Local drug delivery of immunotherapy in head and neck cancer: a narrative review

Introduction

Background

Globally, head and neck squamous cell carcinoma (HNSCC) involves the mucosal lining of the oral cavity, pharynx, and larynx, is the seventh most common cancer with 890,000 new cases and 450,000 deaths annually, and is traditionally associated with tobacco and ethanol exposure as well as human papilloma virus (HPV) infection (HPV-16 or other high-risk HPV, such as HPV-18, HPV-31, HPV-33, and HPV-52) (1,2). The 5-year survival rates for all subsites vary based upon HPV-status and the stage at diagnosis, 86% for localized disease, 69% for locally advanced, and 39% for metastatic disease (1). In comparison to tobacco related HNSCC, HPV⁺ oropharynx patients are uniquely associated with p16 immunohistochemical (IHC) staining; more commonly male; White; non-smokers and non-drinkers; have an elevated lifetime exposure to sexual behaviors (i.e., number of lifetime vaginal and oral sex partners); and usually present with nodal metastasis at initial diagnosis (3). Although these patients have an improved clinical prognosis and survival compared to non-HPV related HNSCC, they still have a 20% mortality at three years despite combined modality therapy (4). While the overall incidence of many cancers, including HPV-unrelated oral cancers, has decreased in the past decades, the incidence rate of HPV mediated oropharyngeal cancer (HPVOPC) continues to increase (5). Standard treatment options for HNSCC include 7 weeks of definitive chemoradiation or surgery plus 6 weeks of risk adapted adjuvant radiation +/− chemotherapy (6). Programmed death receptor-1 inhibitors (PD-1i) pembrolizumab and nivolumab have improved overall survival (OS) in patients with recurrent/metastasis (R/M) HNSCC when administered as monotherapy (7-9) or in combination with cisplatin (2) compared to standard-of-care platinum-based chemotherapy regimens. However, PD-1i response rates are only between 13% and 20% (10). Furthermore, given the significant acute morbidity associated with standard treatment options for HNSCC—7 weeks of definitive chemoradiation, or 6 weeks of adjuvant radiation +/− chemotherapy after surgery, which carry a high rate of grade 3–5 toxicity—and the critical importance of local control in HNSCC, novel treatment approaches are clearly needed to improve survival and decrease treatment-related morbidity (11).

Rationale and knowledge gap

Immunotherapy is expected to activate the immune system and reverse the immune-suppressed tumor microenvironment (TME), ultimately achieving tumor regression and preventing recurrence and metastasis (12). To date, the anti-PD-1 antibody pembrolizumab has been approved by the U.S. Food and Drug Administration (FDA) for use in the R/M HNSCC, irrespective of programmed death-ligand 1 (PD-L1) expression, as monotherapy or in combination with chemotherapy in patients with a combined positive score (CPS) >1 in the first line (13). In addition, the anti-PD-1 antibodies nivolumab and pembrolizumab were approved for second-line cisplatin-resistant therapy to improve OS in patients with R/M HNSCC (14). When combined with chemotherapy, patients receive a higher response rate, however a decreased duration of response has been observed clinically (14). To enhance response rates to immune checkpoint blockade (ICB), numerous combinations with other immune-based and conventional therapies other than chemotherapy are being studied in the laboratory and in clinical trials, including cytokines, chemokines, adoptive cellular therapy (ACT) using tumor infiltrating lymphocytes and T-cell receptor (TCR)-engineered T cells, adjuvants, vaccines, and radiotherapy (15). However, systemic immunotherapy may cause immune-related side effects, some of which are severe. Therefore, immunotherapy strategies that reduce systemic adverse effects and toxicity while enabling the effective administration of immunotherapy combinations are urgently needed to benefit more patients (16).

Objective

Compared to systemic administration, intratumor injections of immune agents have multiple advantages, including directly delivering drugs to the tumor; increasing the intratumoral drug concentration; stimulating tumor antigen-specific immune responses; reversing immunosuppressive mechanisms in the TME; allowing for the combination of multiple drugs; and reducing systemic drug exposure and associated side effects (16,17). In this review, we summarize the clinical studies related to local immunotherapy for HNSCC, provide a brief summary of preclinical cellular and animal models of HNSCC, and research articles on the local delivery of immunotherapy for HNSCC. We present this article in accordance with the Narrative Review reporting checklist (available at https://fomm.amegroups.com/article/view/10.21037/fomm-25-13/rc).

Methods

MEDLINE (accessed via PubMed) and ClinicalTrials.gov were utilized to conduct a review of the papers published between January 1st, 1960, and October 8th, 2025. Head and neck cancer subtypes and various drug dosage forms are queried after permutation and combination (Table 1).

