SCF-mediated mast cell infiltration and activation exacerbate the inflammation and immunosuppression in tumor microenvironment

Abstract

Despite the evidence for the role of inflammation in cancer initiation, promotion, and progression, the precise mechanism by which the inflammation within tumor is orchestrated by inflammatory cells remains to be determined. Here, we report that tumor-infiltrating mast cells remodel tumor microenvironment and promote tumor growth. Mast cell infiltration and activation in tumors were mainly mediated by tumor-derived stem cell factor (SCF) and its receptor c-Kit on mast cells. Low concentrations of SCF efficiently induced the chemotactic migration of mast cells. Tumor-infiltrating mast cells, activated by higher concentrations of SCF, expressed multiple proinflammatory factors and increased IL-17 expression in tumors. The activity of NF-kappaB and AP-1 in tumor cells was intensified in the mast cell-remodeled inflammatory microenvironment. SCF-activated mast cells also exacerbated tumor immunosuppression by releasing adenosine and increasing T regulatory cells, which augmented the suppression of T cells and natural killer cells in tumors. These findings emphasize that the remodeling of the tumor microenvironment can actually be initiated by tumor cell-released SCF and suggest that mast cells are not only a participator but also a critical regulator of inflammation and immunosuppression in the tumor microenvironment.

Figure 1

Mast cells promote tumor growth with SCF/c-Kit–mediated chemotactic migration as the prerequisite. (A) SCF induces the migration of mast cells (MCs). The migration of MCs in transwell assay was determined in the presence of tumor tissues, antibodies, or SCF. *P < .05 compared with tumor tissue group. (B) Infiltration of circulating MCs into the tumor. CFSE-labeled BMMCs with or without antibodies were injected into tumor-bearing mice by the tail vein. The peripheral tumor tissues were surgically excised from mice 24 hours after the injection, and frozen sections were prepared and analyzed by fluorescence microscopy. (C) Mast cells promote tumor growth. BMMCs were injected into tumor-bearing mice by intravenous injection. Bone marrow cells were used as control. The growth of tumor was monitored. (D) Survival rate follow-up after the intravenous injection of BMMCs. The survival period of tumor-bearing mice in the BMMC injection group was significantly shortened, compared with that in the control groups (n = 12; P < .05, Kaplan-Meier analysis). The data were the representative of 2 independent experiments in which the similar results were obtained. (E) Dependence of tumor-promoting effect of MCs on the SCF/c-Kit axis. BMMCs, with or without antibodies, were injected into tumor-bearing mice by the tail vein. Both of 2 antibodies abolished the tumor-promoting effect of MCs. *P < .05, compared with the control group. Error bars represent SD.

Figure 2

Tumor cell–derived SCF is responsible for the infiltration of mast cells into tumor. (A) Assay of SCF expression in H22 tumor or tumor cells. SCF expression was detected by RT-PCR and Western blot, respectively. SCF in the supernatants of the cultured tumor tissue or tumor cells was assayed by ELISA. Mouse monocyte system cell line RAW246.7 was used as the negative control. (B) Assay of SCF expression in tumor cells and tumor tissues. SCF expression in murine tumor cell lines and human tumor cell lines, corresponding murine tumor and specimens from human tumor, and normal tissue adjacent to tumor was detected by RT-PCR and Western blot, respectively. (C) SCF on the surface of different tumor cells was analyzed by flow cytometry. (D) Assay of soluble SCF produced by different tumors. Tumor cell lines, the corresponding tumor tissues, and the adjacent tissues around the tumor were cultured in vitro. SCF in the supernatants was detected by ELISA. (E) Silence of SCF expression in H22 tumor cells by SCF siRNA. SCF expression was detected by RT-PCR and Western blot, respectively. The soluble SCF released from tumor tissue was assayed by ELISA. (F) SCF-knockdown tumor cannot efficiently induce the migration of MCs. SCF-knockdown or control tumor tissues were used for transwell assay of MC migration (left). The infiltration of circulating MCs into SCF-knockdown or control tumor tissue (right) was analyzed using the same protocol as that in Figure 1B. *P < .05, compared with control tumor. Error bars represent SD.

Figure 3

Activation of mast cells by SCF is necessary for their tumor-promoting effect. (A) SCF-knockdown retards tumor growth. Mice (n = 8 per group) were inoculated with SCF-knockdown H22 cells and control WT H22 cells, respectively. The growth of tumor was monitored. (B,C) Mice (n = 8 per group) were inoculated with SCF-knockdown H22 tumor cells. When tumor size reached approximately 5 × 5 mm², the mice received BMMCs either by intravenous (i.v.) injection or by intratumor (i.t.) injection (B), or received the intratumor injection of BMMCs pretreated with different concentrations of SCF and anti–c-kit antibody (20 μg/mL) as indicated (C). The growth of the tumor was promoted only by the intratumor injection of MCs pretreated with a higher concentration of SCF (200 ng/mL), which was abolished by anti–c-kit antibody. Error bars represent SD.

