Abstract
Background
Incomplete radiofrequency ablation (iRFA) in hepatocellular carcinoma
(HCC) often leads to local recurrence and distant metastasis of the
residual tumor. This is closely linked to the development of a tumor
immunosuppressive environment (TIME). In this study, underlying
mechanisms and potential therapeutic targets involved in the formation
of TIME in residual tumors following iRFA were explored. Then,
TAK-981-loaded nanocomposite hydrogel was constructed, and its
therapeutic effects on residual tumors were investigated.
Results
This study reveals that the upregulation of small ubiquitin-like
modifier 2 (Sumo2) and activated SUMOylation is intricately tied to
immunosuppression in residual tumors post-iRFA. Both knockdown of Sumo2
and inhibiting SUMOylation with TAK-981 activate IFN-1 signaling in HCC
cells, thereby promoting dendritic cell maturation. Herein, we propose
an injectable PDLLA-PEG-PDLLA (PLEL) nanocomposite hydrogel which
incorporates self-assembled TAK-981 and BSA nanoparticles for
complementary localized treatment of residual tumor after iRFA. The
sustained release of TAK-981 from this hydrogel curbs the expansion of
residual tumors and notably stimulates the dendritic cell and cytotoxic
lymphocyte-mediated antitumor immune response in residual tumors while
maintaining biosafety. Furthermore, the treatment with TAK-981
nanocomposite hydrogel resulted in a widespread elevation in PD-L1
levels. Combining TAK-981 nanocomposite hydrogel with PD-L1 blockade
therapy synergistically eradicates residual tumors and suppresses
distant tumors.
Conclusions
These findings underscore the potential of the TAK-981-based strategy
as an effective therapy to enhance RFA therapy for HCC.
Graphic Abstract
[44]graphic file with name 12951_2024_2579_Figa_HTML.jpg
Supplementary Information
The online version contains supplementary material available at
10.1186/s12951-024-02579-1.
Keywords: Hepatocellular carcinoma, Radiofrequency ablation, Small
ubiquitin-like modifier 2, TAK-981, Nanocomposite hydrogel
Introduction
Radiofrequency ablation (RFA) stands as a primary treatment for
early-stage hepatocellular carcinoma (HCC), valued for its exceptional
efficiency, minimal invasiveness, lower complication morbidity, and
abbreviated hospital stays [[45]1, [46]2]. However, RFA results are
compromised by high rates of local relapse, which can reach up to 60%
[[47]3]. This recurrence is frequently attributed to the residual
tumors that persist after incomplete RFA (iRFA) [[48]4]. Additionally,
the sublethal heat stimulation induced by iRFA has been observed to
expedite the progression of these residual tumors. Current research
indicates that this sublethal heat stimulation fosters a suppressive
tumor immune microenvironment (TIME) and facilitates the survival and
progression of the residual tumors [[49]5–[50]7]. However, the exact
mechanisms driving these phenomena are indeed complex and still being
explored.
The extensive tumor necrosis and profusion of cellular debris resulting
from RFA might be expected to serve as tumor-specific antigens,
potentially triggering an adaptive antitumor immune response [[51]8].
However, clinical studies indicate that the immune response often falls
short of completely eradicating or managing residual tumors in iRFA
scenarios [[52]9]. This insufficiency implies that antigen
presentation, a crucial component of adaptive antitumor immunity, is
impeded. In this process, dendritic cells (DCs) play a significant role
in the antigen presentation process, promoting the infiltration and
activation of cytotoxic T lymphocytes (CTLs) [[53]10]. They specialize
in antigen capture and processing, which are then presented to
immunocompetent T cells. The Type I Interferon (IFN-1) signal notably
impacts the activation, maturation, migration, and survival of DCs, and
concurrently boosts the activity of CTLs [[54]11]. However, the
IFN-1/DCs/CTLs signal is frequently suppressed within the TIME due to
various mechanisms [[55]12]. Hence, inhibition of the IFN-1 pathway
might contribute to the imbalanced immune response post-iRFA, though
further research is imperative to understand this intricate mechanism
comprehensively.
In this study, we revealed heightened small ubiquitin-like modifier
protein 2 (Sumo2)-mediated SUMOylation activity in residual tumors
after iRFA of HCC. SUMOylation is a post-translational modification
process involving the attachment of SUMO proteins to substrates, is
linked with increased tumorigenic potential and poorer clinical
outcomes [[56]13]. It has been reported to interfere with the IFN-1
signaling by modifying multiple targets. Inhibition of SUMOylation has
shown promise in enhancing DC maturation and activating anti-tumor
immune responses by stimulating IFN-1 production in diverse tumors
during preclinical studies. Multiple ongoing or completed phase 1/2
clinical trials have evaluated the efficacy of small molecule
SUMOylation inhibitors as potential immunotherapy [[57]14, [58]15].
While no direct association exists between targeting SUMOylation and
residual tumors after iRFA in existing studies, this novel approach
shows promise for eliminating HCC residual tumors post-iRFA in future
clinical applications. TAK-981, a novel small molecule inhibitor
specifically designed to target the SUMOylation pathway with remarkable
selectivity and potency [[59]16, [60]17], is expected to be a
compelling candidate for treating residual tumors after iRFA. However,
SUMOylation is essential for preserving cell survival and
functionality, and the systemic administration of SUMOylation
inhibitors may result in potential toxicity [[61]18–[62]20]. TAK-981
has been primarily delivered via intratumoral injections in the case of
solid tumors [[63]21, [64]22]. However, intratumoral injections come
with a set of limitations, including a restricted duration of drug
retention and the occurrence of local reactions [[65]23]. To achieve
optimal efficacy, there is a risk of repeat administration and
overexposure of TAK-981 due to the restricted duration of drug
retention. This scenario may result in potential systemic and local
toxicity stemming from overdosage and off-target delivery[[66]23].
Therefore, it is of significant clinical importance to develop a novel
drug formulation that can locally and sustainably release small
molecule inhibitors, thereby enhancing anti-tumor efficacy while
minimizing side effects.
In-situ forming hydrogels are viewed as potential carriers to address
these challenges [[67]24]. The biodegradable
poly(d,L-lactide)-poly(ethylene glycol)-poly(d,L-lactide)
(PDLLA-PEG-PDLLA; PLEL) triblock copolymer gel exhibits a flowable sol
state at room temperature, which facilitates drug loading and
injection. It undergoes phase transition to form a drug-loaded gel
depot for sustained drug release upon in vivo administration. Moreover,
PLEL hydrogels effectively prolong drug retention time [[68]25,
[69]26]. Nevertheless, the poor solubility of small-molecule drugs
makes it difficult to achieve a uniform dispersion within hydrogels.
Integrating nanomaterials into hydrogels achieves uniform drug
dispersion and avoids drug burst release [[70]27]. Bovine Serum Albumin
(BSA) is widely used in drug delivery systems due to its beneficial
properties, including easy drug loading, good water solubility, and
non-toxicity. Its amphiphilic nature allows it to form nanoparticles
with hydrophobic drugs and remain soluble in aqueous solutions
[[71]28]. In our study, BSA and TAK-981 first self-assembled to form
nanoparticles, subsequently encapsulated within PLEL to obtain
BT-NPs@PLEL nanocomposite hydrogels. The BT-NPs@PLEL was injected into
residual tumor sites following iRFA, and the results indicate that
BT-NPs@PLEL fostered the maturation of DCs, bolstered the infiltration
and activation of CTLs, and effectively suppressed the residual tumors.
