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. 2019 Dec 31;5(1):726–734. doi: 10.1021/acsomega.9b03381

Oxaliplatin-Based Platinum(IV) Prodrug Bearing Toll-like Receptor 7 Agonist for Enhanced Immunochemotherapy

Li Tang †,‡, Demin Cai †, Mian Qin †, Shuo Lu †, Ming-Hao Hu †, Shuangchen Ruan ‡, Guangyi Jin †,§,∥,*, Zhigang Wang †,∥,*
PMCID: PMC6964279  PMID: 31956823

Abstract

graphic file with name ao9b03381_0005.jpg

A combination of platinum drugs with immunotherapy has shown promising anticancer effects, especially in the drug resistance cancer model. Herein, a new type of immunochemotherapeutic was designed by tethering the toll-like receptor 7 (TLR7) agonist on the axial position of oxaliplatin-based platinum(IV) prodrug. The prodrug simultaneously induced immunogenic cell death of 4T1 cancer cells to initiate an immune response and activate dendritic cells (DCs) to secrete proinflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-12, to further enhance the adaptive immunity. The prodrug exhibited better in vivo anticancer effects than oxaliplatin in the 4T1 allograft mouse model, a later stage breast cancer model, which showed poor response to traditional chemotherapy. Mechanism studies revealed that enhanced activation of cytotoxic T cells within tumor contribute to the high in vivo anticancer efficiency of the prodrug. Moreover, the prodrug displayed much lower cytotoxicity to DCs compared with oxaliplatin, indicating its safety to normal cells. These results highlight the potential of the conjugation of TLR7 agonist with oxaliplatin-based Pt(IV) prodrug as an effective anticancer agent to overcome the toxic side effects and drug resistance of traditional platinum chemotherapy.

1. Introduction

Platinum drugs including cisplatin, carboplatin, and oxaliplatin have been widely used in the clinic against many solid tumors, such as testicular, ovarian, colorectal, and non-small-cell lung cancer.1 However, the toxic side effects and inherent or acquired resistance limited the therapeutic effects.2,3 Efforts has been devoted in developing multifunctional platinum drugs that target the cytotoxic pathways of platinum drugs to improve the anticancer effects.4,5 It is generally accepted that the formation of Pt-DNA damage is the main mechanism of action of platinum-based drugs.1,6 However, they also induce off-target effects on the immune system.6−8 Thus, platinum agents targeting the immune system have been emerging as a promising strategy in drug development. Several examples of the combination of platinum drugs with immunotherapy have been reported and showed promising anticancer effects, especially in drug-resistant cells.9−13

Recent studies found that oxaliplatin has an unexpectedly unique mode of action, dramatically different from cisplatin and carboplatin.8,14,15 For example, it has been shown that oxaliplatin is able to cause ribosome biogenesis stress and nucleolar stress, which might lead to its different clinical applications and side effects profiles.14,15 Oxaliplatin is also noted to induce immunogenic cell death (ICD), which is highly associated with the therapeutic outcomes of oxaliplatin.16 Oxaliplatin showed poor anticancer effects in colorectal cancer patients lacking TLR4 gene, a gene responsible for the activation of ICD.17 Thus, modulation of the immune system will affect the anticancer effects of oxaliplatin. Oxaliplatin-induced ICD leads the exposure of calreticulin (CRT) on the cell surface, which provides an “eat-me” signal for antigen-presenting cells (APCs) to phagocytose the dying tumor cell to initiate the adaptive immunity.16 Activation of dendritic cells (DCs) is particularly important in this process since they are the most powerful APCs and play important roles in immune response.18 Therefore, locally enhanced activation of DCs by small molecules may elevate the levels of oxaliplatin-induced immunotherapy, resulting in a better therapeutic outcome, especially in cancers that are resistant to traditional chemotherapy. Nevertheless, the rational design of platinum drugs based on the ICD effects of oxaliplatin has not yet been investigated.

