be transferred to tumor cells. A murine hepatocarcinoma tumor cell line H22 was used to address this question. After 20-h incubation of the above CFSE-labeled splenic MPs with H22 cells, it was found that ∼37% H22 cells were CFSE positive in PMA-MP group versus ∼10% H22 cells in PBS-MP group (Fig. 2A). In contrast, PKH26-labeled MPs (red) and CFSE-labeled H22 cells (green) were coincubated for 20 h. The confocal yellow dots were observed under two-photon laser microscope (Fig. 2B), suggesting that MPs are taken up by, but not adhered to, H22 tumor cells. To clarify the subsequent transfer of immune features to tumor cells, the above H22 cells were stained with fluorophore-conjugated Ab against CD3, CD19, Gr-1, F4/80, or MHC-II molecule and analyzed by flow cytometry. As expected, a panel of immune cell markers was indeed expressed by H22 cells, which were much enhanced in PMA-MP group (Fig. 2C), suggesting that immune cell–derived MPs may transfer immune phenotypes to tumor cells. Intriguingly, this transfer pathway appeared not to be mediated through endosome/lysosome systems, as the analysis by cytofluorescent staining did not show the colocalization of immune MPs with the lysosomes (Fig. 2D). Moreover, those entered MPs did not link up with the ER or Golgi apparatus (Fig. 2E, 2F), suggesting MPs probably end up in the cytoplasm of H22 tumor cells.

FIGURE 2.

MPs transfer immune phenotypes to H22 tumor cells. (A) H22 cells took up MPs. CFSE-labeled splenic cells were treated with or without 50 ng/ml PMA. Twelve hours later, MPs in the supernatants were isolated and incubated with H22 cells for 20 h. CFSE-positive H22 cells were analyzed by flow cytometry. (B) Splenic cells were treated with or without 50 ng/ml PMA. The released MPs were labeled with PKH26 (red) and incubated with CFSE-labeled H22 cells (green). Cells were observed under two-photon confocal microscope. (C) MPs conferred immune phenotypes to tumor cells. Splenic cells were stimulated with or without PMA. The released MPs were incubated with H22 cells for 20 h. CD3, CD19, Gr-1, F4/80, and MHC-II on the surface of H22 cells were analyzed by flow cytometry (#p < 0.001, compared with PBS group). (DF) Splenic MPs were not present in lysosomes, ER, or Golgi in H22 cells. CFSE-labeled splenic cells were stimulated with PMA for 12 h. The isolated MPs were incubated with H22 cells for 20 h, and cells were analyzed with lysosome, ER, and Golgi Red Trackers, respectively, under two-photon confocal microscope. Scale bars, 10 μm.

FIGURE 2.

MPs transfer immune phenotypes to H22 tumor cells. (A) H22 cells took up MPs. CFSE-labeled splenic cells were treated with or without 50 ng/ml PMA. Twelve hours later, MPs in the supernatants were isolated and incubated with H22 cells for 20 h. CFSE-positive H22 cells were analyzed by flow cytometry. (B) Splenic cells were treated with or without 50 ng/ml PMA. The released MPs were labeled with PKH26 (red) and incubated with CFSE-labeled H22 cells (green). Cells were observed under two-photon confocal microscope. (C) MPs conferred immune phenotypes to tumor cells. Splenic cells were stimulated with or without PMA. The released MPs were incubated with H22 cells for 20 h. CD3, CD19, Gr-1, F4/80, and MHC-II on the surface of H22 cells were analyzed by flow cytometry (#p < 0.001, compared with PBS group). (DF) Splenic MPs were not present in lysosomes, ER, or Golgi in H22 cells. CFSE-labeled splenic cells were stimulated with PMA for 12 h. The isolated MPs were incubated with H22 cells for 20 h, and cells were analyzed with lysosome, ER, and Golgi Red Trackers, respectively, under two-photon confocal microscope. Scale bars, 10 μm.

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Next, we tested whether immune cell–derived MPs affected tumor cell metastasis by transferring immune components to tumor cells. Hence, the above MP-treated H22 cells (3 × 105) were injected into mice via their tail veins. Surprisingly, PMA-MP–treated H22 cells formed metastatic tumors in various forms in the mice. The tumors were able to grow in the thorax, abdomen, four limbs, and even genitalia (Fig. 3A). In contrast, control MP-treated H22 cells only caused one mouse tumor formation in six mice (data not shown). Similarly, the injection of untreated H22 cells did not cause the tumor formation (Fig. 3A). In line with these data, mice in both control MP group and untreated group maintained the long-term survival, but most mice in the PMA-MP group died within a short time (Fig. 3B). In line with these in vivo data, PMA-MPs were found to effectively promote tumor cell migration and invasion in vitro. After coincubation with MPs for 20 h, CFSE-labeled H22 cells were added to the Matrigel-precoated upper chamber of a transwell system with serum free in the upper chamber and 20% FBS in the lower chamber. As shown in Fig. 3C, control MPs did not affect the migration of H22 cells. PMA-MPs, however, caused ∼10-fold increases in migration and invasion of H22 cells. A similar result was also observed in B16 cells (Fig. 3D). To exclude the possible effect of PMA, we used splenic cell–free medium containing PMA to incubate with H22 cells for 20 h. We found that this PMA-containing medium did not promote the migration of H22 tumor cells, suggesting that the remnant PMA has no effect on H22 tumor cell metastasis (Supplemental Fig. 1). In addition, in contrast to the above immune-derived MPs, we did not find that nonimmune-derived fibroblast cell MPs had effect on B16 cell migration and invasion (Fig. 3D). Consistently, these nonimmune cell–derived MPs did not affect the long-term survival of mice after inoculation of MP-treated H22 cells (Fig. 3B). In the above metastasis experiments, we used 6 × 106 MPs to incubate with 3 × 105 tumor cells. In this study, we further used 3 × 106 and 3 × 105 MPs, respectively, to incubate with 3 × 105 tumor cells. In line with the above data, we found that the 10:1 ratio resulted in the metastatic tumor formation almost in all the tested mice (7 of 8) and the 1:1 ratio caused the tumor formation in partial mice (3 of 8), suggesting that large number of immune cell MPs might not be requisite to mediate tumor cell metastasis.

