Systemic and Local Strategies for Primary Prevention of Breast Cancer.

One in eight women will develop breast cancer in the US. For women with moderate (15-20%) to average (12.5%) risk of breast cancer, there are few options available for risk reduction. For high-risk (>20%) women, such as BRCA mutation carriers, primary prevention strategies are limited to evidence-based surgical removal of breasts and/or ovaries and anti-estrogen treatment. Despite their effectiveness in risk reduction, not many high-risk individuals opt for surgical or hormonal interventions due to severe side effects and potentially life-changing outcomes as key deterrents. Thus, better communication about the benefits of existing strategies and the development of new strategies with minimal side effects are needed to offer women adequate risk-reducing interventions. We extensively review and discuss innovative investigational strategies for primary prevention. Most of these investigational strategies are at the pre-clinical stage, but some are already being evaluated in clinical trials and others are expected to lead to first-in-human clinical trials within 5 years. Likely, these strategies would be initially tested in high-risk individuals but may be applicable to lower-risk women, if shown to decrease risk at a similar rate to existing strategies, but with minimal side effects.

X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models.

There are still a limited number of primary interventions for prevention of breast cancer. For women at a high risk of developing breast cancer, the most effective intervention is prophylactic mastectomy. This is a drastic surgical procedure in which the mammary epithelial cells that can give rise to breast cancer are completely removed along with the surrounding tissue. The goal of this protocol is to demonstrate the feasibility of a minimally invasive intraductal procedure that could become a new primary intervention for breast cancer prevention. This local procedure would preferentially ablate mammary epithelial cells before they can become malignant. Intraductal methods to deliver solutions directly to these epithelial cells in rodent models of breast cancer have been developed at Michigan State University and elsewhere. The rat mammary gland consists of a single ductal tree that has a simpler and more linear architecture compared to the human breast. However, chemically induced rat models of breast cancer offer valuable tools for proof-of-concept studies of new preventive interventions and scalability from mouse models to humans. Here, a procedure for intraductal delivery of an ethanol-based ablative solution containing tantalum oxide nanoparticles as X-ray contrast agent and ethyl cellulose as gelling agent into the rat mammary ductal tree is described. Delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) by intraductal injection has been described previously in mouse and rat models. This protocol description emphasizes methodological changes and steps that pertain uniquely to delivering an ablative solution, formulation consideration to minimize local and systemic side effects of the ablative solution, and X-ray imaging for in vivo assessment of ductal tree filling. Fluoroscopy and micro-CT techniques enable to determine the success of ablative solution delivery and the extent of ductal tree filling thanks to compatibility with the tantalum-containing contrast agent.

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models.

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

Automated Five-Color Multiplex Co-detection of MicroRNA and Protein Expression in Fixed Tissue Specimens.

microRNAs are an important class of noncoding regulatory RNAs with functional roles in development, physiology, and disease. Visualization of microRNA expression at a single-cell level has contributed to a better understanding of their biological function in animal models and their etiological contribution to human diseases. In addition, several microRNAs have been highlighted as potential biomarkers carrying diagnostic and prognostic information. Co-detection of microRNA expression with that of cell-type-specific proteins can enhance the interpretative power of expression changes during development or altered expression in pathological conditions. Here, we describe an automated fluorescence-based five-color multiplex assay for co-detection of microRNA (e.g., miR-10b, miR-21, miR-205), noncoding RNA (e.g., snRNA U6, 18S rRNA), and protein expression (e.g., cytokeratin 19, vimentin, collagen I) in paraffin-embedded formalin-fixed tissue slides on a Leica Bond Rx staining station. While this protocol uses mainly mouse tissues to demonstrate multiplex detection, it can be generally applied to single-cell expression analysis of other animal models and clinical specimens.

Ductal tree ablation by local delivery of ethanol prevents tumor formation in an aggressive mouse model of breast cancer.

BACKGROUND: Prophylactic mastectomy is the most effective intervention to prevent breast cancer. However, this major surgery has life-changing consequences at the physical, emotional, psychological, and social levels. Therefore, only high-risk individuals consider this aggressive procedure, which completely removes the mammary epithelial cells from which breast cancer arises along with surrounding tissue. Here, we seek to develop a minimally invasive procedure as an alternative to prophylactic mastectomy by intraductal (ID) delivery of a cell-killing solution that locally ablates the mammary epithelial cells before they become malignant. METHODS: After ID injection of a 70% ethanol-containing solution in FVB/NJ female animals, ex vivo dual stained whole-mount tissue analysis and in vivo X-ray microcomputed tomography imaging were used to visualize ductal tree filling, and histological and multiplex immunohistochemical assays were used to characterize ablative effects and quantitate the number of intact epithelial cells and stroma. After ID injection of 70% ethanol or other solutions in cancer-prone FVB-Tg-C3(1)-TAg female animals, mammary glands were palpated weekly to establish tumor latency and examined after necropsy to record tumor incidence. Statistical difference in median tumor latency and tumor incidence between experimental groups was analyzed by log-rank test and logistic mixed-effects model, respectively. RESULTS: We report that ID injection of 70% ethanol effectively ablates the mammary epithelia with limited collateral damage to surrounding stroma and vasculature in the murine ductal tree. ID injection of 70% ethanol into the mammary glands of the C3(1)-TAg multifocal breast cancer model significantly delayed tumor formation (median latency of 150 days in the untreated control group [n = 25] vs. 217 days in the ethanol-treated group [n = 13], p value < 0.0001) and reduced tumor incidence (34% of glands with tumors [85 of 250] in the untreated control group vs. 7.3% of glands with tumor [7 of 95] in the ethanol-treated group, risk ratio = 4.76 [95% CI 1.89 to 11.97, p value < 0.0001]). CONCLUSIONS: This preclinical study demonstrates the feasibility of local ductal tree ablation as a novel strategy for primary prevention of breast cancer. Given the existing clinical uses of ethanol, ethanol-based ablation protocols could be readily implemented in first-in-human clinical trials for high-risk individuals.