Int. J. Biol. Sci. 2023, Vol.

19 2648

Ivyspring
International Publisher
International Journal of Biological Sciences
2023; 19(9): 2648-2662. doi: 10.7150/ijbs.81683
Research Paper

Induction of Immune Responses and Phosphatidylserine

Exposure by TLR9 Activation Results in a Cooperative
Antitumor Effect with a Phosphatidylserine-targeting
Prodrug
Jen-Chih Tseng1, Jing-Xing Yang1, Chia-Yin Lee1,4, Chen-Fu Lo2, Yi-Ling Liu1, Mingzi M. Zhang3, Li-Rung
Huang3, Ko-Jiunn Liu4, Chien-Chia Wang5, Chi-Ying F. Huang6, Yi-Ren Hong7, Lun K. Tsou2,,
Tsung-Hsien Chuang1,5,
1. Immunology Research Center, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan.
2. Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Miaoli, 35053, Taiwan.
3. Institute of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, 35053, Taiwan.
4. National Institute of Cancer Research, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan.
5. Department of Life Sciences, National Central University, Zhongli District, Taoyuan City 32001, Taiwan.
6. Institute of Biopharmaceutical Sciences, College of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei 11221, Taiwan.
7. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.

 Corresponding authors: [email protected] (T.-H. Chuang); [email protected] (L.K. Tsou).

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
See http://ivyspring.com/terms for full terms and conditions.

Received: 2022.12.08; Accepted: 2023.05.05; Published: 2023.05.11

Abstract
Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients.
Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator
(CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic
head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and
BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor
immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in
tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel
function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting
more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056
targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which
enhanced the CpG-272-promoted T cell–mediated tumor-killing effect. These form a positive feed-forward
antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel
strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using
PS-targeting drugs.
Keywords: Cancer immunotherapy, CpG-oligodeoxynucleotide, Immunogenic cell death, Phosphatidylserine-targeting prodrug,
Toll-like receptor, Topoisomerase I inhibitor

Introduction
Head and neck cancers are among the most with HNSCC include radiation, chemotherapy, and
common cancer types with high mortality rates. Head cetuximab. However, patients with cancer suffer from
and neck squamous cell carcinoma (HNSCC) account poor quality of life due to the disease and treatment;
for >90% head and neck cancer cases, often charac- therefore, highly effective and satisfactory therapeutic
terized by an immunosuppressive microenvironment, strategies are desired [1-3].
consistent with the fact that only <20% patients Toll-like receptors (TLRs) are a family of pattern
respond to therapy with immune checkpoint recognition receptors that help detect microbial
inhibitors. Currently, standard treatments for patients pathogens to initiate host responses to infections. Of

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Int. J. Biol. Sci. 2023, Vol. 19 2649

the 13 TLRs (TLR1–13) identified in mammals, 10 loped, including natural products, monoclonal anti-
(TLR1–10) are expressed in humans. TLR9 belongs to bodies, antibody–drug conjugations, and liposomal
a subfamily comprising TLR3, TLR7, TLR8, and TLR9. carriers. Several of these are under investigation in
Distinct from other human TLRs expressed on cell clinical trials as potential monotherapies or combi-
surfaces, these four TLRs are located in intracellular nation drugs for various cancers [22, 23]. Zinc
vesicles, primarily in endolysosomes [4, 5]. The (II)-dipicolylamine (Zn-DPA) reportedly binds
natural TLR9 ligand is microbial unmethylated specifically to PS [24, 25].
CpG-DNA. The ligand ligation of TLR9 activates SN38 (7-ethyl-10-hydroxycamptothecin) is a
nuclear factor (NF)-κB and interferon regulatory topoisomerase I inhibitor with cytotoxicity toward
factors (IRFs), thereby producing inflammatory cancer cells. This agent cannot be directly
cytokines and type I interferons (IFNs), respectively administered to patients owing to its toxicity and
[6, 7]. The proximal signal molecule for TLR9 signal aqueous solubility features; therefore, chemical
transduction is the myeloid differentiation primary modification is required to deliver it more explicitly to
response 88 (MyD88). On activation, MyD88 is tumors [26, 27]. BPRDP056 is a novel PS-targeting
recruited to TLR9, forming a complex with interleukin antitumor drug candidate generated by linking
(IL)-1 receptor–associated kinase-1 (IRAK-1), IRAK-4, Zn-DPA with SN38. It reportedly suppresses the
and tumor necrosis factor (TNF)–associated factor 6 growth of several different types of tumors in cancer
(TRAF6) and transforming growth factor-β–associ- animal models [28]. Nevertheless, to the best of our
ated kinase 1 (TAK1) activation. This cascade activates knowledge, its suppressing effect on head and neck
NF-κB, responsible for the transcription of genes for cancers has not yet been investigated. CpG-2722 is a
proinflammatory cytokines, including TNF-α, IL-1, CpG-ODN exhibiting TLR9-mediated immune-stimu-
IL-6, and IL-12. The dendritic cell (DC) is a major cell latory activities for multiple species [29, 30]. Herein,
type for type I IFN production in response to TLR9 we utilized an orthotopic syngeneic mouse model of
activation. In these cells, the MyD88, IRAK1, IRAK4, HNSCC to investigate the mechanism and function of
and TRAF6 complex activates IRF-7 phosphorylation combined CpG-2722 and BPRDP056 therapy to treat
and translocation into the nucleus for type I IFNs head and neck cancers. The results revealed that
transcription [8-11]. BPRDP056 displayed cytotoxicity in cancer cells.
Synthetic CpG-oligodeoxynucleotides (CpG- TLR9 activation by CpG-2722 activated immune
ODNs) mimic CpG-DNA’s function in TLR9 activa- responses and PS exposure in tumors. CpG-2722 and
tion. Distinct from natural CpG-DNAs containing a BPRDP056 administration alone inhibited tumor
phosphodiester backbone, CpG-ODNs include a growth. The combination treatment with both agents
phosphorothioate backbone that increases resistance further produced a cooperative effect on tumor
to nucleases [12, 13]. TLR9 activation via CpG-ODNs growth suppression.
administration induces an early innate immune
response for priming the subsequent adaptive Materials and methods
immune responses. Innate immune and B cells are
Animal care
activated during the early phase for antigen
presentation and cytokine production. The produced All animal experiments were approved by the
cytokines promote Th1 CD4+ T-cell polarization, Institutional Animal Care and Use Committee of the
thereby expanding antigen-specific CD8+ T cells National Health Research Institute, Taiwan. Both
[14-17]. As these immune responses play a crucial role male and female mice were used after randomization
in cancer cell eradication, the antitumor effect of to the different experimental groups. These mice were
CpG-ODNs has been investigated as both mono- maintained and handled following the stated
therapies and combinational therapies [18, 19]. guidelines.
Phosphatidylserine (PS) is an essential bilayer Chemicals, reagents, and antibodies
cell membrane component. It normally exists in the
CpG-2722 was purchased from Integrated DNA
inner leaflet of the cell; however, it can be externalized
Technologies, Inc. Small molecule compound,
to the external leaflet in response to cell injury or
BPRDP056, BPRDP060, and SN38 were synthesized
apoptosis. The PS exposed on apoptotic cells act as an
and dissolved for treatments as reported previously
engulfment signal for phagocytosis by phagocytic
[28]. Rat antimouse CD8 antibody (clone: 4SM15, Cat.
cells. Recently, PS exposure has also been reported in
No. 14-0808-82) was purchased from Invitrogen.
nonapoptotic forms [20,21]. In tumors, PS exposure is
Rabbit anti-PS antibody (Cat. No. PAB881Ge01) was
significantly increased on tumor cell surface, serving
purchased from Cloud-Clone Corp. Rat anti-mouse
as a targeting marker for diagnosis and therapy.
IFN-γ antibody (MAB485) for neutralizing was
Consequently, PS-targeting agents have been deve-
purchased from R&D. Rat anti-mouse TNF-related

