Abstract
Pulmonary metastasis is a life-threatening complication for patients with hepatocellular carcinoma (HCC) undergoing liver transplantation (LT). In addition to the common mechanisms underlying tumor metastasis, another inevitable factor is that the application of immunosuppressive agents, including calcineurin inhibitors (CNIs) and rapamycin inhibitors (mTORis), after transplantation could influence tumor recurrence and metastasis. In recent years, several studies have reported that mTORis, unlike CNIs, have the capacity to modulate the tumorigenic landscape post-liver transplantation by targeting metastasis-initiating cells and reshaping the pulmonary microenvironment. Therefore, we focused on the effects of immunosuppressive agents on the lung metastatic microenvironment and how mTORis impact tumor growth in distant organs. This revelation has provided profound insights into transplant oncology, leading to a renewed understanding of the use of immunosuppressants after LT for HCC.
keywords
- Liver transplantation
- hepatocellular carcinoma
- lung immune microenvironment
- lung metastasis
- immunosuppressants
Introduction
Hepatocellular carcinoma (HCC) is the sixth most common malignant tumor globally and the third leading cause of cancer-related deaths1. Liver transplantation has become increasingly accepted as the only effective treatment for end-stage liver disease, including HCC2. However, patients undergoing liver transplantation for HCC often develop lung metastases postoperatively. The incidence of pulmonary metastasis is significantly higher than other anatomic sites, accounting for 50%–60% of all recurrences. Hepatic recurrence occurs in 15%–40% of cases, bone metastasis occurs in approximately 25% of cases, and adrenal metastasis occurs in approximately 10% of case3–5. Therefore, post-transplant lung metastasis has become a barrier to liver transplantation for liver cancer patients6. Lung metastasis after liver transplantation for HCC is a complex cascade reaction process involving multiple factors and procedures with a complex and diverse molecular mechanism7,8.
As research into the pulmonary microenvironment deepens, the significance of pulmonary inflammation and the immune microenvironment in lung metastasis after liver transplantation has been shown to be crucial as details about the pulmonary microenvironment have been revealed. Factors in the microenvironment can affect various processes of HCC cells, such as growth, invasion, and metastasis. The lung vasculature provides oxygen and nutrients to support the growth of HCC cells, while immune cells in the lung identify and kill HCC cells7,9. The structure and chemical properties of the extracellular matrix (ECM) in the lung can also affect the movement and growth of HCC cells. Therefore, understanding how the microenvironment affects HCC cell metastasis can guide the development of treatments to inhibit spread to the lung.
However, the pulmonary immune microenvironment exhibits unique characteristics following liver transplantation due to the use of immunosuppressive agents. Currently, the main immunosuppressive agents used after liver transplantation are calcineurin inhibitors (CNIs), such as tacrolimus7, and mammalian target of rapamycin inhibitors (mTORis), such as everolimus6. Numerous studies have shown that CNIs create a permissive environment for tumor growth and increase the risk of HCC recurrence in a dose-dependent manner10. In contrast, mTORis exhibit anti-tumor effects by inhibiting cell proliferation and angiogenesis, albeit with increased adverse reactions as a trade-off6. The effects of CNIs and mTORis on the lung immune microenvironment remain unclear.
In this review we focused on the mechanisms underlying lung metastasis after liver transplantation for HCC, which are influenced by immunosuppressive agents in the immune microenvironment. We hope that this review provides valuable insights for future research on immunosuppressive agents in the field of transplant oncology.
Lung microenvironment: the soil of HCC pulmonary metastasis
In 1889 Stephen Paget9 proposed the “seed and soil” theory of tumor metastasis. Today, with the continuous advances in tumor microenvironment (TME) research, this hypothesis has been significantly refined and supplemented.
A portion of HCC cells penetrate the basement membrane and invade the hepatic blood vessels (usually the hepatic veins). These tumor cells that enter the bloodstream are referred to as circulating tumor cells (CTCs)11. Once in the bloodstream, CTCs travel throughout the body with the flow of blood. CTCs in the blood are the primary cause of pulmonary metastasis after liver transplantation6. In this process, CTCs need to evade immune surveillance and resist various physiologic stresses, such as shear forces from the bloodstream. Upon reaching the lungs, CTCs “roll” and “adhere” to the vessel wall through interactions with endothelial cells12, then cross the vessel wall to enter lung tissue, a process known as “tumor cell extravasation”11.
Once settled in the pulmonary microenvironment, CTCs need to survive and proliferate in the pulmonary microenvironment to form micrometastatic lesions13. This process requires extensive interaction between CTCs and the surrounding microenvironment, including immune cells (T cells, B cells, natural killer cells, macrophages, dendritic cells, and neutrophils), stromal fibroblasts, endothelial cells, pericytes, and components of the ECM (collagen, laminin, fibronectin, and proteoglycans)13,14. After adapting to and manipulating this new environment, CTCs begin to proliferate and eventually form visible pulmonary metastatic lesions.
It is worth noting that although a large number of CTCs enter the bloodstream, the number of CTCs that successfully metastasize and form lesions at distant sites, such as the lungs, is very small14 because CTC survival in the bloodstream and the settling and proliferation in new environments all face significant challenges10,15,16. Indeed CTC metastasis is one of the important topics in current research on the mechanisms underlying tumor metastasis.
