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. 2023 Dec 29;16(1):168.
doi: 10.3390/cancers16010168.

Precision Immunotherapy Utilizing Adapter CAR-T Cells (AdCAR-T) in Metastatic Breast Cancer Leads to Target Specific Lysis

Cansu E Önder  1 , Moustafa Moustafa-Oglou  2 , Sarah M Schröder  3   4 , Andreas D Hartkopf  5 , André Koch  1   5 , Christian M Seitz  2   6   7
Affiliations

Precision Immunotherapy Utilizing Adapter CAR-T Cells (AdCAR-T) in Metastatic Breast Cancer Leads to Target Specific Lysis

Cansu E Önder et al. Cancers (Basel). .

Abstract

A frequent symptom of metastasized breast cancer (BC) includes the development of malignant pleural effusion (MPE), which contains malignant cells derived from the primary tumor site. The poor prognosis of MPE in metastasized BC indicates the necessity for dependable precision oncology and the importance of models representing the heterogenous nature of metastatic BC. In this study, we cultured MPE-derived metastatic tumor cells from four advanced BC patients using organoid technology. We assessed the expression of tumor-associated antigens on MPE-derived organoid lines by flow cytometry (FC). Based on an individual antigen expression pattern, patient-derived organoids were treated with adapter CAR-T cells (AdCAR-T) and biotinylated monoclonal antibodies targeting CD276, HER2, EGFR, TROP2, or EpCAM. Co-culture assays revealed specific organoid lysis by AdCAR-T depending on individual antigen expression patterns. Our results demonstrate that MPE-derived organoids can serve as a reliable tool for assessing the efficacy of AdCAR-T on metastatic BC in a patient-individualized manner. This approach could potentially be applied in a preclinical setting to instruct therapy decisions. Further, our study demonstrates the feasibility of precision immunotherapy utilizing AdCAR-T to target patient-individualized antigen patterns.

Keywords: adapter CAR-T cells; breast cancer; cancer biology; metastasis; organoid culture; patient-derived organoids; pleural effusion; precision immunotherapy.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
AdCAR-T cell design and functional targeting of BC cell lines using adapter molecules (AMs). (A) Schematic illustration of an adapter molecule and AdCAR-T cell receptor. (B) AdCAR-T cells are directed towards the target antigen via LLE-conjugated biotinylated antibodies referred to as AMs. (C,D) Comparison of target cell lysis of MCF-7 and MB-MDA-468 (green bars) to either overtone positive antigen expression (upper blue bars) or mean fluorescence intensity ratio (MFIR) log 10 of antigen expression (lower blue bars). Target cell lysis was determined via luciferin-based cytotoxicity assay after 48 h with an E:T ratio of 2:1. Antigen screening with respective antibodies was performed via flow cytometry (FC) and is represented by mean values (n = 6) ± SD. (E) Correlation between target cell lysis and antigen expression of both cell lines analyzed via linear regression resulted in a Spearman correlation coefficient of 0.66. (F) Analyzing exponential regression for the correlation of target cell lysis and MFIR log 10 of both cell lines showed a Spearman coefficient of 0.77. All data used in (E,F) are depicted by mean values (n = 6).
Figure 2
Figure 2
Cultivation and characterization of metastatic breast cancer (BC) patient-derived organoids (MBC-PDO), originated from malignant pleural effusion (MPE). (A) Schematic overview of isolation of metastatic BC cells from MPEs, as well as cultivation in BME droplets and viral transduction of MBC-PDOs. (B) Brightfield (BF) and fluorescence (FL) images as well as FC analysis of luciferase- and GFP-expressing MCF-7 and MBC-PDO #03, #04, #06, and #07. Scale bar: 200 µm.
Figure 3
Figure 3
AdCAR treatment of MCF-7 organoids expressing luciferase and GFP. (A) Schematic overview of the experimental setup. MCF-7 and MBC-PDOs (green) expressing luciferase and GFP were seeded on BME beds and treated with AdCAR-T cells (red) and corresponding LLE-CD276 mAb. Readouts were performed on luminescence and GFP after 24 h, 48 h, and 72 h using a plate reader and a fluorescence microscope. (B) Brightfield (BF) and fluorescence (FL) images of GFP-expressing MCF-7 organoids treated with AdCAR-T cells without (−) and with (+) LLE-CD276 mAb. Scale bar: 500 µm. (C) Target cell lysis of MCF-7 organoids treated with AdCAR-T cells with (black bars) and without (gray bars) LLE-CD276 mAb over 72 h. Target cell lysis efficiency was determined by luciferase activity of remaining cells. Data shown represent the mean ± SD of biological triplicates (n = 3). Negative values were set to 0. Statistical analysis was performed using paired t-test. ns, not significant. * = p ≤ 0.05; ** = p ≤ 0.01.
Figure 4
Figure 4
AdCAR treatment of luciferase- and GFP-expressing MBC-PDO #07 at various E:T ratios. (A) Fluorescence images of GFP-expressing MBC-PDO #07 treated with AdCAR-T cells of different E:T ratios with the addition of LLE-CD276 mAb over 72 h. Scale bar: 500 µm. (B) Target cell lysis of MBC-PDO #07 treated with AdCAR-T cells with (black bars) and without (gray bars) LLE-CD276 mAb over 72 h. Target cell lysis efficiency was determined by luciferase activity of viable organoids. Data shown represent the mean ± SD of biological triplicates (n = 3). Negative values were set to 0. Statistical analysis was performed using paired t-test. ns, not significant. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001.
Figure 5
Figure 5
AdCAR treatment of MBC-PDO #07 with various LLE-mAbs. (A) Fluorescence images of GFP-expressing MBC-PDO #07 treated with AdCAR-T cells (E:T ratio was set to 1:1) with the addition of LLE-mAbs against CD276, HER2, EGFR, and TROP2. Images were taken after 0 h, 24 h, 48 h, and 72 h of treatment. Scale bar: 500 µm. (B) FC analysis and target cell lysis of MBC-PDO #07 treated with AdCAR-T cells (E:T ratio was set to 1:1) with (colored bars) and without (gray bars) LLE-mAbs over 72 h. Target cell lysis efficiency was determined by luciferase activity of viable organoids. Data shown represent the mean ± SD of biological triplicates (n = 3). Statistical analysis was performed using paired t-test. ns, not significant. * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.
Figure 6
Figure 6
FC analysis and AdCAR treatment of MBC-PDOs with various LLE-mAbs. Treatments were performed on (A) MBC-PDO #03, (B) MBC-PDO #06, and (C) MBC-PDO #04. AdCAR treatment of MBC-PDOs with (colored bars) and without (gray bars) LLE-mAbs was carried out in an E:T ratio of 1:1 for 72 h. Target cell lysis efficiency was determined by luciferase activity of viable organoids. Data shown represent the mean ± SD of multiple biological replicates (n ≥ 3). Statistical analysis was performed using paired t-test. ns, not significant. * = p ≤ 0.05; ** = p ≤ 0.01.
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