TLR4 protein: A core receptor for chemotherapy-induced immunogenic cell death and anti-tumor immune responses

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This article focuses on the molecular characteristics and immune functions of Toll-like receptor 4 (TLR4), systematically elaborating its core role in activating dendritic cell maturation after chemotherapy drug-induced release of damage-associated molecular patterns from colorectal cancer cells, analyzing the driving mechanisms of TLR4-dependent DC activation on Th1 immune responses and in vivo antitumor effects, and exploring the clinical significance of TLR4 as an immune adjuvant pathway linking chemotherapy and antitumor immunity.

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TLR4 Protein: The Core Receptor of Chemotherapy-Induced Immunogenic Cell Death and Antitumor Immune Responses

Summary: This article focuses on the molecular characteristics and immune functions of Toll-like receptor 4 (TLR4), systematically elaborating its core role in activating dendritic cell maturation after chemotherapy drugs induce colorectal cancer cells to release damage-associated molecular patterns, analyzing the driving mechanism of TLR4-dependent DC activation on Th1 immune responses and in vivo antitumor effects, and discussing the clinical significance of TLR4 as an immune adjuvant pathway connecting chemotherapy and antitumor immunity.

I. Immunological Effects of Chemotherapy for Colorectal Cancer and Research Background.

In developed countries, colorectal cancer is the third most common cancer and the third leading cause of cancer death. FOLFOX chemotherapy based on oxaliplatin and 5-fluorouracil is a first-line treatment regimen for patients with advanced colorectal cancer. Chemical drugs mainly kill tumor cells through cytotoxic effects while also killing normal cells, which can lead to lymphopenia and suppress immune system function. Immunotherapy is now becoming a promising treatment approach for colorectal cancer, and the combination of chemotherapy and immunotherapy has attracted widespread attention. Colorectal cancer cells treated with oxaliplatin and 5-fluorouracil may release danger-associated molecular patterns, which play an important role in immunogenic tumor cell death. Both in vivo and in vitro experiments have demonstrated that damage-associated molecular patterns induced by chemical drugs can stimulate dendritic cells to induce anticancer immune responses. Therefore, how to more effectively trigger anticancer immune responses mediated by damage-associated molecular patterns is a scientific question worthy of in-depth exploration.

II. Chemotherapy Drugs Induce Colorectal Cancer Cells to Release Damage-Associated Molecular Patterns.

HMGB1 and HSP70 are typical markers of damage-associated molecular patterns and are released after chemotherapy. The study detected the release of HMGB1 and HSP70 in the mouse colorectal cancer cell line CT-26 and the human colorectal cancer cell line SW480. The study found that compared with the untreated control group, the amounts of HSP70 and HMGB1 released into the cell supernatant were significantly increased 6 hours after treatment with oxaliplatin and/or 5-fluorouracil. In addition, 18, 24, and 30 hours after treatment with the combination of oxaliplatin and 5-fluorouracil, the concentrations of HSP70 and HMGB1 in the cell supernatant were even higher than those after treatment with oxaliplatin or 5-fluorouracil alone. In clinical samples, the researchers investigated changes in serum HMGB1 and HSP70 expression in 11 patients with advanced colorectal cancer receiving FOLFOX chemotherapy and found that the serological concentrations of HSP70 and HMGB1 in patients receiving chemotherapy were significantly increased after chemotherapy. These results indicate that chemotherapy can increase the release of damage-associated molecular patterns in cancer patients, providing a molecular basis for chemotherapy-induced immune responses.

III. TLR4 Mediates the Maturation of Human DCs Induced by the Supernatant of Chemotherapy-Stressed Tumor Cells.

Damage-associated molecular patterns have been shown to be candidate TLR4 agonists and promote the maturation and activation of DCs. To investigate the role of TLR4 in DC maturation mediated by the supernatant of SW480 cells treated with chemical drugs, the researchers observed phenotypic changes in DCs stimulated with the supernatant of chemically stressed SW480 cells. The study found that compared with stimulation with untreated control supernatant, stimulation with oxaliplatin supernatant, 5-fluorouracil supernatant, or oxaliplatin combined with 5-fluorouracil supernatant induced upregulation of HLA-DR, CD80, and CD86 expression on human DCs. Compared with stimulation with oxaliplatin supernatant or 5-fluorouracil supernatant alone, stimulation with the combined supernatant especially caused greater phenotypic changes. When the TLR4 antagonist monoclonal antibody HTA125 was used to block the interaction between TLR4 and damage-associated molecular patterns in the supernatant of chemically treated SW480 cells, the upregulation of HLA-DR, CD80, and CD86 expression on DCs was reversed, indicating that TLR4 on DCs mediated the enhancing effect.

The researchers further investigated whether TLR4 is essential for the secretion of proinflammatory cytokines and chemokines induced by DCs stimulated with the supernatant of chemically stressed SW480 cells. The study found that DCs stimulated with oxaliplatin supernatant, 5-fluorouracil supernatant, or combined supernatant showed increased secretion of proinflammatory cytokines and chemokines, especially when stimulated with the combined supernatant, but these increases were reversed in DCs blocked by the TLR4 antagonist. These results indicate that TLR4 is a key receptor mediating damage-associated molecular pattern-induced DC maturation.

