Natural killer cell recognition of in vivo drug-induced senescent multiple myeloma cells

Abstract

Recognition of tumor cells by the immune system is a key step in cancer eradication. Melphalan is an alkylating agent routinely used in the treatment of patients with multiple myeloma (MM), but at therapeutic doses it leads to an immunosuppressive state due to lymphopenia. Here, we used a mouse model of MM to investigate the ability of in vivo treatment with low doses of melphalan to modulate natural killer (NK) cell activity, which have been shown to play a major role in the control of MM growth. Melphalan treatment was able to enhance the surface expression of the stress-induced NKG2D ligands RAE-1 and MULT-1, and of the DNAM-1 ligand PVR (CD155) on MM cells, leading to better tumor cell recognition and killing by NK cells, as highlighted by NK cell increased degranulation triggered by melphalan-treated tumor cells. Remarkably, NK cell population was not affected by the melphalan dose used, but rather displayed activation features as indicated by CD107a and CD69 expression. Furthermore, we showed that low doses of melphalan fail to induce tumor cell apoptosis, but promote the in vivo establishment of a senescent tumor cell population, harboring high levels of the stress-induced ligands RAE-1 and PVR. Taken together our data support the concept of using chemotherapy in order to boost antitumor innate immune responses and report the possibility to induce cellular senescence of tumor cells in vivo.

Keywords: DNAM-1; NKG2D; immunomodulation; melphalan; multiple myeloma; natural killer cell; senescence.

Figure 1.

Study design. Fifty micrograms of melphalan in PBS (or PBS alone as sham) were intraperitoneally administered every 3 d until day 20 beginning at day 11 after 5TGM1 cell injection, to tumor-bearing and age-matched healthy mice.

Figure 2.

Modulation of RAE-1, MULT-1, and PVR expression on BM MM cells from tumor-bearing mice after melphalan treatment. RAE-1, MULT-1, and PVR surface expression was analyzed as median fluorescence intensity (MFI) by immunostaining and flow cytometry. (A) Representative histograms. Gray histogram and black line represent PBS- and melphalan-treated tumor-bearing mice, respectively; dotted and dashed lines represent PBS- and melphalan-treated tumor-bearing mice isotype controls, respectively. (B) Dot plots and statistical analysis. Each circle or square represents a mouse. The horizontal bar represents the mean value. * p < 0.05, ** p < 0.01.

Figure 3.

BM NK cell number, MM apoptotic rate, and tumor growth after melphalan treatment. (A) The number of BM NK cells from healthy and tumor-bearing mice was evaluated by immunostaining and flow cytometry, NK cells identified as CD3⁻ NK1.1⁺ lymphocytes. (B and C) MM apoptotic rate was evaluated as the percentage of AnnexinV⁺ cells and by the cleavage of PARP1 among the CD138⁺ tumor cells. (D and E) BM tumor growth as evaluated by the number of IgG2b⁺ cells at 3 and 4 weeks after tumor injection. (F) Concentration of soluble IgG2b in serum of PBS- and melphalan-treated tumor-bearing mice as estimated by ELISA assay. Each circle, square, or triangle represents a mouse. The horizontal bar represents the mean value. * p < 0.05, ** p < 0.01.

Figure 4.

Analysis of the senescent phenotype of FACS-sorted CD138⁺ BM MM cells from melphalan- and PBS-treated tumor-bearing mice. (A) Representative histogram of MM cell cycle and (B) statistical analysis of three independent experiments. (C) Percentage of bromodeoxyuridine⁺ (BrdU⁺) cells among the IgG2b⁺ tumor cells after 16 h of BrdU in vivo labeling (three mice per group). Error bar represents SEM. * p < 0.05, ** p < 0.01. (D) SA-β-Gal microscopy analysis. Arrows indicate senescent cells identified as blue-stained. Magnification 400X. (E) Western blot analysis of p53 phosphorylation and p16 expression on FACS-sorted CD138⁺ BM MM cells. Numbers represent relative intensity of p16 versus PBS sample normalized to β-actin.

Figure 5.

RAE-1 and PVR expression on MM cells in association with the SA-β-Gal cytofluorimetric assay. MM cells from melphalan-treated (black line) and PBS-treated (gray line) tumor-bearing mice were stained using the fluorogenic substrate C₁₂FDG. MM cells from melphalan-treated tumor-bearing mice were divided into SA-β-Gal^low and SA-β-Gal^high in respect to the staining of MM cells from PBS-treated tumor-bearing mice. RAE-1 and PVR expression was evaluated as median fluorescence intensity (MFI) by immunostaining and flow cytometry in the two-gated populations. Triangles represent MM cells from PBS-treated tumor-bearing mice, whereas circles and squares represent SA-β-Gal^low and SA-β-Gal^high populations, respectively, from melphalan-treated tumor-bearing mice. The horizontal bar represents the mean value. * p < 0.05, # p = 0.0585.

Figure 6.

Degranulation of NK cells against MM cells. Expression of the lysosomal marker CD107a was evaluated by immunostaining and flow cytometry on IL-15 activated NK cells upon their interaction with (A) FACS-sorted CD138⁺ MM cells from tumor-bearing mice or (B) 5TGM1 MM cells used as target at 1:1 cell ratio. To evaluate NKG2D and DNAM-1 contribution, degranulation assay was also performed by pre-incubating NK cells with anti-NKG2D or anti-DNAM-1 blocking antibodies. Each circle or square represents a mouse. The horizontal bar represents the mean value. Error bar represents SEM. * p < 0.05.

Figure 7.

CD107a and CD69 expression on BM NK cells. (A) CD107a and (B) CD69 surface expression was analyzed by flow cytometry. Each circle, square, or triangle represents a mouse. The horizontal bar represents the mean value. * p < 0.05.