The BA-BCS 2021: An Initial "Trial" for Integrating Basic Science and Medical Progress on Breast Cancer in a Latin-American Country.

The first Buenos Aires Breast Cancer Symposium (BA-BCS) was held in a virtual format, between the 17th and the 21st of May 2021. The main goal of the meeting was to facilitate the interaction among physicians and basic researchers from South America and with peers from the rest of the world. To embrace their different interests and concerns, the congress included not only talks on basic, translational and clinical research, but also round tables to discuss diagnostic methods, research financing and biobank management, as well as virtual poster sessions in which the youngest fellows presented their recent findings. This report provides a brief overview of the talks delivered during the meeting, which addressed a wide variety of vital issues for breast cancer research mostly focused on the accurate diagnosis, prevention and treatment of this illness. The presentations included a wide spectrum of themes including hormone receptors and the relevance of their mutations, immunotherapy, cancer stem cells, mouse models, environmental hazards, genetics and epigenetics, local and systemic therapies, liquid biopsies, the metastatic cascade, therapy resistance and dormancy, among others.

A Heterotypic Tridimensional Model to Study the Interaction of Macrophages and Glioblastoma In Vitro.

BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor, and macrophages account for 30-40% of its composition. Most of these macrophages derive from bone marrow monocytes playing a crucial role in tumor progression. Unraveling the mechanisms of macrophages-GBM crosstalk in an appropriate model will contribute to the development of specific and more successful therapies. We investigated the interaction of U87MG human GBM cells with primary human CD14+ monocytes or the THP-1 cell line with the aim of establishing a physiologically relevant heterotypic culture model. METHODS: primary monocytes and THP-1 cells were cultured in the presence of U87MG conditioned media or co-cultured together with previously formed GBM spheroids. Monocyte differentiation was determined by flow cytometry. RESULTS: primary monocytes differentiate to M2 macrophages when incubated with U87MG conditioned media in 2-dimensional culture, as determined by the increased percentage of CD14+CD206+ and CD64+CD206+ populations in CD11b+ cells. Moreover, the mitochondrial protein p32/gC1qR is expressed in monocytes exposed to U87MG conditioned media. When primary CD14+ monocytes or THP-1 cells are added to previously formed GBM spheroids, both invade and establish within them. However, only primary monocytes differentiate and acquire a clear M2 phenotype characterized by the upregulation of CD206, CD163, and MERTK surface markers on the CD11b+CD14+ population and induce alterations in the sphericity of the cell cultures. CONCLUSION: our results present a new physiologically relevant model to study GBM/macrophage interactions in a human setting and suggest that both soluble GBM factors, as well as cell-contact dependent signals, are strong inducers of anti-inflammatory macrophages within the tumor niche.

iRGD-guided tamoxifen polymersomes inhibit estrogen receptor transcriptional activity and decrease the number of breast cancer cells with self-renewing capacity.

BACKGROUND: Tamoxifen (Tam) is the most frequent treatment for estrogen receptor (ER) positive breast cancer. We recently showed that fibronectin (FN) leads to Tam resistance and selection of breast cancer stem cells. With the aim of developing a nanoformulation that would simultaneously tackle ER and FN/ß1 integrin interactions, we designed polyethylene glycol-polycaprolactone polymersomes polymersomes (PS) that carry Tam and are functionalized with the tumor-penetrating iRGD peptide (iRGD-PS-Tam). RESULTS: Polyethylene glycol-polycaprolactone PS were assembled and loaded with Tam using the hydration film method. The loading of encapsulated Tam, measured by UPLC, was 2.4 ± 0.5 mol Tam/mol polymer. Physicochemical characterization of the PS demonstrated that iRGD functionalization had no effect on morphology, and a minimal effect on the PS size and polydispersity (176 nm and Pdi 0.37 for iRGD-TAM-PS and 171 nm and Pdi 0.36 for TAM-PS). iRGD-PS-Tam were taken up by ER+ breast carcinoma cells in 2D-culture and exhibited increased penetration of 3D-spheroids. Treatment with iRGD-PS-Tam inhibited proliferation and sensitized cells cultured on FN to Tam. Mechanistically, treatment with iRGD-PS-Tam resulted in inhibition ER transcriptional activity as evaluated by a luciferase reporter assay. iRGD-PS-Tam reduced the number of cells with self-renewing capacity, a characteristic of breast cancer stem cells. In vivo, systemic iRGD-PS-Tam showed selective accumulation at the tumor site. CONCLUSIONS: Our study suggests iRGD-guided delivery of PS-Tam as a potential novel therapeutic strategy for the management of breast tumors that express high levels of FN. Future studies in pre-clinical in vivo models are warranted.

