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Abstract Background The five-year prognosis for patients with late-stage high-grade serous carcinoma (HGSC) remains dismal, underscoring the critical need for identifying early-stage biomarkers. This study explores the potential of extracellular vesicles (EVs) circulating in blood, which are believed to harbor proteomic cargo reflective of the HGSC microenvironment, as a source for biomarker discovery. Results We conducted a comprehensive proteomic profiling of EVs isolated from blood plasma, ascites, and cell lines of patients, employing both data-dependent (DDA) and data-independent acquisition (DIA) methods to construct a spectral library tailored for targeted proteomics. Our investigation aimed at uncovering novel biomarkers for the early detection of HGSC by comparing the proteomic signatures of EVs from women with HGSC to those with benign gynecological conditions. The initial cohort, comprising 19 donors, utilized DDA proteomics for spectral library development. The subsequent cohort, involving 30 HGSC patients and 30 control subjects, employed DIA proteomics for a similar purpose. Support vector machine (SVM) classification was applied in both cohorts to identify combinatorial biomarkers with high specificity and sensitivity (ROC-AUC > 0.90). Notably, MUC1 emerged as a significant biomarker in both cohorts when used in combination with additional biomarkers. Validation through an ELISA assay on a subset of benign (n = 18), Stage I (n = 9), and stage II (n = 9) plasma samples corroborated the diagnostic utility of MUC1 in the early-stage detection of HGSC. Conclusions This study highlights the value of EV-based proteomic analysis in the discovery of combinatorial biomarkers for early ovarian cancer detection.

Abstract The current prostate cancer (PCa) screen test, prostate-specific antigen (PSA), has a high sensitivity for PCa but low specificity for high-risk, clinically significant PCa (csPCa), resulting in overdiagnosis and overtreatment of non-csPCa. Early identification of csPCa while avoiding unnecessary biopsies in men with non-csPCa is challenging. We built an optimized machine learning platform (ClarityDX) and showed its utility in generating models predicting csPCa. Integrating the ClarityDX platform with blood-based biomarkers for clinically significant PCa and clinical biomarker data from a 3448-patient cohort, we developed a test to stratify patients’ risk of csPCa; called ClarityDX Prostate. When predicting high risk cancer in the validation cohort, ClarityDX Prostate showed 95% sensitivity, 35% specificity, 54% positive predictive value, and 91% negative predictive value, at a ≥ 25% threshold. Using ClarityDX Prostate at this threshold could avoid up to 35% of unnecessary prostate biopsies. ClarityDX Prostate showed higher accuracy for predicting the risk of csPCa than PSA alone and the tested model-based risk calculators. Using this test as a reflex test in men with elevated PSA levels may help patients and their healthcare providers decide if a prostate biopsy is necessary.

Abstract Background It is well-established that parental obesity is a strong risk factor for offspring obesity. Further, a converging body of evidence now suggests that maternal weight profiles may affect the developing offspring’s brain in a manner that confers future obesity risk. Here, we investigated how pre-pregnancy maternal weight status influences the reward-related striatal areas of the offspring’s brain during in utero development. Methods We used diffusion tensor imaging to quantify the microstructure of the striatal brain regions of interest in neonates (N = 116 [66 males, 50 females], mean gestational weeks at birth [39.88], SD = 1.14; at scan [43.56], SD = 1.05). Linear regression was used to test the associations between maternal pre-pregnancy body mass index (BMI) and infant striatal mean diffusivity. Results High maternal pre-pregnancy BMI was associated with higher mean MD values in the infant’s left caudate nucleus. Results remained unchanged after the adjustment for covariates. Conclusions In utero exposure to maternal adiposity might have a growth-impairing impact on the mean diffusivity of the infant’s left caudate nucleus. Considering the involvement of the caudate nucleus in regulating eating behavior and food-related reward processing later in life, this finding calls for further investigations to define the prognostic relevance of early-life caudate nucleus development and weight trajectories of the offspring.