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Int. J. Biol. Sci. 2023, Vol. 19 2650

apoptosis-inducing ligand (Trail) neutralizing anti- (GM-CSF) (PeproTech). On days three and six, a
body (Cat. No. 109315) and hamster anti-mouse complete medium supplemented with 100 ng/mL
TNF-α neutralizing antibody (Cat. No. 506110) were Flt3-L and 5 ng/mL GM-CSF was added for further
purchased from BioLegend. Mouse IFN-α, and the culture. On day nine, immature DCs were seeded at 2
Trail enzyme-linked immunosorbent assay (ELISA) × 106 cells/well of a noncoated six-well plate and
kit were purchased from R&D. Rabbit anti-TrailR2 stimulated with CpG-2722 at 5 μg/ml for 24 h.
antibody (Cat. No. ab8416) was purchased from
Abcam. Trizol reagent and the SuperScriptTM IV kit
Flow cytometry
were purchased from Invitrogen. The SYBR® Green BMDCs maturation status was assessed by
PCR kit was purchased from Qiagen. fluorescence-activated cell sorting (FACS). Briefly,
BMDCs were harvested and rinsed twice with
Cell culture ice-cold PBS containing 2% FBS and spun at 1500 rpm
SAS is a human tongue squamous cell carcinoma for 5 min at 4°C. The cells were surface-antibody
(SCC) cell line [31]. OECM1 is a human gingival stained for CD11c (Cat.No. 117308, BioLegend), CD40
squamous carcinoma cell line harboring a p53 (Cat. No. 12-0401-82, Invitrogen), CD80 (Cat. No.
missense mutation [32]. M11-1-2 and NHRI-HN1 are 17-0801-82, Invitrogen), and CD86 (Cat. No.
mouse tongue SCC cell lines established previously 25-0862-82, Invitrogen) in ice-cold PBS containing 2%
[33]. SAS, M11-1-2, and NHRI-HN1 were maintained FBS for 30 min. After washing twice with PBS
in Dulbecco’s modified Eagle medium (complete containing 2% FBS, these surface molecules
medium) containing 10% fetal bovine serum (FBS) expression levels on BMDCs were acquired on a
(Hyclone), 2 mM L-glutamine, 1% antibiotic– FACS Canto II and analyzed using FlowJo software.
antimycotic, and 10 mM HEPES (4-(2-hydroxyethyl)- To analyze immune cells in tumors, tumor samples
1-piperazineethanesulfonic acid). OECM1 cancer cells were incubated in gentle collagenase/hyaluronidase
were maintained in RPMI complete medium in DMEM for enzymatic dissociation. After overnight
containing 10% FBS, 1% antibiotic–antimycotic, and incubation, cells were collected after passing them
10 mM HEPES and cultured at 37°C in a 5% CO2 through a 70-μm nylon cell strainer and centrifuged at
incubator. 1500 rpm for 5 min. The cells were surface-antibody
stained as indicated in ice-cold PBS containing 2% FBS
Mouse splenocytes preparation for 30 min. After washing twice with PBS containing
Mouse splenocytes were isolated from 6- to 2% FBS, the immune cells composition in tumors were
8-week-old C57BL/6J mice (National Laboratory acquired on a FACS Canto II and analyzed using
Animal Center, Taiwan). Mouse spleen fragments FlowJo software.
were pounded using a syringe plunger; single cells
were collected after passing them through a 40-μm Cell cytotoxicity assay
nylon cell strainer (BD FalconTM) and centrifuged at Mouse splenocytes and cancer cells were seeded
1500 rpm for 5 min. Red blood cells (RBCs) were onto 96 well plates at 106 and 3 × 103 in 100 μl/well,
removed by resuspending the cell pellet in RBC lysis respectively. The next day, splenocytes and cancer
buffer at room temperature for 2 min, and the lysis cells were treated with the CpG-2722, BPRDP056,
reaction was terminated by adding 30 ml BPRDP060, and SN38 as indicated. Cell cytotoxicity
phosphate-buffered saline (PBS). The isolated was determined using a CellTiter 96 Aqueous One
splenocytes were cultured in RPMI 1640 complete Solution Cell Proliferation Assay kit (Promega)
medium at 37°C in a 5% CO2 incubator. following the manufacturer’s protocol.