Gene expression profile and cancer-associated pathways linked to progesterone receptor isoform a (PRA) predominance in transgenic mouse mammary glands.

BACKGROUND: Progesterone receptor (PR) is expressed from a single gene as two isoforms, PRA and PRB. In normal breast human tissue, PRA and PRB are expressed in equimolar ratios, but isoform ratio is altered during malignant progression, usually leading to high PRA:PRB ratios. We took advantage of a transgenic mouse model where PRA isoform is predominant (PRA transgenics) and identified the key transcriptional events and associated pathways underlying the preneoplastic phenotype in mammary glands of PRA transgenics as compared with normal wild-type littermates. METHODS: The transcriptomic profiles of PRA transgenics and wild-type mammary glands were generated using microarray technology. We identified differentially expressed genes and analyzed clustering, gene ontology (GO), gene set enrichment analysis (GSEA), and pathway profiles. We also performed comparisons with publicly available gene expression data sets of human breast cancer. RESULTS: We identified a large number of differentially expressed genes which were mainly associated with metabolic pathways for the PRA transgenics phenotype while inflammation- related pathways were negatively correlated. Further, we determined a significant overlap of the pathways characterizing PRA transgenics and those in breast cancer subtypes Luminal A and Luminal B and identified novel putative biomarkers, such as PDHB and LAMB3. CONCLUSION: The transcriptional targets identified in this study should facilitate the formulation or refinement of useful molecular descriptors for diagnosis, prognosis, and therapy of breast cancer.

Reversal of the Migratory and Invasive Phenotype of Ras-Transfected Mammary Cells by Photodynamic Therapy Treatment.

Photodynamic therapy (PDT) is a non-thermal technique for inducing tumor damage following administration of a light-activated photosensitizing drug (PS). In a previous work we found that PDT induces cytoskeleton changes in HB4a-Ras cells (human mammary breast carcinoma HB4a cells transfected with the RAS oncogene). In the present work we have studied the migratory and invasive features and the expression of proteins related to these processes on HB4a-Ras cells after three successive cycles of PDT using different PSs: 5-aminolevulinic acid (ALA), Verteporfin (Verte), m-tetrahydroxyphenylchlorin (m-THPC), and Merocyanine 540 (MC). A slight (1.25- to 2-fold) degree of resistance was acquired in cell populations subjected to the three successive PDT treatments. However, complete cell killing was achieved after a light dose increase. Regardless of the PS employed, all the PDT-treated populations had shorter stress fibres than the untreated control HB4a-Ras cells, and the number of dorsal stress fibres was decreased in the PDT-treated populations. E-Cadherin distribution, which was already aberrant in HB4a-Ras cells, became even more diffuse in the PDT-treated populations, though its expression was increased in some of them. The strong migratory and invasive ability of HB4a-Ras cells in vitro was impaired in all the PDT-treated populations, with a behavior that was similar to the parental non-tumoral HB4a cells. MMP-2 and -9 metalloproteinase activities were also impaired in the PDT-treated populations. The evidence presented herein suggests that the cells surviving PDT would be less metastatic than the initial population. These findings encourage the use of PDT in combination with other treatments such as intraoperative or post-surgery therapeutic procedures. J. Cell. Biochem. 118: 464-477, 2017. © 2016 Wiley Periodicals, Inc.

Tamoxifen selects for breast cancer cells with mammosphere forming capacity and increased growth rate.

Using the M05 mouse mammary tumor model and the MCF-7 cell line, we investigated the effect of tamoxifen treatment on the fraction of breast cancer cells with self-renewing capacity both in vitro and in vivo. We found that pretreatment with 4-OH-tamoxifen leads to an increase in cells with the ability of forming mammospheres that express lower levels of ER-α and increased expression of transcription factors associated with pluripotency. Moreover, exposure on plastic to 4-OH-tamoxifen by itself leads to an upregulation of these transcription factors. M05 tumors grown in mice treated with tamoxifen have a higher percentage of cells with self-renewing capacity and this proportion is conserved when tumors are passaged to nontreated mice. Furthermore, interruption of tamoxifen leads to increased tumor growth compared to tumors grown in mice that were never exposed to the antiestrogen. In addition, these tumors are characterized by a higher number of CD24(l)CD29(h) cells compared to tumors grown in nontreated mice. Treatment in vitro with 4-OH-tamoxifen for 5 days leads to a long lasting increase in the proportion of cells with self-renewing capacity even after 1 month of growth in the absence of the antiestrogen. Finally, we compared the mammosphere forming capacity of hormone dependent and independent passages of the M05 tumor and found that hormone independence is associated to an increase in cells with self-renewing capacity. Our results support previous findings that suggest that endocrine treatment selects for cells with stem cell properties.

AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system.

Angiotensin II (AngII), the main effector peptide of the renin-angiotensin system (RAS), participates in multiple biological processes, including cell growth, apoptosis, and tissue remodeling. Since AngII activates, in different cell types, signal transducing pathways that are critical for mammary gland postlactational regression, we investigated the role of the RAS during this process. We found that exogenous administration of AngII in mammary glands of lactating Balb/c mice induced epithelium apoptosis [2.9±0.5% (control) vs. 9.6±1.1% (AngII); P < 0.001] and activation of the proapoptotic factor STAT3, an effect inhibited by irbesartan, an AT(1) receptor blocker. Subsequently, we studied the expression kinetics of RAS components during involution. We found that angiotensin-converting enzyme (ACE) mRNA expression peaked 6 h after weaning (5.7-fold; P<0.01), while induction of angiotensinogen and AT(1) and AT(2) receptors expression was detected 96 h after weaning (6.2-, 10-, and 6.2-fold increase, respectively; P<0.01). To assess the role of endogenously generated AngII, mice were treated with losartan, an AT(1) receptor blocker, during mammary involution. Mammary glands from losartan-treated mice showed activation of the survival factors AKT and BCL-(XL), significantly lower LIF and TNF-α mRNA expression (P<0.05), reduced apoptosis [12.1±2.1% (control) vs. 4.8±0.7% (losartan); P<0.001] and shedding of epithelial cells, inhibition of MMP-9 activity in a dose-dependent manner (80%; P<0.05; with losartan IC(50) value of 6.9 mg/kg/d] and lower collagen deposition and adipocyte invasion causing a delayed involution compared to vehicle-treated mice. Furthermore, mammary glands of forced weaned AT(1A)- and/or AT(1B)-deficient mice exhibited retarded apoptosis of epithelial cells [6.3±0.95% (WT) vs. 3.3±0.56% (AT(1A)/AT(1B) DKO); P<0.05] with remarkable delayed postlactational regression compared to wild-type animals. Taken together, these results strongly suggest that AngII, via the AT(1) receptor, plays a major role in mouse mammary gland involution identifying a novel role for the RAS. angiotensin system.

The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through ß1 integrin.

Tamoxifen resistance has been largely attributed to genetic alterations in the epithelial tumor cells themselves, such as overexpression of HER-2/Neu. However, in the clinic, only about 15-20% of cases of HER-2/Neu amplification has actually been correlated to the acquisition of endocrine resistance, suggesting that other mechanisms must be involved as well. Using the epithelial LM05-E and the fibroblastic LM05-F cell lines, derived from the estrogen dependent spontaneous M05 mouse mammary tumor, as well as MCF-7 cells, we analyzed whether soluble stromal factors or extracellular matrix components protected against tamoxifen induced cell death. Involvement of signaling pathways was determined by using specific inhibitors and western blot, and phosphorylation of the estrogen receptor alpha by western blot and immunofluorescence. Soluble factors produced by the fibroblastic cells protect the epithelial tumor cells from tamoxifen-induced cell death through a mechanism that involves EGFR and matrix metalloproteinases upstream of PI3K/AKT. Exogenous fibronectin by itself confers endocrine resistance through interaction with ß1 integrin and activation of PI3K/AKT and MAPK/ERK 1/2 pathways. The conferred resistance is reversed by blocking ß1 integrin. We show also that treatment with both conditioned medium and fibronectin leads to the phosphorylation of the estrogen receptor at serine-118, suggesting stromal factors as modulators of ER activity. Our results show that the tumor microenvironment can modulate tamoxifen resistance, providing an alternative explanation for why patients become refractory to hormone-therapy.

Buenos Aires Breast Cancer Symposium BA-BCS 2021: Tribute to Dr. Christiane Dosne Pasqualini in her 101 birthday, May, 17-21, 2021.

