Chordoma recruits and polarizes tumor-associated macrophages via secreting CCL5 to promote malignant progression

Abstract

Background: Chordoma is an extremely rare, locally aggressive malignant bone tumor originating from undifferentiated embryonic remnants. There are no effective therapeutic strategies for chordoma. Herein, we aimed to explore cellular interactions within the chordoma immune microenvironment and provide new therapeutic targets.

Methods: Spectrum flow cytometry and multiplex immunofluorescence (IF) staining were used to investigate the immune microenvironment of chordoma. Cell Counting Kit-8, Edu, clone formation, Transwell, and healing assays were used to validate tumor functions. Flow cytometry and Transwell assays were used to analyze macrophage phenotype and chemotaxis alterations. Immunohistochemistry, IF, western blot, PCR, and ELISA assays were used to analyze molecular expression. An organoid model and a xenograft mouse model were constructed to investigate the efficacy of maraviroc (MVC).

Results: The chordoma immune microenvironment landscape was characterized, and we observed that chordoma exhibits a typical immune exclusion phenotype. However, macrophages infiltrating the tumor zone were also noted. Through functional assays, we demonstrated that chordoma-secreted CCL5 significantly promoted malignancy progression, macrophage recruitment, and M2 polarization. In turn, M2 macrophages markedly enhanced the proliferation, invasion, and migration viability of chordoma. CCL5 knockdown and MVC (CCL5/CCR5 inhibitor) treatment both significantly inhibited chordoma malignant progression and M2 macrophage polarization. We established chordoma patient-derived organoids, wherein MVC exhibited antitumor effects, especially in patient 4, with robust killing effect. MVC inhibits chordoma growth and lung metastasis in vivo.

Conclusions: Our study implicates that the CCL5-CCR5 axis plays an important role in the malignant progression of chordoma and the regulation of macrophages, and that the CCL5-CCR5 axis is a potential therapeutic target in chordoma.

Keywords: Sarcoma.

Figure 1

Depict the immune microenvironment atlas of chordoma using spectral flowcytometry and multiplex IF. (A) Markers used to gate the different populations of immune cells. (B) Opt-SNE(a dimensionality reduction analysis method) displays different immune cell clusters and expressions of major markers on the opt-SNE map. (C) The results of opt-SNE dimensionality reduction analysis showed immune infiltration phenotype in five patients. (D) Circle diagram showing the major infiltrating immune cell and its proportion in chordoma. (E) Violin chart showed the 22 immune cell subpopulation proportion. (F) H&E staining and two staining panels were performed for the infiltration of T cells, B cells, NK cells, macrophage cells, and the expression of PD-L1 in 22 chordoma tissue microarrays. A large number of immune cells were infiltrated in the fibrous septa of the chordoma. (G) Representative IF images showing different tumor-infiltrating immune cells in chordoma tissuse. Panel 1: pan macrophage (CD68-positive, green), M2 macrophage (CD163-positive, red), M2 macrophage (CD206-positive, yellow), and M1 macrophage (IRF-positive, pink). Panel 2: T cell (CD3-positive, blue), cytotoxic T cell (CD8-positive, pink), NK cell (CD56-positive, green), B cell (CD20-positive, red), and PD-L1 (yellow). DC, dendritic cell; IF, immunofluorescence; mDC, myeloid dendritic cell; NK, natural killer; IRF, interferon regulatory factor.

Figure 2

Intercommunication between chordoma and M2 macrophages promotes tumor progression. (A, B) Induced THP-1-derived M2 macrophage and PBMC-derived M2 macrophage were verified by microscopic photography and flow cytometry to detect CD206 expression. (C, D) CCK8 and EDU assays showed M2 macrophage enhanced chordoma proliferation viability. (E, F) The results of wound healing showed M2 macrophage accelerated chordoma migration viability. (G). THP-1 derived and PBMC-derived M2 macrophage both facilitated the migration and invasion viability of chordoma. (H). Western blot showed that M2 macrophages resulted in a decrease in epithelial protein E-cadherin and increased the expression of mesenchymal protein N-cadherin and vimentin. (I) Flow cytometry showed chordoma cells induced macrophage into M2 type (CD206-positive). CCK8, Cell Counting Kit-8, IL, interleukin; MDM, monocyte-derived macrophage; PBMC, peripheral blood mononuclear cell.

