Letter Cite This: Nano Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/NanoLett
Nanoenabled Reversal of IDO1-Mediated Immunosuppression Synergizes with Immunogenic Chemotherapy for Improved Cancer Therapy Hua Huang,† Cheng-Tao Jiang,‡ Song Shen,*,‡,§ An Liu,∇ Yun-Jiu Gan,∇ Qi-Song Tong,† Sen-Biao Chen,‡ Zhu-Xin Gao,† Jin-Zhi Du,‡,§ Jie Cao,*,† and Jun Wang*,†,§,∥,⊥,#
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†
Guangzhou First People’s Hospital, School of Biomedical Sciences and Engineering, Guangzhou International Campus, ‡Institutes for Life Sciences and School of Medicine, §National Engineering Research Center for Tissue Restoration and Reconstruction and ∥ Key Laboratory of Biomedical Engineering of Guangdong Province, and Innovation Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, P.R. China ⊥ Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou 510005, P.R. China # Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology, Guangzhou 510006, P.R. China ∇ Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei 230027, P.R. China S Supporting Information *
ABSTRACT: Certain chemotherapeutics (e.g., oxaliplatin, OXA) can evoke effective antitumor immunity responses by inducing immunogenic cell death (ICD). Unfortunately, tumors always develop multiple immunosuppressive mechanisms, such as the upregulation of immunosuppressive factors, to counteract the effects of immunogenic chemotherapy. Indoleamine 2,3-dioxygenase-1 (IDO1), a tryptophan catabolic enzyme overexpressed in tumor-draining lymph nodes (TDLNs) and tumor tissues, plays a pivotal role in the generation of the immunosuppressive microenvironment. Reversing IDO1-mediated immunosuppression may strengthen the ICD-induced immune response. Herein, we developed a nanoenabled approach for IDO1 pathway interference, which is accomplished by delivering IDO1 siRNA to both TDLNs and tumor tissues with the help of cationic lipid-assisted nanoparticles (CLANs). We demonstrated that the contemporaneous administration of OXA and CLANsiIDO1 could achieve synergetic antitumor effects via promoting dendritic cell maturation, increasing tumor-infiltrating T lymphocytes and decreasing the number of regulatory T cells in a subcutaneous colorectal tumor model. We further proved that this therapeutic strategy is applicable for the treatment of orthotopic pancreatic tumors and offers a strong immunological memory effect, which can provide protection against tumor rechallenge. KEYWORDS: Oxaliplatin, immunogenic cell death, indoleamine 2,3-dioxygenase 1, CLANs, cancer chemoimmunotherapy
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engulfment of dying tumor cells by DCs, which increases cancer immunogenicity and boosts antitumor immune response.9 Although immunogenic chemotherapeutics could promote the maturation of DCs and tumor-infiltration of cytotoxic T lymphocytes (CTLs), the clinical therapeutic effect of these chemical agents is limited and unsatisfactory.14−16 Accumulating works indicate that tumors developed multiple immunosuppressive mechanisms, such as the overexpression of immune checkpoint proteins or immunosuppressive factors to counteract
hemotherapy remains one of the mainstays in the treatment of malignant cancer, including colorectal and pancreatic cancers.1−3 Accumulating evidence suggests that additional engagement of the immune response contributes significantly to the overall antitumor efficacy of chemotherapy:4−6 for instance, certain chemotherapeutic agents (e.g., anthracyclines and oxaliplatin) induce immunogenic cell death (ICD) in tumor cells.7−9 Unlike cell apoptosis, ICD is accompanied by the expression of calreticulin (CRT) on the surface of dying tumor cells, which provides an “eat me” signal for antigen-presenting cells (e.g., dendritic cells, DCs).10,11 Furthermore, ATP and high-mobility group box 1 (HMGB-1) released by dying tumor cells provide adjuvant stimuli for the activation of DCs.12,13 In this way, ICD facilitates the © XXXX American Chemical Society
Received: May 2, 2019 Revised: July 5, 2019 Published: July 9, 2019 A
DOI: 10.1021/acs.nanolett.9b01807 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters Scheme 1. CLANsiIDO1-Mediated IDO1 Activity Inhibitiona
a
CLANsiIDO1-mediated IDO1 activity inhibition in tumor-draining lymph nodes (TDLNs) and tumor tissues synergizes with immunogenic chemotherapy by promoting dendritic cell maturation, increasing tumor-infiltrating cytotoxic T lymphocytes, and decreasing numbers of regulatory T cells. Additionally, OXA plus CLANsiIDO1 treatment could generate memory T cells and prevent tumor relapse.