Clinical trials of intratumoral administration of immunotherapy in head and neck cancer

The clinical studies related to the local delivery of immunotherapy for HNSCC can be classified into two types: (I) intratumoral injection of immune agents into solid tumor that can be easily and safely accessed with a needle (Table 2) (18-22), such as interleukins (22), toll-like receptor (TLR) agonists (18,23), stimulator of interferon genes (STING) agonists (24); and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antagonistic antibody (21) and (II) local immunotherapy triggered by tumor-specific antigen released from immunogenic cell death after hypofractionated radiotherapy.

The phase III study NCT01265849 administered interleukins (a natural cytokine mixture) at a dose of 200 IU peritumorally and 200 IU perilymphatically, totaling 400 IU [calculated as interleukin-2 (IL-2) equivalent], continuously for three weeks in the neoadjuvant setting for treatment-naïve, resectable, locally advanced primary squamous cell carcinoma (SCC) of the soft palate and oral cavity. Interleukin injection is the first neoadjuvant immunotherapy in decades to demonstrate an objective early response and improved OS outcomes in the treatment of these patients (22). NCT01984892 is a phase I study among patients with recurrent metastatic disease who failed prior systemic therapy (7 patients with HNSCC). One milligram polyinosinicpolycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Poly-ICLC), a double-stranded RNA complex that directly activates dendritic cells (DCs) and triggers NK cells to kill tumor cells, was injected intratumorally three times a week. Results showed that genes related to antigen presentation were upregulated indicating that Poly-ICLC treatment was well tolerated and induced local and systemic immune activation in patients with advanced solid tumors (18). A phase II trial NCT02521870 evaluated whether SD-101, a synthetic cytosine-phosphate-guanine (CpG) oligonucleotide that triggers TLR9, can increase the antitumor activity of pembrolizumab in patients with R/M HNSCC. Intratumor injection of 2 mg SD-101 in 1–4 lesions or 8 mg SD-101 in one single lesion was given 4 doses weekly and then 7 doses every 3 weeks. A 200 mg dose of the anti-PD-1 antibodies pembrolizumab was intravenously administered every 3 weeks. No treatment-related deaths occurred during treatment and follow-up time. Among 50 patients, 2 had complete response and 10 had partial. Additionally, 16 HPV⁺ patients had a higher response rate than HPV⁻ patients. These suggest that SD-101 combined with pembrolizumab can induce objective responses, particularly in HPV⁺ tumors. This is usually attributed to SD-101, which stimulates DCs to release interferon-α (IFN-α), and thereby activates T cell anti-tumor responses (19). Another recruiting phase I trial is NCT06022029, which is a dose escalation and dose expansion study of intratumoral ONM-501 alone and in combination with cemiplimab in patients with advanced solid tumors, including HNSCC. ONM-501 is a dual-activating STING agonist, where the endogenous STING agonist is encapsulated in polycarbonate-based polymer 7A (PC7A) micelles. The synthetic polymer PC7A has the ability to induce polyvalent STING condensation, and further prolong innate immune activation (20). Finally, in a clinical trial conducted between 2016 and 2019 (NCT02812524), assessing the TME of HNSCC patients who were treated with the local injection of microdoses of anti-CTLA-4 helped define the inhibitory FcγRIIB as a novel immune checkpoint limiting antibody-mediated Treg depletion in the TME after anti-CTLA-4 administration, and demonstrated Fc engineering as an effective strategy to overcome this limitation and improve the efficacy of Treg-targeting antibodies (21).

Preclinical models for HNSCC

Various preclinical animal models mimicking human tumors have been developed to understand tumorigenesis and progression and to test the efficacy of various therapeutic regimens. Here, we will briefly summarize the HNSCC cell and animal models (Figure 1).

Figure 1 A summary of the HNSCC cell and animal models. 2D, two-dimensional; 3D, three-dimensional; HNSCC, head and neck squamous cell carcinoma; HPV, human papilloma virus; MOC, mouse oral cancer; MOSC, murine oral squamous cells; NOD, non-obese diabetic; NSG, non-obese diabetic scid gamma; SCC, squamous cell carcinoma; SCID, severe combined immunodeficiency.

Cellular models

Cell models can be categorized into murine and human HNSCC by source. Murine HNSCC cell lines include mouse oral cancer 1 (MOC1), MOC22, MOC2, and SSCVII (25). Human HNSCC cell lines include immortalized cell lines and patient-derived xenograft (PDX). These models can be categorized into two-dimensional (2D) and three-dimensional (3D) cultures by culture mode. These in vitro cellular studies are the most cost-effective way to explore drug efficacy and molecular mechanisms (26).

