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BACKGROUND Significant disparities in surgical outcomes exist. It is imperative to prepare future doctors to eliminate disparities. Our team of senior medical students developed a surgical clerkship module examining equity in prostate cancer. Student attitudes before and after a facilitated teaching session were assessed. METHODS A surgical equity pilot module was integrated into the core surgical clerkship starting in July 2020. This module was composed of (1) asynchronous preparatory material and (2) a synchronous interactive case discussion regarding disparities in prostate cancer. Discussion sessions were facilitated by upper-level medical students. Participants answered optional anonymous Likert-style and open-ended survey questions before and after the session. Pre- and post-responses were compared. RESULTS One hundred and sixteen students completed the module between July 2020 and January 2021. Pre- and post-survey response rates were 66% and 29%, respectively. At baseline, almost all students (95%) agreed knowledge of disparities would make them a better physician. However, the majority (95%) described their general knowledge of surgical disparities as "nonexistent," "poor," or "average." Most students did not have a framework for assessing causes of surgical disparities (86%) and were not aware of interventions for reducing disparities (90%). After intervention, the majority rated their knowledge of surgical disparities as "good" or "excellent" (71%; P < .001). Most students indicated they had a framework 79%; P < .001) and were aware of effective interventions (62%; P < .001). CONCLUSION We demonstrated a successful pilot of an equity-focused clerkship module. Student attitudes after a single session reflected significant improvement in knowledge of causes and interventions related to surgical disparities. Equity-focused teaching can be incorporated into the surgical clerkship.

Supplementary Figure S1. CONSORT diagram. Supplementary Figure S2. Histologic analysis of the pre-rociletinib biopsy from patient 12 (H&E 400x, left panel) demonstrates characteristic adenocarcinoma with an open chromatin pattern, ample cytoplasm and variable cell density. Supplementary Figure S3. Panel A. The nucleotide sequences of single-cell cloning analysis for six additional clones isolated from the afatinib-resistant cell line MGH176 are shown (see also figure 2B). Panel B. Summary of single-clone analyses performed on two additional patient-derived cell lines. Supplementary Figure S4. Longitudinal quantitative analyses of EGFR mutations in the ctDNA are shown for patients 8 (panel A), 6 (panel B) and 3 (panel C). Supplementary Figure S5. Scatterplot showing the relationship between the allelic fraction of T790M (calculated as the percent of T790M alleles/percent of EGFR activating mutation alleles as quantified by allele-specific PCR or NGS) in the pre-rociletinib tumor biopsy (y-axis) and in the pre-rociletinib plasma (as quantified by BEAMing) (x-axis).

Supplementary Figure Legends. Supplementary Table S1. Patient characteristics. Supplementary Table S2. Tissue genotyping analyses. Supplementary Table S3. Allelic Fraction of T790M and maximum reduction in tumor volume in response to rociletinib.

Top 10 correlated genes to BIM (BCL2L11)

Supplementary Figure 1 Legend. Describing the qPCR assay and variant allele frequency data that are included in the figure.

Supplemental legend

Purpose: A secondary EGFR mutation, T790M, is the most common resistance mechanism in EGFR-mutant adenocarcinomas that have progressed on erlotinib. Third-generation EGFR inhibitors capable of inhibiting mutant EGFR with T790M produce responses in nearly two thirds of patients. However, acquired resistance mechanisms in patients treated with these drugs are yet to be described.Experimental Design: To study acquired resistance to third-generation EGFR inhibitors, T790M-positive cells derived from an erlotinib-resistant cancer were made resistant to a third-generation TKI and then characterized using cell and molecular analyses.Results: Cells resistant to a third-generation TKI acquired an additional EGFR mutation, C797S, which prevented suppression of EGFR. Our results demonstrate that the allelic context in which C797S was acquired may predict responsiveness to alternative treatments. If the C797S and T790M mutations are in trans, cells will be resistant to third-generation EGFR TKIs, but will be sensitive to a combination of first- and third-generation TKIs. If the mutations are in cis, no EGFR TKIs alone or in combination can suppress activity. If C797S develops in cells wild-type for T790 (when third-generation TKIs are administered in the first-line setting), the cells are resistant to third-generation TKIs, but retain sensitivity to first-generation TKIs.Conclusions: Mutation of C797S in EGFR is a novel mechanism of acquired resistance to third-generation TKIs. The context in which the C797S develops with respect to the other EGFR alleles affects the efficacy of subsequent treatments. Clin Cancer Res; 21(17); 3924–33. ©2015 AACR.See related commentary by Ayeni et al., p. 3818