The lung microenvironment can be modified by primary tumors to form a pre-metastatic niche, a favorable environment that helps incoming CTCs survive and proliferate. Formation of this metastatic niche is often facilitated by the recruitment of immune cells, changes in the local vasculature, and the deposition of ECM proteins. The lung microenvironment can also contribute to CTC immune evasion. For example, immune cells in the lung, such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), suppress the immune response against CTCs16, which allows CTCs to survive and establish metastases. The lung microenvironment promotes angiogenesis, the formation of new blood vessels, which provides nutrients and oxygen to the growing metastatic tumors. Growth factors and cytokines present in the lung microenvironment promote the survival and proliferation of CTCs, facilitating the growth of metastatic tumors. The lung is a common site of metastasis for many types of cancer, including HCC. This organotropism is thought to be influenced by the interactions between CTCs and the lung microenvironment.
In the following discussion, we will briefly explore the pulmonary metastasis of liver cancer from the perspective of tumor inflammation and the immune microenvironment.
Differential impact of immunosuppressants on post-transplant prognosis
Currently, the main immunosuppressive agents used after liver transplantation are CNIs, such as tacrolimus, and mTORis, such as everolimus. CNIs inhibit calcineurin dephosphorylation activity, exerting its immunosuppressive effect. CNIs create a permissive environment for tumor growth and increase the risk of HCC recurrence in a dose-dependent manner10,15. In contrast, the mainstream view suggests that mTORis enable a better prognosis for patients with HCC post-liver transplantation. mTORis exhibit anti-tumor effects by inhibiting cell proliferation and angiogenesis10,14,17,18. However, some studies present an opposing view, suggesting that the use of mTORis do not impact the prognosis of transplant recipients19.
Historical investigations have revealed that elevated blood concentrations of CNIs (tacrolimus trough levels > 10 ng/mL or cyclosporine trough levels > 220 ng/mL) are associated with a 5–6-fold increase in the risk of tumor recurrence20. Most studies suggest that mTORis provide a better prognosis for patients with HCC post-liver transplantation. Three multicenter, open-label, randomized controlled trials indicated that liver transplant recipients undergoing mTORi-based immunosuppression had lower tumor recurrence rates compared to liver transplant recipients not receiving such treatment21–24. In a global study involving 525 patients, those treated with sirolimus alone or in combination had better 3-year recurrence-free survival (RFS) and 5-year overall survival (OS) than patients who were not treated with sirolimus. A retrospective clinical study from 2010 indicated that induction with anti-CD25 antibodies combined with sirolimus-based maintenance therapy was associated with improved post-transplant survival rates in HCC patients. Sirolimus showed a trend towards reduced survival rates in non-HCC recipients, suggesting that the potential benefit may be specific to cancer patients17. The most pronounced benefits were observed in low-risk patients < 60 years of age who had not received prior HCC-specific treatment. Notably, sirolimus monotherapy was more effective than combination therapy in these patients24.
mTORis provide a better prognosis for patients with HCC post-liver transplantation. The use of sirolimus improved OS 1 and 2 years after HCC recurrence following liver transplantation (P = 0.035)25. Rapamycin significantly prolongs RFS and OS compared to other immunosuppressive regimens. A prospective study published in 2018 demonstrated that the early introduction of everolimus combined with reduced-dose tacrolimus was non-inferior to standard-dose tacrolimus in terms of efficacy and renal function at 12 months. Notably, HCC recurrence was only observed in the docetaxel, doxorubicin, and cyclophosphamide (TAC)-only control group26. A retrospective study published in 2024 further confirmed this finding27. Some studies hold a negative or neutral view regarding the role of mTORis in anti-tumor activity24,28.
These findings suggest that mTORis inhibitors might improve the prognosis of liver cancer patients post-liver transplantation. It is still premature to assert that a novel post-transplantation immunosuppressive regimen primarily based on mTORis will become the mainstream approach for tumor patients in the future. Therefore, the following sections will focus on how two immunosuppressants or their receptors affect the anti-tumor immune microenvironment in the lungs. Caution is essential when translating basic research into clinical applications. Further studies are encouraged to develop international guidelines for the use of immunosuppressants in post-transplant tumor patients.
CTCs: the origin of distant metastasis after transplantation
mTORis and CNIs have opposing effects on the tumor components. mTORis can reduce the number of CTCs, while CNIs increase the number of CTCs. CTCs flow into the bloodstream from primary and metastatic lesions and provide critical information about tumor progression and metastasis29. The transformation from epithelial-to-mesenchymal cells is a crucial step in CTC-mediated tumor metastasis.
mTORis diminish the metastatic potential of CTCs
mTORis reduce the number of CTCs in the peripheral blood of cancer patients. A clinical study conducted in 2012 showed that treatment with sirolimus reduced the level of CTCs in ovarian cancer patients30. This finding was significant because CTCs are difficult to target with anti-cancer drugs due to an ability to evade immune surveillance by natural killer (NK) cells, which is caused by abnormal HLA-E levels resulting from the engulfment of platelets31. mTOR alters the malignancy of CTCs with more cells exhibiting adhesion in culture media rather than suspension growth when the PI3K/AKT/mTOR pathway is inhibited. Inhibition of the PI3K/AKT/mTOR pathway leads to a reduction in the number of CTCs in models of breast, ovarian, and colon cancer. Furthermore, combining an AKT inhibitor with an mTOR inhibitor has a greater PI3K/AKT/mTOR pathway inhibitory effect than using an mTOR inhibitor alone, providing new insights into post-liver transplantation drug use32. Therefore, we plan to conduct a multicenter clinical trial to determine if mTORi reduces CTC levels in the peripheral blood of patients after liver transplantation for HCC is warranted (Figure 1).
