Targeting SUMOylation with an injectable nanocomposite hydrogel to optimize radiofrequency ablation therapy for hepatocellular carcinoma

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.

Keywords: Hepatocellular carcinoma; Nanocomposite hydrogel; Radiofrequency ablation; Small ubiquitin-like modifier 2; TAK-981.

Fig. 1

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

Fig. 2

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

Fig. 3

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

Fig. 4

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

Fig. 5

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

Fig. 6

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

Fig. 7

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