FIGURE 3.

PMA-induced splenic MPs promote H22 tumor cell metastasis. (A) H22 cells (3 × 105) treated with or without splenic MPs were injected into mice via their tail vein. The typical tumor growth was shown. Tumor sites were indicated by arrows. (B) The long-term survival of tumor-bearing mice was analyzed by Kaplan-Meier method (n = 6 for each group, p < 0.001). Nonimmune cell–derived MPs (3T3-MP) did not affect the long-term survival of mice after inoculation of H22 cells. (C) PMA-stimulated splenic MPs induced H22 hepatocarcinoma cell migration. The migration and invasion of H22 cells in transwell assay were determined in the presence of PMA-MPs, PBS-MPs, or PBS. *p < 0.05 compared with control group. (D) PMA-stimulated splenic MPs induced B16 melanoma cell migration. Transwell assay was conducted as described in 2Materials and Methods. B16 cells that migrated to the lower surface were stained with hematoxylin and counted under microscope. #p < 0.01 compared with control-MP group. The 3T3 fibroblast cell–derived MPs were also assayed as additional control.

FIGURE 3.

PMA-induced splenic MPs promote H22 tumor cell metastasis. (A) H22 cells (3 × 105) treated with or without splenic MPs were injected into mice via their tail vein. The typical tumor growth was shown. Tumor sites were indicated by arrows. (B) The long-term survival of tumor-bearing mice was analyzed by Kaplan-Meier method (n = 6 for each group, p < 0.001). Nonimmune cell–derived MPs (3T3-MP) did not affect the long-term survival of mice after inoculation of H22 cells. (C) PMA-stimulated splenic MPs induced H22 hepatocarcinoma cell migration. The migration and invasion of H22 cells in transwell assay were determined in the presence of PMA-MPs, PBS-MPs, or PBS. *p < 0.05 compared with control group. (D) PMA-stimulated splenic MPs induced B16 melanoma cell migration. Transwell assay was conducted as described in 2Materials and Methods. B16 cells that migrated to the lower surface were stained with hematoxylin and counted under microscope. #p < 0.01 compared with control-MP group. The 3T3 fibroblast cell–derived MPs were also assayed as additional control.

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The above data suggested that immune cell–derived MPs confer certain immune components to tumor cells, leading to tumor cell migration and metastasis. To identify immune molecule(s) in MPs that mediates tumor cell metastasis, we focused on β2 integrin (CD18), the critical molecule in mediating immune cell extravasation (24, 25). As expected, CD18 was found to be expressed in the MPs and upregulated after PMA stimulation (Fig. 4A). Moreover, in the transwell assay, the addition of anti-CD18 blocking Ab to the upper chamber effectively inhibited H22 cells migrating to the low chamber (Fig. 4B). To confirm the specificity of this anti-CD18 Ab, a CD3 blocking Ab was also tested in this study. We found that the addition of this control Ab did not affect the MP-mediated H22 tumor cell migration (Supplemental Fig. 2). To further validate the above data in vivo, BALB/c mice were i.v. injected with PMA-MP–treated H22 cells, and then administrated with 50 μg anti-CD18 Ab twice, 1 h before and 12 h after tumor cell injection. We found that the administration of CD18 Ab effectively inhibited the effect of MPs on tumor development and prolonged the survival of mice (Fig. 4C), suggesting that CD18 is essential for MPs to mediate H22 cell metastasis. In addition, we found that the transferred CD18 molecules were relatively stable on the surface of tumor cells. After 12-h incubation of the MPs with H22 cells (50 μg cytomycin C added in the last half hour), H22 cells were continued to culture and analyzed by flow cytometry at different time points. A high level of CD18 could be maintained on the surface of H22 cells at least for 12 h, and the fluorescence decay could last for 24 h (Fig. 4D). Also, we did not find that the remnant PMA in MPs induced the expression of CD18 or CD11b in H22 tumor cells (Supplemental Fig. 1).

FIGURE 4.

