Background
Triple-negative breast cancer (TNBC), accounting for ~ 15–20% of breast cancers, is a heterogeneous group of tumors with highly metastatic behavior, poor prognosis and an urgent therapeutic need [
1,
2]. The lack of expression of oestrogen receptor, progesterone receptor and epidermal growth factor receptor 2 (HER2) makes TNBC difficult to treat leaving chemotherapy as the solely available option for most patients, both in early and advanced stages of the disease, despite its considerable side effects and limited success [
3‐
5]. Nevertheless, thanks to of the continuous effort in searching molecularly targeted approaches for TNBC, the first targeted therapies have been recently approved. These consist in the PARP inhibitors Olaparib or Talazoparib [
6], which is beneficial for BRCA-mutated patients but efficiently target TNBC cells regardless of the BRCA-status, and the anti-programmed cell death-ligand 1 (PD-L1) Atezolizumab
plus nab-paclitaxel chemotherapy [
7], which applies to patients with unresectable locally advanced or metastatic PD-L1-positive TNBCs.
The rationale for the latter regimen, representing the first immunotherapy to be approved for the treatment of breast cancer, stands on the significant role of the immune system in TNBC [
8]. High PD-L1 expression and amplification of
CD274 (encoding PD-L1) have been found in most TNBC [
9] and, along with the presence of tumor-infiltrating lymphocytes (TILs), has been shown to influence TNBC prognosis [
10]. Thus, by blocking the interaction of PD-L1, on tumor cells, with PD-1 and B7.1 receptors, on tumor-infiltrating T-cells and antigen-presenting cells, the anti-PD-L1 monoclonal antibody (mAb) Atezolizumab causes a reduction of immunosuppressive signals within the tumor microenvironment (TME). This in turn causes the enhancement of T cell-mediated immunity against tumors [
11]. Noteworthy, ongoing clinical studies are exploring combination approaches of various targeting agents together with anti-PD-1/PD-L1 mAbs aimed at maximizing the effectiveness of the treatment, especially for patients with metastatic TNBC, that have only modest response to immune checkpoint inhibitors as monotherapy [
12,
13]. Among these, based on preclinical evidence of therapeutic synergy, clinical trials for TNBC treatment are currently underway, or are in recruitment status, by combining anti-PD-1/PD-L1 mAbs with small-molecules inhibitors of receptor tyrosine kinases (RTKs). Some examples are the inhibitors of Axl (NCT03184558), VEGFR (NCT03394287, NCT03797326) and c-Kit (NCT03855358). Furthermore, there is a rapid increase of the number of studies showing the efficacy of co-blocking PD-1, or its ligand, and RTKs in various human cancers [
14‐
17], including TNBC [
18].
Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane RTK expressed on endothelial and perivascular cells, where it plays an important role in wound healing and tissue repair, inflammation and angiogenesis [
19]. It is well known that overexpression of PDGFRβ on endothelial cells and tumor-associated stromal cells surface occurs in different human cancers, where the receptor establishes complex signaling pathways inducing angiogenesis and tumor progression [
20‐
22]. Moreover, PDGFRβ expression has been shown as a unique feature of tumor cells characterized by a mesenchymal/stem and poorly differentiated phenotype, and it correlates with aggressiveness and resistance to therapy in multiple tumor types [
23‐
30]. Recent findings prove that PDGFRβ is expressed on the surface of tumor cells belonging to a subgroup of mesenchymal TNBC with invasive and stem-like phenotype and contributes to drive the metastatic potential [
31] and vasculogenic mimicry [
31,
32] of these tumors.
In searching efficacious strategies to target PDGFRβ-positive TNBC in alternative to PDGFRβ tyrosine kinase inhibitors, which showed limited clinical activity in TNBC as single agents and severe side effects [
33,
34], we recently tested the Gint4.T nuclease-resistant RNA aptamer, which we previously validated as a high affinity ligand/inhibitor of PDGFRβ in glioblastoma (GBM) [
35,
36] and human bone marrow-derived mesenchymal stem cells (BM-MSCs) [
22]. We found that Gint4.T is a potent theranostic agent in TNBC [
31], as it efficiently detected lung metastases derived from TNBC cells and suppressed their formation when intravenously administrated in a mouse model [
31].
