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

Circulating tumor cells (CTC) can be isolated via a minimally invasive blood draw and are considered a "liquid biopsy" of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small-molecule drug screen to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetyl-L-cysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single-cell expansion. RNA-seq analysis of growing clones and nongrowing clones confirmed the effect by NAC, but also indicates that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with predominant senescent features. Despite the challenge in expanding all CTCs, NAC treatment led to establishment of single CTC clones that have similar tumorigenic features. IMPLICATIONS: Through a small molecule screen and validation study, we found that NAC could improve the success of ex vivo expansion of single CTCs by mitigating the initial stress, with the potential to facilitate the investigation of functional heterogeneity in CTCs.

Author Correction: PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circulating Tumor Cells Exhibit Metastatic Tropism and Reveal Brain Metastasis Drivers.

Hematogenous metastasis is initiated by a subset of circulating tumor cells (CTC) shed from primary or metastatic tumors into the blood circulation. Thus, CTCs provide a unique patient biopsy resource to decipher the cellular subpopulations that initiate metastasis and their molecular properties. However, one crucial question is whether CTCs derived and expanded ex vivo from patients recapitulate human metastatic disease in an animal model. Here, we show that CTC lines established from patients with breast cancer are capable of generating metastases in mice with a pattern recapitulating most major organs from corresponding patients. Genome-wide sequencing analyses of metastatic variants identified semaphorin 4D as a regulator of tumor cell transmigration through the blood-brain barrier and MYC as a crucial regulator for the adaptation of disseminated tumor cells to the activated brain microenvironment. These data provide the direct experimental evidence of the promising role of CTCs as a prognostic factor for site-specific metastasis. SIGNIFICANCE: Interests abound in gaining new knowledge of the physiopathology of brain metastasis. In a direct metastatic tropism analysis, we demonstrated that ex vivo-cultured CTCs from 4 patients with breast cancer showed organotropism, revealing molecular features that allow a subset of CTCs to enter and grow in the brain.This article is highlighted in the In This Issue feature, p. 1.

PIC&RUN: An integrated assay for the detection and retrieval of single viable circulating tumor cells.

Circulating tumor cells (CTCs) shed from solid tumors can serve as a minimally invasive liquid biopsy for monitoring disease progression. Because CTCs are rare and heterogeneous, their biological properties need to be investigated at the single cell level, which requires efficient ways to isolate and analyze live single CTCs. Current methods for CTC isolation and identification are either performed on fixed and stained cells or need multiple procedures to isolate pure live CTCs. Here, we used the AccuCyte-RareCyte system to develop a Protocol for Integrated Capture and Retrieval of Ultra-pure single live CTCs using Negative and positive selection (PIC&RUN). The positive selection module of PIC&RUN identifies CTCs based on detection of cancer surface markers and exclusion of immune markers. Combined with a two-step cell picking protocol to retrieve ultrapure single CTCs, the positive selection module is compatible for downstream single cell transcriptomic analysis. The negative selection module of PIC&RUN identifies CTCs based on a live cell dye and the absence of immune markers, allowing retrieval of viable CTCs that are suitable for ex vivo culture. This new assay combines the CTC capture and retrieval in one integrated platform, providing a valuable tool for downstream live CTC analyses.

Unexpected Friendship: Neutrophils Help Tumor Cells En Route to Metastasis.

Circulating tumor cells (CTCs) contain metastatic precursors that can initiate new metastases. Recent work shows that association with neutrophils provides a proliferative advantage to CTCs, rendering them more competent in metastasis formation. The study identifies molecular players in CTC-neutrophil interactions, providing potential targets for disrupting formation of these deadly metastatic seeds.

TAK1 mediates microenvironment-triggered autocrine signals and promotes triple-negative breast cancer lung metastasis.

Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer that has limited therapeutic options. Thus, developing novel treatments for metastatic TNBC is an urgent need. Here, we show that nanoparticle-mediated delivery of transforming growth factor-ß1-activated kinase-1 (TAK1) inhibitor 5Z-7-Oxozeaenol can inhibit TNBC lung metastasis in most animals tested. P38 is a central signal downstream of TAK1 in TNBC cells in TAK1-mediated response to multiple cytokines. Following co-culturing with macrophages or fibroblasts, TNBC cells express interleukin-1 (IL1) or tumor necrosis factor-α (TNFα), respectively. Compared to TAK1 inhibition, suppressing IL1 signaling with recombinant IL1 receptor antagonist (IL1RA) is less efficient in reducing lung metastasis, possibly due to the additional TAK1 signals coming from distinct stromal cells. Together, these observations suggest that TAK1 may play a central role in promoting TNBC cell adaptation to the lung microenvironment by facilitating positive feedback signaling mediated by P38. Approaches targeting the key TAK1-P38 signal could offer a novel means for suppressing TNBC lung metastasis.

Distinct breast cancer stem/progenitor cell populations require either HIF1α or loss of PHD3 to expand under hypoxic conditions.

