Tumor Cell-Autonomous Pro-Metastatic Activities of PD-L1 in Human Breast Cancer Are Mediated by PD-L1-S283 and Chemokine Axes

Abstract

Therapies targeting the PD-L1/PD-1 axis have recently been introduced to triple-negative breast cancer (TNBC) with limited efficacy, suggesting that this axis promotes tumor progression through mechanisms other than immune suppression. Here, we over-expressed WT-PD-L1 in human TNBC cells (express endogenous PD-L1) and in luminal-A breast cancer cells (no endogenous PD-L1 expression) and demonstrated that cell-autonomous PD-L1 activities lead to increased tumor cell growth, invasion and release of pro-metastatic factors (CXCL8, sICAM-1, GM-CSF). These activities were promoted by PD-1 and were inhibited by mutating S283 in PD-L1. Invasion of WT-PD-L1-cells required signaling by chemokine receptors CXCR1/2, CCR2 and CCR5 through autocrine circuits involving CXCL8, CCL2 and CCL5. Studies with T cell-deficient mice demonstrated that cell-autonomous WT-PD-L1 activities in TNBC cells increased tumor growth and metastasis compared to knock-out (KO)-PD-L1-cells, whereas S283A-PD-L1-expressing cells had minimal ability to form tumors and did not metastasize. Overall, our findings reveal autonomous and PD-1-induced tumor-promoting activities of PD-L1 that depend on S283 and on chemokine circuits. These results suggest that TNBC patients whose tumors express PD-L1 could benefit from therapies that prevent immune suppression by targeting PD-1/CTLA-4, alongside with antibodies to PD-L1, which would allow maximal impact by mainly targeting the cancer cells.

Keywords: CCR2; CCR5; CXCR1/2; PD-1; PD-L1; chemokines; luminal-A breast cancer; triple-negative breast cancer (TNBC).

Figure 1

PD-L1 expression levels, at endogenous levels and after WT-PD-L1 over-expression. (A,B) Endogenous expression of PD-L1 by breast cancer cells. (A) TNBC cells: (A1) BT-549 (BT) cells. (A2) MDA-MB-231 (MDA) cells. (B) Luminal-A cells: (B1) MCF-7 cells. (B2) T47D cells. (C,D) PD-L1 expression by breast tumor cells following infection with PD-L1 or its control vector (CTRL-vector cells). (C) TNBC cells: (C1) BT cells. (C2) MDA cells. (D) Luminal-A cells: (D1) MCF-7 cells. (D2) T47D cells. In all parts, PD-L1 expression was determined by flow cytometry. Non-relevant isotype-matched antibodies served as negative controls (Isotype). MFI, mean fluorescence intensity. In parts (C,D), three cell types are demonstrated for each cell line: (1) “CTRL-vector”—Cells infected to express the control vector of PD-L1, stained by antibodies to PD-L1; these cells demonstrate endogenous PD-L1 expression (BT and MDA cells) or the lack of PD-L1 endogenous expression (MCF-7 and T47D cells), as appropriate; (2) “WT-PD-L1”: Cells infected to over-express WT-PD-L1, stained by antibodies to PD-L1; (3) Isotype—Cells stained by a non-relevant isotype-matched antibody control (no staining was noted for both WT-PD-L1 and CTRL-vector cells stained by isotype control; for simplicity, only one of two histograms is shown). The Figure represents multiple experiments that were performed for each cell type.

Figure 2

The cell-autonomous activities of WT-PD-L1 increase pro-metastatic activities, and are potentiated by cell exposure to PD-1. (A1,A2) Cell growth of WT-PD-L1-TNBC cells and WT-PD-L1-luminal-A cells and their corresponding CTRL-vector cells. (A1) TNBC cells: BT and MDA cells. (A2) Luminal-A cells: MCF-7 and T47D cells. WT-PD-L1-cells of each cell line were plated at similar concentration as the CTRL-vector cells. At days four and five after cell plating, the cells were counted in order to determine cell growth, as described in Section 4. (A3,A4) The impact of exposure to PD-1 on cell growth. (A3) TNBC cells: BT and MDA cells. (A4) Luminal-A cells: MCF-7 and T47D cells. PD-1 or its control (ctrl) (See Section 4 for more details) were added one day after cell plating, at 2 μg/mL for 72 h or 96 h (namely days four and five after cell plating). Cell growth was determined as described in Part (A) of the Figure. (B) The Figures in this part demonstrate the extracellular expression levels of pro-metastatic factors by WT-PD-L1-MDA cells and CTRL-vector-MDA cells. The cells were cultured, and 24 h later, PD-1 or its control (ctrl), were added as stated above. Cleared cell supernatants were collected and ELISA assays were performed to determine the expression of (B1) CXCL8; (B2) sICAM-1; (B3) GM-CSF. Each panel demonstrates the results of a representative experiment out of n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05. NS, Non-significant.

