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Supplemental Figure 1 shows Pancreatic stellate cells (PSCs) promoted tumor cell invasion through close cell-cell interactions.

Pancreatic cancer has an abysmal 5-year survival rate of 8%, making it a deadly disease with a need for novel therapies. Here we describe a multitargeting heparin-based mimetic, necuparanib, and its antitumor activity in both in vitro and in vivo models of pancreatic cancer. Necuparanib reduced tumor cell proliferation and invasion in a three-dimensional (3D) culture model; in vivo, it extended survival and reduced metastasis. Furthermore, proteomic analysis demonstrated that necuparanib altered the expression levels of multiple proteins involved in cancer-driving pathways including organ development, angiogenesis, proliferation, genomic stability, cellular energetics, and invasion and metastasis. One protein family known to be involved in invasion and metastasis and altered by necuparanib treatment was the matrix metalloprotease (MMP) family. Necuparanib reduced metalloproteinase 1 (MMP1) and increased tissue inhibitor of metalloproteinase 3 (TIMP3) protein levels and was found to increase RNA expression of TIMP3. MMP enzymatic activity was also found to be reduced in the 3D model. Finally, we confirmed necuparanib's in vivo activity by analyzing plasma samples of patients enrolled in a phase I/II study in patients with metastatic pancreatic cancer; treatment with necuparanib plus standard of care significantly increased TIMP3 plasma protein levels. Together, these results demonstrate necuparanib acts as a broad multitargeting therapeutic with in vitro and in vivo anti-invasive and antimetastatic activity.

Supplementary Table 2 lists GO Categories significantly altered by necuparanib treatment, assigned to cancer-driving pathways

Supplementary Table 1 lists proteins significantly altered by necuparanib treatment

Supplemental Figure 3 shows MMP1 and MMP7 protein levels were unchanged in the plasma of patients.

Supplemental Figure 2 shows GM6001 reduced spheroid growth and invasion in the 3D co-culture model.

O puerpério é um período que suscita cuidados, pois a mulher encontra-se fragilizada física e emocionalmente. Tal situação ganha contornos especiais quando o puerpério se desenvolve dentro do ambiente hospitalar, devido à hospitalização do recém-nascido, pois causa a quebra de um ciclo esperado pela mãe e demais familiares. Nesse sentido, o presente artigo tem por objetivo relatar a experiência nas rodas de conversa com as puérperas que estão com seus filhos hospitalizados, desenvolvidas pelo projeto de extensão “Prevenção e Promoção da Saúde em grupos de Gestantes e Puérperas”, como forma de auxiliar a mulher, na promoção da saúde e do vínculo com o bebê, aproximação com o ambiente hospitalar e atenção específica no período puerperal. As rodas de conversa, de caráter multidisciplinar, ocorreram no período de maio a dezembro de 2018, nas dependências de um hospital. Realizaram-se registros, tipo diário de campo, para posterior análise. Os assuntos foram agrupados conforme relevância dos achados: Autocuidado e Cuidado de si; Puerpério: modificações corporais; Amamentação; Redes de apoio e os direitos das puérperas e dos recém-nascidos; Ansiedades e medos relacionados ao puerpério e hospitalização do recém-nascido. As rodas de conversa oportunizaram a troca de experiências entre as mulheres e os profissionais, assim contribuindo para minimizar angústias e elucidar questões relativas ao período puerperal e cuidados do recém-nascido. Além disso, o caráter multidisciplinar confere um espaço de comunicação entre diversas áreas e seus saberes, refletindo no âmbito acadêmico em oportunidade para exercitar a integralidade do cuidado. O puerpério é um período que suscita cuidados, pois a mulher encontra-se fragilizada física e emocionalmente. Tal situação ganha contornos especiais quando o puerpério se desenvolve dentro do ambiente hospitalar, devido à hospitalização do recém-nascido, pois causa a quebra de um ciclo esperado pela mãe e demais familiares. Nesse sentido, o presente artigo tem por objetivo relatar a experiência nas rodas de conversa com as puérperas que estão com seus filhos hospitalizados, desenvolvidas pelo projeto de extensão “Prevenção e Promoção da Saúde em grupos de Gestantes e Puérperas”, como forma de auxiliar a mulher, na promoção da saúde e do vínculo com o bebê, aproximação com o ambiente hospitalar e atenção específica no período puerperal. As rodas de conversa, de caráter multidisciplinar, ocorreram no período de maio a dezembro de 2018, nas dependências de um hospital. Realizaram-se registros, tipo diário de campo, para posterior análise. Os assuntos foram agrupados conforme relevância dos achados: Autocuidado e Cuidado de si; Puerpério: modificações corporais; Amamentação; Redes de apoio e os direitos das puérperas e dos recém-nascidos; Ansiedades e medos relacionados ao puerpério e hospitalização do recém-nascido. As rodas de conversa oportunizaram a troca de experiências entre as mulheres e os profissionais, assim contribuindo para minimizar angústias e elucidar questões relativas ao período puerperal e cuidados do recém-nascido. Além disso, o caráter multidisciplinar confere um espaço de comunicação entre diversas áreas e seus saberes, refletindo no âmbito acadêmico em oportunidade para exercitar a integralidade do cuidado.

