Cancer is one of the leading causes of mortality, accounting for ~10 million annual deaths worldwide. According to the latest report, it was estimated that annually, there were ~4,064,000 new cases and ~2,413,500 cancer-associated deaths in China (1). In the US, there were ~1,958,310 new cancer cases and ~609,820 cancer-associated deaths in 2023 (2). In addition, cancer also negatively affects a country's economic growth; the estimated global economic cost of cancer between 2020 and 2050 is $25.2 trillion (3). Of note, the burden of cancer shows a continuously increasing trend worldwide, and as such, cancer prevention and control are becoming an ever more prominent subject. However, the efficacy of treatments with traditional therapeutic strategies is limited, and thus, there is an urgent need to develop novel approaches for cancer treatment.

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary success in cancer-adoptive cellular immunotherapy. Adoptive cellular immunotherapy is a promising strategy for curing cancer completely. Numerous remarkable clinical responses have been observed in treating certain subsets of B-cell leukemia or lymphoma with CAR-T cells; however, several challenges limit their therapeutic efficacy in solid tumors (4). Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cellular immunotherapy in which TILs are harvested from tumor tissues. After amplification in vitro, TILs are infused back into a patient and exert a specific killing effect on cancerous cells (Fig. 1). Since TILs are autogenous lymphocytes that are not genetically modified, they exhibit T-cell receptor (TCR) clone diversity, superior tumor-homing ability and low off-target toxicity, and thus, there are scarcely any adverse reactions. Therefore, TIL-based therapy offers unique advantages in treating solid tumors compared with other adoptive cellular therapies (5,6).

However, in the tumor microenvironment, given the antigen heterogeneity and tumor evolution, the application of TIL-based therapy is still limited. In the present review, the fundamental studies on TIL-based therapy and clinical trials on improving the therapeutic effect of TILs are summarized. Furthermore, the current limitations, challenges and opportunities of TIL-based therapy are also discussed. Finally, the future developmental directions of next-generation TIL-based therapy are highlighted.

In 1980, Yron et al (7) reported that lymphoid cells in suspension derived from tumor tissues were able to expand and grow continuously in the presence of T-cell growth factor without tumor cell proliferation. These lymphoid cells exhibited significant cytotoxic activity towards syngeneic tumor cells and normal fibroblasts grown in culture but did not lyse normal lymphoid cells. After 6 years, they reported a novel approach by adopting immunotherapy towards cancerous cells using TILs. Their results showed that in combination with cyclophosphamide, treatment with TILs and IL-2 cured all mice bearing MC-38-induced colon adenocarcinoma of advanced hepatic metastases, and up to 50% of mice were cured of advanced pulmonary metastases (8). These data provide a rationale for the use of TILs in the treatment of patients with advanced cancer.

In 1988, Topalian et al (9) performed a pilot study to investigate the feasibility and practicality of administering IL-2-expanded TILs in patients with metastatic cancer; two partial responses to therapy were observed and one additional patient with breast cancer experienced a partial regression of disease. In five of six patients with melanoma, TILs demonstrated lytic activity specific to the autologous tumor target. No toxic effects were observed that were directly attributable to TIL infusions. This study represents an initial attempt to use TILs as a means of immunotherapy in patients with cancer. Similarly, in 1994, Rosenberg et al (10) reported that treatment with TILs and IL-2, with or without cyclophosphamide, resulted in objective responses (ORs) in approximately one-third of patients with metastatic melanoma, and the treatment was safely administered. These results demonstrate the practicality of TILs for the treatment of patients with melanoma.

In 2008, Tran et al (11) developed an alternative ‘young’ TILs method; this approach used tumor tissue to rapidly expand TILs for administration without testing for tumor recognition. These younger TILs exhibited higher levels of antigen reactivity, longer telomeres and higher levels of CD27 and CD28. This strategy may facilitate the widespread application of TILs in tumor immunotherapy. In 2011, Itzhaki et al (12) described an efficient and reliable method for generating young-TILs for adoptive transfer therapy. Treatment with these young TILs resulted in an OR rate (ORR) of ~50% in patients with refractory melanoma (12). Thus, this approach may be adopted for the treatment of other types of malignancies.

In 2012, Jin et al (13) described a modified method for the initial culture and rapid expansion of TILs in gas-permeable flasks. TIL initiation and rapid expansion procedure in gas-permeable G-Rex flasks required fewer total vessels, less media, less incubator space and less labor than other approaches. In addition, TIL culture in G-Rex flasks facilitated the production of TILs for the treatment of patients. In the same year, Friedman et al (14) found that at least 20% of metastatic melanomas contained CD4⁺ lymphocytes with specific tumor recognition by interferon-γ release assay. After lymphodepletion, a patient with widespread metastatic disease was administered TILs containing human leukocyte antigen (HLA) class II-restricted tumor activity with high-dose IL-2 therapy that mediated the regression of extensive metastatic disease in the liver and spleen. These results suggest a possible role for CD4⁺ cells in the effectiveness of adoptive cell therapy.