IV. Genetic Validation of TLR4-Mediated Mouse DC Maturation and Cytokine Secretion.

Next, the researchers prepared DCs from wild-type and TLR4 gene knockout C57BL/6 mice and observed the phenotypic changes in these DCs when stimulated with supernatant from chemically stressed CT-26 cells. The study found that stimulation with oxaliplatin supernatant, 5-fluorouracil supernatant, or combined supernatant induced upregulation of Ia b, CD80, and CD86 expression on wild-type DCs, but did not stimulate expression on TLR4-deficient DCs. In wild-type DC mice stimulated with the combined supernatant, the production of proinflammatory cytokines IL-1β, TNF-α, and IL-6 as well as chemokines MIP-1α, MIP-1β, RANTES, and IP-10 was significantly increased; however, these cytokines and chemokines produced by TLR4-deficient DCs stimulated with the combined supernatant were not significantly increased. Taken together, the results indicate that the supernatant of chemically treated colorectal cancer cells induces DC phenotypic maturation and the production of chemokines and proinflammatory cytokines through TLR4, indicating that TLR4 is one of the functional receptors mediating DC activation through chemical supernatant. This genetic validation provides direct evidence for the core position of TLR4 in DC activation.

V. TLR4-Dependent DC Activation Induces Th1 Responses and In Vivo Antitumor Effects.

To evaluate whether TLR4 plays an important role in inducing Th1 responses in vitro by DCs pulsed with damage-associated molecular patterns, the researchers incubated peripheral blood lymphocytes from healthy donors with autologous DCs pulsed with the supernatant of in vitro chemically stressed SW480 cells, and also incubated splenocytes from wild-type or TLR4-deficient C57BL/6 mice and pulsed syngeneic DCs with the supernatant of chemically stressed CT-26 cells. The study found that DCs pulsed with oxaliplatin supernatant, 5-fluorouracil supernatant, or combined supernatant induced a significantly higher number of IFN-γ-producing cells than the untreated control group; however, the TLR4 antagonist HTA125 reduced the number of IFN-γ-producing cells, and no increase in the number of IFN-γ-producing cells was observed in TLR4-deficient mice immunized with syngeneic DCs. These results further indicate that TLR4 serves as a receptor for damage-associated molecular pattern activation of DCs; DCs are activated through TLR4 by the supernatant of chemically stressed colorectal cancer cells containing damage-associated molecular patterns and then more effectively trigger Th1 responses.

The induction of in vivo antitumor immune responses by damage-associated molecular patterns was evaluated in a mouse model with CT-26 tumor challenge. Bone marrow-derived DCs stimulated with the supernatant of chemically stressed CT-26 cells were subcutaneously immunized into Balb/c mice 3 times at 1-week intervals. On the seventh day after the last immunization, mice were subcutaneously challenged with CT-26 cells, and tumor size was measured every two days. Compared with the control group, tumor growth in the combined supernatant group was significantly inhibited, and survival time was significantly prolonged. These data indicate that mice immunized with DCs pulsed with the supernatant of chemically stressed colorectal cancer cells containing damage-associated molecular patterns can more effectively induce antitumor immune responses.

VI. Conclusion.

In TLR4-related basic research and drug development, high-quality TLR4-specific antibodies are core tools for dissecting its ligand recognition mechanisms and evaluating targeted intervention strategies. In response to the needs of TLR4 functional research and exploration of immune regulatory mechanisms, Univ provides Anti-TLR4 Monoclonal Antibody (Paridiprubart). This product is suitable for applications such as blocking analysis of the binding activity between TLR4 and damage-associated molecular patterns, mechanistic research on TLR4-mediated DC maturation and cytokine secretion, regulatory research on TLR4-dependent Th1 immune responses, and development of chemotherapy combined with immunotherapy strategies.

As a core receptor that recognizes damage-associated molecular patterns and activates dendritic cell maturation, TLR4, by virtue of its key role in initiating antitumor immune responses after chemotherapy drug-induced immunogenic death of colorectal cancer cells, has become an important molecular bridge connecting chemotherapy and immunotherapy. From oxaliplatin and 5-fluorouracil inducing the release of HMGB1 and HSP70, to TLR4-dependent DC phenotypic maturation and proinflammatory cytokine secretion, and then to the induction of TLR4-dependent Th1 responses and in vivo antitumor effects, TLR4 continues to drive the in-depth development of the field of chemotherapy combined with immunotherapy. Anti-TLR4 Monoclonal Antibody (Paridiprubart) provides reliable tool support for mechanistic research on the TLR4 signaling pathway and the development of targeted intervention strategies, and will continue to facilitate innovative exploration in the field of tumor immunotherapy.

This article is reviewed and published by the technical expert team of UA

Disclaimer: This article partially utilizes artificial intelligence assistance in its creation. If any content involves copyright or intellectual property issues, please let us know and we promise to verify and remove it as soon as possible.

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