The aim of the present study was to investigate the effectiveness of the combination of the Gint4.T aptamer with anti-PD-L1 antibodies in TNBC since there are no studies including a combined inhibition of both PDGFRβ and PD-1/PD-L1 interaction in the treatment of these tumors. The inhibitory effects of combination of Gint4.T with anti-PD-L1 monoclonal antibodies on tumor cells growth, in monolayer and in co-cultures with lymphocytes, were tested in both human and mouse TNBC cell models. Importantly, we show that the PDGFRβ aptamer augments antitumor immunity and potentiates anti-PD-L1 antibody inhibitory effects on tumor growth and lung metastases formation in 4 T1 TNBC orthotopic mouse model.
Methods
Cell cultures
Growth conditions for human breast cancer MDA-MB-231 and BT-474 cell lines, and murine NIH3T3 fibroblasts (American Type Culture Collection, ATCC, Manassas, VA) were previously reported [
37]. The murine TNBC 4 T1 cells (ATCC) were grown in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Sigma-Aldrich, Milan, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich), in 95% air/5% CO2 atmosphere at 37 °C.
Human peripheral blood mononuclear cells (hPBMCs) were isolated and grown as previously described [
38,
39]. Mouse lymphocytes were isolated from mouse spleen and grown in R10 medium consisting of RPMI-1640 medium, supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 nM L-glutamine, 10 mM HEPES and 50 mM β-mercaptoethanol.
Aptamers and monoclonal antibodies
The sequences of the 2’Fluoro-pyrimidines (2’F-Py) RNA PDGFRβ Gint4.T and scrambled (Scr) aptamer, used as negative control, were previously reported [
22]. Unlabeled and FAM-labeled aptamers were synthesized by TriLink Biotechnologies (San Diego, CA, USA). 5′-biotinylated Gint4.T and Scr were synthesized by LGC Biosearch Technologies (Risskov Denmark). The handling protocols for aptamers, prior to each treatment, were previously described [
31].
Anti-human PD-L1 10_12 mAb, anti-mouse PD-L1 mAb (clone 10F.9G2, BioXcell) and unrelated IgG, used as negative control, were previously reported [
40].
Binding of Gint4.T aptamer to PDGFRβ-positive murine cells
Binding affinity (Kd value) calculation
Binding of Gint4.T to 4 T1 cells was assessed by streptavidin-biotin-based assay, as previously described [
41]. Briefly, 4 T1 cells (2.0 × 10
4 cells/well in clear round bottom 96-well plate) were incubated for 10 min at room temperature (RT) with increasing concentrations of 5′-biotinylated Gint4.T or Scr aptamers (10 nM, 20 nM, 50 nM, 100 nM, 200 nM and 500 nM), diluted in the binding buffer (BB) consisting of BlockAid™ blocking solution (Invitrogen, Carlsbad, CA, USA) with 1 mg/ml yeast tRNA and 1 mg/ml ultrapure™ salmon sperm DNA (Invitrogen), as nonspecific competitors. The binding affinity (Kd value) was calculated as previously reported [
41], by using Scr to determine the nonspecific binding.
Confocal microscopy
To visualize Gint4.T on the surface of PDGFRβ-positive murine cells, 4 T1 and NIH3T3 (1.0 × 105 cells/well in 24-well), previously seeded on a coverslip for 24 h, were incubated with FAM-labeled Gint4.T or FAM-labeled Scr (500 nM-final concentration in BB) for 10 min at RT. PDGFRβ-negative BT-474 cells were treated in the same condition and used as negative control. After three washes in Dulbecco’s phosphate-buffered saline (DPBS), cells were fixed with 4% paraformaldehyde in DPBS for 20 min, washed three times in DPBS and incubated with 1.5 μM 4′,6-Diamidino- 2-phenylindole (DAPI, D9542, Sigma-Aldrich). Finally, coverslips were mounted with glycerol/DPBS. The fluorescence images were taken under a Zeiss LSM 700 META confocal microscopy equipped with a Plan-Apochromat 63x/1.4 Oil DIC objective.