Toll-like receptors (TLRs) are pattern recognition receptors that play an essential role in the innate immune system and serve as a bridge linking early-stage innate responses to adaptive immunity.19,20 TLRs are expressed on many types of immune cells, including DCs and macrophages.21 Recognition of antigens by TLRs in DCs leading to the production of proinflammatory cytokines and elevated antigen presentation to naive T cells, and activation of antigen-specific adaptive immune responses.21 Toll-like receptor 7 (TLR7) is a member of this receptor class and becomes a popular target for drug discovery since it recognizes synthetic small molecules.22,23 Small-molecule based TLR7 agonists have been shown to activate immune cells such as DCs, monocytes, and macrophages, resulting in elevated immunity response. TLR7 agonists have been developed as anticancer immunotherapeutics as single agents or in combination with chemotherapeutics that induce ICD.24,25 Thus, TLR7 agonists will enhance the antitumor immunity of oxaliplatin-induced ICD through the activation of DCs, leading to improved anticancer effects.

To correlate this hypothesis, herein, we designed a novel immunochemotherapeutic agent (TPt) by conjugating a TLR7 agonist (SZU101) to the axial position of an oxaliplatin-based Pt(IV) prodrug scaffold. Pt(IV) complexes are prodrugs, which are kinetically inert and can be activated by endogenous reductants to release the active Pt(II) drug along with the dissociation of the axial ligands.26,27 We reasoned that this prodrug could simultaneously induce ICD by the released oxaliplatin and promote the activation of DCs by the released TLR7 agonist, leading to an enhanced immunochemotherapy (Scheme 1).

Scheme 1. Overview of the Design of the Immunochemotherapeutic.

Scheme 1

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TPt enters cancer cells and is reduced to release oxaliplatin and TLR7 agonist SZU101. The former induces immunogenic cell death of cancer cells to provide tumor antigen, the latter enhances the maturation of DCs, leading to increased production of cytokines and activation of cytotoxic T cells. Lastly, the activated T cells kill the remaining cancer cells.

2. Results and Discussion

An adenine type of TLR7 agonists SZU101 (Scheme 1) was prepared following previously reported methods.24 This small molecule SZU101 is able to stimulate DCs with a higher level of cytokines compared with lipopolysaccharides, a class of larger molecules that act as prototypical endotoxins to induce an immune response. TPt was constructed by conjugation of SZU101 to the axial position of c,t,c-[Pt(DACH)(OH)2ox], DACH = (1R,2R)-1,2-diaminocyclohexane, ox = oxalate, using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) as coupling agent in dimethyl sulfoxide (DMSO) (Scheme S1). The final product was purified by preparative high-performance liquid chromatography (HPLC). The purity of TPt is 96%, as ascertained by analytical liquid chromatography-mass spectrometry (LC-MS) (Figure S1). TPt was well characterized by ESI-HRMS, 1H NMR, 13C NMR, and 195Pt NMR (Figures S2–S5). Since Pt(IV) prodrug is believed to exert its cytotoxicity by its reduced products, we measured the reduction process of TPt in the presence of sodium ascorbate in phosphate buffer saline (PBS, pH = 7.4). As shown in Figures 1A and S6, TPt is stable in PBS buffer with 96% TPt remained after incubation for 24 h. In the presence of 1 mM sodium ascorbate, 43% TPt was reduced to oxaliplatin and SZU101 after 24 h incubation, indicating the reduction process is relatively slow. This result is consistent with other reports that oxaliplatin-based Pt(IV) prodrugs are general not easily to be reduced.28,29

Figure 1.

Figure 1

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(A) Percentage of remained TPt after different incubation time in PBS (containing 0.2% dimethylformamide (DMF)) without or with 1 mM sodium ascorbate (NaAs) at 37 °C; (B) cellular platinum levels of 4T1 cells treated with 10 μM TPt or oxaliplatin for 6 and 12 h; (C, D) surface calreticulin levels of 4T1 cells treated with SZU101 (100 μM), oxaliplatin (100 μM), or TPt (100 μM) for 24 h; (E) extracellular adenosine 5′-triphosphate (ATP) release concentration of 4T1 cells treated with SZU101 (100 μM), oxaliplatin (100 μM), or TPt (100 μM) for 24 h; (F) activation of human TLR7-NF-κB signaling by SZU101, oxaliplatin, or TPt for 18 h.

