The intestinal epithelium comprises diverse cell types and executes many specialized functions as the primary interface between luminal contents and internal organs. A key function provided by the epithelium is maintenance of a barrier that protects the individual from pathogens, irritating luminal contents, and the microbiota. Disruption of this barrier can lead to inflammatory disease within the intestinal mucosa, and, in more severe cases, to sepsis. Animal models to study intestinal permeability are costly and not entirely predictive of human biology. Here we present a model of human colon barrier function that integrates primary human colon stem cells into Draper’s PREDICT96 microfluidic organ-on-chip platform to yield a high-throughput system appropriate to predict damage and healing of the human colon epithelial barrier. We have demonstrated pharmacologically induced barrier damage measured by both a high throughput molecular permeability assay and transepithelial resistance. Using these assays, we developed an Inflammatory Bowel Disease-relevant model through cytokine induced damage that can support studies of disease mechanisms and putative therapeutics.

Autologous cellular therapies have been highly successful in treating hematological cancers and have the potential to be used for a variety of indications. Manufacturing these therapies rapidly and at low cost remains a major challenge. A key bottleneck in cellular therapy manufacturing is genetic modification of target cells, which is often done using viral vectors. Because vectors are expensive to develop and produce, non‐viral gene transfer using electroporation is emerging as a preferred transfection method for next‐generation therapies. However, most commercial electroporation systems are built for research use rather than large‐scale clinical manufacturing. The microfluidic, continuous‐flow electroporation device presented here offers several advantages including large‐scale and high throughput processing, high performance, and the potential for automation. It transfects primary human T cells with Cas9‐guide ribonucleic acid (RNA) ribonucleoprotein complexes (RNP) and messenger RNA (mRNA) with up to 99–100% efficiency and minimal impact on viability. In addition, this device transfects 3.5 kbp plasmid deoxyribonucleic acid with up to 86% efficiency after preliminary optimization studies. A single microchannel can deliver a total cellular processing throughput of up to 9.6 billion per hour. The combination of high throughput and high performance enables the scale of processing required for future “off‐the‐shelf” allogeneic cellular therapies.

Cellullar Therapy Manufacturing T cells moving through a microfluidic device are electroporated in continuous-flow for high-throughput cellular therapy manufacturing, as reported by Vishal Tandon and co-workers in article 2300275. The combination of high throughput and high performance enables the scale of processing required for future “off-the-shelf” allogeneic cellular therapies.

Polygenic risk scores (PRS) for breast cancer may help guide screening decisions. However, few studies have examined whether PRS are associated with risk of short-term or poor prognosis breast cancers. The study purpose was to evaluate the association of the 313 SNP breast cancer PRS with 2-year risk of poor prognosis breast cancer. We evaluated the association of breast cancer PRS with breast cancer overall, ER + and ER- breast cancer, and poor prognosis breast cancer diagnosed within 2 years of a negative mammogram among a cohort of 3657 women using logistic regression adjusted for age, breast density, race/ethnicity, year of screening, and genetic ancestry principal components. Breast cancers were considered poor prognosis if they were metastatic, positive lymph nodes, ER/PR + HER2− and > 2 cm, ER/PR/HER2−, or HER2 + and > 1 cm. Of the 308 breast cancers, 137 (44

