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Monitoring autochthonous lung tumors induced by somatic CRISPR gene editing in mice using a secreted luciferase.
Literature Information
| DOI | 10.1186/s12943-022-01661-2 |
|---|---|
| PMID | 36192757 |
| Journal | Molecular cancer |
| Impact Factor | 33.9 |
| JCR Quartile | Q1 |
| Publication Year | 2022 |
| Times Cited | 1 |
| Keywords | Adenovirus, Autochthonous mouse tumor, CRISPR, Luciferase, Lung cancer |
| Literature Type | Journal Article, Research Support, Non-U.S. Gov't |
| ISSN | 1476-4598 |
| Pages | 191 |
| Issue | 21(1) |
| Authors | Nastasja Merle, Sabrina Elmshäuser, Florian Strassheimer, Michael Wanzel, Alexander M König, Julianne Funk, Michelle Neumann, Katharina Kochhan, Frederik Helmprobst, Axel Pagenstecher, Andrea Nist, Marco Mernberger, André Schneider, Thomas Braun, Tilman Borggrefe, Rajkumar Savai, Oleg Timofeev, Thorsten Stiewe |
TL;DR
This study introduces a novel GLuc reporter mouse model that enables efficient monitoring of tumor growth through the measurement of luciferase secreted into the blood, facilitating early detection of lung tumors induced by CRISPR-mediated gene editing. The findings provide a cost-effective and high-throughput method for longitudinal tracking of autochthonous tumors, enhancing the utility of these models in preclinical cancer research.
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Adenovirus · Autochthonous mouse tumor · CRISPR · Luciferase · Lung cancer
Abstract
BACKGROUND In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies.
METHODS To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes.
RESULTS We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination.
CONCLUSIONS Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.
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Primary Questions Addressed
- What are the implications of using GLuc monitoring for detecting other types of tumors beyond lung tumors?
- How does the efficiency of the GLuc reporter system compare to other existing imaging techniques for tumor monitoring?
- What potential challenges could arise in the translation of this GLuc monitoring method to human clinical trials?
- In what ways could the toolkit for assembling recombinant adenoviruses be optimized for better delivery efficiency?
- How might variations in the tumor microenvironment affect the accuracy of GLuc as a biomarker for tumor growth?
Key Findings
Research Background and Objectives
The study investigates the survival rates of various genetic mouse models (C.Cre, PR.Cre, and PRL.Cre) under specific experimental conditions. The primary objective is to understand the impact of genetic modifications on the longevity of these models, which may have implications for studying disease mechanisms and therapeutic interventions.
Main Methods/Materials/Experimental Design
The research utilized a comparative survival analysis of three genetically modified mouse strains:
- C.Cre: 5 mice
- PR.Cre: 5 mice
- PRL.Cre: 3 mice
The survival rates were monitored over a specified time frame, with a focus on the days until death (or censoring). Statistical analyses were performed to determine the significance of survival differences among the groups.
Key Results and Findings
- Survival Rates: The study found significant differences in survival rates among the different genetic models:
- C.Cre exhibited the highest survival duration, with a mean survival of 325 days.
- PR.Cre had a mean survival of 196 days.
- PRL.Cre had the lowest mean survival, although specific data were not provided.
- Statistical Significance: The differences in survival rates were statistically significant, with p-values indicating strong evidence against the null hypothesis:
- P < 0.0001 for C.Cre vs. PR.Cre
- P = 0.0067 for comparisons involving PRL.Cre
Main Conclusions/Significance/Innovativeness
The findings suggest that genetic modifications significantly influence the survival of mouse models, which could be pivotal in understanding the role of specific genes in longevity and disease. This research highlights the importance of selecting appropriate genetic models for preclinical studies, potentially leading to more effective therapeutic strategies.
Research Limitations and Future Directions
Limitations: The study is limited by the small sample sizes for some genetic models, particularly PRL.Cre, which may affect the robustness of the findings. Additionally, the study only considers survival without delving into the underlying biological mechanisms or the impact of environmental factors.
Future Directions: Further research is warranted to explore the molecular pathways affected by these genetic modifications. Larger cohorts and additional experimental conditions (e.g., varying environmental stresses) should be included to validate the findings and enhance the understanding of genetic influences on survival.
References
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Literatures Citing This Work
- Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis. - Evangelos Pavlakis;Michelle Neumann;Nastasja Merle;Ronja Wieboldt;Michael Wanzel;Viviane Ponath;Elke Pogge von Strandmann;Sabrina Elmshäuser;Thorsten Stiewe - Journal of experimental & clinical cancer research : CR (2023)
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