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. Author manuscript; available in PMC: 2020 Sep 10.
Published in final edited form as: J Control Release. 2019 Jul 29;309:173–180. doi: 10.1016/j.jconrel.2019.07.039

Inhibiting PI3 Kinase-γ in Both Myeloid and Plasma Cells Remodels the Suppressive Tumor Microenvironment in Desmoplastic Tumors

Xueqiong Zhang 1,2,3, Limei Shen 1,3, Qi Liu 1, Lin Hou 1, Leaf Huang 1,4,*
PMCID: PMC6815713  NIHMSID: NIHMS1536247  PMID: 31362079

Abstract

Phosphoinositide-3-kinases (PI3Ks) are part of signal transducing enzymes that mediate key cellular functions in cancer and immunity. PI3K-γ is crucial for cellular activation and migration in response to certain chemokines. PI3K-γ is highly expressed in myeloid cells and promotes their migration and the production of inflammatory mediators. We found that PI3K-γ was also highly expressed in tumor-associated B cells. IPI-549, the only PI3K-γ inhibitor in clinical development, offers a unique approach to enhance the anti-tumor immune response. We encapsulated IPI-549 in targeted polymeric nanoparticles (NP) and tested its activity in both murine pancreatic cancer and melanoma models. IPI-549 NP significantly decreased tumor growth and prolonged host survival in both models. Importantly, IPI-549 NP treatment reduced the suppressive tumor microenvironment by decreasing both suppressive myeloid and plasma cells in the tumor. We concluded that IPI-549 NP delivery could be a promising method for treating pancreatic cancer and other immune-suppressive tumors.

Keywords: IPI-549, nanoparticle, MDSC, regulatory B cell, pancreatic cancer

Graphical abstract:

graphic file with name nihms-1536247-f0007.jpg

INTRODUCTION

Pancreatic cancer is a malignant disease with a high mortality rate. The total number of deaths from pancreatic cancer is steadily increasing, with pancreatic cancer expected to be the second leading cause of cancer death in the USA by 2030 (Rahib et al., 2014). Historically, chemotherapy or radiotherapy did not reach satisfactory survival benefit in advanced pancreatic cancer. Recent studies have revealed that immunosuppression and inflammation are related to oncogenesis, as well as tumor development, invasion, and metastasis in pancreatic adenocarcinoma (PAC). Therefore, immunosuppression associated signaling, especially when it involves immune checkpoint and inflammation, has served as a novel treatment target for PAC. There is a highly immunosuppressive microenvironment regulated by immune cells, stromal cells, and mediators in PAC. This condition may result in its resistance to immune checkpoint therapy (Ino et al., 2013).

Tumor-associated macrophages and myeloid-derived suppressor cells (MDSC) are immunosuppressive, and a high ratio of suppressive cells to CD4+ and CD8+ T cells is related to poor survival of PAC patients (Ino et al., 2013). MDSC levels are increased both in the circulation and in the microenvironment of PAC. Inhibition of MDSC in PAC is a potential method of cancer therapy (Shara et al., 2011). Phosphoinositide-3-kinases (PI3Ks) pertain to signal transducing enzymes that play an important role in cancer and immunity. PI3K-γ signaling is especially pivotal for the function of myeloid cells, where it is downstream of G-protein coupled receptors (GPCRs) (e.g., chemokine receptors) and RAS (Hirsch et al., 2000; Sasaki et al., 2000; Schmid et al., 2011). For instance, murine syngeneic tumors grow slower when transplanted into immune-competent mice where PI3K-γ is genetically inactivated (Schmid et al., 2011; Joshi et al., 2014). This growth reduction occurs because of the abrogation of tumor-associated myeloid cells that are known to promote an immune-suppressive tumor microenvironment (TME) that permits tumor growth (Schmid et al., 2011; Joshi et al., 2014; Gunderson et al., 2016; Rivera et al., 2015). In addition, MDSCs are associated with tumor regrowth after radiation or chemotherapy, and are known to lead to metastatic spread (De Palma and Lewis, 2013). These preclinical studies highlight an important role for PI3K-γ in myeloid cell biology and suggest that PI3K-γ inhibition in MDSC may be effective at inhibiting tumor growth in a variety of settings.

