Article Text

Download PDFPDF

299 3D in vitro tumor microenvironment models for screening CAR-T cell therapy efficacy
  1. Bin Xue1,
  2. Sophie Vermond2,
  3. Ulrike Herbrand2,
  4. David Harris2,
  5. Gemma Moiset2,
  6. Kolin Hribar1 and
  7. Julia Schuler2
  1. 1Cypre Inc., San Francisco, CA, USA
  2. 2Charles River Laboratories International Inc., Leiden, Netherlands


Background T cells that are genetically modified to express chimeric antigen receptors (CARs) show promising results for treating hematological tumors, however CAR-T cell therapy have thus far demonstrated limited anti-tumor activity in solid tumors.1 The immunosuppressive tumor microenvironment (TME)2 and T cell dysfunction, driven by chronic antigen exposure in solid tumor, likely contribute to the CAR-T resistance. In order to advance the CAR-T therapy into patients with solid tumors, we need models which accurately represent the TME to evaluate CAR-T efficacy at the discovery, preclinical and translational stages of R&D.

Methods Using a proprietary 3D hydrogel patterning technology,3 a 3D in vitro tumor model was generated in 96-well plates utilizing breast cancer cells and human dermal fibroblasts to reflect the tumor and stromal compartments, respectively, of the tumor microenvironment. Specifically, the commercially available HER2-positive breast cancer cell line, JIMT-1, and the triple-negative patient-derived xenograft (PDX) cell line MAXFTN 401, were utilized in these 3D TME models and subsequently interrogated with HER2-specific CAR-T cells as well as untransduced T cells. 5,000, 10,000, or 25,000 T cells were added to each well of the 3D in vitro models and apoptosis via Caspase 3/7 staining was analyzed at day 4 endpoint using high content imaging.

Results T cell-mediated killing in the respective models was highly dependent on their HER2 status – HER2-positive JIMT-1 demonstrated a dose dependent effect in apoptosis (Caspase 3/7 marker) and up to 42% of JIMT-1 cells underwent apoptosis in response to HER2-specific CAR-T cells, while less than 5% of HER2-negative MAXFTN 401 showed a response to any therapeutic dose of the CAR-T cells. Moreover, inclusion of fibroblasts in the 3D TME model enhanced the CAR-T mediated tumor killing in JIMT-1 model. Finally, the untransduced T cells demonstrated negligible effects (<2% in the JIMT-1 model), highlighting the specificity of the HER2-targeting CAR-T cells.

Conclusions A novel 3D in vitro tumor model platform has been described for assaying CAR-T efficacy. In this case, the platform demonstrated the highly specific nature of the CAR-T cells in targeting HER2-positive tumors cells in a translationally relevant 3D in vitro TME model.


  1. Rodriguez-Garcia, A, Palazon, A, Noguera-Ortega, E, Powell, DJ, & Guedan, S, CAR-T Cells Hit the Tumor Microenvironment: Strategies to Overcome Tumor Escape. Front Immunol. 2020; 11:1109.

  2. Pitt, JM, Marabelle, A, Eggermont, A, Soria, JC, Kroemer, G, Zitvogel, L, Targeting the tumor microenvironment: Removing obstruction to anticancer immune responses and immunotherapy. Annal Oncol. 2016; 27:1482-1492.

  3. Hribar, KC, Wheeler, CJ, Bazarov, A, Varshneya, K, Yamada, R, Buckley, P, Patil, CG, A simple three-dimensional hydrogel platform enables ex vivo cell culture of patient and PDX tumors for assaying their response to clinically relevant therapies. Mol Cancer Ther. 2019; 18:718-725.

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.