Notably, extensive PD-L1 upregulation in residual tumors was observed
following BT-NPs@PLEL treatment. The combination therapy of BT-NPs@PLEL
and PD-L1 blockade effectively inhibits residual tumors and impedes the
progression of distant tumors after iRFA. These findings underscore the
potential of a TAK-981-based strategy in activating anti-tumor
immunity, eradicating residual tumors, and optimizing RFA treatment.
Result s
Upregulation of Sumo2 and activation of SUMOylation in residual tumors after
iRFA
Research has consistently demonstrated that iRFA is associated with a
rapid progression of residual tumors, and immunosuppression potentially
plays a significant role. However, the source of immunosuppression is
still unclear. To investigate this, we developed iRFA mouse models with
Hepa1-6 cells, as shown in Figure S1A. Following a 21-day observation
period after the iRFA treatment, the tumor growth curve indicates that
the tumor volume in the iRFA-treated group escalated more rapidly
compared to the untreated group (Figure S1B-C). The tumors were
dissected and weighed, revealing a higher weight in the iRFA-treated
group (Figure S1D). Subsequently, the TIME of the residual tumors was
investigated. On day 21 following the iRFA treatment, tumor tissue was
collected for flow cytometry analysis. The results indicate that while
the proportions of total immune cells and DCs were not different across
the two groups, there was a marked decrease in the infiltration of
mature DCs within the iRFA-treated group (Figure S2A-C). DCs are
essential for antigen presentation and stimulating anti-tumor immunity.
Correspondingly, the infiltration of CD8^+ T cells was also decreased
in the iRFA-treated group (Figure S2D). These results suggest that iRFA
may impede antigen presentation in residual tumors.
Suppression of antigen presentation could disrupt the anti-tumor immune
response, potentially enabling residual tumors to persist and progress.
To investigate the underlying mechanism, High-throughput transcriptome
sequencing (RNA-Seq) was conducted on iRFA-derived residual tumor
tissue. The pathway enrichment analysis shows that the cytosolic
DNA-sensing pathway is a top enriched term (Fig. [72]1A). The cytosolic
DNA-sensing pathway has been empirically linked to antigen
presentation-dependent anti-tumor immunity, predominantly via the
synthesis of IFN-1[[73]29]. Consequently, we examined the downstream
interferon-stimulated genes (ISGs) of IFN-1 signaling and discovered
that the majority of ISGs were expressed at lower levels in the
iRFA-treated group (Figure S3). The IFN-1 signal is crucial for
promoting antigen presentation and DC activation. Suppression of IFN-1
signaling was considered responsible for inhibiting antigen
presentation in residual tumors. Moreover, the differentially expressed
genes (DEGs) analysis revealed that Sumo2 was significantly
up-regulated in residual tumor following iRFA in HCC (Fig. [74]1B, C,
Figure S4). The expression levels of the SUMO pathway’s key
components-Sumo1, Sumo3, Uba2, Sae1, and Ube2i-showed no significant
difference between the iRFA-treated group and the untreated group
(Fig. [75]1C). These findings are consistent with another iRFA-related
dataset ([76]GSE138224) in the GEO sequencing database (Figure S5)
[[77]5]. According to the TCGA database, lower SUMO2 expression
correlates with longer overall survival in HCC patients (Figure S6).
The upregulation of SUMO2 protein was also observed in the residual
tumor tissue with IHC staining (Fig. [78]1D, E), and an increased level
of conjugated-SUMO2 was observed in the Western blot test
(Fig. [79]1F). In line with these findings, in vitro simulation of iRFA
using sublethal heat stimulation resulted in upregulated Sumo2
expression and increased conjugated-SUMO2 in mouse Hepa1-6 HCC cells
(Fig. [80]1G, I). A similar trend of upregulated SUMO2 expression and
increased conjugated-SUMO2 was noted in human-derived HCC cells (HepG2
and Hep3B) following exposure to sublethal heat stress (Fig. [81]1H, J,
Figure S7). SUMOylation has been reported to down-regulate the IFN-1
signaling [[82]30]. ELISA testing of IFN-1 in tumor tissue also
revealed impaired IFN-1 secretion following iRFA treatment (Figure S8).
These results indicate that the sublethal heat stimulation induced by
iRFA may inhibit the IFN-1 signal in HCC by activating SUMO2-mediated
SUMOylation, potentially facilitating the development of the
immunosuppressive microenvironment.
Fig. 1.
[83]Fig. 1
[84]Open in a new tab
Upregulation of Sumo2 and activation of SUMOylation in residual tumors
after iRFA. A Significant enrichment of the DEGs of iRFA and untreated
group in KEEG terms (top 20). B Volcano plot of DEGs in RNA-seq
dataset. C Differences in expressions critical regulators in
SUMOylation pathway between iRFA and the untreated group (n = 3). D The
IHC staining of SUMO2 in tumor tissues, scale bar = 20 μm. E The IHC
staining score of SUMO2 (n = 40). F Western blot analysis of
conjugated-SUMO2 in tumor tissues. G The RT-qPCR analysis of the Sumo2
expression in heated Hepa1-6 cells (n=3). H The RT-qPCR analysis of the
SUMO2 expression in heated HepG2 cells (n=3). I Western blot analysis
of conjugated-SUMO2 in heated Hepa1-6 cells. J Western blot analysis of
conjugated-SUMO2 in heated HepG2 cells. ns, not significant, *p < 0.05,
**p < 0.01, ***p < 0.001
Inhibiting the SUMO2-mediated SUMOylation activates the IFN-1 signal in iRFA
HCC cells and promotes BMDCs maturation in vitro
SUMOylation has previously been identified as suppressing the IFN-1
signal by modifying several targets within the cell [[85]30]. In this
study, we further investigated if knocking down Sumo2 and inhibiting
SUMOylation could stimulate the IFN-1 signal in HCC cells. To this end,
we genetically engineered Hepa1-6 cells to interfere with Sumo2 using
short hairpin RNA (shRNA) (Fig. [86]2A). Following the knockdown of
Sumo2, we observed a decrease in conjugated-SUMO2 in Hepa1-6 cells
(Fig. [87]2B), while the cell proliferation rate was not significantly
affected (Fig. [88]2C). Meanwhile, significantly higher Ifnb1
expression and IFN-β secretion were observed in the Sumo2-knockdown
heated HCC cells compared to the control group (Fig. [89]2D, E).
