Investigating feline injection site sarcoma

14 October 2026

In 2025, Dr Lisa Pang at the University of Edinburgh received BSAVA PetSavers funding to support a Student Research Project investigating feline injection site sarcoma. Student Michelle Lai describes her work.

The introduction of vaccination has revolutionized the field of preventing lethal infectious diseases; however, cats have a unique but small risk of developing an aggressive tumour, FISS, at the site of injection. Practitioners may be familiar with the ‘3-2-1 rule’: lumps at the site of injection persisting more than 3 months, with a diameter larger than 2 cm, or increasing in size 1 month after initial appearance, should be considered suspicious and investigated further for FISS.

The pathogenesis of this tumour remains unknown but it is reported that rabies and FeLV vaccine injections are the biggest risk factors for developing FISS. Interestingly and correspondingly, there is a difference between the incidence in the UK and the US, as rabies vaccination is mandatory in the latter. The US incidence is 1–4 cases per 10,000 vaccinated cats,1,2 while in the UK it is 1 in 10,000.3 Hence, most of our knowledge on FISS is derived from US models, which might not fully represent UK cases that are mostly associated with FeLV vaccines, highlighting the need for a comparable UK model.

FISSs are locally invasive, fast-growing, and difficult to eliminate. Even with the standard treatment of radical surgery with wide margins and adjuvant radiotherapy, recurrence is common, leading to high morbidity and mortality. Other adjunctive treatments for more disseminated disease, such as doxorubicin, show very limited efficacy; hence, better treatment options should be explored.

FISS, and tumour recurrence in general, may be largely attributed to the persistence of cancer stem cells (CSC). CSCs have self-renewal properties and can proliferate to repopulate tumors. More importantly, they have been shown to be chemo- and radioresistant in both animal and human models. Without targeted treatment against FISS CSCs, recurrence is likely.

To date, no CSC has been isolated from FISS; therefore, this project attempted to isolate CSCs from US and UK FISS cell lines using a tumour spheroid assay. Isolated CSCs were planned to be investigated for their characteristics, including behaviour that may favour cancer cell survival and chemosusceptibility compared with non-CSCs.

In search of the masterminds of FISS

We used five cell lines isolated from US patients and one primary cell line recently isolated from a UK patient. After validating their identity as sarcoma cells, we separated them into single cell suspensions in a special medium called 3D Tumorsphere Medium XF, and seeded each cell line into a 96-well round bottomed ultra-low-attachment plate at a density of 1500 cells/well. The plates were then incubated at 37°C and 5% CO2.

Tumorspheres from US cell lines were first seen 7 days after seeding, and by 2 weeks >90% of wells contained tumorspheres. We harvested these spheres from the plates and performed real-time quantitative PCR (RT-qPCR) investigating the relative expression of three main embryonic stem cell markers: NANOG, Oct4 and Sox2. Unfortunately, there was no significant difference in the expression of these markers between the tumorspheres collected and the non-enriched parental population across all five cell lines. Hence, we could not conclude that these tumorspheres from the US cell lines were CSCs.

As for the UK cell line, we encountered difficulties with maintaining and expanding the cells.  Being recently established, the cells were fragile. To start with, the growth rate was slow, probably because cells were still adapting to the new microenvironment provided by culture medium and plastic culture vessels. We repeated the same experiment to isolate CSCs. The growth rate was significantly lower and the tumoursphere appearance was only first observed after 10 days and took 21 days for >90% of wells to contain tumourspheres. Morphologically, the UK cell line contained smaller but multiple tumourspheres in each well, while the US cell lines had larger, fewer spheres. The UK FISS tumourspheres were also more adherent to the vessel, making harvesting challenging. Unfortunately, the RNA concentration and quality of the extracted cells were not up to the standards for qPCR.

Further, there was an incident where the incubator malfunctioned so cells were left at a suboptimal temperature overnight, resulting in a lot of floating, unhealthy cells. Since then, even with our greatest efforts, such as grouping cells and changing to a tumour-specific medium, cells failed to thrive. Due to the altered behaviour, the cells could not be used for experiments and we could not attempt to isolate CSC again during the timeframe of this work.

However, the lab will continue working on this project. Our next steps upon establishing a stable culture will be to resume optimizing the CSC isolation protocol. Other options, such as using fluorescence activated cell sorting (FACS) can be explored as well.4

What does this mean for general practitioners

The ultimate goal of this project is to explore new drug options that target FISS CSCs to eliminate residual tumours and prevent recurrence. Currently, there is no drug with proven benefits to treat FISS. Being able to isolate CSCs will mean we can test the relative susceptibility of these cells to chemotherapeutic drugs in comparison with the general tumour population. This could eventually lead to the development of novel treatment methods for FISS patients, which will require clinical trials carried out in both GP and specialist clinics.

Why consider BSAVA PetSavers?

This research was made possible by BSAVA PetSavers who kindly funded our project. They contributed immensely to covering the costs of laboratory consumables and qPCR test kits. I would strongly encourage vet students and vets interested in research, both laboratory and clinical, to consider PetSavers as a means of making your idea possible. PetSavers also offers an opportunity for publishing your findings and sharing them with the wider veterinary community.

The personal experience

I was delighted to return to research as part of the veterinary profession. Finding familiarity with cell culture and biochemical tests was comforting but I certainly underestimated the difficulty of culturing primary cell lines and optimizing new experimental protocols! I am grateful to Dr Lisa Pang, who kindly offered me the opportunity to work on the FISS project in her lab and all the guidance along the entire length of this project. I would also like to thank Hazem Alkhaldi, who patiently guided me through numerous experiments. This project inspired me to continue research during my veterinary career and I look forward to seeing what this project uncovers further down the line!

About the author

Michelle is a vet student currently in her third year of the graduate entry BVM&S programme at the University of Edinburgh. Her primary interests are small animal medicine but she is open to all areas of veterinary medicine. During her undergraduate studies in biomedical science, she joined a molecular biology lab focusing on cell matrix adhesion signalling.

References

  1. Coyne MJ, Reeves NCP and Rosen DK (1997) Estimated prevalence of injection-site sarcomas in cats during 1992. Journal of the American Veterinary Medical Association 210(2), 249–251
  2. Gobar GM and Kass PH (2002) World Wide Web-based survey of vaccination practices, postvaccinal reactions, and vaccine site-associated sarcomas in cats. Journal of the American Veterinary Medical Association 220(10), 1477–1482
  3. Dean RS, Pfeiffer DU and Adams VJ (2013) The incidence of feline injection site sarcomas in the United Kingdom. BMC Veterinary Research 9, 17
  4. 4. Duan et al., 2013