The heterogeneous nature of breast cancer is a result of intrinsic tumor complexity and also of the tumor microenvironment, which is known to be hypoxic. We found that hypoxia expands different breast stem/progenitor cell populations (cells with increased aldehyde dehydrogenase activity (Aldefluor+), high mammosphere formation capacity and CD44+CD24-/low cells) both in primary normal epithelial and tumor cells. The presence of the estrogen receptor (ER) limits hypoxia-dependent CD44+CD24-/low cell expansion.We further show that the hypoxia-driven cancer stem-like cell enrichment results from a dedifferentiation process. The enhanced mammosphere formation and Aldefluor+ cell content observed in breast cancer cells relies on hypoxia-inducible factor 1α (HIF1α). In contrast, the CD44+CD24-/low population expansion is HIF1α independent and requires prolyl hydroxylase 3 (PHD3) downregulation, which mimics hypoxic conditions, leading to reduced CD24 expression through activation of NFkB signaling. These studies show that hypoxic conditions expand CSC populations through distinct molecular mechanisms. Thus, potential therapies that combine current treatments for breast cancer with drugs that target CSC should take into account the heterogeneity of the CSC subpopulations.

FACS Sorting Mammary Stem Cells.

Fluorescent-activated cell sorting (FACS) represents one of the key techniques that have been used to isolate and characterize stem cells, including cells from the mammary gland. A combination of approaches, including recognition of cell surface antigens and different cellular activities, has facilitated the identification of stem cells from the healthy mammary gland and from breast tumors. In this chapter we describe the protocol to use FACS to separate breast cancer stem cells, but most of the general principles discussed could be applied to sort other types of cells.

Respuesta hormonal de las células madre de mama y resistencia a tamoxifeno / Hormone response of breast stem cells and tamoxifen resistance

Objetivo. Determinar la influencia a nivel celular y molecular de varios tratamientos hormonales (estrógeno, tamoxifeno y fulvestrant) sobre las células epiteliales y las células madre de la mama sana y tumoral. Métodos. Se emplearon muestras de tejido mamario sano y tumoral, así como líneas celulares de cáncer de mama y células resistentes a tamoxifeno, para analizar los efectos de las hormonas sobre la proliferación y diferenciación celular. Resultados. Las células epiteliales y las células madre de la mama respondieron de forma diferente a los tratamientos hormonales. Las células resistentes a tamoxifeno presentaban un mayor contenido de células madre cancerosas y expresaban niveles de Sox2 más elevados, mientras que los niveles de expresión del receptor de progesterona eran muy bajos. Las células resistentes a tamoxifeno eran, además, más resistentes al tratamiento con fulvestrant. Conclusiones. El desarrollo de resistencia a tamoxifeno está asociado con un incremento en el contenido de células madre cancerosas. El tratamiento con fulvestrant no parece disminuir la población de células madre cancerosas. Sox2 podría ser un biomarcador de resistencia a tamoxifeno en el cáncer de mama (AU)

Objective. To determine the influence of various hormones (estrogen, tamoxifen and fulvestrant) on cell proliferation and differentiation in normal and cancer breast stem cells. Methods. Primary tissue samples, breast cancer cell lines and tamoxifen-resistant cells were used to analyze the effects of hormones on cell proliferation and differentiation. Results. Breast epithelial cells and stem cells responded differentially to hormone treatments. Tamoxifen-resistant cells showed increased cancer stem cell content and expressed higher Sox2 levels, while progesterone receptor levels were very low. Tamoxifen-resistant cells were resistant to fulvestrant treatment. Conclusions. The development of tamoxifen resistance is associated with an increase in cancer stem cell content. Treatment with fulvestrant does not appear to reduce the cancer stem cell population. Sox2 could represent a biomarker of tamoxifen resistance in breast cancer (AU)

Role of UTX in retinoic acid receptor-mediated gene regulation in leukemia.

Human UTX, a member of the Jumonji C family of proteins, associates with mixed-lineage leukemia 3/4 complexes. Stimulation with retinoic acid leads to the recruitment of UTX-containing complexes to HOX genes, which results in demethylation of histone H3 lysine 27 and concomitant methylation of histone H3 lysine 4. Here, we show that UTX interacts with the retinoic acid receptor α (RARα) and that this interaction is essential for proper differentiation of leukemic U937 cells in response to retinoic acid. UTX occupies the promoters of several RAR target genes and regulates their transcriptional output by modulating ASH2L complex recruitment. Overexpression of UTX in promyelocytic NB4 cells results in enhanced cellular differentiation upon retinoic acid treatment. Our results show that UTX is important for RAR-mediated transcription and provide insight into the critical role of cross talk between histone H3 lysine 4 methylation and histone H3 lysine 27 demethylation during cellular differentiation.

Sox2 promotes tamoxifen resistance in breast cancer cells.

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo­ and radiotherapy. Here, we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen­resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely, ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2­expressing cells, and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure, and also in the primary tumours of these patients, compared to those of responders. Together, these results suggest that development of tamoxifen resistance is driven by Sox2­dependent activation of Wnt signalling in cancer stem/progenitor cells.

Effects of estrogen on the proportion of stem cells in the breast.

There is increasing evidence that breast cancers contain tumor-initiating cells with stem cell properties. The importance of estrogen in the development of the mammary gland and in breast cancer is well known, but the influence of estrogen on the stem cell population has not been assessed. We show that estrogen reduces the proportion of stem cells in the normal human mammary gland and in breast cancer cells. The embryonic stem cell genes NANOG, OCT4, and SOX2 are expressed in normal breast stem cells and at higher levels in breast tumor cells and their expression decreases upon differentiation. Overexpression of each stem cell gene reduces estrogen receptor (ER) expression, and increases the number of stem cells and their capacity for invasion, properties associated with tumorigenesis and poor prognosis. These results indicate that estrogen reduces the size of the human breast stem cell pool and may provide an explanation for the better prognosis of ER-positive tumors.