Murine bone marrow-derived dendritic cells RNA isolation and reverse

preparation transcription-quantitative PCR (RT-qPCR)
Mouse bone marrow–derived dendritic cells analyses
(BMDCs) were prepared from C57BL/6J mice. Bone Total RNA from cells were isolated using the
marrow cells were flushed from the femur and tibia illustraTM RNAspin Mini kit (GE Healthcare). Total
with PBS and resuspended in RPMI-1640 complete RNA from tumors was isolated using Trizol reagent
medium containing 10% FBS, 2 mM L-glutamine, 1% following the manufacturer’s protocol. RNA sample’s
antibiotic–antimycotic, 1% MEM nonessential amino reverse transcription was performed using the
acid solution, 1 mM sodium pyruvate, 10 mM HEPES SuperScript IV First-Strand Synthesis System
supplemented with 100 ng/mL FMS-like tyrosine (Invitrogen). Gene expression levels induced by
kinase 3 ligand (Flt3-L) (PeproTech) and 5 ng/mL CpG-2722 or BPRDP056 were analyzed using the
granulocyte-macrophage colony-stimulating factor QuantiNovaTM SYBR Green PCR kit (Qiagen) for

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Int. J. Biol. Sci. 2023, Vol. 19 2651

qPCR analyses and gene specific primers (Table S1). Statistical analysis
β-actin expression level served as the loading control. Data are expressed as mean ± SEM. All groups
Syngeneic orthotopic animal model of head were from three or more independent experiments as
and neck cancer indicated. Statistical analyses were performed using
Student’s t-test. Differences were considered
Two million NHRI-HN1 cells were mixed with
significant when the p value was less than 0.05.
Matrigel (BD Biosciences) at a 1:1 ratio to a total
Statistically significant p values are abbreviated as
volume of 100 μl per mouse. The NHRI-HN1 cells
follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
were intramucosally injected into the 6- to 8-week-old
C57BL/6J mice through a side of the buccal region to Results
grow the tumor. When the tumor size was
approximately 100 mm3, the mice were intratumorally Cytotoxic effect of BPRDP056 and CpG-2722
injected with the indicated amount of CpG-2722, BPRDP056 is a prodrug generated by
BPRDP056, and their combination every 4 days. All conjugating BPRDP060 to SN38. The BPRDP060
groups contained five mice and five tumors. The comprises Zn-DPA for PS targeting and a linker
tumor volume was measured using the formula: region to bridge SN38 and Zn-DPA (Figure 1) [28]. To
Volume = length × (width) 2 × 0.5. investigate the feasibility of using BPRDP056 alone
and in combination with CpG-2722 on head and neck
ELISA
cancers, we first examined the cytotoxicity of
Mouse splenocytes or tumor tissue lysates BPRDP056 toward several different lines of head and
culture medium was collected. TNF-α, IFN-α, IFN-γ, neck cancer cells, including the human SAS and
and Trail production levels were determined using OECM1 lines as well as the mouse M11-1-2 and
ELISA kits following the manufacturer’s protocol. NHRI-HN1 lines. BPRDP056 exerted different
Immunohistochemistry cytotoxicity toward these cells. Human SAS was more
sensitive than OECM1, and mouse M11-1-2 was more
Paraffin-embedded tumors were sectioned into
sensitive than NHRI-HN1 to BPRDP056 cytotoxicity.
5-μm tissue slides. These tissue slides were rehy-
The cytotoxicity of the different parts of BPRDP056
drated with graded ethanol to PBS concentrations,
and CpG-2722 was also investigated. When compared
and endogenous peroxidase was blocked with 3%
with BPRDP056, SN38 exhibited more powerful
hydrogen peroxide for 5 min. For PS, Trail, and CD8
cytotoxicity. However, the degree of cytotoxicity of
staining, the primary antibody against PS or CD8 was
both BPRDP056 and SN38 was similar across the four
used at a 1:50 dilution, and the Trail antibody was
analyzed cell lines. Conversely, the cytotoxicity of
used at a 1:25 dilution. The tissue slides were
BPRDP060 and CpG-2722 were not observed under
incubated with specific antibodies at room tempera-
the experimental conditions (Figure 2A). These
ture for 1 h. Tissue sections were then incubated with
findings suggest that SN38 is the moiety responsible
horseradish peroxidase–conjugated secondary anti-
for the cytotoxicity of BPRDP056 on these head and
body at room temperature for 30 min after washing
neck cancer cells. The cytotoxicities of BPRDP056,
with PBS solution. Detection was processed using the
BPRDP060, and SN38 were further assessed on mouse
Discovery XT automated IHC/ISH slide staining
splenocytes. Some cytotoxicity of SN38 was observed
system (Ventana Medical System, Inc., Tucson) using
when its concentration reached 0.2 µM. Nevertheless,
an ultraView Universal DAB Detection Kit (Ventana
the cytotoxicity of BPRDP056 on these cells was not
Medical System, Inc., Tucson), according to the
observed even at 10 µM (Figure 2B), suggesting that
manufacturer’s instructions. PS-positive areas
compared with the head and neck cancer cells,
percentage and the number of CD8-positive cells were
splenocytes were less sensitive to BPRDP056.
calculated using ImageJ (RRID:SCR-003070).
Immunostimulatory effect of CpG-2722 and
Phosphatidylserine exposure analysis
BPRDP056
The condition medium collection following
The immunostimulatory activities of CpG-2722
mouse splenonocytes were stimulated with or
and BPRPD056 were compared. Mouse splenocytes
without 5 μg/ml of CpG-2722 for 48 h. NHRI-HN1
were treated with CpG-2722, and the expression of
cells were treatment with DMEM containing 25%
cytokines was analyzed via RT-qPCR. CpG-2722
condition medium for 8 h. The PS exposure were
significantly induced various cytokines expression,
stained by annexin V conjugated APC antibody
including that of TNF-α, IL-1β, IL-6, IL-12A, IL-12B,
(Biolegend) at room temperature for 15 min, and the
NHRI-HN1 PS exposure level were acquired on a IFN-α, IFN-β, and IFN-γ (Figure 3A). Among these,
FACS Canto II and analyzed using FlowJo software. TNF-α and IFN-γ are signature Th1 immune response

