The Enderstruder: An accessible open-source syringe extruder compatible with Ender series 3D printers.

Bioprinting has enabled the precise spatiotemporal deposition of cells and biomaterials, opening new avenues of research in tissue engineering and regenerative medicine. Although several open-source syringe extruder adaptations for bioprinters have been published and adopted by end users, only one has been specifically adapted for the Ender series, an affordable and open-source line of thermoplastic 3D printers. Here, we introduce the Enderstruder, a cost-effective extruder attachment that uses a standard 10 mL BD syringe, positions the stepper motor at the level of the gantry, enhances x-axis stability with a linear rail, and uses the originally included stepper motor, resulting in reduced cost and simplified assembly. Furthermore, we present an iterative process to fine-tune printing profiles for high-viscosity biomaterial inks. To facilitate the implementation of our work by other researchers, we provide fully editable Cura profiles for five commonly used biomaterials. Using these five materials to validate and characterize our design, we employ the Enderstruder to print established calibration patterns and complex shapes. By presenting the Enderstruder and its iterative development process, this study contributes to the growing repository of open-source bioprinting solutions, fostering greater accessibility and affordability for researchers in tissue engineering.

A phase 1/2 clinical trial of the nitric oxide synthase inhibitor L-NMMA and taxane for treating chemoresistant triple-negative breast cancer.

The inducible nitric oxide signaling (iNOS) pathway is associated with poor prognosis in triple-negative breast cancer (TNBC). Prior studies using in vivo models showed that inhibition of the iNOS signaling pathway using the pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) reduced tumor growth and enhanced survival in patients with TNBC. Here, we report a first-in-class phase 1/2 trial of L-NMMA combined with taxane for treating patients with chemorefractory, locally advanced breast cancer (LABC) or metastatic TNBC. We also examined immune cell correlates of chemotherapy response. 35 patients with metastatic TNBC were recruited: 15 in the phase 1 trial and 24 in the phase 2 trial (including 4 recommended phase 2 dose patients from the phase 1 trial). The overall response rate was 45.8% (11 of 24): 81.8% (9 of 11) for patients with LABC and 15.4% (2 of 13) for patients with metastatic TNBC. Among the patients with LABC, three patients had a pathological complete response at surgery (27.3%). Grade ≥3 toxicity was noted in 21% of patients; however, no adverse events were attributed to L-NMMA. Immune cells analyzed by CyTOF indicated that chemotherapy nonresponders showed greater expression of markers associated with M2 macrophage polarization and increased concentrations of circulating IL-6 and IL-10 cytokines. In contrast, chemotherapy responders showed an increase in CD15+ neutrophils in blood, as well as a decrease in arginase (a marker of protumor N2 neutrophils) in tumor biopsies obtained at the end of treatment. L-NMMA combined with taxane warrants further investigation in larger clinical studies of patients with breast cancer.

A randomized, controlled phase II trial of neoadjuvant ado-trastuzumab emtansine, lapatinib, and nab-paclitaxel versus trastuzumab, pertuzumab, and paclitaxel in HER2-positive breast cancer (TEAL study).

BACKGROUND: Neoadjuvant dual human epidermal growth factor receptor (HER2) blockade with trastuzumab and pertuzumab plus paclitaxel leads to an overall pathologic complete response (pCR) rate of 46%. Dual HER2 blockade with ado-trastuzumab emtansine (T-DM1) and lapatinib plus nab-paclitaxel has shown efficacy in patients with metastatic HER2-positive breast cancer. To test neoadjuvant effectiveness of this regimen, an open-label, multicenter, randomized, phase II trial was conducted comparing T-DM1, lapatinib, and nab-paclitaxel with trastuzumab, pertuzumab, and paclitaxel in patients with early-stage HER2-positive breast cancer. METHODS: Stratification by estrogen receptor (ER) status occurred prior to randomization. Patients in the experimental arm received 6 weeks of targeted therapies (T-DM1 and lapatinib) followed by T-DM1 every 3 weeks, lapatinib daily, and nab-paclitaxel weekly for 12 weeks. In the standard arm, patients received 6 weeks of trastuzumab and pertuzumab followed by trastuzumab weekly, pertuzumab every 3 weeks, and paclitaxel weekly for 12 weeks. The primary objective was to evaluate the proportion of patients with residual cancer burden (RCB) 0 or I. Key secondary objectives included pCR rate, safety, and change in tumor size at 6 weeks. Hypothesis-generating correlative assessments were also performed. RESULTS: The 30 evaluable patients were well-balanced in patient and tumor characteristics. The proportion of patients with RCB 0 or I was higher in the experimental arm (100% vs. 62.5% in the standard arm, p = 0.0035). In the ER-positive subset, all patients in the experimental arm achieved RCB 0-I versus 25% in the standard arm (p = 0.0035). Adverse events were similar between the two arms. CONCLUSION: In early-stage HER2-positive breast cancer, the neoadjuvant treatment with T-DM1, lapatinib, and nab-paclitaxel was more effective than the standard treatment, particularly in the ER-positive cohort. TRIAL REGISTRATION: Clinicaltrials.gov NCT02073487 , February 27, 2014.