IPI-549 can reduce the T-cell-suppressive activity of both murine and human myeloid-derived suppressor cells in vitro (De Henau et al., 2016). These findings indicate that IPI-549 increases antitumor immunity by remodeling the tumor-immune microenvironment via blockade of tumor-associated myeloid cells. In addition, the up-regulations of costimulatory and coinhibitory genes with IPI-549 treatment provides a mechanistic rationale for the observed combination activity with immune checkpoint inhibition. IPI-549 is currently in phase I development, both as a single agent and in combination with an anti-PD-1 antibody, in solid tumors (ClinicalTrials.gov ).

B cells have been typical of positive regulators of humoral immune response and are characterized by their ability to terminally differentiate into antibody-secreting plasma cells (DiLillo et al., 2008; LeBien and Tedder, 2008). B-cell inhibition of an immune response was first reported in 1974; spleen B-cells were found to impair delayed type hypersensitivity response in guinea pigs (Katz et al., 1974; Neta and Salvin, 1974). B cells are now regarded as an important ingredient of the immune suppression system. A potential therapeutic strategy for PAC would include targeted B-cell suppression (Gunderson et al., 2016; Lee et al., 2016; Pylayeva-Gupta et al., 2016; Roghanian et al., 2016).

Nanoparticles (NPs) can prolong the half-life of payloads in vivo and passively accumulate in the tumor regions via the enhanced permeability and retention (EPR) effect (Guo and Huang, 2014; Mura et al., 2013; Wang et al., 2012). Numerous anti-tumor or immune-stimulating agents have been delivered by NPs (De Henau et al., 2016; Zhang et al., 2014). For example, a Toll-like-receptor-7 agonist was encapsulated by poly(lactic-co-glycolic) acid (PLGA) (Chen Q et al., 2016). In the current study, targeted IPI-549 NP was designed to improve the bioavailability and therapeutic activity. We hypothesized that tumor-targeted IPI-549 could reshape the tumor immune microenvironment and reverse its immune suppression more effectively than the oral dosage form.

MATERIALS AND METHODS

Materials

IPI-549 was purchased from Chemietek (Indianapolis, IN, USA). Acid-terminated PLGA (lactide/glycolide (50:50)) was purchased from DURECT (Pelham, AL, USA). mPEG3500-NH2⋅HCl and tBOC-PEG3500-NH2⋅HCl were purchased from JenKem Technology USA, Inc. (Allen, TX). PLGA-PEG and PLGA-PEG-AEAA were synthesized according to our previous work, and the structures were confirmed by 1H-NMR (Guo et al., 2014). If not specifically mentioned, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

The mouse KPC cell line (KPC 98027) used in this study was stably transfected with the vector carrying the mCherry red fluorescent protein (RFP), firefly luciferase (Luc), and the puromycin resistance gene; cells were then selected in the presence of puromycin. The cells stably expressed RFP/Luc. Dulbecco’s Modified Eagle’s Medium was used in cell cultivation: Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% Penicillin/Streptomycin at 37 °C and 5% CO2 in a humidified atmosphere.

Murine BRAF-mutant melanoma cell line BPD6 (BRAFV600E, PTEN−/−, syngeneic with C57BL/6 mice) was provided by Brent Hanks (Duke Cancer Institute) as previously reported (Liu et al, 2018) and cultivated in RPMI-1640 Medium (Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin (Invitrogen, Carlsbad, CA) at 37 °C and 5% CO2.

Experimental animals

Six-week-old female C57BL/6 mice were purchased from Charles River Laboratories and maintained under pathogen-free conditions. All animal handling procedures were approved by the University of North Carolina at Chapel Hill’s Institutional Animal Care and Use Committee.

Animal tumor model

Sub-confluent KPC98207 (with or without RFP/Luc) cells were harvested and washed with phosphate buffered saline (PBS). We then injected 1×106 cells into the tail of pancreas to establish the orthotopic allografting KPC model. In brief, eight-week-old C57BL/6 mice were anesthetized by 2.5% isoflurane and placed in supine position. A midline incision was made to exteriorize the spleen and pancreas. Using an insulin-gage syringe, 1×106 cells in 50 μL (PBS + Matrigel) were injected into the tail of pancreas. The abdominal wall and skin were closed with 6-0 polyglycolic acid sutures. The injection site was sealed with a tissue adhesive (3M, St. Paul, MN) and sterilized with 70% alcohol. Tumor growth was monitored via intraperitoneal (i.p.) injection of 100 μL of D-luciferin (10 mg/mL). The bioluminescent analysis was completed using an IVIS Kinetics optical imaging system (BD Pharmingen, CA).