The murine HNSCC cell lines MOC1, MOC22, and MOC2 can form tumors by injection of as few as 10,000 cells and are the primary murine HNSCC cell lines for preclinical studies of HNSCC. Exposure of wild-type C57BL/6 (B6) background mice to the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) induces SCCs that produce multifocal lesions. MOC1 is derived from mucosal lip lesions, MOC22 from buccal lesions, and MOC2 from a mass in the floor of the mouth. MOC1 and MOC22 are inert growth phenotypes, and MOC2 is an aggressive growth phenotype with lymphatic and pulmonary metastases. In addition, the highly immunogenic MOC22 has a higher mutational burden than the low-immunogenic MOC2. Although MOC genomics has less overlap with tobacco-related traits, it contains key driver mutations in human HNSCC, such as TP53 and Notch (25). Of human HNSCC immortalized cell lines, 60% were derived from oral cavity tumors, 12% from pharyngeal tumors, 18% from laryngeal tumors, and 3% from nasal septum tumors (27). There are also six HPV⁺ tumor cell lines available. They can be immortalized by blocking the cell cycle checkpoint pathway through ectopic expression of telomerase, whose catalytic subunit is encoded by the gene telomerase reverse transcriptase (TERT), or through mutant forms of the proteins p53 and pRb. However, they have reduced bio fidelity after clonal selection induced by selective survival pressures inherent to culture conditions. PDX is derived from the real tumor 3D microenvironment in the human body and retains the biology of the original tumor. PDX-derived 2D cultures up to 10 generations old have a very similar heterogeneity and genetic profile to that of human tumors, with a high level of genomic fidelity and ability to predict treatment response (28). In addition, a model of head and neck cancer with poor immunogenicity, low immune infiltration, and dense tumor cell growth can be established after subcutaneous injection of a squamous cell tumor cell line, SSCVII, isolated from a spontaneous carcinoma in C3H/HeJ mice, into the lateral abdomen of mice (29).

2D culture is the main in vitro culture method, but it does not realistically simulate the heterogeneous and dense acidic hypoxic TME consisting of multiple cellular and vascular network interactions (2). On the other hand, 3D cultures are under active development that more closely mimic tumor-specific structures and responses to anticancer regimens than 2D monolayer systems. Spheroids, organoids, and microfluidic systems are three types of 3D cell cultures that reflect the structure of tumor tissues. Spheroids formed by spontaneous aggregation are the most economical and simplest 3D models. Those containing only tumor cells are multicellular tumor spheroids (MCTS), and those containing both tumor cells and stem cells with a heterogeneous tumor environment are tumor-derived spheroids (TDS). Organoids are a proliferative mixture of pluripotent embryonic or induced stem cells (PSCs) or adult stem cells (ASCs) formed in the presence of differentiation factors in an environment that requires large droplets of 3D Matrigel as support. They preserve the 3D structure in vivo and the heterogeneous cell types in the TME, which can be used to test drugs in vitro and to monitor therapeutic response (30). Microfluidic systems have been able to demonstrate the original tissue structure of HNSCC and can provide the environment of controlled culture in small compartments suitable for cancer and immunotherapy research. However, such models are costly, technically challenging, and time-consuming. Despite the high in vivo similarity of these 3D models, they cannot simulate complete physiological regulation in immunotherapy involving remote cells. Therefore HNSCC in vivo modeling remains critical in translational immune-oncology research (31).

Animal models

Mouse models can be classified into PDX models based on immunodeficient mice or human immuno- (32) systematized immunodeficient mice, syngeneic mouse models, transgenic mouse models, and chemically induced mouse models.

PDX models can be obtained by subcutaneously implanting xenografts from human tumor cell lines or patient tumor biopsies into recipient mice. Immunodeficient mouse strains such as non-obese diabetic (NOD)/severe combined immunodeficiency (SCID)/NOD scid gamma (NSG) mice lacking mature T and B cells improved grafting success with these implants and reduced tumor regression (28). Additionally, transplantation of human hematopoietic stem cells or bone marrow cells from patients into immunodeficient mice yields humanized mice, which can be used to study the interaction between immune cells and tumors in a humanized context to avoid the risk of human leukocyte antigen (HLA) mismatch (33). Although the PDX models preserve the original heterogeneity and molecular properties of human tumors, the growth dynamics of the implants are not easily predictable and their development does not fully resemble the behavior of the original tumors. To date, the creation and maintenance of such models remain time-consuming, expensive, and laborious, and there is no standardized protocol for HPV⁺ tumor implantation (26).

