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. 2020 Sep 7;39(1):180.
doi: 10.1186/s13046-020-01694-9.

Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer

Simona Camorani  1 , Margherita Passariello  2   3 , Lisa Agnello  1 , Silvia Esposito  3 , Francesca Collina  4 , Monica Cantile  4 , Maurizio Di Bonito  4 , Ilya V Ulasov  5 , Monica Fedele  1 , Antonella Zannetti  6 , Claudia De Lorenzo  7   8 , Laura Cerchia  9
Affiliations

Aptamer targeted therapy potentiates immune checkpoint blockade in triple-negative breast cancer

Simona Camorani et al. J Exp Clin Cancer Res. .

Abstract

Background: Triple-negative breast cancer (TNBC) is a uniquely aggressive cancer with high rates of relapse due to resistance to chemotherapy. TNBC expresses higher levels of programmed cell death-ligand 1 (PD-L1) compared to other breast cancers, providing the rationale for the recently approved immunotherapy with anti-PD-L1 monoclonal antibodies (mAbs). A huge effort is dedicated to identify actionable biomarkers allowing for combination therapies with immune-checkpoint blockade. Platelet-derived growth factor receptor β (PDGFRβ) is highly expressed in invasive TNBC, both on tumor cells and tumor microenvironment. We recently proved that tumor growth and lung metastases are impaired in mouse models of human TNBC by a high efficacious PDGFRβ aptamer. Hence, we aimed at investigating the effectiveness of a novel combination treatment with the PDGFRβ aptamer and anti-PD-L1 mAbs in TNBC.

Methods: The targeting ability of the anti-human PDGFRβ aptamer toward the murine receptor was verified by streptavidin-biotin assays and confocal microscopy, and its inhibitory function by transwell migration assays. The anti-proliferative effects of the PDGFRβ aptamer/anti-PD-L1 mAbs combination was assessed in human MDA-MB-231 and murine 4 T1 TNBC cells, both grown as monolayer or co-cultured with lymphocytes. Tumor cell lysis and cytokines secretion by lymphocytes were analyzed by LDH quantification and ELISA, respectively. Orthotopic 4 T1 xenografts in syngeneic mice were used for dissecting the effect of aptamer/mAb combination on tumor growth, metastasis and lymphocytes infiltration. Ex vivo analyses through immunohistochemistry, RT-qPCR and immunoblotting were performed.

Results: We show that the PDGFRβ aptamer potentiates the anti-proliferative activity of anti-PD-L1 mAbs on both human and murine TNBC cells, according to its human/mouse cross-reactivity. Further, by binding to activated human and mouse lymphocytes, the aptamer enhances the anti-PD-L1 mAb-induced cytotoxicity of lymphocytes against tumor cells. Importantly, the aptamer heightens the antibody efficacy in inhibiting tumor growth and lung metastases in mice. It acts on both tumor cells, inhibiting Akt and ERK1/2 signaling pathways, and immune populations, increasing intratumoral CD8 + T cells and reducing FOXP3 + Treg cells.

Conclusion: Co-treatment of PDGFRβ aptamer with anti-PD-L1 mAbs is a viable strategy, thus providing for the first time an evidence of the efficacy of PDGFRβ/PD-L1 co-targeting combination therapy in TNBC.