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Int. J. Biol. Sci. 2023, Vol. 19 2652

cytokines. Additionally, TNF-α displays cytostatic tumors reached ~100 mm3, the mice were
and cytotoxic effects against cancer cells [34, 35]. intratumorally injected with 1, 5, or 10 mg/kg/mouse
Therefore, the production of these two cytokines was BPRDP056 every 4 days. A dose-dependent effect of
verified via enzyme-linked immunosorbent assay the BPRDP056 on HNSCC growth suppression was
(ELISA). Consistent with their increased gene observed (Figure 4). The dose of 10 mg/kg
expression profiles, these two cytokine productions BPRDP056/mouse exhibited a higher antitumor effect
increased in parallel with CpG-2722 concentrations than the other two doses, although the tumors
(Figure 3B). Next, BPRDP056 immunostimulatory continued to grow. Therefore, this BPRDP056 dose
activities were investigated. Compared with was used in the subsequent studies to investigate its
CpG-2722, BPRDP056 elicited a milder immune combinational effect with CpG-2722.
response. Weak cytokine induction, including that of
Cooperative effect of CpG-2722 and
IL-6, IL-12A, IL-12B, IFN-β, and IFN-γ, was observed
when the BPRDP056 concentration increased to
BPRDP056 on HNSCC suppression
certain levels. The immunostimulatory activity To investigate the suppression of HNSCC via
disappeared on further increase in concentration combinational therapy involving BPRPD056 and
(Figure 3C). DCs comprise major antigen-presenting CpG‑2722, the mice were continuously intratumorally
cells [36, 37]; thus, CpG-2722 activity on DC was injected with CpG-2722 and BPRPD056 alone or in
investigated. CpG-2722 promoted the expression of combination every 4 days following the schedule
CD40, CD80, and CD86, markers for DC maturation shown in Figure 5A when the tumors reached ~100
and costimulatory molecules of DCs for T-cell mm3. These mice were monitored for tumor growths
activation (Figure 3D) [36, 37]. These results indicate (Figure 5B), euthanized at the end of the CpG-2722
CpG-2722 immune-stimulatory activities and and BPRPD056 treatments, and the tumors were
suggested distinct functional profiles for the removed to analyze their sizes (Figure 5C). It was
antitumor effect of CpG-2722 and BPRDP056. observed that treatment with CpG-2722 or BPRDP056
alone suppressed tumor growth. Combination
Tumor suppressing effect of BPRDP056 on treatment involving CpG-2722 and BPRDP056
HNSCC demonstrated more effective tumor suppression than
The head and neck cancer cell line, NHRI-HN1, treatment with any of these two agents alone (Figure
was established from C56BL/6J-derived oral 5B–D). The collected tumors were fixed and sectioned;
squamous cell carcinoma cells to develop an the tissue was stained with hematoxylin and eosin to
orthotopic syngeneic cancer animal model to study examine the infiltrating leukocytes. The results
the cancer immunobiology of head and neck cancers revealed that CpG-2722 and BPRDP056 alone and in
[33]. We investigated the antitumor activity of the combination increased the amounts of infiltrating
BPRDP056 with this HNSCC animal model. The leukocytes in tumors (Figure 5E). These findings
NHRI-HN1 cells (2 × 106 cell/mouse) were injected suggest that the treatments activated immune
into the mice through the side of the buccal region to responses in the tumors.
develop orthotopic tumors. When the size of the

Figure 1. Structural features of BPRDP056. BPRDP056 is a phosphatidylserine (PS)-targeting prodrug of SN38. BPRDP056 contains three moieties: SN38 for
tumor-cell killing; Zn-DAP for PS targeting; and a linker between SN38 and Zn-DAP. BPRDP060 contains the linker and Zn-DAP regions of BPRDP056. These compounds were
prepared according to the previous report [28]. Briefly, by coupling the pegylated linker that contains the cyclohexyl-para-chlorophenyl functional groups to the Zn-DPA, the
precursor of BPRDP060 was first prepared. Condensation between SN38 and the precursor of BPRDP060 allowed the synthesis of BPRDP056 precursor. Incubation of each
precursor with two equivalents of zinc nitrate resulted in the formation of Zn-DPA conjugates BPRDP060 and BPRDP056.

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Int. J. Biol. Sci. 2023, Vol. 19 2653

Figure 2. Cytotoxic effects of BPRDP056 and CpG-2722. (A) Human tongue squamous cell carcinoma SAS cells, human gingival squamous carcinoma OECM1cells, and
mouse tongue squamous cell carcinoma M11-1-2 and NHRI-HN1 cells, and (B) mouse splenocytes were treated with different concentrations of BPRDP056, BPRDP060, SN38,
or CpG-2722 for 72 h, as indicated. Cell cytotoxicity was then determined using the CellTiter 96 aqueous assay. Data represent the mean ± SEM (n = 3 independent
experiments). *, **, and *** represent statistically significant differences; p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the control vehicle.