Identification of Incidental Germline Mutations in Patients With Advanced Solid Tumors Who Underwent Cell-Free Circulating Tumor DNA Sequencing.

PURPOSE: To determine the potential for detection of incidental germline cancer predisposition mutations through cell-free DNA (cfDNA) analyses in patients who underwent solid tumor somatic mutation evaluation. PATIENTS AND METHODS: Data were evaluated from 10,888 unselected patients with advanced (stage III/IV) cancer who underwent Guardant360 testing between November 2015 and December 2016. The main outcome was prevalence of putative germline mutations identified among 16 actionable hereditary cancer predisposition genes. RESULTS: More than 50 cancer types were studied, including lung (41%), breast (19%), colorectal (8%), prostate (6%), pancreatic (3%), and ovarian (2%). Average patient age was 63.5 years (range, 18 to 95 years); 43% were male. One hundred and fifty-six individuals (1.4%) had suspected hereditary cancer mutations in 11 genes. Putative germline mutations were more frequent in individuals younger than 50 years versus those 50 years and older (3.0% v 1.2%, respectively; P < .001). Highest yields of putative germline findings were in patients with ovarian (8.13%), prostate (3.46%), pancreatic (3.34%), and breast (2.2%) cancer. Putative germline mutation identification was consistent among 12 individuals with multiple samples. Patients with circulating tumor DNA copy number variation and/or reversion mutations suggestive of functional loss of the wild-type allele in the tumor DNA also are described. CONCLUSION: Detection of putative germline mutations from cfDNA is feasible across multiple genes and cancer types without prior mutation knowledge. Many mutations were found in cancers without clear guidelines for hereditary cancer genetic counseling/testing. Given the clinical significance of identifying hereditary cancer predisposition for patients and their families as well as targetable germline alterations such as in BRCA1 or BRCA2, research on the best way to validate and return potential germline results from cfDNA analysis to clinicians and patients is needed.

Correlating mammographic and pathologic findings in clinical decision support using natural language processing and data mining methods.

BACKGROUND: A key challenge to mining electronic health records for mammography research is the preponderance of unstructured narrative text, which strikingly limits usable output. The imaging characteristics of breast cancer subtypes have been described previously, but without standardization of parameters for data mining. METHODS: The authors searched the enterprise-wide data warehouse at the Houston Methodist Hospital, the Methodist Environment for Translational Enhancement and Outcomes Research (METEOR), for patients with Breast Imaging Reporting and Data System (BI-RADS) category 5 mammogram readings performed between January 2006 and May 2015 and an available pathology report. The authors developed natural language processing (NLP) software algorithms to automatically extract mammographic and pathologic findings from free text mammogram and pathology reports. The correlation between mammographic imaging features and breast cancer subtype was analyzed using one-way analysis of variance and the Fisher exact test. RESULTS: The NLP algorithm was able to obtain key characteristics for 543 patients who met the inclusion criteria. Patients with estrogen receptor-positive tumors were more likely to have spiculated margins (P = .0008), and those with tumors that overexpressed human epidermal growth factor receptor 2 (HER2) were more likely to have heterogeneous and pleomorphic calcifications (P = .0078 and P = .0002, respectively). CONCLUSIONS: Mammographic imaging characteristics, obtained from an automated text search and the extraction of mammogram reports using NLP techniques, correlated with pathologic breast cancer subtype. The results of the current study validate previously reported trends assessed by manual data collection. Furthermore, NLP provides an automated means with which to scale up data extraction and analysis for clinical decision support. Cancer 2017;114-121. © 2016 American Cancer Society.

Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells.

BACKGROUND: Developing novel strategies against treatment-resistant triple negative breast cancer (TNBC) cells remains a significant challenge. The ErbB family, including epidermal growth factor receptor (EGFR), plays key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSC) which are believed to be responsible for tumor initiation and maintenance. Ixabepilone is a new generation microtubule-stabilizing agent, which has been expected to be more efficacious than conventional taxanes. Here we aim to investigate whether the EGFR monoclonal antibody Cetuximab, in combination with Ixabepilone, is more effective in eliminating CSC populations compared to chemotherapy alone in TNBC. METHODS: Representative TNBC cell lines (MDA-MB-231 and SUM159) were used to evaluate breast CSC populations. We used fluorescence-activated cell sorter analysis (CD44(+) and CD24(-/low), or Aldefluor(+)) and a self-renewal assay called mammosphere formation efficiency (MSFE) to measure CSC population size after treatment with Cetuximab, or Cetuximab plus Ixabepilone in vitro. RESULTS: Although there was no significant decrease in cell viability, Cetuximab reduced MSFE and the CSC population in breast cancer cells in vitro and in vivo through inhibition of autophagy. Also, SUM159 and MDA-MB-231 orthotopic tumors demonstrated partial response to Centuximab or Ixabepilone monotherapy; however, the effect of the combination treatment was significant only in SUM159 tumors (p <0.0001), when compared to Ixabepilone alone. CONCLUSIONS: Overall, our findings demonstrate that EGFR-targeted therapy by Cetuximab effectively reduces the CSC population in TNBC tumors. However, combination therapy with Ixabepilone may be effective only in a small subset of TNBCs, warranting further investigation of alternative approaches to target multiple pathways for TNBC treatment.

Cell-free DNA as a molecular tool for monitoring disease progression and response to therapy in breast cancer patients.

Due to the spatial and temporal genomic heterogeneity of breast cancer, genomic sequencing obtained from a single biopsy may not capture the complete genomic profile of tumors. Thus, we propose that cell-free DNA (cfDNA) in plasma may be an alternate source of genomic information to provide comprehensive data throughout a patient's clinical course. We performed a retrospective chart review of 100 patients with stage 4 or high-risk stage 3 breast cancer. The degree of agreement between genomic alterations found in tumor DNA (tDNA) and cfDNA was determined by Cohen's Kappa. Clinical disease progression was compared to mutant allele frequency using a two-sided Fisher's exact test. The presence of mutations and mutant allele frequency was correlated with progression-free survival (PFS) using a Cox proportional hazards model and a log-rank test. The most commonly found genomic alterations were mutations in TP53 and PIK3CA, and amplification of EGFR and ERBB2. PIK3CA mutation and ERBB2 amplification demonstrated robust agreement between tDNA and cfDNA (Cohen's kappa = 0.64 and 0.77, respectively). TP53 mutation and EGFR amplification demonstrated poor agreement between tDNA and cfDNA (Cohen's kappa = 0.18 and 0.33, respectively). The directional changes of TP53 and PIK3CA mutant allele frequency were closely associated with response to therapy (p = 0.002). The presence of TP53 mutation (p = 0.0004) and PIK3CA mutant allele frequency [p = 0.01, HR 1.074 (95 % CI 1.018-1.134)] was excellent predictors of PFS. Identification of selected cancer-specific genomic alterations from cfDNA may be a noninvasive way to monitor disease progression, predict PFS, and offer targeted therapy.

Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. METHODS: iNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models. RESULTS: High endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor ß was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal. CONCLUSIONS: Considering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.

Chloroquine eliminates cancer stem cells through deregulation of Jak2 and DNMT1.

Triple negative breast cancer (TNBC) is known to contain a high percentage of CD44(+) /CD24(-/low) cancer stem cells (CSCs), corresponding with a poor prognosis despite systemic chemotherapy. Chloroquine (CQ), an antimalarial drug, is a lysotropic reagent which inhibits autophagy. CQ was identified as a potential CSC inhibitor through in silico gene expression signature analysis of the CD44(+) /CD24(-/low) CSC population. Autophagy plays a critical role in adaptation to stress conditions in cancer cells, and is related with drug resistance and CSC maintenance. Thus, the objectives of this study were to examine the potential enhanced efficacy arising from addition of CQ to standard chemotherapy (paclitaxel) in TNBC and to identify the mechanism by which CQ eliminates CSCs in TNBCs. Herein, we report that CQ sensitizes TNBC cells to paclitaxel through inhibition of autophagy and reduces the CD44(+) /CD24(-/low) CSC population in both preclinical and clinical settings. Also, we are the first to report a mechanism by which CQ regulates the CSCs in TNBC through inhibition of the Janus-activated kinase 2 (Jak2)-signal transducer and activator of transcription 3 signaling pathway by reducing the expression of Jak2 and DNA methyltransferase 1.

