Protocol for delivery of intraoperative immunotherapy to mice by surgical debulking of subcutaneous tumors.

Pre-clinical studies developing novel therapies to prevent cancer recurrence require appropriate surgical models. Here, we present a protocol for surgical debulking of subcutaneous tumors in mice, which allows for intraoperative application of immunotherapy-loaded biomaterials. We describe steps for inoculating tumor cells, anesthetizing mice, and performing surgery. We then detail procedures for applying biomaterial, bandaging mice, and data collection and analysis. The optimized bandaging regimen resolves the issue of wound dehiscence after surgery, for C57BL/6 mice, which interfere with surgical sites. For complete details on the use and execution of this protocol, please refer to Rwandamuriye et al.1.

A surgically optimized intraoperative poly(I:C)-releasing hydrogel prevents cancer recurrence.

Recurrences frequently occur following surgical removal of primary tumors. In many cancers, adjuvant therapies have limited efficacy. Surgery provides access to the tumor microenvironment, creating an opportunity for local therapy, in particular immunotherapy, which can induce local and systemic anti-cancer effects. Here, we develop a surgically optimized biodegradable hyaluronic acid-based hydrogel for sustained intraoperative delivery of Toll-like receptor 3 agonist poly(I:C) and demonstrate that it significantly reduces tumor recurrence after surgery in multiple mouse models. Mechanistically, poly(I:C) induces a transient interferon alpha (IFNα) response, reshaping the tumor/wound microenvironment by attracting inflammatory monocytes and depleting regulatory T cells. We demonstrate that a pre-existing IFN signature predicts response to the poly(I:C) hydrogel, which sensitizes tumors to immune checkpoint therapy. The safety, immunogenicity, and surgical feasibility are confirmed in a veterinary trial in canine soft tissue tumors. The surgically optimized poly(I:C)-loaded hydrogel provides a safe and effective approach to prevent cancer recurrence.

Abacavir inhibits but does not cause self-reactivity to HLA-B*57:01-restricted EBV specific T cell receptors.

Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions.

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies.

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant or neo-adjuvant therapies. This occurs when surgical margins are insufficiently tumor-free, resulting in residual cancer cells. From a biological and immunological perspective, surgery is not a null event; the wound healing environment is known to induce both pro- and anti-tumorigenic pathways. As a consequence, preclinical models for drug development aimed at preventing local relapse should incorporate surgical resection when testing new (neo)adjuvant therapies, to model the clinical settings in patients treated with surgery. Here, we describe a mouse model of incomplete surgical resection of WEHI 164 soft tissue sarcoma that allows testing of (neo)adjuvant therapies in the setting of a wound healing response. In this model, 50% or 75% of the tumor is removed, leaving behind some cancer tissue in situ to model gross residual disease after surgery in the clinical setting. This model allows testing therapies in the context of surgery while also considering the wound healing response, which may affect the efficacy of (neo)adjuvant treatments. The incomplete surgical resection results in reproducible regrowth of the tumor in all mice in the absence of adjuvant therapy. Adjuvant treatment with checkpoint blockade results in reduced tumor regrowth. This model is thus appropriate for testing therapies in the context of debulking surgery and its associated wound healing response and can be extended to other types of solid cancer.