Abstract Background: Molecular characterization of breast tumors has revealed four subtypes which differ in expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Breast cancer subtypes have different prognosis and unique risk factors. Breast cancer risk assessment models mainly reflect risk of ER/PR+ HER2- tumors, the most common subtype, and may not reflect risk of other subtypes. The study objective was to compare associations of established breast cancer risk factors across invasive breast cancer subtypes. Methods: The study population included women aged 40-84 years who had a screening mammogram at Massachusetts General Hospital, Newton Wellesley Hospital, or the University of Pennsylvania from 2006-2015. Patients completed a risk factor questionnaire and additional risk factors were ascertained from clinical records. Women with prior breast cancer, breast implants, or BRCA1/2 mutations were excluded. Women diagnosed with breast cancer within 6 months were excluded to remove those with cancer at the time of risk assessment. Tumor characteristics were obtained from linkage with hospital and state cancer registries. For invasive tumors, subtype was defined based on immunohistochemistry as ER and/or PR+ HER2-, ER and/or PR+HER2+, ER and PR- HER2+, or ER and PR and HER2- (triple negative breast cancer, TNBC). Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for breast cancer using time from mammogram to cancer diagnosis or censoring. Separate Cox models were fit for each subtype, and patients who developed DCIS or another subtype, died, or were alive and cancer free on December 31, 2017 were censored. Age, biopsy history, atypical hyperplasia, age at menarche, age at first live birth, family history, race, BMI, and breast density at the time of screening were included in the models. Results: The study population (N=197,836) had a mean age of 54 years and 74% of patients were white, 15% were Black, and 11% were other races. During a mean follow-up of 6.3 years, 4,510 (2.3%) women developed breast cancer. Of these cancers, 1068 (24%) were DCIS. Of the invasive cancers, 2675 (77%) were ER/PR+HER2-, 290 (8%) were ER/PR+HER2+, 108 (3%) were ER/PR-HER2+, 264 (8%) were TNBC and 105 (3%) had missing subtype. For ER/PR+HER2- cancers, all risk factors were consistent with the literature and statistically significant. Breast density was associated with increased risk of all four subtypes compared to women with less dense breasts. Atypical hyperplasia was strongly associated with HER2+ cancers (HR=2.97 CI 1.63-5.40 p<0.01), less strongly associated with ER/PR+HER2- cancers, and not significantly associated with TNBC. Black women had higher risk of TNBC than white women (HR=2.61 CI 1.91-3.57 p<0.01). Conclusion Our results highlight both similarities and heterogeneity in risk factors across breast cancer subtypes. Prior diagnosis of atypical hyperplasia was more strongly associated with HER2+ compared to HER2- tumors. While it is well known that Black women have higher risk of TNBC, it is striking that the more than two-and-a-half-fold increased risk persisted even with comprehensive adjustment for breast cancer risk factors in a screened population. These results suggest that additional factors, such as genetics, biomarkers, and environmental exposures should be included in risk assessment to better capture risk of less common breast cancer subtypes such as TNBC. Risk factors for breast cancer subtypes among 197,836 women undergoing screening mammography*ER/PR+ HER2- N=2674ER/PR+ HER2+ N=290ER/PR-HER2+ N=108ER/PR/HER2- N=264HR,95% CIpHR,95% CIpHR,95% CIpHR,95% CIpBlack vs. white unadjusted0.67,0.58-0.77<0.010.73,0.48-1.090.120.91,0.49-1.680.772.61,1.96-3.46<0.01Black vs. white multivariate*0.72,0.63-0.83<0.010.75,0.49-1.150.191.23,0.65-2.330.532.61,1.91-3.57<0.01Atypical Hyperplasia*1.38,1.02-1.870.042.77,1.42-5.42<0.013.89,1.03-14.70.040.43,0.06-3.140.401 FDR** vs. none1.46,1.32-1.63<0.011.16,0.82-1.650.391.93,1.18-3.160.011.11,0.75-1.630.602 FDR** vs. none2.12,1.67-2.71<0.011.17,0.44-3.150.751.90,0.47-7.760.372.81,1.44-5.49<0.01BMI ≥25 vs. <25 kg/m2*1.37,1.25-1.50<0.011.35,1.04-1.750.031.07,0.71-1.640.741.29,0.97-1.730.08Dense vs. non-dense breasts1.55,1.42-1.69<0.011.75,1.34-2.29<0.011.97,1.25-3.10<0.011.65,1.26-2.17<0.01*Adjusted for all factors in the table and additionally age, biopsy, age at menarche, age at first live birth**FDR= first degree relative with breast cancer Citation Format: Anne Marie McCarthy, Tara Friebel, Wei He, Michaela Welch, Sarah Ehsan, Kevin Hughes, Alan Semine, Jinbo Chen, Despina Kontos, Susan Domchek, Emily Conant, Aditya Bardia, Constance Lehman, Katrina Armstrong. The relationship of established breast cancer risk factors with breast cancer subtypes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-02.

