Tuftsin-tailored fusion protein inhibits the growth of circulating gastric tumor cells associated with macrophage phagocytosis

Abstract

Circulating tumor cells (CTCs) are a major cause of tumor metastasis and resistance to anticancer therapies. To date, no effective low-toxicity chemotherapeutic agents or antibodies have exhibited significant clinical activity against CTCs. Macrophages are important mediators of antitumor immunity. Tuftsin (TF), a tetrapeptide located at residues 289-292 of the CH2 domain of the Fc region of the IgG heavy chain, binds to Nrp-1, a receptor on the surface of macrophages that promotes phagocytosis and induces nonspecific activation of the immune system against tumors. Lidamycin (LDM) is an antitumor chemotherapy agent that is strongly cytotoxic to tumors and can dissociate into an apoprotein (LDP) and active enediyne (AE) in vitro. We previously constructed the fusion protein LDP-TF through genetic engineering and inserted the chromophore AE to produce LDM-TF, which can target macrophages to promote their phagocytic and cytotoxic activity against tumor cells. Preliminary experiments confirmed the anti-tumor activity of LDM-TFs. In this study, we found that LDM-TF effectively inhibited the growth of CTCs of gastric cancer origin and enhanced macrophage phagocytosis both in vivo and in vitro. Tumor cell expression of CD47, which helps to evade phagocytosis by macrophages, was substantially downregulated by LDM-TF. Notably, our in vitro experiments demonstrated that the combination of LDM-TF and anti-CD47 antibodies promoted phagocytosis more than either component alone. Our findings demonstrate the significant inhibitory effect of LDM-TF on the growth of CTCs of gastric cancer origin and suggest that the combination of LDM-TF and anti-CD47 antibodies may exhibit synergistic effects, thereby providing a new option for the clinical treatment of patients with advanced tumors that have metastasized.

Keywords: CD47; Circulating tumor cells; Fusion protein; Lidamycin; Macrophage; Tuftsin.

Fig. 1

(A) Diagram of the NdeI/XhoI gene fragments encoding for the protein LDP-TF. (B) The diagram indicates the components of the LDM-TF fusion protein and its reconstituted enediyneintegrated analog LDM-TF. (C) Representative images of mouse macrophages phagocytosing FITC-labled BSA following treatment with fusion proteins. Arrows point to phagocytosed BSA. Cell morphology through microscope (10 × original magnification). (D) Bar graph shows the mean fluorescence intensity of FITC-labeled BSA after being primed by BSA, LDP-TF, LDP or tuftsin for the 2 h time point. Fold change is expressed as a ratio of mean fluorescence intensity level of the different fusion proteins relative to the mean fluorescence intensity level of the BSA protein. *p < 0.05. (E) Graphs show flow-cytometry-based quantification of phagocytosis of CTC cell lines in the presence of LDP-TF, LDP or BSA treatment. Shown are mean ± s.d. of three experimental replicates. (F) CTC-141 (left) cells or CTC-105 (right) cells treated with tested samples (100 μg), including LDP, LDP-TF, LDM (1 nM), and LDM-TF (1 nM), respectively.

Fig. 2

Percentage of CTC cells treated with various concentrations of drugs. (A–D) Percentage of CTC cells treated with various concentrations of LDM, LDM-TF, FU, and oxaliplatin, respectively. € IC50 values of LDM, LDM-TF, FU, and oxaliplatin. One-way ANOVA and Dunnett's multiple comparison tests were used. *p < 0.05, ***p < 0.001 (LDMP-TF vs LDM, FU, or oxaliplatin). (F–H) Percentage of live cells treated with various concentrations of LDP-TF, cetuximab, and trastuzumab, respectively.

Fig. 3

In vivo efficacy of tuftsin-based fusion proteins. (A) Graphs show flow-cytometry-based quantification of phagocytosis of CTC-141 cell lines in the presence of LDP-TF, anti-CD47, LDP-TF + anti-CD47, or BSA treatment. Shown are mean ± s.d. of three experimental replicates. (B) Antitumor effects of LDP-TF alone on CTC-141 xenograft. Triangles, the day of injection (day 6). (C) Mean body weights of mice in each group are shown. (D) The mean tumor weight after treatment is shown. (E) Histopathological examination of tumors (H&E staining, magnification × 200) of CTC-141 xenograft-bearing mice. (F) The Kaplan–Meyer survival curve (endpoints defined as tumor load of 400 mm³) demonstrated that compared with other groups, the probability of survival (i.e., probability of maintaining a tumor volume <400 mm³) of mice treated with LDP-TF (20 mg/kg) was significantly improved (p < 0.05). (G) Production of cytokine in blood serum treated with fusion proteins. Blood samples were taken 24 h after the injection of drugs. Results are presented in histograms IL-10, IL-12, IFN-γ, and TNF-α per milliliter ± SD. Asterisks (*) indicate a significant difference (p < 0.05) between the untreated tumor group and drugs treated groups.

Fig. 4

In vivo efficacy of enediyne-energized tuftsin-based fusion proteins. (A) Antitumor effects of LDM-TF on CTC-141 xenograft. Triangles, the day of injection (day 5). (B) Mean body weights of mice in each group are shown. (C) The mean tumor weight after treatment is shown. (D) The Kaplan–Meyer survival curve (endpoints defined as tumor load of 400 mm³) demonstrated that compared with other groups, the probability of survival (i.e., probability of maintaining a tumor volume <400 mm³) of mice treated with LDM-TF (0.15 mg/kg) was significantly improved (p < 0.05). (E) Expression of CD47, ZEB-1, AKT, and p-AKT in CTC-141 cells detected by Western blot. (F) Representative IHC images showing the detection of macrophages (anti-F4/80).