Building on the understanding that Signal Transducer and Activator of
Transcription 1 (STAT1) plays a pivotal role in the maturation and
differentiation of DCs, we observed activation of STAT1 in Bone
Marrow-Derived Dendritic Cells (BMDCs). This occurred following their
co-culture with Hepa1-6 cells, in which Sumo2 had been knocked down and
subsequently heated (Fig. [90]2F). Furthermore, ISGs, exemplified by
Isg15, were significantly upregulated in BMDCs following the same
co-culture process (Fig. [91]2G, Figure S9). These results underscore
the response of BMDCs to signals from interferons. Then the heated
Hepa1-6 cells were cultured with an increased concentration of TAK-981,
and we observed a gradual reduction in the conjugated-SUMO2.
Concurrently, there was an elevation in the expression of Ifnb1 and
secretion of IFN-β in these heated Hepa1-6 cells (Fig. [92]2H, I, J).
Similarly, STAT1 was activated (Fig. [93]2K), and ISGs were
significantly up-regulated in BMDCs when co-culturing with heated
Hepa1-6 cells treated with TAK-981 sequentially (Fig. [94]2L, Figure
S10). These findings provide strong evidence of a negative correlation
between SUMO2-mediated SUMOylation and the IFN-1 signaling pathway in
HCC cells. They also demonstrate that BMDCs could be affected by the
IFN-1 secreted by HCC cells when co-cultured together. Therefore, this
co-culture system of HCC cells and BMDCs was further utilized to
simulate the in vivo TIME of the iRFA tumors, and the flow cytometry
analysis showed that both knockdown Sumo2 and treating Hepa1-6 cells
with TAK-981 resulted in increased proportions of matured DCs. However,
when IFN-β was neutralized with an antibody, this “pro-maturation
effect” was reversed (Fig. [95]2M, N). These results emphasize the
potential of targeting SUMO2-mediated SUMOylation as a treatment
approach to enhance antigen presentation in residual tumors after iRFA.
Fig. 2.
[96]Fig. 2
[97]Open in a new tab
Knockdown of Sumo2 or inhibition of SUMOylation effectively activates
the IFN-1 pathway. A The RT-qPCR analysis of Sumo2 in Sumo2-knockdown
Hepa1-6 cells (n=3). B Western blot analysis of conjugated- SUMO2 in
Sumo2-knockdown Hepa1-6 cells. C The relative proliferation rate in
Sumo2-knockdown Hepa1-6 cells (n=3). D The RT-qPCR analysis of the
Ifnb1 gene in heated Sumo2-knockdown Hepa1-6 cells (n=3). E ELISA
analysis of the IFN-β in the medium supernatant of heated
Sumo2-knockdown Hepa1-6 cells (n=3). F Western blot analysis of
STAT1/pSTAT1 in BMDCs co-cultured with heated Hepa1-6 cells. G The
relative expression of Isg15 in BMDCs co-cultured with heated Hepa1-6
cells (n=3). H Western blot analysis of conjugated-SUMO2 in Hepa1-6
cells treated with TAK-981. I The RT-qPCR analysis of the Ifnb1 gene in
Hepa1-6 cells treated with TAK-981 (n=3). J ELISA analysis of the IFN-β
in the medium supernatant of Hepa1-6 cells treated with TAK-981 (n=3).
K Western blot analysis of STAT1/pSTAT1 in BMDCs co-cultured with
heated Hepa1-6 cells treated with TAK-981. L The relative expression of
Isg15 in BMDCs co-cultured with heated Hepa1-6 cells treated with
TAK-981 (n=3). M The representative flow cytometry plots and
statistical analysis of mature DCs rate after co-cultured with heated
Sumo2-knockdown Hepa1-6 cells (n=6). N The representative flow
cytometry plots and statistical analysis of mature DCs rate after
co-cultured with heated Hepa1-6 cells treated with TAK-981. ns, not
significant (n=6), *p < 0.05, **p < 0.01, ***p < 0.001
Preparation and characterization of BT-NPs@PLEL
To enhance the therapeutic efficacy of TAK-981 in treating residual
tumors post-iRFA, we engineered a nanocomposite thermosensitive
hydrogel known as BT-NPs@PLEL. The synthesized PLEL block compounds
were first analyzed by Fourier transform infrared spectroscopy (FTIR).
In the FTIR spectrum (Fig. [98]3A), a strong C = O stretching band
appeared at 1747 cm^−1, attributed to the ester carbonyl bond. The
absorption band at 1082 cm^−1 corresponds to the C–O–C asymmetric
stretching vibration of the ether group in PEG. The peak at 2872 cm^−1
and 1451 cm^−1 correspond to the –CH[2] asymmetric stretching vibration
of both PDLLA and PEG, consistent with previous reports [[99]31].
Meanwhile, the hydrodynamic size of triblock copolymer PLEL micelle
solution (1 wt%) at 25 °C was measured to be about 25 nm (Figure S11),
which remained nearly unchanged at room temperature over 24 h
(Fig. [100]3B). BSA nanoparticles carrying TAK-981 were synthesized via
the self-assembly method by hydrophilic and hydrophobic interactions.
The transmission electron microscopy (TEM) revealed the uniform
morphology of TAK-981@BSA, and the hydrodynamic size of TAK-981@BSA NPs
was measured to be approximately 128nm (Fig. [101]3C). Subsequently,
the TAK-981@BSA NPs were encapsulated into PLEL to construct the final
composite gel (BT-NPs@PLEL). In addition, the peak of BT-NPs@PLEL in
FTIR was consistent with PLEL (Fig. [102]3A), indicating that the
introduction of TAK-981@BSA NPs did not change the chemical structure
of PLEL. The Lower Critical Gelation Temperature (LCGT) of PLEL and
BT-NPs@PLEL hydrogels is influenced by molecular weight and polymer
concentration [[103]26]. As PLEL concentration increases, the LCGT
decreases (Figure S12). However, below 25 wt% PLEL, the LCGT of
BT-NPs@PLEL exceeds body temperature, which makes it challenging to
guarantee that the sol-gel transition occurs upon injection into the
body temperature. Higher PLEL concentrations slow degradation,
potentially causing inflammation [[104]32]. Therefore, we selected a 25
wt% concentration for the nanocomposite hydrogels. To further
strengthen the finding between embedded contents and gelling abilities,
the temperature of sol–gel transition of PLEL and BT-NPs@PLEL was
measured through the rheological behavior test. The sol–gel transition
is defined as the point where the storage modulus (G′) exceeds the loss
modulus (G′′). PLEL and BT-NPs@PLEL were present in the sol state at
room temperature (25 °C) and turned into the gel state at body
temperature (37° C) (Fig. [105]3D). Meanwhile, the macroscopic views of
the gelation process of PLEL and BT-NPs@PLEL (25 wt%) are shown in
Fig. [106]3E. Then, we examined the in vivo gelation and biodegradation
behavior of BT-NPs@PLEL (mixed with the blue dye to be visualized).
Gelation occurred swiftly following subcutaneous injection, most of
which degraded within 14 days after the injection (Fig. [107]3F). In
our subsequent experiments, we evaluated the effectiveness of drug
delivery systems in facilitating the slow release of the drug.
Remarkably, about 80% of the encapsulated TAK-981 could be released
sustainably over 14 days in a PBS buffer at 37° C (Figure S13).