ZEB1 binding sites found in 1975R2 cells compared to 1975 parental cells

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it.Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance.Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes.Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Supplementary Figure 1. EGFR amplification status and variant allele frequencies of T790M and C797S mutations for MGH121 parental and MGH121 resistant #1 cells.

BACKGROUND Study groups are multicenter collaborations aimed at improving orthopaedic decision-making through higher-powered, more generalizable studies. New research is disseminated through peer-reviewed literature and academic meetings, including the Pediatric Orthopaedic Society of North America (POSNA) annual meeting, which brings together academic and medical professionals in pediatric orthopaedics. The goal of this study was to identify patterns in podium presentations (PP) at the POSNA annual meeting resulting from multicenter study groups during a 15-year period. METHODS A total of 2065 PP from the 2006 to 2020 POSNA annual meetings were identified. The abstracts of each PP were reviewed to determine if they resulted from a multicenter study group and for characteristics including subspecialty focus. PP from 2006 to 2018 were further reviewed for publication in academic journals. Pearson correlation was used to assess change in the number of PP resulting from study groups overtime. Univariate analysis was used to compare characteristics of PP based on study group involvement (significance P

Geriatric trauma patients who require an unplanned ICU admission (UIA) may experience worse outcomes. As such, the American College of Surgeons initiated the Trauma Quality Improvement Program which tracks UIA as a quality benchmark. We sought to determine the overall rate and impact of UIA in our geriatric trauma population and to identify predictive risk factors.All geriatric trauma patients (≥65) admitted to an urban, level I trauma center from January 2012 to June 2018 were identified. A retrospectively collected administrative database was queried for demographics, comorbidities, injury characteristics, and outcomes. UIA were identified and medical records were queried. Univariate analysis followed by binary logistic regression analysis were performed (P 0.05 = significant).Of the 2923 geriatric patients identified, 95 (3.3%) patients experienced UIA, most commonly secondary to respiratory (34.7%) and cardiac (22.1%) events. Patients with UIA were older (81 versus 78, P = 0.04), and had higher injury severity score (10 versus 9, P 0.01) and Charlson comorbidity indices (5 versus 4, P = 0.02). On logistic regression, age (OR 1.027, P = 0.04) and injury severity score (OR 1.032, P 0.01) were predictive of unplanned ICU admission. Of the UIA, 69.4% were readmissions, or "bounce backs". Patients initially admitted to the ICU had 2.5 increased odds of requiring UIA. Patients with UIA experienced longer hospital stays (15 versus 5, P 0.01), more days in the ICU (6 versus 1, P 0.01), and higher rates of mortality (11.6% versus 5.0%, P = 0.02).Despite relatively low injury severity, geriatric trauma patients requiring UIA have a significant increase in morbidity and mortality. Those initially admitted to the ICU are at especially high risk for UIA, suggesting the benefit of strategies to provide an extra layer of care post-ICU.