We first evaluated the cancer cell killing effects of TPt as well as oxaliplatin and SZU101 alone in 4T1 murine breast cancer cell line. This cell line has been widely employed in antitumor studies, as a model mimics stage IV human breast cancer, which shows poor response to chemotherapy.30−33 Cells were treated with complexes for 48 h, and the cell viability was measured by CCK 8 assay. As shown in Table 1, oxaliplatin showed moderate cytotoxicity against 4T1 cells with an IC50 value of 7.2 ± 2.7 μM. SZU101 itself exhibited low direct cytotoxicity towards the cell lines with IC50 values larger than 50 μM. SZU101 is a TLR7 agonist, which kills cancer cells through activation of immune cells, so its direct cytotoxicity to cancer cells is low. TPt demonstrated lower activity against 4T1 cells compared with oxaliplatin with an IC50 value of 48.0 ± 1.9 μM. The lower cytotoxicity of TPt might due to its slow reduction process within cells, which was also observed in other reported oxaliplatin-based Pt(IV) prodrugs.34,35 A physical mixture of oxaliplatin with SZU101 (1:1 mole ratio) showed comparable cytotoxicity with that of oxaliplatin as expected. The cytotoxicity of these complexes against immune cells was also investigated, a higher cell killing effect to immune cells might inhibit the immune response. Bone marrow-derived dendritic cells (BMDCs) were used for the measurement. Notably, oxaliplatin is very toxic to BMDCs with an IC50 value of 2.7 ± 0.2 μM. While SZU101 and TPt were less toxic with IC50 values larger than 50 μM. These data suggest that TPt showed lower cytotoxicity to both cancer cells and DCs.

Table 1. Cell Viability of 4T1 Cell Lines and BMDCs Treated with Oxaliplatin, SZU101, and TPt.

  IC50/μM
cell lines oxaliplatin SZU101 TPt oxaliplatin + SZU101
4T1 7.2 ± 2.7 >50 48.0 ± 1.9 6.5 ± 0.8
BMDCs 2.7 ± 0.2 >50 >50 NDa
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a

ND is defined as not determined.

The cytotoxicity of platinum-based anticancer drugs is highly correlated with cellular platinum levels. We measured the cellular uptake of TPt and oxaliplatin in 4T1 cells. 4T1 cells were treated with 10 μM platinum complexes for 6 or 12 h, and the platinum contents in cells were analyzed using inductively coupled plasma-mass spectrometer (ICP-MS). As shown in Figure 1B, the platinum level in TPt-treated cells are higher than that by oxaliplatin. For example, after 6 h treatment, the values are 1.03 ± 0.52 and 0.53 ± 0.23 ng Pt per 106 cells respectively. The results indicated that ligation of SZU101 facilitated the drug uptake, and the lower cytotoxicity of TPt towards 4T1 cells mainly resulted from its slow activation process within cells.

The induction of ICD of cancer cells is the first step to initiate antitumor immunity of the designed immunochemotherapeutic. We tested the ability of TPt to induce the ICD of 4T1 tumor cells by measuring calreticulin (CRT) exposure. Calreticulin is a protein that is widely distributed in the endoplasmic reticulum (ER). Cell surface exposure of calreticulin determines the immunogenicity of cancer cell death.36 We measured the CRT exposure of 4T1 cells treated with TPt as well as oxaliplatin and SZU101 by flow cytometry. Cells were treated with complexes for 24 h and stained with Alexa Fluor 647 conjugated anti-CRT antibody, the dead cells were gated out by using Sytox Green. As shown in Figures 1C and 1D, treatment with oxaliplatin-induced CRT exposure on the surface, which is consistent with previous reports.37 SZU101 did not induce noticeable CRT exposure. As expected, TPt also induced CRT exposure with a comparable mean value of CRT fluorescence with that of oxaliplatin. In addition, studies indicated the additional in vitro detection of ICD marker ATP was also critical for the immune response.38 Chemiluminescent assay was used to assess the extracellular release of ATP in the culture medium of 4T1 cells after 24 h treatment with TPt, oxaliplatin or SZU101 at a concentration of 100 μM. The extracellular ATP concentration was detected to be 14 nM after treatment with TPt, while the values are 11 and 2.8 nM after oxaliplatin and SZU101 treatment respectively (Figure 1E). These data suggest that TPt is able to induce immunogenic cell death as its active parent Pt(II) drugs, thereby stimulating the engulfment of dying tumor cells-associated antigen by immature DCs.