Inhibition of murine cells migration by Gint4.T aptamer
4 T1 and NIH3T3 cells were serum starved overnight in the presence of 500 nM Gint4.T or Scr. Then, cells (4 T1, 5 × 104/well; NIH3T3, 2 × 105/well) were seeded into the upper chamber of a 24-well transwell (Transwell filters 8 μm pore size; Corning Incorporate, Corning, NY) in the presence of 500 nM Gint4.T or Scr and exposed to medium containing PDGF-BB (50 ng/ml, R&D Systems, Minneapolis, MN), as inducer of migration. After incubation for 24 h at 37 °C in a humidified incubator in 5% CO2, the migrated cells were visualized by staining with 0.1% crystal violet in 25% methanol and photographed. Stained cells were lysed in 1% sodium dodecyl sulfate and absorbance at 595 nm was measured on a microplate reader.
PDGF-BB stimulation of murine cells
NIH3T3 cells (1.5 × 10
5 cells/well in 6-well) were mock-treated or serum-starved for 18 h and then left untreated or stimulated for 10 min with 20 ng/mL PDGF-BB. Cell lysates preparation and immunoblotting analyses with anti-phospho-PDGFRβ (Tyr771, indicated as p-PDGFRβ) (Cell Signaling Technology Inc.) primary antibodies were performed as previously reported [
42] by using anti-vinculin (N-19) (Santa Cruz Biotechnology, Santa Cruz, CA) as loading control. Blots shown are representative of at least three independent experiments.
Cell growth inhibition by Gint4.T aptamer/anti-PDL1 mAb combined treatment
MDA-MB-231 (5.0 × 103 cells/well) and 4 T1 (3.0 × 103 cells/well) were plated in 96-well and, after 16 h at 37 °C, were either untreated or treated for 96 h with 200 nM Gint4.T and 100 nM human anti-PD-L1 10_12 (MDA-MB-231) or murine anti-mPD-L1 (4T1), used alone or in combination. Unrelated IgG (100 nM) and Scr (200 nM) were used as negative controls. The treatment with the aptamers was renewed after 72 h. Cell counts were measured by the trypan blue exclusion test.
Effects of combinatorial treatments on co-cultures of tumor cells and lymphocytes
MDA-MB-231 (1.5 × 104 cells/well) or 4 T1 (1.5 × 104 cells/well) cells, previously seeded in 96-well flat-bottom plates for 16 h at 37 °C, were co-cultured with human or murine lymphocytes, respectively, at effector:target cells ratio 10:1, in the absence or presence of 200 nM Gint4.T or 100 nM anti-PD-L1 10_12 (human setting) or anti-mPD-L1 (mouse setting), used alone or in combination. Unrelated IgG (100 nM) and Scr (200 nM) were used as negative controls. After 24 h incubation at 37 °C in a humidified incubator in 5% CO2, lymphocytes were removed and adherent cells were washed and counted by the trypan blue exclusion test.
For determination of tumor cell lysis, the release of lactate dehydrogenase (LDH) in the cellular co-culture supernatants was measured by a LDH detection kit (Thermofisher Scientific, Meridian Rd., Rockoford, IL, USA), as previously described [
38,
39].
The concentration of interleukin-2 (IL-2) or interferon gamma (IFN-γ) cytokines secreted in the cellular co-cultures supernatant was measured by ELISA assays (DuoSet ELISA, R&D Systems, Minneapolis, MN, USA), as previously described [
38,
39].
Binding of Gint4.T aptamer to human and mouse lymphocytes
Binding of 5′-biotinylated Gint4.T to human or mouse activated lymphocytes was assessed as previously described [
38] by using increasing concentrations (50 nM, 100 nM and 200 nM) of 5′-biotinylated Gint4.T or Scr aptamers.