Transplantation of tumor tissue or cells from a mouse, such as MOC cells, into another mouse with a similar genetic background results in a syngeneic mouse model with full immune competence (34,35). This model is mechanistically capable of probing tumor-host environment interactions to a higher degree and is suitable for evaluating new immunotherapies (32). Currently, SCCVII cells, MOC1 and MOC2 from DMBA-treated mice, and HPV⁺ SCC cells designated MEER, which would produce tumors after implanted into the flanks of mice of the corresponding genotypes, can be used to establish a syngeneic HNSCC model (36).

However, this particular system fails to replicate human disease (32). Transgenic mice are obtained by modulation of proto-oncogenes and oncogenes. A total of 30–35% of HNSCC patients have heterozygous deletions of SMAD4, 4% have pure deletions of KLF4 and SMAD4, and 0.2% have HRAS and KRAS mutations, the latter of which are most commonly regulated in genetically engineered models of HNSCC. Double transgenic models such as KrasG12/Trp53 deletion, KrasG12/Klf4 deletion, or KrasG12/Smad4 deletion are more common than KrasG12D overexpressed in oral epithelium in SL-KrasG12D mice that had a higher prevalence of oral tumor formation (37-39). These double transgenic models could produce tongue cancer more successfully within two weeks of induction. In addition, transgenic HPV⁺ oral tumors that mimic all stages of cancer from initiation to local invasion and metastasis have been established (40). However, the alteration of all tissue genes in the genetically engineered mouse model leads to the disruption of other physiological processes, and the frequency and outgrowth of tumors are so low that the results are usually unpredictable (26).

Highly successful HNSCC models induced by carcinogens can be obtained in mice and rats after exposure to low doses of the carcinogens 4-nitroquinoline 1-oxide (4NQO) or DMBA for approximately 40 weeks. This chemically induced model mimics human oral cancer from tumor origin to development in an immunocompetent environment and is of high value in assessing the value of immunotherapy (41). To improve tumorigenic efficiency, HNSCC mouse models developed using both gene modification and chemical carcinogens have been used to achieve 100% tumor production in mice (26).

Preclinical in vivo animal models of HNSCC include mouse models that have been standardized, controlled, and widely used (42). Although the development of HNSCC disease in the mouse model is similar to its human counterpart, differences in oral anatomy and immune background limit the use of these models to fully recapitulate human disease (43).

Metastasis models

Local and regional recurrence is the main cause of eventual treatment failure. The incidence of metastasis in conventional subcutaneous xenograft models is only 1.3%, which is not representative of advanced local or distant metastatic disease in human disease. There are differences in biological behavior between subcutaneous and in situ tumor growth, with in situ transplantation models more closely mimicking the host microenvironment (26,44). Wang et al. (36) established a syngeneic in situ 4NQO-induced murine oral squamous cells (MOSC) by in situ transplantation of 4NQO-induced primary tumors in the tongue of C57BL/6 mice into the tongue of immunocompetent C57BL/6 mice. Histological identification of the established tumors was HNSCC. SigProfiler analysis of exon DNAseq results showed that the 4NQO-induced SCC lesions had 93.9% similarity to the tobacco mutant landscape significantly. And it possessed abundant potential for lymph angiogenesis and lymph node metastasis (36). In addition, 4NQO was more effective in reflecting the human tobacco-associated HNSCC mutational landscape compared to the widely used tobacco carcinogen DMBA-induced mutational landscape with only 39.7% similarity to human HNSCC (45).

Three types of animal models of head and neck cancer metastasis, MOC, MOSC, and xenograft in situ transplantation, now driven by intrinsic or extrinsic factors, mimic the growth and spread of the disease with the aid of innovations in cellular labeling techniques and small animal imaging that contribute to better understanding of the mechanisms of local and regional metastasis of HNSCC as well as the mechanism of action and therapeutic efficacy of novel antitumor drugs. These models are the most suitable for testing the effects of local immunotherapy. However, the techniques used to successfully establish these models and avoid other lesions or even death in animals are difficult to replicate (26,44).