Promoting immune cell accumulation in the 2722. Conversely, the profile of magnitude of immune
tumor microenvironment via CpG-2722 and cell accumulations induced by the combination of
BPRDP056 treatment CpG-2722 and BPRDP056 was between that induced
The mechanism underlying the cooperative by either CpG-2722 or BPRDP056 alone (Figure 6),
antitumor effect of CpG-2722 and BPRDP056 was suggesting that the CpG-2722 played a major role in
further investigated. The accumulation of different promoting immune cell accumulation in tumors. The
immune cell types in tumors derived from different infiltration of immune cells in tumors was further
groups of mice treated with CpG-2722 or BPRDP056 investigated via RT-PCR as this method is more
alone and their combinations were investigated via sensitive and can detect more cell types in tumors. For
flow cytometry. The results revealed that CpG-2722 the DC subsets, the results showed that BST2+ pDCs
induced the accumulation of CD45+ leukocytes, increased in the tumors of mice treated with
CD11c+ CD11b+ DC cells, BST2+MHCII− pDC cells, CpG-2722 alone and combination of CpG-2722 and
and CD8+ T cells in tumors. BPRDP056 moderately BPRDP056; CD205+, Clec9a+, and CD86+ cDC1s
induced CD11c+ CD11b+ DC cells, BST2+MHCII− pDC increased in the tumors of mice treated with
cells, CD8+ T cells accumulation; however, it induced CpG-2722 or BPRDP056 alone and their combination;
more CD4+ T cells in tumors compared with CpG- however, the levels of Clec10a+ and SIRPα+ cDC2s did

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Int. J. Biol. Sci. 2023, Vol. 19 2654

not change in the tumors of mice treated with any of levels of Ly6G+ granulocytes remained unaffected by
these treatments (Figure S1A). For the macrophage the different treatments (Figure S1C). For the T-cell
subsets, treatment with CpG-2722 alone and subsets, CD45+ leukocytes and CD3+ and CD8+ T cells
combination of CpG-2722 and BPRDP056 promoted were detected in the tumors of mice treated with
F4/80+ macrophage and iNOS+ M1 macrophage CpG-2722 alone and combination of CpG-2722 and
accumulation in tumors; conversely, no treatment BPRDP056; in contrast, CD4+ T-cell accumulation in
affected the level of ARG-1 M2 macrophages in the tumors was promoted via BPRDP056 alone treatment
tumor (Figure S1B). Additionally, treatment with and not via the other treatments (Figure S1D). These
BPRDP056 alone and combination of CpG-2722 and results together with those obtained from flow
BPRDP056 promoted NKp46+ NK cell accumulation cytometric analysis indicate that CpG-2722 plays a
in tumors, and BPRDP056 alone treatment increased significant role in promoting immune cell accumu-
CD20+ B cell accumulation in tumors. Conversely, the lation in the tumors.

Figure 3. Immune-stimulatory activities of CpG-2722 and BPRDP056. Mouse splenocytes were treated with different concentrations of CpG-2722 as indicated. (A)
After 4 h, expressions of cytokines were analyzed by RT-qPCR. The expression level of actin was used as a loading control. (B) After 24 h, cytokines secreted into the cell culture
medium were measured by ELISA, as indicated. (C) Mouse splenocytes were treated with different concentrations of BPRDP056 as indicated. After 4 h, expressions of cytokines
were analyzed by RT-qPCR. (D) Bone marrow-derived dendritic cells (BMDCs) were treated with 5 µg/ml of CpG-2722 for 24 h. Left panels: expressions of cluster of
differentiation (CD)40, CD80, and CD86 were analyzed by flow cytometry. Right panels: a set of representative histograms for flow cytometric analyses. Data represent the
mean ± SEM (n = 3; independent experiments). *, **, and *** represent statistically significant differences; p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the
control.

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Int. J. Biol. Sci. 2023, Vol. 19 2655

Figure 4. Inhibitory effects of BPRDP056 on head and neck squamous cell carcinoma (HNSCC). C57BL/6J mice were orthotopically injected with 2 × 106
NHRI-HNC1 cells to establish HNSCC. When the tumors reached approximately 100 mm3, the mice were intratumorally injected with a control vehicle and different doses of
BPRDP056 every 4 days, as indicated. (A) The tumor size was measured every 4 days (each group contained five mice and five tumors). (B) The endpoint of tumor growth in
every group. (C) The body weight of each mouse was measured every 4 days (n = 5). Data represent the mean ± SEM. *, **, and *** represent statistically significant differences;
p < 0.05, p < 0.01, and p < 0.001, respectively, between different groups, as indicated.

Figure 5. Cooperative effect of BPRDP056 and CpG-2722 on suppressing the growth of head and neck squamous cell carcinomas (HNSCC). (A) C57BL/6J
mice were orthotopically injected with 2 × 106 NHRI-HNC1 cells to grow HNSCC. When the tumors reached approximately 100 mm3, the mice were intratumorally injected
with a control vehicle, 10 mg/kg BPRDP056 or 50 µg CpG-2722 alone and in combination every 4 days with the injection of CpG-2722 one day ahead of the BPRDP056 injection,
as illustrated. (B) The growth of tumors was measured (n = 5). (C) The endpoint of tumor growth in every group. (D) The body weight of each mouse was measured (n = 5).
(E) Tumor samples were visualized by H&E staining for leukocyte infiltrations. Scale bar represents 100 μm. Leukocyte infiltrations were counted using ImageJ. Data represent
the mean ± SEM. *, **, and *** represent statistically significant differences; p < 0.05, p < 0.01, and p < 0.001, respectively, between different groups, as indicated.

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Int. J. Biol. Sci. 2023, Vol. 19 2656

Figure 6. CpG-2722 or BPRDP056 alone and in combination promote the accumulation of immune cells in head and neck squamous cell carcinoma
(HNSCC). Tumor-bearing mice treated with CpG-2722 or BPRDP056 alone or in combination in the experiment in Figure 5 were euthanized at the end point to collect tumor
samples. Tumor cells were dissociated and stained with surface-antibody for different immune cells as indicated. The immune cells composition was acquired on a FACS Canto
II and analyzed using FlowJo software. Left panel: a representative set of histograms. Right panel bar figures: Data represent the mean ± SEM (n = 5). *, **, and *** represent
statistically significant differences; p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the control or as indicated.

