Spatial Targeting of Tumor-Associated Macrophages and Tumor Cells

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Spatial Targeting of Tumor-Associated Macrophages and Tumor Cells with a pH-Sensitive Cluster Nanocarrier for Cancer Chemo-Immunotherapy Song Shen, Hong-Jun Li, Kai-Ge Chen, Yucai Wang, XianZhu Yang, Zhe-Xiong Lian, Jinzhi Du, and Jun Wang Nano Lett., Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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Spatial Targeting of Tumor-Associated Macrophages and Tumor Cells with a pH-Sensitive Cluster Nanocarrier for Cancer Chemo-Immunotherapy Song Shen,†,‡,II Hong-Jun Li,†,‡,II Kai-Ge Chen,‡ Yu-Cai Wang,‡ Xian-Zhu Yang,†,§,⊥ Zhe-Xiong Lian, †Jin-Zhi Du,*,†,§,⊥ and Jun Wang*,†,‡,§, ⊥ †

Institutes for Life Sciences, School of Medicine, South China University of

Technology, Guangzhou, Guangdong 510006, China ‡

CAS Center for Excellence in Nanoscience, School of Life Sciences, University of

Science and Technology of China, Hefei, Anhui 230027, China §

National Engineering Research Center for Tissue Restoration and Reconstruction,

Guangzhou, Guangdong 510006, China ⊥

Key Laboratory of Biomedical Materials of Ministry of Education, South China

University of Technology, Guangzhou 510641, China II

S. Shen and H. Li contributed equally to this work.

*Address correspondence to: [email protected] (J. Wang), Tel: 020-39380992; [email protected] (J. Du), Tel: 020-39380992

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ABSTRACT Chemo-immunotherapy, which combines chemotherapeutics with immune-modulating agents, represents an appealing approach for improving cancer therapy. To optimize its therapeutic efficacy, differentially delivering multiple therapeutic

drugs

to

target

cells

is

immunostimulatory nanocarrier (denoted as tumor-associated

macrophages

chemo-immunotherapy.

BLZ-945

desirable.

Here

we

developed

an

BLZ-945

(TAMs)

SCNs/Pt) that could spatially target

and

tumor

cells

for

cancer

SCNs/Pt undergo supersensitive structure collapse in the

prevascular regions of tumor tissues, and enable simultaneous release of platinum (Pt)-prodrug conjugated small particles and BLZ-945, a small molecule inhibitor of colony stimulating factor 1 receptor (CSF-1R) of TAMs. The released BLZ-945 can be preferentially taken up by TAMs to cause TAMs depletion from tumor tissues, while the small particles carrying Pt-prodrug enable deep tumor penetration as well as intracellularly specific drug release to kill more cancer cells. Our studies demonstrate that

BLZ-945

SCNs/Pt outperform their monotherapy counterparts in multiple tumor

models. The underlying mechanism studies suggest that the designer pH-sensitive co-delivery nanocarrier not only induces apoptosis of tumor cells, but also modulates the tumor immune environment to eventually augment the antitumor effect of CD8+ cytotoxic T cells through TAMs depletion. KEYWORDS:

tumor

microenvironment,

cancer

chemo-immunotherapy,

tumor-associated macrophages (TAMs), pH-responsive, spatial targeting


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Chemotherapy remains a major treatment option for various cancers.1,2 Increasing evidence indicates that the effectiveness of chemotherapy is strongly regulated by the tumor microenvironment.3,4 For example, intelligent nanomaterials mediated depletion of cancer-associated fibroblasts