the antitumor efficacy of immunogenic chemotherapeutics.17,18 Inhibition of negative immune regulation represents one of the most effective strategies to reactivate the antitumor immune response.19 Indeed, exciting clinical outcomes have confirmed the efficacy of monoclonal antibodies against checkpoint inhibitors (e.g., CTLA-4, PD-1, and PD-L1).20,21 Indoleamine 2,3-dioxygenase-1 (IDO1), a tryptophan catabolic enzyme overexpressed in tumor-draining lymph nodes (TDLNs) and tumor tissues, has gained emerging attention in the last several years, as IDO1 activity is associated with the generation of immunosuppressive microenvironments. IDO1 exerts its immunoregulatory functions by affecting the activity of DCs, inducing CTL apoptosis and increasing the number of regulatory T (Treg) cells.22−25 Considering that certain chemotherapeutics would lead to IDO1 upregulation, it is rational to predict that suppressing IDO1 activity in combination with chemotherapy could achieve synergetic antitumor efficacy.26,27 Preclinical studies have now provided evidence that small molecule inhibitors of IDO1 (e.g., NLG919, 1-methyl-Dtryptophan, indoximod) can prevent T cell anergy and exert antitumor effects.28,29 Studies also confirmed the magnified therapeutic effects of ICD inducers resulting from cooperation with IDO1 inhibitors.26,30 Furthermore, accumulating nanocarriers have been exploited to overcome the limitation of immunomodulator and to enhance the efficacy of cancer immunotherapy.31−33 For instance, Lu and colleagues demonstrated that mesoporous silica nanoparticles, mediating contemporaneous delivery of oxaliplatin and indoximod, could boost antitumor immunity in a pancreatic ductal adenocarcinoma (PDAC) model.34 Additionally, Chen et al. proved that the codelivery of paclitaxel and NLG919 to tumor tissues could achieve improved antitumor efficacy in multiple tumor models.27 To our knowledge, simultaneously downregulating/ inhibiting IDO1 activity in both tumor tissues and TDLNs has not been reported yet; however, IDO1 protein is critically involved in immunosuppression in both tissues. At TDLNs, where antigen-presenting cells (APC) primarily localize, excessive tryptophan catabolism inhibits APC activity and leads to reduced numbers of antigen-specific T cells and increased numbers of Treg cells.35 Additionally, high expression
of IDO1 TDLNs was associated with poor clinical outcomes.36,37 At tumor sites, the depletion of tryptophan and the increase in immunosuppressive metabolites affect the survival and function of cytotoxic T cells.23,38 We posited that the immunogenic effect of certain chemotherapeutics could be significantly enhanced if immunosuppressive effects of IDO1 were reversed in both TDLNs and tumor tissues, and we speculated that proper nanocarriers could increase the accumulation of anti-IDO1 agents (e.g., inhibitor, small interfering RNA) in both tissues. In the present study, we first proved that IDO1 was overexpressed in human colorectal cancer samples and that treatment with ICD-inducer OXA could upregulate IDO1 levels in both TDLNs in a murine colorectal tumor model. We then encapsulated small interfering RNA targeting IDO1 (siIDO1) using cationic lipid-assisted nanoparticles (CLANs)39,40 and confirmed that CLANsiIDO1 could be effectively enriched in both tissues and downregulate the target gene. As expected, CLANs-mediated IDO1 inhibition significantly enhanced the antitumor efficacy of OXA in mouse models of colorectal and pancreatic cancer via promoting DCs maturation in TDLNs, increasing abundance of CTLs and decreasing numbers of Treg cells in tumor tissues. In addition, the combination therapy could produce long-term memory efficacy and prevent tumor recurrence (Scheme 