Inducing cytokine production and PS exposure treatments on PS exposure in tumors were

in tumors via CpG-2722 and BPRDP056 investigated. The sections of tumors from the different
groups of mice were stained using an anti-PS
treatments
antibody. Treatment with CpG-2722 or BPRDP056
Cytokines, including IL-12 and IFN-γ, play a alone increased PS exposure in tumors, and their
crucial role in maintaining a favorable microenviron- combination further enhanced PS exposure (Figure
ment for cancer immune therapy [38]. Thus, the 7B).
induction of cytokine expressions in the tumors of
mice treated with CpG-2722 and BPRDP056 alone and CpG-2722 increases PS exposure in cancer
their combination was investigated. RT-qPCR cells by inducing cytokine release from
analyses revealed that treatment with CpG-2722 alone immune cells
and combination of CpG-2722 and BPRDP056 BPRDP056 reportedly causes cancer cell death
promoted the expression levels of IL-12A, IFN-γ, Trail, and PS exposure owing to its cytotoxicity [28].
and Trail receptor 2 (TrailR2) in tumors. CpG-2722 Conversely, PS exposure in tumors due to CpG-ODN
alone and not the other treatments activated TNF-α treatment is unexpected and to the best of our
and IL-12 B expressions, and BPRDP056 alone knowledge, has not yet been reported. Therefore, the
increased IL-12A, Trail, and TrailR2 expressions in mechanism underlying this function of CpG-2722 was
tumors (Figure 7A). The level of PS exposure in tumor further investigated. Whether CpG-2722 directly
is crucial for targeting BPRD056 to the tumor, activated PS exposure in cancer cells or indirectly
therefore the effects of CpG-2722 and BPRDP056 activated the exposure via immune cell–mediated

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Int. J. Biol. Sci. 2023, Vol. 19 2657

mechanisms was first investigated. The NHRI-HN1 blocked via neutralizing antibodies against TNF-α,
cancer cells were incubated with/without CpG-2722 IFN-γ, and Trail or their combination (Figure 8D).
or incubated with condition media from splenocytes These results suggest that CpG-2722 can induce PS
treated with/without CpG-2722 and analyzed via exposure in tumors by inducing TNF-α, IFN-γ, and
flow cytometry. The results revealed that the Trail production from immune cells.
condition media from the CpG-2722-stimulated Thus, as illustrated in Figure 9, the results of this
splenocytes showed activated PS exposure in cancer study indicate that the TLR9 activator, CpG-2722,
cells. Conversely, direct treatment with CpG-2722 did increases immune cell accumulation and induces
not induce PS exposure in cancer cells (Figure 8A). In inflammatory cytokine expression in tumors,
addition to their proinflammatory activity, cytokines resulting in a favorable microenvironment for tumor
such as TNF-α, IFN-γ, and Trail have always been eradication via the immune system. Additionally,
reported to increase PS exposure in cancer cells [39, CpG-2722 can promote PS exposure in tumors by
40]. CpG-2722 induced TNF-α and IFN-γ expressions inducing cytokines, such as TNF-α, IFN-γ, and Trail,
in splenocytes (Figure 3A). TNF-α, IFN-γ, and Trail from the immune cells to facilitate the PS targeting of
induction in the tumors of the mouse groups BPRDP056 to release its payload SN38 in tumors.
subjected to different treatments was measured via These account for the observed cooperative antitumor
ELISA. CpG-2722 alone treatment increased TNF-α, effect of CpG-2722 and BPRDP056.
IFN-γ, and Trail protein production, consistent with
the increased expression of these genes in the tumors Discussion
of CpG-2722-treated mice (Figures 7A and 8B). PS Head and neck cancers are a group of malignan-
exposure induction in cancer cells via these cytokines cies comprising oral, oropharyngeal, nasopharyngeal,
was verified by the treatment of the NHRI-HN1 hypopharyngeal, laryngeal, sinus, salivary gland, and
cancer cells with TNF-α, IFN-γ, and Trail and analysis thyroid cancers. Alcohol consumption and smoking
via flow cytometry (Figure 8C). Further, PS exposure constitute the major causes of these cancers. Other risk
due to the treatment of cancer cells with the condition factors include human papillomavirus infections and
media of CpG-2722-activated splenocytes was betel nut consumption. These cancers are a common

Figure 7. CpG-2722 or BPRDP056 alone and in combination promote cytokine expression and phosphatidylserine exposure in head and neck squamous
cell carcinomas (HNSCC). Tumor-bearing mice treated with CpG-2722 or BPRDP056 alone or in combination in the experiment in Figure 5 were euthanized at the end
point to collect tumor samples. (A) Total RNA samples from the tumors were isolated by Trizol reagent. Expressions of different cytokines were analyzed by RT-qPCR. The
expression level of β-actin was used as a loading control. (B) Tissue sections were immunohistochemically stained to detect PS exposure in tumors (upper left panel: 100× and
bottom left panel: 200×). Scale bar represents 100 μm. PS exposure was quantified using Image J (right panel). *, **, and *** represent statistically significant differences; p < 0.05,
p < 0.01, and p < 0.001, respectively, compared with the control.