CD11b/CD18 mediates H22 tumor cell metastasis. (A) Splenic cells were stimulated with 50 ng/ml PMA for 12 h. CD18 expression on the surface of released MPs was detected by flow cytometry. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments. #p < 0.001 compared with PBS group. (B) CD11b and CD18 molecules were involved in MP-mediated H22 tumor cell migration. The migration of CFSE-labeled H22 cells in transwell assay was determined in the presence of either anti-CD18 or anti-CD11b blocking Ab. (C) CD11b/CD18 was involved in MP-mediated H22 tumor cell metastasis in vivo. PMA-MP–treated H22 cells were i.v. injected to BALB/c mice. The mice were administrated with 30 μg anti-CD18 or anti-CD11b Ab twice, 1 h before and 12 h after the tail vein injection. The long-term survival was analyzed. The results were combined from two reproducible experiments (n = 6 for each group, p < 0.001). (D) CD18 on H22 cells was relatively stable. After 12-h incubation of PMA-MPs with H22 cells (50 μg cytomycin C added in the last half hour), H22 cells were continued to culture for 4, 12, and 24 h, respectively. The expression of CD18 by H22 cells was analyzed by flow cytometry.

FIGURE 4.

CD11b/CD18 mediates H22 tumor cell metastasis. (A) Splenic cells were stimulated with 50 ng/ml PMA for 12 h. CD18 expression on the surface of released MPs was detected by flow cytometry. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments. #p < 0.001 compared with PBS group. (B) CD11b and CD18 molecules were involved in MP-mediated H22 tumor cell migration. The migration of CFSE-labeled H22 cells in transwell assay was determined in the presence of either anti-CD18 or anti-CD11b blocking Ab. (C) CD11b/CD18 was involved in MP-mediated H22 tumor cell metastasis in vivo. PMA-MP–treated H22 cells were i.v. injected to BALB/c mice. The mice were administrated with 30 μg anti-CD18 or anti-CD11b Ab twice, 1 h before and 12 h after the tail vein injection. The long-term survival was analyzed. The results were combined from two reproducible experiments (n = 6 for each group, p < 0.001). (D) CD18 on H22 cells was relatively stable. After 12-h incubation of PMA-MPs with H22 cells (50 μg cytomycin C added in the last half hour), H22 cells were continued to culture for 4, 12, and 24 h, respectively. The expression of CD18 by H22 cells was analyzed by flow cytometry.

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Functionally, CD18 combines with integrin αL (CD11a) to form LFA-1 on T cells or B cells, or bind integrin αM (CD11b) to form Mac-1 on macrophages or other innate cells (26). We therefore separated CD3+ T cells, CD19+ B cells, and CD3CD19 innate immune cells from spleen to clarify the roles of LFA-1 and Mac-1 in MP-mediated tumor metastasis. After PMA stimulation, T, B, or innate cell–derived MPs were used for the transwell assay. Intriguingly, neither T cell– nor B cell–generated MPs had the effect on H22 cell migration (Fig. 5A). In contrast, CD3CD19 innate MPs were capable of promoting H22 cell migration similar to bulk MPs (Fig. 5A). We further tested the above MPs in vivo. We found that T cell– and B cell–generated MPs did not result in the tumor formation, but CD3CD19 innate MPs caused the tumor formation in the tested mice (Supplemental Fig. 3). Corroboratively, the blockade of CD11b resulted in the inhibition of MP-mediated H22 tumor cell migration in vitro and H22 tumor metastasis in vivo (Fig. 4A, 4B). In addition, the expression of CD11b was found to be upregulated in innate MPs, but not in T or B cell MPs, although the expression of CD18 was upregulated in both innate MPs and T or B cell MPs (Fig. 5B). Consistently, H22 cells, only when incubated with innate MPs rather than T or B cell MPs, could simultaneously express CD11b and CD18 on their surfaces (Fig. 5C). These findings together suggested that innate immune cell–generated MPs are capable of promoting H22 tumor cell metastasis.

FIGURE 5.

Innate immune cells are the source of MPs that mediate H22 tumor cell migration. (A) CD3+ T, CD19+ B, and CD3CD19 innate immune cells were separated from splenic cells and stimulated with PMA. The isolated MPs were used to treat H22 cells for the transwell assay. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments (#p < 0.01 compared with untreated H22 cell group). (B) The expressions of CD11b and CD18 by MPs derived from T, B, or innate immune cells were detected by flow cytometry. (C) MPs transferred CD11b/CD18 to H22 cells. H22 cells were incubated with different MPs for 20 h. The expressions of CD11b and CD18 on H22 cells were detected by flow cytometry.

FIGURE 5.

Innate immune cells are the source of MPs that mediate H22 tumor cell migration. (A) CD3+ T, CD19+ B, and CD3CD19 innate immune cells were separated from splenic cells and stimulated with PMA. The isolated MPs were used to treat H22 cells for the transwell assay. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments (#p < 0.01 compared with untreated H22 cell group). (B) The expressions of CD11b and CD18 by MPs derived from T, B, or innate immune cells were detected by flow cytometry. (C) MPs transferred CD11b/CD18 to H22 cells. H22 cells were incubated with different MPs for 20 h. The expressions of CD11b and CD18 on H22 cells were detected by flow cytometry.