Abstract Background There is an unmet clinical need for minimally invasive diagnostic tests to improve the detection of grade group (GG) ≥3 prostate cancer relative to prostate antigen‐specific risk calculators. We determined the accuracy of the blood‐based extracellular vesicle (EV) biomarker assay (EV Fingerprint test) at the point of a prostate biopsy decision to predict GG ≥3 from GG ≤2 and avoid unnecessary biopsies. Methods This study analyzed 415 men referred to urology clinics and scheduled for a prostate biopsy, were recruited to the APCaRI 01 prospective cohort study. The EV machine learning analysis platform was used to generate predictive EV models from microflow data. Logistic regression was then used to analyze the combined EV models and patient clinical data and generate the patients' risk score for GG ≥3 prostate cancer. Results The EV‐Fingerprint test was evaluated using the area under the curve (AUC) in discrimination of GG ≥3 from GG ≤2 and benign disease on initial biopsy. EV‐Fingerprint identified GG ≥3 cancer patients with high accuracy (0.81 AUC) at 95% sensitivity and 97% negative predictive value. Using a 7.85% probability cutoff, 95% of men with GG ≥3 would have been recommended a biopsy while avoiding 144 unnecessary biopsies (35%) and missing four GG ≥3 cancers (5%). Conversely, a 5% cutoff would have avoided 31 unnecessary biopsies (7%), missing no GG ≥3 cancers (0%). Conclusions EV‐Fingerprint accurately predicted GG ≥3 prostate cancer and would have significantly reduced unnecessary prostate biopsies.

Extracellular vesicles (EVs) are highly abundant in human biofluids, containing a repertoire of macromolecules and biomarkers representative of the tissue of origin. EVs released by tumours can communicate key signals both locally and to distant sites to promote growth and survival or impact invasive and metastatic progression. Microscale flow cytometry of circulating EVs is an emerging technology that is a promising alternative to biopsy for disease diagnosis. However, biofluid‐derived EVs are highly heterogeneous in size and composition, making their analysis complex. To address this, we developed a machine learning approach combined with EV microscale cytometry using tissue‐ and disease‐specific biomarkers to generate predictive models. We demonstrate the utility of this novel extracellular vesicle machine learning analysis platform (EVMAP) to predict disease from patient samples by developing a blood test to identify high‐grade prostate cancer and validate its performance in a prospective 215 patient cohort. Models generated using the EVMAP approach significantly improved the prediction of high‐risk prostate cancer, highlighting the clinical utility of this diagnostic platform for improved cancer prediction from a blood test.

Traditional external light-based Photodynamic Therapy (PDT)’s application is limited to the surface and minimal thickness tumors because of the inefficiency of light in penetrating deep-seated tumors. To address this, the emerging field of radiation-activated PDT (radioPDT) uses X-rays to trigger photosensitizer-containing nanoparticles (NPs). A key consideration in radioPDT is the energy transfer efficiency from X-rays to the photosensitizer for ultimately generating the phototoxic reactive oxygen species (ROS). In this study, we developed a new variant of pegylated poly-lactic-co-glycolic (PEG-PLGA) encapsulated nanoscintillators (NSCs) along with a new, highly efficient ruthenium-based photosensitizer (Ru/radioPDT). Characterization of this NP via transmission electron microscopy, dynamic light scattering, UV-Vis spectroscopy, and inductively coupled plasma mass-spectroscopy showed an NP size of 120 nm, polydispersity index (PDI) of less than 0.25, high NSCs loading efficiency over 90% and in vitro accumulation within the cytosolic structure of endoplasmic reticulum and lysosome. The therapeutic efficacy of Ru/radioPDT was determined using PC3 cell viability and clonogenic assays. Ru/radioPDT exhibited minimal cell toxicity until activated by radiation to induce significant cancer cell kill over radiation alone. Compared to protoporphyrin IX-mediated radioPDT (PPIX/radioPDT), Ru/radioPDT showed higher capacity for singlet oxygen generation, maintaining a comparable cytotoxic effect on PC3 cells.

Cell migration regulates diverse (patho)physiological processes, including cancer metastasis. Here the authors show that the chloride ion channel SWELL1 and the ion exchanger NHE1 are preferentially enriched at the trailing and leading edges, respectively, of migrating cells and regulate cell volume to propel confined cells, favouring breast cancer cell extravasation and metastasis.

