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Cancer Immunology, Immunotherapy
Published in: Cancer Immunology, Immunotherapy 12/2023

Open Access 05-10-2023 | Lung Cancer | Methodology

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1

Authors: Haibin Deng, Huixiang Ge, Christelle Dubey, Tereza Losmanova, Michaela Medová, Georgia Konstantinidou, Seyran Mathilde Mutlu, Fabienne Esther Birrer, Tess Melinda Brodie, Deborah Stroka, Wenxiang Wang, Ren-Wang Peng, Patrick Dorn, Thomas Michael Marti

Published in: Cancer Immunology, Immunotherapy | Issue 12/2023

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Abstract

Background

The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure.

Results

The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT.

Conclusion

Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles.
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Literature
1.
Ahmadi-Noorbakhsh S, Abbasi MF, Ghasemi M, Bayat G, Davoodian N, Sharif-Paghaleh E, Poormoosavi SM, Rafizadeh M, Maleki M, Shirzad-Aski H, Jahromi HK, Dadkhah M, Khalvati B, Safari T, Behmanesh MA, Khoshnam SE, Houshmand G, Talaei SA (2022) Anesthesia and analgesia for common research models of adult mice. Lab Anim Res 38:40PubMedPubMedCentralCrossRef
2.
Albrecht M, Henke J, Tacke S, Markert M, Guth B (2014) Effects of isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl on physiological variables continuously measured by telemetry in Wistar rats. BMC Vet Res 10:198PubMedPubMedCentralCrossRef
3.
4.
5.
Berezowska S, Galvan JA (2017) Immunohistochemical detection of the autophagy markers LC3 and p62/SQSTM1 in formalin-fixed and paraffin-embedded tissue. Methods Mol Biol 1560:189–194PubMedCrossRef
6.
Birrer F, Brodie T, Stroka D (2023) OMIP-088: twenty-target imaging mass cytometry panel for major cell populations in mouse formalin fixed paraffin embedded liver. Cytometry A 103:189–192PubMedCrossRef
7.
Blessing D, Vachey G, Pythoud C, Rey M, Padrun V, Wurm FM, Schneider BL, Déglon N (2019) Scalable production of AAV vectors in orbitally shaken HEK293 cells. Mol Ther-Methods Clin Dev 13:14–26PubMedCrossRef
8.
Buitrago S, Martin TE, Tetens-Woodring J, Belicha-Villanueva A, Wilding GE (2008) Safety and efficacy of various combinations of injectable anesthetics in BALB/c mice. J Am Assoc Lab Anim Sci 47:11–17PubMedPubMedCentral
9.
Chandler RJ, Sands MS, Venditti CP (2017) recombinant adeno-associated viral integration and genotoxicity: insights from animal models. Hum Gene Ther 28:314–322PubMedPubMedCentralCrossRef
10.
Collins DE, Reuter JD, Rush HG, Villano JS (2017) Viral vector biosafety in laboratory animal research. Comp Med 67:215–221PubMedPubMedCentral
11.
Deng H, Gao Y, Trappetti V, Hertig D, Karatkevich D, Losmanova T, Urzi C, Ge H, Geest GA, Bruggmann R, Djonov V, Nuoffer J-M, Vermathen P, Zamboni N, Riether C, Ochsenbein A, Peng R-W, Kocher GJ, Schmid RA, Dorn P, Marti TM (2022) Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage. Cell Mol Life Sci 79(8):445PubMedPubMedCentralCrossRef
12.
DuPage M, Dooley AL, Jacks T (2009) Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 4:1064–1072PubMedPubMedCentralCrossRef
13.
Dupage M, Cheung AF, Mazumdar C, Winslow MM, Bronson R, Schmidt LM, Crowley D, Chen J, Jacks T (2011) endogenous t cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression. Cancer Cell 19:72–85PubMedPubMedCentralCrossRef
14.
Dupage M, Mazumdar C, Schmidt LM, Cheung AF, Jacks T (2012) Expression of tumour-specific antigens underlies cancer immunoediting. Nature 482:405–409PubMedPubMedCentralCrossRef
15.
Erhardt W, Henke J, Lendl C (2002) Narkose-Notfälle. Haug Fachbuchverlag, Karl F
16.
Fitzgerald B, Connolly KA, Cui C, Fagerberg E, Mariuzza DL, Hornick NI, Foster GG, William I, Cheung JF, Joshi NS (2021) A mouse model for the study of anti-tumor T cell responses in Kras-driven lung adenocarcinoma. Cell Rep Methods 1(5):100080PubMedPubMedCentralCrossRef
