Unlocking the role of tumor-associated macrophages (TAMs) in feline injection-site sarcomas – a path to immunotherapy

Abstract

Feline injection site sarcomas (FISSs) are tumors frequently found at injection sites in domestic cats associated with vaccines and other pharmaceutical substances. The most accepted theory suggests that chronic inflammatory reactions at the injection site trigger these tumors. This study analyzed 58 cases of FISS in cats to investigate the role of tumor-associated macrophages (TAMs). Immunohistochemistry for MAC387+ macrophages was performed via the Novolink™ polymer detection system. TAMs were quantified and categorized into low, moderate, and extensive infiltration groups. Most tumors showed sparse macrophage infiltration (29 out of 58 cases), with moderate macrophage infiltration (18 out of 58), and 11 cases out of 58 showed high infiltration. Significant associations were found between TAM infiltration and the degree of differentiation (p<0.001), degree of necrosis (p=0.033), mitotic index (p= 0.003), and histological degree of malignancy (p<0.001). This study revealed that TAM density is correlated with tumor aggressiveness in the FISS, suggesting a fundamental role for macrophages in the tumor microenvironment and a promising marker for prognosis.

Introduction

Vaccination is one of the most effective strategies for preventing infectious diseases, but like any medical intervention, it can have adverse effects (Hartmann et al. 2015). These include the occurrence of vaccine-site sarcomas in cats, known as Feline Injection-Site Sarcomas (FISS). In recent years, there has been an increase in the number of cases of tumors at vaccine application sites in cats (Hartmann et al. 2015, 2023).

Vaccine-associated adverse effects (VAAEs) can be triggered by an excessive immune response, whether innate or adaptive, or by exaggerated reactions at the site of application. In addition, errors in the administration technique, when the product's instructions are not strictly followed, can also contribute to the appearance of these events. Although VAAEs can occur in different species, dogs and cats are particularly susceptible (Dobromylskyj 2022; Hartmann et al. 2023).

The most severe adverse effect in cats is the formation of invasive sarcomas, often fibrosarcomas, at the vaccination sites. These tumors, called FISSs, also known as "feline injection-site sarcomas," are common in domestic cats and rarely observed in other species, making them a species-specific concern (Hauck 2003; Porcellato et al. 2017; Dobromylskyj 2022).

The FISS tends to develop at classic vaccination and injection sites, such as the region between the scapulae, the lateral wall of the thorax or abdomen, the lumbar area and the regions of the semimembranosus and semitendinosus muscles of the hind limbs (Kirpensteijn et al. 2006; Porcellato et al. 2017; Zabielska-Koczywąs et al. 2017; Carneiro et al. 2019). Although their occurrence is more frequent in the subcutaneous tissue, they can also manifest themselves intramuscularly (Porcellato et al. 2017; Zabielska-Koczywąs et al. 2017; Carneiro et al. 2019). These tumors can appear anywhere from four months to two or three years after the vaccine is administered. They are characterized by the proliferation of anomalous spindle cells and often by the presence of multinucleated giant cells (Porcellato et al. 2017; Zabielska-Koczywąs et al. 2017; Carneiro et al. 2019). Most cases are fibrosarcomas, but other types, such as rhabdomyosarcoma, myxosarcoma, chondrosarcoma, osteosarcoma, and histiocytic sarcoma, can also occur (Porcellato et al. 2017; Zabielska-Koczywąs et al. 2017; Carneiro et al. 2019).

Despite research efforts, there is still no definitive causal explanation for the occurrence of FISSs or a direct and unequivocal link with vaccination. The most accepted theory suggests that persistent chronic inflammation at the injection site can create conditions for malignant transformation of the cells (Hartmann et al. 2023). The latency period can vary depending on the progression of these tumors. Although FISSs have a low rate of metastasis, they are known for their rapid growth and locally aggressive behavior (Séguin 2002; Hauck 2003; Martano et al. 2011). The FISS shows histological and behavioral characteristics reminiscent of those of musculoskeletal sarcomas observed in humans, which share similarities in the histological structure of fibroblasts and aggressive clinical behavior (Day et al. 1999; Martano et al. 2011, 2019).

Cancer is currently considered a complex process that involves constant, dynamic, and bidirectional interactions between tumor cells and the tumor microenvironment (Xiao and Yu 2021). The tumor microenvironment (TME) comprises different cell types, such as endothelial, stromal, and immune cells, which communicate continuously, influencing the maintenance of homeostasis or tumor progression (Vitale et al. 2019; Arneth 2019).

Within this context, tumor-associated macrophages (TAMs) stand out as central elements in the TME and are the most abundant cells involved in the infiltration of many human and animal (dogs and cats) malignancies (Arneth 2019; Wu et al. 2020; Nascimento et al. 2022; Brady and Thamm 2023). They are highly varied and highly plastic (Arneth 2019; Wu et al. 2020; Nascimento et al. 2022; Brady and Thamm 2023). Although the exact role of macrophages in the tumor microenvironment has not yet been fully elucidated, they are considered promising therapeutic targets because of their significant influence on the TME and ability to both promote and suppress tumor growth (Arneth 2019; Wu et al. 2020; Xiang et al. 2021). The protumor functions of TAMs include promoting inflammation, stimulating angiogenesis, and facilitating the circulation of tumor cells, favoring metastasis to different organs (Xiao and Yu 2021).