Dual HER2 blockade: preclinical and clinical data.

The estrogen receptor and human epidermal growth factor receptor (HER) signaling pathways are the dominant drivers of cell proliferation and survival in the majority of human breast cancers. Not surprisingly, targeting these pathways provides the most effective therapies in appropriately selected patients. However, de novo and acquired resistance remain major obstacles to successful treatment. By increasing the understanding of the molecular mechanisms of combined HER2-targeted therapies, we aim to be better able to select patients who would respond to these treatments and understand some of the mechanisms of resistance to HER2-targeted treatments. Recent studies have demonstrated an increased effectiveness of dual targeted HER2 therapies against HER2-amplified breast cancer as compared with single blockade. These studies have resulted in the recent US Food and Drug Administration approval of the combination of taxane chemotherapy with pertuzumab and trastuzumab in the first-line metastatic setting as well as an accelerated approval in the neoadjuvant setting. Another mechanism for overcoming resistance to HER2 targeted therapies is the antibody-drug conjugate trastuzumab-emtansine, which targets the HER2 receptor conjugated to the potent antimicrotubule agent mertansine, allowing for intracellular release of the cytotoxic drug. Studies evaluating the efficacy of dual blockade with antibody-drug conjugate are currently ongoing. This article reviews recent data on different combinations of anti-HER2 treatments as well as ongoing and future research in this area.

Identification of prognosis-relevant subgroups in patients with chemoresistant triple-negative breast cancer.

PURPOSE: Patients with triple-negative breast cancer (TNBC) and residual disease after neoadjuvant chemotherapy generally have worse outcome; however, some patients with residual tumor after neoadjuvant chemotherapy do not relapse. We hypothesize that there are subgroups of patients with chemoresistant TNBC with different prognosis. EXPERIMENTAL DESIGN: Forty-nine chemoresistant cases from 111 patients with TNBC treated with neoadjuvant chemotherapy (M.D. Anderson Cancer Center, Houston, TX) constituted the discovery cohort, and 25 chemoresistant samples from 47 neoadjuvant chemotherapy-treated TNBC (The Methodist Hospital, Houston, TX) were chosen for validation. Extended validation was carried out in 269 operable TNBC predicted to be chemoresistant by expression pattern from published datasets. RESULTS: We established a seven-gene prognostic signature using dChip and gene set enrichment analyses. In the independent validation cohort, the classifier predicted correctly with positive predictive value of 75.0% and negative predictive value (i.e., relapse-free survival; RFS) of 76.9% at 3 years. Those predicted to relapse had a HR of 4.67 [95% confidence interval (CI): 1.27-17.15] for relapse in 3 years. In extended validation, patients predicted not to relapse exhibited 3-year RFS of 78.9%, whereas the 3-year RFS was 48.5% for patients predicted to relapse, with HR of 2.61 (95% CI: 1.52-4.49). The TNBC subgroup that predicted to have relatively favorable prognosis was characterized by high expression of "luminal-like" genes [androgen-receptor (AR) and GATA3], whereas the subgroup with worse prognosis was characterized by expression of cancer stem-cell markers. CONCLUSION: We developed a clinically relevant signature for patients with chemoresistant TNBC. For these women, new therapeutic strategies like targeting AR activation or cancer stem cells may need to be developed.

Multicenter phase II study of neoadjuvant lapatinib and trastuzumab with hormonal therapy and without chemotherapy in patients with human epidermal growth factor receptor 2-overexpressing breast cancer: TBCRC 006.

