Tumor-associated macrophages regulate tumorigenicity and anticancer drug responses of cancer stem/initiating cells

Abstract

Recent evidence has unveiled the critical role of tumor cells with stem cell activities in tumorigenicity and drug resistance, but how tumor microenvironments regulate cancer stem/initiating cells (CSCs) remains unknown. We clarified the role of tumor-associated macrophages (TAMs) and their downstream factor milk-fat globule-epidermal growth factor-VIII (MFG-E8) in the regulation of CSC activities. Bone marrow chimeric systems and adoptive cell transfers elucidated the importance of MFG-E8 from TAMs in conferring to CSCs with the ability to promote tumorigenicity and anticancer drug resistance. MFG-E8 mainly activates signal transducer and activator of transcription-3 (Stat3) and Sonic Hedgehog pathways in CSCs and further amplifies their anticancer drug resistance in cooperation with IL-6. Thus, the pharmacological targeting of key factors derived from tumor-associated inflammation provides a unique strategy to eradicate therapy-resistant tumors by manipulating CSC activities.

Fig. 1.

CSCs induce MFG-E8 expression from TAM. (A) F4/80⁺CD11b⁺ macrophages were isolated from tumors, tumor-draining lymph nodes (TDLs), and splenocytes in mice bearing CD44⁺ALDEFLOUR⁺ MC38-CSCs, or CD133⁺ALDEFLOUR⁺ 3LL-CSCs, or their non-CSC counterparts (n = 3 per group). Murine MFG-E8 mRNA was quantified by RT-PCR. The results are shown as fold induction of target genes relative to a reference gene (GAPDH). (B) MFG-E8 expression in F4/80⁺CD11b⁺ macrophages isolated from MC38-CSCs or non-CSCs was evaluated by intracellular flow cytometry. The splenic macrophages from CSC-bearing mice (spleen) serve as negative control. (C) MC38-CSCs or non-CSCs, bulk MC38 cells were cultured with F4/80⁺ splenic macrophages (Mac) for 24 h, and MFG-E8 in macrophages (F4/80⁺) or MC38 tumor cells (F4/80⁻) were quantified by flow cytometry. Splenic macrophages or MC38 tumor cells without coculture served as a negative control. The expression was shown as mean fluorescence intensity (MFI). (D) MFG-E8 in CD68⁺ macrophages (Mac) isolated from pleural effusion (PE) or peripheral blood (PBMC) of two nonsmall cell lung cancer patients (Pt-1 and Pt-2). The MFG-E8 mRNA (Left) or protein (Right) levels were analyzed by RT-PCR or flow cytometry, respectively. Data are representative of three independent experiments.

Fig. 2.

TAMs contribute to triggering tumorigenicity and anticancer drug resistance in an MFG-E8–dependent manner. (A) MC38-CSCs or 3LL-CSCs were inoculated into wild-type (WT) or MFG-E8–deficient (MFG-E8 KO) mice for 1 mo, and the frequencies of CSCs were determined in established tumors by quantifying each CSC marker. Representative dot plots (Upper) and data from three experiments (Lower) are shown. (B) TAMs were isolated from tumors of wild-type (WT) or MFG-E8–deficient CD45.1⁺ mice (MFG-E8 KO). MC38-CSCs or non-CSCs were injected into CD45.2⁺ MFG-E8–deficient mice (CSCs or non-CSCs→MFG-E8 KO; n = 4 per group) either alone or with TAM, treated with CDDP after tumor inoculation, and tumor growth was measured on the indicated days. (C) CD44⁺ALDH1⁺ MC38-CSCs were treated with CDDP in the presence or absence of TAM supernatant (1:10 dilution) from wild-type (WT) or MFG-E8–deficient (MFG-E8 KO) mice, or splenic macrophages infected with control, or MFG-E8 retrovirus. After 24 h of treatment, cell viability was quantified by cleaved caspase-3 intensities by colorimetric assay. Data are representative of three independent experiments.

Fig. 3.