Antibodies.

Primary antibodies, fluorescence-conjugated primary and secondary antibodies used for immunostaining (IF), and flow cytometry (flow cytr) are listed in Table S2.

Tumor growth inhibition

Treatments on the KPC98027 RFP/Luc allografts bearing mice were initiated on day 18. Thirty mg/kg oral IPI-549 suspension was used as a control. The IPI-549 was dissolved at 5% 1-methyl-2-pyrrolidone in polyethylene glycol 400. Mice were then randomized into three groups (n = 8-10) as follows: untreated group (PBS), oral IPI-549 suspension, and IPI-549 nanoparticle. Intravenous injections with IPI-549 nanoparticle were administered every three days at 15 mg/kg. Tumor growth was monitored using IVIS Kinetics optical imaging system (Perkin Elmer, CA) every three days. The increases of tumor volumes were calculated as the radiance of the intensities and standardized with the initial tumor volume (Vt/V0).

Flow cytometry assay

Tumor-infiltrating immune cells were analyzed using flow cytometry. In brief, tumor tissues were harvested and digested with collagenase A and DNase at 37 °C for 60 min. After red blood cell lysis, cells were resuspended in 5 mL of PBS. Two million cells were stained with the fluorescein-conjugated antibodies for surface marker expression. After staining, cells were fixed with 300 μL 4% PFA and analyzed via FACS (BD LSR II). Cytokine production by tumor-infiltrating cells were determined via intracellular staining. Cells were stained with surface marker, washed, fixed, and permeabilized using the cytofix/cytoperm kit from BD Pharmingen. Intracellular stains were performed using anti-IFN-gamma antibody. Cells were analyzed on an 18-color flow cytometer (LSRII), and the data were analyzed with FlowJo 8.6 software (TreeStar).

Immunostaining

After deparaffinizing, antigen retrieval and permeabilization, tissue sections were blocked in 1% bovine serum albumin (BSA) at room temperature for 1 h. Primary antibodies conjugated with fluorophores (BD, eBioscience, abcam) were incubated overnight at 4 °C. Nuclei were counterstained with DAPI containing mounting medium (Vector Laboratories Inc., Burlingame, CA). All antibodies were diluted according to the manufacturer’s manual. Images were taken using Zeiss 880 confocal microscopy (Germany). Three random microscopic fields were selected and quantified by ImageJ software.

Quantitative real-time PCR (qPCR) assay.

Total RNA was extracted from the tumor tissues using a RNeasy Microarray Tissue Mini Kit (Qiagen). cDNA was reverse-transcribed using the iScript™ cDNA Synthesis Kit (BIO-RAD). One hundred fifty ng of cDNA was amplified with the iScript™ Reverse Transcription Supermix for RT-qPCR (BIO-RAD). GAPDH was used as the endogenous control.

Reactions were conducted using the 7500 Real-Time PCR System, and the data were analyzed with the 7500 Software.

Statistical analysis

Data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed via PRISM software using Students’ t-test when only two value sets were compared, and one-way analysis of variance (ANOVA) with a Dunnette’s test for post hoc analysis when the data involved three or more groups. Kaplan-Meier analysis was used for determination of the survival study, in which the log-rank (Mantel-Cox) test was applied to determine significance between treatment groups. *, **, ***, or **** denotes p < 0.05, 0.01, 0.001, and 0.0001, respectively; these were considered significant and are shown in figure or figure legend.

