There are limitations to using laboratory animals to advance our understanding of cancer and evaluate new drugs and/or therapies. With their homogenous environments, a smaller overall size, and induced tumors—laboratory animals are unable to mirror the complex genetic, metabolic and epigenetic alterations associated with cancer (1, 2). Like humans, naturally occurring cancer in companion animals (dogs and cats) is one of the leading causes of death (1–5). Living in the same environments, humans and companion animals share many of the same environmental and socioeconomic factors that can predispose cancer development. These risk factors include, but are not limited to, air pollution, pesticides, age and obesity (1–4). The large number of companion animals affected by cancer is an underutilized resource to bridge the translational gap. Naturally occurring cancer in dogs and cats can provide an intermediary step between artificially induced laboratory models and human patients.

Dogs, cats and humans develop cancers with similar presentation, common phenotypes, clinical pathologies (receptors, copy number), progression and treatment response (1–5). There are many examples of comparative cancers including oral cavity squamous cell carcinoma, mammary (breast) cancer, bladder and prostate cancer, lymphoma, melanoma and pulmonary adenocarcinoma (1–5). With the relatively short life span of companion animals, in comparison to humans, cancer progresses quicker, allowing for timely assessment of novel therapies and continuity of long-term data; endpoints [disease-free interval (DFI) and median survival time (MST) (i.e., 1–2 years vs. 5–10 years) (1–3)]. In addition, identical diagnostic and imaging modalities can streamline the interpretation and comparison of results. There is also relative ease for companion animal trials as regulations are more flexible than human medicine, and the overall cost is much lower (1, 4). Moreover, increasing sentiments of companion animals as valuable household members and a desire to extend their lifespans have led to an increasing demand for improved human-level care and advanced techniques through veterinary clinical research (5–8). Due to all of these factors, there has been an increasing interest in including companion animals in the therapy development pathway (1–5, 9).

Surgical excision of a tumor(s) and lymphadenectomy is the first-line treatment for many cancer types. Nodal involvement has a significant bearing on tumor staging, treatment, and prognosis. The sentinel lymph node (SLN) is a tumor’s primary or first draining lymph node(s). Understanding the SLN allows for decreased surgical dissection, more accurate lymph node extirpation, and better overall outcomes (10, 11). Comparative cancers in dogs, cats, and humans tend to have similar lymphatic metastasis rates, making them a good model for comparison. While there are many similarities in the lymphatic anatomy and function between species, several notable differences exist. Firstly, dogs and cats have an overall fewer number of lymph nodes. Dogs and cats have only 1–2 axillary lymph nodes per side, while it is estimated that there are 20–30 axillary lymph nodes in the human body (12, 13). Secondly, unique anatomical species differences can influence the identity of the SLN. For example, dogs and cats have 8–10 mammary glands evenly distributed on their ventral abdomen, while humans have 2 mammary glands closer to the forearms. In over 90% of clinically node-negative human breast cancer patients, SLN mapping and biopsies are regarded as crucial diagnostic tests due to the large number of axillary lymph nodes (10, 11). In dogs with mammary cancer, the SLN was determined to be the axillary or accessory axillary lymph node(s) when the tumor was in the first, second, or third mammary gland and the superficial inguinal and medial iliac lymph node(s) when the tumor was in the fourth mammary gland (14). Interestingly, there seems to be wide variation and a lack of clear pattern for the tumors located at the fifth or inguinal mammary gland (14–17). Therefore, in companion animals, the SLN could be the axillary or superficial inguinal lymph node, depending on the location of the affected mammary gland, compared to a human patient with breast cancer where the SLN is typically the axillary lymph node (10, 11, 16, 18).

This review will focus on comparative aspects of SLN mapping as a reference for the potential inclusion of companion animals in future developmental pipelines for targeted SLN mapping agents.

While SLN mapping has become the standard of care for many human tumor types, it is a relatively new concept to veterinary medicine, becoming more prevalent in the last 10–15 years. These techniques often involve a combination of preoperative and intraoperative methods. Preoperative modalities include indirect lymphography using radiography or computed tomography (CT), lymphoscintigraphy and contrast-enhanced ultrasound (CEUS). Commonly used intraoperative techniques include gross visualization following an injection of blue dyes [such as methylene blue (MB)], or near-infrared fluorescence (NIRF) imaging with fluorescent dyes such as indocyanine green (ICG).