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We then further addressed the question of whether TIL-derived MPs also promote tumor metastasis. TILs, isolated from H22 tumors, were cultured in the presence or absence of frozen-thawed H22 cell supernatants to generate MPs. We found that these TIL-derived MPs also expressed CD11b and CD18, which could be transferred to H22 cells after incubation (Fig. 6A, 6B). Similar to splenic MPs above, the unstimulated TIL-derived MPs just had a minimal influence on H22 tumor cell migration, whereas the stimulated TIL-generated MPs significantly increased (∼11-fold) cell migration (Fig. 6C). Consistently, blocking CD18 or CD11b by the Ab led to the impairment of cell migration (Fig. 6C). To verify the influence of TIL-derived MPs on tumor metastasis in vivo, H22 cells were treated with TIL-derived MPs and i.v. injected to BALB/c mice. As expected, MPs from rest TILs did not enhance the formation of metastatic tumors; however, MPs from stimulated TIL facilitated H22 cells to grow tumors at unsettled sites (Fig. 6D). In addition, the long-term survival experiment showed that mice survived very well in the rest TIL-MP group and died within 50 d in the stimulated TIL-MP group. Consistent with the previous data, the administration of anti-CD18 or anti-CD11b Ab prevented more than half of the mice from death (Fig. 6E).

FIGURE 6.

TIL-derived MPs promote H22 cell metastasis. (A) The isolated TILs were cultured in frozen-thawed H22 supernatant (FTS) for 12 h. The released MPs were isolated to analyze the expressions of CD11b and CD18 by flow cytometry. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments. (B) TIL-MPs transferred CD11b/CD18 to H22 cells. H22 cells were incubated with TIL-MPs for 20 h and analyzed the expressions of CD11b and CD18 by flow cytometry (#p < 0.05, compared with FTS group). (C) FTS-induced TIL-MPs promoted H22 cell migration. Transwell assay, as described in 2Materials and Methods. CD11b or CD18 blocking Ab was added to the upper chamber. CFSE-labeled H22 cells that migrated to the lower chamber were counted using a fluorescence microscope. (D) CD11b/CD18 mediated tumor cell metastasis in vivo. A total of 3 × 105 H22 cells treated with or without TIL-derived MPs was injected into mice via their tail veins (n = 6 per group). The mice were treated with 50 μg anti-CD18 or anti-CD11b Ab twice, 1 h before and 12 h after the tail vein injection. The typical tumor growth was shown. Tumors were indicated by arrows. (E) The long-term survival of the mice was analyzed. The results were combined from two reproducible experiments (n = 6 for each group, p < 0.001).

FIGURE 6.

TIL-derived MPs promote H22 cell metastasis. (A) The isolated TILs were cultured in frozen-thawed H22 supernatant (FTS) for 12 h. The released MPs were isolated to analyze the expressions of CD11b and CD18 by flow cytometry. The left shown was the representative of three independent experiments, and the right shown was the combination of those experiments. (B) TIL-MPs transferred CD11b/CD18 to H22 cells. H22 cells were incubated with TIL-MPs for 20 h and analyzed the expressions of CD11b and CD18 by flow cytometry (#p < 0.05, compared with FTS group). (C) FTS-induced TIL-MPs promoted H22 cell migration. Transwell assay, as described in 2Materials and Methods. CD11b or CD18 blocking Ab was added to the upper chamber. CFSE-labeled H22 cells that migrated to the lower chamber were counted using a fluorescence microscope. (D) CD11b/CD18 mediated tumor cell metastasis in vivo. A total of 3 × 105 H22 cells treated with or without TIL-derived MPs was injected into mice via their tail veins (n = 6 per group). The mice were treated with 50 μg anti-CD18 or anti-CD11b Ab twice, 1 h before and 12 h after the tail vein injection. The typical tumor growth was shown. Tumors were indicated by arrows. (E) The long-term survival of the mice was analyzed. The results were combined from two reproducible experiments (n = 6 for each group, p < 0.001).

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Finally, we verified whether immune cell–derived MPs were taken up by tumor cells so to transfer immune phenotypes to tumor cells in vivo. For this purpose, CFSE-labeled H22 cells and PKH26-labeled immune cells were mixed and i.p. or i.m. injected to BALB/c mice. H22 cells were isolated from peritoneal cavity or muscle tissue 8 or 24 h after coinjection and analyzed by flow cytometry. We found that 16% versus 7% CFSE-H22 cells were PKH26 positive in i.p. versus i.m. injected mice (Fig. 7A), suggesting that immune phenotypes can be transferred to tumor cells in vivo. Hence, we further i.p. or i.m. injected BALB/c mice with the mixture of CFSE-labeled H22 cells and PKH26-labeled immune cell–derived MPs. Eight or 24 h later, H22 cells in peritoneal cavity or muscle tissue were used for flow cytometric analysis. Again, ∼21% CFSE-H22 cells were found to be PKH26 positive in i.p. injection group and ∼8% CFSE-H22 cells showed PKH26 positive in i.m. injection group (Fig. 7A). Consistently, the expressions of CD11b and CD18 were found on the H22 tumor cells (Fig. 7B). Therefore, there is potential for immune MPs to transfer immune phenotypes to tumor cells in tumor microenvironment.