Local drug delivery of immunotherapy in head and neck cancer

In reviewing the literature on local drug delivery of immunotherapy in head and neck cancer, we found the following therapeutic strategies injected into head and neck tumors (Figure 2) involving head and neck subsites have been reported in preclinical studies: cytokines such as interleukin (a natural cytokine mixture) (22), IL-2 (46) and IL-12 (47,48); chemokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) (49); IFNs such as IFN-β (50); TLR agonists such as polyinosinic:polycytidylic (polyI:C) (51); resiquimod (R848) (52); CMP-001 (53); and CpG (51,54); antibodies such as immunoglobulin M (IgM) (55) and anti-CD47 antibodies (56); peptides such as E7 long peptide (57); immune cells such as lymphokine (IL-2)-activated killer (LAK) cells (58); DCs (59); and chimeric antigen receptor T cells (CAR-T cells) (60,61); plasmids such as MEDI0457 (62); and drugs such as OK-432 (63), glucose oxidase and a carbonic anhydrase inhibitor (64), Celastrol (65); and talimogene laherparepvec (66). Until recently, most agents for immunotherapy were injected in liquid form without consistent dosing. But more recently, biomaterials like glatiramer acetate (GA) were utilized to decrease the flowability of the liquid drug, trying to increase the retention time of the drug within the local tumors (51). In the latest research-based articles, gels and microneedles are being developed to deliver reproducible doses of immunotherapy to solid tumors (67). The results showed that local drug delivery of immunotherapy in head and neck cancer is a promising way to realize the regression of the local tumors with lower systemic toxicity and fewer metastases (51).

Figure 2 Local drug delivery of immunotherapy in HNC. GM-CSF, granulocyte-macrophage colony-stimulating factor; HNC, head and neck cancer; IFN, interferon; IL, interleukin; PolyI:C, polyinosinic-polycytidylic acid; TLR, toll-like receptor.

Intratumoral delivery of immunotherapy in head and neck cancer

IL-12 is a pro-inflammatory type 1 cytokine that can reverse tumor-induced immunosuppression. Local delivery systems for IL-12 are being developed due to the dose-limiting toxicity of its systemic administration (68). Wei et al. transduced a lentiviral vector expressing IL-12 into the SCCVII head and neck cancer cell line and injected IL-12-expressing SCCVII as an autologous tumor cell vaccine into the flanks of syngeneic mice C3H/HeJ. They found that when the total amount of IL-12 produced was consistent, tumors could be killed by tumor cell mixtures containing small amounts of high-producing IL-12, but not by tumor cell mixtures containing large amounts of low-producing IL-12. They verified that cancer cells are effective vectors for delivering IL-12, activating the local and systemic immunity needed to control SCCVII and avoid the severe toxicity and low response rate that systemic injection of IL-12 would cause (47). Lee et al. administered messenger RNA lipid nanoparticles (mRNA-LNPs) encoding HPV16 E7, GM-CSF, and IL-12 via peritumoral subcutaneous injection in MEER tumor-bearing C57BL/6 mice, demonstrating the antitumor and antimetastatic effects of the mRNA vaccine expressing both antigen and cytokines (48). In a Phase Ib/II study, Aggarwal et al. locally delivered MEDI0457, a novel immunotherapy combining VGX-3100 and INO-9012, via an electroporation device called CELLECTRA^®. VGX-3100 are synthetic DNA sequences of HPV 16/18 E6 and E7 which can trigger E6/E7 specific cellular responses, while, INO-9012 are encoding plasmids of recombinant IL-12. The data examined that MEDI0457 can lead to strong HPV16/18 antigen-specific tumor immune responses in the HPV-relevant patients with PD-1/PD-L1 inhibition (62).

The transmembrane receptor family TLRs, of which 10 types are expressed in humans, are viable therapeutic targets for activating antigen-presenting cells (69). Sato-Kaneko et al. injected TLR7 and TLR9 agonists intratumorally in combination with anti-PD-1 administered via intraperitoneal injection to three syngeneic mouse models carrying the HPV-negative MOC1 and SCC7, and HPV-positive MEER, respectively. They clarified that this combined therapy can increase the ratio of M1/M2 tumor-associated macrophages and enhance the efficacy of checkpoint inhibitors, which in turn inhibited primary tumor growth and prevented metastasis (70). Additionally, Cheng et al. found that in situ injection of CMP-001, another novel TLR9 agonist, induced antitumor immune responses both locally and distantly (abscopal effect), and enhanced anti-PD1 therapy in HPV-positive tumor mouse models where C57BL/6 mice bear mEERL HNSCC tumors (53). In order to increase the retention time of these active substances within the tumor after local injection, some biomaterials have been used to reduce the fluidity of these liquid formulations (51,52). Negatively charged TLR3 agonist polyI:C and TLR9 agonist CpG were respectively mixed with a positive charged peptide GA forming the complexes polyI:C-GA and CpG-GA by Pressnall et al. These complexes were then injected into floor of mouth tumors generated by AT84 cells. Even though the slowest tumor growth was seen in the group treated with intratumoral CpG injection, the CpG-GA injection group had a dramatically lower level of systemic proinflammatory cytokines. This demonstrated that CpG-GA complexes have potential to alleviate the systemic immune-related adverse events of CpG while also triggering local anti-tumor immune responses (51). Xu et al. established an HPV-positive orthotopic mouse model by subcutaneously implanting TC-1 cells into the intracheek region of female C57BL/6 mice. They loaded the immunoadjuvant CpG and the E749-57 (RAHYNIVTF) into self-assembled particles formed by salcaprozate-modified chitosan. Intranasal mucosal administration of these particles led to significant tumor regression (54). Systemic administration of Resiquimod, a TLR7/8 agonist, induces strong immune-related toxicity leading to its limited therapeutic efficacy against tumors. Lu et al developed a precursor-based nanocarrier delivery system, where R848 was conjugated with α-tocopherol to make R848-toco prodrug and then formulated with tocopherol-modified hyaluronic acids (HA-Toco) as a polymer nanosuspension. When injected subcutaneously in a HNSCC model established by AT84 cells in C3H mouse, the nanosuspension formed a reservoir at the injection site, inducing a localized immune response, recruiting immune cells, and inhibiting tumor growth and systemic expansion (52).