MFG-E8 plays a critical role in tumor self-renewal. (A) MC38 cells were treated with TAM supernatant (1:10 dilution) from wild-type (WT) or MFG-E8–deficient (MFG-E8 KO) mice or recombinant murine MFG-E8 protein (1 or 10 μg/mL) in ultra-low attachment plates. The cells were then cultured for three passages, and the numbers of formed spheres generated per 10,000 cells were determined. (B) In vivo serial tumor passages were performed to evaluate the frequency of CSCs. TAM from wild-type or MFG-E8–deficient mice was injected along with MC38-CSCs s.c. into MFG-E8–deficient mice (n = 4 per group) at 1 × 10⁵ per mouse. After 30 d, single cell suspensions were prepared from growing tumors and further transplanted with each TAM into tumor-free MFG-E8–deficient mice using the indicated number of cells. Tumor growth was measured on the indicated days. (C) EpCAM⁺CD133⁺ tumor cells obtained from patients with advanced NSCLC (1 × 10² per mouse) were inoculated into clodronate-pretreated NOD-SCID mice (n = 4 per group) with or without autologous TAM or peripheral blood macrophages (PBMs) in the presence of isotype control Ig or antihuman MFG-E8 Ab. The growth curves of each tumors (Left) and representative results (Right) are shown. Similar results were obtained in three independent experiments.

Fig. 4.

Oncogenic signals induced by MFG-E8. (A) Immunoblot for pSTAT3, STAT3, and SMO on lysates from CD44⁺ALDH1⁺ MC38 (CSC) or their non-CSC counterparts 2 h after stimulation with wild-type TAM supernatant (TAM) or not (−). (B) MC38-CSCs were treated with TAM supernatant or splenic macrophages (SPMs) from wild-type (WT) or MFG-E8–deficient (MFG-E8 KO) mice for 6 h. The expression of phospho-stat3 and shh were evaluated by flow cytometry. (C) Target gene expression for stat3 (SOCS3, MCL, and VEGFA) and shh (GLi1, PRCH1, and GAS1) in CSCs stimulated with TAM supernatant from wild-type (WT) or MFG-E8–deficient mice (MFG-E8 KO). (D) MC38-CSCs were infected with siRNA specific for Stat3, Sonic Hedgehog (shh), or both and treated with WT or MFG-E8–deficient TAM supernatant (1:10 dilution) in the presence of anti–MFG-E8 or isotype-matched control IgG for 24 h. The cell viability was quantified by cleaved caspase-3 intensities with colorimetric assay. *P < 0.05 compared with scrambled siRNA. (E) The chimeric mice reconstituted with wild-type (WT→MFG-E8 KO) or MFG-E8 KO (MFG-E8 KO→MFG-E8 KO) bone marrow into MFG-E8-deficient host (n = 4 per group) were generated. Eight weeks after transplantation, MC38-CSCs were inoculated s.c. into each chimeric mice, and mice were then treated with CDDP with or without AG490, cyclopamine, or both on days 10, 12, 14, and 16 after tumor challenge. Tumor growth was measured on the indicated days. Similar results were obtained in two independent experiments, and representative results are shown.

Fig. 5.

MFG-E8 and IL-6 coordinately trigger CSC chemoresistance in human CSCs. (A) Primary NSCLC-CSCs were treated with recombinant MFG-E8 (50 ng/mL), IL-6 (10 ng/mL), or both for 16 h. Immunoblotting for Stst3, pStat3, and SMO proteins was performed on the cell lysates. (B) Primary NSCLC-CSCs were treated with TAM or PBM supernatant (1:10 dilution) in the presence of anti–MFG-E8 Ab and/or anti–IL-6 mAb in ultra-low attachment plates. The cells were then cultured for three passages, and the numbers of formed spheres generated per 10,000 cells were determined. (C) Primary NSCLC-CSCs were treated with CDDP and the TAM or PBM supernatant (1:10 dilution) in the presence or absence of anti–MFG-E8 Ab or anti–IL-6 mAb for 24 h, and the cell viability was quantified by cleaved caspase-3 intensities with colorimetric assay. (D) Primary NSCLC-CSCs were inoculated into clodronate-pretreated NOD-SCID mice (n = 4 per group) with CD68⁺ macrophages isolated from primary tumors (TAM), treated with isotype-matched IgG (Isotype), anti–MFG-E8 Ab and/or anti–IL-6 mAb, or both, and tumor growth was measured on the indicated days. The NSCLC-CSCs without TAM serve as a negative control (−). The differences in tumor growth were compared between monotherapy (either anti–MFG-E8 or anti–IL-6 alone) and combined regimens. Data are representative of three independent experiments.