In 2014, Tran et al (15) used a whole-exome sequencing-based method to demonstrate that TILs from a patient with metastatic cholangiocarcinoma contained CD4⁺ T helper type 1 (Th1) cells recognizing a mutation in the erb-b2 receptor tyrosine kinase 2 interacting protein frequently expressed in cancer. Adoptive transfer mutation-specific polyfunctional CD4⁺ Th1 cells may help a patient achieve prolonged stabilization of disease. Patients with cancer progression treated with CD4⁺ Th1 cells have also been shown to exhibit tumor regression. These results provide novel evidence that CD4⁺ T cells recognize and respond to mutated antigens, and this can be exploited to promote the regression of metastatic epithelial cancer.

In 2015, Zhang et al (16) genetically engineered TILs to secrete single-chain IL-12 selectively at the tumor site. The IL-12 encoding gene was driven by a nuclear factor of the activated T-cell promoter. In this first-in-man trial, administration of genetically engineered TILs mediated tumor responses in the absence of IL-2 administration using cell doses 10- to 100-fold lower than conventional TIL-based treatment. However, due to the toxicity of secreted IL-12, further improvements are necessary before this approach can be safely used in the therapy of patients with cancer. In addition, Beane et al (17) used zinc finger nucleases (ZFNs) directed against the gene encoding human programmed cell death 1 (PD-1) to genetically edit melanoma TILs, which resulted in a 76% reduction in PD-1 surface expression. Of note, the genetically edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile compared to unmodified TILs. Furthermore, all donor cells displayed an effector memory phenotype and expanded on a large scale in vitro. However, the efficiency and safety of PD-1 gene-edited TILs for the treatment of metastatic melanoma requires further study.

The adoptive transfer of neoantigen-reactive TILs can result in tumor regression in patients with cancer. Thus, neoantigen-specific TCR identification is a key technology for improving the clinical efficacy of TILs. In 2017, Parkhurst et al (18) identified 27 TCRs from 6 patients that specifically recognized 14 neoantigens expressed by autologous tumor cells. In 2018, Lu et al (19) developed a new TCR identification approach, which included the co-culture of TILs with tandem minigene-transfected or peptide-pulsed autologous antigen-presenting cells and identification of paired TCR sequences using single-cell RNA sequencing analysis. Transduced T cells with these TCRs specifically recognized the neoantigens. This strategy provides an efficient procedure to identify neoantigen-specific TCRs for use in future clinical applications for patients with cancer.

In 2019, Nguyen et al (20) used a modified TIL protocol with a lower dose of IL-2 in a single-institution phase II clinical trial in patients with unresectable, metastatic melanoma. Their results showed that this novel protocol of low-dose IL-2 following adoptive cell transfer of TILs was feasible and clinically effective (20).

In 2020, Krishna et al (21) identified a memory-progenitor CD39-negative stem-like phenotype (CD39⁻CD69⁻) by using high-dimensional analysis of human adoptive cell therapy products. These TILs were associated with complete cancer regression and TIL persistence. In addition, these TILs were capable of self-renewal, expansion, persistence and a superior antitumor response in vivo.

In 2021, Sinicrope et al (22) reported that lower circulating adiponectin levels were not only associated with an increase in TIL densities in colon cancer but also with an enhanced antitumor immune response.

In 2022, Chamberlain et al (23) used clustered regularly interspersed short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) gene editing to knock out PD-1 in TILs; an 87.53% reduction in cell surface PD-1 expression was observed, and PD-1 knockout did not affect the final fold expansion of TILs. These results demonstrated that a non-viral, non-plasmid-based CRISPR-Cas9 gene editing method can be feasibly adopted into a TIL-based adoptive cell transfer therapy protocol to produce treatment products without any evident negative effects.

In 2023, Forsberg et al (24) produced CAR-TILs through transducing a lentiviral vector encoding an anti-HER2 CAR construct. CAR-TILs were able to eradicate melanoma in patient-derived xenograft mouse models, even in the absence of antigen presentation by HLA. Furthermore, the tolerable and anti-tumor activity of CAR-TILs was also observed in four companion dogs. These results demonstrated that CAR-TIL therapy is a promising approach for improving the tumor-targeting capacity of TILs.

Representative techniques and significant findings in the development of TIL-based therapies are summarized in Fig. 2.

TCRs are heterodimers comprised of an α and β chain; the TCR repertoire is highly diverse and allows T cells to specifically recognize multiple types of major histocompatibility complex I-presenting tumor antigens. After interaction with tumor antigens, TCRs complex with CD3ϵ/γ/δ/ζ subunits to ensure signal transduction and drive the antigen-specific immune response to the cancer cell (25,26).