BACKGROUND Women are underrepresented across most surgical specialties and especially in the field of orthopaedic surgery. Despite pediatric orthopaedic surgery being one of the most gender diverse subspecialties in orthopaedics, women may still face barriers to academic advancement. Research presentations at national meetings and publication record are important drivers of advancement in the field of academic orthopaedic surgery. The aim of this study is to assess whether research abstracts authored by women are less likely to be published than abstracts authored by men. METHODS Abstracts from research podium presentations given at the Pediatric Orthopaedic Society of North America annual meetings from 2006 to 2020 were reviewed to assess research quality and determine basic study characteristics. For each abstract, author gender was determined through a search of institutional websites and professional networking websites for gender-specific pronouns. Resulting publications corresponding to the 2006 to 2018 were identified using a systematic search of PubMed and Google Scholar databases. Kaplan-Meier inverse survival analysis with log rank test were used to determine differences in publication rates based on whether the last (senior) author was female versus male. Multivariate, binary logistic regression was performed to assess factors predictive of eventual publication. RESULTS One thousand five hundred and eighty-one of 1626 (97.2%) of abstracts from 2006 to 2020 had an identifiable last author gender, with 17.8% (281/1581) female. No differences in study quality were identified across genders including sample size, level of evidence, or impact factor of journal if leading to publication. Women were more likely to author abstracts in foot, ankle, or lower extremity surgery [17.8% (50/281) vs. 12.9% (168/1300), P=0.032] and less likely to author abstracts focusing on the hip [11.0% (31/281) vs. 17.1% (222/1300), P=0.012]. Abstracts with women as the last author were significantly less likely to be published compared with abstracts with men as the last author [59.6% (143/240) vs. 67.9% (783/1154), P=0.013]. Multivariate analysis demonstrated that last author female gender was predictive of a lower likelihood of publication (odds ratio: 0.684, 95% confidence interval: 0.513-0.912, P=0.010). Women were less likely to be the last author of abstracts presented by study groups [2.1% (6/281) vs. 5.5% (71/1300), P=0.019]. CONCLUSION In pediatric orthopaedic surgery, abstracts authored by women are less likely to reach publication, despite no identifiable differences in study quality. Reasons for this discrepancy must be explored including insufficient mentorship, exclusion from study group participation, or potential bias against female researchers in the field of orthopaedic surgery. LEVEL OF EVIDENCE Level IV.

Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-β signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it. Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance. Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance “free” cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes. Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197–208. ©2017 AACR.

Purpose: A secondary EGFR mutation, T790M, is the most common resistance mechanism in EGFR-mutant adenocarcinomas that have progressed on erlotinib. Third-generation EGFR inhibitors capable of inhibiting mutant EGFR with T790M produce responses in nearly two thirds of patients. However, acquired resistance mechanisms in patients treated with these drugs are yet to be described. Experimental Design: To study acquired resistance to third-generation EGFR inhibitors, T790M-positive cells derived from an erlotinib-resistant cancer were made resistant to a third-generation TKI and then characterized using cell and molecular analyses. Results: Cells resistant to a third-generation TKI acquired an additional EGFR mutation, C797S, which prevented suppression of EGFR. Our results demonstrate that the allelic context in which C797S was acquired may predict responsiveness to alternative treatments. If the C797S and T790M mutations are in trans, cells will be resistant to third-generation EGFR TKIs, but will be sensitive to a combination of first- and third-generation TKIs. If the mutations are in cis, no EGFR TKIs alone or in combination can suppress activity. If C797S develops in cells wild-type for T790 (when third-generation TKIs are administered in the first-line setting), the cells are resistant to third-generation TKIs, but retain sensitivity to first-generation TKIs. Conclusions: Mutation of C797S in EGFR is a novel mechanism of acquired resistance to third-generation TKIs. The context in which the C797S develops with respect to the other EGFR alleles affects the efficacy of subsequent treatments. Clin Cancer Res; 21(17); 3924–33. ©2015 AACR. See related commentary by Ayeni et al., p. 3818