Figure 2

The cell-autonomous activities of WT-PD-L1 increase pro-metastatic activities, and are potentiated by cell exposure to PD-1. (A1,A2) Cell growth of WT-PD-L1-TNBC cells and WT-PD-L1-luminal-A cells and their corresponding CTRL-vector cells. (A1) TNBC cells: BT and MDA cells. (A2) Luminal-A cells: MCF-7 and T47D cells. WT-PD-L1-cells of each cell line were plated at similar concentration as the CTRL-vector cells. At days four and five after cell plating, the cells were counted in order to determine cell growth, as described in Section 4. (A3,A4) The impact of exposure to PD-1 on cell growth. (A3) TNBC cells: BT and MDA cells. (A4) Luminal-A cells: MCF-7 and T47D cells. PD-1 or its control (ctrl) (See Section 4 for more details) were added one day after cell plating, at 2 μg/mL for 72 h or 96 h (namely days four and five after cell plating). Cell growth was determined as described in Part (A) of the Figure. (B) The Figures in this part demonstrate the extracellular expression levels of pro-metastatic factors by WT-PD-L1-MDA cells and CTRL-vector-MDA cells. The cells were cultured, and 24 h later, PD-1 or its control (ctrl), were added as stated above. Cleared cell supernatants were collected and ELISA assays were performed to determine the expression of (B1) CXCL8; (B2) sICAM-1; (B3) GM-CSF. Each panel demonstrates the results of a representative experiment out of n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05. NS, Non-significant.

Figure 3

The cell-autonomous activities of WT-PD-L1 increase breast tumor cell invasion, are potentiated by cell exposure to PD-1, and are led via molecular pathways that include Gαi- and Ras-mediated signaling and activation of chemokine receptors. (A) Tumor cell invasion. (A1) Invasion of WT-PD-L1-TNBC cells: BT and MDA cells. (A2) Invasion of WT-PD-L1-luminal-A MCF-7 cells. Cell invasion was determined in matrigel-coated transwells, for the following time points: BT cells: 8 h; MDA cells: 11 h; MCF-7 cells: 21.5 h. (B) Tumor cell invasion following exposure to PD-1 or its control (ctrl). (B1) BT and MDA cells. (B2) MCF-7 cells. T47D cells were not analyzed due to lack of invasive abilities under the experimental conditions that were assayed. WT-PD-L1-over-expressing cells of each cell line were cultured, and 24 h later, PD-1 or its control (ctrl) were added at 2 μg/mL for additional 72 h. Then, cell invasion was determined in matrigel-coated transwells, at the time points indicated above. Note: In BT and MDA experiments, the number of cells loaded in the transwells was lower than in Part (A), in order to enable determination of increment in invasion by the addition of PD-1 when membranes are not over-loaded with cells. (C) The Figure demonstrates the effects of different pathway/receptor antagonists on invasion of WT-PD-L1-MDA cells. The cells were grown with the following inhibitors for 48 h: (1) i-Gαi—PTx; (2) i-Ras—FTS (Salirasib); (3) i-CXCR1/2—Reparixin; (4) i-CCR2—CAS 445479-97-0; (5) i-CCR5—Maraviroc. DMSO was used as a vehicle control (Vehicle). Cell invasion was determined in matrigel-coated transwells. Inhibitor concentrations (provided in Section 4) that did not affect cancer cell viability were selected based on titration analyses (see a comment on i-CCR2 in Section 4). HPF, High power field. Images (Bar, 50 μm) and graph demonstrate the results of a representative experiment out of n = 3. *** p < 0.001 for comparisons between the different treatments and the vehicle control.