1). These results verified our hypothesis that nanocarrier-mediated interference of the immunosuppressive IDO1 pathway in TDLNs and tumor tissues offers the benefit of synergistically enhancing antitumor immune response induced by immunogenic chemotherapy, and this combination therapy represents a promising strategy against aggressive and difficult-to-treat cancers. Results and Discussion. Oxaliplatin (OXA) Treatment Induces Overexpression of IDO1 in Tumor Tissues and TDLN. IDO1 is a rate-limiting catalyzing enzyme in the catabolism of tryptophan along the kynurenine pathway, and an increasing numbers of works have indicated that the constitutive overexpression of IDO1 in human tumors (e.g., colorectal cancer and pancreatic cancer) is associated with a reduction in tumor-infiltrating lymphocytes and response to antitumor therapeutics.41,42 In the present study, the expression of IDO1 protein in tumor tissues and adjacent normal tissues of B
DOI: 10.1021/acs.nanolett.9b01807 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Oxaliplatin (OXA) treatment induces overexpression of IDO1 in vitro and in vivo. (a) Immunohistochemical staining of IDO1 in colorectal cancer tissues and adjacent normal tissues. The representative images are shown. Scale bar, 50 μm. (b) Percentages of CRT positive cells treated with different concentrations of OXA. CRT expression on the surface of treated CT26 cells was detected by flow cytometry after staining with anti-CRT primary antibody and Alexa Fluor 488 conjugated secondary antibody. The percentages of CRT-positive cells in OXA-treated groups were normalized to the PBS group. The error bars represent the means ± SD (*P < 0.05; **P < 0.01 versus PBS group). (c) Vaccination approach in vivo. OXA-treated dying CT26 cells were incubated subcutaneously, followed by injection of untreated cells in the opposite flank 7 days later. The growth of second tumors was recorded. (d) IDO1 expression after treatment with PBS or OXA. CT26 cells were treated with OXA (50 μM) for 24 h. The error bars represent the means ± SD (***P < 0.001 versus PBS group). Detection of IDO1 expression in tumor-draining lymph nodes (e) and tumor tissues (f) of mice bearing CT26 tumors via real-time PCR (left, upper), Western blot (left, lower), and immunohistochemistry (right). Tumor-bearing mice were treated with OXA (1.25 mg/kg) 3 times every 3 days. Scale bar, 50 μm. The error bars represent the means ± SD (*P < 0.05 versus PBS group).
colorectal cancer patients (Table S1) was first examined using immunohistochemistry (IHC) analysis. As shown in Figure 1a and Figure S1 in contrast to adjacent normal tissues, high IDO1 expression was observed in tumor sites, indicating immunosuppression in colorectal cancer. Before detecting whether oxaliplatin, one of the four components in the FOLFIRINOX chemotherapy regimen used in colorectal cancer,43 would result in the upregulation of IDO1 expression, we first confirmed that OXA could induce ICD in colorectal cancer cells via calreticulin (CRT) examination and a vaccination approach.44 As a distinct biomarker of ICD, the surface-exposed CRTs on the OXAtreated CT26 colon cancer cells were evaluated using flow cytometry and confocal microscopy. As shown in Figure 1b and Figure S2, OXA induced the surface expression of CRT in a dosage-dependent manner, compared to cisplatin, which only induces apoptosis but not ICD of tumor cells (Figure S3). Increased release of HMGB1 and ATP, two other important markers of ICD, was also observed after treated with OXA (Figure S4). Furthermore, an in vivo vaccination approach was performed for confirming ICD. Dying tumor cells generated by exposure to OXA were subcutaneously (s.c.) injected into one flank of male BALB/c mice, and the mice were subsequently challenged with live CT26 cells injected into the contralateral flank 7 days later. As shown in Figure 1c and Figure S5, vaccination with PBS or CDDP-treated cells has a negligible effect on tumor growth on the contralateral side, while OXAtreated cells exert a “vaccine” function and prevent (or delay) the tumor growth of a second injection: notably, 4 out of 5 mice in the OXA-treated group survived tumor-free through the end of