Owing to their remarkable plasticity, TAMs are a key component of the immunosuppressive microenvironment of tumors. They can be classified into two main types, M1 and M2, with the ability to be reprogrammed to the M1 phenotype, which is antitumor. Thus, therapeutic strategies targeting TAMs are being developed to reduce monocyte recruitment and direct the activation and function of TAMs (Xiao and Yu 2021).

The main objective of this study was to investigate inflammatory infiltration by TAMs in injection site-associated sarcomas in cats.

Main text

Clinical presentation and epidemiological correlations

The analyzed feline injection-site sarcomas (FISSs) manifested as nodular or ulcerated lesions (0.5–17.5 cm; mean 4.9 cm) localized to vaccine/injection sites (scapular/neck regions), which is consistent with prior reports (Romanelli et al. 2008). A female predominance (63.8% vs. 36.2% males) was observed, which aligns with the findings of some studies (Kass et al. 2003) but contrasts with claims of no sex predisposition (Porcellato et al. 2017). European breed cats predominate (81%), although breed-specific risk remains debated (Cecco et al. 2019; Pinnelo et al. 2022) The average age of affected cats is 10.5 years (±3.027), corroborating established age trends (Romanelli et al. 2008).

Histopathological grading & TAM infiltration dynamics

Histological grading revealed 25.9% Grade 1 (highly differentiated), 48.2% Grade 2 (moderately differentiated), and 25.9% Grade 3 (poorly differentiated) tumors. Necrosis (51.7% moderate, 25.9% extensive) and elevated mitotic indices (53.4% Index 1, 20.7% Index 3) paralleled the degree of malignancy. Notably, TAM infiltration intensity was significantly correlated with aggressive features:

• Differentiation: Increased TAM density in poorly differentiated tumors (p < 0.001)

• Necrosis: Increased TAMs in necrotic regions (p = 0.033)

• Mitotic activity: Positive association with the mitotic index (p = 0.003)

• Malignancy grade: Grade III tumors exhibited predominant moderate/high TAM infiltration (81.2%), in contrast with Grade I tumors (100% low infiltration; p < 0.001) (Fig. 1).

Fig. 1

TAM immunolabeling (MAC387+) in Feline Injection-Site Sarcomas (FISS) was performed via immunohistochemistry. A shows a Grade 3 FISS with high TAM infiltration, while B shows a Grade 2 FISS with moderate macrophage infiltration; scale bar =50 μm

These findings mirror observations in canine carcinomas (Monteiro et al. 2021) and feline transitional cell carcinomas (Nascimento et al. 2022), reinforcing the role of TAMs in tumor progression. Mechanistically, TAMs promote immunosuppression via PD-L1 expression, cytokine secretion (e.g., IL-6 and TGF-β), and recruitment of regulatory T cells (Tregs) via CCL22, dampening CD8+ T-cell activity (Chen et al. 2021; Yan & Wan 2021).

TAM polarization & therapeutic implications

The dual M1/M2 polarization of TAMs underscores their functional plasticity. While M1 macrophages promote antitumor immunity via IL-12 and TNF-α, M2-polarized TAMs dominate aggressive tumors, driving angiogenesis and immune evasion through IL-10 and ARG1 expression (Chen et al. 2021; Zhang & Sioud 2023). Our data highlight M2-skewed TAM dominance in high-grade FISSs, which is consistent with their protumorigenic role in human sarcomas (Ganjoo et al. 2011).

Emerging therapies targeting TAMs include CAR-M engineering, which reprograms M2 TAMs toward the tumoricidal M1 phenotype while blocking PD-L1-mediated immunosuppression (Zhang et al. 2023). In combination with checkpoint inhibitors, such strategies may overcome TME-driven resistance, offering a promising avenue for FISS management.

Prognostic value & translational relevance

TAM quantification serves as a robust prognostic biomarker, with high infiltration (>20/field) predicting advanced malignancy (Fig. 2). This aligns with human oncology paradigms, where TAM abundance correlates with poor outcomes in patients with leiomyosarcoma and urothelial carcinoma (Lopez-Beltran et al. 2007; Ganjoo et al. 2011)  Files et al. 2024). In veterinary medicine, TAM profiling could guide treatment stratification, particularly for tumors refractory to conventional chemotherapy (Brady & Thamm 2023).

Fig. 2

Distribution of tumor-associated macrophage (TAM) scores: low, moderate, and high across feline injection site sarcomas of different histological grades of malignancy (grades I, II, and III)

This study establishes TAM infiltration as a hallmark of FISS aggressiveness, which is linked to key histopathological markers. By elucidating TAM-driven immunosuppression and plasticity, we identified actionable targets (e.g., CAR-Ms and PD-L1 blockade) to increase therapeutic efficacy. Further research should validate these mechanisms in vivo and explore combinatorial regimens to improve outcomes in feline oncology.