Figure 3

The cell-autonomous activities of WT-PD-L1 increase breast tumor cell invasion, are potentiated by cell exposure to PD-1, and are led via molecular pathways that include Gαi- and Ras-mediated signaling and activation of chemokine receptors. (A) Tumor cell invasion. (A1) Invasion of WT-PD-L1-TNBC cells: BT and MDA cells. (A2) Invasion of WT-PD-L1-luminal-A MCF-7 cells. Cell invasion was determined in matrigel-coated transwells, for the following time points: BT cells: 8 h; MDA cells: 11 h; MCF-7 cells: 21.5 h. (B) Tumor cell invasion following exposure to PD-1 or its control (ctrl). (B1) BT and MDA cells. (B2) MCF-7 cells. T47D cells were not analyzed due to lack of invasive abilities under the experimental conditions that were assayed. WT-PD-L1-over-expressing cells of each cell line were cultured, and 24 h later, PD-1 or its control (ctrl) were added at 2 μg/mL for additional 72 h. Then, cell invasion was determined in matrigel-coated transwells, at the time points indicated above. Note: In BT and MDA experiments, the number of cells loaded in the transwells was lower than in Part (A), in order to enable determination of increment in invasion by the addition of PD-1 when membranes are not over-loaded with cells. (C) The Figure demonstrates the effects of different pathway/receptor antagonists on invasion of WT-PD-L1-MDA cells. The cells were grown with the following inhibitors for 48 h: (1) i-Gαi—PTx; (2) i-Ras—FTS (Salirasib); (3) i-CXCR1/2—Reparixin; (4) i-CCR2—CAS 445479-97-0; (5) i-CCR5—Maraviroc. DMSO was used as a vehicle control (Vehicle). Cell invasion was determined in matrigel-coated transwells. Inhibitor concentrations (provided in Section 4) that did not affect cancer cell viability were selected based on titration analyses (see a comment on i-CCR2 in Section 4). HPF, High power field. Images (Bar, 50 μm) and graph demonstrate the results of a representative experiment out of n = 3. *** p < 0.001 for comparisons between the different treatments and the vehicle control.

Figure 4

The cell-autonomous activities of WT-PD-L1 act via chemokine receptors to upregulate the expression of their corresponding chemokines. (A1,B1,C1) The extracellular expression of pro-metastatic chemokines by WT-PD-L1-MDA cells compared to their CTRL-vector-MDA counterparts, determined by ELISA. (A1) CXCL8 (the experiment shown here is different from the one presented in Figure 2(B1); the results are presented again for readers’ convenience). (B1) CCL2. (C1) CCL5. (A2,B2,C2) The effects of different pathway/receptor inhibitors on the expression of pro-metastatic chemokines by WT-PD-L1-MDA cells. DMSO was used as a vehicle control (Vehicle). The extracellular expression of CXCL8 (A2), CCL2 (B2) and CCL5 (C2) was determined in cleared supernatants by ELISA. Selection of Inhibitor concentrations and doses used are the same as in Figure 3C. The data demonstrate the extracellular levels of the chemokines, normalized to cell numbers. Each panel demonstrates the results of a representative experiment out of n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05 and NS, Non-significant for comparisons between the different treatments and the vehicle control.

Figure 5

The S283 residue of PD-L1 is required for optimal cell-autonomous and PD-1-induced tumor cell growth. (A) Analyses of MDA cells. (A1) MDA cells expressing WT-PD-L1 or S283A-PD-L1. As MDA cells endogenously express PD-L1 (Figure 1(A2)), PD-L1 in these cells was knocked-out by the Alt-R CRISPR-Cas9 method and validated for the lack of PD-L1 expression by flow cytometry, as described in Figure S5. Then, these KO-PD-L1 cells were analyzed to guarantee an out-of-frame sequence of PD-L1 and were infected to express WT-PD-L1 or S283A-PD-L1; this step was followed by validation of PD-L1 cell surface expression in these two cell types by flow cytometry analyses, as shown in Figure 1. Control MDA cells in which PD-L1 was knocked out were infected by a sham vector; they did not express PD-L1 as shown in the Figure (called herein KO-PD-L1-MDA cells). (A2) Determination of MDA cell growth. (A2a) WT-PD-L1-MDA cells and S283A-PD-L1-MDA cells were plated at similar concentrations. At days four and five after cell plating, the cells were counted in order to determine cell growth. (A2b) One day after culturing of S283A-PD-L1-MDA cells, PD-1 or its ctrl were added at 2 μg/mL for 72 h or 96 h (namely, days four and five after cell plating); then, the cells were counted in order to determine cell growth. (B,C) Analyses of MCF-7 and T47D cells, respectively. (B1,C1) MCF-7 and T47D cells, which do not express PD-L1 constitutively (Figure 1(B1,2), respectively), were infected to express WT-PD-L1 (as shown in Figure 1D) or S283A-PD-L1 and were then analyzed by flow cytometry for the expression of PD-L1 at the cell surface. (B2,C2) Determination of cell growth. (B2a,C2a) WT-PD-L1-MCF-7 and WT-PD-L1-T47D cells vs. S283A-PD-L1-MCF-7 cells and S283A-PD-L1-T47D cells, respectively. (B2b,C2b) The effects of PD-1 on cell growth of S283A-PD-L1-MCF-7 cells and S283A-PD-L1-T47D cells. The experimental design was similar to that of Part Figure 5(A2). Panels (A1,B1,C1): Non-relevant isotype-matched antibodies served as negative controls (Isotype). MFI, mean fluorescence intensity. Panels (A2,B2,C2) demonstrate the results of a representative experiment out of n = 3. ** p < 0.01, * p < 0.05. NS, Non-significant.

