4. Discussion
Currently, HCC is still one of the primary causes of cancer deaths, although the survival rates and prognosis of HCC have been greatly improved by liver transplantation and surgical resection [2]. TACE procedures are currently widely used for various cases of HCC, mostly patients at BCLC stage B. Gelatin sponge microparticle (GSM) is an effective embolization agent, and a study reported that when compared with the ethiodized oil which is a permanent embolization agent, GSMs had many advantages, such as a higher tumor response rate at 1 month after treatment, especially in large tumors [13]. Our previous clinical practices have confirmed the safety and efficacy of GSMs-TACE [5, 14, 15]. It was found that 350-560 μm GSMs combined with single chemotherapy drug TACE procedures was effective and safe in treating elderly HCC without surgical resection, and more elderly patients may have better prognosis [5]. GSMs combined with trans-arterial p53-gene-embolization also achieved satisfactory efficacy in treating BCLC stage B HCC as evidenced by favorable survival rates (100% for both 6 and 12 months) and no significant complications [14]. Therefore, GSMs were adopted in this study, and imaging scan confirmed the efficacy of GSMs-TACE. DSA angiography during the procedure revealed that after GSMs embolization, the blood flow velocity of tumor feeding arteries, slowed down, and the tumor staining basically disappeared. Moreover, 4 days after GSMs-TACE, CT plain scan showed some honeycomb-like low-density changes in tumors compared with before the procedure. In addition, in a previous study, after analyzing the data of 37 patients who received GSMs‑TACE for liver metastases after gastrointestinal tumor surgery, it was observed that the treatment produced various degrees of necrosis and shrinkage of lesions, and even 2 patients achieved a complete response [16]. Considering the close relationship between tumor metastasis and the immunosuppressive TME, these findings prompted us to wonder whether tumor necrosis after GSMs-TACE improved the therapeutic efficacy via regulating the immune-mediated elimination of tumor cells.
Regulatory T (Treg) cells are a subset of CD4+ T cells in immune system known to function as “immune-suppressor”. It is well recognized that Treg cells play an important role in protecting against excessive inflammation and immune response in physiological or some pathological conditions. Furthermore, it has been noticed that Treg cells are involved in liver diseases including HCC [17]. The infiltration of Treg cells into tumor sites can exert a suppressive effect on host anticancer immunity and thereby become an obstacle to curative anticancer therapy [10, 18, 19]. In the current study, we first compared the Treg cell proportion in peripheral blood between HCC patients and healthy volunteers, and it was found that there was an increased proportion of Treg cells in peripheral blood of patients with HCC (11.26 ± 1.31% and 7.32 ±1.63%, respectively), which was in accordance with the previous study [19]. Besides, we analyzed the association between the Treg cell proportion and various clinical indicators of HCC, and it was found that the Treg cell proportion in peripheral blood of HCC patients was closely related to tumor stage, AFP content, and tumor diameter, which further confirmed the association between the Treg cell proportion and HCC. However, the mechanism of the association between peripheral Treg cells and HCC pathophysiological characteristics has not been fully elucidated. Shi et al.proposed that Tregs could modify HCC in the way that potentiates the metastasis since it was found that Treg cell proportions in HCC patients were significantly correlated with biomarkers of tumor cell metastasis such as E-cadherin, vimentin and SNAIL, although these findings were based on the analysis of limited cases [20]. In addition, tumor encapsulation was an important clinicopathological marker of HCC invasiveness, and it was observed that the high Treg cell proportion in peripheral blood of HCC patients was significantly correlated with the presence of tumor encapsulation, which was inconsistent with the published literature, where Gao et al. reported that a high Tregs proportion was associated with the absence of tumor encapsulation [21]. Nevertheless, these await further studies.
The 28 patients enrolled in this study received GSMs-TACE, and the imaging techniques confirmed the success of the procedure. To testify our hypothesis that TACE procedures may also have an impact on anti-cancer immunity, the Treg cell proportion in peripheral blood of these 28 HCC patients was determined before and at 10 and 30 days after TACE. It was found that the peripheral Treg cell proportion at 10 days after TACE was significantly lower than that of before the procedure, and the percentage slightly increased 30 days after TACE but was still significantly lower than that of before the procedure. These results indicated that GSMs-TACE had a positive regulatory effect on immune function. The possible reason is that after the effective embolization of tumor feeding artery, the tumor cell necrosis is induced significantly in a short time, and the tumor load is also significantly reduced, which is related to the significant reduction of the immunosuppressive effect on the body. It is well recognized that many tumors bear tumor antigens and can induce T-cell cytotoxic responses [22]. The release of tumor antigens then led to local microenvironment immune response and a subsequent reduction of Treg cells in peripheral blood. Our results showed that the Treg cells proportion in peripheral blood at 10 days after operation was the lowest, which indicated that it may be the optimal time for immunoadjuvant therapy. The improvement of immune function can be maintained within one month, but with the further proliferation of tumor cells, the immune function gradually recovers to the state of inhibition. Therefore, the follow-up treatment including TACE should be carried out subsequently to further reduce the burden of tumors in order to better restore and enhance the body’s anti-tumor immunity.
In recent years, tumor immunotherapies have gained promising results. Programmed cell death protein 1 (PD‐1), expressed by various activated immune cells, is an immune checkpoint and can protect against autoimmune responses. It has been revealed that in cancer immunity, tumor cells express PD-L1, the ligand of PD-1, and combine with PD-1 in T cells, thereby suppressing T cell activities and promoting the differentiation of Treg cells [23]. As a result, the anticancer immune responses are inhibited in tumor microenvironment. The immunotherapies targeting PD-1/PD-L1 have successfully improved the outcomes of various types of cancer worldwide, including HCC [24]. A pioneering study analyzed the PD-L1 expression in 240 HCC patients who received surgical resection, and it was found that patients who were positive for PD-L1 showed significant shorter disease-free survival or overall survival than those PD-L1 negative patients [25]. Antibodies of PD-1 or PD-L1 in HCC immunology have shown promising efficacies [26]. However, there are still unmet clinical needs considering that many patients show no response. Since one of the important mechanisms of PD-1/PD-L1 pathway is the promotion of Treg cells differentiation, an interesting and promising suggestion was drawn from this study that GSMs-TACE has the potential to be used in combination with immune adjuvant therapies such as PD-1/PD-L1 pathway targeting therapies to increase the efficacy of HCC treatment.
In conclusion, the Treg cell proportion in peripheral blood of HCC patients was higher than that of healthy controls and was closely related to the clinical characteristics of HCC. The GSMs-TACE procedure significantly reduced the peripheral Treg cell proportions at 10 and 30 days after operation. These results indicated that GSMs-TACE could reduce the proportion of Treg cells in peripheral blood after operation and exert a positive regulatory effect on the immune function of HCC patients. This study provides a piece of preliminary evidence that GSMs-TACE has the potential to be used in combination with immune adjuvant therapies such as therapies targeting PD-1 or PDL-1 to increase the efficacy of HCC treatment.
However, there are also some limitations of this study, and the expression of other molecules or biomarkers involved in tumor immunity should be assessed along with the Treg cell proportion to better explain the mechanism of GSMs-TACE procedures affecting tumor immunity, so as to provide references for the treatment design of HCC patients.