Lung metastasis is the most common site of metastasis in patients undergoing LT for HCC and different immunosuppressants have varying effects on post-transplant lung metastasis. CNIs promote the differentiation of monocytes in the lung microenvironment into the M2 phenotype, preventing the shift to the M1 phenotype. Additionally, CNIs simultaneously act directly on tumor cells to enhance angiogenesis and the EMT. In summary, CNIs promote processes that contribute to distant tumor metastasis. In contrast, mTORis inhibit distant tumor metastasis by promoting macrophage polarization toward the M1 phenotype, enhancing the anti-tumor functions of neutrophils and dendritic cells (DCs) and suppressing Treg differentiation. Moreover, mTORis boost neutrophil-mediated support of NK cell functions and mTORis also inhibit pro-tumor angiogenesis, reinforcing the anti-metastatic properties. This differential modulation of the tumor microenvironment highlights the opposing roles of CNIs and mTORis in cancer progression. While CNIs foster a pro-tumor environment by facilitating immune evasion and vascular development and mTORis strengthen anti-tumor immunity and inhibit angiogenesis, effectively suppressing metastasis. mTORis, mechanistic target of rapamycin inhibitors; CNIs, calcineurin inhibitors; M1, classically activated macrophage; M2, alternatively activated macrophage; EMT, epithelial-to-mesenchymal transition; DC, dendritic cell; NK cell, natural killer cell; Treg, regulatory T cell; mTORC1, mechanistic target of rapamycin complex 1; mLST8, mammalian lethal with SEC13 protein 8; PRAS40, proline-rich AKT substrate of 40 kDa.
However, CNIs increase the number of CTCs in the peripheral blood of cancer patients. Early studies reported that tacrolimus promotes tumor progression by enhancing the expression of transforming growth factor-beta 1 (TGF-β1)33. In recent years it has been shown that TGF-β acts as a tumor suppressor by inducing cell cycle arrest and apoptosis in normal and pre-cancerous cells34. However, in later stages, when cancer cells acquire oncogenic mutations and/or lose tumor suppressor gene function, cancer cells become resistant to TGF-β-induced growth arrest35. TGF-β then exerts tumor-promoting effects by stimulating tumor cells to undergo epithelial-to-mesenchymal transition (EMT)36. EpCAM serves as a key effector in this process, in which tacrolimus increases the number of CTCs in the peripheral blood of patients after transplantation (Figure 1). In contrast, it has long been known that sirolimus prevents the growth and metastatic progression of HCC and inhibits VEGF synthesis and secretion through downregulation of HIF-1α expression. In recent years it has also been shown that the HIF-1α/VEGF axis regulates HCC stemness and angiogenesis. It has also been shown that mTORI is able to inhibit HCC via this mechanism37,38 In contrast, CNIs have a diametrically opposite role because CNIs regulate IFI27/VEGFA, and therefore tumor growth, metastasis, and angiogenesis by inhibiting RCAN39. In addition, CNIs promote the CTC EMT process by activating the TGF-β/SMAD signaling pathway40,41 (Figure 2).
mTORis inhibit tumor metastasis to the lungs by suppressing EMT, tumor proliferation, and angiogenesis, whereas CNIs exert the opposite effect. (1) mTORis first binds to FKBP12, forming an mTORi-FKBP12 complex, which then inhibits mTORC1 activity. This inhibition upregulates EpCAM expression and downregulates HIF1a and VEGF activity. (2) CNIs bind to FKBP12 to form a CNI-FKBP12 complex, which inhibits calcineurin activity. CNIs increase ROS levels, thereby activating the TGF-β/SMAD signaling pathway. Inhibition of calcineurin also activates the IFI27 and VEGFA pathways. (3) mTORi-induced upregulation of EpCAM suppresses EMT, while downregulation of HIF1α and VEGF inhibits angiogenesis and tumor proliferation. Conversely, the CNI-activated TGF-β/SMAD pathway promotes EMT and the IFI27 and VEGFA pathways enhance angiogenesis and tumor proliferation. FKBP12, FK506-binding protein 12; EpCAM, epithelial cell adhesion molecule; HIF1α, hypoxia-inducible factor 1-alpha; VEGF, vascular endothelial growth factor; ROS, reactive oxygen species; SMAD, Sma and Mad related protein; TGF-β, transforming growth factor beta; IFI27, interferon alpha-inducible protein 27; VEGFA, vascular endothelial growth factor A.
Immunosuppressants affect CTCs by disrupting EMT/mesenchymal-to-epithelial transition (MET)
CTCs flow into the bloodstream from primary and metastatic lesions, providing crucial information about tumor progression and metastasis. CTCs promote tumor metastasis by undergoing EMT9,42. The switch between EMT and MET may be reversible. Contrary to the physiologic process of EMT, cells with a mesenchymal phenotype transition to an epithelial phenotype by restoring intercellular adhesion and decreasing mobility during MET, enabling the cells to establish distant colonization and metastasis43. EMT has a role in the first step of CTC formation, and after CTCs survive in circulation the CTCs undergo MET upon arrival at appropriate organ sites to acquire appropriate characteristics needed for establishing a pre-metastatic niche and promoting tumor progression.