Figure 3

CCL5 secreted by chordoma was the key cytokine involved in cellular communication between chordoma cells and macrophage cells. (A) Heat-map representation of the differentially secretion in MUG, macrophage single culture systems versus MUG–macrophage coculture system. (B) Heat-map of qRT-PCR results showed the expression levels of IL-6, IL-8, CCL5, IL-1β, and IL-13 were all upregulated in cocultured MUG cells. (C) IL-6, IL-8, and CCL5 were significantly elevated in the coculture system from both RNA and secreted protein expression levels. (D) The secretion of CCL5 by MUG cells was the most drastically increased at 4.745-fold using ELISA assay. (E, F) ELISA assay results showed the secretion of CCL5 in MUG, macrophage supernatants, MUG–macrophage coculture supernatant, and cocultured MUG or macrophage supernatant. (E). Secretions of MUG and THP-1-DMs were at a low level but were obviously increased in the coculture system. Cocultured THP-1-DMs did not secrete more CCL5, but an obvious increase in CCL5 secretion by MUG cells was shown. (F) Secretions of MUG and MDMs were at a low level but were obviously increased in the coculture system. A modest increase in CCL5 secretion was seen in cocultured MDMs, and an intense increase in CCL5 secretion was shown by cocultured MUG cells. (G, H) Transwell assay showed CCL5 obviously increased chordoma cell migration viability at 5, 10, 20, and 10 ng/mL CCL5 caused the most significant functional change. (I, J) Healing wound assay showed CCL5 significantly enhanced chordoma healing at different concentrations, and 10 ng/mL CCL5 caused the most obvious functional change. (K) Western blot results indicated CCL5 induced chordoma epithelial–mesenchymal transition in a concentration-dependent effect and increased MMP2 expression. (L, M) CCL5 expression level was validated by qRT-PCR (L) and ELISA assay (M). After siRNA treatment (N, P), Transwell (N) and wound healing assays (P) showed chordoma cell migration viability was decreased by si-CCL5 transfection and reversed after adding exogenous CCL5 or coculture with macrophage. (O–Q) Statistical analysis results of Transwell and wound healing assays. (R) Western blot showed E-cadherin expression was increased, and N-ca, vimentin, and MMP2 were decreased after CCL5 reduction, and the protein expressions were inverted by adding exogenous CCL5 or coculture with macrophage. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. IL, interleukin; MDM, monocyte-derived macrophage; ns, not significant; THP-1-DM, THP-1-derived macrophage.

Figure 4

CCL5 educated macrophage into M2 macrophage and facilitated macrophage chemotaxis. (A) CD206 expression on macrophage surface was increased by adding CCL5 at different concentrations, and 10 ng/mL CCL5 caused the most obvious macrophage polarization, which was tested by flow cytometry. (B). Western blot showed exogenous CCL5 increased SIRPα expression on macrophage in a concentration-dependent effect. (C) Transwell assay indicated macrophage cells chemotaxis viability was increased by CCL5, and the most significant chemotaxis viability change was seen at the concentration of 10 ng/mL. (D) Statistical analysis results of macrophage chemotaxis. (E) Flow cytometry results showed that coculture with MUG cells promoted the M2 macrophage polarization, and coculture with siCCL5–MUG cells did not affect macrophage CD206 expression, which was increased by adding CCL5. (F) The results of western blot showed immune checkpoint SIRPα on marcophage was significantly increased with coculture with MUG, while with coculture with siCCL5–MUG cells, SIRPα expression was not obviously changed. Adding exogenous CCL5 into siCCL5–MUG–macrophage coculture system just lightly increased SIRPα expression. (G, H) Transwell assays results showed that preplating MUG cells promoted the macrophages chemotaxis, and preplating siCCL5–MUG cells did not affect macrophage chemotaxis, which was enhanced by exogenous CCL5. *P<0.05, **P<0.01,***P＜0.001,****P＜0.0001. ns, not significant; SIRPα, signal-regulatory protein α.