Background Necuparanib, a rationally engineered low-molecular-weight heparin, combined with gemcitabine/nab-paclitaxel showed an encouraging safety and oncologic signal in a phase Ib trial. This randomised multicentre phase II trial evaluates the addition of necuparanib or placebo to gemcitabine/nab-paclitaxel in untreated metastatic pancreatic ductal adenocarcinoma (PDAC). Patients and methods Eligibility included 18 years, histologically or cytologically confirmed metastatic PDAC, measurable disease and Eastern Co-Operative Oncology Group performance status of 0–1. Patients were randomly assigned to necuparanib (5 mg/kg subcutaneous injection once daily) or placebo (subcutaneous injection once daily) and gemcitabine/nab-paclitaxel on days 1, 8 and 15 of 28-day cycles. The primary end-point was median overall survival (OS), and secondary end-points included median progression-free survival, response rates and safety. Results One-hundred ten patients were randomised, 62 to necuparanib arm and 58 to placebo arm. The futility boundary was crossed at a planned interim analysis, and the study was terminated by the Data Safety Monitoring Board. The median OS was 10.71 months (95% confidence interval [CI]: 7.95–11.96) for necuparanib arm and 9.99 months (95% CI: 7.85–12.85) for placebo arm (hazard ratio: 1.12, 95% CI: 0.66–1.89, P-value: 0.671). The necuparanib arm had a higher incidence of haematologic toxicity relative to placebo patients (83% and 70%). Conclusion The addition of necuparanib to standard of care treatment for advanced PDAC did not improve OS. Safety was acceptable. No further development of necuparanib is planned although targeting the coagulation cascade pathway remains relevant in PDAC. NCT01621243

Objetivo: Desenvolver recomendações de consenso atualizadas com perspectivas voltadas para injeção de toxina botulínica e preenchedores dérmicos a base de ácido hialurônico durante e pós-pandemia COVID-19. Revisão bibliográfica: Os tratamentos minimamente invasivos continuam sendo os procedimentos estéticos mais procurados em todo o mundo. Portanto, as diretrizes de consenso são extremamente importantes por abordar as precauções de segurança sobre a injeção de toxina botulínica (TXB-A) e procedimentos de preenchimento de ácido hialurônico (AH). Principalmente na conjuntura atual, a qual se vive risco eminente de infecção e transmissão do vírus SARS-CoV-2 entre o paciente e o profissional em clínicas estéticas. As mudanças que se fazem hoje são necessárias para proteger os pacientes das complicações dos tratamentos minimamente invasivos, já que as características fisiopatológicas e imunológicas do coronavírus ainda são incomuns. Considerações finais: A pandemia COVID-19 redefiniu a atuação dos profissionais e das clínicas estéticas exigindo na conjuntura atual adequação as recomendações conforme as apresentadas nessa diretriz. Esses esforções incluem o distanciamento social, anamnese e triagem de risco para Covid-19, aferição de temperatura e controle da higienização, investigação do estado de saúde do paciente por meio de exames laboratoriais e paramentação adequada.

O objetivo deste trabalho é expor ao meio acadêmico e a comunidade as ações desenvolvidas pelo projeto de extensão intitulado “Prevenção e Promoção da Saúde em Grupos de Gestantes e Puérperas” no âmbito hospitalar, evidenciando sua importância diante da experiência da mulher no ciclo gravídico-puerperal. O planejamento das ações ocorreu a parti r das fragilidades encontradas nas gestantes e puérperas hospitalizadas, sendo então organizado rodas de conversas semanais de cunho multiprofissional, com a colaboração de profissionais e acadêmicos das áreas de enfermagem, serviço social e psicologia. As temáticas abordadas nos encontros foram: ansiedade e medos relacionados à gestação; parto e puerpério; rede de apoio e direitos das gestantes e puérperas; pintura em ventre gravídico; modificações puerperais; aleitamento materno; o que tem na mala do bebê?, prevenção de acidentes com crianças. As ações desenvolvidas no projeto de extensão proporcionam trocas entre a equipe multiprofissional, acadêmicos, mulheres no período gravídico-puerperal e seus familiares, qualificando o cuidado ofertado. As rodas de conversas, nesse senti do, disparam reflexões acerca do cuidado materno e infantil, possibilitando a identificação das participantes entre si. Tal reconhecimento amplia o diálogo e gera maior conforto, o que minimiza a sensação de solidão e promove a construção da parentalidade.