Keywords: Antitumor immunity; Aptamer; Metastases; PD-L1 monoclonal antibody; PDGFRβ; TNBC; Tumor microenvironment.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Effects of combinatorial treatments on human MDA-MB-231 cells in the absence or presence of lymphocytes. a Cell growth inhibition of MDA-MB-231 cells, untreated or treated with the anti-PD-L1 (10_12) mAb or the PDGFRβ aptamer, used alone (dark grey bars) or in combination (black bars), for 96 h at 37 °C at the indicated concentrations. Untreated cells (white bars) or cells treated with an unrelated IgG or Scr (light grey bars) were used as negative controls. Data were expressed as percentage of viable treated cells with respect to untreated cells. b-e MDA-MB-231 cells were co-cultured with human lymphocytes and left untreated or treated for 24 h, as indicated. b Cell counts were expressed as percentage of viable treated cells with respect to untreated co-cultured cells. c Cell lysis was expressed as measure of LDH release and reported as percentage of lysis of treated cells with respect to the untreated cells, used as a control. d IL-2 and e IFN-γ secretion levels were measured by ELISA on supernatants of cells treated, as indicated. a-e Error bars depict mean ± SD (n = 3). ####P < 0.0001, ### P < 0.001, ## P < 0.01 relative to IgG; °P < 0.05 relative to Scr; ****P < 0.0001, *** P < 0.001, ** P < 0.01, *P < 0.05; one-way ANOVA followed by Tukey’s multiple comparison test
Fig. 2
Fig. 2
Gint4.T aptamer specifically binds to and inhibits migration of PDGFRβ-positive murine cells. a Binding curve of 5′-biotinylated PDGFRβ Gint4.T aptamer to 4 T1 cells for calculation of the apparent Kd of aptamer-cell interaction. The background binding value for 5′-biotinylated Scr sequence was subtracted from each data point. Data shown are mean ± SD (n = 3). b Representative confocal images of 4 T1, NIH3T3 and BT-474 cells incubated with FAM-Gint4.T or FAM-Scr, as indicated. FAM-aptamers and nuclei are visualized in green and blue, respectively. All digital images were captured at the same setting to allow for direct comparison of staining patterns. Magnification 63 ×, scale bar = 20 μm. c Left, 4 T1 and NIH3T3 cell migration toward PDGF-BB was analyzed by transwell migration assay in the presence of Gint4.T or Scr. Photographs of a representative experiment are shown. Magnification 5 ×, scale bar = 500 μm. Right, data were presented as percentage of migrated cells in the presence of Gint4.T with respect to cells treated with Scr. Bars depict mean ± SD (n = 3). *** P < 0.001, ** P < 0.01 relative to Scr; unpaired t-test
Fig. 3
Fig. 3
Effects of combinatorial treatments on murine 4 T1 cells in the absence or presence of lymphocytes. a Cell growth inhibition of 4 T1 cells, untreated or treated with the anti-mPD-L1 mAb or the PDGFRβ aptamer, used alone (dark grey bars) or in combination (black bars), for 96 h at 37 °C at the indicated concentrations. Untreated cells (white bars) or cells treated with an unrelated IgG or Scr (light grey bars) were used as negative controls. Data were expressed as percentage of viable treated cells with respect to untreated cells. b-e 4 T1 cells were co-cultured with mouse lymphocytes and treated for 24 h, as indicated. b Cell counts were expressed as percentage of viable treated cells with respect to untreated co-cultured cells. c Cell lysis was measured by LDH release in the medium after incubation with the indicated compounds. d IL-2 and e IFN-γ secretion levels were measured by ELISA on supernatants of cells treated as indicated. a-e Error bars depict mean ± SD (n = 3). #### P < 0.0001, ## P < 0.01 relative to IgG; °°°° P < 0.0001, °° P < 0.01, ° P < 0.05 relative to Scr; **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05; one-way ANOVA followed by Tukey’s multiple comparison test
Fig. 4
Fig. 4
Effects of combinatorial treatments on tumor growth. a Mice bearing 4 T1 orthotopic xenografts were injected intravenously with PDGFRβ Gint4.T aptamer (at day 0, 2, 4, 7 and 9) and intraperitoneally with anti-mPD-L1 mAb (at day 0, 4 and 9), alone and in combination. Mice treated with Scr aptamer were used as the control group (Ctrl). Tumor growth was monitored by calipers over time and experimental raw data (expressed as fold increase) were interpolated with no curve fitting or regression analysis. Treatment schedule is shown. Day 0 marks the start of treatments. # P < 0.05 relative to Ctrl; one-way ANOVA followed by Tukey’s multiple comparison test; * P < 0.05. b Mice body weight was measured at the indicated days and the mean weight of each group is shown. a-b The mean ± SEM (n = 5) were calculated for all the groups. c Shown are images from one representative tumor sample for each treatment group stained with Ki-67 antibody. Ki-67 proliferation index was calculated as percentage of Ki-67 positive cells/total cell count for 5 randomly selected 40 × microscopic fields considering the Ctrl-group as 100%. Magnification 40 ×, scale bar = 50 μm. d-e Left, lysates from recovered tumors were immunoblotted with the indicated antibodies. Equal loading was confirmed by immunoblot with anti-vinculin antibody. One representative tumor sample per group is shown. Molecular weights of indicated proteins are reported. Middle and Right, quantification of immunoblot analysis for p-Akt, p-ERK1/2, PDGFRβ and PD-L1 normalized to the loading control vinculin. Bars depict mean ± SD (five mice for each group). c-e #### P < 0.0001, ### P < 0.001, ## P < 0.01 relative to Ctrl;** P < 0.01; * P < 0.05; one-way ANOVA followed by Tukey’s multiple comparison test
Fig. 5
Fig. 5
Effects of combinatorial treatments on tumor-infiltrating lymphocytes. Shown are images from one representative tumor sample for each treatment group stained for H&E (a) and immunostained with anti-CD8 (b), anti-GRZB (c) and anti-FoxP3 (d) antibodies. Magnification 40 ×, scale bar = 50 μm. e CD8+, GRZB+ and FoxP3+ cell counts in the tumors of anti-mPD-L1, PDGFRβ aptamer and PDGFRβ aptamer plus anti-mPD-L1 groups expressed relative to cell counts in Ctrl-tumors. f RNA extracted from recovered tumors was analyzed by RT-qPCR for the IL-2 and INF-γ genes and mRNA relative expression was reported. Bars depict mean ± SD (n = 4). e-f *** P < 0.001; ** P < 0.01, * P < 0.05; one-way ANOVA followed by Tukey’s multiple comparison test
Fig. 6
Fig. 6
Effects of combinatorial treatments on lung metastases formation. Shown are images from one representative lung sample for each treatment group stained for H&E (Magnification 2 ×). Metastatic foci (indicated by the arrows) in H&E lung sections were counted under a light microscope (Magnification 10 ×). Bars depict mean ± SD. #### P < 0.0001, ### P < 0.001 relative to Ctrl; * P < 0.05; one-way ANOVA followed by Tukey’s multiple comparison test
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