More than 830,000 people worldwide each year are diagnosed with head and neck cancers, with head and neck squamous cell carcinomas (HNSCCs) accounting for 90% of these new cases¹. Most cases are associated with tobacco and alcohol use, but a sizable minority are caused by infection with human papillomavirus (HPV)^9,10. HPV+ HNSCC has a more favorable prognosis, as these tumors are more sensitive to radiotherapy and show much higher rates of overall survival than HPV-HNSCC^9,10. HNSCC is further categorized as oral cavity, laryngeal, nasopharyngeal, oropharyngeal, and hypopharyngeal, with higher income nations seeing greater prevalence of oropharyngeal cancers in comparison to developing countries⁹. As with most cancers, early detection greatly improves long-term survival outcomes; however, most cases (approximately two-thirds) are diagnosed in the advanced stages of the disease, causing challenges in treatment^9,10.

Due to their location in the head and neck, HNSCC tumors are prone to metastasis into the lymph nodes of the neck¹. Metastasis can make treatment more difficult, as the movement of tumor cells to a new environment selects for more aggressive clones that are more likely to resist treatment¹¹. Metastasis to the lymph nodes presents a major challenge in the clinic, as lymph nodes play a central role in the body’s immune response, yet can be hijacked by tumor cells to create a permissive immune microenvironment for further metastasis. Tumor cells secrete cytokines to stimulate lymphangiogenesis, suppress immune function, and increase lymphatic flow¹⁵. These changes allow invading tumor cells to evade an adaptive immune response, specifically through signaling from cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or programmed death ligand 1 (PD-L1) during the priming process that inhibits the antigen-specific T cell response to cancer cells¹¹. As such, the status of the cervical lymph nodes in HNSCC patients is considered the most important factor in overall prognosis, as evidence of lymph node metastasis is correlated with a dramatic reduction in survival rate¹⁷.

To prevent metastasis, it is common practice for HNSCC patients to undergo surgical lymph nodes resection or ablation through radiotherapy. In a randomized clinical trial of patients with lateralized stage T1 or T2 oral SCC, patients who underwent elective neck dissection had improved rates of both overall survival and disease-free survival, likely due to this prevention of metastasis¹. Because of its major benefits to patients in terms of long-term survival, this practice of lymphoablation has become the standard practice, alongside chemotherapy and radiation treatments. However, new treatments that modulate the immune system or target cancer cell signaling have the potential to revolutionize patient care and improve overall survival outcomes–but only if they are implemented in the correct circumstances.

Immunotherapies function by enhancing the immune system’s ability to identify cancer cells, enabling the patient’s immune cells to fight their cancers from within¹⁴. Novel immunotherapies can target inhibitory mechanisms in a process called checkpoint inhibition. The checkpoint inhibitor of interest for this review is the anti-CTLA-4 antibody. The CTLA-4 surface receptor naturally inhibits T-cell proliferation by outcompeting the CD28 receptor responsible for T-cell activation, weakening antigen-specific immune activation^15,16. Blockade of CTLA-4 eliminates the receptor’s competition against CD28, enabling uninterrupted T-cell activation. While more research is needed to pinpoint the exact location, timing, and mechanism of anti-tumor T-cell priming, these observations indicate that the CTLA-4 blockade indirectly and specifically promotes the activation of antitumor T cells¹⁶.

To better understand the effects of anti-CTLA-4 therapy on HNC, the Gutkind lab at the UCSD Moores Cancer Center recently characterized a 4NQO-induced murine orthotopic model that displays a mutational signature with a 94% similarity to that of human tobacco-induced HNSCC⁵. Named 4MOSC1, this model displays unique binary sensitivity to anti-CTLA-4 checkpoint blockade treatment in a CD8-dependent manner⁵. However, a similar complete response to anti-CTLA-4 treatment has not been demonstrated in clinical trials for HNSCC. For example, in a phase III study (“EAGLE”) comparing the combination therapy of PD-1 and CTLA-4 checkpoint inhibitors to SOC for R/M HNSCC patients, it was concluded that there were no statistically significant improvements on overall survival⁴. As such, we hypothesize that the contradiction between the results of the phase III EAGLE study and the success of anti-CTLA-4 therapy seen in both late stage melanoma and the 4MOSC model may be attributed to the surgical removal of lymph nodes that is standard practice for HNSCC patients.