Animal and human tumors have shown complete or partial responses to adoptive cellular therapy (ACT) with tumor infiltrating lymphocytes and gene engineered T cells since the 1980s (71). In 1991, Sacchi et al. realized that direct delivery of cells or cytokines, or both, to tumors or tumor regions has the potential to avoid the toxicity caused by high-dose systemic administration of cytokines, which are used to activate adoptive immune effector cells. The local delivery of IL-2 and 10⁶in vitro-enriched CD3⁻CD56⁺ effector cells effectively inhibited tumor growth and achieved local therapeutic cure in human HNSCC PCI-1 tumors established in nude mice for 3 days and 7 days (72). In 2004, Ahmed et al. demonstrated that, in C3H/HeN mice bearing SCCVII, the oral administration of the fluoropyrimidine anticancer drug TS-1 followed by intratumoral injection of the immunotherapeutic agent OK-432 and bone marrow-derived DCs resulted in significant CD8⁺ T cells infiltration, tumor growth inhibition and prolonged survival. These phenomena could not be observed in the TLR4-deficient mouse model, demonstrating that intratumoral injection of the immunotherapeutic agent OK-432 activated the TLR4 signaling pathway which likely explained the efficacy of this treatment (73). Inoue et al. developed a novel surgically implanted immunotherapy, termed “immuno-flap”. They resected a round area of skin first, then a rat head and neck cancer cell line SCC-158 cells were loaded into the surface of the resection, and finally, the mature DCs were injected into the groin skin flap. After 2 weeks, the level of IL-2 and IFN-γ increased and tumor volume decreased significantly in the “immuno-flap” group, indicating that the “immuno-flap” is promising to prevent local metastasis in head and neck cancer (59). However, immunosuppression and immunologic tolerance limit the therapeutic efficacy of DC vaccines. Moyer et al. combined the DCs injection with systemic chemotherapy and local radiation [3 gray (Gy) × 5 days] in a mouse HNSCC model where SCCVII cell line was injected into the C3H/HeNCr MTV-mice. They observed complete tumor regression in 30% of the mice and improved survival rate compared to control. Thus, a combination of the novel adoptive cell therapy with traditional chemoradiotherapy may improve the therapeutic efficacy (74). Importantly, a similar approach involving direct intratumoral injection of engineered CAR-T cells has been tested in clinical trials, demonstrating safety in advanced HNSCC patients (60,75). Ito et al. gave two intratumoral injections of EPH receptor B4 (EPHB4)-CAR-T cells into NOD SCID gamma mice where oral SCC (OSCC) PDX tumors or SAS OSCC cell line-derived tumors had been subcutaneously implanted, resulting in reduced EPHB4 expression levels in the tumor tissue (61).

In 1994, Kumazawa et al. encapsulated OK-432 and attenuated Streptococcus pyogenes into a fibrinogen gel and injected the OK-432 loaded gel directly into 15 patients with HNSCC. Local immune responses were triggered indicating that the hydrogel in solid form overcomes the limitation of liquid drugs, which rapidly disappear in the tumor area after local injection (63). Baird et al. loaded STING ligands in the Matrigel (Corning Inc.) and implanted it in the tumor resection site in the SCCVII (C3H mice), MOC1 (C57BL/6 mice), and MOC2 (C57BL/6 mice) HNSCC models. The treatment protected the mice from metastasis, compared to a control group of blank gel (76). Similarly, Leach et al. developed an injectable hydrogel delivering STING agonists cyclic dinucleotides (CDNs), termed “STINGel”. STINGel was intratumorally injected into the wild-type C57BL/6 female mice with MOC2-E6E7 tumor cells. The OS was increased because STINGel improved the distribution of CDNs in the tumor significantly (77). Wu et al. developed an injectable hydrogel based on the frame PLGA-PEG-PLGA. A peptide-based proteolysis-targeting chimera (PROTAC) of BMI1 and paclitaxel was loaded with mesoporous silica nanoparticles, and then the nanoparticles were coated with a cancer cell membrane. CaCO₃ nanoparticles were also made to encapsulate Resiquimod. And the two nanoparticles were delivered to the head and neck tumors by the novel injectable hydrogel. The combined immunotherapy and chemotherapy succeeded in inhibiting the tumor growth and metastasis (78). Nie et al. developed a self-healing and pH dual-responsive hydrogel loaded with glucose oxidase and a carbonic anhydrase inhibitor. Local injection of this hydrogel effectively inhibited tumor growth in the MOC-1 tumor-bearing mouse model. In the MOC-1 metastatic mouse model, combination therapy using this hydrogel and anti-PD-L1 treatment effectively suppressed tumor progression and potential metastasis through a triple ferroptosis mechanism (64).