Zheng et al.44 reported that tumor cells undergoing gradual or complete EMT (E, E/m, M/e, and M) are associated with slow proliferation, loss of expression of the epithelial cell adhesion molecule, EpCAM, and increased migration. Tumor cells undergoing EMT have enhanced intravasation into the lymphatic and vascular systems in the metastasis cascade. Tumor cells migrate into the vasculature, then become CTCs. E-type and E/m-type CTCs have enhanced adhesion and extravasation abilities to distant sites (Figure 2).
mTORis downregulate the expression of EpCAM, inhibiting the EMT/MET process in tumor cells (Figure 1). The mechanism underlying this effect may be due to the impact of mTOR inhibition on the EMT and MET processes of tumors. A study showed that downregulation of the PI3K/AKT/mTOR pathway leads to decreased expression of EpCAM, a transmembrane glycoprotein that has a role in cell adhesion, stem cell signaling, cell migration, proliferation, and differentiation, which inhibits tumor proliferation and invasion and increased sensitivity to chemotherapy45. Lipoprotein C2 is a key activating agent of lipoprotein lipase in the metabolic process and lipid metabolism has an essential role in EMT46. This role is also an important mechanism by which mTORis reduce the CTC level in patients blood, demonstrating the superiority of mTORis compared to CNIs.
In contrast, CNIs induce the EMT process in HCC cells. A 2016 study showed that tacrolimus induces EMT in human proximal tubule epithelial cells, primarily by activating the transforming growth factor-beta (TGF-β)/SMAD signaling pathway47, which also has a crucial role in EMT of HCC cells48. Moreover, a 2020 randomized clinical trial showed that patients taking tacrolimus exhibited substantially accelerated EMT progression after 3 years compared to patients receiving imatinib49.
In conclusion, the contrasting effects of mTORis and CNIs on CTCs and the EMT/MET process highlight the potential of mTORis as a superior therapeutic strategy in managing HCC post-liver transplantation49. By reducing CTC levels and inhibiting EMT, mTORis may significantly decrease the risk of metastasis and recurrence, offering a promising approach for improving patients outcomes. Further multicenter clinical studies are warranted to confirm these findings and explore the full potential of mTORis in this context.
Immunosuppressants degrade ECM
The ECM, which is composed of collagen, proteoglycans, and glycosaminoglycans, is a major component of the TME and mediates interactions between cancer and stromal cells to promote tumor seeding and progression50.
mTORis enhance the ability of tumor cells to degrade the ECM. Previous research showed that suppression of the mTOR signaling pathway mediated by SIRT1 enhances transcription factor EB (TFEB) transcription and upregulates autophagy51, thereby resulting in ECM degradation. Some studies showed that FGF21 triggers the SIRT1-mTOR signaling pathway to relocate TFEB into the nucleus, thereby attenuating ECM degradation. Tumor cells exploit invadopodia under the control of MTORC1 and targets the initiation of widespread ECM degradation via a protease-dependent process. Thus, mTORis unleash tumor cells ECM degradation aptitude, indicating limitations of anti-mTOR therapies52,53.
CNIs inhibit the generation of ECM. Research on CNIs and ECM has been ongoing for a long time54. As early as 2008 a study revealed that tacrolimus decreased ECM deposition by suppressing the expression of plasminogen activator inhibitor type 1 (PAI-1)55. The most recent research showed that tacrolimus inhibits activation of the JAK3/STAT2 signaling pathway by targeting JAK2, thereby suppressing M2 macrophages and reducing profibrotic factors56, which in turn slows the excessive proliferation of fibroblasts and the irregular remodeling of ECM. However, an early study showed that tacrolimus decreased the degradation of ECM and protected ECM integrity in vivo57, but the research objects were articular cartilage58.
Bone marrow immune cells: mediators of mTORi anti-tumor capability
The bone marrow serves as a vital source of immune cells, including macrophages, dendritic cells (DCs), and neutrophils. Immune cells contribute to the process of liver cancer lung metastasis through various functions. Macrophages are immune cells capable of engulfing and degrading pathogens and abnormal cells59. Macrophages recognize and phagocytose metastatic liver cancer cells in the lungs, eliminating the liver cancer cells by releasing lysosomal enzymes and cytokines.
DCs are a type of immune cells with antigen-presenting capabilities. DCs capture tumor antigens and present tumor cells to T cells, initiating specific immune responses to enhance immune killing of liver cancer cells. NK cells, in contrast, recognize and eliminate liver cancer cells or CTCs, inhibiting spread to the lungs. NK cells directly attack and kill tumor cells by releasing cytotoxic molecules and cytokines, reducing survival and metastatic potential. Additionally, NK cells modulate immune responses, enhance the activity of other immune cells, and coordinate immune surveillance and anti-tumor functions in the lungs.
mTORis boost DC potency
In addition to NK cells, DCs have been shown to be associated with the mTOR signaling pathway in recent years. Wang et al.58. concluded that DC cell apoptosis is reduced when treated with mTOR inhibitors and DCs derived from bone marrow mononuclear cells (BMMs) have better antigen-presenting ability. Moreover, e7-specific cytotoxic CD8+ T lymphocytes activated by these DCs exhibit greater antitumor activity60. Therefore, mTORis enhance the efficacy of tumor immunotherapy by prolonging the lifespan of DCs and improving antigen-presenting and antigen-processing capabilities.
