Transcriptome analysis of heterogeneity in mouse model of metastatic breast cancer.

BACKGROUND: Cancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are complicated by the underlying heterogeneity of the cell types involved. The purpose of this study was to examine the transcriptomes of metastatic breast cancer cells using the well-established MMTV-PyMT mouse model. METHODS: Organ-derived metastatic cell lines were harvested from 10 female MMTV-PyMT mice. Cancer cells were isolated and sorted based on the expression of CD44low/EpCAMhigh or CD44high/EpCAMhigh surface markers. RNA from each cell line was extracted and sequenced using the NextSeq 500 Illumina platform. Tissue-specific genes were compared across the different metastatic and primary tumor samples. Reads were mapped to the mouse genome using STAR, and gene expression was quantified using RSEM. Single-cell RNA-seq (scRNA-seq) was performed on select samples using the ddSeq platform by BioRad and analyzed using Seurat v3.2.3. Monocle2 was used to infer pseudo-time progression. RESULTS: Comparison of RNA sequencing data across all cell populations produced distinct gene clusters. Differential gene expression patterns related to CD44 expression, organ tropism, and immunomodulatory signatures were observed. scRNA-seq identified expression profiles based on tissue-dependent niches and clonal heterogeneity. These cohorts of data were narrowed down to identify subsets of genes with high expression and known metastatic propensity. Dot plot analyses further revealed clusters expressing cancer stem cell and cancer dormancy markers. Changes in relevant genes were investigated across pseudo-time and tissue origin using Monocle2. These data revealed transcriptomes that may contribute to sub-clonal evolution and treatment evasion during cancer progression. CONCLUSIONS: We performed a comprehensive transcriptome analysis of tumor heterogeneity and organ tropism during breast cancer metastasis. These data add to our understanding of metastatic progression and highlight targets for breast cancer treatment. These markers could also be used to image the impact of tumor heterogeneity on metastases.

Nicotine e-cigarette vapor inhalation and self-administration in a rodent model: Sex- and nicotine delivery-specific effects on metabolism and behavior.

E-cigarettes, which deliver vaporized nicotine, have dramatically risen in popularity in recent years, despite many unanswered questions about safety, efficacy in reducing dependence, and overall impact on public health. Other factors, such as sex, also play an important role in determining behavioral and neurochemical responses to drugs of abuse. In these studies, we sought to develop a protocol for vaporized e-cigarette nicotine self-administration in rats, as a foundation to better understand the differing effects of nicotine exposure routes on behavior and physiological function. We report a novel method that elicits robust nicotine vapor self-administration in male and female rats. Our findings indicate that 5-mg/ml nicotine vape solution provides a high level of consistency in lever-pressing behavior for both males and females. Moreover, in male rats, we find that such e-cigarette nicotine vapor induces similar blood levels of nicotine's main metabolite, cotinine, as that found with intravenous nicotine self-administration. Therefore, the breathing pattern during vapor exposure in males leads to similar levels of titrated nicotine intake as with intravenous nicotine self-administration. Interestingly, a differential effect was found in the females, in which the same conditions of vapor exposure led to decreased cotinine levels with vapor compared to intravenous self-administration. Finally, differences in nicotine-mediated locomotion provide further support of the physiological effects of e-cigarette vapor inhalation. Taken together, our findings reveal important sex differences in nicotine intake based on the route of exposure, and we further establish a protocol for nicotine vapor self-administration in rats.