the study (30 days). Though OXA-based therapy may benefit from ICD-induced activation of the body’s immune system, the antitumor immune response may be attenuated by several immune escape mechanisms, such as the overexpression of inhibitor receptor (and/or ligand) or IDO1. The relationship between IDO1 expression and OXA treatment was explored here: as depicted in Figure 1d, OXA incubation markedly increases the IDO1 mRNA expression in CT26 cells in vitro. For the in vivo examination, BALB/c mice bearing CT26 tumors (in the flank) were administered vehicle or OXA (1.25 mg/kg), and the IDO1 expression levels in inguinal TDLNs and tumor tissues were detected via real-time PCR, Western blot, and immunohistochemistry 24 h after the third treatment; as shown in Figure 1e,f, the IDO1 expression levels were greatly upregulated after OXA treatment in both tissues. Overall, it is rational to hypothesize that blunting OXA-induced IDO1 upregulation in TDLNs and tumor tissues may ameliorate the clinical applications of OXA. siRNA-Encapsulated CLANs Effectively Accumulated in TDLNs and Tumor Tissues Resulting in IDO1 Suppression in Both Tissues. In the above work, we have revealed that OXA, an ICD inducer, led to the upregulation of IDO1 in the TDLNs and tumor tissues; the simultaneous suppression of IDO1 levels in both tissues is meaningful, but also challenging. Our group developed cationic lipid-assisted nanoparticles (CLANs) fabricated with biocompatible poly(ethylene glycol)-blockpoly(lactic-co-glycolic acid) (PEG-b-PLGA) block copolymer and cationic lipid for the delivery of nucleic acids (e.g., small interfering RNA and CRISPR/Cas9).40 As CLANs have been C
DOI: 10.1021/acs.nanolett.9b01807 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 2. siRNA-encapsulated CLANs effectively accumulated in TDLNs and tumor tissues and suppressed IDO1 expression in both tissues. (a) Schematic illustration showed the preparation of CLANsiIDO1 using a double emulsion method. (b) The size distribution of CLANsiIDO1 analyzed by DLS. (c,d) The capability of CLANsiIDO1 to knockdown IDO1 was detected via real-time PCR and Western blot, respectively. CT26 cells were transfected with Lipo/siIDO1 (50 nM), Lipo/siN.C. (50 nM), CLANsiN.C. (100 nM), and CLANsiIDO1 (100 nM). The mRNA and protein levels of IDO1 were examined 24 and 48 h post-transfection, respectively. The error bars represent the means ± SD (***P < 0.001 versus PBS group). (e) Distribution and tumor accumulation of Cy5-siRNA in CT26 tumor-bearing mice receiving intravenous injection of CLANCy5‑siRNA detected by IVIS system. (f) Major organs (including heart, liver, spleen, lung, kidney, and pancreas), inguinal TDLNs and tumor tissues were excised at 48 h for fluorescence signal detection. (g) Fluorescent signal of Cy5 siRNA in TDLNs and tumor tissues after administration of CLANCy5‑siRNA was captured by confocal microscopy. (h) Knockdown efficacy of CLANsiIDO1 in vivo. BALB/c mice bearing CT26 tumors were injected intravenously three times with CLANsiIDO1 at the dosage of 1 mg/kg, and then the TDLNs (left) and tumor tissues (right) were collected for the examination of IDO1 expression at the mRNA and protein levels. The error bars represent the means ± SD (***P < 0.001; ****P < 0.0001 versus PBS group).