In vivo experiments
4 T1 cells (3 × 10
4) were re-suspended in 0.1 ml of 1:1 mix of physiological saline and Matrigel (BD Biosciences, Franklin Lakes, NJ) and orthotopically injected into the mammary fat pads of five-week-old Female Balb/c mice, which weighed about 20–22 g (Charles River, Milan, Italy). Once tumors became approximately 150 mm
3 [volume = 0.5 × long diameter × (short diameter)
2], mice were randomized into four groups (five mice for each group): Ctrl (1400 pmol Scr/intravenous injection, at day 0, 2, 4, 7 and 9); Gint4.T aptamer (1400 pmol Gint4.T/intravenous injection, at day 0, 2, 4, 7 and 9); anti-mPD-L1 (200 μg/intraperitoneal injection, at day 0, 4 and 9); Gint4.T
plus anti-mPD-L1 (1400 pmol Gint4.T/intravenous injection at day 0, 2, 4, 7 and 9
plus 200 μg anti-mPD-L1/intraperitoneal injection, at day 0, 4 and 9). The long and short diameters of the tumors were measured using slide calipers up to day 11 (2 days after the last treatment) and the body weight was also measured. At day 11, mice were euthanized. Treatment schedule is schematized in Fig.
4a.
Ex vivo analyses
After sacrificing mice, tumors from each animal were excised and cut into two pieces for sample processing: one piece was stored in 10% neutral buffered formalin for immunohistochemistry analyses, and the other piece was frozen, using liquid nitrogen, for RNA extraction and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and protein lysis for immunoblotting analyses. Lung from each animal were harvested and stored in 10% neutral buffered formalin for immunohistochemistry analyses.
RT-qPCR
RNA extraction and RT-qPCR were performed as previously described [
31] on tumors from four animals per group. Primers used were: IL-2, Fwd 5′-TTGTCGTCCTTGTCAACAGC-3′, Rev. 5′- CTGGGGAGTTTCAGGTTCCT-3′; IFN-γ, Fwd, 5′-AGCGGCTGACTGAACTCAGATTGTAG-3′, Rev. 5′-GTCACAGTTTTCAGCTGTATAGGG-3′. The following primers were used for internal control: Glucose-6-phosphate dehydrogenase, Fwd 5′-TTATCATCATGGGTGCATCG-3′, Rev. 5′-GCATAGCCCACAATGAAGGT-3′.
Immunoblotting analyses on tumor lysates
Tumor lysates preparation and immunoblotting analyses were performed as previously reported [
42]. Filters were probed with the indicated primary antibodies: anti-PDGFRβ, anti-phospho-44/42 MAPK (extracellular signal-regulated kinase 1/2, ERK1/2, indicated as p-ERK1/2), anti-phospho-Akt (Ser473, indicated as p-Akt), anti-Akt (Cell Signaling Technology Inc.), anti-PD-L1/CD274 (Proteintech Group, Inc.), anti-ERK1 (C-16) and anti-vinculin (N-19) (Santa Cruz Biotechnology, Santa Cruz, CA). Blots shown are representative of at least three independent experiments.
Immunohistochemistry
Formalin-fixed tumors and lungs were paraffin-embedded and sectioned (4 μm) and three samples per group were stained with hematoxylin and eosin (H&E) or immunostained as reported [
31]. The primary antibodies used were: anti-CD8 alpha antibody (clone D4W2Z, #98941S, dilution 1:500, 1 h 4 °C incubation, Cell Signaling Technology Inc.); anti-FoxP3 (clone D608R, #12653S, dilution 1:150, 1 h 4 °C incubation, Cell Signaling); anti-Granzyme B (GRZB ab4059; dilution 1:150 diluted, 4 °C RT, Abcam, Cambridge, MA); anti-Ki-67 (dilution 1:75, overnight 4 °C incubation, Invitrogen); anti-PDGFRβ (diluition 1:50, 1 h 4 °C incubation, Cell signaling Technology Inc.); anti-PD-L1 (diluition 1:50, 1 h 4 °C incubation, Proteintech Group).