Session 1: Tumor heterogeneity and breast cancer therapy. Session 2: From hormone receptors to the immune system: the evolution of therapeutic targets in breast cancer. Session 3: Cancer stem cells and de-differentiated phenotype. Session 4: Mouse models for studying breast cancer initiation and progression. Session 5: Round Table 1 - Genomics Platforms. Session 6: Genetics and Epigenetics of Breast Cancer. Session 7: Understanding the metastatic cascade to learn how to inhibit tumor progression. Session 8: Round Table 2 - Biorepositories and sample management. Session 9: Estrogen receptors: their involvement in endocrine resistance and dormancy. Session 10: Novel targets in the era of precision medicine. Session 11: Round Table 3 - Interaction among government, non-government agencies, and industry for funding and promoting breast cancer translational research. Session 12: Local and systemic therapies. Session 13: New developments in diagnosis and epidemiology of breast cancer.

Learning deep features for dead and living breast cancer cell classification without staining.

Automated cell classification in cancer biology is a challenging topic in computer vision and machine learning research. Breast cancer is the most common malignancy in women that usually involves phenotypically diverse populations of breast cancer cells and an heterogeneous stroma. In recent years, automated microscopy technologies are allowing the study of live cells over extended periods of time, simplifying the task of compiling large image databases. For instance, there have been several studies oriented towards building machine learning systems capable of automatically classifying images of different cell types (i.e. motor neurons, stem cells). In this work we were interested in classifying breast cancer cells as live or dead, based on a set of automatically retrieved morphological characteristics using image processing techniques. Our hypothesis is that live-dead classification can be performed without any staining and using only bright-field images as input. We tackled this problem using the JIMT-1 breast cancer cell line that grows as an adherent monolayer. First, a vast image set composed by JIMT-1 human breast cancer cells that had been exposed to a chemotherapeutic drug treatment (doxorubicin and paclitaxel) or vehicle control was compiled. Next, several classifiers were trained based on well-known convolutional neural networks (CNN) backbones to perform supervised classification using labels obtained from fluorescence microscopy images associated with each bright-field image. Model performances were evaluated and compared on a large number of bright-field images. The best model reached an AUC = 0.941 for classifying breast cancer cells without treatment. Furthermore, it reached AUC = 0.978 when classifying breast cancer cells under drug treatment. Our results highlight the potential of machine learning and computational image analysis to build new diagnosis tools that benefit the biomedical field by reducing cost, time, and stimulating work reproducibility. More importantly, we analyzed the way our classifiers clusterize bright-field images in the learned high-dimensional embedding and linked these groups to salient visual characteristics in live-dead cell biology observed by trained experts.

2-C-methyluridine modified hammerhead ribozyme against the estrogen receptor.

A new synthesis of 2'-C-methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2'-hydroxyl group. Comparison to previous protecting strategies and analysis of stability under 5'-DMTr removing conditions are discussed. The synthetic incorporation of this modified nucleoside into the catalytic core of a hammerhead ribozyme against the estrogen receptor alpha protein (ER-alpha), and transfection experiments in MCF-7 cell line are also presented.

Alterations in Progesterone Receptor Isoform Balance in Normal and Neoplastic Breast Cells Modulates the Stem Cell Population.

To investigate the role of PR isoforms on the homeostasis of stem cells in the normal and neoplastic mammary gland, we used PRA and PRB transgenic mice and the T47D human breast cancer cell line and its derivatives, T47D YA and YB (manipulated to express only PRA or PRB, respectively). Flow cytometry and mammosphere assays revealed that in murine breast, overexpression of PRB leads to an increase in luminal and basal progenitor/stem cells. Ovariectomy had a negative impact on the luminal compartment and induced an increase in mammosphere-forming capacity in cells derived from WT and PRA mice only. Treatment with ICI 182,780 augmented the mammosphere-forming capacity of cells isolated from WT and PRA mice, whilst those from PRB remained unaltered. T47D YB cells showed an increase in the CD44+/CD24Low/- subpopulation; however, the number of tumorspheres did not vary relative to T47D and YA, even though they were larger, more irregular, and had increased clonogenic capacity. T47D and YA tumorspheres were modulated by estrogen/antiestrogens, whereas YB spheres remained unchanged in size and number. Our results show that alterations in PR isoform balance have an impact on normal and tumorigenic breast progenitor/stem cells and suggest a key role for the B isoform, with implications in response to antiestrogens.