Previous literature links maternal pregnancy-specific anxiety (PSA) with later difficulties in child emotional and social cognition as well as memory, functions closely related to the amygdala and the hippocampus. Some evidence also suggests that PSA affects child amygdalar volumes in a sex-dependent way. However, no studies investigating the associations between PSA and newborn amygdalar and hippocampal volumes have been reported. We investigated the associations between PSA and newborn amygdalar and hippocampal volumes and whether associations are sex-specific in 122 healthy newborns (68 males/54 females) scanned at 2–5 weeks postpartum. PSA was measured at gestational week 24 with the Pregnancy-Related Anxiety Questionnaire Revised 2 (PRAQ-R2). The associations were analyzed with linear regression controlling for confounding variables. PSA was associated positively with left amygdalar volume in girls, but no significant main effect was found in the whole group or in boys. No significant main or sex-specific effect was found for hippocampal volumes. Although this was an exploratory study, the findings suggest a sexually dimorphic association of mid-pregnancy PSA with newborn amygdalar volumes.

Metastasis is the primary cause of cancer patient death and the elevation of SLC2A5 gene expression is often observed in metastatic cancer cells. Here we evaluated the importance of SLC2A5 in cancer cell motility by silencing its gene. We discovered that CRISPR/Cas9-mediated inactivation of the SLC2A5 gene inhibited cancer cell proliferation and migration in vitro as well as metastases in vivo in several animal models. Moreover, SLC2A5-attenuated cancer cells exhibited dramatic alterations in mitochondrial architecture and localization, uncovering the importance of SLC2A5 in directing mitochondrial function for cancer cell motility and migration. The direct association of increased abundance of SLC2A5 in cancer cells with metastatic risk in several types of cancers identifies SLC2A5 as an important therapeutic target to reduce or prevent cancer metastasis.

Thymic stromal lymphopoietin (TSLP) is an epithelium-derived pro-inflammatory cytokine involved in lung inflammatory responses. Previous studies show conflicting observations in blood TSLP in COVID-19, while none report SARS-CoV-2 inducing TSLP expression in bronchial epithelial cells. Our objective in this study was to determine whether TSLP levels increase in COVID-19 patients and if SARS-CoV-2 induces TSLP expression in bronchial epithelial cells. Plasma cytokine levels were measured in patients hospitalized with confirmed COVID-19 and age- and sex-matched healthy controls. Demographic and clinical information from COVID-19 patients was collected. We determined associations between plasma TSLP and clinical parameters using Poisson regression. Cultured human nasal (HNEpC) and bronchial epithelial cells (NHBEs), Caco-2 cells, and patient-derived bronchial epithelial cells (HBECs) obtained from elective bronchoscopy were infected in vitro with SARS-CoV-2, and secretion as well as intracellular expression of TSLP was detected by immunofluorescence. Increased TSLP levels were detected in the plasma of hospitalized COVID-19 patients (603.4 ± 75.4 vs 997.6 ± 241.4 fg/mL, mean ± SEM), the levels of which correlated with duration of stay in hospital (β: 0.11; 95% confidence interval (CI): 0.01–0.21). In cultured NHBE and HBECs but not HNEpCs or Caco-2 cells, TSLP levels were significantly elevated after 24 h post-infection with SARS-CoV-2 (p < 0.001) in a dose-dependent manner. Plasma TSLP in COVID-19 patients significantly correlated with duration of hospitalization, while SARS-CoV-2 induced TSLP secretion from bronchial epithelial cells in vitro. Based on our findings, TSLP may be considered an important therapeutic target for COVID-19 treatment.

