Quantifying CAR T-cell mediated tumor killing using image-based cytometry.

While CAR T-cell therapy has shown promising results in hematologic malignancies, translating that success to solid tumors remains challenging. A major barrier is the immunosuppressive tumor microenvironment (TME), which promotes T-cell exhaustion and limits sustained antitumor activity. Achieving a clearer understanding of the TME is therefore a critical step in successfully developing new therapeutic targets in immunotherapy.

Clear cell renal cell carcinoma (ccRCC) is the most prevalent sub-type of RCC, accounting for approximately 75% of all cases.¹ In ccRCC, a counterintuitive relationship exists where higher CD8+ T cell infiltration correlates with unfavorable prognosis. In advanced disease, the TME contains increased exhausted CD8+ T cells and M2-like macrophages that form a dysfunctional immune circuit promoting further T-cell exhaustion and immunosuppression, ultimately leading to poorer outcomes.

Humanized models enable immune-restoring strategies

Previous immunotherapy research in ccRCC has been limited by the availability of appropriate mouse models that accurately reflect human immune responses. In a recent study, Wang and Cho et al.² employed a humanized mouse model to demonstrate how immune-restoring CAR T cells exhibit antitumor effects and reverse immunosuppression in the TME.

Notably, G36-PDL1 CAR T cells successfully restored antitumor characteristics through three key mechanisms:

1. Activating tumor-killing cytotoxicity
2. Reducing immunosuppressive cell populations
3. Promoting T follicular helper (Tfh)-B cell crosstalk

Quantifying CAR T-cell efficacy with image cytometry

The researchers used the Celigo™ image cytometer to evaluate the cytotoxic effects of the immune-restoring CAR T cells targeting the humanized mouse model of ccRCC. The high-throughput platform successfully quantified tumor cell killing with an image-based cytotoxicity assay³ to measure the effectiveness of different CAR T-cell constructs against skrc-59 cancer cells.

The CAR T cells were engineered to target carbonic anhydrase IX (CAIX), a protein highly expressed in ccRCC, and to secrete anti-PD-L1 monoclonal antibodies (CAR G36-PDL1). Approximately 3,000 skrc-59 tumor cells were seeded in 96-well plates and incubated for 12 hours. Imaging was performed using brightfield and far-red fluorescence to detect the mCardinal reporter, with endpoint analysis performed 48 hours after introducing the G36-PDL1 CAR T cells.

At an effector:target (E:T) ratio of 10:1, G36-PDL1 CAR T cells achieved approximately 95% target cell killing in vitro, which was significantly higher than control cells. The Celigo assay provided unbiased, quantitative data to evaluate CAR T-cell efficacy, supporting the study's conclusion that anti-CAIX G36-PDL1 CAR T cells secreting anti-PD-L1 mAb exhibited superior tumor control.

Building on prior strategies

These findings build on previous work showing that CAIX-specific CAR T cells incorporating a 4-1BB signaling domain outperform other designs,³ and that CAR T cells secreting immune checkpoint inhibitors can reduce CAR T-cell exhaustion in solid tumor models.

The Celigo image cytometer proved to be a critical screening tool for identifying the optimal CAR T constructs for downstream experiments on the IVIS™ Spectrum In Vivo Imaging system. This workflow offers a powerful approach to identify and advance the most promising CAR T-cell designs for ccRCC and other solid tumors.