The lymphatic network plays a crucial role in the immune system: antigen-presenting cells display antigens to lymphocytes at particular sites within the network, which initiates clonal expansion of lymphocytes and their secretion of antibodies into the lymph. Lymphatic vessels are essential to the circulation and transportation of immune cells throughout the body, as well as the enabling of immune surveillance of surrounding blood vessels and tissues². By surgically removing lymph nodes in HNSCC patients, the main sites of immune priming and primary organs for housing T-cell populations are lost. Consequently, from data gathered through murine models, we hypothesize that the mechanisms by which anti-CTLA-4 based therapy is successful are lost to this SOC treatment.

To rescue anti-CTLA4 response after lymphatic resection, the field of tissue engineering has become a promising avenue with recent advancements that support an engineered artificial lymph node as a viable solution. Tissue engineering aims to recreate the structure and function of native tissues using a combination of molecular signals, cellular components, and biomaterial scaffolds¹⁷. The most notable achievements in tissue engineering are those involved in developing therapies to regenerate skin, blood vessels, bone, cartilage and even more complex tissues like the trachea, bladder, liver, and pancreas^17,18.

While lymphatic tissue engineering itself is a newer discipline, it still stands to benefit from existing research on different organ systems due to similarities in organ structure and the development of fundamental tissue engineering techniques¹⁸. For example, lymphatic vasculature is relatively similar to blood vessels in its utilization of a valve system to prevent backflow and their composition of similar endothelial cells, which may indicate that certain aspects of creating artificial blood vessels could be translated to create artificial lymphatic vessels^17,18. Furthermore, several established biomaterials, bioreactors, and cell and molecular delivery systems used in engineering tissue have shown promising results in stimulating lymphatic regeneration^17-20. In one case, a disposable bioreactor that consisted of sheets of porous agarose and polymer fibers was seeded with primed dendritic cells and cultured for two weeks under controlled medium exchange¹⁸. This setup allowed for the creation of an ideal environment that promotes lymphatic growth, which was confirmed with clusters of lymphocytes and signs of T-cell activation within the inner mesh of the bioreactor, thus exemplifying the possibility of engineering lymphatic tissues from environmental and biochemical controls¹⁸.

The extracellular environment plays an important role in determining cellular function, gene expression, differentiation, proliferation, migration, and morphology²¹. As a result, careful choice of biomaterial in designing a tissue engineered lymph node is essential in ensuring that the implant will be conducive to the specific microenvironment and biochemical cues needed to stimulate immune activity and regulate cell behavior²¹. Cells naturally exist in the extracellular matrix (ECM), which contains a plethora of different proteins, such as collagen, hyaluronic acid, and hundreds of others²¹. Thus, it can be difficult to mimic the varied composition and complex biochemical cues of the ECM using scaffolding composed of just a few biomaterials. As a result, decellularized tissue has gained popularity as a biological scaffold, as it is able to preserve cellular binding sites and tissue-specific growth factors²². Two effective methods of decellularization use either sodium dodecyl sulfate (SDS) or organic acid to remove cellular materials from a piece of tissue, leaving behind just the protein scaffold^{22, 23}. These protocols are deemed successful based on their ability to preserve the ECM architecture and biomolecules while sufficiently removing nuclear material^22,23. Furthermore, a decellularized ECM proved effective in vivo to deliver leukocytes and dendritic cells in separate studies without triggering a negative antigenic response^22,23. Decellularized ECM is a promising scaffold that can be used to deliver cellular or molecular therapies to regenerate the lymph nodes.

Both biomaterial scaffolding and decellularized ECM have shown promising results in studies attempting to simulate the environment of the lymph node, creating tissues that have structural resemblance to native secondary lymphatic organs and recover some degree of function^24,25,26. These tissues, deemed lymphoid organoids, have established several markers of success^25,26. For example, one lymphoid organoid made of a spongy collagen scaffold seeded with LTɑ-expressing stromal cells and dendritic cells was transplanted into the renal subcapsular space in mice^25,27. Three weeks after transplant, the lymphoid organoids showed distinct B and T cell clustering similar to normal CD4+ to CD8+ ratios, formation of follicular dendritic cell networks, and active proliferation of B cells²⁵. These results proved successful enough for Watanabe et al. to obtain a patent for creating an artificial lymph node for the purpose of treating cancer²⁷.

Another technique that has been considered to engineer an artificial lymph node for the purpose of treating cancer include a biomaterial scaffold containing chemokines. This involved the creation of an artificial tertiary lymph node from a combination of a collagen scaffold and Medgel without the use of live cells²⁸. This was then demonstrated to successfully recreate a semblance of native lymphoid tissue in vivo²⁸. By slowly releasing an appropriate chemokine profile, the originally cell-free lymph node replacement recruited dendritic cells, clusters of B and T cells, and proper signaling networks, which allowed it to exhibit an immune response²⁸.