We then tested the agonistic activity of TPt by using a HEK-Blue hTLR7 reporter cell system. Stimulation of TLR7 will activate NF-kB, followed by the expression of secreted embryonic alkaline phosphatase (SEAP), which will be detected by using HEK-blue as the substrate. The 50% of maximal effective concentration was calculated for each complex. As indicated in Figure 1F, SZU101 activated TLR7 with an EC50 value of 0.93 ± 0.17 μM, while oxaliplatin itself did not stimulate TLR7. TPt significantly activated TLR7 with an EC50 value of 0.14 ± 0.01 μM. The TLR7 stimulation ability of TPt increased 6.6-fold compared with that of SZU101. TLR7 is expressed on both cell surface and intracellular endoplasmic reticulum.39 The negative charge of SZU101 might reduce its cellular accumulation, conjugation of SZU101 with platinum improved its cellular uptake. The high concentration of TPt within cells might contribute to its improved agonistic activity compared with SZU101. These data suggest that TPt is a strong agonist to human TLR7, which will stimulate DCs to generate proinflammatory cytokines.

The stimulation of DCs by TPt was further measured. Activation of BMDCs was confirmed via the levels of cytokines released in the media by cytometric bead array (CBA) assay. TPt was incubated with BMDCs for 18 h, oxaliplatin and SZU101 were used as controls. As shown in Figures 2 and S7, oxaliplatin itself did not induce the secretion of proinflammatory cytokines, suggesting that oxaliplatin itself does not elicit the DCs response directly. Treatment with SZU101 leads to the secretion of cytokines at a moderate level. Notably, TPt effectively induced the secretion of proinflammatory cytokines with high amplitude. TPt showed approximately 2-fold higher potencies, as assessed by IFN-γ, TNF-α, IL-6, and IL-12 release, compared with SZU101. In addition, upon treatment with SZU101 or TPt, the upregulation of antiinflammatory cytokine IL-10 was negligible (Figure 2E). The significantly enhanced secretion of proinflammatory cytokines by DCs upon treatment of TPt will promote the proliferation and activity of cytolytic T cells, resulting in an elevated antitumor immune response.

Figure 2.

Figure 2

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Proinflammatory cytokine levels in the culture media of BMDCs treated with SZU101 (2 μM), oxaliplatin (2 μM), or TPt (2 μM) for 18 h. (A) IFN-γ; (B) TNF-α; (C) IL-6; (D) IL-12; (E) IL-10. Statistical analysis was carried out using Student’s t test. **P < 0.01, n = 3.

The above in vitro results prompted us to address whether TPt induced tumor cell killing and cytokines production activity can efficiently inhibit tumor growth in vivo. The murine mammary adenocarcinoma 4T1 allograft models were established by subcutaneous inoculation with 2 × 105 4T1 cells in the right flank of BALB/c mice. 4T1 tumor-bearing mice were intratumorally injected with the tested complexes every three days for a total administration of three times when the tumors were palpable on day 7. As indicated in Figure 3A–C, oxaliplatin or SZU101 alone weakly inhibited the tumor growth. However, TPt significantly inhibited tumor growth with a 55% reduction in tumor volume and 64% reduction in tumor weight, which is significantly more effective than oxaliplatin with a 15% reduction in tumor volume and 26% reduction in tumor weight respectively. In addition, the body weight of the mice treated with TPt did not change significantly (Figure 3D), indicating the safety of the complex. Lastly, 32 days after tumor inoculation, mice were sacrificed and cellular infiltrates in treated tumors were analyzed. Compared to the PBS group, TPt largely increased the intratumoral infiltration of CD8+ T cells. The frequency of tumor-infiltrating CD8+ T cells in the TPt-treated group was comparable with that in the SZU101 or oxaliplatin treated group with a slightly high mean value (Figure 4A). Similar results were also found within splenocytes (Figure 4B). The antitumor immune response elicited by TPt was further confirmed by immunofluorescence assay (Figure 4C). We found that TPt treatment instigated extensive CD8+ T cell infiltration within the tumor, whereas less tumor-infiltrating CD8+ T cells were observed in the other groups. In addition, a large fraction of the tumor-infiltrating CD8+ T cells secreted IFN-γ, indicating the ability of TPt to promote activated CD8+ cytotoxic T lymphocytes infiltration into tumors and induce antitumor immunity. These results further confirm the anticancer efficacy of the prodrug in vivo, and activation of T cells partially contributed to this effect, which verified our strategy to enhance the anticancer effects of oxaliplatin by stimulation of DCs followed by activation of T cells. Despite the prodrug showed low direct cytotoxicity to 4T1 cells as indicated by the in vitro assay, the prodrug significantly inhibited in vivo tumor growth compared with oxaliplatin, which highlights the advantages of activation of the immune system to kill cancer cells.

