Qin Huang formula enhances the effect of Adriamycin in B-cell lymphoma via increasing tumor infiltrating lymphocytes by targeting toll-like receptor signaling pathway

Non-Hodgkin lymphoma (NHL) is a malignancy that mainly infringes the lymphoid system, with extranodal sites often affected as well. It has arisen to the top 10 most common cancers in 2020 [1]. Among all the subtypes of NHLs, diffuse large B cell lymphoma (DLBCL) is the most common subtype and accounts for 25-30% of diagnoses [2]. Albeit aggressive, most DLBCL is chemo-sensitive, and R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) is the standard regimen. However, due to resistance to R-CHOP, there are still ~ 30% of patients suffering from refractory or relapse (r/r) disease, and their prognosis is rather poor [3, 4]. Such treatment plight has prompted the emergence of novel treatment options, such as therapeutics targeting immune checkpoints, tumor microenvironment (TME), signaling pathways, and cellular immunotherapy [5]. As a cellular immunotherapy, chimeric antigen receptor (CAR) T-cell therapy has shown a promising future in the management of B cell lymphomas. Two CAR T-cell products are now approved for the treatment of relapsed/refractory large B-cell lymphoma [6]. However, its acute and late toxicities, as well as on-target off-tumor effect should be addressed [7]. PD-1/PD-L1 blockade, as an immune checkpoint inhibitor (ICI), aims to protect cytotoxic T cells and restore their function from PD-1/PD-L1 mediated immune evasion. However, its efficacy in DLBCL remains unsatisfactory compared to classical Hodgkin Lymphoma (cHL) [8, 9]. Research accumulated that the pre-existing immune landscape within the TME may influence the response to immunotherapies [10‐12] and R-CHOP [13, 14]. Therefore, it is pivotal to explore agents that can modulate TME. TME is an extremely intricate environment that harbors diverse cells, structures, and matters, with one component interacting with many others, forming complex regulatory networks. Drugs that have multiple targets might be a promising combination therapy to modulate TME, especially those that could elicit both innate and adaptive immune responses against cancers.

Traditional Chinese medicine (TCM) has a long history in treating various diseases and ailments. Formula, a prescription that contains multiple drugs with different but related modes of action, is a representative of combination therapy in the system of TCM [15]. With the guidance of this philosophy, the Qin Huang formula (QHF), was grouped and used as adjuvant therapy for treating lymphoma in our hospital (Patent No.:2015103646392). Containing Scutellaria baicalensis Georgi. (Lamiaceae) (SR), Astragalus membranaceus (Fisch) Bunge (AM), Prunella vulgaris L. (Lamiaceae) (PV), and Curcuma Longa L. (Zingiberaceae) (CL), the 4-herb formula conforms to the theology of TCM in treating malignancies. Huang et al. [16] found that baicalin, an ingredient of SR, could down-regulate PI3K/Akt signaling pathway and induce apoptosis in the Burkitt lymphoma cell line. Kumagai et al. [17] demonstrated that SR and its major components, baicalein, and wogonin, could exert anti-proliferation and induce apoptosis mediated by mitochondrial damage in lymphocytic leukemia, Burkitt lymphoma, and myeloma cell lines. Studies [18‐20] also showed that PV and CL have a strong antiproliferation effect upon Raji, a Burkitt lymphoma cell line, and other solid tumors. Meanwhile, it is reported that AM and its extract could protect human epithelial and endothelial cells from LPS-induced apoptosis [21] and exert a cardioprotective effect against Adriamycin-induced cardiotoxicity [22]. Thus, as a mixture of the four herbs, QHF was expected to exert an antitumor effect in B cell lymphoma as well. And our previous work [23] found that QHF (also referred to as Qin Huang mixture) has a modulating effect on the systemic immune function of lymphoma patients who have completed R-CHOP chemotherapy. Besides, we also found that when combined with R-CHOP, QHF can significantly lower the overall side effect frequencies and improve the short-term effects of R-CHOP in treating initial onset DLBCL patients [24]. In addition, we explored the effects of several major flavonoids of QHF in vitro and found that wogonin, luteolin, kaempferol, quercetin, and silymarin can inhibit the proliferation and induce apoptosis of the Raji, SU-DHL-4, and A20 cell lines (data not published), which is consistent with other studies [25‐28]. Therefore, we hypothesized that QHF can enhance the antitumor effects of chemotherapy by modulating immune functions, especially the tumor immune microenvironment (TIME), of B cell NHL. In this study, a systemic pharmacological analysis was carried out to predict the targets and mechanisms of QHF in treating B cell lymphoma. Then, the antitumor effect of QHF in combination with Adriamycin (ADM) was evaluated on a murine model induced by the mouse B cell lymphoma cell line A20. Moreover, the TME of the mouse B cell lymphoma was further evaluated via IHC staining, ELISA, as well as western blot to confirm our hypothesis. Our extensive study provides experimental evidence regarding the antitumor effect of QHF + ADM combination therapy and revealed its underlying mechanisms.