FIGURE 7.

H22 tumor cells take up MPs in vivo. (A) A total of 3 × 105 CFSE-labeled H22 cells was mixed with 1 × 108 PKH26-labeled immune cells or 6 × 106 MPs derived from 1 × 108 PKH26-labeled splenocytes. The mixture was i.p. or i.m. injected to BALB/c mice. H22 cells were isolated from peritoneal cavity or muscle tissue 8 or 24 h after coinjection and analyzed by flow cytometry. (B) CD11b and CD18 were expressed by H22 cells in vivo. H22 cells and immune cell–derived MPs were mixed, respectively, and i.p. or i.m. injected to BALB/c mice. H22 cells were isolated from peritoneal cavity or muscle tissue 8 or 24 h after coinjection. The expression of CD11b and CD18 on H22 cells was analyzed by flow cytometry.

FIGURE 7.

H22 tumor cells take up MPs in vivo. (A) A total of 3 × 105 CFSE-labeled H22 cells was mixed with 1 × 108 PKH26-labeled immune cells or 6 × 106 MPs derived from 1 × 108 PKH26-labeled splenocytes. The mixture was i.p. or i.m. injected to BALB/c mice. H22 cells were isolated from peritoneal cavity or muscle tissue 8 or 24 h after coinjection and analyzed by flow cytometry. (B) CD11b and CD18 were expressed by H22 cells in vivo. H22 cells and immune cell–derived MPs were mixed, respectively, and i.p. or i.m. injected to BALB/c mice. H22 cells were isolated from peritoneal cavity or muscle tissue 8 or 24 h after coinjection. The expression of CD11b and CD18 on H22 cells was analyzed by flow cytometry.

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Next, we explored how innate MPs influence tumor cell behavior. The above data showed that tumor cells were conferred an enhanced capability of metastasis after acquiring integrin CD11b/CD18 function by MPs. Given the adhesion function of CD11b/CD18, we tested the attachment of tumor cells to endothelium, a key step for tumor cell metastasis. We found that, after PMA-MP treatment, H22 cells effectively adhered to endothelial cells; however, the addition of CD18 blocking Ab impaired this process (Supplemental Fig. 4A), suggesting that the uptake of MPs may enhance attachment of tumor cells to endothelium. Whether the uptake of MPs affects tumor cell metabolic phenotype such as glucose metabolism was also explored. A panel of glucose metabolic enzymes, including hexokinase 2, phosphofructokinase 1, pyruvate kinase M2, lactate dehydrogenase A, pyruvate dehydrogenase α1, citrate synthase, succinate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase, was analyzed by RT-PCR. We did not find the altered expression in these tested genes (Supplemental Fig. 4B), suggesting that the uptake of MPs by tumor cells appears not to influence the metabolism, at least for glucose metabolism.

Notwithstanding the generally accepted concept that innate immune cells are capable of promoting tumor metastasis in tumor microenvironment (27), the underlying cellular and molecular mechanisms still remain incompletely understood. The present study revealed that innate immune cells release MPs upon stimulation, acting as a new pathway to mediate tumor metastasis in a murine hepatocarcinoma model.

Cancer metastasis is a succession of cell-biological events (28, 29), including the following: 1) local invasion of surrounding tissue; 2) entry into the lymph and blood circulatory systems (intravasation); 3) survival and translocation to distant microvessels; 4) exit from the bloodstream (extravasation); and 5) survival and growth in the new microenvironment of distant tissues (colonization). Despite the intensive research, metastasis still to date remains one of the most enigmatic aspects of malignancies. Gene mutations that cause tumorigenesis have also been thought to result in cancer cell metastasis (30). Recently, epithelial mesenchymal transition theory is highlighted to explain cancer metastasis (31, 32). The explanation of metastasis by mutation and epithelial mesenchymal transition is supported by experimental data and is at the center of cancer research. Alternatively, cell–cell fusion theory, initially proposed by German pathologist Otto Aichel in 1911, was revisited recently. The core of this idea is that metastasis is generated by the fusion of tumor cells with tumor-associated leukocytes. Although the fusion theory is speculative, it is very intriguing. Laboratory studies by Rachkovsky et al. (33) found that when fused with macrophages, malignant melanoma cells are more likely to metastasize. In multiple myeloma patients, osteoclasts were found to fuse with myeloma cancer cells (34), even if the biological significance remained elusive. Corroboration can be found where lymphocytes, neutrophils, NK cells, and even tumor cells have been shown to reside temporarily within tumor cells, a phenomenon called cell cannibalism or cell-eat-cell (3537). The present study interprets the cell fusion theory from a new conceptual angle by showing that it is not innate immune cells, but their derived MPs that fuse with and promote tumor cell metastasis.

