Construction of the systemic anticancer immune environment in tumour-bearing humanized mouse by using liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated progesterone

Abstract

Immune checkpoint inhibitors highlight the importance of anticancer immunity. However, their clinical utility and safety are limited by the low response rates and adverse effects. We focused on progesterone (P4), a hormone produced by the placenta during pregnancy, because it has multiple biological activities related to anticancer and immune regulation effects. P4 has a reversible immune regulatory function distinct from that of the stress hormone cortisol, which may drive irreversible immune suppression that promotes T cell exhaustion and apoptosis in patients with cancer. Because the anticancer effect of P4 is induced at higher than physiological concentrations, we aimed to develop a new anticancer drug by encapsulating P4 in liposomes. In this study, we prepared liposome-encapsulated anti-programmed death ligand 1 (PD-L1) antibody-conjugated P4 (Lipo-anti-PD-L1-P4) and evaluated the effects on the growth of MDA-MB-231 cells, a PD-L1-expressing triple-negative breast cancer cell line, in vitro and in NOG-hIL-4-Tg mice transplanted with human peripheral blood mononuclear cells (humanized mice). Lipo-anti-PD-L1-P4 at physiological concentrations reduced T cell exhaustion and proliferation of MDA-MB-231 in vitro. Humanized mice bearing MDA-MB-231 cells expressing PD-L1 showed suppressed tumor growth and peripheral tissue inflammation. The proportion of B cells and CD4+ T cells decreased, whereas the proportion of CD8+ T cells increased in Lipo-anti-PD-L1-P4-administrated mice spleens and tumor-infiltrated lymphocytes. Our results suggested that Lipo-anti-PD-L1-P4 establishes a systemic anticancer immune environment with minimal toxicity. Thus, the use of P4 as an anticancer drug may represent a new strategy for cancer treatment.

Keywords: breast cancer; humanized mouse; immune environment; liposome; progesterone; programmed death ligand 1.

Figure 1

Effect of P4 on human T cells and cancer cell lines. (A) Peripheral blood mononuclear cells (PBMCs) were stimulated with toxic shock syndrome toxin 1 in the presence of 0, 2, 20, and 200 μM of P4. T cell activation was analyzed using flow cytometry. Upper panels: CD4+ T cells; lower panels: CD8+ T cells. The levels of activated CD25+/PD-1+ T cells are shown. (B) JEG-3, BeWo, MDA-MB-231, HEK-293, and Karpas 707H cells cultured with P4 (left panels) or cortisol (right panels). Solid black lines: 0 μM; broken black lines: 2 μM; solid gray lines: 20 μM; broken gray lines: 200 μM. Data are presented as the mean ± standard deviation of three independent experiments. P4, progesterone.

Figure 2

Effect of atezolizumab on the expression of immune checkpoint molecules. (A) PD-L1 and (B) PD-1 expression on T cells. P4 (0–200 μM) was added to human PBMCs and stimulated with TSST-1 for 72 h. PD-L1 expression in CD3-gated cells are shown as histograms. The culture with/without atezolizumab is shown as [ATZ(+)] or [ATZ (–)]. “Day 0” means the PBMCs before culture and “0μM” means the TSST-1 treatment without P4. Percentages shown in each panel of T cells are the PD-L1(A)- and PD-1(B)-positive cell ratios. Numbers shown in each panel of ATZ, atezolizumab; P4, progesterone; PD-1, programmed death 1; PD-L1, programmed death ligand 1. (C) PD-L1/PD-1 expressing cells (%). (D) MFI of PD-L1 or PD-1 in the CD3+ cells. Data are presented as mean ± standard deviation (S.D.) **P < 0.01 (one-way analysis of variance or Student’s t-test) [n = 5].

Figure 3

Preparation of Lipo-anti-PD-L1-P4. (A) Protocol for the preparation of Lipo-anti-PD-L1-P4. (B) Representative result of liposome size distribution. Average size: 44 nm; polydispersity index: 0.22; ζ-potential: –20.1 kV; P4 concentration: 78.5 μg/mL. (C) P4 leakage test. DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DSPE-PEG-Maleimide, N-[(3-maleimide-1-oxopropyl)aminopropyl polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine; EDTA, ethylenediaminetetraacetic acid; Lipo-anti-PD-L1-P4, liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated P4; P4, progesterone; PBS, phosphate-buffered saline; r.t., room temperature.