Pancreatic cancer has an abysmal 5-year survival rate of 8%, making it a deadly disease with a need for novel therapies. Here we describe a multitargeting heparin-based mimetic, necuparanib, and its antitumor activity in both in vitro and in vivo models of pancreatic cancer. Necuparanib reduced tumor cell proliferation and invasion in a three-dimensional (3D) culture model; in vivo, it extended survival and reduced metastasis. Furthermore, proteomic analysis demonstrated that necuparanib altered the expression levels of multiple proteins involved in cancer-driving pathways including organ development, angiogenesis, proliferation, genomic stability, cellular energetics, and invasion and metastasis. One protein family known to be involved in invasion and metastasis and altered by necuparanib treatment was the matrix metalloprotease (MMP) family. Necuparanib reduced metalloproteinase 1 (MMP1) and increased tissue inhibitor of metalloproteinase 3 (TIMP3) protein levels and was found to increase RNA expression of TIMP3. MMP enzymatic activity was also found to be reduced in the 3D model. Finally, we confirmed necuparanib's in vivo activity by analyzing plasma samples of patients enrolled in a phase I/II study in patients with metastatic pancreatic cancer; treatment with necuparanib plus standard of care significantly increased TIMP3 plasma protein levels. Together, these results demonstrate necuparanib acts as a broad multitargeting therapeutic with in vitro and in vivo anti-invasive and antimetastatic activity.

Some epithelial cancers can be induced to revert to quiescent differentiated tissue when combined with embryonic mesenchyme; however, the mechanism of this induction is unknown. Here we combine tissue engineering, developmental biology, biochemistry and proteomics approaches to attack this problem. Using a synthetic reconstitution system, we show that co-culture of breast cancer cells with embryonic mesenchyme from early stage (E12.5–13.5) mammary glands decreases tumor cell proliferation while stimulating acinus differentiation, whereas cancer-associated fibroblasts (CAFs) fail to produce these normalizing effects. When insoluble extracellular matrices (ECMs) were isolated from cultured early stage (E12.5–13.5) embryonic mammary mesenchyme cells or E10 tooth mesenchyme and recombined with mammary tumor cells, they were found to be sufficient to induce breast cancer normalization including enhanced expression of estrogen receptor-α (ER-α). In contrast, ECM from later stage (E14.5) mammary mesenchyme and conditioned medium isolated from mesenchymal cell cultures were ineffective. Importantly, when the inductive ECMs produced by early stage embryonic mammary mesenchyme were scraped from dishes and injected into fast-growing breast tumors in mice, they significantly inhibited cancer expansion. Proteomics analysis of the detergent insoluble ECM material revealed several matrix components that were preferentially expressed in the embryonic ECMs. Analysis of two of these molecules previously implicated in cancer regulation – biglycan and tenascin C – revealed that addition of biglyan can mimic the tumor normalization response, and that siRNA knockdown of its expression in cultured embryonic mesenchyme results in loss of the ECM's inductive activity. These studies confirm that embryonic mesenchyme retains the ability to induce partial breast cancer reversion, and that its inductive capability resides at least in part in the ECM protein biglycan that it produces.

Differentiation therapies that induce malignant cells to stop growing and revert to normal tissue-specific differentiated cell types are successful in the treatment of a few specific haematological tumours. However, this approach has not been widely applied to solid tumours because their developmental origins are less well understood. Recent advances suggest that understanding tumour cell plasticity and how intrinsic factors (such as genetic noise and microenvironmental signals, including physical cues from the extracellular matrix) govern cell state switches will help in the development of clinically relevant differentiation therapies for solid cancers.

Lessons LearnedDespite the compelling preclinical rationale of evaluating the genetically engineered heparin derivative, necuparanib, combined with standard therapy in metastatic pancreas adenocarcinoma, the results were ultimately disappointing. Safety was documented, although dose escalation was limited by the number of subcutaneous injections, the potential for skin toxicity (cellulitis), and low-level anticoagulant effect. Nonetheless, the hypothesis of targeting prothrombotic pathways in pancreas adenocarcinoma remains compelling.BackgroundNecuparanib is derived from unfractionated heparin and engineered for reduced anticoagulant activity while preserving known heparin-associated antitumor properties. This trial assessed the safety, pharmacokinetics (PK), pharmacodynamics, and initial efficacy of necuparanib combined with gemcitabine ± nab-paclitaxel in patients with metastatic pancreatic cancer.MethodsPatients received escalating daily subcutaneous doses of necuparanib plus 1,000 mg/m2 gemcitabine (days 1, 8, 15, and every 28 days). The protocol was amended to include 125 mg/m2 nab-paclitaxel after two cohorts (following release of the phase III MPACT data). The necuparanib starting dose was 0.5 mg/kg, with escalation via a modified 3 + 3 design until the maximum tolerated dose (MTD) was determined.ResultsThirty-nine patients were enrolled into seven cohorts (necuparanib 0.5, 1 mg/kg + gemcitabine; necuparanib 1, 2, 4, 6, and 5 mg/kg + nab-paclitaxel + gemcitabine). The most common adverse events were anemia (56%), fatigue (51%), neutropenia (51%), leukopenia (41%), and thrombocytopenia (41%). No deaths and two serious adverse events were potentially related to necuparanib. Measurable levels of necuparanib were seen starting at the 2 mg/kg dose. Of 24 patients who received at least one dose of necuparanib + nab-paclitaxel + gemcitabine, 9 (38%) achieved a partial response and 6 (25%) achieved stable disease (63% disease control rate). Given a cellulitis event and mild activated partial thromboplastin time increases at 6 mg/kg, the 5 mg/kg dose was considered the MTD and selected for further assessment in phase II.ConclusionAcceptable safety and encouraging signals of activity in patients with metastatic pancreatic cancer receiving necuparanib, nab-paclitaxel, and gemcitabine were demonstrated.