could significantly modulate the tumor

microenvironment and improve drug delivery in solid tumors.5,6 Tumor-associated macrophages (TAMs) are prominent components and critical modulators of the tumor microenvironment. They play a crucial role in tumor development, invasion, and metastases.7,8 More importantly, TAMs have been shown to protect tumor cells against chemotherapy, and suppress the immune response of cytotoxic T cells by releasing abundant growth factors, cytokines and proteases (e.g., MFG-E8, IL-6, EGF and IL-10).9-13 Studies have suggested that TAMs accumulation in tumors correlates with poor clinical outcomes.14,15 These discoveries highlight the essential of targeting TAMs for cancer treatment.16 Colony stimulating factor 1 (CSF-1) and its receptor, CSF-1R, is viewed as the primary signaling pathway for function maintenance of TAMs.12,17,18 BLZ-945, a hydrophobic small molecule, is a highly selective inhibitor of CSF-1R, and will not affect the proliferation of tumor cells.19,20 CSF-1R inhibition by BLZ-945 is capable of greatly reducing the abundance of macrophages in tumor site, and facilitating tumor infiltration of CD8+ T cells, which collectively contribute to tumor growth inhibition.21 Unfortunately, simply targeting TAMs by BLZ-945 only demonstrated marginally tumor growth suppression.18,22 Integrated chemo-immunotherapy that simultaneously targets TAMs and tumor cells represents an attractive and promising approach for improving cancer therapy.14 However, BLZ-945 and most chemotherapeutics are poorly water soluble molecules, which pose a major challenge in their therapeutic delivery.23,24 More importantly, TAMs and tumor cells are two subpopulation cells with different distribution patterns in tumor tissues.25,26 TAMs are more likely enriched in well-perfused areas, such as perivascular regions, where they are known to potentiate tumor progression and invasion, while tumor cells spread throughout the bulk tumor mass.27 Differentially delivering BLZ-945 and chemotherapeutics to these two types of cells turns out to be more challenging. Nanotechnology-based delivery


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systems are powerful in accommodating multiple cargos, and improving their delivery profiles,28,29 which have also been implemented for cancer immunotherapy.30-32 Many co-delivery systems have been explored for combination cancer therapy.33-36 However, these conventional co-delivery systems are designed to simultaneously deliver different payloads into one type of cells.37,38 A nanocarrier that is capable of carrying two different payloads, but differentially delivering them into corresponding cells has rarely been reported.39 In this study, we present a nanomedicine-based strategy to spatially deliver BLZ-945 and platinum (Pt)-based prodrug to TAMs and tumor cells for cancer chemo-immunotherapy. To take advantage of the distribution features of TAMs and tumor cells, we developed tumor microenvironment sensitive cluster nanoparticles (SCNs) to concurrently load BLZ-945 and Pt-based prodrug to form co-delivery nanoparticles

BLZ-945

SCNs/Pt.

In

BLZ-945

SCNs/Pt,

BLZ-945

was

physically

encapsulated in the hydrophobic domain, while Pt prodrug was covalently conjugated to the particle. Our previous results have demonstrated that SCNs are able to rapidly disassemble into small particles once they deposit in tumor site through their ultrahigh pH-sensitive hydrophobic-hydrophilic transition towards tumor acidity.40 We propose that this property could be exploited to trigger the rapid release of BLZ-945 in the perivascular regions, which is then preferentially taken up by TAMs to cause effective TAMs depletion, while the Pt-prodrug conjugated small particles would penetrate into deep tumor tissues, and release active Pt drugs intracellularly to kill more cancer cells, eventually realizing synergistic antitumor effect of chemo- immunotherapy (Figure 1A). Characterization of BLZ-945SCNs/Pt. SCNs/Pt were self-assembled from a Pt-prodrug conjugated amphiphilic copolymer according to our recent study.40 We have demonstrated that SCNs/Pt have ultrahigh sensitivity towards acidic tumor microenvironment. Such responsiveness is able to trigger rapid structure collapse to release the Pt-prodrug containing small particles. In this study,

BLZ-945

SCNs/Pt were

prepared following similar procedures with adding hydrophobic BLZ-945 during the self-assembly process. BLZ-945 was encapsulated in the hydrophobic core of the


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nanocarrier via hydrophobic-hydrophobic interactions. To test the pH-responsiveness, BLZ-945

SCNs/Pt were incubated with a series of PBS solutions with pH values in the

range of 6.2 to 7.6. We observed a sharp size transition from ∼90 nm at pH 6.8 or above to ∼10 nm at pH 6.7 or below (Figure 1B), which is consistent with previous results.40-42 Dynamic light scattering (DLS) histogram data and transmission electron microscope (TEM) images at pH 7.4 (Figure 1C) and pH 6.7 (Figure 1D) clearly demonstrated the tumor pH-triggered size transition of control groups

BLZ-945

BLZ-945

SCNs/Pt. For two

SCNs and SCNs/Pt, similar size-switching feature was observed

(Figure S1). It was found that the loading of BLZ-945 and Pt-prodrug didn’t affect the pH-sensitivity, morphology, or size of the nanoparticles. Next, we studied the spatial distribution of TAMs and tumor cells within the tumor microenvironment. Considerable evidence has emerged that TAMs preferentially locate in well-perfused regions of the tumor, such as perivascular areas, where they potentiate cancer progression and invasion.25 In our study, distribution patterns of TAMs and tumor cells in orthotopic murine 4T1 breast cancer model were observed by confocal laser scanning microscope (CLSM). As shown in Figure 1E, F4/80-positive TAMs (green) were more likely emerged in the region around CD31-labelled blood vessels (red), while less TAMs appeared in poorly vascularized areas. By contrast, tumor cells can distribute throughout the bulk tumor mass. To achieve spatial delivery, the release profiles of BLZ-945 and the Pt drug were controlled through loading methods. BLZ-945 was loaded via physical entrapment, while Pt-prodrug was covalently conjugated to the particles. As indicated (Figure 1F), almost 100% of BLZ-945 was released from

