Nov 27, 2023
In vivo CAR T cell tumor control assay
- Sean R. McCutcheon1,
- Adam M. Swartz1,
- Michael C. Brown1,
- Alejandro Barrera1,
- Christian McRoberts Amador1,
- Keith Siklenka1,
- Lucas Humayun1,
- Maria A. ter Weele1,
- James M. Isaacs1,
- Andrea R Daniel1,
- Timothy E. Reddy1,
- Andrew S. Allen1,
- Smita K. Nair1,
- Scott J. Antonia1,
- Charles A. Gersbach1
- 1Duke University
- Andrea R Daniel: This protocol is adapted from the M. Brown lab at Duke University.
- Gersbach Lab
Protocol Citation: Sean R. McCutcheon, Adam M. Swartz, Michael C. Brown, Alejandro Barrera, Christian McRoberts Amador, Keith Siklenka, Lucas Humayun, Maria A. ter Weele, James M. Isaacs, Andrea R Daniel, Timothy E. Reddy, Andrew S. Allen, Smita K. Nair, Scott J. Antonia, Charles A. Gersbach 2023. In vivo CAR T cell tumor control assay . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3qj6l3p/v1
Manuscript citation:
McCutcheon, S.R., Swartz, A.M., Brown, M.C. et al. Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens. Nat Genet (2023). https://doi.org/10.1038/s41588-023-01554-0
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 27, 2023
Last Modified: November 27, 2023
Protocol Integer ID: 91460
Keywords: CAR T cells, CD8+ T cells, HER2, HCC1954, in vivo tumor model
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This protocol describes methods for a HCC1954 cell implanted orthotopic tumor model in mice with delivery of T cells expressing HER2 CAR. Tumor volumes are measured to evaluate tumor control by CAR T cell therapy.
HCC1954 orthotopic tumor model
HCC1954 orthotopic tumor model
Six- to 8-week-old female immunodeficient NOD/SCID gamma (NSG) mice were obtained from Jackson Laboratory and then housed in 12-h light/dark cycles, at an ambient temperature (21 ± 3 °C) with relative humidity (50 ± 20%) and handled in pathogen-free conditions
HCC1954s were maintained in DMEM/F12 supplemented with 10% FBS, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin.
Resuspend cells in 2ml PBS (5x10^7 cells/ml), then diluted 1:1 with Matrigel (2.5e7/ml)
thus 2.5e6 per 100ul.
2.5x 106 HCC1954 cells are implanted orthotopically into the mammary fat pad of NSG mice in 100μL volume of 50:50 (v:v) PBS:Matrigel.
Upon detection tumor volumes were calculated based on caliper measurements using the formula volume = ½ (Length × Width2). Measure tumors every 4-6 days.
CAR T cell delivery
CAR T cell delivery
Human donor T cells were expanded for 9-11 days post-transduction with HER2-CAR-2A-GFP
and HER2-CAR-2A-BATF3 constructs before delivery to tumor bearing mice.
Transduction rates were measured on the day of treatment using flow cytometry and transduction rates exceed 80%.
T cells were resuspended at 50 x 106 CAR+ cells mL-1 in 1X PBS and serially diluted to the appropriate cell concentrations for 200 μL injections of either 10 x 106, 2 x 106, 5 x 105,
2.5 x 105, or 1 x 105 HER2 CAR+ T cells.
21 days after tumor implantation randomize mice into groups for CAR T cell injections. Deliver CAR T cells intravenously by tail vein injection.
Measure the length and width of tumors every 4-6 days using calipers.
Mice are euthanized before reaching a tumor volume of 2,000 mm3, the upper threshold defined by the Duke Institutional Animal Care and Use Committee.