Introdução: Durante o atendimento odontológico, o desconhecimento por parte do cirurgião-dentista quanto à condição de seu paciente, principalmente se este possuir algum distúrbio hemorrágico, pode colocar em risco a saúde do paciente. Objetivo: O propósito deste estudo é, através de revisão de literatura, abordar os principais distúrbios hemorrágicos e sua relação com a odontologia. Metodologia: Os dados foram coletados através da análise de prontuários de pacientes atendidos na disciplina de Clínica Integral II do curso de Odontologia da ULBRA de Canoas entre 2007/2 e 2008/1, verificando a prevalência de casos de alterações hemorrágicas. Resultados: Ao analisar os resultados, verificamos que dos 416 pacientes, 35 pacientes apresentaram histórico de hemorragia diagnosticada, com uma maior prevalência no gênero feminino. Ao analisar a distribuição das ocorrências hemorrágicas quanto a faixa etária, verificamos uma maior prevalência dos 40 aos 60 anos. Quanto ao histórico familiar de hemorragia diagnosticada, 21 pacientes apresentaram relato positivo. Dos pacientes que apresentaram alteração hemorrágica, 16 apresentaram alterações sistêmicas que predispõem a alterações hemorrágicas como hipertensão, disfunção renal, disfunção hepática, anemia e/ou HIV. Conclusões: Os resultados demonstraram que para detectar pacientes de risco hemorrágico, uma anamnese detalhada é de fundamental importância, pois através dela verifica-se o histórico de hemorragia, a utilização de medicamentos que induzem a alterações hemorrágicas, bem como alterações sistêmicas que interferem na capacidade de hemostasia do paciente.

With advances in screening, the incidence of detection of premalignant breast lesions has increased in recent decades; however, treatment options remain limited to surveillance or surgical removal by lumpectomy or mastectomy. We hypothesized that disease progression could be blocked by RNA interference (RNAi) therapy and set out to develop a targeted therapeutic delivery strategy. Using computational gene network modeling, we identified HoxA1 as a putative driver of early mammary cancer progression in transgenic C3(1)-SV40TAg mice. Silencing this gene in cultured mouse or human mammary tumor spheroids resulted in increased acinar lumen formation, reduced tumor cell proliferation, and restoration of normal epithelial polarization. When the HoxA1 gene was silenced in vivo via intraductal delivery of nanoparticle-formulated small interfering RNA (siRNA) through the nipple of transgenic mice with early-stage disease, mammary epithelial cell proliferation rates were suppressed, loss of estrogen and progesterone receptor expression was prevented, and tumor incidence was reduced by 75%. This approach that leverages new advances in systems biology and nanotechnology offers a novel noninvasive strategy to block breast cancer progression through targeted silencing of critical genes directly within the mammary epithelium.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

370 Background: The Phase 1 portion of a Phase 1/2 trial of Necuparanib (“Necu”) combined with nab-paclitaxel (nabP) + gemcitabine (gem) in patients with metastatic pancreatic cancer (ClinicalTrials.gov Identifier NCT01621243) showed acceptable safety and tolerability and encouraging signals of activity and established a dose for the randomized, placebo (PBO)-controlled Phase 2 portion. Methods: In Phase 2, patients received daily s.c. injections of either 5 mg/kg Necu daily or PBO, combined with i.v. 125 mg/m2 nabP and 1000 mg/m2 gem (Days 1, 8, 15 of each 28-day cycle). The primary endpoint was overall survival (OS); other endpoints included progression-free survival (PFS), response rates, safety, and CA19.9 levels. An interim futility analysis was conducted in July 2016 once 57 deaths (50% of the target number of 114 events required for trial completion) had occurred. Results: The analysis was conducted on data from 120 randomized patients (62 Necu, 58 PBO). The Z-score for futility was -0.42 (prespecified boundary of -0.148 was crossed as actual score was lower). Median OS was Necu = 10.71 and PBO = 9.99 months; hazard ratio (HR) = 1.12 (favoring PBO); OS curves were intertwined. PFS was Necu = 5.52 and PBO = 6.93 months; HR = 0.97. RECIST response rates were comparable between arms: complete response, Necu = 0%, PBO = 3%; partial response, Necu = 26%, PBO = 26%; stable disease, Necu = 31%, PBO = 34%; disease control rate, Necu = 56%, PBO = 64%. The most common Grade 3+ adverse events (AEs) were neutropenia (Necu = 33%, PBO = 33%), thrombocytopenia (Necu = 27%, PBO = 5%), and anemia (Necu = 22%, PBO = 11%). There were lower rate of serious AEs with Necu (48%) vs. PBO (60%). Modest increases in APTT, AST, and ALT were noted following Necu relative to PBO. 23% of Necu and 5% of PBO patients were IgG positive with an anti-heparin/PF4 antibody titer of ≥ 0.4 at any time. There were no treatment differences for decreases in CA19.9. Conclusions: No new safety signals were observed and the toxicity profile was considered manageable; however, Necu in combination with nabP and gem did not show a sufficient level of efficacy in metastatic pancreatic cancer to warrant continued enrollment. Clinical trial information: NCT01621243.