Cells, upon stimulation or apoptosis, may change their cytoskeletal structure and result in plasma membranes encapsulating cytosolic elements and shedding them into the extracellular space. These specialized subcellular vesicles are called MPs (21, 38). It is not surprising that abundant immune cell–derived MPs are present in tumor microenvironment, considering numerous immune stimulatory signals and apoptotic cues in tumor microenvironment (39, 40). To date, how and whether these immune MPs interact with tumor cells remains unclear. In this study, we showed that immune MPs can be taken up by tumor cells, and this uptake pathway seems not to be mediated through the classical endo/lysosome pathway, because MPs did not colocalize with endo/lysosomes in tumor cells (Fig. 2D). Additionally, we also showed that the entered MPs are not linked with ER or Golgi apparatus (Fig. 2E, 2F). Recently, MPs were reported to be Triton X-100 resistant, but proteinase K sensitive (41). Based on these findings, we propose that innate MPs are taken up by tumor cells through certain unclear pathways, localize in the cytoplasm, are lysed by proteinase, and partially translocated to cell membranes, leading to the transfer of immune molecules to tumor cells that mediates tumor cell metastasis.

Although innate immune cells and their derived MPs contain numerous immune molecules, in this study, we clearly show that CD11b/CD18 is the key to mediate H22 tumor cell metastasis. CD11b and the common integrin β2 subunit CD18 together form the functional αMβ2 integrin. αMβ2 is expressed on the surface of most innate immune cells, such as monocytes, granulocytes, macrophages, and NK cells (26). Functionally, it mediates cell adhesion and migration as well as chemotaxis, phagocytosis, and respiratory burst activity (42, 43). Interestingly, CD11b self seems to be capable of mediating cell adhesion and spreading, but not cell migration, unless it is assisted by CD18 (44). In line with this, in this study, the blockade of either CD11b or CD18 resulted in similar consequences in both in vitro and in vivo experiments. Although CD11b/CD18 is identified to mediate H22 tumor cell metastasis, CD11b/CD18 on rest innate MPs does not have such effect. This difference might be ascribed to the following: high levels of CD11b/CD18 are required and the expressions of CD11b/CD18 are upregulated on MPs upon stimulation; and other molecules are also upregulated upon stimulation, which synergize with CD11b/CD18 to promote tumor cell metastasis. Therefore, other migration-related molecules in innate MPs are worthy to be identified to better understand the mechanisms of tumor cell metastasis.

Besides the acquired ability to migrate, tumor cells have to escape immune attack during metastatic processes. How and whether innate MPs also transfer immune suppressive signals to tumor cells so to facilitate tumor immune evasion and metastasis remain intact in this study, but are worthy to investigate. It is known that activated immune cells may upregulate inhibitory receptor(s) or signal molecules to avoid over immune responses and keep immune homeostasis. Typically, T cells upregulate inhibitory receptors such as PD-1 and CTLA-4 after activation. In parallel, the activated dendritic cells may upregulate IDO (45). Therefore, in this study, H22 tumor cells might usurp inhibitory molecules of MPs by activated innate immune cells to escape immune attack. If this is the case, it at least partly explains why only activated rather than rest immune cells are capable of generating metastasis-promoting MPs. In fact, this study has shown that activated immune cells resulted in H22 tumor cells upregulating MHC class II through MP pathways (Fig. 1D). Intriguingly, MHC class II may engage with lymphocyte activation gene-3 negatively regulating the functions of T cells, NK cells, and dendritic cells (46). Whether MHC class II plays a role in mediating H22 tumor cell immune evasion is currently under study.

In summary, data in this study show that activated innate immune cell–derived MPs, by virtue of their biological formation and consequent biochemical features, can act as a ferry to transfer innate molecules to tumor cells, leading to tumor metastasis. This study possibly opens a new aspect of MP biology and may lead to discovery of new mechanisms underlying innate immune cell–promoted tumor metastasis.

This work was supported by National Basic Research Program of China Grant 2012CB932500, National Science Fund for Distinguished Young Scholars of China Grant 81225021, and National Natural Science Foundation of China Grant 81101508.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ER

endoplasmic reticulum

MP

microparticle

TIL

tumor-infiltrating leukocyte.