RESULTS

PI3K-γ in B cells

During inflammation and cancer, PI3K-γ controls a critical switch between immune stimulation and suppression. Previous studies have shown that PI3K-γ is highly expressed in myeloid cells (Kaneda et al., 2016). As shown in Fig.1, we demonstrated that PI3K-γ was also highly expressed in B cells associated with both KPC pancreatic cancer and BPD6 melanoma models. Both tumor models contained extensive stroma structure (Fig. S1), which is typical of an immune-suppressive microenvironment (Moffitt et al., 2015). BRAF mutation is very common in cutaneous melanoma, mutated BRAF (V600E) protein is highly activated comparing to wild-type, because of a conformational transformation in protein structure (Liu et al., 2017). Fig. 1 also indicates that B cells represented a significant portion of PI3K-γ expressing cells in the tumor. Most of these cells are regulatory B (Breg) cells which are CD138, CD1d, and CD5 positive (Shen et al., 2014). Non-B cells that are PI3K-γ positive, which includes MDSCs, are relatively few. We hypothesized that IPI-549, the only PI3K-γ inhibitor in clinical development, should significantly remodel the suppressive microenvironment in these tumors.

Figure 1.

Figure 1.

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PI3K-γ was detected in tumor associated B cells. Mice were sacrificed, and single cell suspension were prepared for flow cytometry. PI3K- γ in B cells were detected (CD19+ B220+PI3Kγ+, CD19 in BV605, CD138 in PE-cy7 and PI3k- γ in PE) and quantified via flow cytometry (n = 3). Upper line are KPC, lower line are BPD6.

Preparation and characterization of IPI-549 NP.

Nanoparticles can prolong the half-life of payloads in vivo and passively accumulate in the tumor regions (Chen et al., 2016). Nanoparticles modified with the aminoethylanisamide (AEAA) exhibit a sigma-receptor-dependent cellular uptake mechanism (Goodwin and Huang, 2016). Sigma receptor is highly expressed in PAC and tumor-associated fibroblasts (TAFs) (Guo et al., 2014).

We have formulated IPI-549 in poly-lactic-glycolic-acid (PLGA) nanoparticles. The IPI-549 NP was prepared according to the solvent displacement method (Guo et al., 2014). Further details of preparation were given in supplementary information. The particle size of nanoparticles was measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). The NP was negatively charged (~ -15 mV) with a relatively small size (~ 70 nm) (Fig. S2A). The morphology of nanoparticle was observed by transmission electron microscopy. The NP exhibited a spherical morphology with smooth surfaces (Fig. S2B). The size was approximately 20-50 nm, smaller than the size measured by DLS. IPI-549 can be encapsulated into PLGA-polyethylene glycol (PEG) nanoparticles with high encapsulation efficiency (91.2 ± 3.6%) and loading (13.1 ± 2.1%). The in vitro release behaviors of IPI-549 from NP were studied. As shown in Fig. S2C, IPI-549 was slowly released from NPs at 37 °C. IPI-549 taken up by cells in the tumor was expected to spread to other cells in the tumor because of the hydrophobicity of the drug. The pharmacokinetic parameters of IPI-549 NPs are shown in Table S1. For IPI-549 NPs, the area under the concentration time curve (AUC0-∞) and half-life (T1/2) were 103.0 μg⋅/mL, 4.5 h, which were higher compared with oral IPI-549 of 3.1 μg⋅/mL and 2.1 h, respectively. The results showed that PLGA-PEG NPs are a promising formulation to improve the pharmacokinetic properties of IPI-549.

The distribution of IPI-549 NPs and free IPI-549 in the tumor, heart, liver, spleen, lungs, and kidney is shown in Fig. S3. For the IPI-549 NPs group, the IPI-549 level in the tumor reached 6.6 μg/g at 4 h, which was significantly higher than for the free IPI-549 group. The IPI-549 levels in the spleen and kidney of the mice treated with oral IPI-549 were higher than the IPI-549 NPs group at 4, 8, and 12 h, respectively. Nanoparticles could avoid undesired uptake by RES and improve circulation of their encapsulated drugs in the blood, compared to free drug after modification. Nanoparticles modified with AEAA exhibit a sigma-1 receptor-dependent cellular uptake mechanism. Sigma receptors are well-known membrane-bound proteins that show high affinity for neuroleptics and are overexpressed on many human tumors (Goodwin and Huang, 2016).