Furthermore, free Cy5.5 (substitution for TAK-981) or Cy5.5@Gel
(substitution for BT-NPs@PLEL) was injected subcutaneously and
monitored by an in vivo fluorescence imaging system (PerkinElmer IVIS
Lumina III) at different time points post-injection. It was found that
the fluorescence signal from Cy5.5 rapidly diminished at the injection
site injected with free Cy5.5, demonstrating that free drugs could
diffuse quickly. In contrast, for the Cy5.5@Gel group, the fluorescence
signal was maintained at high levels within tumors for 14 days
(Fig. [108]3G, H), demonstrating the significantly prolonged retention
of drugs with the help of the nanocomposite hydrogel.
Fig. 3.
[109]Fig. 3
[110]Open in a new tab
Preparation and Characterization of BT-NPs@PLEL. A FTIR spectra
analysis of TAK-981, BSA, PLEL, and BT-NPs@PLEL. B The hydrodynamic
diameter and polymer dispersity index of PLEL-Sol (1 wt%) within 24 h.
C The hydrodynamic diameter and the TEM image of TAK-981@BSA
nanoparticles, scale bar = 200 nm. D The rheological behavior of PLEL
(25 wt%), BT-NPs@PLEL (25 wt%) in dependent of temperature. G′, storage
modulus; G″, loss modulus. E Photographs showing the macroscopic
thermo-sensitive sol–gel translation of PLEL and BT-NPs@PLEL (25 wt%).
F In vivo gelation and degradation behavior of BT-NPs@PLEL (25 wt%) at
different time points. G, H IVIS images and statistical analysis of
fluorescence signal recorded at different times after injection of
Cy5.5 and Cy5.5@Gel (n=3). ***p < 0.001
BT-NPs@PLEL activates anti-tumor immunity and suppresses residual tumors
after iRFA
Driven by the promising in vitro results regarding the maturation of
DCs through SUMOylation targeting and the prolonged retention of
TAK-981 in BT-NPs@PLEL, the anti-tumor efficacy and the capacity to
activate adaptive anti-tumor immunity were further investigated in iRFA
mouse models. This investigation was conducted in accordance with the
outlined therapeutic schedule (Fig. [111]4A). Hepa1-6-bearing mice were
intratumorally injected with Saline, PLEL, TAK-981, and BT-NPs@PLEL
after iRFA treatment. The tumor growth curve revealed that TAK-981
somewhat curtailed tumor growth when compared to the saline group, but
no notable tumor suppression was seen in the PLEL groups. On the other
hand, the residual tumor growth of BT-NPs@PLEL group was significantly
inhibited (Fig. [112]4B). Additionally, an evaluation of the anti-tumor
efficacy of BT-NPs@PLEL in residual tumors, excised from mice on day
21, revealed a significant reduction in both tumor volume and weight in
the group treated with BT-NPs@PLEL, compared to other groups (Figure
S14, Fig. [113]4C). In line with these results, the Kaplan–Meier
survival curve indicates that the group treated with BT-NPs@PLEL
exhibited a more extended survival period than the other groups
(Fig. [114]4D). Crucially, no significant variations were observed in
the body weight changes of the treated mice during the experiment
(Fig. [115]4E). Besides, the serum biochemistry assay, complete blood
panel test, and H&E staining of organ sections were performed, which
further indicated no apparent systemic toxicity induced by BT-NPs@PLEL
(Figure S15, Fig. [116]4F). These findings collectively suggest that
BT-NPs@PLEL significantly improved the effectiveness of TAK-981 in
treating iRFA residual tumor while ensuring safety. We also performed
SUMOylation analysis on residual tumor tissues after treatment. Given
that SUMOylation primarily occurs in the nucleus [[117]17], we carried
out immunohistochemical staining on the post-treatment residual tumor
tissue. Our findings showed a notable decrease in SUMO2 staining within
the nucleus of the residual cancer tissues after treatment
(Fig. [118]4G). The results confirmed that BT-NPs@PLEL effectively
blocked SUMOylation in residual tumor after iRFA.
Fig. 4.
[119]Fig. 4
[120]Open in a new tab
Inhibition of residual tumor after iRFA in vivo and blocking
SUMOylation of the BT-NPs@PLEL. A Schematic representation of treatment
of residual tumor after iRFA in C57BL/6 mice. B Tumor volume of
residual tumors in different groups (n=6). C The weight of residual
tumors post-iRFA on day 21 after different treatments (n=6). D Survival
analysis of experimental mice in different groups (n=6). E The body
weight changes of mice during treatment. F Images of H&E staining of
tissue sections in essential organs after treatment with BT-NPs@PLEL on
day 1, day 10, and day 30, scale bar: 50 µm. G Immunohistochemical
analysis of SUMO2 in nucleus of residual tumor tissue after different
treatments, scale bar: 10 µm. ns, not significant, *p < 0.05,
**p < 0.01, ***p < 0.001
To shed light on the primary immune mechanisms contributing to the
anti-tumor therapeutic efficacy observed in this study, we assessed
adaptive anti-tumor immunity following various treatments. As expected,
the proportions of mature DCs in the BT-NPs@PLEL group were
significantly higher than in other groups (Fig. [121]5A).
Correspondingly, there was a significant increase in the proportions of
CD8^+ T cells and their expression of Granzyme-B (Fig. [122]5B, C).
Immunohistochemical staining also confirmed a substantial increase in
CD8^+ T cells infiltration following BT-NPs@PLEL treatment
(Fig. [123]5D). TAK-981 treatment alone already showed considerable
effectiveness in enhancing the infiltration of mature DCs and CD8^ + T
cells, showing a greater effect than either the saline or PLEL groups.
The BT-NPs@PLEL displayed an improved efficacy. Additionally, ELISA
detection indicated that the group administered with BT-NPs@PLEL saw an
increase in pro-inflammatory cytokines, IFN-γ and TNF-α, within the
residual tumors. These were significantly higher than in the other
groups (Fig. [124]5E). Nevertheless, it’s worth noting that earlier
studies have shown a link between exposure to TAK-981 and a broad
increase in PD-L1 within the TIME [[125]21]. Consequently, we conducted
an additional evaluation of PD-L1 expression and noted an increase in
PD-L1 expression of the residual tumors following BT-NPs@PLEL treatment
(Fig. [126]5F). This sets the stage for a combination of anti-PD-L1
therapy and BT-NPs@PLEL.
Fig. 5.
[127]Fig. 5
[128]Open in a new tab
BT-NPs@PLEL activates anti-tumor immunity of residual tumor after iRFA.
A Representative flow cytometry plots and proportions of mature DCs on
day 7 (n = 6). B Representative flow cytometry plots and proportions of
CD8^+ T cells on day 7 (n = 6). C Representative flow cytometry plots
and proportions of Granzyme B^+ on day 7 (n = 6). D IHC staining of CD8
in residual tumor tissue, scale bar: 20 µm. E ELISA assay to detect the
amount of IFN-γ and TNF-α in tumor tissue (n = 6). F The flow cytometry
validation of PD-L1 overlay histogram and the mean fluorescence of the
cells of tumor tissue after different treatments (n = 6). ns, not
significant, *p < 0.05, **p < 0.01, ***p < 0.001
Combining BT-NPs@PLEL with anti-PD-L1 treatment enhanced anti-tumor effects
in residual tumors after iRFA
The efficacy of the combination of BT-NPs@PLEL and anti-PD-L1 treatment
was further investigated. The experimental design follows the
illustrated therapeutic schedule (Fig. [129]6A). Significantly stronger
inhibition of tumor progression was observed in the combined group
compared with that of the BT-NPs@PLEL and αPD-L1-treated group, as
indicated by bioluminescence intensity measurements (Fig. [130]6B, C).