CLANCy5‑siRNA group compared with the mice administered PBS (Figure 2e). Mice were sacrificed and major organs were harvested 48 h postadministration for ex vivo observation. As demonstrated in Figure 2f, strong Cy5 fluorescent signals in TDLNs and tumor tissues were detected in the CLANCy5‑siRNA group, and confocal observation also confirmed the enrichment of nanoparticles in both tissues (Figure 2g). Furthermore, mice bearing CT26 tumors were treated three times with CLANsiIDO1 (1 mg per kg body weight) every 3 days, and remarkable downregulation of IDO1 in both inguinal TDLNs and tumor tissues were detected 24 h after final injection (Figure 2h). In Vivo Antitumor Activity of OXA Plus CLANsiIDO1 in CT26 Tumor Model. Encouraged by the capability of CLANsiIDO1 to suppress IDO1 expression in TDLNs and tumor tissues, we further assessed whether CLANsiIDO1 could effectively potentiate the therapeutic efficacy of OXA. Subcutaneous CT26 tumor models were established, and when the volumes reached approximately 50−100 mm3, mice were randomly divided into PBS control, OXA, CLANsiIDO1 and OXA plus CLANsiIDO1 groups (five mice per group); mice were then i.v. administered corresponding formulations four times every 3 days, the injection doses of OXA and IDO1 siRNA were 1.25 mg and 1.0 mg per kg body weight, respectively, and the tumor growth was monitored with the traditional caliper measurement method (Figure 3a). As depicted in Figure 3b,c, OXA and CLANsiIDO1 monotherapies only moderately inhibited tumor growth with
proven to be efficient vectors of nucleic acids and can effectively accumulate in tumor tissues, liver, adipose tissue and other tissues, we asked whether CLANs could be enriched in TDLNs. To test this, CLANs encapsulating siIDO1 (CLANsiIDO1) were constructed using a double emulsion method (Figure 2a), and the encapsulation efficacy could reach about 98% with the assistance of cationic lipid DOTAP. As shown in Figure 2b and Figure S6, the hydrodynamic average diameters of CLANsiIDO1 were approximately 120 nm, as measured by dynamic light scattering (DLS), and CLANsiIDO1 could remain stable after being incubated with FBS-containing complete medium for 2 days. Before investigating the in vivo behavior of CLANsiIDO1, the capability to downregulate the target gene was evaluated. As shown in Figure 2c,d, the IDO1 mRNA and protein expression levels were downregulated to approximately 50% in the CT26 cells transfected with CLANsiIDO1 (100 nM siIDO1) compared with PBS control, which is similar to the positive control Lipofectamine 2000/siIDO1, whereas the CLANsiN.C. had an ignorable influence on IDO1 expression. To visualize the biodistribution of CLANs and verify whether CLANs could simultaneously deliver siRNA to TDLNs and tumor tissue after systemic administration, mice bearing CT26 tumors in the right flank were i.v. injected with PBS or Cy5-labeled siRNA encapsulated CLANs (CLANCy5‑siRNA), and the fluorescent signals of Cy5-siRNA were acquired by IVIS system every 24 h: high fluorescent signals were visualized at tumor sites in the D
DOI: 10.1021/acs.nanolett.9b01807 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. In vivo antitumor activity of OXA plus CLANsiIDO1 in CT26 colon cancer model. (a) Schematic illustration showing the treatment details in vivo. The CT26 tumors were established by s.c. injecting tumor cells into the flanks of male BALB/c mice, OXA (1.25 mg/kg) and CLANsiIDO1 (1 mg/ kg) were administered four times every 3 days when tumor volumes reached approximately 50 mm3, and the tumor growth was recorded during the antitumor experiment. (b) Spaghetti curves show the CT26 tumor growth in groups receiving different formulations. (c) CT26 tumor growth curves after treatment with PBS, OXA, CLANsiIDO1, or OXA plus CLANsiIDO1. The error bars represent the means ± SD (*P < 0.05, OXA group versus OXA group; **P < 0.01, OXA plus CLANsiIDO1 group versus OXA group). (d) Tumor images from each group after the animals were sacrificed.
approximately 51.6 ± 12.1% and 44.9 ± 16.3% inhibition rates versus the PBS group at the end of treatment. In marked contrast, the tumor growth of the mice treated with OXA plus CLANsiIDO1 was distinctly delayed compared with other groups. At the end of therapy, mice receiving combination therapy had 70.4% and 65.9% smaller tumors when compared with those receiving OXA and CLANsiIDO1 treatments, respectively, and a clear synergetic antitumor effect (combination index