Results were interpreted using Olympus BX43 light microscope (Olympus, Center Valley, PA). Each slide was reviewed blinded and, to ensure accuracy, the number of metastases and of Ki-67, CD8, GRZB and Foxp3-positive cells was determined by two independent counts.
Statistical analysis
Statistical values were defined using GraphPad Prism version 8.00 by unpaired t-test (two variables) or one-way ANOVA followed by Tukey’s multiple comparison test (more than two variables). P value < 0.05 was considered significant for all analyses.
Discussion
Although immunotherapy approaches based on immune checkpoint inhibitors such as anti-PD-1 or anti-PD-L1 antibodies have shown convincing results in multiple cancers, they are active in only a minority of patients [
59]. Thus, multiple combination approaches currently aim to improve the efficacy of PD-1/PD-L1 blockade counteracting the immunosuppressive effect of the TME and avoiding the occurrence of therapeutic resistance. However, this often requires the use of cytotoxic chemotherapy, as in the case of metastatic TNBC, which may cause negative systemic side effects. In order to address this shortcoming, the ideal anti-cancer treatment alongside immunotherapy should include tumor-specific targeting agents that exhibit significant therapeutic effects and high tissue penetration. In that regards, one of the latest trends in oncotherapy is the use of aptamers, representing one of the most promising compounds able to specifically target tumor markers [
60‐
62]. They are single-stranded oligonucleotides that, resembling antibodies, utilize their tridimensional shape for target recognition [
63,
64]. Active tumor targeting by aptamers, while preserving affinity and specificity similar to mAbs, presents several advantages over them, including smaller size, higher stability, cheaper cost for synthesis, minimal inter-batch variability and lack of immunogenicity [
61,
65‐
67]. While aptamers are generally functionalized with cytotoxic payloads for cancer therapy, it has been also demonstrated their ability to be antagonistic agents independent of drug conjugation, exerting significant potential as anti-cancer therapeutics [
31,
37,
68]. PDGFRβ has been shown as an ideal target for antagonistic therapy development in aggressive human cancers, including TNBC [
23‐
31]. Herein, the PDGFRβ antagonist Gint4.T aptamer [
22,
31,
35] strengthens anti-PD-L1 antibody therapeutic efficacy in both human and murine TNBC cell cultures and in a well-established mouse model for TNBC. Our results clearly show that there are multiple mechanisms through which PDGFRβ aptamer could potentiate anti-PD-L1 antibody effects. It indeed causes inhibition of tumor growth and metastases formation by a direct effect on tumor cells, and also augments tumor immunity, which might secondarily facilitate the anti-tumor activity of anti-PD-L1 antibodies. The mechanism of action of the Gint4.T aptamer as both high specific targeting agent of human PDGFRβ expressed on the surface of tumor cells and inhibitor of receptor activation and downstream dependent ERK1/2 and PI3K/Akt signaling pathways in GBM [
35,
36,
42] and TNBC [
31], has been previously clarified and reported in literature and here confirmed for the first time in murine cells. Indeed, we show that Gint4.T is able to bind to murine PDGFRβ-positive cells thus hampering cell growth and migration. Accordingly, a strong reduction of tumor growth and lung metastases was observed in orthotopic 4 T1 mouse xenografts in syngeneic BALB/c mice, which was accompanied by a strong inhibition of both ERK1/2 and Akt signaling molecules in tumor samples. Also, we suggest that Gint4.T acts on immune populations causing both the depletion of Treg cells and the increase of CD8
+ T cells tumor infiltration together with an increase of GRZB, thus heightening the antibody-dependent antitumor immunity. Consistently, in both in vitro human and murine co-cultures of TNBC cells and lymphocytes and in vivo 4 T1 xenograft model, the aptamer potentiates the anti-PD-L1 antibody-induced T cell stimulation [
46], as demonstrated by the increase in the levels of IL-2 and IFN-γ cytokines. Accordingly, it has been shown that PDGF may act directly on certain lymphocyte subsets [
47,
69,
70]. Further, the increase of Tregs in breast tumors correlates with an invasive phenotype and a poor prognosis, and aggressive TNBC is considerably associated with high expression of Tregs [
71]. Notably, small molecules inhibitors of PDGFRβ have been reported to augment tumor immunity by depletion of FoxP3-expressing Tregs and increase in CD8
+ T cells in humans and advanced tumor-bearing mice [
72‐
74].