Qin et al. studied the effect of recombinant vaccinia virus expressing IL-2 (rvv-IL-2) that was injected intratumorally on the 5th day after tumor implantation in the HNSCC model SCC VII/SF. The mice in the treatment group lived longer than those in the control group and expression of GM-CSF, IL-10, TGF-β, and NO synthetase was higher in the experimental group. Thus, rvv-IL-2 is a promising therapeutic vaccine for HNSCC (79). Adappa et al. performed intratumoral injection of viral vectors advCMV- IL-12/GM-CSF and advCMV-IL-12 to produce IL-12 and GM-CSF in combination with systemic Ig-4-1BB ligand in orthotopic models of HNSCC. The combination therapy increased the survival in experimental mice compared to controls (80). To treat unresectable tumors in tongue cancer, Albertoni et al. injected 90Y-ST2210 intravenously into nude mice and then injected intratumorally AvidinOX into the orthotopically transplanted human OSC19 tongue cancer. In their therapeutic regimen, the tongue’s integrity and functionality were preserved and antitumor activity was observed (81). Leach et al. developed SynerGel, an injectable biomaterial-based hydrogel, which can inhibit inducible nitric oxide synthase (iNOS) and control release antitumor CDNs. Median survival was increased to 67.5 days in the treatment group from 44 days in the no-treatment group in the murine treatment-resistant oral tumors model (82). Gilardi et al. developed an anti-CTLA-4 antibody loaded soluble microneedle to locally deliver the immunotherapy to the tumors directly. They found immune-related adverse events that occurred in mice undergoing systemic administration of anti-CTLA-4 antibodies were alleviated in Foxp3-GFP^DTR transgenic mice (67). In a postoperative model of SCC7 oral cancer cells, Chen et al. utilized an in situ hydrogel containing δ-aminolevulinic acid and anti-CD47 antibodies loaded CaCO₃ nanoparticles. This system facilitated both photodynamic therapy and photothermal therapy, promoted the polarization of tumor-associated macrophages from the M2 to the M1 phenotype, and ultimately inhibited tumor recurrence and metastasis (56). Yao et al. encapsulated the poorly soluble drug Celastrol in a RADA16-I hydrogel, which enabled the treatment of OSCC by enhancing immunogenic cell death (65).

In addition, there was a special case of a female patient with a history of autoimmune disease and breast cancer who was diagnosed with oral palatal mucosal melanoma, a rare form of malignant melanoma occurring in the head and neck region. Local injection of talimogene laherparepvec, which is approved for the treatment of cutaneous melanoma, led to a complete local remission in this patient (66).

Intratumoral immunotherapy delivery to enhance abscopal effect with radiation in HNSCC

At radiation doses of 8 Gy and above, cancer cells experience immunogenic cell death associated with the release of DAMPs. The induced release of IFN can facilitate DC maturation and promote T-cell activation. The resulting antitumor immune response also has the potential to act on distal unirradiated tumors and significantly increase the incidence of distal effects. These events lead to T-cell initiation, trafficking, infiltration, and immunogenic killing. Based on the strong supporting preclinical data on the immunobiology of the 8 Gy × 3 dose fraction, it is considered the standard graded regimen for immunotherapy combinations (83,84). NCT03283605 is a Phase I/II trial of safety and efficacy test of durvalumab plus tremelimumab and stereotactic body radiotherapy (SBRT) for 33 patients with oligometastatic HNSCC (2–10 lesions). Durvalumab (anti-PD-L1, 1,500 mg) and Tremelimumab (anti-CTLA-4, 75 mg) were administered for 4 months, followed by 8 months of Durvalumab and SBRT at 2–5 lesions. SBRT is a high-precision radiation therapy technique where a single high dose of radiation results in localized cancer cell killing and cancer cell debris release, which may stimulate the immune system and have immunotherapeutic effects (85). Only one case of serious side effects occurred and the trial was terminated, with the remaining patients reaping the benefits of a response rate that achieved a progression-free survival (PFS) of six months beyond expectations (86). Since the 8 Gy × 3 regimen is commonly used for immunomodulation (Table 3) (87-93), we summarize clinical trials in which a single dose of 8 Gy and above was used for HNSCC treatment. However, the immune effects of radiation therapy exhibit different dose-response profiles, which means that one dose-fraction regimen cannot provide optimal immunomodulation in all cases. In addition, different chronological planning of radiotherapy immunomodulation in combination with conventional therapy influences the results, so an elaborate design of the overall regimen is needed for optimal systemic therapeutic effect (83).