Indirect radiographic lymphography (IRL) has been successfully applied in healthy and diseased dogs. Contrast is introduced through a series (most often a quadrant pattern) of intradermal/subcutaneous injections around the tumor (Figure 1) (19, 20). Contrast agents reported in veterinary patients include both water-soluble (iopamidol, iohexol) and lipid-based (lipiodol) solutions (19, 21–23). Advantages of IRL include its excellent safety profile, low cost, simple injection technique, and widespread availability of radiography in most veterinary clinics (14, 19, 20). Furthermore, IRL can be performed in animals with sedation rather than general anesthesia (14, 20). Mayer et al. reported uniform contrast uptake in associated lymph nodes following injections of lipid-based contrast in 8 different locations (head, neck, ventral abdomen and limbs) in 16 healthy purpose-bred dogs (19). A similar study by De Bonis et al. reported an SLN detection rate of 90% using lipiodol in 23 dogs diagnosed with 26 MCTs (23). Haas et al. evaluated IRL with water-soluble contrast in 53 dogs with 59 mast cell tumors (MCT), reporting a diagnostic rate of 77.9% (20). The SLN of canine cutaneous and subcutaneous mast cell tumors of the trunk and limbs are most investigated due to the lack of agreement between the locoregional lymph node and the SLN, with disagreement rates ranging between 28 to 63% (24). In a study published by Annoni et al., where SLN mapping was performed in 80 dogs with 138 mast cell tumors, 84% originated from the trunk and limbs (25). The SLN was found to differ from the locoregional lymph node in 57% of the cases, and IRL successfully mapped the SLN in 95% of cases.

The main limitation of IRL is its lower sensitivity for SLN detection compared to CT. Despite Haas et al. reporting a high diagnostic rate of 77.9% via IRL, 20.3% of the cases were considered only partially diagnostic due to the reported visibility of the lymphatic tract but not the SLN (20). In CT lymphography (CTL), water-soluble iodinated contrast is injected directly into the lymphatics, peri or intratumorally, followed by a series of scans (Figure 2) (21). The increased resolution of CT allows for better visibility of the lymphatic vessel system, making preoperative CTL superior to radiographic lymphography (21). Sentinel lymph node detection rates using CTL are highly variable in veterinary medicine, with studies reporting rates ranging between 60 and 95%, compared to human literature where detection rates are between 95.8 and 100% (26–29). In addition, the type of contrast used for SLN mapping may affect the sensitivity of CTL. Mahieu et al. compared the use of lymphoscintigraphy and CTL in human patients with oral cancer using a lipid-based contrast and reported poor sensitivity (55%) and high negative predictive value (75%) using CTL (30). For veterinary patients, CTL is performed under general anesthesia and is typically only available in a tertiary referral hospital setting, making it a less accessible and more costly option (31). Overall limitations with both preoperative methods include the lack of real-time visual guidance and the increased exposure of hospital staff and patients to radiation.

Lymphoscintigraphy involves peritumoral injections of radiocolloids and is considered the gold standard for SLN mapping in human medicine (31–33). Lymphoscintigraphy is reported to be superior to IRL, with successful SLN detection rates of 100 and 41%, respectively. Manfredi et al. evaluated the use of preoperative lymphoscintigraphy in dogs with a malignant primary tumor (e.g., MCT, mammary adenocarcinoma, oral melanoma, thyroid carcinoma, sublingual SCC, parotid adenocarcinoma, etc.) without metastasis and the SLN was detected in 90.5% (n = 57/60) of tumors in 53 dogs (34). Similarly, an SLN detection rate of 91% was reported in 30 dogs with 34 MCTs that received peritumoral injections of radiocolloids and MB (35). However, the use of radiocolloids for SLN mapping is limited and is rarely applied in veterinary medicine due to its high cost and lack of accessibility (36).

Contrast-enhanced ultrasound (CEUS) is an alternative method for preoperative SLN mapping and involves peritumoral or subdermal injections of microbubbles, a high molecular weight gas that is enclosed in a lipid shell, which functions as a sonographic contrast agent (37). The use of CEUS has been documented in healthy dogs, pigs, rabbits and monkeys with success in identifying the SLN at a detection rate of 91.3–100% (38–40). Fournier et al. reported a SLN detection rate of 95.2% when CEUS was used for SLN mapping in dogs with MCT (37). While a promising alternative to other techniques, CEUS can only be performed on a superficial level and is limited by the depth of the lymphatic tracts. When comparing CEUS and ICG-NIRF, ICG-NIRF is a better technique due to its capability to identify the afferent lymphatic tracts, which are deeper within the tissues (41). In addition, radiology expertise and specialized training are required, which makes accessibility of this technique limited in veterinary practice.