BACKGROUND:Identifying women at risk for advanced interval cancers would allow better targeting of mammography and supplemental screening. The authors assessed risk factors for advanced breast cancer within 2 years of a negative mammogram.METHODS:The authors included 293,520 negative mammograms performed from 2006 to 2015 among 74,736 women. Breast cancers were defined as advanced if they were >2 cm, were >1 cm and triple-negative or human epidermal growth factor receptor 2-positive, had positive lymph nodes, or were metastatic. Cox proportional hazards modeling was used to evaluate associations of age, breast density, menopause, mammogram type, prior breast biopsy, body mass index (BMI), and a family history of breast cancer with a cancer diagnosis within 2 years of a negative mammogram. Models were stratified by year since a negative mammogram.RESULTS:Among 1345 breast cancers, 357 were advanced (26.5%), and 988 (73.5%) were at an early stage. Breast density, prior biopsy, and family history were associated with an increased risk of both advanced and early-stage cancers. Overweight and obese women had a 40% higher risk of early-stage cancer only in year 2 (overweight hazard ratio [HR], 1.41; 95% confidence interval [CI], 1.19-1.67; P < .001; obese HR, 1.41; 95% CI, 1.17-1.70; P < .001). Obese women had a 90% increased risk of advanced cancer in year 1 (HR, 1.90; 95% CI, 1.14-3.18; P = .014), and both overweight and obese women had a 40% or greater increased risk in year 2 (overweight HR, 1.55; 95% CI, 1.14-2.07; P = .005; obese HR, 1.42; 95% CI, 1.00-2.01; P = .051).CONCLUSIONS:A higher BMI was associated with an advanced breast cancer diagnosis within 2 years of a negative mammogram. These results have important implications for risk assessment, screening intervals, and use of supplemental screening.

Mesenchymal stem cell derived extracellular vesicles (MSC-EVs) are bioactive particles that evoke beneficial responses in recipient cells. We identified a role for MSC-EV in immune modulation and cellular salvage in a model of SARS-CoV-2 induced acute lung injury (ALI) using pulmonary epithelial cells and exposure to cytokines or the SARS-CoV-2 receptor binding domain (RBD). Whereas RBD or cytokine exposure caused a pro-inflammatory cellular environment and injurious signaling, impairing alveolar-capillary barrier function, and inducing cell death, MSC-EVs reduced inflammation and reestablished target cell health. Importantly, MSC-EV treatment increased active ACE2 surface protein compared to RBD injury, identifying a previously unknown role for MSC-EV treatment in COVID-19 signaling and pathogenesis. The beneficial effect of MSC-EV treatment was confirmed in an LPS-induced rat model of ALI wherein MSC-EVs reduced pro-inflammatory cytokine secretion and respiratory dysfunction associated with disease. MSC-EV administration was dose-responsive, demonstrating a large effective dose range for clinical translation. These data provide direct evidence of an MSC-EV-mediated improvement in ALI and contribute new insights into the therapeutic potential of MSC-EVs in COVID-19 or similar pathologies of respiratory distress.

AbstractBackgroundBreast cancer is a heterogeneous disease, divided into subtypes based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Subtypes have different biology and prognosis, with accumulating evidence of different risk factors. The purpose of this study was to compare breast cancer risk factors across tumor subtypes in a large, diverse mammography population.MethodsWomen aged 40–84 without a history of breast cancer with a screening mammogram at three United States health systems from 2006 to 2015 were included. Risk factor questionnaires were completed at mammogram visit, supplemented by electronic health records. Invasive tumor subtype was defined by immunohistochemistry as ER/PR+HER2−, ER/PR+HER2+, ER, and PR−HER2+, or triple‐negative breast cancer (TNBC). Cox proportional hazards models were run for each subtype. Associations of race, reproductive history, prior breast problems, family history, breast density, and body mass index (BMI) were assessed. The association of tumor subtypes with screen detection and interval cancer was assessed using logistic regression among invasive cases.ResultsThe study population included 198,278 women with a median of 6.5 years of follow‐up (IQR 4.2–9.0 years). There were 4002 invasive cancers, including 3077 (77%) ER/PR+HER2−, 300 (8%) TNBC, 342 (9%) ER/PR+HER2+, and 126 (3%) ER/PR−HER2+ subtype. In multivariate models, Black women had 2.7 times higher risk of TNBC than white women (HR = 2.67, 95% CI 1.99–3.58). Breast density was associated with increased risk of all subtypes. BMI was more strongly associated with ER/PR+HER2− and HER2+ subtypes among postmenopausal women than premenopausal women. Breast density was more strongly associated with ER/PR+HER2− and TNBC among premenopausal than postmenopausal women. TNBC was more likely to be interval cancer than other subtypes.ConclusionsThese results have implications for risk assessment and understanding of the etiology of breast cancer subtypes. More research is needed to determine what factors explain the higher risk of TNBC for Black women.

