At present, a large number of studies have confirmed that thymosin drugs in combination with other drugs or monotherapy have the effect of inhibiting the growth of LC (28–30, 33–35). Both in vitro and in vivo experiments have demonstrated that Tα1 monotherapy can significantly inhibit the growth of NSCLC (28). Chemoprevention, a method, and strategy to reduce cancer incidence and mortality, is defined as the use of natural or synthetic substances, biological agents, and other pharmacological agents in the early stages of cancer to achieve the purpose of impeding, arresting, or even reversing the development of cancer (41). Chemoprevention has a wide range of applications in the field of LC and is one of the potential therapeutic options to reduce the risk of LC (42). According to studies, Tα1 can be used as a chemopreventive drug that can effectively prevent the occurrence of LUAD, and Tα1 also has a direct inhibitory effect on the growth of LUAD (29, 30). In addition to the monotherapy of thymosin drugs, many scholars have also paid attention to the effect and mechanism of thymosin drugs combined with other drugs on LC, these studies shown that Tα1 in combination with interferon (IFN) α/β or interleukin-2 (IL-2) can inhibit LUAD growth and that Tα1 has a synergistic effect with IFNα/β or IL-2, and the combination has a better effect of inhibiting tumor growth than Tα1 monotherapy (33–35). The dysregulation of the JAK/STAT pathway is closely related to LC and other cancers, STAT3 plays an important role in the JAK-STAT signaling pathway, and STAT3 is primarily involved in tumor growth, differentiation, migration, and invasion regulation (43, 44). A kind of matrix metalloproteinase named MMP2 can degrade type IV and type V collagen and elastic fibers, thereby destroying the basement membrane and extracellular matrix, and promoting tumor invasion and metastasis (45). Bo et al. discovered that Tα1 inhibited the transcription of MMP2 by inhibiting phosphorylation of STAT3, resulting in a decrease in MMP2 protein expression and inhibiting the migration and invasion of NSCLC cells with programmed cell death ligand 1 (PD-L1) high-expressing, which suggests that Tα1 may inhibit the STAT3-MMP2 signaling pathway by stimulating PD-L1 (31). One of the main components produced by oxidative stress in the body is reactive oxygen species (ROS), which has the role of driving the development of cancer and other diseases, and the increase of ROS can promote the growth, migration, and invasion of tumors (46). ROS is also one of the main causes of LC occurrence and progression (47). A study has confirmed that Tα1 has the effect of inhibiting the proliferation and migration of LUAD cells, and Tα1 also can inhibit the antioxidant activity of LUAD cells by reducing ROS levels and increasing the activity of antioxidant enzymes like catalase, superoxide dismutase, and glutathione peroxidase (32). Thymosin β4 (Tβ4) is also thought to be associated with LC in addition to Tα1. Tβ4, a polypeptide with 43 amino acids that is distributed in various tissues of the human body and is one of the main actin regulators in the human body, makes up the largest proportion of β-thymosin (48, 49). According to Huang et al., Tβ4 was overexpressed in NSCLC, and after Tβ4 was silenced, the activation of Notch1 was inhibited, which inhibited the proliferation and migration, and invasion of LUAD cells (40).

Chemotherapy is a common treatment for patients with advanced LC and those with early-stage, high-risk LC (69, 70). Compared to the control group of NSCLC patients receiving only ifosfamide chemotherapy, patients receiving Tα1 and low-dose interferon-α in addition to chemotherapy had higher response rates (33% vs. 10%) and longer times to progression (TTP) (p=0.0059), according to a phase II clinical study. In the chemotherapy group, CD4⁺, CD8⁺, and NK cell counts significantly declined. Combination therapy with Tα1 reduced the toxicity of chemotherapy while leaving lymphocyte counts essentially unchanged from before chemotherapy (62). The effectiveness of thymosin in combination with chemotherapy was assessed in a meta-analysis of 10 randomized controlled trials (RCTs) in a total of 724 patients with advanced (stage III, IV) NSCLC regimens, primarily cisplatin plus vinorelbine (NP) regimens and gemcitabine plus cisplatin (GP) regimens. Thymosin plus NP was found to increase overall response rate (ORR) (odds ratio (OR) 1.86) compared with the control group using NP-only chemotherapy, and the addition of thymosin also increased tumor control rate (TCR) (OR 3.06), 1-year survival rate (OR 3.05) and improved the quality of life (QOL) (OR 3.39). Thymosin and GP-containing chemotherapy showed superior ORR (OR 1.67), TCR (OR 2.38), and QOL (OR 3.84) compared to GP-only chemotherapy. These findings imply that for patients with advanced NSCLC, thymosin plus NP/GP chemotherapy is preferable to NP/GP chemotherapy alone (63). Another meta-analysis included 27 RCTs involving a total of 1925 patients with