Consistent with these results was indicated by the tumor growth curve
(Fig. [131]6D). After a 21 day treatment, the combination therapy
demonstrated superior effectiveness in treating residual tumors after
iRFA. Remarkably, 1 out of 6 tumors was completely eradicated. In
contrast, treatments with either BT-NPs@PLEL or anti-PD-L1 alone showed
comparatively moderate effectiveness, with no instances of complete
tumor eradication (Fig. [132]6E, Figure S16). This outcome was further
corroborated by the survival analysis, which showed that mice subjected
to the combination therapy experienced extended survival durations in
the context of iRFA (Fig. [133]6F). Flow cytometry analysis of residual
tumors was conducted on day 7 after different treatments. The results
revealed that the group treated with the combination therapy had a
significantly higher proportion of CD8^+ T cells (Fig. [134]6G).
Furthermore, these CD8^+ T cells exhibited a higher expression of
Granzyme B than those treated with BT-NPs@PLEL or anti-PD-L1 alone
(Fig. [135]6H). Consistently, the residual tumors from the combination
treatment group exhibited higher levels of pro-inflammatory cytokines,
including IFN-γ and TNF-α (Figure S17). These findings suggest that
BT-NPs@PLEL treatment and anti-PD-L1 therapy complement each other,
leading to a combination therapy that enhances anti-tumor immunity.
Fig. 6.
[136]Fig. 6
[137]Open in a new tab
Combining BT-NPs@PLEL with anti-PD-L1 treatment for inhibition in
residual tumors after iRFA. A Schematic representation of treatment of
residual tumor after iRFA in C57/BL6 mice. B Bioluminescence images of
mice with residual tumors after iRFA after different treatments on days
0, 10, and 20 (n = 3). C Bioluminescence signals of mice in each group
on day 0, 10, and 20 (n = 3). D Tumor volume of residual tumors after
iRFA in different groups(n = 6). E The weight of residual tumors on day
21 after different treatments (n = 6). F Survival analysis of
experimental mice in different groups (n = 6). G Representative Flow
cytometry plots and proportions of CD8^+ T cells on day 7 (n = 6). H
Representative flow cytometry plots and proportions of Granzyme B^+
cells on day 7 (n = 6). ns, not significant *p < 0.05, **p < 0.01,
***p < 0.001
Combining BT-NPs@PLEL with anti-PD-L1 treatment enhanced effectiveness in
inhibiting distant tumors
Occasionally, residual tumors after iRFA may coexist with distant
metastasis or concurrent tumors [[138]33]. Biomaterials-assisted local
treatments have the potential to stimulate systemic tumor-specific
immunological responses. These responses can be further enhanced when
combined with Immune Checkpoint Blockade (ICB) therapy, which has the
capability to fight metastatic cancer cells [[139]34]. Consequently,
BT-NPs@PLEL holds promise for augmenting the anti-tumor effectiveness
of αPD-L1 therapy and suppressing distant tumors. Our study employed a
bilateral tumor model to assess the systemic anti-tumor response, as
depicted in the therapeutic schedule (Fig. [140]7A). As indicated by
the bioluminescence imaging, the combination therapy notably suppressed
the growth of distant tumors, while the efficacy of BT-NPs@PLEL and
anti-PD-L1 treatments was comparatively moderate (Fig. [141]7B, C).
Consistent with these results was indicated by the tumor growth curve
(Fig. [142]7D). Following a 21 day treatment period, the combination
therapy showed superior effectiveness in treating the distant tumor, as
evidenced by a reduction in both tumor volume and weight (Fig. [143]7E,
Figure S18).This result was further validated by the survival analysis,
demonstrating that mice treated with the combination therapy had
prolonged survival periods in scenarios involving iRFA with the
presence of a distant tumor (Fig. [144]7F). To investigate whether the
BT-NPs@PLEL and anti-PD-L1 combination suppressed the distant tumors by
triggering a systemic anti-tumor response, we first evaluated the
immune response in the spleens. Spleens from mice in the iRFA models
were gathered on day7 following various treatments for flow cytometry
analysis. We observed that the proportions and cytolytic function of
CD8^+ T cells were significantly increased in the combination treatment
group (Figure S19), suggesting an activated adaptive systemic immune
response. At the same time, the flow cytometry analysis of distant
tumors showed that the proportions and cytolytic function of CD8^+ T
cells were significantly increased in mice receiving the combination
treatment, while a weaker trend was observed in the BT-NPs@PLEL and
anti-PD-L1 group, respectively (Fig. [145]7 G, H). Consistently, higher
levels of cytokines (TNF-α, IFN-γ) were detected in the combination
treatment group (Figure S20). These results indicate that the
synergistic use of BT-NPs@PLEL and αPD-L1 can stimulate a heightened
systemic anti-tumor immune response in residual tumor after iRFA.
Fig. 7.
[146]Fig. 7
[147]Open in a new tab
Combining BT-NPs@PLEL with anti-PD-L1 treatment for distant tumor
inhibition. A Schematic representation of the treatment of residual
tumors after iRFA and distant tumors in C57/BL6 mice. B Bioluminescence
images of mice with residual tumors after iRFA and distant tumors after
different treatments on days 0, 10, and 20 (n = 3). C Bioluminescence
signals of mice in each group on day 0, 10, and 20. D Tumor volume of
distant tumors in different groups (n = 6). E The weight of distant
tumors post-iRFA on day 21 after different treatments (n = 6). F
Survival analysis of experimental mice in different groups (n = 6). G
Representative flow cytometry plots and proportions of CD8^+ T cells in
distant tumors on day 7 (n = 6). H Representative flow cytometry plots
and proportions of Granzyme B^+ cells in distant tumors on day 7
(n = 6). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001
Discussion
In this study, the acceleration of residual tumor growth following iRFA
was demonstrated in a murine model. The residual tumors exhibited a
suppressive TIME, diminished IFN-1 signaling, and decreased DCs and
CTLs. Concurrently, there was an increase in Sumo2 expression and
activation of SUMOylation in these residual tumors following iRFA. Upon
further investigation, we found that that SUMOylation, mediated by
Sumo2, obstructed IFN-1 signaling in HCC cells following iRFA. This
obstruction, in turn, hindered the anti-tumor immunity mediated by DCs
and CTLs. Both the knockdown of Sumo2 and the pharmacological
inhibition of SUMOylation resulted in the activation of IFN-1 signaling
in HCC cells post-iRFA and subsequently promoted DC maturation in a
co-culture system. However, this effect was reversed upon
administration of an IFN-1 antibody. Given that DCs play a pivotal role
in antigen presentation and the activation of CTLs [[148]11], we
posited that targeting SUMOylation could activate anti-tumor immunity
and effectively treat residual tumors after iRFA. Thus, a small
molecule SUMOylation inhibitor TAK-981-based nanocomposite hydrogel was
devised to effectively target SUMO2-mediated SUMOylation within the
residual tumors, named BT-NPs@PLEL. The in vivo results suggest that
BT-NPs@PLEL shows superior efficacy in promoting the maturation of DCs
and the infiltration and activation of CTLs, which result in enhanced
suppression of residual tumors. Significantly, extensive PD-L1
upregulation was observed after BT-NPs@PLEL treatment in the residual
tumors, setting the stage for the combination strategy with anti-PD-L1
treatment. Combining BT-NPs@PLEL with anti-PD-L1 treatment demonstrated
a potent synergistic effect in activating the antitumor immune
response, eliminating residual tumors after iRFA, and suppressing
distant tumors. Collectively, this study identifies an innovative
therapeutic target and develops a new strategy to treat RFA residual
tumors, offering potential for eliminating remnant tumors and
preventing HCC recurrence post-iRFA.