Figure 3.

Figure 3

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In vivo antitumor activity of the complexes against 4T1 allograft tumors. (A) The tumor growth curve. The complex (5 μmol/kg) were administrated at day 7, 10, 13; (B) excised tumor weight at the endpoint; (C) photograph of the excised tumor at the endpoint; (D) the body weight of the mice. Arrows show the days with drug injection. Statistical analysis was carried out using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, n = 4.

Figure 4.

Figure 4

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(A) Percentage of CD3+CD8+ T cells in the tumor; (B) the percentage of CD3+CD8+ T cells in spleen; (C) representative images of tumors after immunofluorescence staining (400×). Statistical analysis was carried out using Student’s t test. *P < 0.05, **P < 0.01, n = 4.

3. Conclusions

In summary, we report the first example of an oxaliplatin-based Pt(IV) prodrug bearing TLR7 agonist as an immunochemotherapeutic agent against breast cancer. The prodrug induced ICD of cancer cells compared with the parent oxaliplatin. Remarkably, the prodrug significantly activated TLR7 and simulated BMDCs to secrete proinflammatory cytokines, including IFN-γ, TNF-α, IL-6, and IL-12 to further activate cytotoxic T cells to kill cancer cells. A combination of these effects leads to enhanced in vivo antitumor effects of the prodrug against stage IV breast cancer model compared with that of oxaliplatin. In addition, the prodrug showed very lower cytotoxicity to BMDCs compared with oxaliplatin. These results suggest that the prodrug hold great promise to overcome the systemic side effects and drug resistance issues of traditional platinum chemotherapy. We believe this strategy will shed light on the design of Pt-based immune-therapeutics with improved antitumor efficacy.

4. Experimental Section

4.1. General Methods

Oxaliplatin was obtained from Boyuan Technology (Shandong, China). All the reagents and solvents were bought from commercial suppliers and used as received without further purification. NMR spectra were collected on a Bruker Ascend AVANCE III 600 MHz spectrometer. Chemical shifts are reported in parts per million relative to residual solvent peaks. ESI-HRMS were conducted on a Thermo Scientific LTQ Orbitrap XL. LC-MS analysis was performed on an Agilent Infinitylab LC/MSD 1260 system, equipped with a reversed-phase C18 column (YMC 100 × 2.10 mm, 2 μm). The samples were monitored by UV absorbance at 254 and 370 nm and ESI-MS at scan mode. Solvent A (H2O with 5% acetonitrile and 0.1% HCOOH) and solvent B (acetonitrile with 5% H2O and 0.1% HCOOH) were used for a gradient elution at a flow rate of 0.35 mL/min. The samples were eluted by using a program as follows: 0% B (0 min) → 20% B (3 min) → 100% B (6 min) → 100% B (12 min). For measurement of the reduction of TPt, the system equipped with another C18 column (YMC, 250 × 4.6 mm, 5 μm), and the samples were eluted by using a program as follows: 0% B (0 min) → 40% B (15 min) → 60% B (20 min) at a flow rate of 1.0 mL/min. Preparative HPLC was conducted on an Agilent Infinitylab LC/MSD 1260 system, equipped with a reversed-phase C18 column (YMC 300 × 20 mm, 5 μm). Pt content was measured by an inductively coupled plasma-mass spectrometer (ICP-MS) (PE Nexion 2000).