stage IIIa-IV NSCLC, of whom 976 received combination therapy with synthetic thymic peptides (sTPs) (including Tα1 and thymopentin) and chemotherapy and 949 received chemotherapy alone to evaluate the clinical effects of sTPs plus chemotherapy (64). In comparison to chemotherapy, the use of sTPs significantly improved QOL [relative risk (RR) = 2.05, P<0.00001], ORR [RR = 1.28, P = 0.0001], and one-year overall survival (OS) rate [RR = 1.43, P = 0.001], as well as increased disease control rate (DCR) [RR = 1.10, P = 0.02]. The combination of sTPs and chemotherapy demonstrated that the combination therapy was clinically effective. Combination therapy with sTPs and chemotherapy increased CD3⁺T cell levels [P<0.00001], CD3⁺CD4⁺T cell levels [P<0.00001], NK cell numbers [P<0.00001], and the proportion of CD4⁺/CD8⁺T cells [P<0.00001] at the lymphocyte level, and it can lessen the immunotoxic effects of chemotherapy when combined with sTPs. Additionally, the risk of chemotherapy-related side effects like neutropenia (RR=0.75, P=0.04), thrombocytopenia (RR=0.68, P=0.0002), and gastrointestinal reactions (RR=0.62, P<0.00001) was decreased by sTPs. To compare the two sTPs of Tα1 and thymopentin, further subgroup analysis was done. The results showed that Tα1 could significantly increase ORR [RR: 1.32, P=0.0003] and DCR [RR: 1.11, P=0.02], but that there were no statistically significant differences in OR and DCR in the thymopentin subgroup. Tα1 and thymopentin could also increase the level of peripheral blood lymphocytes. It was demonstrated that Tα1 could boost clinical effectiveness while also boosting immune function.

Although radiation therapy is one of the most effective treatments for advanced lung cancer, it invariably results in side effects, such as immune response (71). An RCT study analyzed the effect of synthetic Tα1 on immune recovery properties in NSCLC patients after radiotherapy (65). The findings revealed that thymosin treatment following radiotherapy significantly improved OS (P=0.009) and disease-free survival (DFS) (P=0.04) compared with placebo, and thymosin restored the decrease in T helper cell levels brought on by radiotherapy (p=0.04). This proved that Tα1 can both enhance the efficacy of radiotherapy and reverse immunosuppression brought on by radiotherapy. The experimental group received two regimens of subcutaneous injection twice a week with Tα1 900 mg/m2 (body surface area) or continuous thymosin 900 mg/m2 subcutaneous injection once daily for 14 days, followed by twice-weekly subcutaneous injection. The study evaluated the difference in efficacy between different dosing regimens, with the control group receiving a placebo subcutaneous injection regimen. The results showed that the control group showed shoddy results. In the experimental group, there was no discernible difference between the two regimens, indicating that both regimens might be practicable.

For stage III unresectable NSCLC, consolidation immunotherapy followed by concurrent chemoradiotherapy (CCRT) is the gold standard of care (72). Radiation pneumonitis (RP) and pulmonary fibrosis are the two stages of radiation lung injury (RILI), a common side effect of radiation therapy (RT) in the chest (73, 74). One of the major barriers to the use of consolidation immunotherapy following CCRT is RP, which has a detrimental effect on survival and quality of life in patients with locally advanced NSCLC (LANSCLC). The GASTO-1043 study, a Phase II clinical trial, was explored the efficacy of Tα1 in the treatment of RP in patients with LANSCLC receiving CCRT (66). 138 patients with LANSCLC in total were enrolled in the study, 69 of whom received CCRT alone in the control group and 69 of whom received Tα1 treatment from the start of CCRT to 2 months after it ended in the experimental group. Compared to the control group, the experimental group had a lower incidence of grade 2 or higher RP (36.2% vs. 53.6%, P=0.040), lower incidence of G3 to G4 lymphopenia (19.1% vs. 62.1%, P<0.001), and significantly higher total lymphocyte count (TLC) (0.51 k/mL vs. 0.30 k/mL, P<0.001). Patients with maximum C-reactive protein (CRP) ≥ 100 mg/L (13.8% vs. 29.7%, P = 0.029) and patients with 2 to 3 weeks during CCRT CRP ≥ 10 mg/L (23.5% vs. 48.5%, P = 0.003) were significantly less common in the experimental group. Patients with LANSCLC who used Tα1 during and after CCRT showed lower rates of grade 2 RP and G3 to G4 lymphopenia compared to the control group, as well as higher TLC levels and lower CRP, demonstrating that Tα1 can treat side effects and immune dysfunction brought on by CCRT. Additionally, patients receiving Tα1 had a longer median progression free survival (PFS) (14.4 months vs. 10.7 months, P=0.131) and a longer median OS (34.8 months vs. 28.7 months in the control group, P=0.062). Despite being superior to the control group in both OS and PFS, however, the Tα1 experimental group did not achieve a statistically significant difference, which may be related to the smaller sample size.