The TCR and CD3 complex transduces extracellular signals to intracellular effectors and thus has an irreplaceable role in the activation of TILs and their anti-tumor properties. The activated TILs secrete various cytokines to improve the killing effect on tumor cells. For example, activated TILs upregulate the expression of CD107a on the cell surface, which is associated with cytotoxity and degranulation. Furthermore, the cellular signals increase the release of IFN-γ, a key effector of TIL anti-tumor activity (27). Importantly, IFN-γ can induce apoptosis of both Fas-high and Fas-low cancer cells by upregulating apoptosis-related proteins, such as caspase-3, Bak and Fas (28). Activated TILs produce more TNF-α, perforin and granzyme, which further enhance the anti-tumor function of TILs (29–31). TNF-α initiates cancer-cell apoptosis through p53-dependent pathways by upregulating death receptor-4 and death receptor-5 (32). Interestingly, TNF-α can also improve the sensitivity of cells to Fas ligand (FasL)-induced cell death (33). Granzyme and perforin are released by TILs and have both been shown to exert anti-cancer properties in several types of cancer. Perforin and granzyme B prevent the formation of cancer cell foci and induce cancer-cell apoptosis (34,35). Mechanistically, granzyme B induces the accumulation of reactive oxygen species, which results in dysregulated mitochondrial function and cytochrome c release, and therefore, accelerated cancer cell apoptosis (36,37).

The interaction between TCRs and tumor antigens upregulates the expression of death ligands on the surface of TILs, including TNF-related apoptosis-inducing ligand (TRAIL) and FasL. TRAIL and FasL can recognize TRAIL receptor and Fas on cancer cells, respectively, and induce cancer-cell apoptosis (38–40). Binding of TRAIL to its receptor activates caspase family-dependent apoptosis signaling, such as caspase-8, −10 and −3. The activated caspase-8 leads to mitochondrial outer membrane permeabilization via the Bid-Bax/Bak pathway, which results in mitochondrial disorder and cytochrome C release, accelerating cancer-cell apoptosis (41). Furthermore, FasL binds Fas to induce cancer-cell apoptosis through the MAPK, NF-κB and caspase-8 signaling pathways (42).

Taken together, TILs show powerful anti-tumor properties via multiple mechanisms (Fig. 3).

The anti-tumor properties of TILs have innate advantages, such as high safety, TCR diversity and superior tumor-homing ability, amongst other benefits (5,6). However, there remain several challenges regarding their clinical use. Thus, innovative therapeutic strategies are required to improve the anti-tumor properties of TILs.

Successful cancer therapy remains a formidable challenge worldwide. Currently, traditional therapeutic strategies fall short in achieving a cure for cancer, resulting in substantial economic losses.

TIL therapy is an advanced immunotherapy approach for the treatment of cancer, which involves the collection and expansion of autologous TILs followed by their reinfusion into patients to specifically target and eliminate tumor cells. Currently, TIL therapy has demonstrated significant efficacy in treating malignant tumors, such as melanoma and colorectal cancer (44,45). Adoptive TIL therapy represents a promising strategy for cancer treatment, as TILs exhibit remarkable diversity in their TCR repertoire, possess tumor-homing capabilities and exert potent cytotoxic effects. Multiple clinical trials have consistently demonstrated the safety and efficacy of this approach (5,6).

However, TIL therapy also encounters several challenges and issues, including the acquisition of an adequate quantity and quality of immune cells, optimization of cell preparation processes, as well as addressing concerns related to immune evasion and drug resistance. These key issues necessitate urgent resolution. Furthermore, from a clinical perspective, immune toxicity resulting from the targeting of normal tissues is a significant concern due to the potential for immune cells to inadvertently harm healthy tissue during their attack on tumor cells. From a production standpoint, the protracted manufacturing cycle, exorbitant costs and challenging pricing structure associated with cell therapy impede patient accessibility and present commercial obstacles for this emerging treatment. Besides that, the infusion of TILs is ineffective in a subset of patients with cancer due to tumor heterogeneity and the intricate nature of the tumor microenvironment (Fig. 4).

Therefore, further enhancement of the anti-tumor properties of TILs in multiple dimensions is imperative, including the enrichment of functional TILs, gene editing techniques, and combination therapies. Gene editing represents one of the most efficacious strategies for modifying TIL characteristics and augmenting their tumor-killing potential, thereby paving the way for future clinical applications and commercialization of TILs. TIL infusion is a highly promising strategy for cancer treatment. Addressing clinical unmet needs and developing innovative TIL-based therapies will accelerate the growth of the TIL industry in tumor treatment.