BLZ-945

SCNs/Pt when they were

incubated at pH 6.7 or lower pHs within 30 seconds. Since we have confirmed that BLZ-945

SCNs/Pt

underwent supersensitive

disassembly

to

release

Pt-prodrug

conjugated small particles at tumor pH, it is reasonable that BLZ-945 was concurrently released as the hydrophobic interactions between BLZ-945 and polymer disappeared. By contrast, less that 10% Pt drug could be detected even after 4 h incubation at pH 7.4 and 6.7 (Figure 1G). Our previous study has demonstrated that the Pt-prodrug were stable to pH variations, but could be specifically activated by


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intracellular reduction environment of cancer cells.43 Moreover, the DLS data indicated that the drug-loaded SCNs were quite stable in DMEM medium (containing 10% FBS, pH 7.4) at 37 °C. (Figure S2)

Figure 1. Tumor pH triggered release of BLZ-945 and Pt-prodrug conjugated small particles for spatial delivery to TAMs and tumor cells. (A) Schematic illustration showing the mechanism of spatial delivery of BLZ-945 and Pt-prodrug to TAMs and tumor cells. (i)

BLZ-945

SCNs/Pt

extravasate from blood vessels to deposit at the perivascular region, and are triggered to release BLZ-945 and Pt-prodrug conjugated small particles by extracellular acidic tumor pH. BLZ-945 can be taken up preferentially by TAMs distributed in nearby region and disturbs their CSF-1/CSF-1R signaling pathway by binding to the transmembrane CSF-1R and decreasing CSF-1R phosphorylation. (ii) Pt-prodrug conjugated small particles with a size less than 10 nm penetrate to poorly vascularized areas and induce the apoptosis of tumor cells. (B) pH-dependent size change of

BLZ-945

SCNs/Pt analyzed by DLS. (C, D) DLS (left panel) and TEM (right panel)

measurements of BLZ-945SCNs/Pt at pH 7.4 (C) and pH 6.7 (D). (E) Spatial distributions of TAMs


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and tumor cells within the tumor microenvironment of 4T1 breast tumor model. TAMs were stained by F4/80 antibody (green, marked by white arrow), while blood vessels were labeled by CD31 antibody (red, circled by dash line). DAPI (blue) was used to stain cell nuclei and label total cells. Scale bar, 200 µm. (F) pH-dependent BLZ-945 release from

BLZ-945

SCNs/Pt analyzed by

HPLC. (G) Cumulative release of Pt drug at pH 7.4 and 6.7 analyzed by ICP-MS.

In vitro Therapeutic Efficacy of

BLZ-945

SCNs/Pt. Next, we determined the

effectiveness of BLZ-945 in blocking the CSF-1/CSF-1R signaling pathway of TAMs at different pH values and time periods. We generated bone marrow-derived macrophages (BMDMs) from wild-type mice as a model of TAMs, which has been widely used in in vitro studies.13 BMDMs were treated with BLZ-945SCNs at pH 7.4 or 6.7 for 2 h, 6 h and 12 h, and then CSF-1R phosphorylation (pCSF-1R) was examined using Western Blot. As showed in Figure 2A,

BLZ-945

SCNs could effectively disturb

the formation of pCSF-1R at pH 6.7 after even 2 h incubation, indicating that the blocking process occurred very quickly and effectively. With incubation time increase to 6 h and 12 h, more dramatic reduction of pCSF-1R was observed. In contrast, pCSF-1R down-regulation only occurred after 6 h treatment at pH 7.4, and obvious pCSF-1R reduction was only achieved until 12 h, which was still less effective than that at pH 6.7. Considering BLZ-945 is released at pH 6.7, while encapsulated within the particles at pH 7.4, these pH-dependent blockade data further confirmed that the released BLZ-945, which works like free BLZ-945, is more potent than its encapsulated counterpart in suppressing the expression of pCSF-1R (Figure 2A, and Figure S3). The phenomenon, in turn, corroborates our initial design of releasing BLZ-945 in the extracellular environment to allow them to exert its inhibitory effect more vigorously in tumor tissues.