Figure 4

Effect of Lipo-anti-PD-L1-P4 on cell proliferation. PD-1 and PD-L1 expression on T cells (A, B) and (C) cancer cells. (A) Left panel: Percentage of PD-1 and PD-L1 expressing cells in CD3+ T cells. Right panels: PD-1 and PD-L1 mean fluorescence intensities of CD3+ T cells. (B) Left panel: percentage of PD-1 and PD-L1 expressing cells in CD4+ T cells; right panel: percentage of PD-1 and PD-L1 expressing cells in CD8+ T cells. (C) MDA-MB-231, JEG-3, and Karpas 707H cells were cultured in the presence or absence of P4 (open bars) or Lipo-anti-PD-L1-P4 (solid bars). Vertical lines represent the cell number after 3 d of culture. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way analysis of variance or two-sided Student’s t-test). Data are presented as the mean ± standard deviation of four independent experiments. DN, double-negative; DP, double-positive; Lipo-anti-PD-L1-P4 (Lipo-P4), liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated P4; MFI, mean fluorescence intensity; PD-1, programmed death 1; PD-L1, programmed death ligand 1.

Figure 5

Effect of Lipo-anti-PD-L1-P4 on tumor growth in tumor-bearing humanized NOG-hIL-4-Tgmice. (A) Protocol for establishing the NOG-hIL-4-Tg mouse model. PD-L1 expression in MDA-MB-231 cells was analyzed using flow cytometry. (B) Kinetics of tumor growth in NOG-hIL-4-Tg mice. Tumor growth was measured from PBMC transplantation (Day 0). The mean volume ± standard error was calculated for each week. After four weeks, the mice were sacrificed. **P < 0.01 (one-way analysis of variance or Student’s t-test) (PBS [n = 11], ATZ [n = 9], Lipo-P4 [n = 7], Lipo-Emp [n = 3]). (C) Representative immunohistochemistry of CD3 expression in tumor tissues. Indicated bars in the tissue sections represent 100 μm. (D) Representative cytokine production patterns of CD3 T cells purified from NOG-hIL-4-Tg mouse spleens and stimulated with CD3/CD28. ATZ, atezolizumab; FCM, flow cytometry; FCS, fetal calf serum; IHC, immunohistochemistry; Lipo-anti-PD-L1-P4 (Lipo-P4), liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated P4; Lipo-Emp, empty liposomes; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; PD-L1, programmed death ligand 1.

Figure 6

Profile of human lymphocytes in the spleen of tumor-bearing humanized mise. (A) Typical patterns of human lymphocytes in the NOG-hIL-4-Tg mouse spleen. Cells were gated as shown in Supplementary Figure S1 . The gated fractions are shown above each panel. Vertical and horizontal lines represent the markers analyzed using flow cytometry. (B) Box-and-whisker plots of lymphocyte subsets. *P < 0.05; **P < 0.01 (one-way analysis of variance or Student’s t-test) (PBS [n = 11], ATZ [n = 9], Lipo-P4 [n = 7], Lipo-Emp [n = 3]). ATZ [A], atezolizumab; Lipo-Emp, empty liposome; Lipo-anti-PD-L1-P4 (Lipo-P4), liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated P4; MA, MDA-MB-231 cells transplanted with atezolizumab; ME, MDA-MB-231 cells transplanted with Lipo-Emp; ML, MDA-MB-231 cells transplanted with Lipo-anti-PD-L1-P4; MP, MDA-MB-231 cells transplanted with PBS; PBS [P], phosphate-buffered saline.

Figure 7

Profiles of spleen and bone marrow lymphocytes of tumor-bearing humanized NOG-hIL-4-Tg mice. The proportions (%) of human CD45+ cells/lymphocyte gated-cells, CD19+/CD45+ cells, CD3+/CD45+ cells, CD4+/CD3+ cells, and CD8+/CD3+ cells in the spleen (horizontal lines) and bone marrow (vertical lines) of NOG-hIL-4-Tg mice are plotted. Approximate straight lines and correlation coefficients are shown (PBS [n = 8], ATZ [n = 6], Lipo-P4 [n = 6]). ATZ, atezolizumab; Lipo-P4, liposome-encapsulated anti-programmed death ligand 1 antibody-conjugated P4; PBS, phosphate-buffered saline. Lower two panels of Lipo-P4 present the same graphs shown in the upper panels, but the vertical axis was expanded. Correlation lines and correlation coefficients (R²) are shown in the panels.