Title of the project
Solid Tumor Model Pilot Study - Raji Tumor Cell Line
NTS Identifier
NTS-NO-622531 v.1, 09-11-2023
NTS National Identifier
Field will not be published.
EU Submission
Field will not be published.
Yes
Project duration expressed in months.
48
Keywords
cancer
Cancer immunotherapy
solid tumor model
Purpose(s) of the project
Translational and applied research: Human Cancer
Describe the objectives of the project (for example, addressing certain scientific unknowns, or scientific or clinical needs).
While cellular immunotherapies have made significant inroads when it comes to treating haematological cancers, this success has not extended to solid tumors. Our lab, the Karl-Johan Malmberg group, has a focus on engineering NK cells to enhance their persistence and efficacy as cellular cancer immunotherapies for both liquid and solid tumors. In the context of solid tumors, we are particularly interested in engineering NK cells with enhanced tumor homing capabilities and cells that are able to overcome the immunosuppressive nature of many solid tumor microenvironments. The overall goal of these pilot studies is to establish a subcutaneous solid tumor model to evaluate our NK cell therapies. Raji cells (isolated from a Burkitt's Lymphoma patient, which naturally express CD19) will be used for this model. We will use NK cells with CD19-directed CARs to specifically target the tumors and evaluate how our other modifications to these cells affect their function in the context of a solid tumor. This pilot study will assess tumor growth rates and mouse health as a function of initial Raji cell dose delivered subcutaneously in Matrigel in immunocompromised NSG/NXG mice, enabling us to determine the optimal tumor cell dose for our future solid tumor studies.
What are the potential benefits likely to derive from this project? Explain how science could be advanced, or humans, animals or environment may ultimately benefit from the project. Where applicable, differentiate between short-term benefits (within the duration of the project) and long-term benefits (which may accrue after the project is finished).
While cellular immunotherapies (especially CAR T cells) have made significant progress in the treatment of liquid tumors, to date this success has not translated to solid tumors. Thus good models of solid tumors to test CAR-based and other immunotherapy platforms are needed to better understand their limitations in the solid tumor setting. Furthermore, determining which engineered NK cell therapeutics have the highest probability of being therapeutically beneficial in the context of solid tumors is critical to successful clinical translation of these cell products.
In what procedures will the animals typically be used (for example, injections, surgical procedures)? Indicate the number and duration of these procedures.
Earmarking/biopsy according to SOP "ID-merking og uttak av biopsi" ved KPM/OUS, s.c. injection of Raji umor cells, i.p injection of Luciferin for bioluminescence imaging, inhalation anesthesia, biolumminescence imaging, tumor measurment with calipers.
S.c.: Mice will be anesthetized using sevoflurane inhalation anesthesia. WT Raji-fluc will be injected subcutaneously into the dorsal hind flank. Prior to injection the cell suspension (in PBS) will be mixed 1:1 into 100uL of cold Matrigel (Corning) as a delivery vehicle (200uL total solution will be injected s.c.).
I.p.: D-Luciferin (substrate) 100ul (15mg/ml stock) per 10 grams of mouse body weight.
BLI Imaging: The mice will be anesthetized by inhalation of sevoflurane before/during IVIS imaging. IVIS imaging will be performed on days 1, 4, 7, 10, 14, 21, 28, 35, 42, 49, 56 following injection of Raji cells. Tumors will be measured using calipers weekly (also under sevoflurane anesthesia to get accurate measurments). When possible, this will be done in conjunction with IVIS imaging.
What are the expected impacts/adverse effects on the animals, for example pain, weight loss, inactivity/reduced mobility, stress, abnormal behaviour, and the duration of those effects?
The experimental procedures (s.c. injection, anesthesia, bioluminescent imaging) and phenotype of the GMO NSG and NXG mice are mild. The majority of the disease progression is expected to be mild to moderate, but the disease progresses very quickly at the end during the critical period. If the animals enters the advanced stage of the disease, they can experience short-term severe symptoms including lack of responsiveness, hind-limb paralysis, ascietes, tumor ulceration, and loss of over 20% of their body weight. These severe symptoms represent immediate humane endpoints and if mice exhibit any of these symptoms they will be immediately euthanized. All animals engrafted with tumors will be terminated within 8 weeks (56 days) of tumor innoculation. The overall severity category is severe as animals will be allowed to progress until they hit a HEP if it occurs before the 56 day end point of the study. Metastasis is expected, especially to the bone marrow and central nervous system. Bioluminescent imaging will be used to monitor metastasis, although it may be difficult to see metastases over the brightness of the main tumor. Potential severe symptoms discussed above include symptoms related to metastasis, thus the monitoring will not be different for metastatic disease.