Rociletinib is a third-generation EGFR inhibitor active in lung cancers with T790M, the gatekeeper mutation underlying most first-generation EGFR drug resistance. We biopsied patients at rociletinib progression to explore resistance mechanisms. Among 12 patients with T790M-positive cancers at rociletinib initiation, six had T790–wild-type rociletinib-resistant biopsies. Two T790–wild-type cancers underwent small cell lung cancer transformation; three T790M-positive cancers acquired EGFR amplification. We documented T790–wild-type and T790M-positive clones coexisting within a single pre-rociletinib biopsy. The pretreatment fraction of T790M-positive cells affected response to rociletinib. Longitudinal circulating tumor DNA (ctDNA) analysis revealed an increase in plasma EGFR-activating mutation, and T790M heralded rociletinib resistance in some patients, whereas in others the activating mutation increased but T790M remained suppressed. Together, these findings demonstrate the role of tumor heterogeneity when therapies targeting a singular resistance mechanism are used. To further improve outcomes, combination regimens that also target T790–wild-type clones are required. Significance: This report documents that half of T790M-positive EGFR-mutant lung cancers treated with rociletinib are T790–wild-type upon progression, suggesting that T790–wild-type clones can emerge as the dominant source of resistance. We show that tumor heterogeneity has important clinical implications and that plasma ctDNA analyses can sometimes predict emerging resistance mechanisms. Cancer Discov; 5(7); 713–22. ©2015 AACR. See related commentary by Ichihara and Lovly, p. 694. This article is highlighted in the In This Issue feature, p. 681

Despite significant advances in targeted therapies for lung cancer, nearly all patients develop drug resistance within 6-12 months and prognosis remains poor. Developing drug resistance is a progressive process that involves tumor cells and their microenvironment. We hypothesize that microenvironment factors alter tumor growth and response to targeted therapy. We conducted in vitro studies in human EGFR-mutant lung carcinoma cells, and demonstrated that factors secreted from lung fibroblasts results in increased tumor cell survival during targeted therapy with EGFR inhibitor, gefitinib. We also demonstrated that increased environment stiffness results in increased tumor survival during gefitinib therapy. In order to test our hypothesis in vivo, we developed a multimodal optical imaging protocol for preclinical intravital imaging in mouse models to assess tumor and its microenvironment over time. We have successfully conducted multimodal imaging of dorsal skinfold chamber (DSC) window mice implanted with GFP-labeled human EGFR mutant lung carcinoma cells and visualized changes in tumor development and microenvironment facets over time. Multimodal imaging included structural OCT to assess tumor viability and necrosis, polarization-sensitive OCT to measure tissue birefringence for collagen/fibroblast detection, and Doppler OCT to assess tumor vasculature. Confocal imaging was also performed for high-resolution visualization of EGFR-mutant lung cancer cells labeled with GFP, and was coregistered with OCT. Our results demonstrated that stromal support and vascular growth are essential to tumor progression. Multimodal imaging is a useful tool to assess tumor and its microenvironment over time.

Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFR(T790M) gatekeeper mutation can occur either by selection of pre-existing EGFR(T790M)-positive clones or via genetic evolution of initially EGFR(T790M)-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFR(T790M); treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic.

Tyrosine kinase inhibitors are effective treatments for non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of 1 year of continuous treatment. A fundamental histological transformation from NSCLC to small-cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumour samples and cell lines derived from resistant EGFR mutant patients revealed that Retinoblastoma (RB) is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared with resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC., Resistance to tyrosine kinase inhibitors occurs in treatments of non-small-cell lung cancers (NSCLCs) with EGFR mutations but the mechanisms underlying this acquired resistance are unknown. Here the authors examine the molecular changes that occur in resistant cancers that transition from NSCLC to small-cell lung cancer phenotype and implicate loss of retinoblastoma in this process.