B cell non-Hodgkin lymphoma is a highly heterogeneous neoplasm, and its most common subtype is diffuse large B cell lymphoma. Although it is curable to most patients, there are still 30-40% recurrence or refractory cases [53]. And cancer immunotherapies are initially used in these settings. The TME, especially the immune landscape within it, is found to be crucial in the responses to the immunotherapies [54]. The TME of B-cell lymphoma mainly consists of T cells, macrophages, natural killer (NK) cells, stromal cells, blood vessels, and extracellular matrix [55, 56]. A combination therapy targeting TME has proven efficacy and displayed great potential for novel immunotherapies. As a traditional formula, QHF consists of four different herbs with various functions. In this study, we verified the tumoricidal effect of QHF in combination with ADM using a murine model and explored its underlying mechanisms with the guidance of systemic pharmacology.

Systemic pharmacology is a comprehensive approach to clarify the active compounds and drug targets, as well as to predict the mechanisms of traditional Chinese medicinal formulas. Our work displayed that QHF has 541 significant targets in B cell lymphoma, among which MAPK1/ERK2 and AKT1 are mostly enriched. Based on KEGG pathway enrichment analysis, we discovered that the TLR signaling pathway, which plays a critical role in activating both innate and adaptive immune responses, was one of the major pathways that QHF might trigger. In addition, we filtered out five active components of QHF, i.e., quercetin, luteolin, kaempferol, wogonin, and stigmasterol, that might be the most promising ingredients to represent QHF. The molecular docking technique also authenticated the ligation of the five components and targets.

Our murine model built with an A20 cell line showed that compared with ADM monotherapy, the QHF + ADM group could significantly inhibit the tumor growth. Meanwhile, the tumor-infiltrating CD4+ and CD8+ T cells of the QHF + ADM group were remarkably increased. Research demonstrated that anti-CD20 mAb, such as rituximab, could achieve a long-lasting “vaccinal effect” with the presence of both CD4+ and CD8+ T cells [57, 58], indicating the potential value of QHF as an add-on therapy to the R-based therapy for lymphoma patients. In this study, one mouse from each group was picked out to receive the ¹⁸F-FDG PET-CT scan before and after the treatment, respectively. Notably, although the posttreatment tumor volume of the combination group is smaller than the ADM group, the SUV_max of the combination group is higher. What’s more, after the treatment was discontinued for 1 week, the SUV_max of the combination group decreased to a level lower than that of pretreatment. Evidence showed that SUV_max is positively related to the TILs and TAMs [59, 60]. And a provoking study published in Nature this year has proven that it is the myeloid cells, followed by T cells, that uptake most FDG rather than tumor cells [61]. Therefore, the dynamic changes of SUV_max of the combination group might indicate the level changes of infiltrating T cells. The ¹⁸F-FDG PET-CT scan provided an additional description of the increased infiltrating T cells in the QHF + ADM group.