Ghanian et al. evaluated the feasibility and safety of the combination therapy of radiation therapy (8 Gy × 4) and resiquimod, a product developed by CureBiotech Inc., and formulated in a proprietary hydrogel-based injectable formulation, in three dogs with head and neck cancer (94). Akutsu et al. subcutaneously injected 10⁵ SCCVII cells into the chest tissue and left femur of C3H/He mice. A single dose of 4 or 10 Gy of ionizing radiation and intratumoral injection of DCs (10⁶) was administered to the femur tumor site only. Data showed that the combination of radiation and DCs not only inhibited growth of the treated tumor, but also the untreated tumor in the chest indicating that the systemic antitumor response can be triggered (95). Pieper et al. demonstrated that local radiotherapy of 8/12 Gy, in combination with a CD122-preferred IL-2 pathway agonist, and ICB triad significantly improved survival and primary tumor response in a MOC2 model (96). Additionally, in 1997, Borchardt et al. labeled IgMγ CR4E8, a human monoclonal antibody for treating the SCC of the neck, with indium-111 (¹¹¹In) or yttrium-90 (⁹⁰Y) and injected these radiolabeled IgM to the nude mice with subcutaneous HNSCC cell lines 886. The results showed that compared to intravenous injection, the intralesional injection caused a higher biodistribution rate in the tumor than normal tissues, which is not related to radio markers. This is a promising method to improve patient tolerance of increased radiation dose with less normal tissue toxicity (55).

Conclusions

Local drug delivery of immunotherapy in head and neck cancer is a promising approach with the advantages of low systemic toxicity and a high distribution rate within or around the tumor compared to systemic administration (Figure 3). Hydrogel and microneedle patches are potential tools to increase the retention time and facilitate predictable drug dosing. Additionally, these biomaterials facilitate novel immunomodulatory therapies to treat HNSCC, alone or in combination with traditional chemotherapy, radiation and surgery, and have the potential to improve efficacy and minimize toxicity associated with the systemic administration of immunotherapy and conventional treatment.

Figure 3 Ideal responses to intratumoral administration of immunotherapy in HNSCC. HNSCC, head and neck squamous cell carcinoma.

Our review centers on immunotherapy, categorizing local immunotherapeutic approaches into cytokines, chemokines, IFNs, TLR agonists, antibodies, peptides, immune cell therapies, plasmids, and other agents. It also highlights the role of radiotherapy in both preclinical and clinical settings of immunotherapy. Compared to existing reviews, our article is more focused on immunotherapy and provides a more detailed classification. Furthermore, we integrate and compare clinical and preclinical studies of local immunotherapy for HNSCC, emphasizing opportunities for clinical translation. Although we categorized local immunotherapy for HNSCC based on the functional types of immunotherapeutic agents, some preclinical studies involve combined treatments using components from different categories, resulting in overlap between categories. This may affect readers’ understanding of certain strategies. Besides, we have attempted to include all preclinical studies of local immunotherapy for HNSCC in this review. However, some immunotherapeutic agents have not been validated through multiple experiments. Therefore, it is possible that future clinical treatments for HNSCC may not fully benefit from certain immunotherapies discussed in this review. We believe that the closer the in vitro and in vivo experimental models resemble human HNSCC tumors, the more reliable the conclusions drawn from them are.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://fomm.amegroups.com/article/view/10.21037/fomm-25-13/rc

Peer Review File: Available at https://fomm.amegroups.com/article/view/10.21037/fomm-25-13/prf

Funding: This work was supported by Providence funding.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://fomm.amegroups.com/article/view/10.21037/fomm-25-13/coif). R.B.B. serves as an unpaid editorial board member of Frontiers of Oral and Maxillofacial Medicine from August 2025 to July 2027. R.B.B. receives grant funding and consulting fees from Merck, Regeneron, AdelaBio, Brightpeak Pharma, and Macrogenics. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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