Immunosuppressants regulate tumor-associated macrophage (TAM) differentiation
TAMs are a type of cell belonging to the monocyte-macrophage lineage that can be divided into two groups (M1 and M2). M1 macrophages are characterized by the expression of IL-1, IL-12, TNF-α, and inducible nitric oxide synthase (iNOS)59,60, which can prolong lung cancer patient survival. In contrast, M2 macrophages produce anti-inflammatory cytokines, such as IL-1061, downregulate the expression of iNOS and inhibit antigen presentation and T cell proliferation. Indeed, M2 macrophages promote the development of various tumors.
It has been shown that mTORis, such as rapamycin, induce apoptosis in M0/M2 macrophages62, reduce M2 polarization, and enhance the M1 phenotype, thereby promoting the transition of macrophages from M2-to-M1. Moreover, mTORis have been shown to suppress the expression of M2 macrophage phenotypes and the types of cytokines M2 macrophages secrete63, thereby achieving a therapeutic effect in human diseases associated with M2 macrophages.
In a recent study64 researchers identified a microRNA that reduces the activity of the REDD1/mTOR pathway in TAMs and lowers the anaerobic respiration levels when downregulated under hypoxic conditions. It was subsequently observed that the function and differentiation of M1 macrophages were inhibited64. Taken together, these findings indicate that the role of mTOR in tumors does not have a simple one-dimensional pattern. The mTOR pathway is essential for M2 macrophages but is not necessarily required for M1 macrophage survival (M1 macrophages rely on aerobic glycolysis and lipid synthesis programs, while M2 macrophages depend on increased glucose utilization, upregulation of fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS)65.
mTOR serves as a sensor for external nutrient conditions and directly or indirectly regulates cell metabolism (Figure 3). This mechanism provides an important homeostatic function to control the number and activity of M1 and M2 macrophages based on the underlying nutritional status. Therefore, mTORis significantly suppress M2 macrophages in the TME of malignant tumors, while the abundance of M1 macrophages is relatively restricted. This finding is a significant reason why mTORis should replace CNIs.
The mechanisms underlying mTOR regulation in macrophage differentiation. TLRs on macrophages activate mTORC1 via the PI3K/AKT signaling pathway. Once activated, mTORC1 inhibits PI3K/AKT activity to prevent mTORC1 hyperactivation. Additionally, PI3K/AKT activates mTORC2 via the TSC1/TSC2 complex. Activated mTORC1 serves as a peripheral sensor for oxygen, amino acids, and glucose and functions as a peripheral sensor for oxygen and glucose. Under sufficient oxygen and nutrient levels, mTORC1 and mTORC2 upregulate HIF-1α and glycolysis, thereby driving M0 macrophage differentiation toward the M1 phenotype. In contrast, under oxygen and nutrient deficiency mTORC1 and mTORC2 upregulate FAO and OXPHOS processes, thereby facilitating the differentiation of M0 macrophages into the M2 phenotype. mTORis modulate macrophage differentiation within the tumor immune microenvironment, influencing immune responses and potentially affecting tumor progression via this mechanism. mTORC2, mechanistic target of rapamycin complex 2; TLR, toll-like receptor; TSC1, tuberous sclerosis complex 1; TSC2, tuberous sclerosis complex 2; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; FAO, fatty acid oxidation; OXPHOS, oxidative phosphorylation; mSIN1, mammalian stress-activated protein kinase-interacting protein 1.
The specific impact of CNIs on TAMs is not clear but some studies indicate that CNIs inhibit the antigen-presenting function of macrophages. For example, tacrolimus has been shown to suppress cytokine production and antigen presentation functions of macrophages. However, these studies primarily focused on the role of macrophages in immune responses66,67, not the role within the TME. Currently, there is relatively limited research on the specific effects of CNIs on TAMs. Theoretically, CNIs may influence the phenotype and function of TAMs by inhibiting macrophage activation and function67, which warrants further investigation.
mTORis attenuate the inflammatory responses of neutrophils
Research from early studies revealed that mTORis, specifically EVE and SIR, exert maximal or near-maximal suppressive effects on the pro-inflammatory responses of neutrophils. However, the pro-inflammatory actions of neutrophils in cancer are multifaceted. Neutrophils secrete monocyte chemoattractant protein-1 (CCL2), CCL17, VEGF, and IL-8, which recruit monocytes and Tregs, enhance tumor angiogenesis, and mediate tumor resistance, respectively. These functions collectively support tumor progression (pro-tumor). In contrast, neutrophils stimulated by IFN-γ secrete IL-18, recruiting and further activating NK cells, thereby exerting anti-tumor effects. mTORis are most effective in reducing the release of VEGF and IL-8, thus contributing to anti-tumor activity. IL-1β, IL-6, VEGF, and GM-CSF have been shown to induce the recruitment of CD11b+ Gr1+ neutrophils in murine tumors, leading to T-cell suppression and ultimately contributing to the formation of a pro-tumor microenvironment68.