Figure 6

The cell-autonomous and PD-1-induced expression of CXCL8 and sICAM-1 depend on the integrity of the S283 residue of PD-L1. The extracellular expression levels of (A) CXCL8 and (B) sICAM-1 were determined in cell supernatants of WT-PD-L1-MDA and S283A-PD-L1-MDA cells by ELISA assays. PD-1 or its control (ctrl) were added at 2 μg/mL for 72 h. Each panel demonstrates the results of representative experiment out of n = 3. p values are demonstrated in the Figure. NS, Non-significant.

Figure 7

The cell-autonomous and PD-1-induced activities of PD-L1 that promote tumor cell invasion depend on the integrity of the S283 residue of PD-L1. Invasion of (A) MDA and (B) MCF-7 cells that over-expressed WT-PD-L1 or S283A-PD-L1 was determined in transwell assays, after exposure to PD-1 or its control (ctrl). PD-1 or its ctrl were added one day after cell plating, at 2 μg/mL, for 72 h. Cell invasion was determined in matrigel-coated transwells for the time points indicated in Figure 3A. HPF, High power field. Images (Bar, 50 μm) and graphs, in both panels (A,B) demonstrate the results of a representative experiment out of n = 3. p values are demonstrated in the Figure. NS, Non-significant.

Figure 8

The cell-autonomous activities of PD-L1 shorten the lag period of tumor appearance in vivo and depend on the integrity of the S283 residue of PD-L1. (A) Description of the experimental procedure taken in order to determine the cell-autonomous roles of PD-L1 and its S283 residue in regulating tumor growth and metastasis. Tumor cells were administered to the mammary fat pad of female nude mice, deficient in T cell activities. This part of the Figure demonstrates the time points in which the mice of each group were sacrificed and analyzed for tumor volumes and metastasis formation. All cells were infected to express mCherry; tumor growth was determined by caliper every 3–4 days and was validated at different time points also by intravital imaging of the mammary region. Two independent experiments were performed, with the following mouse groups: (1) Group 1 = KO-PD-L1-MDA cells, with a total of n = 10 mice in the two experiments together. To follow up on the regulations of the Ethics Committee of Animal Use, the experiments of this group of mice were terminated at day 56 after tumor cell inoculation. Of note, as indicated in Figure 5(A1), in these cells the expression of endogenous PD-L1 was knocked out and the cells were infected with the control vector of PD-L1; thus, these cells do not express PD-L1 at all. (2) Group 2 = WT-PD-L1-MDA cells (generated as indicated in Figure 5(A1)), with a total of n = 9 mice in the two experiments together; the experiments of this group were terminated at day 49. (3) Group 3 = S283A-PD-L1-MDA cells (generated as indicated in Figure 5(A1)), with a total of n = 9 mice in the two experiments together, followed up until day 77. Follow up of five of the nine mice was continued until day 111. (B) Kinetics of tumor cell appearance. p values are demonstrated in the Figure.

Figure 9

The cell-autonomous activities of PD-L1 lead to increased tumor volumes in vivo and depend on the integrity of the S283 residue of PD-L1. Tumor cells were administered to the mammary fat pad of female nude mice, as described in Figure 8A, including the same three groups of mice. The current Figure shows tumor volumes at different time points along the process, determined by caliper, measured every 3–4 days (validated at some of the time points by intravital imaging mCherry signals). (A) Averages of tumor volumes in each group of mice ± SEM, at each time point. (B) Each panel shows a different mice group, where tumor volume in each individual mouse is demonstrated along tumor progression. p values of comparisons between tumor volumes are indicated in the Figure.

Figure 10

The cell-autonomous activities of PD-L1 promote metastasis in vivo and depend on the integrity of the S283 residue of PD-L1. (A) Incidence of metastasis-bearing mice. Metastases were determined in the three groups of mice at the time points that they were sacrificed: (1) Group 1 = KO-PD-L1-MDA cells: at day 56, in n = 10; (2) Group 2 = WT-PD-L1-MDA cells: at day 49, in n = 9; (3) Group 3 = S283A-PD-L1-MDA cells, at day 77 for four mice and at day 111 for five mice in which follow up was extended. Statistical analysis was performed for the three groups of mice at day 77 (in Group 3, analysis included 4 of the mice); of note, no metastases were detected in the five mice whose follow up was extended until day 111. p value is demonstrated in the Figure. (B) The incidence of mice bearing metastases in different organs, calculated in mice with metastases only.