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Bilateral murine tumor models for characterizing the response to immune checkpoint blockade

Abstract

The therapeutic response to immune checkpoint blockade (ICB) is highly variable, not only between different cancers but also between patients with the same cancer type. The biological mechanisms underlying these differences in response are incompletely understood. Identifying correlates in patient tumor samples is challenging because of genetic and environmental variability. Murine studies usually compare different tumor models or treatments, introducing potential confounding variables. This protocol describes bilateral murine tumor models, derived from syngeneic cancer cell lines, that display a symmetrical yet dichotomous response to ICB. These models enable detailed analysis of whole tumors in a highly homogeneous background, combined with knowledge of the therapeutic outcome within a few weeks, and could potentially be used for mechanistic studies using other (immuno-)therapies. We discuss key considerations and describe how to use two cell lines as fully optimized models. We discuss experimental details, including proper inoculation technique to achieve symmetry and one-sided surgical tumor removal, which takes only 5 min per mouse. Furthermore, we outline the preparation of bulk tissue or single-cell suspensions for downstream analyses such as bulk RNA-seq, immunohistochemistry, single-cell RNA-seq and flow cytometry.

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Fig. 1: Schematic of experimental design.
Fig. 2: Tumor models with symmetrical, dichotomous responses to ICB.
Fig. 3: Intermediate responses are not consistently symmetrical.
Fig. 4: Response rates are influenced by day of dosing.
Fig. 5: Analysis of surgically removed tumor.
Fig. 6: Analysis of bulk RNA-seq of responders versus non-responders.
Fig. 7: Flow cytometric analysis of responders versus non-responders.
Fig. 8: Mouse handling for symmetric tumor inoculation.
Fig. 9: Removal of the right-side tumor.

Data availability

The raw RNA-seq data used to generate Fig. 6 are available from the Gene Expression Omnibus data repository (accession no. GSE117358).

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Acknowledgements

We acknowledge the facilities, as well as the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility funded by the University and State and Commonwealth governments. We thank P. Deng and C. Rinaldi for technical assistance. This work was funded by grant 1103980 from the National Health and Medical Research Council (NHMRC). W.J.L. was funded by an NHMRC RD Wright Fellowship and a Fellowship from the Cancer Council of Western Australia.

Author information

Authors and Affiliations

Authors

Contributions

R.M.Z. optimized the mouse models, performed and interpreted all mouse experiments and flow cytometry experiments, prepared samples for RNA-seq analysis, and wrote the paper. V.S.F., C.F., and T.H.C. provided technical assistance with surgery and in vivo treatment experiments. E.d.J., T.L. and A.B. interpreted the bulk RNA-seq analyses. L.B. provided critical reagents. R.A.L., M.J.M., and A.K.N. were involved in experimental design and interpretation of all in vivo studies. R.A.L. and V.S.F provided critical revision of the manuscript. W.J.L. designed and developed the models, interpreted the data and supervised the project. R.M.Z and W.J.L. wrote the manuscript with input from all authors. All authors gave final approval of the version to be published.

Corresponding authors

Correspondence to Rachael M. Zemek or W. Joost Lesterhuis.

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Competing interests

A patent application pertaining to aspects of this work has been filed by R.M.Z., W.J.L., R.A.L., and A.B. (PCT/AU2019/050259, “Method for immunotherapy drug treatment”). A.K.N., M.J.M., A.B., R.A.L., and W.J.L. have received research funding and consultancy fees from Douglas Pharmaceuticals. W.J.L. has received research funding from AstraZeneca and consultancy fees from Merck Sharp & Dohme. A.K.N. declares consultancy or advisory board membership for Boehringer Ingelheim, Bayer Pharmaceuticals, Roche Pharmaceutics, Merck Sharp Dohme, Pharmabcine, Atara Biotherapeutics and Trizell, and research funding from AstraZeneca. M.J.M. has served on advisory boards for Merck Sharp & Dohme, Bristol-Myers Squibb, Roche, and AstraZeneca.

Additional information

Peer review information Nature Protocols thanks Gottfried Baier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Zemek, R. M. et al. Sci. Transl. Med. 11, eaav7816 (2019): https://doi.org/10.1126/scitranslmed.aav7816

Lesterhuis, W. J. et al. Sci. Rep. 5, 12298 (2015): https://doi.org/10.1038/srep12298

Integrated supplementary information

Supplementary Figure 1 Response to ICB in the B16-F10 tumor model.

a) Growth curves of C57BL/6 mice bearing subcutaneous B16-F10 tumours, treated with i.p. PBS or (b) with ICB (anti-CTLA4 day 5 and anti-PD-L1 on days 5,7 and 9 post-inoculation) when tumours were at least 9mm2 (n=15 per arm, combined data from 3 experiments).

Supplementary Figure 2 Flow cytometry gating strategy.

Gating strategy used to delineate immune cell subsets in mouse tumour and lymph nodes.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Supplementary Table 1.

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Zemek, R.M., Fear, V.S., Forbes, C. et al. Bilateral murine tumor models for characterizing the response to immune checkpoint blockade. Nat Protoc 15, 1628–1648 (2020). https://doi.org/10.1038/s41596-020-0299-3

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