Changes in extracellular matrix (ECM) structure or mechanics can actively drive cancer progression; however, the underlying mechanism remains unknown. Here we explore whether this process could be mediated by changes in cell shape that lead to increases in genetic noise, given that both factors have been independently shown to alter gene expression and induce cell fate switching. We do this using a computer simulation model that explores the impact of physical changes in the tissue microenvironment under conditions in which physical deformation of cells increases gene expression variability among genetically identical cells. The model reveals that cancerous tissue growth can be driven by physical changes in the microenvironment: when increases in cell shape variability due to growth-dependent increases in cell packing density enhance gene expression variation, heterogeneous autonomous growth and further structural disorganization can result, thereby driving cancer progression via positive feedback. The model parameters that led to this prediction are consistent with experimental measurements of mammary tissues that spontaneously undergo cancer progression in transgenic C3(1)-SV40Tag female mice, which exhibit enhanced stiffness of mammary ducts, as well as progressive increases in variability of cell-cell relations and associated cell shape changes. These results demonstrate the potential for physical changes in the tissue microenvironment (e.g., altered ECM mechanics) to induce a cancerous phenotype or accelerate cancer progression in a clonal population through local changes in cell geometry and increased phenotypic variability, even in the absence of gene mutation.

Pancreatic cancer is one of the most aggressive types of cancer, with only about 5% of patients surviving 5 years past the initial diagnosis. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal due to the cancer's complex pathology. Novel multi-targeted agents or therapeutic combinations able to disrupt the aggressive fibrotic microenvironment and restrict tumor proliferation are a current focus in the field. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, and adhesion molecules in cancer progression and metastasis and as such has been shown to modulate multiple pathways in tumors. In order to further explore how necuparanib affects the tumor and its microenvironment, a 3-dimensional (3D) culture system mimicking pancreatic cancer was developed containing a co-culture of human pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. Morphological and histological assessment of the 3D tumor spheroids demonstrated that necuparanib inhibits a) pancreatic tumor cell growth, b) pancreatic tumor cell invasion, and c) pancreatic stellate cell growth. Necuparanib's activity in these assays was shown to be dose-dependent. Moreover, using a negative control for necuparanib engineered to lack the ability to bind growth factors, we established that necuparanib's activity is growth factor binding dependent. The ability to inhibit both tumor and stromal cells demonstrates that necuparanib is a very promising multi-targeting agent for difficult-to-treat cancers such as desmoplastic pancreatic tumors. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support the current Phase 2 safety and efficacy study being performed in patients with metastatic pancreatic cancer. Citation Format: Amanda MacDonald, Michelle Priess, Jennifer Curran, Silva Krause. Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B22.

Here we describe a combined microfluidic-micromagnetic cell separation device that has been developed to isolate, detect and culture circulating tumor cells (CTCs) from whole blood, and demonstrate its utility using blood from mammary cancer-bearing mice. The device was fabricated from polydimethylsiloxane and contains a microfluidic architecture with a main channel and redundant ‘double collection’ channel lined by two rows of dead-end side chambers for tumor cell collection. The microdevice design was optimized using computational simulation to determine dimensions, magnetic forces and flow rates for cell isolation using epithelial cell adhesion molecule (EpCAM) antibody-coated magnetic microbeads (2.8 μm diameter). Using this device, isolation efficiencies increased in a linear manner and reached efficiencies close to 90% when only 2 to 80 breast cancer cells were spiked into a small volume (1.0 mL) of blood taken from wild type mice. The high sensitivity visualization capabilities of the device also allowed detection of a single cell within one of its dead-end side chambers. When blood was removed from FVB C3(1)-SV40 T-antigen mammary tumor-bearing transgenic mice at different stages of tumor progression, cells isolated in the device using anti-EpCAM-beads and magnetically collected within the dead-end side chambers, also stained positive for pan-cytokeratin-FITC and DAPI, negative for CD45-PerCP, and expressed SV40 large T antigen, thus confirming their identity as CTCs. Using this isolation approach, we detected a time-dependent rise in the number of CTCs in blood of female transgenic mice, with a dramatic increase in the numbers of metastatic tumor cells appearing in the blood after 20 weeks when tumors transition to invasive carcinoma and exhibit increased growth of metastases in this model. Importantly, in contrast to previously described CTC isolation methods, breast tumor cells collected from a small volume of blood removed from a breast tumor-bearing animal remain viable and they can be easily removed from these devices and expanded in culture for additional analytical studies or potential drug sensitivity testing.

Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, the development of defined three-dimensional (3D) models is essential. In the present study, we have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo. Cocultures of a human mammary epithelial cell line MCF10A and human mammary fibroblasts obtained from reduction mammoplasties embedded in either a type I collagen or a mixed Matrigel-collagen matrix were carried out for up to 6 weeks. Histological and ultrastructural analysis confirmed the formation of ductal and alveolar structures. The importance of the stromal cells was apparent in both matrices; in the collagen gels the presence of reduction mammoplasty fibroblasts accelerated the initial formation of epithelial structures, and in the mixed Matrigel-collagen gels the presence of those fibroblasts was necessary for the formation of ductal structures. These models provide an excellent system to study tissue organization, epithelial morphogenesis, and breast carcinogenesis.

Pancreatic cancer is one of the most aggressive and deadliest cancers, with only about 5% of patients surviving 5 years past the initial diagnosis. In over 80% of patients the cancer has already spread to distant organs at the time of diagnosis, making it difficult to treat. Most alarmingly, pancreatic cancer is the only cancer for which deaths are predicted to increase over the next few years. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal and require novel therapeutic approaches. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, adhesion molecules and enzymes involved in cancer progression and metastasis and as such modulates multiple elements of the tumor microenvironment. In order to further explore the mechanism of action for epithelial-stromal interactions affected by necuparanib, a 3-dimensional culture system mimicking pancreatic cancer was developed containing a co-culture of pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. In this culture system, pancreatic cancer cells form a tumor spheroid embedded in a matrix that is a mixture of type I collagen and basement membrane extract and contains pancreatic stellate cells. Notably, pancreatic cancer cells required the co-culture with pancreatic stellate cells to become invasive in vitro. Conditioned medium derived from pancreatic stellate cells could not mimic this effect indicating that soluble factors are not sufficient to cause invasion into surrounding matrix. Necuparanib inhibited the invasive behavior in a dose-dependent manner. The effect of pancreatic stellate cells on tumor cell invasion and the effect of necuparanib on stromal cells were further explored in this culture system. We showed an effect of necuparanib on proliferation, migration and invasion of pancreatic tumor cells. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support biomarker discovery in the ongoing Phase 2 efficacy study in metastatic pancreatic cancer. Citation Format: Silva Krause, Amanda Weyers, Katie Loveluck, Birgit Schultes. Necuparanib affects tumor progression and invasion in a 3D co-culture system of pancreatic cancer cells and stellate cells. [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 5499. doi:10.1158/1538-7445.AM2015-5499

During carcinoma progression, tumor cells often undergo changes similar but not identical to epithelial-mesenchymal transitions in embryonic development. In this study I have demonstrated that experimental stimulation of hyaluronan synthesis in normal epithelial cells is sufficient to induce mesenchymal and transformed characteristics. Using recombinant adenoviral expression of hyaluronan synthase 2, 1 showed that increased hyaluronan production promotes anchorage- independent growth and invasiveness, induces gelatinase production, and stimulates phosphoinositide-3-kinase/Akt pathway activity in phenotypically normal MDCK canine kidney and MCF-lOA human mammary epithelial cells. Cells infected with hyaluronan synthase 2 adenovirus also acquire mesenchymal characteristics, including up-regulation of vimentin', dispersion of cytokeratin, and loss of organized adhesion proteins at intercellular boundaries. Furthermore, I showed that transforming effects of two well-described agents, hepatocyte growth factor and P-catenin, are dependent on byaluronan-cell interactions. Thus increased expression of hyaluronan is sufficient to induce epithelial to mesenchymal transition and acquisition of transformed properties in phenotypically normal epithelial cells. In addition I have shown that the extracellular matrix metalloproteinase inducer emmprin, promotes anchorage-independent growth and stimulates ErbB2, phosphoinositide 3-kinase /Akt, MAP kinase and focal adhesion kinase cell survival signaling pathways in a hyaluronan-dependent manner.

The contribution of specific genes to the development and progression of mammary tumors has been hampered by the inability to silence these genes locally in the mammary gland. Localized intraductal chemotherapy treatments have been explored and offer significant advantages in terms of reduced toxicity and side effects. Therefore, we wanted to explore the possibility of gene knockdown using siRNA delivery via intraductal injection. FVB C3(1)-SV40Tag transgenic female mice spontaneously develop hyperplastic mammary lesions that progress to invasive mammary tumors. Hyperplastic ducts form as early as 12 weeks of age and progress into DCIS-resembling tumors by 16 weeks of age. These further develop into invasive tumors that invade locally into adipose tissue and muscle and occasionally form metastases to lung by 18 to 20 weeks of age. We intraductally injected nanoparticle-formulated siRNA targeted to the SV40 T-antigen transgene beginning at 8 weeks of age through 23 weeks of age into the mammary gland twice a month. As controls, transgenic females were either left untreated or intraductally injected with a non-targeting siRNA. Tumor formation was monitored using ultrasound technology that allows for tumor detection as early as 0.4mm in diameter. At 23 weeks of age, when 100% of the control mice developed tumors, only 1 of 8 mice that received siRNA to the transgene had developed a small tumor. Histological analysis of the mammary glands confirmed development of early lesions but tumor progression was successfully prevented. Mammary lesions were analyzed for proliferation, apoptosis and expression of various markers of differentiation, including hormone receptor status. We showed that siRNA was not detectable in any other organs of the mouse, including the liver. Furthermore, we confirmed that frequent intraductal siRNA injection did not interfere with subsequent pregnancy or lactation, a period in which the mammary gland undergoes extensive remodeling to allow for milk production. Our long-term goal is to leverage the information we uncovered through this effort to target key genes we identified through gene network analysis aiming to prevent the progression of DCIS to invasive tumors. Moreover, this new technology can be utilized to explore the role of genes during specific times during development and tumor formation. Citation Format: Silva Krause, Amy Brock, Michael Goldberg, Donald E. Ingber. Prevention of mammary tumor progression via intraductal injection of nanoparticle-formulated siRNA. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2025. doi:10.1158/1538-7445.AM2013-2025