In vivo anti-tumor efficacy

IPI-549 is an oral drug candidate for immuno-oncology in clinical development. In our work, IPI-549 was dissolved at 5% 1-methyl-2-pyrrolidone in polyethylene glycol 400 and administered by oral gavage at 30 mg/kg every three days. IPI-549 NP was administered via IV at a lower dose, (i.e. 15 mg/kg) every three days. As shown in Fig. 2A, IPI-549 NP exhibited a significant reduction in KPC tumor growth. It showed greater anti-tumor efficacy than oral IPI-549. IPI-549 NP treatment also led to tumor growth inhibition in the BPD6 melanoma tumor model (Fig. 2B)

Figure 2. Tumor growth inhibition in KPC and BRAF-mutant melanoma tumor-bearing mice.

Figure 2.

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Tumor inhibition curve of KPC (n = 8-10). Tumor inhibition curve of BRAF-mutant melanoma (n = 5). Arrows indicate time of dosing. *p < 0.05 and **p < 0.01.

IPI-549 NP reduced PI3K-γ

After IPI-549 NP treatment, PI3K-γ+ Breg cells decreased significantly in both KPC and BPD6 melanoma tumor-bearing mice (Fig. 3), which was consistent with our hypothesis that PI3K-γ inhibitor could reduce the Breg cell population. Patients with a low PI3K-γ own a better survival in lung, and head and neck cancers (Kaneda et al., 2016). Pharmacological or genetic blockade of p110γ subunit of PI3Kγ inhibited inflammation, growth, and metastasis of implanted and spontaneous tumors (Schmid et al., 2011). The result indicated that PI3K-γ inhibition is promising in cancer therapy. By means of the production of interleukin-10 (IL-10), IL-35, and transforming growth factor ² (TGF-²), Breg cells suppress immunopathology by preventing the expansion of pathogenic T cells and other pro-inflammatory lymphocytes (Zhang et al., 2015). The number and suppressive ability of Breg cells are related to inflammation; the class I PI3K family was implicated in inflammatory responses. Tumor-derived chemokines, growth factors, and cytokines accelerate tumor inflammation and progression by activating p110γ in inflammatory cells (Schmid et al., 2011).

Figure 3. PI3K-γ+ B cells were reduced after IPI-549 NP treatment in both KPC and BPD6 tumor bearing mice.

Figure 3.

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Mice were sacrificed at the end of the experiment. Single cell suspension was prepared for flow cytometry. PI3K-γ in Breg cells was significantly reduced (CD19+ Per-cp, CDld+FITC, CD5+Pacific Blue, PI3K-γ+ Alex 555) subsets were quantified via flow cytometry (n = 4). Data are expressed as mean ± s.d., calculated from samples. * p<0.05, **p < 0.01, respectively, compared to corresponding PBS control.

IPI-549 NP changed the TME structure

Tumor growth relies on the relationship between multiple inter-dependent cells. Among these different cells, TAFs have drawn increasing attention as pro-tumorigenic signals due to an established source of classical growth factors known to possess a tumor-promoting role, for example EGF and TGF-β (Roghanian et al., 2016). Pancreatic cancer is characterized by a dense background of TAFs. Therefore, we evaluated the density of TAFs in the KPC model after treatment. As expected, the IPI-549 NP treatment group exhibited the lowest density of α-SMA+ TAFs (19.2 ± 5.1% vs 10.9 ± 2.3% vs 12.1 ±2.9% in PBS, NP, and oral, respectively, P < 0.05) (Fig. 4). TAFs in the PBS control group showed bundled stroma morphology, typical of a desmoplastic tumor. TAFs in the drug-treated groups, both in NP and oral, showed disorganized morphology, indicating a lower level of fibroblast activation. By CD31 immunostaining, the tumor micro-vessels were also transformed from collapsed slender shape (white arrowheads), indicating high interstitial fluid pressure, in the PBS control to the round and relaxed shape (red arrowheads) in the drug-treated groups. These results indicate that inhibition of PI3K-γ+ cells induced a profound change in TME in favor of normalized fibroblast and vessel structures.

Figure 4. Tumor microenvironment changes after various treatments.

Figure 4.

