Macrophages eat cancer cells using their own calreticulin as a guide: roles of TLR and Btk

Abstract

Macrophage-mediated programmed cell removal (PrCR) is an important mechanism of eliminating diseased and damaged cells before programmed cell death. The induction of PrCR by eat-me signals on tumor cells is countered by don't-eat-me signals such as CD47, which binds macrophage signal-regulatory protein α to inhibit phagocytosis. Blockade of CD47 on tumor cells leads to phagocytosis by macrophages. Here we demonstrate that the activation of Toll-like receptor (TLR) signaling pathways in macrophages synergizes with blocking CD47 on tumor cells to enhance PrCR. Bruton's tyrosine kinase (Btk) mediates TLR signaling in macrophages. Calreticulin, previously shown to be an eat-me signal on cancer cells, is activated in macrophages for secretion and cell-surface exposure by TLR and Btk to target cancer cells for phagocytosis, even if the cancer cells themselves do not express calreticulin.

Keywords: Bruton's tyrosine kinase; Toll-like receptor; immunosurveillance; programmed cell removal; “eat me” signal.

Fig. 1.

Activation of TLR signaling leads to enhanced PrCR of living cancer cells. (A, Left) Schematic showing PrCR of living tumor cells by macrophages. Blockade of CD47 leads to an imbalance of eat-me over don’t-eat-me pathways, which elicits phagocytosis of tumor cells, either Fc-dependent (elicited by Fc–FcR interaction) or Fc-independent (labeled in red, representing cancer-specific eat-me signals other than Fc). (Right) A phagocytosis assay showing blockade of CD47-induced phagocytosis, with SW620 cells [control IgG-treated, anti-CD47 antibody (B6H12)–treated, or CD47^KO] as target cells and BMDMs from RAG2^−/−, γc^−/− mice. Fc receptor blocker (FcRB) reversed phagocytosis of B6H12-treated cells to the same level as inf CD47^KO cells. **P < 0.01, t test; ns, not significant. (B) A phagocytosis assay showing a screen of TLR agonists, with SW620 cells [PBS-treated, anti-CD47 antibody (Hu5F9-G4)–treated, or CD47^KO] as target cells and BMDMs from BALB/c mice. TLR agonists used in the screen were Pam3CSK4 (Pam, TLR1/2), heat-killed Listeria monocytogenes (HKLM, TLR2), poly (I:C) HMW [poly (I:C), TLR3)], lipopolysaccharide (LPS, TLR4), flagellin from Salmonella typhimurium (FLA-ST, TLR5), Pam2CGDPKHPKSF (FSL-1, TLR6/2), imiquimod (Imi, TLR7), and class B CpG oligonucleotide (ODN 1826, TLR9). Dashed lines indicate twofold phagocytosis of each condition [PBS-treated, anti-CD47 antibody (Hu5F9-G4)-treated, or CD47^KO] in the control macrophage group. Error bars represent SD.

Fig. 2.

Btk is the key signaling molecule regulating PrCR of cancer cells. (A) A phagocytosis assay showing a screen with combined TLR agonists and various inhibitors targeting downstream signaling molecules, with SW620 cells (control or CD47^KO) as target cells and BMDMs from RAG2^−/−, γc^−/− mice. Inhibitors used in the screen were PD98059 (PD, MEK inhibitor), LY294002 (LY, PI3K inhibitor), ibrutinib (Ibr, Btk inhibitor), and YVAD (vaspase-1 inhibitor). **P < 0.01 (t test; comparison between samples in control or CD47^KO groups, Imi-ctrl vs. other conditions). (B) Immunoblots showing the phosphorylation of Btk induced by TLR agonists [Poly (I:C) HMW, LPS, imiquimod]. When cells were treated simultaneously with TLR agonists and ibrutinib, the induction of Btk phosphorylation was attenuated. Total Btk showed no change. (C and D) Temporal effects of Btk activator (imiquimod) (C) and inhibitor (ibrutinib) (D) on phagocytosis, with SW620 cells^CD47KO as target cells and BMDMs from NSG mice. Error bars in A, C, and D represent SD.

Fig. 3.

Btk controls cell-surface exposure of CRT on macrophages to regulate PrCR of cancer cells. (A) The expression of CRT on macrophages was examined by a cell-surface biotinylation assay. Immunoblots showed that cell-surface CRT increased upon Btk activation and decreased upon Btk inhibition. Ibr, ibrutinib; Imi, imiquimod. (B) Increased cell-surface exposure of CRT on macrophages induced by TLR agonists [poly (I:C) HMW, LPS, imiquimod], as examined by flow cytometry analyses. (C) A phagocytosis assay showing CRT antibody inhibited phagocytosis of cancer cells, with SW620 cells [PBS-treated, anti-CD47 antibody (Hu5F9-G4)-treated or CD47^KO] as target cells and BMDMs from RAG2^−/−, γc^−/− mice. Enhancement of phagocytosis induced by Poly (I:C) or imiquimod was reversed by CRT blockade. Dashed lines indicate normalized phagocytosis of each condition [PBS-treated, anti-CD47 antibody (Hu5F9-G4)-treated or CD47^KO] in the control macrophage group. (D) Overexpression of CRT in J774 cells promoted phagocytosis. Expression of CRT was examined by immunoblotting. SW620 cells [control IgG- or anti-CD47 antibody (B6H12)–treated] were used as target cells. *P < 0.05, **P < 0.01 (t test). Error bars in C and D represent SD.

Fig. 4.

CRT is a key effector on macrophages in mediating PrCR of cancer cells. (A) A phagocytosis assay showing the effects of blocking CRT on macrophages or cancer cells. (Left) A schematic showing the design of the experiments. Macrophages, target cells, or both were pretreated with CRT antibody and then subjected to the phagocytosis assay. (Right) A phagocytosis assay showing CRT on macrophages is necessary for phagocytosis of cancer cells, with SW620 cells (control or CD47^KO) as target cells and BMDMs from RAG2^−/−, γc^−/− mice. (B) Phagocytic ability of macrophages with differential surface CRT expression levels. Definitions of CRT^Low, CRT^Medium, and CRT^High populations are given in Fig. S8 A and B. (C) Normalized tumor cell phagocytosis (y axis) was plotted against normalized cell-surface CRT expression (Log2; x axis) on macrophages with SW620 cells (CD47^KO) as target cells and BMDMs from RAG2^−/−, γc^−/− or NSG mice. ■, BMDMs from NSG mice treated with imiquimod for 0, 1, 6, 16, or 24 h; ▲, BMDMs from RAG2^−/−, γc^−/− mice (CRT^Low, CRT^Medium, CRT^High, and bulk populations); ●, BMDMs from NSG mice (CRT^Low, CRT^Medium, CRT^High, and bulk populations). Error bars in A and B represent SD.