Protocol for specific induction of apoptosis or necroptosis in established murine tumors

Sep 29, 2026

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Protocol describing in vivo cancer-cell death studies using mouse tumor models with inducible dimerizer-based cell lines that enable selective “pure” apoptosis or necroptosis. The workflow covers lentiviral transduction and flow cytometry-guided optimization of the death induction schedule, followed by optional intradermal vaccination and antigen-specific CD8+ T cell tracking to assess immune consequences. The system targets downstream effector mechanisms in established tumors and emphasizes verifying the induced death modality.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages21

Reading Time53 min

AccessFree to download

Summary

Before you begin

Rationale and design of the inducible dimerizer system

Applicable tumor models and verification needs

Institutional permission

Key resources table

Innovation

ll OPEN ACCESS Protocol Protocol for specific induction of apoptosis ornecroptosis in established murine tumors Kay Ha¨nggi, Brian Ruffell [kay.hanggi@moffitt.org](kay.hanggi@moffitt.org)[ ](kay.hanggi@moffitt.org)(K. H.) [brian.ruffell@moffitt.org](brian.ruffell@moffitt.org)[ ](brian.ruffell@moffitt.org)(B. R.) Highlights Steps to generate tumors allowing induction of apoptosis ornecroptosis Details to study the impact of cell death induction in established tumors Vaccination approach to compare the immune response to ex vivo induction of cell death Optional steps for the addition of fluorescent markers and/or model antigens Ha¨nggi & Ruffell, STAR Protocols 6 , 104185 December 19, 2025 © 2025 The Author(s) . Published by Elsevier Inc. [https://doi.org/10.1016/j](https://doi.org/10.1016/j). xpro.2025.104185 ll OPEN ACCESS Protocol Protocol for specific induction of apoptosis ornecroptosis in established murine tumors Kay Ha¨nggi 1,3,* and Brian Ruffell 1,2,4,* 1Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA 2Department of Breast Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA 3Technical contact 4Lead contact *Correspondence: [kay.hanggi@moffitt.org](kay.hanggi@moffitt.org) (K. H.), [brian.ruffell@moffitt.org](brian.ruffell@moffitt.org) (B. R.) [https://doi.org/10.1016/j.xpro.2025.104185](https://doi.org/10.1016/j.xpro.2025.104185) SUMMARY We present a protocol to study cancer cell death in vivo using mouse tumor models by generating cell lines that allow for the induction of “pure’’ apoptosis or necroptosis via an inducible dimerizer system. We describe steps for lentiviral transduction and flow cytometry-based optimization of the death induction schedule. We then detail optional procedures for “intradermal vaccination’’and for tracking antigen-specific CD8+ T cell responses. For complete details on the use and execution of this protocol, please refer to H¨anggi et al.1 BEFORE YOU BEGIN Inducing cell death in cancer cells is the primary goal of most therapies, but how different forms of cell death regulate the immune response and can be rationally combined with immunotherapies remains poorly understood. The extensive crosstalk between cell death pathways, cytotoxic treatments (e. g. chemotherapy) or inflammatory environments (e. g. cytokines like TNFα) often lead toa mixed population of cell death pathways being executed, making it difficult to interpret the consequences of a particular death modality on the immune response. We developed a protocol using the ‘dimerizer inducible’ cell death execution system, routinely used in vitro,2 ,3 allowing the induction of either apoptosis or necroptosis in tumors. The apoptosis construct used here utilizes a caspase 8 fusion protein in which the endogenous dimerization domain of caspase 8 is replaced with a DmrB domain, allowing inducible dimerization with the BB Homodimerizer compound. The necroptosis construct similarly uses a DmrB-RIPK3 fusion protein for inducible RIPK3 necrosome formation, leading to RIPK3 to phosphorylate MLKL. Alternatively, necroptosis can be induced by expression of a phospho-mimetic version of MLKL (MLKLS345D), listed in the key resource table but not described in the protocol.1 Note that the constructs used induce apoptosis or necroptosis in the absence of upstream receptor signaling induction. The protocol described herein is based upon the PyMT-B6 orthotopic tumor model, but the same approach has also been successfully applied in the subcutaneous MC38 and LLC1 tumor models.1 Other groups have used the dimerizing system and/or similar strategies to induce death in NIH3T3, CT26, B16-F10, and LLC1 .2 ,4–6 It is expected that the system can be applied to a wide range of cell types and tumor models provided that the downstream effector molecules are present in the cell of interest. However, as outlined in the proto

FAQs

What is the main purpose of this protocol?

To study cancer cell death in vivo by generating mouse tumor model cell lines that allow inducible, selective apoptosis or necroptosis, enabling downstream analysis of immune responses.

How is selective apoptosis induced in this system?

The apoptosis construct uses a caspase 8 fusion protein where the endogenous dimerization domain is replaced with a DmrB domain, enabling inducible dimerization with the BB Homodimerizer compound.

How is necroptosis induced and what is recommended for validation?

Necroptosis is induced using a DmrB-RIPK3 fusion for inducible necrosome formation leading to MLKL phosphorylation, or via expression of an MLKL phospho-mimetic variant. The protocol recommends verifying that the correct form of cell death is being induced.

Sep 29, 2026

Uploaded by Rizky