We utilize a computational systems biology approach to identify gene targets with the potential to serve as targets for therapeutic RNAi. Inferred gene regulatory network models were constructed using whole transcriptome expression profiling for progressive stages of mammary tumorigenesis in FVB C3(1)-SV40Tag mice. These computational models predicted key mediator transcription factors that are critical for hyperplasia and ductal filling in the early stages of disease progression. Putative mediator transcription factors were targeted by RNAi first in a mammary spheroid assay. Based on their ability to restore lumen formation and normal apical-basal polarization to mammary tumor cells, two transcription factors, Hoxa1 and Hoxb13, were selected for validation in vivo. siRNA targeting each of these transcription factors was formulated with lipidoid nanoparticles and delivered to the mammary gland via intraductal injection. Silencing Hoxa1 resulted in a significant reduction in tumor incidence in FVB C3(1)-SV40Tag. This represents a novel strategy to achieve localized silencing of a target gene in the mammary epithelium. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-05-04.

Male breast cancer accounts for about 1% of all breast cancers and even though it is a rare disease, the incidence rate has increased steadily during the past 20 years. Study of the male form of the disease has been hampered by a lack of relevant animal models as it is commonly believed that male mammary glands do not contain ducts. Female FVB C3(1)-SV40Tag transgenic mice spontaneously develop hyperplastic mammary lesions that progress to invasive mammary tumors, whereas most of the transgenic males develop prostatic hyperplasia that progresses to adenocarcinoma. However, upon closer examination, we discovered that approximately 55% of transgenic males develop mammary tumors by 40 weeks of age, often prior to developing prostate tumors. The course of male mammary development is similar to that observed in the females but with a later onset. Hyperplastic ducts form as early as 15 weeks of age and progress into DCIS-resembling tumors before developing into invasive tumors that invade locally into adipose tissue and muscle and occasionally form metastases to lung and other organs. We then examined male mammary development in the FVB background strain (wild type) and showed that all males develop mammary ducts that continue to grow and branch throughout adulthood. The morphology of these ducts was similar to that of females with a central lumen surrounded by concentric layers of luminal epithelium, myoepithelium, a basement membrane and surrounding stroma. A study of CD1 male mice confirmed that male mammary development was not specific to the FVB strain but that CD1 males showed similar mammary ductal development. Male mammary development in FVB wild type animals and tumor progression in the transgenic males was characterized in terms of cell proliferation, apoptosis and expression of various markers of differentiation including hormone receptor status. In summary, our results demonstrate that FVB C3(1)-SV40Tag transgenic males represent a highly robust and reproducible model for the study male breast cancer. Our long-term goal is to leverage the information we uncovered through this effort to design novel therapies for breast cancer treatment in men. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3273. doi:1538-7445.AM2012-3273

The majority of breast cancer therapeutics aim at slowing tumor expansion through selective killing of tumor cells. Given the enormous plasticity of cells (highlighted by recent advances in the iPS field), we instead sought to induce a cell state transition from a mammary tumor cell phenotype to a normal mammary epithelial cell state. We have tackled the critical problem of identifying target genes for the normalization or differentiation of mammary tumor cells by employing a systems biology approach that integrates computational modeling with in vivo and in vitro studies. In vivo gene expression data of mammary tumor progression in the FVB C3(1) SV40Tag transgenic mouse model were collected at multiple time points as the disease progressed from normal gland to DCIS-like lesions to invasive carcinoma. Gene regulatory network models predicted novel transcription factor targets that appeared critical to the rise of the tumor phenotype. These putative gene targets were then assessed in a 3D spheroid assay. RNAi inhibition of two computationally predicted transcription factors reverted mammary tumor cells to a normalized mammary epithelial cell phenotype, with appropriate apical/basal polarization, lumen formation and reduced proliferative index. This integrated strategy has pinpointed transcription factors for RNAi therapeutic targeting in vivo but importantly, the method is not limited to breast cancer and may be useful for many different tumor types. In summary, our major advance has been the demonstration that a gene regulatory network reverse engineering approach can identify genes that are causatively involved in cancer progression and that represent viable therapeutic differentiation targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4920. doi:1538-7445.AM2012-4920