1
Talmadge
J. E.
,
Donkor
M.
,
Scholar
E.
.
2007
.
Inflammatory cell infiltration of tumors: Jekyll or Hyde.
Cancer Metastasis Rev.
26
:
373
400
.
2
Qian
B. Z.
,
Pollard
J. W.
.
2010
.
Macrophage diversity enhances tumor progression and metastasis.
Cell
141
:
39
51
.
3
DeNardo
D. G.
,
Johansson
M.
,
Coussens
L. M.
.
2008
.
Immune cells as mediators of solid tumor metastasis.
Cancer Metastasis Rev.
27
:
11
18
.
4
Shaked
Y.
,
Voest
E. E.
.
2009
.
Bone marrow derived cells in tumor angiogenesis and growth: are they the good, the bad or the evil?
Biochim. Biophys. Acta
1796
:
1
4
.
5
Murdoch
C.
,
Muthana
M.
,
Coffelt
S. B.
,
Lewis
C. E.
.
2008
.
The role of myeloid cells in the promotion of tumour angiogenesis.
Nat. Rev. Cancer
8
:
618
631
.
6
Liu
J.
,
Zhang
Y.
,
Zhao
J.
,
Yang
Z. S.
,
Li
D. P.
,
Katirai
F.
,
Huang
B.
.
2011
.
Mast cell: insight into remodeling a tumor microenvironment.
Cancer Metastasis Rev.
30
:
177
184
.
7
Joyce
J. A.
,
Pollard
J. W.
.
2009
.
Microenvironmental regulation of metastasis.
Nat. Rev. Cancer
9
:
239
252
.
8
Kaplan
R. N.
,
Riba
R. D.
,
Zacharoulis
S.
,
Bramley
A. H.
,
Vincent
L.
,
Costa
C.
,
MacDonald
D. D.
,
Jin
D. K.
,
Shido
K.
,
Kerns
S. A.
, et al
.
2005
.
VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.
Nature
438
:
820
827
.
9
Kaplan
R. N.
,
Psaila
B.
,
Lyden
D.
.
2006
.
Bone marrow cells in the ‘pre-metastatic niche’: within bone and beyond.
Cancer Metastasis Rev.
25
:
521
529
.
10
McIntyre
T. M.
,
Prescott
S. M.
,
Weyrich
A. S.
,
Zimmerman
G. A.
.
2003
.
Cell-cell interactions: leukocyte-endothelial interactions.
Curr. Opin. Hematol.
10
:
150
158
.
11
Smith
A.
,
Stanley
P.
,
Jones
K.
,
Svensson
L.
,
McDowall
A.
,
Hogg
N.
.
2007
.
The role of the integrin LFA-1 in T-lymphocyte migration.
Immunol. Rev.
218
:
135
146
.
12
Ross
G. D.
2002
.
Role of the lectin domain of Mac-1/CR3 (CD11b/CD18) in regulating intercellular adhesion.
Immunol. Res.
25
:
219
227
.
13
Pawelek
J.
,
Chakraborty
A.
,
Lazova
R.
,
Yilmaz
Y.
,
Cooper
D.
,
Brash
D.
,
Handerson
T.
.
2006
.
Co-opting macrophage traits in cancer progression: a consequence of tumor cell fusion?
Contrib. Microbiol.
13
:
138
155
.
14
Lazova
R.
,
Chakraborty
A.
,
Pawelek
J. M.
.
2011
.
Leukocyte-cancer cell fusion: initiator of the Warburg effect in malignancy?
Adv. Exp. Med. Biol.
714
:
151
172
.
15
Pawelek
J. M.
,
Chakraborty
A. K.
.
2008
.
The cancer cell-leukocyte fusion theory of metastasis.
Adv. Cancer Res.
101
:
397
444
.
16
Koido
S.
,
Hara
E.
,
Homma
S.
,
Fujise
K.
,
Gong
J.
,
Tajiri
H.
.
2007
.
Dendritic/tumor fusion cell-based vaccination against cancer.
Arch. Immunol. Ther. Exp.
55
:
281
287
.
17
Pluskota
E.
,
Woody
N. M.
,
Szpak
D.
,
Ballantyne
C. M.
,
Soloviev
D. A.
,
Simon
D. I.
,
Plow
E. F.
.
2008
.
Expression, activation, and function of integrin alphaMbeta2 (Mac-1) on neutrophil-derived microparticles.
Blood
112
:
2327
2335
.
18
Tang
K.
,
Liu
J.
,
Yang
Z. S.
,
Zhang
B.
,
Zhang
H. F.
,
Huang
C. M.
,
Ma
J. W.
,
Shen
G. X.
,
Ye
D. Y.
,
Huang
B.
.
2010
.
Microparticles mediate enzyme transfer from platelets to mast cells: a new pathway for lipoxin A4 biosynthesis.
Biochem. Biophys. Res. Commun.
400
:
432
436
.
19
Wolf
P.
1967
.
The nature and significance of platelet products in human plasma.
Br. J. Haematol.
13
:
269
288
.
20
Hugel
B.
,
Martínez
M. C.
,
Kunzelmann
C.
,
Freyssinet
J. M.
.
2005
.
Membrane microparticles: two sides of the coin.
Physiology
20
:
22
27
.
21
Ratajczak
J.
,
Wysoczynski
M.
,
Hayek
F.
,
Janowska-Wieczorek
A.
,
Ratajczak
M. Z.
.
2006
.
Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.
Leukemia
20
:
1487
1495
.
22
Mause
S. F.
,
Weber
C.
.
2010
.
Microparticles: protagonists of a novel communication network for intercellular information exchange.
Circ. Res.
107
:
1047
1057
.
23
VanWijk
M. J.
,
VanBavel
E.
,
Sturk
A.
,
Nieuwland
R.
.
2003
.
Microparticles in cardiovascular diseases.
