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- N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses

# N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses

Teng T, Kamal M, Iriondo O, Amzaleg Y, Luo C, Thomas A, et al.

Molecular Cancer Research . 2021;19(3):441-450. DOI [10.1158/1541-7786.MCR-20-0482](https://doi.org/10.1158/1541-7786.MCR-20-0482). PMID 33303691. PMCID PMC7925378.

How to cite

### AMA

Teng T, Kamal M, Iriondo O, Amzaleg Y, Luo C, Thomas A, et al. N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses. Mol Cancer Res . 2021;19(3):441-450. doi:10.1158/1541-7786.MCR-20-0482. Epub 2020 Dec 10.

### APA

Teng, T., Kamal, M., Iriondo, O., Amzaleg, Y., Luo, C., Thomas, A., et al. (2021). N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses. Molecular Cancer Research , 19(3), 441-450. https://doi.org/10.1158/1541-7786.MCR-20-0482

### BibTeX

@article{teng2021nacetyl,
title = {N-Acetyl-L-cysteine Promotes Ex Vivo Growth and Expansion of Single Circulating Tumor Cells by Mitigating Cellular Stress Responses},
author = {Teng, Teng and Kamal, Mohamed and Iriondo, Oihana and Amzaleg, Yonatan and Luo, Chunqiao and Thomas, Amal and others},
journal = {Molecular Cancer Research},
volume = {19},
number = {3},
pages = {441-450},
year = {2021},
doi = {10.1158/1541-7786.MCR-20-0482}
}

Circulating tumor cells break away from a tumor and travel in the blood, and studying them could show how a cancer spreads. The trouble is that they are extremely rare and fragile, and growing even a single one in the lab almost always fails. The isolation itself, along with the loss of contact with neighboring cells, floods the cell with oxidative stress.

This study screened hundreds of small molecules to find ones that help a lone tumor cell survive and divide. It found that N-acetylcysteine, a common antioxidant, was the most reliable, easing that early stress so more cells make it through the critical first days of culture.

The effect held for tumor cells taken straight from a breast-cancer patient's blood, not only from established laboratory lines.

[Read publication at Molecular Cancer Research](https://pmc.ncbi.nlm.nih.gov/articles/PMC7925378/)

## Key findings

- A screen of 317 small molecules pointed to antioxidant and stress-reducing pathways as the strongest promoters of single-CTC growth. Among 130 compounds that outperformed controls, 6 of 11 antioxidants, 7 of 12 COX inhibitors, and 4 of 4 AMPK activators improved the expansion of single circulating tumor cells.

- N-acetylcysteine at 300 μM was the one compound that helped across every patient-derived line tested. Alone or combined with the COX inhibitor P1C2, 300 μM NAC consistently and significantly increased single-CTC expansion in all 4 breast-cancer CTC lines, including two lines that are otherwise very hard to grow from single cells.

- The effect carried from cell lines to a fresh patient sample: NAC revived a dying single-CTC clone that then grew for about 5 more weeks. Live single CTCs were isolated from 12 metastatic breast-cancer patients with the AccuCyte separation workflow, and copy-number profiling of 8 cells from the rescued clone confirmed a common cancer-cell origin.

## AccuCyte in the methods

“Single CTCs were detected and retrieved using the negative selection protocol of PIC&RUN assay as previously described( 17 ). Briefly, 7.5 ml of blood was added to each AccuCyte Separation Tube (RareCyte) and tubes were centrifuged twice in a special device to collect buffy coats in 1 ml of CTC media. Buffy coats were stained with a cocktail of immune cell markers (IM) antibodies (CD45, CD14 and CD16) and Cell-Tracker green for 30 min at 37 °C. Stained buffy coats were seeded on polyhema coated cyteslides and scanned semi-automatically using RareCyte fluorescence. When a CTC was detected, the Rarecyte ceramic tip needle was operated semi-automatically to pick up the cell of interest in a draw volume of 50 nL to 0.5 μL, and deposit it in a PCR tube contains 50 μL of media.”

— Teng et al., Molecular Cancer Research (2021), Materials and Methods, “Isolation of single CTCs”

## Why it matters for AccuCyte users

If you work with circulating tumor cells, the isolation step decides what is possible afterward. This study used the AccuCyte separation workflow to pull live single tumor cells from just 7.5 mL of a patient's blood without first enriching by an epithelial surface marker, so fragile cells that a marker-based step might have discarded stayed available for culture. That matters because everything downstream — expanding a clone, profiling its genome, testing a drug on it — depends on the cell arriving intact and alive. Here the AccuCyte-isolated cells from a breast-cancer patient were viable enough that one, once rescued with an antioxidant, grew into a colony over several weeks and was confirmed by copy-number profiling to be a true cancer clone. The lesson for your own work is that a gentle, antigen-agnostic isolation is the foundation, and the bottleneck that remains is the stress a single cell feels right after it is retrieved — a bottleneck this paper shows can be eased rather than accepted.

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