The overexpression of Sumo2 and activating of SUMOylation were first
reported in residual tumors following iRFA and were revealed to play a
critical role in shaping suppressive TIME in this study. A similar
pattern of Sumo2 expression could also be found in the GEO database
associated with iRFA ([149]GSE138224) [[150]5], signifying that this
phenomenon is consistent rather than a random event. Indeed, SUMO
mediated SUMOylation activation has long been linked to heat stress
[[151]35]. Studies have demonstrated that SUMO-2/3-mediated SUMOylation
is upregulated after heat shock, a mechanism essential for cellular
survival during stressful conditions. Our findings and the GEO database
data consistently demonstrate a significant increase solely in Sumo2
expression within residual tumors after iRFA, with no differential
expression observed in other genes associated with SUMOylation
signaling. The precise underlying mechanism for this specificity
remains elusive. Research has proposed a potential connection involving
Heat Shock Protein 27 (HSP27), which has been documented to augment the
number of cellular proteins modified by SUMO2/3. It has also been
linked to the SUMO2/3 modification of Heat Shock Factor 1 (HSF1),
thereby modulating the activity of this transcription factor [[152]36].
Further investigation is warranted to ascertain whether HSP27 regulates
the translational expression of SUMO2.
In our investigation, we observed a notable inhibition of the IFN-1
signaling pathway within RFA residual tumors. Upon knocking down Sumo2
or inhibiting SUMOylation, we observed a significant upregulation of
IFN-1 signaling in the HCC cells post-iRFA. This finding aligns with
existing research that suggests SUMO proteins can modify several
targets critical for IFN-1 signaling, including, cGAS, IRF3, IRF7, and
the enhancer element of IFN-β [[153]14, [154]37, [155]38]. These
studies collectively highlight that SUMOylation of these targets
negatively regulates IFN-1 production. Our findings indicated a poor
infiltration of mature DCs and CD8^+ T cells, suggesting inhibited
antigen presentation in residual tumors after iRFA. Given the crucial
role of the IFN-1 pathway in DC functions and antigen presentation for
the host’s antitumor immune response, targeting SUMOylation emerges as
a promising approach to activate DCs. Indeed, our results suggest that
inhibiting SUMOylation effectively activated DCs in both in vivo and in
vitro models. In this study, we primarily explored the
SUMOylation/IFN-1 mechanism in HCC cells under heat stress, as the
sequencing results primarily reflected the overall state of the iRFA
tumor. Additionally, previous research by Lu C et al. has highlighted
the pivotal role of IFN-1 signaling within tumor cells for DC-mediated
adaptive antitumor immunity, while Wang Z et al. reported that
activating IFN-1 signaling in tumor cells contributes to the activation
of dendritic cells and subsequent T cells [[156]39, [157]40]. These
cumulative findings emphasize the significance of IFN-1 signaling
within tumor cells in orchestrating the anti-tumor immune response and
underscore the potential of targeting SUMOylation as a therapeutic
strategy for treating residual tumors after iRFA.
TAK-981, a SUMOylation inhibitor, was explored as a potential treatment
for residual HCC after iRFA. TAK-981 has demonstrated promising
preclinical efficacy in activating anti-tumor immunity [[158]20,
[159]41–[160]44], prompting its evaluation in several phase 1/2
clinical trials for various kinds of tumors (#[161]NCT03648372,
#[162]NCT04074330, #[163]NCT04776018, and #[164]NCT04381650), but not
for HCC. However, the broad application of TAK-981 is challenged by
important factors that need to be considered, particularly the
potential toxicity risks associated with TAK-981 due to the widespread
presence of SUMO modification. Systemic and local injections of TAK-981
can induce undesirable inflammation in normal tissues, leading to
adverse events such as diarrhea and ulceration [[165]20]. Additionally,
its poor solubility and low bioavailability further limit its clinical
utility. Another challenge is the inability of systemic or local
injections to maintain sustained, high local drug concentrations, which
is essential for the effective eradication of residual tumors in iRFA
cases. To overcome these limitations, we have developed a novel
injectable drug delivery system that combines BSA-based nanoparticles
with a thermosensitive PLEL hydrogel. This innovative method ensures
the encapsulation and localized, sustained release of TAK-981,
enhancing its effectiveness against RFA residual tumors. The system’s
key advantage lies in its ability to undergo a spontaneous sol–gel
transition upon heating, minimizing systemic drug exposure. Although
hydrogels are inherently hydrophilic, posing challenges in loading
hydrophobic agents due to their high water content [[166]45], our
strategy utilizes BSA-based nanoparticles to overcome these challenges.
These nanoparticles effectively encapsulate TAK-981, elevating drug
loading capacity, enhancing solubility, and circumventing the obstacles
associated with integrating hydrophobic agents into hydrogel systems.
Immunotherapy, particularly anti-PD-1/PD-L1 therapy, has revolutionized
the landscape of cancer treatment over the past decade. However, it’s
worth noting that the response rate for advanced HCC stands at a modest
18.3% [[167]46]. The efficacy of anti-PD-1/PD-L1 therapy relies
significantly on the expression of PD-1/PD-L1 in TIME [[168]44,
[169]47–[170]50]. Our research, corroborated by other studies, has
substantiated that administering TAK-981 treatment results in an
augmented expression of PD-L1 at the cellular level within tumor
tissues [[171]21]. The heightened PD-L1 expression potentially
influences the treatment efficacy of TAK-981 nanocomposite hydrogel,
aligning with the prerequisites for anti-PD-1/PD-L1 treatment. Our data
suggest that combining BT-NPs@PLEL with anti-PD-L1 treatment
significantly enhances the efficacy in treating residual tumors after
iRFA and distant tumors, indicating a potential for an extended
application of BT-NPs@PLEL.