Figure 5

Treatment of MVC suppressed malignant progession of chordoma and macrophage polarization. (A) MUG, THP-1-DMs, and MDMs were stained with DAPI (blue) and CCR5 (red). (B) Chordoma tissue microarrays were assessed immunohistochemically in CCR5 expression, and the results showed CCR5 was highly expressed on chordoma cells. (C) Triple fluorescent staining with DAPI (blue), anti-CCR5 antibody (red), and anti-CD68 antibody (red) showed macrophages expressed CCR5 in chordoma microenvironment. (D) The viability of MUG cells with the treatment of MVC from the concentration of 0–128 µM. The 48-hour IC₅₀ value of MUG is 50.53 µM. (E, F) Transwell assay results demonstrated that blocking the CCL5–CCR5 axis by adding MVC into the upper chamber of Transwell plates inhibited chordoma cell migration. (G, H) Wound healing assay results showed MVC reversed the increased chordoma cell healing rate caused by CCL5. (I) Western blot showed EMT-related protein E-cadherin was increased, and N-ca and vimentin expressions were decreased after MVC treatment. (J) Western blot showed MVC reduced the increased immune checkpoint SIRPα expression on macrophages induced by CCL5. (K, L). The results of flow cytometry showed MVC decreased M2 polarizated macrophages caused by CCL5. (M, N) Transwell assay results indicated MVC reversed the increased macrophage migrating vialibity caused by CCL5. (O) Xenograft photographs were taken after 14-day administration. (P) Results of tumor weights on the day the mice were euthanized. (Q) Results of tumor volume on the day the mice were euthanized. (R) Representative images of lungs harvested from mice. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. EMT, epithelial-to-mesenchymal transition; MDM, monocyte-derived macrophage; MVC, maraviroc; ns, not significant; SIRPα, signal-regulatory protein α; THP-1-DM, THP-1-derived macrophage.

Figure 6

Blocking the CCL5–CCR5 axis inhibited tumor malignant progression in chordoma organoid. (A) Schematic illustration for chordoma organoid established, validation and drug test. (B) Chordoma organoids were visualized by brightfield in 0 day and 5 days. Scale bar, 200 μm. (C) H&E staining on both the parent tumor tissues sections and organoid showed chordoma morphological features were well conserved. (D) IHC staining showed Brachyury was highly expressed in chordoma tissues and organoids; organoids preserved features of the parent tumor. (E) Genome-wide distributions in chromosomes of parental tumor tissues and organoids. (F) CNV was compared between tumor tissues and organoids, and the results indicated no significant difference was shown. (G) The IC₅₀ values of anlotinib and MVC in organoid patients. Anlotinib was used as the positive control of chordoma treatment. (H) CCL5 and CCR5 IHC staining in the parent tumor tissues. The results showed CCL5 and CCR5 expressions were correlated with sensitivity to MVC. CNV, copy number variation; IHC, immunohistochemistry; MVC, maraviroc.

Figure 7

CCL5 and CCR5 are highly expressed and correlated with tumor recurrence. (A) The results of PCR showed CCL5 expression level was correlated with chordoma recurrence. (B) Statistical analysis of CCL5 expression in primary chordoma and recurrence chordoma tissues.(C) CCL5 and CCR5 were both shown with robust expressions in patients with chordoma. (D) CCL5 IRS score distribution in low (0–4) (n=3), medium (5–8) (n=14), and high (9–12) (n=24) classes. (E) The correlation of CCL5 expression and CCR5 expression. (F) Statistical analysis of CCLR5 expression in primary chordoma and recurrence chordoma sections. (G) Schematic diagram illustrating the hypothetical mechanisms of chordoma secreted CCL5 to recruit and polarize tumor-associated macrophages to promote chordoma malignant progression. *P<0.05, **P<0.01. EMT, epithelial-to-mesenchymal transition; SIRPα, signal-regulatory protein α.