Cardiovasc. Res.
59
:
277
287
.
24
Arnaout
M. A.
1990
.
Structure and function of the leukocyte adhesion molecules CD11/CD18.
Blood
75
:
1037
1050
.
25
von Andrian
U. H.
,
Chambers
J. D.
,
McEvoy
L. M.
,
Bargatze
R. F.
,
Arfors
K. E.
,
Butcher
E. C.
.
1991
.
Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo.
Proc. Natl. Acad. Sci. USA
88
:
7538
7542
.
26
Tan
S. M.
2012
.
The leucocyte β2 (CD18) integrins: the structure, functional regulation and signalling properties.
Biosci. Rep.
32
:
241
269
.
27
Lorusso
G.
,
Rüegg
C.
.
2008
.
The tumor microenvironment and its contribution to tumor evolution toward metastasis.
Histochem. Cell Biol.
130
:
1091
1103
.
28
Steeg
P. S.
2006
.
Tumor metastasis: mechanistic insights and clinical challenges.
Nat. Med.
12
:
895
904
.
29
Chaffer
C. L.
,
Weinberg
R. A.
.
2011
.
A perspective on cancer cell metastasis.
Science
331
:
1559
1564
.
30
Muller
P. A.
,
Vousden
K. H.
,
Norman
J. C.
.
2011
.
p53 and its mutants in tumor cell migration and invasion.
J. Cell Biol.
192
:
209
218
.
31
Micalizzi
D. S.
,
Farabaugh
S. M.
,
Ford
H. L.
.
2010
.
Epithelial-mesenchymal transition in cancer: parallels between normal development and tumor progression.
J. Mammary Gland Biol. Neoplasia
15
:
117
134
.
32
Kraljevic Pavelic
S.
,
Sedic
M.
,
Bosnjak
H.
,
Spaventi
S.
,
Pavelic
K.
.
2011
.
Metastasis: new perspectives on an old problem.
Mol. Cancer
10
:
22
.
33
Rachkovsky
M.
,
Sodi
S.
,
Chakraborty
A.
,
Avissar
Y.
,
Bolognia
J.
,
McNiff
J. M.
,
Platt
J.
,
Bermudes
D.
,
Pawelek
J.
.
1998
.
Melanoma × macrophage hybrids with enhanced metastatic potential.
Clin. Exp. Metastasis
16
:
299
312
.
34
Pawelek
J. M.
,
Chakraborty
A. K.
.
2008
.
Fusion of tumour cells with bone marrow-derived cells: a unifying explanation for metastasis.
Nat. Rev. Cancer
8
:
377
386
.
35
Krajcovic
M.
,
Overholtzer
M.
.
2012
.
Mechanisms of ploidy increase in human cancers: a new role for cell cannibalism.
Cancer Res.
72
:
1596
1601
.
36
White
E.
2007
.
Entosis: it’s a cell-eat-cell world.
Cell
131
:
840
842
.
37
Clarke
R.
2011
.
Cannibalism, cell survival, and endocrine resistance in breast cancer.
Breast Cancer Res.
13
:
311
.
38
Al-Nedawi
K.
,
Meehan
B.
,
Rak
J.
.
2009
.
Microvesicles: messengers and mediators of tumor progression.
Cell Cycle
8
:
2014
2018
.
39
Townson
J. L.
,
Naumov
G. N.
,
Chambers
A. F.
.
2003
.
The role of apoptosis in tumor progression and metastasis.
Curr. Mol. Med.
3
:
631
642
.
40
Huang
B.
,
Zhao
J.
,
Unkeless
J. C.
,
Feng
Z. H.
,
Xiong
H.
.
2008
.
TLR signaling by tumor and immune cells: a double-edged sword.
Oncogene
27
:
218
224
.
41
Tang
K.
,
Zhang
Y.
,
Zhang
H. F.
,
Xu
P. W.
,
Liu
J.
,
Ma
J. W.
,
Lv
M.
,
Li
D. P.
,
Katirai
F.
,
Shen
G. X.
, et al
.
2012
.
Delivery of chemotherapeutic drugs in tumour cell-derived microparticles.
Nat. Commun.
3
:
1282
.
42
Ahn
G. O.
,
Tseng
D.
,
Liao
C. H.
,
Dorie
M. J.
,
Czechowicz
A.
,
Brown
J. M.
.
2010
.
Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment.
Proc. Natl. Acad. Sci. USA
107
:
8363
8368
.
43
van Spriel
A. B.
,
Leusen
J. H.
,
van Egmond
M.
,
Dijkman
H. B.
,
Assmann
K. J.
,
Mayadas
T. N.
,
van de Winkel
J. G.
.
2001
.
Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.
Blood
97
:
2478
2486
.
44
Solovjov
D. A.
,
Pluskota
E.
,
Plow
E. F.
.
2005
.
Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2.
J. Biol. Chem.
280
:
1336
1345
.
45
Von Bubnoff
D.
,
Scheler
M.
,
Wilms
H.
,
Fimmers
R.
,
Bieber
T.
.
2011
.
Identification of IDO-positive and IDO-negative human dendritic cells after activation by various proinflammatory stimuli.
J. Immunol.
186
:
6701
6709
.
46
Liang
B.
,
Workman
C.
,
Lee
J.
,
Chew
C.
,
Dale
B. M.
,
Colonna
L.
,
Flores
M.
,
Li
N.
,
Schweighoffer
E.
,
Greenberg
S.
, et al
.
2008
.
Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II.
J. Immunol.
180
:
5916
5926
.

The authors have no financial conflicts of interest.