17.
Foster WK, Ford NL (2011) Investigating the effect of longitudinal micro-CT imaging on tumour growth in mice. Phys Med Biol 56:315–326PubMedCrossRef
18.
Gallaher SD, Berk AJ (2013) A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results. J Virol Methods 192:28–38PubMedPubMedCentralCrossRef
19.
Haines BB, Bettano KA, Chenard M, Sevilla RS, Ware C, Angagaw MH, Winkelmann CT, Tong C, Reilly JF, Sur C, Zhang W (2009) A quantitative volumetric micro-computed tomography method to analyze lung tumors in genetically engineered mouse models. Neoplasia 11:39–47PubMedPubMedCentralCrossRef
20.
Harno E, Ec C, White A (2013) Metabolic pitfalls of CNS Cre-based technology. Cell Metab 18:21–28PubMedCrossRef
21.
Henke J, Müller D (2017) Vollstandig antagonisierbare lnjektionsnarkose bei Kleinsaugern (Nager, Meerschweinchen, Kaninchen). www.​docplayer.​org. online: docplayer
22.
23.
Herter-Sprie GS, Korideck H, Christensen CL, Herter JM, Rhee K, Berbeco RI, Bennett DG, Akbay EA, Kozono D, Mak RH, Mike Makrigiorgos G, Kimmelman AC, Wong K-K (2014) Image-guided radiotherapy platform using single nodule conditional lung cancer mouse models. Nat Commun 5:5870PubMedCrossRef
24.
Jackson EL, Olive KP, Tuveson DA, Bronson R, Crowley D, Brown M, Jacks T (2005) The differential effects of mutant p53 alleles on advanced murine lung cancer. Cancer Res 65:10280–10288PubMedCrossRef
25.
Jackson EL, Willis N, Mercer K, Bronson RT, Crowley D, Montoya R, Jacks T, Tuveson DA (2001) Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15:3243–3248PubMedPubMedCentralCrossRef
26.
Jaynes JM, Sable R, Ronzetti M, Bautista W, Knotts Z, Abisoye-Ogunniyan A, Li D, Calvo R, Dashnyam M, Singh A, Guerin T, White J, Ravichandran S, Kumar P, Talsania K, Chen V, Ghebremedhin A, Karanam B, Bin Salam A, Amin R, Odzorig T, Aiken T, Nguyen V, Bian Y, Zarif JC, de Groot AE, Mehta M, Fan L, Hu X, Simeonov A, Pate N, Abu-Asab M, Ferrer M, Southall N, Ock CY, Zhao Y, Lopez H, Kozlov S, de Val N, Yates CC, Baljinnyam B, Marugan J, Rudloff U (2020) Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses. Sci Transl Med 12(530):eaax6337PubMedPubMedCentralCrossRef
27.
Kersten K, Visser KE, Miltenburg MH, Jonkers J (2017) Genetically engineered mouse models in oncology research and cancer medicine. EMBO Mol Med 9:137–153PubMedCrossRef
28.
Kim H, Kim M, Im SK, Fang S (2018) Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res 34:147–159PubMedPubMedCentralCrossRef
29.
Kirsch DG, Dinulescu DM, Miller JB, Grimm J, Santiago PM, Young NP, Nielsen GP, Quade BJ, Chaber CJ, Schultz CP, Takeuchi O, Bronson RT, Crowley D, Korsmeyer SJ, Yoon SS, Hornicek FJ, Weissleder R, Jacks T (2007) A spatially and temporally restricted mouse model of soft tissue sarcoma. Nat Med 13:992–997PubMedCrossRef
30.
Kirsch DG, Grimm J, Guimaraes AR, Wojtkiewicz GR, Perez BA, Santiago PM, Anthony NK, Forbes T, Doppke K, Weissleder R, Jacks T (2010) Imaging primary lung cancers in mice to study radiation biology. Int J Radiat Oncol Biol Phys 76:973–977PubMedPubMedCentralCrossRef
31.
32.
Loonstra A, Marc Vooijs H, Beverloo B, Allak BA, Van Drunen E, Kanaar R, Berns A, Jonkers J (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci 98:9209–9214PubMedPubMedCentralCrossRef
33.
Marino S, Vooijs M, van der Gulden H, Jonkers J, Berns A (2000) Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev 14:994–1004PubMedPubMedCentralCrossRef
34.
Marjanovic ND, Hofree M, Chan JE, Canner D, Katherine Wu, Trakala M, Hartmann GG, Smith OC, Kim JY, Evans KV, Hudson A, Ashenberg O, Porter CBM, Bejnood A, Subramanian A, Pitter K, Yan Y, Delorey T, Phillips DR, Shah N, Chaudhary O, Tsankov A, Hollmann T, Rekhtman N, Massion PP, Poirier JT, Mazutis L, Li R, Lee J-H, Amon A, Rudin CM, Jacks T, Regev A, Tammela T (2020) Emergence of a high-plasticity cell state during lung cancer evolution. Cancer Cell 38:229–246PubMedPubMedCentralCrossRef