In EGFR mutant lung adenocarcinomas, the activating mutation to EGFR is the founder alteration, present in every cancer cell. Most patients with this genetic alteration respond to EGFR tyrosine kinase inhibitors, although the average duration of the response is only one year before the cancer develops acquired resistance. Efforts to identify the mechanisms underlying the resistance in patients have led to the identification of a resistance mechanism in the majority of cases. However, preliminary data is emerging indicating that multiple resistance mechanisms can be present within an individual patient's disease, and thus, classifying resistant disease with a single mechanism is an oversimplification. Here, utilizing single cell approaches on patient samples, we demonstrate heterogeneity within individual biopsies with respect to resistance mechanisms, epigenetic state, and sensitivity to TKI. Additionally, we observe that the fraction of T790M-positive cells is predictive of response to 3rd generation EGFR inhibitor treatment, suggesting that the degree of heterogeneity has important clinical implications. As therapies designed to target singular resistance mechanisms become more widely implemented, assessing heterogeneity will become increasingly critical in order to choose the most effective therapy for the patient. Citation Format: Matt J. Niederst, Huidan Zhang, Haichuan Hu, Naiwen Cui, Yamei Cai, Hillary Mulvey, Zofia Piotrowska, Lecia Sequist, David A. Weitz, Jeffrey A. Engelman. Single cell analyses reveal resistance heterogeneity in EGFR mutant NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 291.

Despite the success of targeted cancer therapies, acquired drug resistance remains a significant clinical challenge, as in the case of EGFR mutant non-small cell lung cancers (NSCLC) treated with EGFR inhibitor therapy. Although molecular mechanisms of acquired resistance to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. In some cases, clones with clinically validated genetic resistance mechanisms may exist prior to drug exposure and are simply selected by treatment. Alternatively, it has been hypothesized that drug tolerant (or “persister”) cells without bona fide resistance mechanisms may survive initial drug treatment by epigenetic adaptations, and undergo further evolution over time to acquire validated genetic resistance mechanisms. Although this would have immediate implications for new therapeutic strategies to prevent resistance, there has not been any direct evidence that drug tolerant cells can undergo such evolution. Using EGFR mutant NSCLC models, we observe that acquired resistance caused by the T790M gatekeeper mutation, which is observed in 50% of patients with acquired resistance to EGFR inhibitor therapy, can occur by either selection of pre-existing T790M clones or via evolution of T790M-negative drug tolerant cells that develop the mutation during months of drug treatment. Additionally, the path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells have a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR and can be sensitized by BCL-XL inhibitors. In total, these findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to new therapeutic opportunities to prevent or overcome resistance in the clinic. Citation Format: Aaron N. Hata, Matthew Niederst, Maria Gomez-Caraballo, Hannah Archibald, Faria Siddiqui, Hillary Mulvey, Haichuan Hu, Lecia Sequist, Jeffrey Engelman. Dynamic evolution of resistance to EGFR blockade from drug tolerant cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B53.

The development of acquired drug resistance limits the effectiveness of targeted therapies for lung cancer. In 50% of patients with EGFR mutant non-small cell lung cancer (NSCLC), acquired resistance to EGFR inhibitors can be attributed to the development of a secondary T790M mutation that prevents drug binding. Third generation irreversible EGFR inhibitors that effectively inhibit EGFR with T790M are currently under clinical development. Using patient-derived EGFR mutant NSCLC models of T790M+ acquired resistance, we observed that the presence of a mesenchymal phenotype was associated with a decreased response to subsequent irreversible EGFR inhibitor therapy. Epithelial-to-mesenchymal transformation (EMT) led to suppression of pro-apoptotic signaling and a blunted apoptotic response to EGFR inhibition. Combination BCL-XL and irreversible EGFR inhibitor therapy restored the apoptotic response and resulted in regressions of T790M+ resistant tumors that exhibited poor response to single agent irreversible EGFR inhibitors. This suggests that combining BCL-XL and irreversible EGFR inhibitors may be an effective treatment strategy for T790M+ resistant EGFR mutant lung cancers with suppressed apoptotic signaling due co-acquisition of a mesenchymal phenotype. Citation Format: Aaron N. Hata, Matthew J. Niederst, Hannah L. Archibald, Hillary Mulvey, Jungoh Ham, Maria Gomez-Caraballo, Anuj Kalsy, Anthony C. Faber, Jeffrey Engelman. Co-acquisition of T790M and EMT in resistant EGFR mutant non-small cell lung cancer can be overcome by combined irreversible EGFR and BCL-XL inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2845. doi:10.1158/1538-7445.AM2015-2845

Background Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. Methods EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB. Results This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Conclusions Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.