4.2. Synthesis of TPt

[Pt(DACH)(OH)2(ox)] and SZU101 were prepared following reported methods.24,40 [Pt(DACH)(OH)2(ox)] (50 mg, 0.115 mmol) was stirred in DMSO (3 mL) with SZU101 (51 mg, 0.115 mol, 1 equiv), triethylamine (14 mg, 19.4 μL, 0.138 mmol, 1.2 equiv), and TBTU (44.2 mg, 0.138 mmol, 1.2 equiv) overnight at room temperature (RT). The mixture was centrifuged at 5000g, the supernatant was collected, and DMSO was removed by lyophilization. The raw product was further washed three times with water and then subjected to HPLC for purification. Solvent A (H2O) and solvent B (acetonitrile) were used for a gradient elution at a flow rate of 5 mL/min. The samples were eluted by using a program as follows: 20% B (0 min) → 100% B (12 min) → 100% B (20 min). The final product was collected at time 15.2–15.8 min, the solvent was removed under high vacuum to yield a white powder (yield 26%). 1H NMR (600 MHz, DMSO-d6): δ 9.96 (s, 1H), 8.43 (t, J = 10.0 Hz, 1H), 8.28 (t, J = 5.9 Hz, 1H), 8.08 (s, 1H), 7.81 (s, 1H), 7.24 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 7.05 (s, 1H), 6.46 (s, 2H), 4.82 (s, 2H), 4.25 (t, J = 9.5 Hz, 2H), 4.23–4.14 (m, 2H), 3.58 (t, J = 9.5 Hz, 2H), 3.27 (s, 3H), 2.61–2.50 (m, 1H), 2.47–2.44 (m, 1H), 2.44–2.41 (m, 1H), 2.41–2.37 (m, 1H), 2.37–2.34 (m, 1H), 2.32–2.27 (m, 1H), 2.02 (t, J = 15.1 Hz, 2H), 1.42 (d, J = 10.9 Hz, 2H), 1.27 (q, J = 13.4, 12.8 Hz, 1H), 1.05 (q, J = 12.3, 11.8 Hz, 2H). 13C NMR (151 MHz, DMSO-d6) δ 181.1, 171.3, 163.7, 163.7, 159.7, 152.1, 149.0, 147.6, 138.7, 135.5, 127.4, 127.2, 98.2, 70.1, 65.2, 60.9, 59.9, 58.0, 42.0, 41.6, 31.9, 31.1, 30.8, 30.5, 23.6, 23.3. 195Pt NMR (129 MHz, DMSO-d6): δ 1410.4 (s). ESI-HRMS (positive ion mode) m/z: [M + H]+ calcd. for C28H39N8O11Pt 858.23805, found 858.23816.

4.3. Cell Culture

4T1 mouse breast carcinoma was purchased from ATCC and cultured in complete Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin (HyClone), and 100 μg/mL streptomycin (HyClone). HEK-Blue hTLR7 reporter cells were purchased from InvivoGen. The cell lines were maintained and subcultured in growth medium (DMEM, 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 100 mg/mL Normocin, 2 mM l-glutamine) supplemented with 10 μg/mL of blasticidin and 100 μg/mL of Zeocin. Bone marrow-derived dendritic cells (BMDCs) were harvested from 8-week-old Balb/c mice. BMDCs were cultured in BMDC primary media: RPMI 1640 (HyClone), 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 20 ng/mL GM-CSF (Sino Biological), and 10 ng/mL IL-4 (Sino Biological). All cell lines and assay cultures were maintained at 37 °C and 5% CO2. Cell lines were tested regularly for mycoplasma contamination, and none used tested positive at any point.