Patients with LUAD who have an EGFR target mutation that is positive can benefit from EGFR-tyrosine kinase inhibitors (TKI) targeted therapy, which is now one of the standard treatments for LC (75). In patients with IV-stage LUAD and EGFR mutations, some researchers have compared the clinical effects of EGFR-TKI monotherapy and EGFR-TKI plus thymosin therapy (67). To balance the baseline differences, propensity score matching (PSM) was carried out in a 2:1 ratio between the control group and the experimental group, with 130 LUAD patients being treated with first-generation EGFR-TKI (gefitinib/erlotinib/icotinib) and as part of the experimental group 65 LUAD patients being treated with EGFR-TKI plus thymosin (including Tα1 and thymopentin). The results revealed that the median PFS (14.4 months vs. 9.2 months, HR=0.433, P<0.0001) and median OS (29.5 months vs. 19.8 months, HR=0.430, P<0.0001) in the EGFR-TKI plus thymosin group were both significantly longer than those in the EGFR-TKI group, respectively. These findings demonstrated that EGFR-TKI plus thymosin was extremely effective in lowering the risk of relapse and improving prognosis in patients with LUAD compared with EGFR-TKI alone. The safety of EGFR-TKI plus thymosin was acceptable because there was no significant difference in ORR and DCR between the two groups, nor was there any significant difference in side effects. The number of CD3⁺T cells and the number of CD3⁺CD4⁺T and CD3⁺CD8⁺T subsets after treatment were significantly reduced in the EGFR-TKI monotherapy group (P<0.05), but there was no difference between these parameters in the EGFR-TKI plus thymosin group, indicating that the addition of thymosin based on targeted therapy has some effect on restoring the body’s immune activity. Further subgroup analysis revealed that in most subgroups, including age, EGFR mutation type (exon 19 deletion or exon 21 L858R mutation), presence or absence of bone metastases, central nervous system metastases, adrenal metastases, multiple lung metastases, pleural metastases, gefitinib or erlotinib, EGFR-TKI plus thymosin demonstrated a superior PFS advantage over EGFR-TKI monotherapy. There was no statistically significant difference in OS or PFS between the EGFR-TKI plus thymosin group and the single-agent group in patients with ECOG score of 2 who had liver metastases, received icotinib, and received EGFR-TKI as second-line therapy. Male patients could benefit from combination therapy in OS, and patients with LUAD who received radiotherapy solely for bone metastases benefited from combination therapy in PFS(P=0.041), while patients with LUAD who received radiotherapy for the remaining causes did not benefit in terms of OS or PFS. A comparison of the two thymosin medications revealed no statistically significant difference in PFS or OS between Tα1 and thymopentin, suggesting that the use of both thymosin medications in combination with an EGFR-TKI may be possible.

Because it boosts human immunity and enhances patients’ quality of life after LC surgery, Tα1 is frequently used in clinical practice as adjuvant therapy for postoperative LC patients. The long-term survival effects of Tα1 as postoperative immunomodulatory therapy were examined in a large, real-world study involving 5746 patients with NSCLC who underwent surgery and achieved R0 resection (68). The study compared 1027 operable (stage I–IIIA) NSCLC patients who received thymosin treatment to 1027 patients who did not receive thymosin treatment using PSM analysis. When compared to the control group without Tα1 treatment, the use of Tα1 in postoperative patients can lower the risk of recurrence and death in NSCLC patients. Even after multivariate analysis, Tα1 treatment still showed very significant differences in DFS (HR=0.655, P<0.0001) and OS (HR=0.548, P<0.0001). Additionally, following PSM, the Tα1 treatment group outperformed the control group in terms of 5-year DFS (77.3% vs. 64.7%, P<0.0001) and 5-year OS (83.3% vs. 72.7%, P<0.0001). Tα1 therapy may be an efficient postoperative adjuvant therapy in the early stage, especially in patients with stage IA NSCLC, as evidenced by the fact that the benefits of OS and DFS in patients with stage IA were more advantageous than those of all other stages in terms of staging (P<0.0001). According to pathological classification, other non-adenocarcinoma and non-squamous cell carcinoma subtypes were independently linked to worse OS (HR=2.019, P=0.0009) and DFS (HR=1.706, P=0.0038). The results of the subgroup analysis showed that patients who used Tα1 for more than 24 months had the best OS and DFS efficacy. The treatment effect was proportional to the length of treatment, with the best effect for more than 24 months, followed by treatment for 12–24 months, and the worst effects for less than 12 months. The 5-year DFS rates of the three groups were 84.7%, 81.0%, and 66.1%, respectively, and the 5-year OS rates were 92.2%, 83.7%, and 64.5%. This suggests that to reap the greatest benefits, patients with NSCLC may use Tα1 for longer than 24 months. Tα1 could show efficacy in OS and DFS in patients of any age, sex, Charlson Comorbidity Index (CCI), smoking status, and any stage of pathologic TNM, as well as patients with non-squamous cell carcinoma NSCLC who did not receive targeted therapy and BMI<28 kg/m². This was confirmed by subgroup analysis. Tα1 only demonstrated OS efficacy in postoperative NSCLC patients who received chemoradiotherapy, and it did not affect OS or DFS in patients with a BMI>28 kg/m², the pathological type of squamous cell carcinoma, targeted therapy, or combination therapy (including targeted therapy plus chemotherapy, targeted therapy plus chemoradiotherapy). Tα1 can be used as an adjuvant therapy after surgery to lower the risk of recurrence and increase the long-term survival of LC patients as well as an immunomodulatory therapy to enhance the postoperative quality of life of LC patients.