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Figure 2. In vitro therapeutic efficacy of

BLZ-945

SCNs/Pt. (A) pCSF-1R expression in BMDMs

following incubation with BLZ-945SCNs for 2 h, 6 h or 12 h at pH 7.4 or pH 6.7. The concentration of BLZ-945 was 500 nM. GAPDH was used as the loading control. The signal intensities were quantified using software ImageJ (Version 1.47) (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001. (B) CLSM images of in vitro penetration of fluorescence-labeled SCNs/Cy5 and BLZ-945SCNs/Cy5 in 4T1 multicellular spheroids (MCSs). The MCSs were incubated with Cy5-labeled (red) SCNs/Cy5 or

BLZ-945

SCNs/Cy5 for 4 h at designated pHs. Cell nucleus (blue) was stained with

DAPI. Scale bar, 100 µm. (C) Apoptosis of digested MCS cells after 24 h SCNs/Pt or BLZ-945

SCNs/Pt treatment, and measured by flow cytometry. (D) Scheme of co-culture assay of

4T1 cells and BMDMs. (E, F) Percentage of apoptotic BMDMs (E) and tumor cells (F) with different formulations at pH 6.7 or 7.4. BMDMs and tumor cells were divided by APC-F4/80 antibody during FACS detection, and apoptotic cells were shown as Annexin-V+. The concentration of BLZ-945 and platinum was 500 nM and 5 µM, respectively. * P < 0.05; ** P < 0.01.

We have confirmed in our previous study that SCNs/Pt could release small particles


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at tumor acidity and the released small particle showed improved tumor penetration in pancreatic tumor model.40 However, the penetration capability of BLZ-945 loaded SCNs in breast 4T1 tumor model is still not clear. To test this, we established 4T1 derived multicellular spheroids (MCSs) as an in vitro model and employed CLSM and flow cytometry to assess the pH-dependent penetration capability and cell killing efficiency. BLZ-945

For

CLSM

observations,

Cy5-labeled

BLZ-945

SCNs (denoted as

SCNs/Cy5) were incubated with MCSs for 4 h at pH 7.4 or 6.7. At pH 7.4, we

observed that the red fluorescence of Cy5 was mostly located on the periphery of the MCSs, indicating that poor penetration capability of larger nanoparticles (~ 90 nm). In contrast, deep penetration and uniform distribution of

BLZ-945

SCNs/Cy5 was observed

at pH 6.7, owning to the rapid disintegration of SCNs and release of small particles. SCNs/Cy5 without BLZ-945 loading exhibited comparable penetration behavior (Figure 2B). We further examined the cell apoptosis of digested MCSs after treated with SCNs/Pt or

BLZ-945

SCNs/Pt at designed pH conditions. As shown in Figure 2C,

SCNs/Pt and BLZ-945SCNs/Pt resulted in similar total cell apoptosis (∼12%) at pH 7.4, while at pH 6.7 nanoparticles treatment led to significantly higher total cell apoptosis (∼27%), which can be ascribed to the enhanced penetration and cellular uptake at pH 6.7. To investigate the effect of

BLZ-945

SCNs/Pt on mutual interaction of TAMs and

tumor cells in vitro, we established a co-culture assay of 4T1 cells and syngeneic BMDMs (Figure 2D). The co-cultured cells were treated with different formulations at pH 6.7 or 7.4 for 48 h, and the cell death was measured by flow cytometry. Macrophages and tumor cells were gated using APC-F4/80 antibody, with F4/80+ as BMDMs and F4/80- as tumor cells. An Annexin-V-FITC protocol was employed to identify the percentage of Annexin-V+ cells in each subgroup. For F4/80+ BMDMs (Figure 2E), both

BLZ-945

SCNs and

BLZ-945

SCNs/Pt induced significantly higher

BMDMs death at pH 6.7 than at pH 7.4, which were consistent with pCSF-1R down-regulation results in Figure 2A. For F4/80- 4T1 tumor cells, we did not observe difference of cell apoptosis between PBS and BLZ-945 loaded nanoparticles at both


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pH 7.4 and 6.7, presumably due to that CSF-1R is predominantly expressed in TAMs and BMDMs, while tumor cells (Figure S4A) and other tumor-infiltrating leukocytes (Figure S4B) only express CSF-1R in low levels. More importantly, we observed that BLZ-945