Intraoperative SLN mapping using blue dyes, ICG, or other agents provides real-time visual guidance. Traditionally, blue dye is injected peritumorally in four quadrants surrounding the tumor, and the SLN is located based on gross visualization of blue staining (Figure 3). Using blue dye alone has been most frequently reported in human breast cancer patients, with a high identification rate of 89–91% (42, 43). In contrast to human literature, SLN detection rates in veterinary medicine are highly variable when blue dye is used alone. Ferrari et al. reported an SLN identification rate of 91% in 34 dogs with MCTs, while Wan et al. reported an SLN detection rate of 50.8% in dogs with oral cancer (22, 35). Methylene blue (MB) is a commonly reported blue dye as it is widely available and cost-effective; however, the use of MB alone heavily relies on the knowledge of the location of the SLN with respect to the tumor as blue staining is not easily visible percutaneously (42–44). Given that the success of SLN detection using MB alone is highly variable, recent literature has recommended either a shift from using MB entirely, combining MB with ICG-NIRF, or combining preoperative indirect lymphography using CT with intraoperative MB-ICG/NIRF to maximize SLN detection rates (22, 45, 46).

Compared to MB alone, ICG-NIRF has a higher SLN detection rate, greater tissue penetration, and better visualization of the afferent lymphatic tracts (22, 41, 47). Despite NIRF-ICG being a more expensive modality to MB, it is known to be less invasive, cost-effective, and has a higher sensitivity and specificity compared to lymphoscintigraphy in human literature (48). Arz et al. reported success in identifying SLNs using ICG-NIRF as small as 0.9 cm in a cat diagnosed with a low-grade MCT in the left buccal region. Indocyanine green also has an excellent safety profile and has been demonstrated to have no significant side effects in companion animals (49). Applications have included SLN mapping of the oral, head and neck region, caudal abdomen and limbs in healthy and diseased dogs and head and neck region, trunk, perineum, and limbs in healthy and diseased cats (Figure 3) (22, 29, 41, 46, 47, 50). As a result, this technique is gaining popularity in the veterinary field; however, limitations include the accessibility of NIRF equipment and a lack of protocol standardization.

An optimal SLN mapping imaging agent enables rapid clearance from the injection site, has increased uptake and retention within the SLN, and has low drainage to higher-tier lymph nodes (51) (Figure 4). The previously mentioned techniques for SLN mapping employ passive strategies. These preoperative and intraoperative imaging modalities exhibit unsatisfactory sensitivity and specificity rates, decreasing the fidelity of these resections (52).

This pitfall can be overcome by developing novel ligand-receptor or antigen–antibody binding imaging probes (51). Theoretically, any imaging agent targeting tumor cells can detect both primary tumors and metastatic lymph nodes (53). In recent years, the concept of precision medicine has had unparalleled growth in human oncology; however, it is relatively new in veterinary oncology. While precision medicine encompasses many elements, particularly valuable for SLN mapping is the identification of genomic markers and tumor characteristics.

Several targeted imaging agents have been developed for biomarkers expressed in human cancers and many of these are undergoing clinical testing or have gained regulatory body approval. Dogs and cats spontaneously develop many of the same cancers as humans with similar histological subtypes and tumorigenic genes and pathways, which is fundamental for the translation of targeted therapeutics (1, 2, 9, 54). Building upon the foundation of human cancer research, comparative companion animal cancers can aid in theoretically applying or studying targeted imaging agents for SLN mapping.

Companion animals are often an integral part of our lives, sharing our environments and, subsequently, our risk factors for developing cancer. Current research has determined genomic and tumor similarities within the naturally occurring cancers between humans, dogs and cats, which can be used to develop targeted lymph node mapping agents. Cross-species use increases accessibility and enhances early adoption in clinical trials. The synergistic relationship between animal and human cancer research is vital for the future of translational research and advancements in precision medicine.

MO: Conceptualization, Writing – original draft, Writing – review & editing. HL: Writing – original draft, Writing – review & editing. AR: Writing – original draft, Writing – review & editing, Conceptualization, Visualization. CM: Conceptualization, Writing – review & editing, Writing – original draft, Visualization.