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The KPC bearing mice were divided into three groups and treated with either PBS, IPI-549 NP, or oral IPI-549. At the end of treatment, mice were euthanized, and tumor tissues were harvested. Tumor tissues were assayed for αSMA / CD31 cells, Foxp3+CD4+ Treg cells, and CD11b+Gr-1+MDSC cells with immunofluorescence staining. First line showed blood vessel (CD31 in red) and tumor associated fibroblast (αSMA in green). The second line showed CD4+Foxp3+ Treg (CD4 in red FOXp3 in green). Third line showed Masson’s Trichrome (MT) staining for collagen (in dark blue). Lowest line showed MDSC (Gr-1+ in red, CD11b+ in green). White arrowheads indicate collapsed vessels(first line in PBS group), red arrowheads showed the round and relaxed vessels( in treated groups) in the top panels. Yellow arrowheads indicate collagen morphology. Black arrow indicates normal pancreas region. * p<0.05, ** p<0.01 compared with PBS control.

In pancreatic cancer, Tregs and MDSC stymie the anti-tumor immune response from the premalignant stage to established cancer and are a signal of poor prognosis (Hiraoka et al., 2006). IPI-549 NP and oral treatment groups exhibited fewer Treg cells and MDSC than the control group. Collagen is one of the major extracellular matrices secreted by fibroblasts. The IPI-549 NP treatment group exhibited the lowest collagen (11.9 ± 2.3% vs 1.9 ± 0.4% vs 4.8 ± 1.1%, in PBS, NP, and oral, respectively, p < 0.05).

IPI-549 NP modified immune cell populations against KPC allografts and activated innate immune cells

Changes of the immune cell populations within the tumor and the therapeutic efficacy of this strategy were observed via flow cytometry and bioluminescence analysis. The results demonstrate that NP administration indeed changed the TME (Fig. 5). Inhibition of MDSC in PAC is a promising cancer therapy method. PAC cells express PD-L1 producing inhibitory signal by binding PD-1 on T cells; PD-L1 is another mechanism through which effector cells are suppressed (Feig et al., 2013). IPI-549 NP could significantly decrease MDSC, M2, and PD-L1 in tumor (Fig. 5D, E, F). Dendritic cells (DCs) are antigen-presenting cells, as one of the main regulators of antitumor immune response (Hiraoka et al., 2006; Kimura et al., 2012; Kobayashi et al., 2014; Davis et al., 2012). Tumor-infiltrating CD8+ cells, together with CD4+ cells served as a favorable prognostic factor (Fukunaqa et al., 2004), and high densities of CD8+ T cells in the juxta-tumoral area showed better survival in patients with PAC (Ene-Obong et al., 2016). NP significantly enhanced both DC and CD8+ T cells.

Figure 5. IPI-549 NP reduced immunosuppressive cells in the TME.

Figure 5.

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Mice were sacrificed at the endpoint of the experiment (day 38). Immune subsets were quantified via flow cytometry (n = 3). Data are expressed as mean ± s.d., calculated from samples. *, **, or *** denotes p < 0.05, 0.01, or 0.001, respectively.

As shown in Fig. S4, IPI-549 NP also reduced Breg and M2 in BRAF-mutant melanoma-bearing mice.

Changes in transcription factor and cytokine levels in the TME

We found that the IL-4 expression was significantly lower after NP treatments in KPC-bearing mice, while the IFN-γ expression was significantly enhanced. It is reported that the Th1 immune response in the peripheral blood of pancreatic cancer patients seems to be indirectly suppressed by tumor cell-derived TGF-β and IL-10; however, an overall Th1 response might be inhibited by increased production of IL-4 and decreased generation of IFN-γ and IL-12 from stimulated peripheral blood mononuclear cells (Bellone et al., 1999; Von Bernstorff et al., 2011; Gabitass et al., 2011). Moreover, a variety of studies certified evidence for a general Th2 shift in pancreatic cancer (De Monte et al., 2011; Tassi et al., 2008). Therefore, a predominance of Th2 cytokines (IL-5, IL-6 and IL-10), especially IL-13, was detected in the plasma of pancreatic cancer patients. In the present study, IPI-549 NP administration reduced Th2 response and enhanced Th1 response in the TME, which exhibited superior antitumor efficacy in the KPC tumor-bearing mouse model. Foxp3 was introduced as a key transcription factor for development and function of Treg (Fontenot et al., 2003; Hori et al., 2003). IPI-549 NP reduced mRNA expression of Foxp3 and therefore increased the antitumor response.

Safety Evaluation

As shown in Fig. S5, there is no significant damage in the heart, kidney, spleen, lung, or liver after IPI-549 treatment. However, there is tumor metastasis to liver, and spleen damage in the PBS control group.