SCNs/Pt treatment at pH 6.7 significantly increased 4T1 cell death compared

with SCNs/Pt treatment (57.6% versus 46.2% at pH 6.7, Figure 2F), indicating that the co-delivery of BLZ-945 could induce the death of BMDMs and improve the efficacy of Pt chemotherapy in vitro. In addition, the MTT assay indicated that drug-free SCNs did not show cytotoxicity to BMDMs or 4T1 cells until the concentration of SCNs up to 1.0 mg/mL (Figure S5). In vivo Antitumor Activity of in

BLZ-945

by the in vitro promising results of

SCNs/Pt 4T1 Tumor Models. Encouraged

BLZ-945

SCNs/Pt, we decided to evaluate their in

vivo antitumor activities in 4T1 murine breast cancer model. Before the therapeutic experiment, we first investigated the in vivo dose effect of

BLZ-945

SCNs on CSF-1R

phosphorylation (pCSF-1R) and TAMs abundance in tumor tissues in 4T1 tumor-bearing mice. BLZ-945SCNs with BLZ-945 doses varying from 0.5 to 5.0 mg per kg body weight were intravenously administrated every other day for 5 total injections. Twenty-four hours after the last injection, pCSF-1R and TAMs in tumor tissues were analyzed by Western Blot and flow cytometry, respectively. As shown in Figure 3A, compared with PBS control, all the

BLZ-945

SCNs treatments showed remarkable

suppression of pCSF-1R expression. Furthermore, the relative abundance of TAMs to tumor infiltrating immune cells (marked as DAPI-CD45+) in tumor tissue has already shown significant decline when the injection dose at 1.0 mg/kg (Figure 3B). Further increasing the injection dose, more significant decrease of TAMs abundance was observed. It could be found that 2.5 mg/kg produced comparable results to 5.0 mg/kg. Therefore, 2.5 mg/kg was chosen for subsequent tumor suppression study.


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Figure 3. In vivo antitumor activity of tissues. (A, B) Dose effect of

BLZ-945

BLZ-945

SCNs/Pt, and analysis of immune cells in tumor

SCNs on CSF-1R phosphorylation (A) and TAMs abundance

in tumor tissues (B). (C) Inhibition of tumor growth by various formulations in 4T1 tumor-bearing BALB/c mice (n = 6). The injection dose of BLZ-945 and platinum were 2.5 mg/kg and 1.0 mg/kg, respectively.

BLZ-945

SCNs/Pt versus

BLZ-945

SCN, *** P < 0.001;

BLZ-945

SCNs/Pt versus SCNs/Pt,

*** P < 0.001. (D-F) Relative abundance of various immune cells in 4T1 tumor tissues at the end of treatment by flow cytometry. These cells included CD45+CD11b+Gr1-F4/80+ TAMs (D), CD45+CD11b- CD8+ T cells and CD45+CD11b- CD4+ T cells (E), and Treg cells (F). (G) The ratio of CD8+ cells/Treg cells at the end of treatment. The error bars represent means ± S.D. (** P < 0.01; *** P < 0.001; n = 3).

Tumor-bearing BALB/c mice were randomly divided into four groups (6 mice per group), and treated with PBS,

BLZ-945

SCNs, SCNs/Pt, and

BLZ-945

intravenous injection every two days. As depicted in Figure 3C,

SCNs/Pt through BLZ-945

SCNs and

SCNs/Pt monotherapies only moderately inhibited tumor growth, with approximately


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33.1% and 62.0% inhibition rate versus PBS group at the end of treatment. By contrast, BLZ-945SCNs/Pt showed 88.7% tumor suppression under the same conditions, in which a clear synergistic antitumor effect (C.I. < 1) was observed. At the end of therapy, mice receiving

BLZ-945

SCNs/Pt treatment had 83.3% and 70.6% smaller

tumors when compared with that receiving

BLZ-945

SCNs and SCNs/Pt monotherapy

treatment, respectively. We also photographed the tumor mass and recorded the weights of these tumor tissues at the end of treatment (Figure S6), which visually confirmed that BLZ-945SCNs/Pt was the most effective in suppressing tumor growth. In addition, the treatments did not significantly affect the body weights of the mice. Histological analyses also indicated that

BLZ-945

SCNs/Pt was able to markedly reduce

cell proliferation while increasing cell apoptosis (Figure S8). We have done the experiments of comparing the antitumor efficacy of BLZ-945