According to the immunophenotype of TME, tumors can be divided into “hot” tumors and “cold” tumors. Hot tumors present with rich TILs, whose initial immune response is obstructed by upregulation of immune checkpoints or immunosuppressive cells. Anti-PD-1/PD-L1 antibodies block tumor immune evasion by protecting CD8+ T cells from PD-1 mediated cell death [62, 63]. However, such ICI has limited efficacy in ‘cold’ tumors, where immune cells are less abundant or completely absent in TME [64]. Mullins et al. [65] found that TLR agonists tuned TME to inflamed immunophenotype by activating CD8+ T cells, thus enhancing the antitumor effect of ICIs. This has highlighted the promising value of TLR agonists. TLR is a big family consisting of TLR2, TLR4, TLR7, TLR8, and other receptors. TLR signaling pathway starts with TLR receptor and ligand recognition, followed by TLR dimerization and a signaling cascade to produce inflammatory molecules. In this study, we first predicted that the active components of QHF bind to TLR2 with hydrogen bonds via molecular docking methods. In theory, when the components of QHF bind to TLR2 and activate the TLR2 signaling pathway, the constitutive MAPK or NF-κB signaling pathway will be triggered to express various cytokines like IL-6, TNF-α, and IFNs. It is also reported that activation of TLR2 can reverse mouse Treg suppression, promote and generate efficient memory T cells by increasing IFN-γ level, and enhance the cytotoxic activity of CD8+ T cells [66]. Besides, ADM can activate p38 to regulate the expression of topoisomerase II, inducing apoptosis of lymphoma cells. Therefore, QHF + ADM could enhance the antitumor effect via different mechanisms theoretically. In the present study, the IL-6 level was up-regulated in the QHF + ADM group, but the levels of TNF-α and IFN-γ were not remarkably changed. As the IL-6 production regulation is controlled through both transcriptional and posttranscriptional mechanisms [67], it remains elusive whether the increased IL-6 is the result of TLR signaling pathway activation or not. But the increase in CD4+ and CD8+ T cells in TME, as well as up-regulated CXCL10 and TLR2 in the combination group, indicated QHF + ADM could modulate TME by targeting TLR2.

As aforementioned, the immune cells within TME can interact with the tumor cells directly or through chemokines and cytokines to impact the behavior of the tumor and response rate [68]. A variety of chemokines are expressed by immune cells, including CCR7, CXCL9, CXCL10, CCL2, and IFN-γ. Secreted by effector T cells, IFN-γ can induce the expression of CXCL10, which in return attracts even more T cells into the TME, thus forming a positive feedback loop [69‐73]. Despite IFN-γ, TLR agonists also elicit cytokines and chemokines expression in multiple cells [65]. In a melanoma model, TLR2/6 agonists + IFN-γ can induce CXCL10 production, leading to more T cells migrating into TME [74]. In our study, the combination group displayed upregulated levels of TLR2 and CXCL10, indicating that QHF might target TLR signaling pathway to promote CXCL10 expression with the presence of IFN-γ, and consequently increased T cell infiltration and IFN-γ expression in return. Transforming growth receptor beta (TGF-β), expressed by both tumor cells and tumor-associated cells, directly inhibits T cell proliferation and activation while promoting the survival of immunosuppressive Tregs. Therefore, in the context of TME, upregulated TGF-β may lead to T cell dysfunction and limited efficacy of cancer immunotherapies [75]. Moreover, with the presence of IL-6, TGF-β may also promote CD4+ T cell differentiation along a Th17 pathway, which was predicted in the KEGG enrichment analysis as mentioned before. Although IL-6 is associated with lymphoma proliferation and poor prognosis, it is reported that IL-6 can prevent apoptosis of T cells by upregulation of Bcl-2 and promote T cell proliferation via the TCR pathway [76]. Also, evidence shows that increased IL-6 is associated with highly functional CAR T cells and their expansion [77]. Consistent with these findings, we observed a downregulated level of TGF-β, as well as an upregulated level of IL-6 in the combination group, suggesting that QHF may aid to restore T cell function and survival in the TME. Taken together, we speculated that QHF took effect through modulating the TIME via targeting the TLR signaling pathway.

QHF in combination with ADM improves the anti-lymphoma effect of ADM by modulating TME. Besides, with a method of systematic pharmacology, the active components of QHF were identified. This research established a scientific method for analyzing chemical components and verifying predicted mechanisms of Chinese herbal medicine.