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Int. J. Biol. Sci. 2023, Vol. 19 2658

cancer type worldwide. In the USA, they accounted PS-targeting cytotoxic prodrug (BPRDP056). The
for 66,000 cases and 15,000 deaths in 2022. Globally, tumor-bearing mice were treated via intratumoral
they account for >660,000 new cases and 325,000 administration, as this route reportedly increases the
deaths annually. In addition to being life threatening, antitumor effect of CpG-ODN, and cancer therapies
head and neck cancers severely impact the quality of with intratumoral injection of CpG-ODN are
life of patients and their families [1-3]. Therefore, currently being intensively investigated in clinical
therapeutic agents and strategies are continuously trials. Furthermore, the development of image-guided
developed. Herein, we utilized an NHRI-NH1 cell injection techniques has rendered intratumoral
line–based syngeneic orthotopic head and neck cancer injection in different organs feasible [41-44]. We
animal model to investigate the efficacy and observed a cooperative antitumor effect between the
functional mechanism of a combinational therapy CpG-2722 and BPRDP056.
involving TLR9 activator (CpG-2722) and

Figure 8. CpG-2722 treatment increases PS exposure on cancer cells through induction of cytokines from immune cells. (A) Mouse splenocytes were
stimulated with/without 5 μg/ml of CpG-2722 for 48 h, and collected the condition medium (C.M.). NHRI-HN1 cells were treatment with/without 5 μg/ml of CpG-2722 or
with/without medium containing 25% C.M. for 24 h as illustrated in the top panel. Phosphatidylserine (PS) exposure on the NHRI-HN1 cells were stained by annexin V conjugated
APC antibody, and acquired on a FACS Canto II and analyzed using FlowJo software. Bottom left panel: a representative set of histograms. Bottom right panel: Data represent
the mean ± SEM (n = 3). (B) Cytokine levels of TNF-α, IFN-γ, and Trail in tumors from the experiment in Figure 5 were measured by ELISA. Data represent the mean ± SEM
(n = 5). (C) NHRI-HN1 cells were treated with 100 ng/ml of TNF-α, IFN-γ, or Trail for 24 h. Levels of PS exposure were stained by annexin V conjugated APC antibody and
acquired on a FACS Canto II. Left panel: a representative set of histograms. Right panel: Data represent the mean ± SEM (n = 3). (D) Mouse splenocytes were stimulated
with/without 5 μg/ml of CpG-2722 for 48 h, and the C.M. was collected. NHRI-HN1 cells were incubated with medium containing 25% of the C.M. in the presence of 1 μg/ml of
neutralizing antibody to TNF-α, IFN-γ, Trail or their combination as indicated for 24 h. Phosphatidylserine (PS) exposure on the NHRI-HN1 cells were stained by annexin V
conjugated APC antibody, and acquired on a FACS Canto II and analyzed using FlowJo software. Bottom left panel: a representative set of histograms. Bottom right panel: Data
represent the mean ± SEM (n = 3). *, **, and *** represent statistically significant differences p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the control or as
indicated.

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Int. J. Biol. Sci. 2023, Vol. 19 2659

Figure 9. Cooperative antitumor effects of CpG-2722 and BPRDP056. In the tumors, TLR9 activation by CpG-2722 induces immune responses, including cytokine
production and immune cell accumulation to increase T cell–mediated tumor killing. Conversely, BPRDP056 directly exhibits a cytotoxic effect on cancer cells. Additionally, TLR9
activation leads to increased phosphatidylserine (PS) exposure in cancer cells through inducing the production of TNF-α, IFN-γ, and Trail. This exposure attracts more
BPRDP056 to the tumor site for cancer cell killing. Killed cells further expose PS in tumor for BPRDP056 targeting. Antigens released from the dead cells can be taken up by
antigen-presenting cells, thereby enhancing the T cell–mediated tumor-killing effect promoted by CpG-2722. Thus, the actions of CpG-2722 and BPRDP056 form a positive
feed-forward antitumor effect, and contribute to the cooperative antitumor effect of CpG-2722 and BPRDP056.

NHRI-NH1 is one of the few cell lines available with this, we observed the activity of CpG-2722 in the
for establishing a syngeneic head and neck cancer induction of DC maturation. Additionally, CpG-2722
animal model. Others include the TC-1, MOC1/2, and induced the expression of various inflammatory
4MOSC cell lines. Nevertheless, TC-1 was derived cytokines in splenocytes. The type I IFN produced
from primary lung cells via immortalization with the plays a crucial role in linking innate immune
retroviral transduction of HPV16 E6/E740, and the responses to T cell–mediated antitumor response [48,
MOC1/2 cell lines were generated from gene- 49]. IL-12 and IFN-γ are two cytokines critical for
deficient mice [45, 46]. 4MOSC is a 4-nitroquinoline antitumor responses. IL-12 is produced chiefly by
1-oxide (4NQO)-induced murine oral squamous cell innate immune cells, including natural killer cells,
line [47]. Conversely, NHRI-NH1 cells are generated DCs, monocytes, and macrophages, and play a
by enriching the stemness of M11-1-2 HNSCC cells, significant role in inducing IFN-γ production from T
which were immortalized from 4-NQO/arecoline- cells. IFN-γ is a signature cytokine for the Th1 immune
induced tumors in C57BL/6J mice. The M11-1-2 cells response. It regulates T-cell differentiation and
can cause tumor growth in immune-deficient mice but activation [34, 50]. Further, CpG-2722 induces
cannot develop tumors in immune-competent mice immune cell accumulation, including that of pDCs,
[33]. As NHRI-HN1 cells are stemness-enriched cDC1, M1 macrophages, and CD8 T cells, and
cancer cells, they are probably more malignant and activates cytokine production, including that of
resistant to BPRDP056 than M11-1-2 cells (Figure 2). TNF-α, IL-12, and IFN-γ, in tumors. These processes
The NHRI-HN1 cells showed gene expression and create a tumor microenvironment favorable for
signaling profiles similar to those of human oral SCC antitumor immune responses.
tissue [33]. Therefore, animal cancer models generated The antitumor activity of BPRDP056 depends
using this cell line are reliable for studying HNSCC. mainly on its cytotoxicity. BPRDP056 is a prodrug of
The cooperative antitumor effect of CpG-2722 SN38, a topoisomerase I inhibitor [27, 28]. Topoiso-
and BPRDP056 can arise from their distinct and merase I plays a crucial role in DNA replication and
complementary antitumor functions. CpG-2722 transcription and is essential for cell proliferation.
induced antitumor immune responses, and Therefore, the cytotoxicity of topoisomerase I
BPRDP056 directly exerted cytotoxicity toward cancer inhibitors mainly arises from their antiproliferative
cells. CpG-ODNs can trigger TLR9-mediated innate effects, and these inhibitors are antitumor drug
immune responses, such as inducing the expression of candidates because cancer cells have a proliferation
costimulatory molecules and cytokine production in rate higher than that of normal cells [51, 52]. A
antigen-presenting cells. These responses facilitate prodrug of SN38, irinotecan (also known as CPT-11),
Th1 immune responses, thereby expanding tumor- was approved for the treatment of non-Hodgkin’s
specific T cells for tumor-cell killing [14-17]. In line lymphoma, cervical cancer, small cell lung cancer, and