SCNs/Pt with cisplatin. Mice bearing 4T1 xenograft were injected with

BLZ-945

SCNs/Pt with the platinum dose of 1.0 mg/kg body weight and varying doses of

cisplatin (with platinum dose at 1.0, 2.0, or 3.0 mg/kg body weight). Tumor volume and body weight were measured every three days. As shown in Figure S9A, the antitumor efficacy increases with the increase of platinum dose. More importantly, BLZ-945

SCNs/Pt combination therapy exhibits comparable tumor suppression effect to

cisplatin treatment with platinum dose of 3.0 mg/kg. However, the body weights of the mice receiving cisplatin at this high dosage decrease remarkably due to serious side effect, whereas no significant body weight changes were observed for the mice receiving BLZ-945SCNs/Pt treatment (Figure S9B). To understand the role of immune response in

BLZ-945

SCNs/Pt mediated antitumor

effect, we studied the immune cell populations in the tumor tissues after various treatments one day after the last treatment. Tumor tissues were dissociated into single-cell suspension and a variety of immune cell populations were gated and analyzed by multicolor flow cytometry following the procedures in Figure S10. TAMs were identified as DAPI-CD45+CD11b+Gr1-F4/80+ cells. As shown in Figure 3D, it was clearly found that SCNs/Pt treatment significantly increased the ratio of TAMs in tumor-infiltrating leukocytes compared with PBS group (47.6% versus 43.1%),


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indicating that Pt drug treatment increased the recruitment of TAMs in tumor site. This is in well agreement with previous reports that conventional chemotherapy could increase TAMs infiltration in tumor tissues, which in turn blunted the effectiveness of chemotherapy.12,14,44 On the contrary,

BLZ-945

SCNs treatment significantly reduced

TAMs infiltration (23.8% versus 47.6%). More interestingly, the combination treatment with

BLZ-945

comparison with

SCNs/Pt were capable of further lowering TAMs abundance in

BLZ-945

SCNs treatment (14.2% versus 23.8%). This is probably due

to that BLZ-945SCNs/Pt is able to release BLZ-945 efficiently in the perivascular region to disturb the CSF-1/CSF-1R signaling pathway, which could either block the recruitment of TAMs precursor or induce TAMs depletion from tumor tissues. Accumulating studies have demonstrated that TAMs suppress the immune response of T cells by releasing immunosuppressive factors.45 Depletion of TAMs is believed to switch the immunosuppressive environment to an immunostimulatory one. To test this, we analyzed the relative abundance of CD8+ T cells, CD4+ T cells and regulatory T cells (Treg cells) in tumor tissues at the end of treatment using flow cytometry. As indicated in Figure 3E, tumors receiving

BLZ-945

SCNs/Pt treatment showed a

pronounced increase of CD8+ T and CD4+ T cells infiltration in comparison with other treatments. The fraction of tumor-promoting Treg cells in CD4+ T cells also decreased significantly in comparison with PBS and SCNs/Pt treatments (Figure 3F). Another crucial indicator of an immunostimulatory environment is the ratio of CD8+ T cells/Treg cells. As suggested in Figure 3G, tumors receiving BLZ-945SCNs/Pt treatment showed significant increase of CD8+ T cells/Treg cells ratio, indicating a positive shift of T cells towards cytotoxic effector T cells and decrease of tumor-promoting Treg cells. Effect of

BLZ-945

SCNs/Pt Treatment on 4T1 Tumor Metastasis. Clinical data have

revealed that > 90% of cancer-related mortality is attributable to tumor metastasis (mainly lung, liver, brain, etc.).46 It is believed that TAMs-derived proteolytic enzymes (e.g., matrix metalloproteinase 9 (MMP9)) and epidermal growth factors (e.g., vascular endothelial growth factor A (VEGF-A)) are able to enhance tumor cell invasion and metastasis.47,48 Our data have demonstrated that


BLZ-945

SCNs/Pt could

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more effectively decrease the abundance of TAMs. Its influence on the expression level of TAMs-derived MMP9 and VEGF-A was analyzed from whole-tumor lysates at the end of therapy. As indicated,

BLZ-945

SCNs/Pt treatment led to significant

decrease in the mRNA expression levels of VEGF-A (Figure 4A) and MMP9 (Figure 4B) in comparison with other treatments.

Figure 4. (A, B) Relative mRNA expression of metastasis associated growth factor VEGF-A (A) and proteolytic enzyme MMP9 (B). (C) Representative lung metastatic nodule specimens (upper) and haematoxylin-eosin (H&E) staining of lung metastases from mice treated with different formulations. The boxed regions in the middle panels are shown at higher magnification in the lower panels. Scale bars, 200 µm (middle) or 100 µm (lower). (D) The average numbers of the pulmonary metastatic nodules in 4T1 tumor-bearing mice at the end of treatment. (n = 3).