4.3.1. CCK 8 Assay

The viability of cancer cells exposed to the complexes was evaluated by CCK 8 assay. Cells were seeded in 96-well plates at a density of 3000 cells per well and incubated until the cell confluency reached 30%. Then, the medium was removed and replaced with fresh medium containing different concentrations of complexes with 0.5% DMF. After 48 h incubation, the medium was aspirated and serum-free medium containing CCK 8 was added. After 2 h additional incubation, the absorbance at 450 nm of each well was measured by using a Bio-Tek Quant microplate spectrophotometer.

4.3.2. Cellular Uptake Assay

Cells were seeded in a 6-well plate and incubated until the cell confluency reached 80%. The medium was changed to fresh medium containing 10 μM complex with 1% DMF. After 6 or 12 h exposure, cells were washed twice with ice-cold PBS and collected by trypsinization. The cells were subsequently washed with ice-cold PBS and resuspended in 1 mL PBS. The cell number was counted by hemocytometer. Then, the cells were centrifuged and the cell pellets were digested by 65% nitric acid and 15% H2O2 overnight at 65 °C. The lysate was diluted to a final volume of 2 mL, and the Pt content was measured by ICP-MS. The platinum levels in cells were expressed as ng Pt per million cells.

4.3.3. Fluorescence Detection of Cell Surface Calreticulin

CRT exposure induced by TPt was evaluated by flow cytometry. 4T1 cells were treated with SZU101, oxaliplatin or TPt at a dose of 100 μM for 24 h. Then, the cells were collected, incubated with Alexa Fluor 647-CRT antibody (Abcam) for 30 min, and stained with Sytox Green (Abcam). The samples were analyzed by flow cytometer (Attune NxT, ThermoFisher Scientific Inc.). The permeabilized cells (Sytox Green positive cells) were excluded from the analysis, and the mean fluorescence intensity (MFI) for CRT was analyzed.

4.3.4. Extracellular ATP Assay

The extracellular release of ATP post TPt treatment was determined with an ATP chemiluminescence assay kit (Beyotime, Shanghai, China). 4T1 cells were plated at 15 000 cells/well overnight and then treated with SZU101, oxaliplatin or TPt at a dose of 100 μM for 24 h. After incubation, the cell media were collected and centrifuged at 2500 rpm × 10 min at 4 °C. The level of ATP in the supernatants was measured by the commercial kit according to manufacturer’s protocol. The final chemiluminescence was recorded on a Synergy H4 Hybrid Multi-Mode Microplate Reader (Bio-Tek).

4.3.5. TLR7 Stimulation Determined Using HEK-Blue Assay

HEK-Blue hTLR7 reporter cells were plated at 2.2 × 105 cells/mL density (180 μL) in 96-well plates using HEK-Blue Detection medium (InvivoGen) according to the manufacturer’s instructions. HEK-Blue hTLR7 reporter cells were incubated individually with 20 μL of each tested complex for 16 h at 37 °C in a CO2 incubator. The absorbance (620 nm) was measured using a Bio-Tek Quant microplate spectrophotometer.

4.4. Animal Assays

Female Balb/c mice (6 weeks, 16–20 g) were purchased from the Guangdong Medical Laboratory Animal Center (Guangdong, China). All animals were bred and housed under specific pathogen-free conditions at Shenzhen University. All animal work was conducted under the approval of the Animal Ethics Committee of Shenzhen University in accordance with the declaration of Helsinki and Shenzhen University animal facility guidelines.

4.4.1. BMDC Harvest and Culture

Femur bones were removed from 8-week-old Balb/c mice, and the bone marrow was extracted into PBS buffer. The extract was made into a homogeneous solution using a pipette and subsequently filtered through a 70 μm cell strainer (BD Falcon). The filtrate was centrifuged at 1200 rpm for 5 min at RT, and the supernatant was removed. ACK lysing buffer (2 mL) was added to the cell pellet and incubated for 3 min at RT. Then, PBS buffer (10 mL) was added to the cell suspension, and the cell solution was centrifuged at 1200 rpm for 5 min at RT. The cell pellet was resuspended in RPMI 1640 and centrifuged at 1200 rpm for 5 min at RT. The cell pellet was resuspended in BMDC primary media. Harvested cells were then counted and plated at 1 million cells per well in 24-well plate (1 mL total media per well, day 0 of cell culture). On day 2 and 4, a three quarters media change was performed. On day 5, the BMDCs were collected using a pipette, centrifuged at 1200 rpm for 5 min at RT, and replated in 24-well plates at 1 × 106 cells/mL density for cytokine profile flow cytometry experiments.