Recent investigations have shown that upon entering the circulation, CTCs are rapidly shielded by platelets, which serves as a protective mechanism. In the context of a sepsis model, it has been shown that neutrophil extracellular traps (NETs) ensnare CTCs, promoting early adhesion in metastasis, consequently facilitating tumor recurrence and metastasis. Ren and colleagues69 have shown that during hepatic ischemia/reperfusion (I/R), NETs capture CTCs more effectively than individual cancer cells, accelerating subsequent tumor metastasis. Inflammation in the liver enhances the binding of CTCs to platelets, making CTCs more susceptible to capture by NETs, which in turn promotes metastasis. The TLR4-ERK5 signaling pathway has a pivotal role in this process. A 2023 study on NETs showed that PD-L1 regulates autophagy via the PI3K/Akt/mTOR pathway, sustaining the release of NETs.
Lymphocytes, the cornerstone of the immunosuppressants mechanism
Lymphocytes are a crucial type of immune cell, including T cells, B cells, and NK cells70. Lymphocytes have distinct roles at different stages of liver cancer lung metastasis.
During the circulation and before liver cancer cells enter the lungs, activated T cells recognize and eliminate circulating liver cancer cells, exerting inhibitory effects.
Upon arrival of liver cancer cells in the lungs, lymphocytes in the lung microenvironment identify and kill these cells71–73. Specifically, activated T and NK cells have cytotoxic capabilities against tumor cells, restraining tumor growth and metastasis by releasing cytotoxic molecules or inducing apoptosis in tumor cells72.
Furthermore, T and B cells activate and modulate immune responses, enhancing immune surveillance in the lungs and further restricting the growth and metastasis of liver cancer cells.
mTORis and CNIs differ primarily in Tregs
CD8+ T cells in the immune microenvironment of lung cancer exhibit a wide range of functional dysregulation, primarily due to exclusion by tumor mechanisms74, lack of maintenance cytokines75, and the presence of excessive immune inhibitory cells72. These mechanisms lead to CD8+ T cell dysfunction, resulting in cancer cell escape.
Treg cells suppress abnormal or excessive immune responses to maintain homeostasis76. However, Treg cells often assist tumor cells evade the immune response in the immune microenvironment of the lung77. Studies have shown that Treg cells infiltrate the TME in multiple mouse and human tumors. Tregs are chemotactically attracted to the TME via chemokine gradients, such as CCR4-CCL17/22, CCR8-CCL1, CCR10-CCL28, and CXCR3-CCL9/10/11. Then, Tregs are activated and suppress the anti-tumor immune response77.
mTORis reduce the production of Tregs and enhance the function of CD8+ T cells. MTORC1 deficiencies trigger a mechanism that produces reactive oxygen species and NOS during T-cell differentiation78. This mechanism results in the breakdown of αβ T-cell differentiation and an increase in γδ T-cell differentiation (Table 1). A major study in 2023 showed that chronic T-cell activation and depletion within the effector memory compartments of CD4+ and CD8+ T-cells were decreased with sirolimus treatment. The plasma CXCL9 and CXCL10 levels were also decreased, which was consistent with these changes80 (Table 1). Recent studies have also shown that downregulating the PI3K/mTOR pathway not only inhibits the differentiation and activation of conventional T cells (Figure 4) but also reduces the formation of Tregs (Figure 4), which are known to suppress anti-tumor immune responses by producing IL-10 and blocking IL-2 (Figure 4). As a result79, mTORis prevent recurrence by reducing the number of Tregs (Figure 4).
Role of mTORis and CNIs on different T cell types
mTORis reduce the number and function of Tregs via multiple mechanisms to inhibit tumor progression. The canonical PI3K-Akt-mTORC1 signaling cascade is initiated when PI3K activates and phosphorylates downstream Akt. Activated Akt then inhibits TSC1/2, thereby lifting TSC1/2 inhibition on Rheb. The activated Rheb subsequently activates mTORC1. mTOR inhibitors regulate CD4+ T cell differentiation by inhibiting mTORC1, thereby restricting CD4+ T cell differentiation and subsequently reducing Treg populations. Additionally, mTORis inhibit mTORC1, which leads to decreased FOXP3 expression, reduced glycolysis, and enhanced fatty acid oxidation, ultimately resulting in reduced Treg populations. These Tregs promote distant tumor metastasis through increased IL-10 secretion and inhibited IL-2 production. Through this pathway of reducing Treg populations via mTORC1 inhibition, mTORis effectively inhibit tumor progression. Rheb, Ras homolog enriched in brain; Treg, regulatory T cell; CD4+ T cell, cluster of differentiation 4 T cell; Th17 T cell, T helper 17 cell; FOXP3, forkhead box P3; IL-10, interleukin 10; IL-2, interleukin 2.
CNIs have been shown to diminish the overall functionality of T cells. Tacrolimus acts on T cells by inducing cell cycle arrest at the G0/G1 phase through the expression of cyclin-dependent kinase 4 and cyclin D184, activating caspase-3 to induce cell apoptosis, leading to nuclear fragmentation. NIM811, a CsA analog that strongly binds to and inhibits cyclophilin but is devoid of calcineurin-inhibiting activity, also inhibit Treg activity81. This inhibitory effect is enhanced with prolonged exposure to tacrolimus and increased dosage. In addition to inhibiting the NF-AT pathway, tacrolimus may exert the immunosuppressive effect by blocking the activation of c-Jun N-terminal kinase and p38 pathways85 (Table 1). The tacrolimus inhibitory effect on cytokine secretion is dose-dependent. Tacrolimus also has an inhibitory effect on the expression of certain cytokine receptors and T cell activation molecules86. Tacrolimus inhibits the expression of positive co-stimulatory molecules, such as CD28 and inducible co-stimulatory molecules, while promoting the expression of negative regulatory molecules, such as cytotoxic T-lymphocyte antigen-4, mediating immunosuppression. The tacrolimus immunosuppressive effect is mainly exerted by promoting high expression of the CD1521 molecule82. CNIs inhibit the activity of calcineurin, preventing signal transduction in T cells after antigen stimulation, thereby inhibiting the activation and proliferation of CD8+ T cells (Table 1).