The therapeutic effect of BLZ-945SCNs/Pt on tumor metastasis was further examined. Both observation of the whole lung and examination of H&E staining showed less and smaller lung metastasis after

BLZ-945

SCNs/Pt treatment (Figure 4C). The

pulmonary metastatic nodules were counted at the end of treatment. As shown in Figure 4D, approximately 20 nodules were observed in mice receiving PBS treatment, while all the nanoparticulate formulations led to decreased lung metastasis.


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Nano Letters

BLZ-945

SCNs/Pt group generated the best metastasis inhibition with less than 5 nodules

per lung, which was significantly less than BLZ-945SCNs and SCNs/Pt treatments. In vivo Antitumor Studies in CT26 and B16 Tumor Model. To verify the broad applicability of

BLZ-945

SCNs/Pt in cancer chemo-immunotherapy, we further

investigated its antitumor activities in CT26 colon cancer and B16 melanoma models. In CT26 model, different formulations showed remarkably different antitumor activities (Figure 5A). Compared with PBS control,

BLZ-945

SCNs exhibited minimal

tumor growth inhibition (∼25.4%), whereas SCNs/Pt showed a better effect with 60.1% inhibition. By contrast,

BLZ-945

SCNs/Pt treatment showed ∼95% tumor growth

inhibition, which was significantly more effective than

BLZ-945

SCNs and SCNs/Pt. By

analyzing the tumor tissues after treatment, we also observed significant decrease of TAMs abundance (Figure 5B), and increase of CD8+ T cells infiltration (Figure 5C) in tumors receiving BLZ-945SCNs/Pt treatment in comparison with other formulations. The antitumor effect was further examined in a highly aggressive and metastatic B16 melanoma model. The mice were treated with different formulations, and their survival curves were recorded. Compared with PBS group, all other treatments showed improved median survival time. In particular, the

BZL-945

SCNs/Pt treatment

improved survival time by 96.1%, with significantly longer time to end point (Figure 5D).


B

Tumor volume (mm3)

A

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** ***

100 100

2500

PBS BLZ-945SCNs

2000

SCNs/Pt

1500

BLZ-945SCNs/Pt

***

1000 500

**

0 0

3

6

9

12

15

18

21 24

80 80 60 60 40 40 20 20

00

Days after first injection

D

*

100 100

*

66 44 22 00

Fraction survival (%)

88

CD8+ T cells in DAPI-CD45+ cells (%)

C

80 80 60 60

PBS BLZ-945SCNs

40 40

SCNs/Pt BLZ-945SCNs/Pt

20 20

** *

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TAMs in DAPI-CD45+ cells (%)

Nano Letters

00 0 0

10 10

20 20

30 30

40 40

50 50

60 60

Days after first injection

Figure 5. In vivo antitumor activity of models. (A) Antitumor effect of

BLZ-945

BLZ-945

SCNs/Pt in CT26 colon cancer and B16 melanoma

SCNs/Pt and other formulations in the tumor model of

murine colon cancer cell CT26 (n = 5). (B, C) Percentage of TAMs (B) and CD8+ T cell (C) in the DAPI-CD45+ tumor-infiltrating leukocytes examined by flow cytometery in CT26 tumor tissues after treatment. * P < 0.05; ** P < 0.01; *** P < 0.001. (D) Kaplan–Meier plots of the animal survival in B16 tumor models (n = 10). Mice were treated with different formulations via intravenous administration every other day for 20 days after tumor inoculation.

BLZ-945

SCNs/Pt

versus SCNs/Pt, * P < 0.05; BLZ-945SCNs/Pt versus BLZ-945SCNs, ** P < 0.01.

In summary, we demonstrated in this study a cancer chemo-immunotherapy through nanomedicine-based spatial delivery of BLZ-945 and a Pt-based chemotherapeutic drug to TAMs and tumor cells. The therapeutic efficacy of SCNs-mediated combination treatment strategy was investigated in multiple tumor models (e.g., murine breast cancer, colon cancer and melanoma), which demonstrated that the concurrent delivery system could more effectively suppress tumor growth, inhibit metastasis and prolong the survival of tumor-bearing mice, compared with its monotherapy counterparts. More importantly, our study suggests that simultaneous