4.4.2. Assays of Stimulated BMDCs Cytokine Concentration

BMDCs were incubated individually with each complex for 18 h. The culture supernatants were collected and the IL-6, IL-10, IFN-γ, TNF-α, and IL-12p70 levels were determined by the multiplexed cytometric bead array (Mouse/Rat Soluble Protein Master Buffer Kit, CBA; BD Biosciences), as described in the manufacturer’s protocol. Flow cytometry data were acquired by using Attune NxT Flow Cytometer (ThermoFisher Scientific Inc.) and analyzed with the FlowJo V10 software.

4.4.3. Tumor Inoculation and Intratumoral Tumor Therapy

An inoculum of 2 × 105 4T1 cells was injected s.c. on the right flank of mice in 100 μL sterile PBS. Mice were randomized into four treatment groups on day 7 following tumor inoculation when the diameter of the tumors reached 2–4 mm. PBS, SZU101, oxaliplatin, and TPt were administered on days 7, 10, 13 at a dose of 5 μmol/kg (2 mg/kg for oxaliplatin, 2.44 mg/kg for SZU101, 4.3 mg/kg for TPt) intratumorally. Mice weight, tumor length, and width were recorded every three days, and tumor volumes were calculated using the formula: volume (mm3) = ([width]2 × length)/2.

4.4.4. Analysis of Tumor-Infiltrating Immune Cells

4T1-bearing mice were sacrificed on day 32. Tumors were resected, weighed, and digested with collagenase type I (Sigma) and DNase I (Sigma) to generate a single-cell suspension. The suspensions were isolated by Percoll (GE) gradient centrifugation to obtain tumor-infiltrating leukocytes. Cells were stained by incubation with the following fluorochrome-conjugated antibodies: CD45 (30-F11), CD3 (17A1), CD4 (RM4-5), and CD8 (53-6.7) (all from Biolegend) at RT for 15 min. Attune NxT flow cytometer was used for cell acquisition and data analysis was carried out using FlowJo software.

4.4.5. Immunofluorescence Assay of Immune Response

The excised tumors were fixed with 4% paraformaldehyde at room temperature for 48 h, then dehydrated, embedded in paraffin, and cut into thick slides. The slides were blocked with BSA and stained with first antibodies: CD8 (YTS169.4, from Cell signaling technology) and IFN-γ at 4 °C overnight. The slides were washed and stained with fluorochrome-conjugated antibodies (from Servicebio in China) at RT for 50 min. The slides were further stained with 4′,6-diamidino-2-phenylindole prior to observation at fluorescence microscope (NIKON ECLIPSE C1).

4.5. Statistical Analysis

All results are expressed as means ± standard error (sem). Statistical analysis was performed using the Prism statistical package (GraphPad Software, Inc.). Normally distributed data were analyzed using one-way analysis of variance with a p-test. A value of P < 0.05 was deemed statistically significant.

Acknowledgments

This work was supported by the Natural Science Foundation of Shenzhen University (2018022), the Shenzhen Science and Technology Innovation Commission (JCYJ20170818141916158, JCYJ20160328144942069, and JSGG20160331161046511), and Scientific Research Foundation of Guangdong Province (2016A020215207).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03381.

  • Synthetic route of TPt (Scheme 1); LC-MS analysis of TPt (Figure S1); 1H NMR, 13C NMR, 195Pt NMR, and ESI-HRMS spectra of TPt (Figures S1–S5); LC-MS analysis of the stability of TPt in PBS buffer with or without sodium ascorbate (Figure S6); cytokine levels in the culture media of BMDCs treated with SZU101, oxaliplatin, or TPt (Figure S7) (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b03381_si_001.pdf (582.6KB, pdf)

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Supplementary Materials

ao9b03381_si_001.pdf (582.6KB, pdf)

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