What species and numbers of animals are expected to be used? What are the expected severities and the numbers of animals in each severity category (per species)?
Species
Total number
Estimated numbers per severity
Non recovery
Mild
Moderate
Severe
What will happen to the animals kept alive at the end of the procedure?
Species
Estimated numbers of animals to be reused, to be returned to habitat/husbandry system or to be rehomed
Reused
Returned
Rehomed
Please provide reasons for the planned fate of the animals after the procedure.
The fate of the animals is euthanasia. They will not be re-homed or re-used.
1. Replacement
State which non-animal alternatives are available in this field and why they cannot be used for the purposes of the project.
We have performed extensive in vitro experiments with our products and studied the killing properties,cytokine production, viability, long term cultures, extensive phenotyping by flow cytometry to characterize thecell therapy product as thorough as possible. To address the important questions of long term survival and function in vitro studies are inadequate. To be able to move towards clinical implementation of iPSC-NK cell therapy products, we would like to see that they sustain their function in vivo. These questions can only be addressed in animal models. One alternative is to use large animal models (as primate models) that we would like to avoid as far as possible. Xenograft mouse models is a good alternative and we consider this to be the best option for our studies. To make sure we are updated on recent developments, we have performed searches for alternative methods in different databases as: Google, Pubmed, Ovid, Medline. We also searched for literature by the The Web of Science queries below without finding any new, relevant information about optional models. https://www.webofscience.com/wos/woscc/ summary/b6943e8f-857e-4158-b149-5e8c7ceb76f1-27887d3b/relevance/1. https://www.webofscience.com/ wos/woscc/summary/e3cc55b6-90c3-4398-9748-3309a2c2e258-27883bcd/relevance/1
2. Reduction
Explain how the numbers of animals for this project were determined. Describe steps that have been taken to reduce the number of animals to be used, and principles used to design studies. Where applicable, describe practices that will be used throughout the project to minimise the number of animals used consistent with scientific objectives. Those practices may include e.g. pilot studies, computer modelling, sharing of tissue and reuse.
This pilot study will be run with the minimal amount of mice necessary to achieve statistically significant results. Developing a baseline for how the subcutaneous Raji model affects mice in our hands, in terms of tumor growth rate and effects on mouse health, will enable us to more rationally design our future experiments, thus reducing the overall number of mice used in all of our experiments.
3. Refinement
Give examples of the specific measures (e.g., increased monitoring, post-operative care, pain management, training of animals) to be taken, in relation to the procedures, to minimise welfare costs (harms) to the animals. Describe the mechanisms to take up emerging refinement techniques during the lifetime of the project.
The procedures are standard methods performed with the goal to minimize stress and discomfort for the animals. Anesthesia will be administered before subcutaneous tumor injections and before bioluminescence imaging. We will minimize the time the animals are away from their cage and as far as possible allow the same caged animals stay together to reduce stress. We will use as few animals as possible while still providing representative, meaningful results to avoid unnecessary repetitions. Animals will be monitored closely and euthanized immediately when they hit the outlined euthanasia criteria. The personnel will have all the necessary skills and training to handle the animals and do the procedures professionally and correct.
Explain the choice of species and the related life stages.
Xenograft tumor models in immunocompromized mice are currently considered to be the gold standard with regards to assessment of CAR therapies in vivo according to international research (Mhaidly and Verhoeyen, 2020). See also website of Jax mice, https://www.jax.org/strain/005557 and Janvier mice, https://janvier-labs.com/en/fiche_produit/1-nxg-immunodeficient-mouse/
Project selected for RA?
Yes
Deadline for RA
07-03-2028
Reasons for retrospective assessment
Contains severe procedures
Yes
Explanation of the other reason for retrospective assessment
The Norwegian Food Safety Authority must retrospectively assess all severe experiments. You are therefore required to submit necessary information making it possible for us to evaluate 1) whether the objectives of the project were achieved, 2) the harm inflicted on the animals, and 3) any elements that may contribute to further implementation of 3 R in future studies. This information must be submitted via FOTS.