Tumor development and progression are promoted by the establishment of a favorable TME, including PDGFRβ-positive mesenchymal stem cells (MSCs), tumor-associated fibroblasts, angiogenic endothelial cells, and infiltrating immune cells, through a cytokine network [
75‐
77]. It is likely that, by acting on these cell components, Gint4.T modifies TME to ultimately potentiate the anti-PD-L1 responses. At this regard, we previously showed that Gint4.T aptamer binds to and inhibits PDGFRβ on BM-MSCs, thus hampering their homing into TNBC TME and consequently counteracting bone marrow-derived MSCs role to enhance lung metastases formation [
22]. Also, we showed that the aptamer is able to transcytose the blood-brain barrier (BBB), by binding to PDGFRβ highly expressed on endothelial cells of vessels that vascularize the tumor [
36]. We could thus speculate that the aptamer may interfere with tumor vessels formation thereby inhibiting tumor growth and metastatic potential. Furthermore, because the preferential expression of PDGFRβ by cancer cells with stem-like characteristics and/or that have undergone epithelial-mesenchymal transition (EMT) [
31,
61], it would be interesting to assess whether the proposed aptamer-based strategy results in reducing the proportion of mesenchymal, stem-like cells. In that case it would also counteract the recently evidenced detrimental effects of chemotherapy on tumor relapse and metastasis promotion due to induction of EMT and stemness phenotype [
78].
These findings provide rationale for the combined therapeutic targeting of PDGFRβ and PD-L1 in TNBC, where immune-checkpoint blockade as single therapy has met limited success so far.
Further, they lay the ground to construct a new bispecific immunoconjugate, made up of anti-PD-L1 antibody covalently linked to Gint4.T aptamer, thus optimizing the efficacy of the combination therapy by increasing their co-targeting at the tumor site while dispensing lower doses of either single agents and overcoming the limits related to the rapid clearance of the aptamers. We previously developed three different bispecific conjugates consisting of EGFR aptamer linked to either anti-HER2, anti-PD-L1 or anti-CTLA-4 mAbs [
38,
39]. By this strategy the advantages of Gint4.T aptamer (nuclease resistance, rapid tumor uptake, durable tumor retention [
31] and anti-PD-L1 antibodies (longer half-life in circulation, immunomodulatory activity) could be combined in one single molecule with improved therapeutic effectiveness and pharmacokinetic/pharmacodynamic properties over the parental moieties. At this regard, it would be intriguing to assess whether the conjugation of the PDGFRβ aptamer with a fragment smaller than the entire antibody, such as a Fab lacking the Fc region, preserves the aptamer’s ability to deliver the antibody cargo through the BBB. In such a case the construct could exert therapeutic benefit on brain tumors or brain metastases of breast cancer, while avoiding the side effects previously seen with antibodies entering the brain [
79], thanks to the lower dose of the antibody dispensed in a conjugated form.
It is still debated whether BRCA1- and BRCA2-deficiency, which causes genomic instability and increased tumor burden, could increase immunosensitivity in breast cancer and predict clinical benefit from immunotherapy [
80,
81]. In the present study, we used two BRCA1- and BRCA2-proficient cell lines [
82,
83]. Upcoming studies with a larger number of TNBC cell lines, either wild-type or mutated for the BRCA genes, will be helpful to answer whether BRCA1- and BRCA2-mutations might influence our combined aptamer-immunotherapy.
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