Conclusions
In summary, this study represents a significant breakthrough by
unveiling the role of SUMO2-mediated SUMOylation, induced by iRFA, in
hampering IFN-1 production and impeding antigen presentation in
residual tumors. This discovery introduces a novel therapeutic target,
suggesting that targeting SUMOylation might enhance immune surveillance
and offer a potential avenue to prevent HCC recurrence post-RFA
treatment. Additionally, the development of an injectable drug delivery
hydrogel (BT-NPs@PLEL) shows promising prospects to amplify the
efficacy and broaden the clinical applications of the SUMOylation
inhibitor. The exploration of the combined strategy of BT-NPs@PLEL and
anti-PD-L1 presents opportunities to expand the application of RFA and
enhance the response rate of immunotherapy.
Reagents and methods
Reagent and equipment
TAK-981(S8829) was purchased from Selleck Co Ltd. SUMO-2/3 (#4974) and
STAT1 (14994 T) antibodies were purchased from Cell Signaling
Technology (USA), Rabbit Anti-Sumo2 antibody (bs-15494R) was purchased
from Bioss Antibodies, and Phospho-STAT1 (Tyr727) antibody (ab109461)
was purchased from Abcam plc (USA). PDLLA-PEG-PDLLA (R-PL1054) was
purchased from Xi’an Ruixi Biological Technology Co Ltd. InVivoMab
anti-mouse PD-L1 (B7-H1) (BE0101) was purchased from BioXcell
Technologies, Inc (USA). VIVA RFA system (VRS01, STARmed) with a
straight RFA probe was used for RFA treatment.
Cell lines and animals
The Hepa1-6, Hep3B, HepG2 cells were originally obtained from the
American Type Culture Collection and were grown in Dulbecco’s Modified
Eagle Medium (DMEM) supplemented with 1% Penicillin–Streptomycin and
10% fetal bovine serum (FBS). The cells were maintained under standard
conditions at 37 °C with 5% CO[2]. Female C57/BL6 mice, aged 5–6 weeks,
were procured from the Medical Laboratory Animal Center of Guangdong
Province. All experiments involving mice were carried out in accordance
with the guidelines set forth by the Institutional Animal Care and Use
Committee of The Fifth affiliated hospital of Sun Yat-Sen University.
Sumo2 gene knockout in Hepa1-6
The Sumo2 gene was knocked down by short hairpin RNA (shRNA) in Hepa1-6
cells. The shRNA and vector design and construction were performed by
IGE Biotechnology LTD. In brief, shRNAs were designed against the
mSumo2 gene (Mus musculus, [172]NC_000077.7) using CHOPCHOP. Lentivirus
packaging was carried out, and Hepa1-6 cells were transduced according
to the abm protocol. Puromycin selection was completed two days after
transduction.
Total RNA extraction and real-time PCR
Total RNA was collected and isolated from tissues and cells with an RNA
isolater Total RNA Extraction Reagent kit (Vazyme Biotech Co., Ltd,
Nanjing, China). Reverse transcription was performed using the HiScript
III 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd, Nanjing,
China). The PCR was performed by a ChamQ Blue Universal SYBR qPCR
Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China), according to the
manufacturer’s instructions. The primers were designed according to the
National Center for Biotechnology Information (NCBI) database using the
Primer Premier 5.0 software (Palo Alto, CA, USA), and all the primer
sequences are listed in Table [173]1. The mRNA expression was measured
using a real-time PCR machine, ABI 7500 (Applied Biosystems, USA). Fold
changes expression of each gene was calculated by the comparative
threshold cycle (Ct) method using the formula [174]2^− (ΔΔCt).
Table 1.
Selection of primer sequences of genes for RT-qPCR Analysis
Gene The primer sequence of gene for RT-qPCR analysis The version of
nucleotide sequence
Primer sequence(5'-3')
Sumo2
Forward: CCCggTgCCTCTTTTgTgAA
Reverse: gACTCCTTCCTTgggTTTCTCg
[175]NM_133354.2
SUMO2
Forward: gACgAAAAgCCCAAggAAggAg
Reverse: CCATTTCCAACTgTCgTTCACA
[176]NM_001005849.2
Ifnb1
Forward:gCAAgAggAAAgATTgACgTgg
Reverse: AggCgTAgCTgTTgTACTTCAT
[177]NM_010510.2
Isg15
Forward: CAgCAATggCCTgggACCTAAA
Reverse: gCACACCAATCTTCTgggCAAT
[178]NM_015783.3
Ifit3
Forward: TCAgCCCACACCCAgCTTTT
Reverse: CTTCCAgAgATTCCCggTTgAC
[179]NM_010501.2
Mx1
Forward: gCAgAAgTACggTgCAgACATA
Reverse: ACggTTTCCTgTgCTTgTATCA
[180]NM_010846.1
Cxcl10
Forward: CCgTCATTTTCTgCCTCATCCT
Reverse: TTCCCTATggCCCTCATTCTCA
[181]NM_021274.2
Il6
Forward: ACAgAggATACCACTCCCAACA
Reverse: gCCATTgCACAACTCTTTTCTCA
[182]NM_001314054.1
Gapdh
Forward: ATgACATCAAgAAggTggTg
Reverse: CATACCAggAAATgAgCTTg
[183]NM_001289726.2
GAPDH
Forword: ACAACTTTggTATCgTggAAgg
Reverse: gCCATCACgCCACAgTTTC
[184]NM_001256799.3
[185]Open in a new tab
RNA-sequencing (RNA-seq) and bioinformatics analysis
RNA-seq libraries were performed by Majorbio Corporation (Shanghai,
China), and the data were expressed as the means displayed in the
center of the heatmaps. The fold-change was calculated and converted to
log2. All mRNA sequencing data were downloaded from the TCGA data
portal.
Western blot analysis
An equivalent of 30–50 μg total cellular protein was separated by a
10–15% gradient in SDS-PAGE (Bio-Rad Laboratories). The proteins were
transferred to nitrocellulose membrane (Pall Corporation, Ann Arbor,
MI, USA), and the membranes were blocked with 5% milk powder in TBST
for 60 min. Then, the blots were probed in 0.1% casein/TBS-T with
Primary antibody overnight at 4° C. Subsequently, the blots were
incubated with infrared-labeled secondary Abs at 1:5000 at room
temperature for 1 h. The immunoreactive bands were visualized using an
Invitrogen iBright^™ CL750 Gel Imaging System (Thermo Fisher
Scientific, USA).
Immunohistochemistry (IHC) staining
Immunohistochemistry (IHC) staining of paraffin-embedded tumor tissue
sections was performed using the anti-SUMO2 (bs-15494R, Bioss)
antibodies, according to the manufacturer’s instructions. For
statistical analysis, the DAB staining score was calculated by Image J
with the IHC Profiler plugin [[186]51]. The score of the zone is
assigned as 4 for the high positive zone, 3 for the positive zone, 2
for the low positive zone and 1 for the negative zone. The score
calculation following the algebraic formula:
[MATH:
scor<
mi>e=Number of pixels in a zoneScore of the
zoneTota<
/mi>lnumberofpixelsintheimage :MATH]
1
Transwell experiment on DCs stimulation in vitro
BMDCs were obtained from the marrow of the tibia and femur of C57BL/6
mice by flushing with RPMI 1640 medium. The red blood cells were then
lysed, and the remaining cells were cultured in a medium containing
20 ng/mL GM-CSF and 10 ng/mL IL4 for 7 days. The expression of CD11c
was assessed before the experiment. Heated Hepa1-6 cells were cultured
in the upper compartment of the transwell system, while the BMDCs were
co-seeded in the lower compartment. After various treatments, BMDCs
stained with APC-anti-CD45, PE/Dazzle-anti-CD11c, Percp/Cy5.5anti-CD86
PE, and PE/Cy7-anti-CD80 were analyzed by flow cytometry.
