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Nanoluciferase-CD63 labeling reveals extracellular vesicle kinetics in a mouse intraductal model of ductal carcinoma in situ

ABSTRACT: Background. Ductal carcinoma in situ (DCIS) is a non-invasive precursor to breast cancer that increases lifetime risk for invasive disease and, in rare cases, causes distant metastases without local recurrence. Tumor-derived small extracellular vesicles (sEVs), or exosomes, mediate tumor progression, systemic signaling, and metastasis; however, the timing of release and biodistribution of sEVs from early preinvasive lesions remains unclear. Methods. Here, we investigated the release kinetics and systemic dissemination of sEVs secreted by DCIS cells confined to the mammary ducts using an orthotopic mouse mammary intraductal xenograft model with MCF10DCIS cells engineered to express NanoLuc (NLuc)-tagged CD63, an sEV marker. Tumor growth was monitored using bioluminescent imaging (BLI) in vivo and ex vivo; DCIS outgrowth and focal invasion were confirmed by fluorescent immunohistochemistry; and NLuc activity was assayed in tissue lysates. Next-generation RNA sequencing of brain tissue was used to evaluate gene expression changes at 28 days post-implantation (dpi). Results. Longitudinal BLI revealed CD63-NLuc signals in mammary glands (P < 0.05), and distant organs, including spleen (P < 0.05), liver (P < 0.05), lungs (P < 0.01) and brain (P < 0.01) by day 14 followed by detection in heart tissue (P < 0.001) and in the circulation by day 21, preceding histological evidence of invasion. Further, CD63-NLuc activity was significantly and positively correlated with BLI levels in mammary glands and plasma (P < 0.05). Immune, metabolic, and stress response pathways were downregulated in brain tissue at 28 dpi compared to 3 dpi. Conclusions. These findings demonstrate systemic dissemination of tumor-derived sEVs during DCIS progression, suggesting roles in early microenvironmental conditioning and metastatic preconditioning prior to overt invasion.

ORGANISM(S): Mus musculus

PROVIDER: GSE317178 | GEO | 2026/01/26

REPOSITORIES: GEO

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Project description:BACKGROUND: Ductal carcinoma in situ (DCIS) is the earliest stage of breast cancer. During DCIS, tumor cells remain inside the mammary duct, growing under a microenvironment characterized by hypoxia, nutrient starvation, and waste product accumulation; this harsh microenvironment promotes genomic instability and eventually cell invasion. However, there is a lack of biomarkers to predict what patients will transition to a more invasive tumor or how DCIS cells manage to survive in this harsh microenvironment. </br> METHODS: In this work, we have developed a microfluidic model that recapitulates the DCIS microenvironment. In the microdevice, a DCIS model cell line was grown inside a luminal mammary duct model, embedded in a 3D hydrogel with mammary fibroblasts. Cell behavior was monitored by confocal microscopy and optical metabolic imaging. Additionally, metabolite profile was studied by NMR whereas gene expression was analyzed by RT-qPCR. </br> FINDINGS: DCIS cell metabolism led to hypoxia and nutrient starvation; revealing an altered metabolism focused on glycolysis and other hypoxia-associated pathways. In response to this starvation and hypoxia, DCIS cells modified the expression of multiple genes, and a gradient of different metabolic phenotypes was observed across the mammary duct model. These genetic changes observed in the model were in good agreement with patient genomic profiles; identifying multiple compounds targeting the affected pathways. In this context, the hypoxia-activated prodrug tirapazamine selectively destroyed hypoxic DCIS cells. </br> INTERPRETATION: The results showed the capacity of the microfluidic model to mimic the DCIS structure, identifying multiple cellular adaptations to endure the hypoxia and nutrient starvation generated within the mammary duct. These findings may suggest new potential therapeutic directions to treat DCIS. In summary, given the lack of in vitro models to study DCIS, this microfluidic device holds great potential to find new DCIS predictors and therapies and translate them to the clinic.

Project description:Pathologic and epidemiologic evidence has led to a histologic model of breast cancer progression that involves advancement through specific morphologic stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Numerous observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these different stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after up to a year post-injection. In order to develop a more rapid, readily manipulatable in vitro assay for examining the biologic differences between these cell lines, we have made use of a 3D Matrigel system. When grown in 3D Matrigel, we have found characteristic morphologies of the three cell lines in which quantifiable aspects of the stage-specific in vivo behaviors (i.e. differences in acinar structure formation, cell polarization, cell cohesiveness, cell proliferation, cell invasion) are re-capitulated in a reproducible fashion. Gene expression profiling has revealed a characteristic pattern for each of the three cell lines. Interestingly, WNT pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasiveness phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth. Keywords: DCIS or IDC vs ADH RNA derived from three biological replicates of a cell line representing atypical ductal hyperplasia (ADH), Ductal carcinoma in situ (DCIS), and Invasive ductal carcinoma (IDC)

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Nanoluciferase-CD63 labeling reveals extracellular vesicle kinetics in a mouse intraductal model of ductal carcinoma in situ

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