Background: Serine-arginine protein kinase 1 (SRPK1) has been implicated in prostate cancer (PCa) progression. However, its prognostic value and association with ERG and PTEN expression, two of the most common genetic alterations, have not been explored fully. Objective: We assessed the prognostic value of SRPK1 in association with ERG and PTEN in a cohort of patients managed nonsurgically by androgen deprivation therapy (ADT) for advanced disease. Design, setting, and participants: The study cohort consisted of men diagnosed with PCa by transurethral resection of the prostate (TURP; n = 480). The patients were divided into three main groups: incidental (patients with Gleason score [GS] ≤7 with no prior ADT), advanced (patients with GS ≥8 with no prior ADT), and castrate-resistant PCa (patients with prior ADT). Outcome measurements and statistical analysis: A total of 480 TURP samples were assessed by immunohistochemistry for SRPK1, ERG, and PTEN, and results were correlated with Gleason grade group (GG), overall survival (OS), and PCa-specific mortality (PCSM). Results and limitations: High SRPK1 expression was noted in 105/455 (23%) available patient cores. Expression of SRPK1 was associated with Gleason grade grouping (p

Extracellular vesicles (EVs) are secreted vesicles of diverse size and cargo that are implicated in the cell-to-cell transmission of disease-causing-proteins in several neurodegenerative diseases. Mutant huntingtin, the disease-causing entity in Huntington’s disease, has an expanded polyglutamine track at the N terminus that causes the protein to misfold and form toxic intracellular aggregates. In Huntington’s disease, mutant huntingtin aggregates are transferred between cells by several routes. We have previously identified a cellular pathway that is responsible for the export of mutant huntingtin via extracellular vesicles. Identifying the EV sub-populations that carry misfolded huntingtin cargo is critical to understanding disease progression. In this work we expressed a form of polyglutamine expanded huntingtin (GFP-tagged 72Qhuntingtinexon1) in cells to assess the EVs involved in cellular export. We demonstrate that the molecular chaperone, cysteine string protein (CSPα; DnaJC5), facilitates export of disease-causing-polyglutamine-expanded huntingtin cargo in 180–240 nm vesicles as well as larger 10–30 μm vesicles.

After 5 months of age, infants begin to prioritize attention to fearful over other facial expressions. One key proposition is that amygdala and related early-maturing subcortical network, is important for emergence of this attentional bias – however, empirical data to support these assertions are lacking. In this prospective longitudinal study, we measured amygdala volumes from MR images in 65 healthy neonates at 2–5 weeks of gestation corrected age and attention disengagement from fearful vs. non-fearful facial expressions at 8 months with eye tracking. Overall, infants were less likely to disengage from fearful than happy/neutral faces, demonstrating an age-typical bias for fear. Left, but not right, amygdala volume (corrected for intracranial volume) was positively associated with the likelihood of disengaging attention from fearful faces to a salient lateral distractor (r = .302, p = .014). No association was observed with the disengagement from neutral or happy faces in equivalent conditions (r = .166 and .125, p = .186 and .320, respectively). These results are the first to link the amygdala volume with the emerging perceptual vigilance for fearful faces during infancy. They suggest a link from the prenatally defined variability in the amygdala size to early postnatal emotional and social traits.

Background: Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling. Methods: In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo. Findings: CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ). Interpretation: CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling. Funding: Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research. Keywords: Breast cancer metastasis, Gamma subunits, L-type channels, Voltage-gated calcium channels (VGCCs)

Maternal psychological distress during pregnancy (PPD)11 prenatal psychosocial distress has been associated with changes in offspring amygdalar and hippocampal volumes. Studies on child amygdalae suggest that sex moderates the vulnerability of fetal brains to prenatal stress. However, this has not yet been observed in these structures in newborns. Newborn studies are crucial, as they minimize the confounding influence of postnatal life.We investigated the effects of maternal prenatal psychological symptoms on newborn amygdalar and hippocampal volumes and their interactions with newborn sex in 123 newborns aged 2–5 weeks (69 males, 54 females). Based on earlier studies, we anticipated small, but statistically significant effects of PPD on the volumes of these structures. Maternal psychological distress was measured at gestational weeks (GW)22 gestational weeks 14, 24 and 34 using Symptom Checklist-90 (SCL-90, anxiety scale)33 Symptom Checklist-90 and Edinburgh Postnatal Depression Scale (EPDS)44 Edinburgh Postnatal Depression Scale questionnaires.Newborn sex was found to moderate the relationship between maternal distress symptoms at GW 24 and the volumes of left and right amygdala. This relationship was negative and significant only in males. No significant main effect or sex-based moderation was found for hippocampal volumes.This newborn study provides evidence for a sex-dependent influence of maternal psychiatric symptoms on amygdalar structural development. This association may be relevant to later psychopathology.