DISCUSSION

Breg cells produce large amounts of cytokines such as IL-10, TGF-β, and IL-35, which suppress the differentiation of pro-inflammatory lymphocytes, such as tumor necrosis factor α (TNF-α)-producing monocytes, IL-12-producing dendritic cells, Th17 cells, Th 1 cells, and cytotoxic CD8+ T cells (Matsumoto et al., 2014; Sun et al., 2005; Tian et al., 2001). Breg cells can also result in the differentiation of immune-suppressive T cells, Foxp3+ T cells, and T regulatory 1 (Tr1) cells (Carter et al., 2011; Flores-Boija., 2013). According to previous studies (Henau et al., 2016; Kaneda et al., 2015), PI3K-γ is highly expressed in myeloid cells. We demonstrated that PI3K-γ is also highly expressed in B cells, especially Breg cells. Breg cells were decreased after IPI-549 NP treatment, both in KPC and in BPD6 bearing mice. This indicated that PI3K-γ inhibitor IPI-549 remodels the immune-suppressive TME through its effects on B cells, in addition to the myeloid cells, resulting in enhanced anti-tumor activity and cytokine production, all of which play critical roles in immune response.

The PI3K-γ isoform is expressed in immune cells and has restricted expression in normal or malignant epithelial cells and connective tissue cells (Sasaki et al., 2000). PI3K-γ is crucial for cellular activation and migration in response to certain chemokines (Sasaki et al., 2000; Hirsch et al., 2000). It is reported that murine syngeneic tumors grow slower when transplanted into immune-competent mice when PI3K-γ is genetically inactivated (Schmid et al., 2011; Joshi et al., 2014). This growth reduction is closely associated with the incapability of tumor-associated myeloid cells, which are known to lead to an immune suppressive TME and accelerates tumor growth (Gunderson et al., 2016; Rivera et al., 2015). It is reported that PI3K-γ inhibition using IPI-549 is mainly effective in tumor landscape rich in suppressive myeloid cells and allows for more precise delineation of patients in which it will potentially yield the greatest activity (Kaneda et al., 2016). IPI-549 nanoparticle was effective in inhibiting the growth of pancreatic cancer model and BRAF-mutant melanoma model. In pancreatic cancer, myeloid-derived suppressor cells (MDSC) and macrophages are recruited to the TME (Sanford et al., 2013; Bayne et al., 2012; Pylayeva-Gupta et al., 2012), while stromal-associated fibroblasts help to recruit regulatory B cells (Breg) into the TME. These Bregs produce IL35, which drives PDAC progression (Pylayeva-Gupta et al., 2016; Nicholl et al., 2014). IPI-549 NP can induce the reduction of immune suppressive cells such as Breg (Fig. 5D), and MDSC (Fig. 5E). Meanwhile, the phenotype of DC (expression of CD11c) was analyzed in TME after treatment. IPI-549 NP could enhance the expression of CD11c significantly (Fig. 5A). Two recent in vivo studies provided opinion into the course of such events: the release of IFN-γ by NK cells in TME was beneficial in the stimulation and maturation of dendritic cell (DC), which promoted a powerful and protective anti-turnor CD8+ T-cell response (Guillerey and Smyth, 2016; Morandi et al., 2012). IFN-γ production of CD8+ T cells was increased significantly after IPI-549 NP treatment.

In conclusion, we found that tumor-targeted IPI-549 NP exhibited more significant tumor inhibition effect than oral IPI-549 in the KPC tumor-bearing mice. IPI-549 NP significantly decreased both the number and percentage of MDSC and Breg cells. IPI-549 remodels the immune-suppressive TME through its effects on both plasma cells and myeloid cells.

Supplementary Material

1

Figure 6. IPI-549 NP reduced Th2 response but enhanced Th1 response in the TME.

Figure 6.

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Mice were sacrificed at the endpoint of the experiment. Tumors were analyzed for the indicated mRNA level by RT-PCR. n = 5. *p < 0.05, **p < 0.01 and not significant (n.s.).

ACKNOWLEDGMENTS

This work was supported by NIH grant CA198999 and Eshelman Institute for Innovation.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes five figures and two tables.

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