Figure 4

SCF-stimulated mast cells augment the release of SCF from tumor cells. (A) SCF stimulates the production of active MMP-9 by mast cells (MCs). BMMCs were cultured for 24 hours in the presence of different concentrations of SCF and anti–c-Kit (10 μg/mL). The production of MMP-9 was detected by RT-PCR and gelatin zymography. (B) MC-derived MMP-9 increased the release of SCF from H22 tumor cells. BMMCs were treated with 5 ng/mL of SCF for 4 hours in the absence or presence of 10 μg/mL anti–c-Kit antibody. H22 cells and SCF-treated BMMCs were cultured alone or in 2 chambers separated by semipermeable membrane. SCF in the supernatants was detected by ELISA (left). SCF mRNA was detected by real-time RT-PCR (right). *P < .05, compared with the H22/MC group. (C) Assay of SCF in tumor tissues. Tumor-bearing mice received the intraperitoneal injection of MMP-9 inhibitor and the intratumor (i.t.) injection of MCs, or received the intravenous (i.v.) injection of MCs with anti–c-Kit antibody. The tumor tissues were excised 48 hours after MC injection and cultured in vitro. SCF in the supernatants was detected by ELISA (left). The mRNA level of SCF in tumor tissues was detected by real-time RT-PCR (right). *P < .05, compared with the MC injection groups. Error bars represent SD.

Figure 5

SCF/c-Kit signal induces the mast cell–mediated remodeling of tumor inflammatory microenvironment. (A) Expression of proinflammatory genes in mast cells. Mast cells were cultured in the presence or absence of SCF and anti–c-Kit antibody. The levels of IL-6, TNF-α, VEGF, Cox-2, iNOS, and CCL2 mRNAs were detected by real-time PCR. (B-E) Expression of proinflammatory genes in tumor and the activities of NF-κB and AP-1 in tumor cells. The mice bearing WT H22 tumor received the intravenous injection of mast cells and anti–c-Kit antibody as indicated. The mice bearing SCF-knockdown H22 tumor received the intratumor injection of mast cells. The levels of IL-6, TNF-α, VEGF, Cox-2, iNOS, CCL2, and IL-17 mRNAs in tumor tissues were detected by real-time PCR (B,C). IL-17 expression (IL-17⁺) cells in immune cells from tumor were analyzed by flow cytometry (D). Numbers on plots are percentages of total cells gated. Tumor cells were isolated from tumor tissue as described in “Assay of the activities of NF-κB and AP-1.” (E). *P < .05, compared with control tumor cells or WT tumor control. Error bars represent SD.

Figure 6

SCF/c-Kit signal activates mast cells to exacerbate the immunosuppression in tumor microenvironment. When tumor size reached approximately 5 × 5 mm², the mice bearing WT H22 tumor received the intravenous injection of mast cells (MCs) and anti–c-Kit antibody as indicated, and the mice bearing SCF-knockdown tumor received the intratumor injection of MCs. (A) The expression of Foxp3 and cytokine genes in tumor. The levels of Foxp3, IL-10, TGF-β, and IL-2 mRNAs in tumor tissues were detected by real-time PCR 72 hours after the injection of mast cells. (B) Treg cells (Foxp3⁺) in T cells (gated CD3⁺ cells) from tumor were analyzed by flow cytometry. Numbers on plots are percentages of total cells gated. (C,D) Mast cells intensify the suppression of T cells and NK cells in tumor. Seventy-two hours after the injection of mast cells, T cells and NK cells were isolated from the tumor. The proliferation of T cells (C) and the production of IFN-γ by NK cells (D) were determined as described in “Assay of soluble SCF and IFN-γ by enzyme-linked immunosorbent assay.” T cells and NK cells isolated from normal spleen were used as control. *P < .05, compared with WT tumor control. Error bars represent SD.

Figure 7

SCF-activated mast cells release adenosine to suppress the immune response. (A) Assay of adenosine released by BMMCs after the stimulation with SCF. BMMCs were cultured in the absence or presence of SCF for 48 hours. The adenosine in the supernatant was assayed as described in “Assay of adenosine.” (B) Assay of adenosine in tumor tissues. The mice bearing WT H22 tumor received the intravenous injection of mast cells and anti-c-Kit antibody as indicated. The mice bearing SCF-knockdown H22 tumor received the intratumor injection of mast cells. Seventy-two hours later, the adenosine in tumor tissues was assayed as described in “Assay of adenosine.” *P < .05, compared with the 0 ng/mL SCF group or the WT tumor group. (C,D) Mast cell–produced adenosine inhibits T cells and NK cells. Splenic T cells and NK cells were cultured with the culture supernatant of SCF-stimulated mast cells or control SCF medium in the presence or absence of adenosine receptor A_2A antagonist SCH-58261 (C). The T cells and NK cells from tumor were isolated from the mice bearing WT H22 tumor 72 hours after the intratumor injection of mast cells or control bone marrow cells with or without SCH-58261 (D). The proliferation of T cells (left) and the production of IFN-γ by NK cells (right) were determined as described in “Assay of soluble SCF and IFN-γ by enzyme-linked immunosorbent assay.” *P < .05, compared with the 0 ng/mL SCF, control, or WT tumor groups; #P < .05, compared with the no-SCH-58261 group. Error bars represent SD.