Conclusion

In feline injection-site sarcomas (FISSs) with high malignancy, increased infiltration of macrophages was observed, suggesting a significant role for these cells in tumor progression. This finding underscores the potential of macrophages as future markers of aggressiveness, as their presence and quantity in the tumor microenvironment could help predict tumor severity and behavior. Future studies should focus on characterizing the specific phenotypes of macrophages (e.g., M1 vs. M2) in the FISS, elucidating their molecular pathways to better understand their contribution to tumor dynamics, and incorporating prognostic studies to validate their clinical relevance. Additionally, research should investigate how targeting these immune cells through immunotherapy might influence tumor progression and therapeutic outcomes.

Our results also highlight a promising avenue for integrating immunotherapeutic approaches for treating these tumors, offering new possibilities to improve the prognosis and improve the quality of life of affected animals.

Methods

Study design and ethical compliance

This retrospective study evaluated 58 archival feline injection-site sarcoma cases from the University of Trás-os-Montes and Alto Douro Histopathology Laboratory. All the samples were diagnostic samples collected without experimental animal manipulation, with surgical procedures conducted under anesthesia following veterinary guidelines. Owner consent for research use was documented via laboratory request forms (supplementary material).

Data collection and histopathology

Clinical parameters (tumor size, breed, age, sex) were systematically recorded. Tissue sections (3 μm) stained with hematoxylin and eosin (H&E) were subjected to blinded evaluation by two pathologists (IP, JP) adhering to the WHO classification criteria for animal tumors. Microscopic imaging was performed via a Nikon Eclipse E600 microscope with a DXM1200 digital camera (Nikon Instruments Inc., Melville, NY, USA).

The tumors were classified into three histological grades of malignancy (1-3) on the basis of the following criteria: degree of differentiation, presence or absence of necrosis, mitotic index and inflammatory infiltrate. In addition, the presence or absence of giant cells was assessed. This classification was established by (Couto et al. 2002) and is detailed in Table 1. The characteristics analyzed were given a specific score according to Table 2. to determine the degree of malignancy. The sum of the scores of the analyzed characteristics defines the grade of malignancy of the tumor: Grade I (total score of 3-4), Grade II (score of 5-6), and Grade III (score of 7-9).

Table 1 Association of TAMs and histopathological characteristics in feline injection-site sarcomas

Table 2 Tumor classification criteria and respective classifications (Couto et al. 2002)

Immunohistochemical staining procedure

Tumor-associated macrophages (TAMs) were detected via MAC387 immunohistochemical staining via the Novolink polymer detection system (Leica Biosystems). Briefly, paraffin-embedded sections were deparaffinized with xylene (15 min) and rehydrated through a graded ethanol series (100−70%). Antigen retrieval was performed in citrate buffer via microwave irradiation (750 W, 20 min) with distilled water replenishment. After cooling (30 min, RT) and washing with PBS, endogenous peroxidase activity was blocked with 3% H₂O₂ (30 min).

The sections were incubated with protein block (5 min) followed by incubation with the primary antibody MAC387 (AbDSerotec, Clone MCA 874G; 1:100 dilution in PBS) overnight at 4°C in a humid chamber. After washing with PBS, detection was achieved via the following Novolink Polymer System components: secondary antibody (30 min), polymer reagent (30 min), and DAB chromogen (10-−12 min). Counterstaining was performed with Gill's hematoxylin (1 min), followed by dehydration through graded ethanol (95%−100%) and xylene clearing. The slides were mounted with Entellan resin (Merck) for microscopic analysis.

Quantification of TAMs

A positive result was considered when brown staining was observed in the cell cytoplasm, and only cells with macrophage morphology were counted.

Using a 10⨯ objective, three "hot" areas with the highest number of positive cells were selected, avoiding ulcerated regions. All positive macrophages were counted in the three observed areas, totaling ten high-power fields, via a 40 ⨯ objective. The total number of macrophages was then divided into three different categories: sparse infiltration (tumors with fewer than 5 macrophages), moderate infiltration (6 to 20 macrophages), and extensive infiltration (more than 20 macrophages).

Statistical analysis

Statistical analysis was performed via IBM SPSS Statistics version 21 (Statistical Package for the Social Sciences, IL, USA), where tables were created to cross-reference the results among the six characteristics studied (necrosis, infiltration, degree of differentiation, mitotic index, malignancy, and giant cells) and MAC expression. The chi-square test of independence was applied to examine the associations between MAC expression and the other variables. The observations were assumed to be independent. The expected frequencies were checked to ensure that each cell contained a value of at least 5 to validate the chi-square test. For contingency tables where the expected frequencies were less than 5, the chi-square test was adjusted via a Monte Carlo approximation to ensure accurate p values. A significance level of p<0.05 was applied to determine statistical significance.