PURPOSE: We previously reported the eradication of human epidermal growth factor receptor 2 (HER2)- amplified human xenografts in mice by inhibition of the HER2 pathway with lapatinib and trastuzumab to block all homo- and heterodimer signaling as well as by blockade of estrogen receptor (ER) when expressed. In this clinical trial, we sought to translate these findings to patients using targeted therapy without chemotherapy. PATIENTS AND METHOD: Women with stages II to III HER2-positive breast cancers were eligible. They received trastuzumab once per week (4 mg/kg loading, then 2 mg/kg) and lapatinib 1000 mg once per day for 12 weeks. Women with ER-positive tumors also received letrozole (plus a luteinizing hormone-releasing hormone [LHRH] agonist if premenopausal). Pathologic response was assessed by ER status. Biopsies were obtained at baseline, weeks 2 and 8, and time of surgery. RESULTS: Sixty-six patients were enrolled, and 64 were eligible and evaluable for response. Median tumor size was 6 cm (range, 1.5 to 30 cm). Adverse events were mainly grades 1 to 2 (GI, 63%; skin, 46%). Grade 3 metabolic, GI, and liver (18%; 12 patients) and grade 4 liver toxicities (one patient) were also observed. Overall, in-breast pathologic complete response (pCR; ypT0-is) was 27% (ER positive, 21%; ER negative, 36%). The rate of low-volume residual disease (ypT1a-b) was 22% (ER positive, 33%; ER negative, 4%). CONCLUSION: In patients with locally advanced HER2-positive breast cancer, our approach of targeted therapy only resulted in a high pCR rate without chemotherapy. Our data support the hypothesis that selected patients with HER2-positive tumors may not need chemotherapy, and more-complete blockade of HER receptors and ER is an effective strategy worthy of further study.

Prognostic markers for invasive micropapillary carcinoma of the breast: a population-based analysis.

BACKGROUND: Invasive micropapillary carcinoma (IMPC) is a rare and distinct variant of breast carcinoma with a high propensity for regional lymph node involvement. Because of its lymphotropic nature, IMPC is considered to have an unfavorable prognosis when compared with invasive ductal carcinoma (IDC). PATIENTS AND METHODS: This study of 624 patients diagnosed with breast IMPC (2001-2008) listed in the National Cancer Institute's Surveillance, Epidemiology, and End Results (NCI SEER) database was performed to evaluate prognostic factors for disease-specific survival (DSS) and overall survival (OS). RESULTS: The 5-year DSS and OS for patients with IMPC were 91.9% and 83.8%, respectively. Of those with known estrogen receptor (ER) status, 84.8% were ER-positive (ER(+)), which was associated with better DSS (hazard ratio [HR], 0.27; P < .0002) and OS (HR 0.45; P < .006). At presentation, 52.9% of the patients with lymph node examinations had nodal involvement and 4.1% had distant metastases. Patients with 4 or more positive lymph nodes had worse DSS (HR 6.43; P = .0013) and OS (HR 3.47; P = .00067) than did patients with node-negative disease, but those with 1 to 3 positive lymph nodes had DSS and OS similar to those of patients with node-negative disease. CONCLUSION: Although IMPC has a high propensity for lymph node metastasis, it has a DSS and overall prognosis comparable to those of IDC. Patients with ER-negative (ER-) disease or those with 4 or more positive lymph nodes have the worst prognosis. This is the largest study of IMPC to date, and these findings will help address some of the inconsistencies regarding this rare histologic variant of breast cancer.

Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells.

BACKGROUND: Since the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, transplantation of these cells into the mammary fat pad yields aggressive tumors that self-renew as evidenced by serial xenograft passages through transplantation. Herein we fully characterize cancer stem cell-like features in 293T human embryonic kidney cells. RESULTS: 293T cells can be readily cultured and passaged as spheres in serum-free stem cell promoting culture conditions. Cells cultured in vitro as three-dimensional spheres (3D) were shown to contain higher ALDH1 and CD44+/CD24- population compared to monolayer cells. These cells were also resistant to radiation and upregulate stem cell survival signaling including beta-catenin, Notch1 and Survivin in response to radiation. Moreover, 3D spheres generated from the 293T cells have increased expression of mesenchymal genes including vimentin, n-cadherin, zeb1, snail and slug as well as pro-metastatic genes RhoC, Tenascin C and MTA1. In addition, microRNAs implicated in self-renewal and metastases were markedly reduced in 3D spheres. CONCLUSIONS: 293T cells exhibit a cancer stem cell-like phenotype when cultured as 3D spheres and represent an important research tool for studying the molecular and biological mechanisms of cancer stem cells and for testing and developing novel targets for cancer therapy.