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targeting of TAMs and tumor cells using such a designer pH-sensitive nanocarrier can be a powerful means to synergistically treat cancers, mediated by not only inhibiting the proliferation of tumor cells, but also significantly modulating the tumor immune microenvironment to eventually augment the antitumor effect of CD8+ cytotoxic T cells through the depletion of TAMs. Compared with conventional co-delivery systems that simultaneously and indistinguishably deliver their payloads to one type of cells, the idea of differential delivery of multiple therapeutics to tumor cells and target immune cells by making use of their distinct distribution feature within the tumor microenvironment may represent a novel strategy to optimize the treatment efficacy of chemo- and immuno- combination therapy. ASSOCIATED CONTENT

Supporting Information Detailed materials, methods and additional figures are provided in supporting information, including the synthesis of polymers, preparation and characterization of BLZ-945

SCNs/Pt, stimuli-responsive BLZ-945 and Pt drug release from BLZ-945SCNs/Pt,

in vitro penetration in multicellular spheroids, in vitro co-culture of tumor cells and BMDMs, antitumor activities in varying tumor models, analysis of tumor-infiltrating leukocytes, histological studies and statistics. AUTHOR INFORMATION

Corresponding Author Address correspondence to: [email protected] (J. Wang), and [email protected] (J. Du). Author Contributions II

S. Shen and H. Li contributed equally to this work.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This research was supported by the National Basic Research Program of China (2015CB932100, 2013CB933900), the National Natural Science Foundation of China (51390482, 51633008), and the Fundamental Research Funds for the Central Universities. REFERENCES (1) DeVita, V. T., Jr.; Chu, E. Cancer Res. 2008, 68, 8643-8653. (2) Montero, J.; Sarosiek, K. A.; DeAngelo, J. D.; Maertens, O.; Ryan, J.; Ercan, D.; Piao, H.; Horowitz, N. S.; Berkowitz, R. S.; Matulonis, U.; Janne, P. A.; Amrein, P. C.; Cichowski, K.; Drapkin, R.; Letai, A. Cell 2015, 160, 977-989. (3) Klemm, F.; Joyce, J. A. Trends Cell Biol. 2015, 25, 198-213. (4) McMillin, D. W.; Negri, J. M.; Mitsiades, C. S. Nat. Rev. Drug Discovery 2013, 12, 217 (5) Ji, T. J.; Zhao Y.; Ding Y. P.; Nie G. J. Adv. Mater. 2013, 25, 3508-3525. (6) Ji, T. J.; Ding Y. P.; Zhao Y.; Wang J.; Qin H.; Liu X. M.; Lang J. Y.; Zhao R. F.; Zhang Y. L.; Shi J.; Tao N.; Qin Z. H.; Nie G. J. Adv. Mater. 2015, 27, 1865-1873. (7) Franklin, R. A.; Liao, W.; Sarkar, A.; Kim, M. V.; Bivona, M. R.; Liu, K.; Pamer, E. G.; Li, M. O. Science 2014, 344, 921-925. (8) Liu, Y.; Cao, X. Cell. Mol. Immunol. 2015, 12, 1-4. (9) Mantovani, A.; Allavena, P. J. Exp. Med. 2015, 212, 435-445. (10) Jinushi, M.; Chiba, S.; Yoshiyama, H.; Masutomi, K.; Kinoshita, I.; Dosaka-Akita, H.; Yagita, H.; Takaoka, A.; Tahara, H. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 12425-12430. (11) Mitchem, J. B.; Brennan, D. J.; Knolhoff, B. L.; Belt, B. A.; Zhu, Y.; Sanford, D. E.; Belaygorod, L.; Carpenter, D.; Collins, L.; Piwnica-Worms, D.; Hewitt, S.; Udupi, G. M.; Gallagher, W. M.; Wegner, C.; West, B. L.; Wang-Gillam, A.; Goedegebuure, P.; Linehan, D. C.; DeNardo, D. G. Cancer Res. 2013, 73, 1128-1241. (12) Nakasone, E. S.; Askautrud, H. A.; Kees, T.; Park, J. H.; Plaks, V.; Ewald, A. J.; Fein, M.; Rasch, M. G.; Tan, Y. X.; Qiu, J.; Park, J.; Sinha, P.; Bissell, M. J.; Frengen,


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Table of Contents Graphic BLZ-945 SCNs/Pt

CSF-1 CSF-1R

i.v. injection

i

P

P

P

P

depleting TAMs

tumor pH ii BLZ-945

Pt-prodrug conjugated particle

killing tumor cells


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Spatial Targeting of Tumor-Associated Macrophages and Tumor Cells

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