To be effective, a tissue engineered lymph node must demonstrate reconnection to native lymphatic architecture. As such, this project will focus on the use of IMARIS imaging software to achieve this aim. IMARIS is an interactive software developed by Oxford Instruments that allows researchers to create 3D/4D visualizations and perform analysis of microscopy images. IMARIS provides both automated and semi-automated algorithms for tracking, segmenting, and calculating statistical characteristics of various cell types and networks. Some of these models, like the surface and filament models, have been used to collect data on blood vessels and neuronal networks, and can also be applied to lymphatic vasculature mapping. From these constructed models, researchers can measure characteristics such as cell volumes, distances from membranes to cell centers, and surface areas of individual cell bodies.

One of the main challenges of engineering a lymph node de novo resides in its complexity²⁹. A lymph node has a unique microenvironment consisting of a myriad of cell types, a highly organized structure, cellular motility, and increased cell density all within a very small scale²⁹. These challenges alone make it very difficult to successfully reproduce the 3D structure and viability of a lymph node in vitro compared to a native lymph node in vivo. In addition to the physical problems that the procedure faces, the viability of the artificial lymph node also depends on biochemical factors, such as hypoxia within the lymph node and its generated microenvironment²⁹. Successful methods have overcome these challenges using a variety of mechanisms^18,25,29.

Visualizing the lymphatic system also poses a significant challenge to the goal of using IMARIS for 3D modeling and analysis because of the lack of development in lymphatic imaging technology. Attaining a complete visualization of an entire lymphatic network is difficult because lymphatic vessels carry lymph fluid in only one direction, so a single injection of dye can only stain the lymphatics from the point of injection onwards. This issue in lymphatic imaging has not yet been addressed because the lymphatic system was not perceived to be crucial in understanding the patients’ development of and response to diseases until relatively recently, compared to the circulatory system. As such, there is still a need to develop advanced lymphatic imaging techniques and dyes to address these issues³⁰.

The most significant challenge to engineering such a device is the lack of clinical testing in humans. There are significant safety, efficacy, and manufacturing hurdles that must be cleared before moving from in vitro to in vivo studies, and again from in vivo models into patients. To our knowledge, there currently is no confirmation of artificial lymph node efficacy in treating human cancers. While all of these techniques have shown promise in multiple mouse models, human lymph nodes differ in size and structure, which may alter their potential for success in the clinic. However, by combining some of these methods with proven SOC procedures for lymphatic cancers, we hope to increase the success rate of treatment and provide reassurance that the overall treatment regimen will benefit, rather than hurt, the patient.

Standard of care treatment for HNSCC:

As mentioned before, the SOC for observable tumors, defined by being greater than 2 mm in diameter, is lymphadenectomy to prevent metastasis. The first line of treatment involves 5-fluoro-uracil (5FU) chemotherapy in combination with cetuximab (anti-EGFR), followed by cetuximab with methotrexate³². For recurrent or metastatic and unresectable tumor development, pembrolizumab (anti-PD1 immunotherapy) is introduced³². Clinical trials must be compared to SOC treatment options to ensure that no patient is denied proper care. As such, the testing of this scaffold in a clinical trial must involve comparisons to those treated solely with SOC in the R/M or neoadjuvant setting. Should a tissue engineered scaffold successfully rescue the antitumor immune response after removal of lymphatics, this design may redefine what becomes standard treatment for patients with HNSCC.

Imaging Standards:

Imaging standards that apply to the IMARIS software have been developed by the ISO. It is crucial that we follow these standards to allow us to eventually introduce this model as a future reference of what an intact, healthy lymphatic system in mice should look like for researchers in the lymphatic field. The ISO 12052:2017 defines the Digital Imaging and Communications in Medicine (DICOM) standards researchers must follow when creating, publishing, and transferring images for health informatic purposes. These standards serve to guide diagnostic medical imaging fields, such as radiology, pathology, and cardiology.

Decellularization Protocols:

Our plan to tissue engineer a decellularized lymph node scaffold follows the decellularization of tissues standard set by ASTM F3354-19, which provides guidance on ways to evaluate the efficiency of extracellular matrix decellularization process, as well as related ISO standards like ISO22442-1. We then use reagents and kits produced by scientific material suppliers to verify and characterize our scaffold, and these companies follow standards to manufacture all their products³².