35.
Mayers JR, Wu C, Clish CB, Kraft P, Torrence ME, Fiske BP, Yuan C, Bao Y, Townsend MK, Tworoger SS, Davidson SM, Papagiannakopoulos T, Yang A, Dayton TL, Ogino S, Stampfer MJ, Giovannucci EL, Qian ZR, Rubinson DA, Ma J, Sesso HD, Gaziano JM, Cochrane BB, Liu S, Wactawski-Wende J, Manson JE, Pollak MN, Kimmelman AC, Souza A, Pierce K, Wang TJ, Gerszten RE, Fuchs CS, Vander Heiden MG, Wolpin BM (2014) Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development. Nat Med 20:1193–1198PubMedPubMedCentralCrossRef
36.
Jie M (2019) Refining humane endpoints in mouse models of disease by systematic review and machine learning-based endpoint definition. Altex 36(4):555–571
37.
Rambhai HK, Ashby FJ, Qing K, Srivastava A (2020) Role of essential metal ions in AAV vector-mediated transduction. Mol Ther-Methods Clin Dev 18:159–166PubMedPubMedCentralCrossRef
38.
39.
Ahlers, J., Althviz, D., Amsalem, O., Anderson, A., Bokota, G., Boone, P., Bragantini, J., Buckley, K., Burt, A., Bussonnier, M., Can Solak, A., Caporal, C., Doncila Pop, D., Evans, K., Gaifas, L., Freeman, J., Gohlke, C., Gunalan, K., Har-Gil, H., Harfouche, M., Harrington, K., Hilsenstein, V., Hutchings, K., Lambert, T., Lauer, J., Lichtner, G., Liu, Z., Liu, L., Marconato, L. & Martin, S. et al. Napari: a multi-dimensional image viewer for python, Zenodo, https://​doi.​org/​10.​5281/​zenodo.​8115575 (2023).
40.
Tippimanchai DD, Nolan K, Poczobutt J, Verzosa G, Li H, Scarborough H, Huang J, Young C, DeGregori J, Nemenoff RA, Malkoski SP (2018) Adenoviral vectors transduce alveolar macrophages in lung cancer models. OncoImmunology 7:e1438105PubMedPubMedCentralCrossRef
41.
Der Loo V, Johannes CM, Fraser Wright J (2016) Progress and challenges in viral vector manufacturing. Hum Mol Genet 25:R42–R52PubMedCrossRef
42.
Velazquez ER, Parmar C, Jermoumi M, Mak RH, Van Baardwijk A, Fennessy FM, Lewis JH, De Ruysscher D, Kikinis R, Lambin P, Aerts HJ (2013) Volumetric CT-based segmentation of NSCLC using 3D-Slicer. Sci Rep 3(1):3529PubMedPubMedCentralCrossRef
43.
44.
Walters RW, Duan D, Engelhardt JF, Welsh MJ (2000) Incorporation of adeno-associated virus in a calcium phosphate coprecipitate improves gene transfer to airway epithelia in vitro and in vivo. J Virol 74:535–540PubMedPubMedCentralCrossRef
45.
Wang D, Tai PWL, Gao G (2019) Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discovery 18:358–378PubMedCrossRef
46.
Westcott PMK, Sacks NJ, Schenkel JM, Ely ZA, Smith O, Hauck H, Jaeger AM, Zhang D, Backlund CM, Beytagh MC, Patten JJ, Elbashir R, Eng G, Irvine DJ, Yilmaz OH, Jacks T (2021) Low neoantigen expression and poor T-cell priming underlie early immune escape in colorectal cancer. Nat Cancer 2:1071–1085PubMedPubMedCentralCrossRef
47.
Windhager J, Bodenmiller B, Eling N (2021) An end-to-end workflow for multiplexed image processing and analysis. In: bioRxiv
48.
Xie H, Hanai J, Ren JG, Kats L, Burgess K, Bhargava P, Signoretti S, Billiard J, Duffy KJ, Grant A, Wang X, Lorkiewicz PK, Schatzman S, Bousamra M 2nd, Lane AN, Higashi RM, Fan TW, Pandolfi PP, Sukhatme VP, Seth P (2014) Targeting lactate dehydrogenase–a inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor-initiating cells. Cell Metab 19:795–809PubMedPubMedCentralCrossRef
49.
Metadata
Title
An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1
Authors
Haibin Deng
Huixiang Ge
Christelle Dubey
Tereza Losmanova
Michaela Medová
Georgia Konstantinidou
Seyran Mathilde Mutlu
Fabienne Esther Birrer
Tess Melinda Brodie
Deborah Stroka
Wenxiang Wang
Ren-Wang Peng
Patrick Dorn
Thomas Michael Marti
Publication date
05-10-2023
Publisher
Springer Berlin Heidelberg
Published in
Cancer Immunology, Immunotherapy / Issue 12/2023
Print ISSN: 0340-7004
Electronic ISSN: 1432-0851
DOI
https://doi.org/10.1007/s00262-023-03542-z

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