Cancer cell motility is a key driver of metastasis. Although the intravasation of cancer cells into the blood stream is highly dependent on their motility and metastatic dissemination is the primary cause of cancer related deaths, current therapeutic strategies do not target the genes and proteins that are essential for cell motility. A primary reason for this is because the identification of cell motility-related genes that are relevant in vivo requires the visualization of metastatic lesions forming in an appropriate in vivo model. The cancer research community has lacked an in vivo and intravital metastatic cancer model that could be imaged as motility developed, in real-time. To address this, we developed a novel quantitative in vivo screening platform based on intravital imaging in shell-less ex ovo chick embryos. We applied this imaging approach to screen a human genome-wide short hairpin RNA library (shRNA) versus the highly motile head and neck cancer cells (HEp3 cell line) introduced into the chorioallantoic membrane (CAM) of chick embryos and identified multiple novel in vivo cancer cell motility-associated genes. When the expression of several of the identified genes was inhibited in the HEp3 tumors, we observed a nearly total block of spontaneous cancer metastasis.

Tumour metastasis is dependent on tumour cell motility. Here, the authors investigate genes required for tumour cell motility by establishing a quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos.

Prostate cancer is a leading cause of cancer-associated deaths in men over 60 years of age. Most patients are killed by tumor metastasis. Recent evidence has implicated a role of the tumor microenvironment and urokinase plasminogen activator (uPA) in cancer cell migration, invasion, and metastasis. Here, we examine the role of the Na+/H+ exchanger isoform 1 (NHE1) and uPA in DU 145 prostate cancer cell migration and colony formation. Knockout of NHE1 reduced cell migration. The effects of a series of novel NHE1/uPA hexamethylene-amiloride-based inhibitors with varying efficacy towards NHE1 and uPA were examined on prostate cancer cells. Inhibition of NHE1—alone, or with inhibitors combining NHE1 or uPA inhibition—generally did not prevent prostate cancer cell migration. However, uPA inhibition—but not NHE1 inhibition—prevented anchorage-dependent colony formation. Application of inhibitors at concentrations that only saturate uPA inhibition decreased tumor invasion in vivo. The results suggest that while knockout of NHE1 affects cell migration, these effects are not due to NHE1-dependent proton translocation. Additionally, while neither NHE1 nor uPA activity was critical in cell migration, only uPA activity appeared to be critical in anchorage-dependent colony formation of DU 145 prostate cancer cells and invasion in vivo.

Lipoma preferred partner (LPP) mediates TGFβ-induced breast cancer cell migration and invasion. Here the authors show that LPP is a Src-family kinase substrate that regulates the formation of protrusions of the plasma membrane -called invadopodia- required for breast cancer metastasis.

The gross anatomy of the infant brain at term is fairly similar to that of the adult brain, but structures are immature, and the brain undergoes rapid growth during the first 2 years of life. Neonate magnetic resonance (MR) images have different contrasts compared to adult images, and automated segmentation of brain magnetic resonance imaging (MRI) can thus be considered challenging as less software options are available. Despite this, most anatomical regions are identifiable and thus amenable to manual segmentation. In the current study, we developed a protocol for segmenting the amygdala and hippocampus in T2-weighted neonatal MR images. The participants were 31 healthy infants between 2 and 5 weeks of age. Intra-rater reliability was measured in 12 randomly selected MR images, where 6 MR images were segmented at 1-month intervals between the delineations, and another 6 MR images at 6-month intervals. The protocol was also tested by two independent raters in 20 randomly selected T2-weighted images, and finally with T1 images. Intraclass correlation coefficient (ICC) and Dice similarity coefficient (DSC) for intra-rater, inter-rater, and T1 vs. T2 comparisons were computed. Moreover, manual segmentations were compared to automated segmentations performed by iBEAT toolbox in 10 T2-weighted MR images. The intra-rater reliability was high ICC ≥ 0.91, DSC ≥ 0.89, the inter-rater reliabilities were satisfactory ICC ≥ 0.90, DSC ≥ 0.75 for hippocampus and DSC ≥ 0.52 for amygdalae. Segmentations for T1 vs. T2-weighted images showed high consistency ICC ≥ 0.90, DSC ≥ 0.74. The manual and iBEAT segmentations showed no agreement, DSC ≥ 0.39. In conclusion, there is a clear need to improve and develop the procedures for automated segmentation of infant brain MR images.