Recent research has shown that mTORC1 is involved in CD8+ T-cell function, while mTORC2 regulates memory function83. This suggests that changes in mTORC1 activity may affect mTORC2 activity through a mechanism that requires further investigation. Therefore, mTORis are far superior to CNIs in preventing recurrence after liver transplantation for HCC.
Immunosuppressants inhibit B cells
Several studies have suggested that B cells possess the ability to induce and sustain anti-tumor immunity87, while other studies have suggested that B cells may have pro-tumor effects due to various immunosuppressive subtypes88. It has been shown that tumor-infiltrating B cells in cancer patients present tumor antigens to CD4 T cells89, thereby promoting proliferation and differentiation. Additionally, activated B cells have been shown to directly lyse tumor cells by secreting granulysin B and TRAIL90, thereby exerting cytotoxic effects on liver cancer cells. B cells that promote tumor growth have now been defined as Bregs91, which weaken NK cell function and promote Treg function by secreting cytokines, such as IL-10. A high frequency of CD19IL-10 Bregs has also been identified in lung cancer tissues of patients92, and studies have suggested that CD19IL-10 Bregs induce tumor angiogenesis and immunosuppression through IL-1093.
CNIs also lead to the obstruction of precursor B cell differentiation. Previous studies have long been devoted to proving that compared to sirolimus94, tacrolimus does not have an inhibitory effect on B cell proliferation and differentiation95. However, in recent years it has been shown that either tacrolimus or sirolimus inhibit the differentiation of helper T cells96, leading to reduced B cell activation, although the effect of tacrolimus is weaker.
mTORis reduce the number of B cells by obstructing the differentiation of precursor B cells. Some research has shown95 that early mTOR defects lead to a decrease in the proliferation ability of B cells and a significant increase in cell death. This finding was due to the genetic ablation of raptor, a specific component of mTORC1, which hinders B cell differentiation at the pre-B cell stage, resulting in a decrease in immune responsiveness and antibody production. Recent research has also revealed the role of mTOR in B cells97,98, showing that BCR-dependent mTOR signaling is necessary for the production of high-affinity antibodies. Interestingly, while the mTOR signaling pathway regulates BCR-dependent B cell responses, the mTOR signaling pathway is dispensable for LPS-induced B cell responses.
mTORis enhance NK cell function
CNIs have been implicated in the modulation of NK cell functionality and population dynamics99. Evidence from various studies suggest that CNIs, including tacrolimus and tacrolimus, may exert inhibitory effects on NK cell cytotoxicity and proliferation. However, these effects are not uniform and can be influenced by individual variability and dosage, leading to disparate findings across different studies100. CNIs may also exert influence over the number of NK cells. Long-term administration of CNIs has been associated with a decrease in the NK cell count in some patient cohorts. However, the clinical implications of this observation remain ambiguous, given that the NK cell count do not always correlate directly with the functional capacity. In essence, the interplay between CNIs and NK cells represents a multifaceted issue, warranting further investigation for a comprehensive understanding101. Sirolimus enhances NK cell function by modulating the mTORC1 and mTORC2 pathways. Li et al.99 demonstrated that mTORC2 negatively regulates NK cell function mainly by inhibiting the signal transducer and activator of transcription 5 (STAT5)/solute carrier family 7 member 5 (SLC7A5) axis. In contrast, mTORC1 positively regulates NK cell activity by maintaining the CD2-mediated interleukin (IL)-12 signaling pathway101.
Systemic effects of mTORis and CNIs
Renal protective effects of mTORis
Inhibition of mTOR by sirolimus has a protective effect on the kidneys. mTOR signaling is involved in cellular metabolism and proliferation, including kidney cells102,103. By reducing mTOR activity, sirolimus helps prevent hyperproliferation and fibrosis in the renal tissues, thereby reducing nephrotoxicity103. This mechanism explains why sirolimus is considered renal-sparing compared to other immunosuppressants104,105.
However, mTOR inhibition also impacts other tissues. For example, the mTOR pathway regulates lipid metabolism105, so mTOR pathway inhibition by sirolimus leads to hyperlipidemia106. The pathway role in cellular growth and repair also explains the sirolimus side effects, such as delayed wound healing and pulmonary toxicity7,106.
Nephrotoxicity of CNIs
The calcineurin pathway is also active in renal cells, especially in regulating the balance of electrolytes and the function of renal tubular cells107,108. Inhibiting this pathway with tacrolimus disrupts these processes, leading to nephrotoxicity. The chronic vasoconstriction and interstitial fibrosis induced by calcineurin inhibition are key contributors to the long-term kidney damage associated with tacrolimus109.
CNIs also affect neurologic tissues, leading to the neurotoxic side effects of tacrolimus, such as tremors and seizures110,111. Additionally, calcineurin has a role in regulating vascular tone and calcineurin inhibition can contribute to hypertension and associated cardiovascular risks112,113.