Construction and of TAK-981@BSA nanoparticles
With minimal alteration, the TAK-981 loaded BSA nanoparticles were
created using a self-assembly technique in accordance with earlier
research [[187]52]. In brief, dimethyl sulfoxide (DMSO) solution of
100 μL TAK-981(30 mg/mL) was added into the solution of BSA (9 mg/mL,
10 mL, pH 8.0, adjusted with 0.1 M NaOH) and stirred vigorously for
2 h. The resulting nanoparticles were gathered through gradient
centrifugation. To remove the excess unbound drug, we performed
centrifugal filtration using an Amicon filter (MWCO = 10 kDa) and
washed the sample three times with PBS until no drug was detected in
the filtrate. The morphology of TAK-981@BSA NPs was characterized with
a transmission electron microscope (FEI Tecnai F20). The diameters of
BT-NPs@PLEL were detected by Zeta sizer Nano-ZS (Malvern Instruments,
UK).
Fabrication of BT-NPs@PLEL composite hydrogel
The prepared TAK-981@BSA nanoparticles were dispersed into a 100 μL
solution and were incorporated with a 400 μL PLEL solution to construct
the final BT-NPs@PLEL composites (PLEL, 25 wt%). Additionally, the
initial TAK-981 loading was determined by measuring the concentration
in the discarded supernatant and comparing it to the concentration
prior to the formation of NPs.
The accumulative releaseof drugs from BT-NPs@PLEL
The accumulative drug release behavior was measured using a transwell
system in PBS containing 20% fetal bovine serum at 37° C to mimic the
physiological environment. The BT-NPs@PLEL (25 wt%) encapsulated with
TAK-981 (0.5 mg) was loaded into the upper chamber, and the released
TAK-981 was collected from the bottom of chamber at various time points
and quantified using a PerkinElmer Lambda 750 UV–vis–NIR
spectrophotometer.
Phase diagram of PLEL and BT-NPs@PLEL
The precursor solution of PLEL and BT-NPs@PLEL with concentrations of
25 wt% was prepared, then 1 mL of each solution was added into a vial
and equilibrated in the water bath for 10 min at each temperature. The
gel state was determined by inverting the vial and observing whether
the solution flowed within 30 s.
The in situ gelation and degradation behavior of PLEL
For an in situ gelation experiment, the precursor solution of
BT-NPs@PLEL with a concentration of 25 wt% was injected subcutaneously.
Then, the skins of mice were sacrificed and imaged. For an in vivo
degradation experiment, 100 μL BT-NPs@PLEL (mixed with blue dye) (25 wt
%) was injected into the flank of the mouse. Then the skins of mice
were sacrificed and imaged on day 1, 7, and 14 for records of in vivo
biodegradability.
Flow cytometry analysis
Single cells were obtained by homogenizing the tumors and spleen on day
7 after treatment. Then, the cells were blocked with anti-CD16/32
(Elabscience, E-AB-F0997A) antibodies to avoid nonspecific adsorption
and then stained with the following antibodies according to the
specification: APC-anti-CD45 (BioLegend, 103112), PE/Dazzle-anti-CD11c
(BioLegend, 117347), PE/Cy7-anti-CD80 (BioLegend, 104733),
Percp/Cy5.5-anti-CD86 (BioLegend, 105027), PE-anti-CD86 (BioLegend,
105008), anti-CD45 (BioLegend, 103132), APC/Cy7-anti-CD3 (BioLegend,
100222), BV605-anti-CD4 (BioLegend, 100548), BV510-anti-CD8 (BioLegend,
100751), APC-anti- Granzyme B (BioLegend, 372204). 5.0 × 10^3 events in
the CD45 flow chart in each sample were collected using a CytoFLEX LX
Flow Cytometer (Beckman Coulter, USA) and analyzed by FlowJo 10.8.1.
The gating strategy is given in Figure S21, Supplementary material.
In Vivo experiment
To build the HCC model, 1.0 × 10^^6 Hepa1-6 cells were subcutaneously
inoculated on the right flank or bilateral of each C57BL/6 mouse.
14 days later (Tumor volume about 300 mm^3), the mice were randomly
divided into different groups to perform in vivo experiments. To study
the drug retention in tumors, equal amounts of free Sulfo-Cyanine5.5
dye and Cy5.5@Gel were injected subcutaneously, and the fluorescence
signal was monitored by a fluorescence imaging system (PerkinElmer,
Lumina). For RFA treatment, the radiofrequency needle was pierced into
a non-central location of the tumor to achieve partial necrosis of the
target tumors (Figure S1A), and the power of radiofrequency was 7 Walt,
and the tumor was heated to 60° C for 0.5 min. Then, the equivalent
TAK-981 (150 μg per mouse) and gel (85 μL, 25 wt% per mouse) were
injected into the tumor in the corresponding group. The tumor growth
curves were monitored by calculating the volume using the following
formula:
[MATH: V=(w2×L)2 :MATH]
2
All animal experiments are conducted after administering inhalation
anesthesia with isoflurane.
In Vivo biosafety evaluation
To evaluate the biosafety of BT-NPs@PLEL in vivo, four groups of
healthy mice were injected subcutaneously with BT-NPs@PLEL (TAK-981,
7.5 mg/kg) and sacrificed on day 1, day 10, and day 30, respectively,
while the control group received PBS injection alone. Blood items
including white blood cells (WBC), red blood cells (RBC), platelets
(PLT), Total protein (TP), serum albumin (ALB), urea nitrogen (Urea),
creatinine (Crea), aspartate aminotransferase (AST), alanine
aminotransferase (ALT) and albumin (ALB) were tested.
Cytokine detection
The tumors were harvested and homogenized thoroughly in RIPA lysis
buffer at 4° C on day 7 and centrifuged (12,000 rpm) to remove the
sediment 3 times. The concentration of cytokines was detected by IFN-γ
(MIKX, SZ1095), and TNF-α (MIKX, SZ1098) mouse ELISA assay kit
according to the manufacturer’s instructions.
Statistical analysis
Statistical analysis was performed via GraphPad Prism 9.0. All data are
presented as the mean ± standard error of the mean (s.e.m.). The
Two-tailed Student’s t-test was used for two group comparisons, One-way
ANOVA (for multiple groups) and two-way ANOVA (for multiple groups and
factors) were used for multiple group comparisons, as appropriate,
followed by Tukey’s post hoc multiple comparisons test. The threshold
for statistical significance was ns, not significant, *p < 0.05,
**p < 0.01, ***p < 0.001.
Supplementary Information
[188]Supplementary material 1.^ (2.7MB, docx)
Acknowledgements