Background Several breast cancer risk assessment models exist. Few studies have evaluated predictive accuracy of multiple models in large screening populations. Methods We evaluated the performance of the BRCAPRO, Gail, Claus, Breast Cancer Surveillance Consortium (BCSC) and Tyrer-Cuzick models in predicting risk of breast cancer over six years among 35,921 women aged 40-84 who underwent mammography screening at Newton-Wellesley Hospital from 2007-2009. We assessed model discrimination using the area under the receiver operating characteristic curve (AUC) and assessed calibration by comparing the ratio of observed-to-expected (O/E) cases. We calculated the square root of the Brier Score and positive and negative predictive values of each model. Results Our results confirmed good calibration and comparable moderate discrimination of the BRCAPRO, Gail, Tyrer-Cuzick, and BCSC models. The Gail model had slightly better O/E ratio and AUC (O/E=0.98, 95% CI:0.91-1.06, AUC=0.64, 95% CI:0.61-0.65) than BRCAPRO (O/E=0.94, 95% CI:0.88-1.02, AUC=0.61, 95% CI:0.59-0.63) and Tyrer-Cuzick (version 8, O/E=0.84, 95% CI:0.79-0.91, AUC=0.62, 95% CI:0.60-0.64) in the full study population, and the BCSC model had the highest AUC among women with available breast density information (O/E=0.97, 95% CI:0.89-1.05, AUC=0.64, 95% CI:0.62-0.66). All models had poorer predictive accuracy for HER2+ and triple negative breast cancers than hormone receptor positive HER2- breast cancers. Conclusions In a large cohort of patients undergoing mammography screening, existing risk prediction models had similar, moderate predictive accuracy and good calibration overall. Models that incorporate additional genetic and non-genetic risk factors and estimate risk of tumor subtypes may further improve breast cancer risk prediction.

Implementation of gene editing technologies such as CRISPR/Cas9 in the manufacture of novel cell-based therapeutics has the potential to enable highly-targeted, stable, and persistent genome modifications without the use of viral vectors. Electroporation has emerged as a preferred method for delivering gene-editing machinery to target cells, but a major challenge remaining is that most commercial electroporation machines are built for research and process development rather than for large-scale, automated cellular therapy manufacturing. Here we present a microfluidic continuous-flow electrotransfection device designed for precise, consistent, and high-throughput genetic modification of target cells in cellular therapy manufacturing applications. We optimized our device for delivery of mRNA into primary human T cells and demonstrated up to 95

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.

Introduction The relationships among PIK3CA mutations, medication use and tumor progression remains poorly understood. Aspirin use post-diagnosis may modify components of the PI3K pathway, including AKT and mTOR, and has been associated with lower risk of breast cancer recurrence and mortality. We assessed time to metastasis (TTM) and survival with respect to aspirin use and tumor PIK3CA mutations among women with metastatic breast cancer. Methods Patients with hormone receptor positive, HER2 negative (HR+/HER2-) metastatic breast cancer treated in 2009–2016 who received tumor genotyping were included. Aspirin use between primary and metastatic diagnosis was extracted from electronic medical records. TTM and survival were estimated using Cox proportional hazards regression. Results Among 267 women with metastatic breast cancer, women with P IK3CA mutated tumors had longer TTM than women with PIK3CA wildtype tumors (7.1 vs. 4.7 years, p = 0.008). There was a significant interaction between PIK3CA mutations and aspirin use on TTM ( p = 0.006) and survival ( p = 0.026). PIK3CA mutations were associated with longer TTM among aspirin non-users (HR = 0.60 95% CI:0.44–0.82 p = 0.001) but not among aspirin users (HR = 1.57 0.86–2.84 p = 0.139). Similarly, PIK3CA mutations were associated with reduced mortality among aspirin non-users (HR = 0.70 95% CI:0.48–1.02 p = 0.066) but not among aspirin users (HR = 1.75 95% CI:0.88–3.49 p = 0.110). Conclusions Among women who develop metastatic breast cancer, tumor PIK3CA mutations are associated with slower time to progression and mortality only among aspirin non-users. Larger studies are needed to confirm this finding and examine the relationship among aspirin use, tumor mutation profile, and the overall risk of breast cancer progression.