Breast cancer is one of the most prominent cancers in women with approximately 40,000 women dying each year of the disease in the United States alone. Although cancer has a strong genetic basis, the tissue microenvironment also plays a significant role in tumor formation and progression. To explore this process in greater detail, we analyzed tissue structure, cellular biochemistry and gene expression change during tumor progression in a previously developed FVB C3(1)-SV40Tag transgenic mouse model in which the females spontaneously develop mammary tumors that undergo changes in cell tissue morphology that closely mimic those observed during human breast cancer progression. Histological characterization of breast tissues isolated from 8, 12, 16 and 20 week old female mice confirmed that this model is extremely robust with normal glandular morphology being observed at 8 weeks, ductal carcinoma in situ (DCIS) morphology appearing at 12 weeks and 16 weeks and large tumors with metastatic lesions by 20 weeks of age. More careful analysis revealed that tumor formation did not scale directly with expression of SV40 large T antigen that is the drive gene in this transgenic model, and instead appeared to be more closely related to variations in the tissue microenvironment. This was supported by the observation that the number of myoepithelial cells and expression levels of known breast cancer differentiation markers, including estrogen and progesterone receptors, varied among ducts within tissues at each time point even though the majority of epithelial cells in these ducts expressed T antigen. Gene microarray analysis of mammary tissues from these time points revealed consistent changes in genome-wide expression profiles, and several interesting candidates are currently being analyzed regarding their potential involvement in early microenvironmental changes that might drive the tumor progression. In summary, our preliminary results showed that despite expression of constitutively active large T antigen in the breast epithelial cells of these transgenic mice, we observe heterogeneous tumor formation within mammary tissues. These local variations in tumor progression may be related to regional changes in stromal and extracellular matrix components that are currently being analyzed by immunohistochemical analysis. Our long-term goal is to leverage the information we uncover through this effort to design differentiation therapies that suppress tumor cell growth, induce differentiation and hence, reverse breast cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 520. doi:10.1158/1538-7445.AM2011-520

Cancer is a developmental disease, requiring the concerted switching of multiple cellular gene programs. Here we ask whether we can use computational network inference models in a systems biology-driven approach to identify changes in connectivity within the underlying gene regulatory network during cancer progression, as well as novel key mediator genes that are central to altered disease state networks. To investigate this hypothesis, we reverse engineered the gene regulatory networks of mammary glands within normal mice and transgenic C3(1)-SV40Tag mice that undergo mammary gland transformation and tumor progression. RNA was isolated from the mammary glands of transgenic mice at 8, 12, 16 and 20 weeks of age; wild type mice were analyzed at 8 weeks. Whole genome microarray profiles were obtained for each stage. A network inference algorithm was trained on a set of 3000 genome-wide gene expression signatures from public databases. A Mode-of-action Network Inference (MNI) algorithm was used to identify key mediator genes at each stage of the gene regulatory network during different stages of cancer progression in the transgenic mouse mammary gland based on z-score significance. From this initial set of candidates, we focused on key mediator genes classified by gene ontology analysis as transcription factors. To experimentally assess the functional relevance of genes identified by this computational approach, key mediator genes were silenced in vitro in three-dimensional (3D) cultures of mouse mammary tumor epithelial cells derived from the transgenic animals. In contrast to normal mammary epithelial cells, tumor cells failed to form polarized structures containing a lumen in 3D spheroid culture. Importantly, siRNA knockdown of key mediator genes induced these cancer cells to revert to a normalized phenotype in 3D culture. These data demonstrate the feasibility of using genome reverse engineering approaches to identify genes that can potentially induce cancer reversal. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4924. doi:10.1158/1538-7445.AM2011-4924

Background Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D) in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. Methods The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks), the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. Results Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. Conclusions Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to induce near-complete tumor phenotype reversion. These human breast 3D tissue morphogenesis models promise to become reliable tools for studying tissue interactions, therapeutic screening and drug target validation.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2017-06-05T16:38:26Z No. of bitstreams: 1 1_v.9_3.pdf: 592429 bytes, checksum: 99b5782f5399dd8c66162b4211ee5e62 (MD5) Made available in DSpace on 2017-06-05T16:38:26Z (GMT). No. of bitstreams: 1 1_v.9_3.pdf: 592429 bytes, checksum: 99b5782f5399dd8c66162b4211ee5e62 (MD5) Previous issue date: 2010-09 O tratamento ortodôntico-cirúrgico das deformidades dento-faciais pode ser estimulado por motivação estética, funcional ou ambas. Contudo, devido à característica agressiva do procedimento cirúrgico ortognático, conhecer e entender os motivos que levam um paciente a submeter-se a esse tratamento se torna relevante. O objetivo deste estudo foi apresentar as motivações estéticas de 20 pacientes atendidos na Faculdade de Odontologia da ULBRA – Canoas, que buscavam por cirurgia ortognática. Salvador