Immunotherapy, an emerging therapeutic approach in recent years, has not only improved patient prognosis but also significantly altered the initial treatment regimen of LC patients. In the field of LC, immune checkpoint inhibitors (ICIs) that represent PDL1/PD-1 have the most applications and the best efficacy (76). The gold standard for immunotherapy in patients with advanced NSCLC is high PDL1 expression (PD-L1 Tumor Proportion Score 50%). Patients with NSCLC with high PDL1 expression can achieve better immunotherapy outcomes (77), this has also been confirmed by many RCTs (78, 79). Patients with high PDL1 expression in their NSCLC may respond better to immunotherapy. After long-term follow-up, a study of patients with metastatic melanoma who were given Tα1 discovered that those who also received the CTLA-4 ICI ipilimumab had an advantage in overall survival (80), this suggests that Tα1 and ICIs work in concert. It is expected that the combination of Tα1 and ICIs, represented by PD-1 inhibitors, may also have a beneficial synergistic effect on the treatment of LC. The prognosis of LC patients can be significantly improved by ICIs, but their clinical use is constrained by a few drawbacks. First off, a large number of LC patients are unable to respond to ICIs treatment effectively because some LC patients belong to cold immune tumors (such as immune-desert type and immune-excluded type), which lack lymphocyte infiltration (81, 82). If cold immune tumors can be transformed into hot immune tumors, this will greatly expand the clinical application range of ICIs and increase their clinical efficacy. The degree of immune cell infiltration in the tumor microenvironment (TME) is the primary distinction between cold immune tumors and hot immune tumors. NK cells and CD3⁺, CD4⁺, and CD8⁺T lymphocytes are currently thought to be the primary immune cells in the TME, with CD8⁺T lymphocytes playing a significant role in tumor killing (83, 84). Tα1, as an immunomodulator of thymic hormones, belongs to active immunity (18). As previously stated, using Tα-1 to treat LC can increase CD8⁺T cell counts, restore NK cell activity, and activate T lymphocyte immune function by increasing CD86 expression. When chemotherapy was combined with thymosin, more CD4⁺, CD8⁺, CD3⁺, and NK cells were present. Additionally, the CD4⁺/CD8⁺T cell ratios were higher than when chemotherapy was used alone. Thymosin also has the effect of increasing the helper T cell counts that have been decreased as a result of radiotherapy. Thymosin was also added to address the negative effects of EGFR-TKI treatment on the quantity of CD3⁺T cells produced by LC patients as well as the number of CD3⁺CD4⁺T and CD3⁺CD8⁺T subsets. In addition to improving Tα1 targeting, Tα1-RGDR promoted CD4⁺ and CD8⁺T cell infiltration of LC tissues and increased the immune activity of T cells by raising levels of IL-2 and IFN-γ. These findings imply that Tα1 can increase CD4⁺T, CD8⁺T, CD3⁺CD4⁺T, and NK cell numbers in LC patients. Tα1 has the potential to convert LC patients with cold immune tumors to hot immune tumors by increasing the cellular level and cell activity of immune cells like CD8⁺T cells, enhancing the clinical effectiveness of ICIs. Similarly, Tα1 can inhibit the migration and invasion of NSCLC with high expression of PD-L1 (31) and a favorable effect on immune cells, suggesting that the combination of Tα1 and ICIs may enhance the clinical effect of ICIs and even increase the proportion of LC patients who have an effective response to ICIs. Second, immunotherapy with ICIs is associated with adverse effects such as colitis and/or diarrhea (85). Studies have shown that Tα1 can enhance the small intestine tissue structure and barrier function of mice with cystic fibrosis (86). Additionally, a study has shown that Tα1 can successfully prevent ICIs-induced colitis in preclinical models by fostering the immune pathway of indoleamine 2,3-dioxygenase 1-dependent tolerance (87). These studies demonstrate that Tα1 has reduced the side effects of ICIs on the digestive system.