Anti-tumor effects of mycophenolate mofetil (MMF)
Although the use of MMF in organ transplantation is not as widespread as CNIs and mTORis, MMF remains an important source of immunosuppressive therapy. Some aforementioned clinical trials involved the combined use of MMF and several clinical studies have demonstrated the anti-tumor effects of MMF114. Both in vivo and in vitro experiments have shown that MMF inhibits HCC. This effect may be due to the MMF ability to upregulate two p53-inducible genes (TP53I3 and TP53INP1) as well as the p53 protein114–116. However, these data are complicated by the fact that MMF is often used in combination with other immunosuppressants19. Therefore, the design of new transplant protocols involving different combinations of immunosuppressants for tumor patients must be approached with caution. Efforts should focus on finding an optimal balance between the degree of immunosuppression and the risk of tumor recurrence.
Summary and outlook
Transplant oncology is an emerging field that addresses oncologic challenges in transplantation. Because technologies have rapidly advanced in recent years, research on the immune microenvironment of cancer metastasis has experienced explosive growth, which has also brought vitality to the development of transplant oncology. Colorectal cancer liver metastasis and prostate cancer bone metastasis have been focal points of recent research endeavors. In the context of liver cancer post-liver transplantation, pulmonary metastasis not only shares molecular mechanisms akin to these metastases but also has several distinctive features. First, the surgical procedure induces stimulation in the lungs, leading to a heightened inflammatory state. Second, the postoperative utilization of immunosuppressive agents is noteworthy, Moreover, alterations in the unique hemodynamics of the liver during surgery may have a significant role in promoting pulmonary metastasis117. Given that the lungs are organs with significant immune cell infiltration, the profound alterations in the microenvironment due to immunosuppressants are self-evident. Lung tumor metastasis is more common in patients who have received liver transplantation than in those who have undergone liver resection. In addition to baseline factors, such as tumor staging, the reasons lung tumor metastasis is more common in patients who have received liver transplantation may include the use of postoperative immunosuppressive agents, ischemia-reperfusion injury during liver transplantation, and abnormal inflammatory signal pathway activation caused by immune rejection reactions of allogeneic liver.
The clinical use of immunosuppressants in oncology patients currently faces two major challenges (balancing dosage to prevent both recurrence and rejection, and mitigating the side effects of these drugs). We believe that with the advances in genomics and metabolomics, precision medicine offers new hope for individualized immunosuppressive therapy. Genetic testing can predict a patient’s ability to metabolize drugs, like tacrolimus or sirolimus, enabling the development of more comprehensive immunosuppressive management and monitoring systems118,119. These systems should ensure optimal therapeutic drug levels, minimizing the risks of both tumor recurrence and organ rejection. By integrating advanced monitoring technologies with personalized treatment strategies, we can move closer to long-term success in liver transplantation for HCC patients, ultimately improving patient outcomes and quality of life.
Combining multiple immunosuppressive agents has emerged as a promising approach to managing immunosuppressant side effects. Studies on the combination of tacrolimus and sirolimus suggest that this strategy may reduce tumor recurrence rates while mitigating the toxic effects of monotherapy. Additionally, the development of new immunomodulators used in combination with existing drugs holds potential to further optimize long-term outcomes in transplant recipients. The development of novel immunosuppressants is also crucial.
Looking ahead, the creation of more targeted immunosuppressive delivery systems, fueled by advances in materials engineering, will be pivotal in enhancing the precision of post-transplant care in HCC patients.
Therefore, this review described the composition of the pulmonary immune microenvironment and the effects of two mainstream post-liver transplantation immunosuppressive agents on various components in this environment. The purpose of this review was to provide suggestions for future research on immunosuppressive agents and the development of drug regimens.
Conflict of interest statement
No potential conflicts of interest are disclosed.
Author contributions
Wrote the manuscript: Jinyan Chen, Huigang Li, Jianyong Zhuo.
Made the figures: Zhihang Hu, Rongsen Wang, Yiru Jin, Jiancai Sun, Zuyuan Lin, Zhanyi Lin.
Revised the manuscript: Xiang Wu, Chiyu He, Renyi Su, Xuyong Wei, Yiyang Sun.
Reviewed the literature: Shusen Zheng, Di Lu, Xiao Xu.
Acknowledgments
We thank all members of the Key Laboratory of Integrated Oncology and Intelligent Medicine of Zhejiang Province for helpful advice about the manuscript.
Footnotes
↵*These authors contributed equally to this work.
- Received July 28, 2024.
- Accepted November 25, 2024.
- Copyright: © 2024 The Authors
This work is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License.
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In this issue
Jump to section
- Article
- Abstract
- Introduction
- Lung microenvironment: the soil of HCC pulmonary metastasis
- Differential impact of immunosuppressants on post-transplant prognosis
- CTCs: the origin of distant metastasis after transplantation
- Bone marrow immune cells: mediators of mTORi anti-tumor capability
- Lymphocytes, the cornerstone of the immunosuppressants mechanism
- Systemic effects of mTORis and CNIs
- Anti-tumor effects of mycophenolate mofetil (MMF)
- Summary and outlook
- Conflict of interest statement
- Author contributions
- Acknowledgments
- Footnotes
- References
- Figures & Data
- Info & Metrics
- References
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