Microtubules are hollow cylindrical polymers composed of the highly negatively-charged (~23e), high dipole moment (1750 D) protein α, β- tubulin. While the roles of microtubules in chromosomal segregation, macromolecular transport, and cell migration are relatively well-understood, studies on the electrical properties of microtubules have only recently gained strong interest. Here, we show that while microtubules at physiological concentrations increase solution capacitance, free tubulin has no appreciable effect. Further, we observed a decrease in electrical resistance of solution, with charge transport peaking between 20−60 Hz in the presence of microtubules, consistent with recent findings that microtubules exhibit electric oscillations at such low frequencies. We were able to quantify the capacitance and resistance of the microtubules (MT) network at physiological tubulin concentrations to be 1.27 × 10−5 F and 9.74 × 104 Ω. Our results show that in addition to macromolecular transport, microtubules also act as charge storage devices through counterionic condensation across a broad frequency spectrum. We conclude with a hypothesis of an electrically tunable cytoskeleton where the dielectric properties of tubulin are polymerisation-state dependent.

A small corpus callosum (CC) is one of the most replicated neurobiological findings in autism spectrum (AS). However, its effect on interhemispheric (IH) communication is unknown. We combined structural (CC area and DWI), functional (task-related fMRI activation and connectivity analyses) as well as behavioral (Poffenberger and Purdue tasks) measures to investigate IH integration in adult AS individuals of typical intelligence. Despite similar behavioral IH transfer time and performances in bimanual tasks, the CC sub-regions connecting frontal and parietal cortical areas were smaller in AS than in non-AS individuals, while those connecting visual regions were similar. The activation of visual areas was lower in AS than in non-AS individuals during the presentation of visual stimuli. Behavioral IH performances were related to the properties of CC subregions connecting motor areas in non-AS individuals, but to the properties of posterior CC regions in AS individuals. Furthermore, there was greater functional connectivity between visual areas in the AS than in the non-AS group. Levels of connectivity were also stronger in visual than in motor regions in the autistic subjects, while the opposite was true for the non-autistic group. Thus, visual IH transfer plays an important role in visuo-motor tasks in AS individuals. These findings extend the well established enhanced role of perception in autistic cognition to visuo-motor IH information transfer.

Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation in vivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively to invadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process.

Previous studies indicate that breast cancer cells with high aldehyde dehydrogenase (ALDH) activity and CD44 expression (ALDHhiCD44+) contribute to metastasis and therapy resistance, and that ALDH1 correlates with poor outcome in breast cancer patients. The current study hypothesized that ALDH1 functionally contributes to breast cancer metastatic behavior and therapy resistance. Expression of ALDH1A1 or ALDH1A3 was knocked down in MDA-MB-468 and SUM159 human breast cancer cells using siRNA. Resulting impacts on ALDH activity (Aldefluor® assay); metastatic behavior and therapy response in vitro (proliferation/adhesion/migration/colony formation/chemotherapy and radiation) and extravasation/metastasis in vivo (chick choroiallantoic membrane assay) was assessed. Knockdown of ALDH1A3 but not ALDH1A1 in breast cancer cells decreased ALDH activity, and knockdown of ALDH1A1 reduced breast cancer cell metastatic behavior and therapy resistance relative to control (p < 0.05). In contrast, knockdown of ALDH1A3 did not alter proliferation, extravasation, or therapy resistance, but increased adhesion/migration and decreased colony formation/metastasis relative to control (p < 0.05). This is the first study to systematically examine the function of ALDH1 isozymes in individual breast cancer cell behaviors that contribute to metastasis. Our novel results indicate that ALDH1 mediates breast cancer metastatic behavior and therapy resistance, and that different enzyme isoforms within the ALDH1 family differentially impact these cell behaviors.