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Int. J. Biol. Sci. 2023, Vol. 19 2660

colon cancer. Nevertheless, irinotecan causes adverse cancer cells would facilitate BPRDP056 targeting in
effects at a higher dose, thereby limiting its these cells. Additionally, both BPRDP056 and its
therapeutic usage [53]. Therefore, BPRDP056 was payload SN38 reportedly induce cancer cell apoptosis
designed using a linker to connect Zn-DAP with the [Figure 7B, 28, 59, 60], leading to the accumulation of
cytotoxic SN38 for this drug candidate to have a more PS in the tumor following BPRDP056 targeting.
PS-dependent tumor-targeting mechanism. This Thefore, the actions between CpG-2722 and
design reduces the cytotoxicity of SN38 by prohibiting BPRDP056 may cooperatively induce the PS exposure
the premature release of SN38 before reaching the as that observed in Figure 7B to increase PS targeting
tumor [28]. involved in tumor killing.
BPRDP056 induced immune responses in Thus, this study found that combining TLR9
splenocytes and tumors, although the responses were activator and PS-binding cytotoxic prodrug, CpG-
milder than those induced by CpG-2722. The mecha- 2722 and BPRDP056, respectively, results in a
nism underlying the immunogenicity of BPRDP056 cooperative antitumor effect. CpG-2722 triggers
remains unclear. Topoisomerase I inhibitors, immune responses and PS exposure in tumors, while
including topotecan, camptothecin, irinotecan, and BPRDP056 releases SN38 to kill cancer cells. PS
SN38, reportedly induce immunogenic cell death, exposure in dying cancer cells attracts more
eliciting an immune response in tumors. In this BPRDP056 to the tumor site, and tumor antigens
process, exposure to damage-associated molecular released from the dead cells are taken up by
patterns (DAMPs), such as high-mobility group box 1, antigen-presenting cells, thereby enhancing the
ATP, and calreticulin, from the cells killed by these CpG-2722-promoted T cell–mediated tumor-killing
inhibitors promotes the recruitment of immune cells effect. Together, CpG-2722 and BPRDP056 form a
and production of cytokines in the tumor [54, 55]. As feed-forward mechanism that helps kill cancer cells
BPRDP056 is a prodrug of SN38, it probably follows a more effectively. This study also suggests a novel
similar mechanism to activate immune responses strategy of utilizing the PS-inducing function of TLR9
through SN38 release. The immunogenic cell death agonists for combinational treatments. Future
caused by BPRDP056 may be enhanced by decreased research can explore the potential of integrating this
clearance of apoptotic cells due to the binding of its concept to facilitate various cancer diagnoses and
Zn-DAP part to the apoptotic cells. Generally, PS treatment with PS-targeting drugs. This approach
exposure facilitates apoptotic cell clearance via could also enable the development of novel
phagocytes. If apoptotic cells are not efficiently PS-targeting drugs in a more promising direction.
cleared, they may undergo necrosis and release
inflammatory DAMPs. In this regard, previous Abbreviations
studies have reported that blocking PS by annexin V PS: phosphatidylserine; HNSCC: Head and neck
inhibits apoptotic cancer cell clearance, initiates squamous cell carcinoma; TLR: Toll-like receptors;
necrosis, and renders apoptotic cells immunogenic, NF-κB: nuclear factor-κB; IRF: interferon regulatory
resulting in increased antitumor response [56-58]. factor; IFN: interferon; MyD88: myeloid differenti-
Therefore, although the immunostimulatory effect of ation primary response 88; IRAK: interleukin-1
BPRDP056 is not as strong as that of CpG-2722, this receptor-associated kinase; TRAF6: tumor necrosis
effect may still contribute to the antitumor effect of factor-associated factor 6; TAK1: transforming growth
this prodrug. factor-β-associated kinase 1; TNF-α: tumor necrosis
Cancer cells expose PS on their surface; factor (TNF)-α; IL: interleukin; DC: dendritic cell;
therefore, PS is a marker for tumor diagnosis, CpG-ODNs: CpG-oligodeoxynucleotides; Zn-DPA:
imaging, and targeting for drug treatment [22, 23]. Zinc (II)-dipicolylamine; Trail: TNF-related apoptosis-
Herein, we discovered a novel function and inducing ligand; ELISA: enzyme-linked immunosor-
mechanism of TLR9 activation, which increased PS bent assay; SCC: squamous cell carcinoma; BMDC:
exposure in cancer cells. CpG-2722 induced the bone marrow–derived dendritic cell; Flt3-L: FMS-like
production of cytokines, including TNF-α, Trail, and tyrosine kinase 3 ligand; GM-CSF: granulocyte-
IFN-γ, from immune cells, which in turn activated PS macrophage colony-stimulating factor; TrailR2: Trail
exposure on cancer cells. In addition to its ability to receptor 2.
induce antitumor immune responses, PS exposure
induction in tumors via CpG-2722 likely plays a Supplementary Material
pivotal role in the cooperative antitumor effects of Supplementary figure and table.
CpG-2722 and BPRDP056. As BPRDP056 is designed https://www.ijbs.com/v19p2648s1.pdf
to preferentially bind to PS on cancer cells, the
function of CpG-2722 in increasing PS exposure in

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Int. J. Biol. Sci. 2023, Vol. 19 2661

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