Estrogen and progesterone receptors have distinct roles in the establishment of the hyperplastic phenotype in PR-A transgenic mice.

INTRODUCTION: Expression of the A and B forms of progesterone receptor (PR) in an appropriate ratio is critical for mammary development. Mammary glands of PR-A transgenic mice, carrying an additional A form of PR as a transgene, exhibit morphological features associated with the development of mammary tumors. Our objective was to determine the roles of estrogen (E) and progesterone (P) in the genesis of mammary hyperplasias/preneoplasias in PR-A transgenics. METHODS: We subjected PR-A mice to hormonal treatments and analyzed mammary glands for the presence of hyperplasias and used BrdU incorporation to measure proliferation. Quantitative image analysis was carried out to compare levels of latency-associated peptide and transforming growth factor beta 1 (TGFbeta1) between PR-A and PR-B transgenics. Basement membrane disruption was examined by immunofluorescence and proteolytic activity by zymography. RESULTS: The hyperplastic phenotype of PR-A transgenics is inhibited by ovariectomy, and is reversed by treatment with E + P. Studies using the antiestrogen ICI 182,780 or antiprogestins RU486 or ZK 98,299 show that the increase in proliferation requires signaling through E/estrogen receptor alpha but is not sufficient to give rise to hyperplasias, whereas signaling through P/PR has little impact on proliferation but is essential for the manifestation of hyperplasias. Increased proliferation is correlated with decreased TGFbeta1 activation in the PR-A transgenics. Analysis of basement membrane integrity showed loss of laminin-5, collagen III and collagen IV in mammary glands of PR-A mice, which is restored by ovariectomy. Examination of matrix metalloproteases (MMPs) showed that total levels of MMP-2 correlate with the steady-state levels of PR, and that areas of laminin-5 loss coincide with those of activation of MMP-2 in PR-A transgenics. Activation of MMP-2 is dependent on treatment with E and P in ovariectomized wild-type mice, but is achieved only by treatment with P in PR-A mice. CONCLUSIONS: These data establish a link between hormonal response, proliferation, modulation of MMP activity and maintenance of basement membrane integrity that depend on a balance in the expression levels of PR-A and PR-B isoforms. Notably, concomitant increased proliferation, due to inhibition of TGFbeta1 activation, and loss of basement membrane integrity, via increased MMP-2 activity, appear to be prerequisites for the PR-A hyperplastic phenotype.

A spontaneous estrogen dependent, tamoxifen sensitive mouse mammary tumor: a new model system to study hormone-responsiveness in immune competent mice.

Currently, an in vivo spontaneous model of estrogen dependent, tamoxifen sensitive breast cancer does not exist. We show here the characterization of the M05 mammary tumor that appeared spontaneously in a 1-year-old virgin female BALB/c mouse in our animal facility. The M05 tumor is a semi-differentiated adenocarcinoma that expresses estrogen and progesterone receptors. When it was transplanted to either male or ovariectomized female mice it did not grow. Moreover, ovariectomy or treatment with tamoxifen of tumor bearing mice led to a halt in tumor growth. Treatment of ovariectomized mice that had been inoculated with the M05 tumor showed that only estradiol, but not progesterone, promoted the re-growth of the tumor. Finally, after passage nine, tumor growth was achieved in male and ovariectomized female mice suggesting that the tumor had progressed to hormone independence. However, like often found in the clinic, expression of estrogen and progesterone receptors was maintained. This model mimics the biology of estrogen receptor positive breast cancer in humans and presents itself as an invaluable tool for the study of endocrine resistance in a physiologically relevant setting.

Establishment of an in vitro estrogen-dependent mouse mammary tumor model: a new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal-epithelial interactions.

Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for alpha-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.

The Tumor Microenvironment as a Regulator of Endocrine Resistance in Breast Cancer.

Estrogen receptor positive breast neoplasias represent over 70% of diagnosed breast cancers. Depending on the stage at which the tumor is detected, HER2 status and genomic risk, endocrine therapy is combined with either radio, chemo and/or targeted therapy. A growing amount of evidence supports the notion that components of the tumor microenvironment play specific roles in response to treatment and that strategies targeting these key interactions with tumor cells could pave the way to a new generation of therapies. In this review, we analyze the evidence suggesting different components of the tumor microenvironment play a role in hormone receptor positive breast cancer progression. In particular we focus on the immune system, carcinoma associated fibroblasts and the extracellular matrix. Further insight into the cross talk between these constituents of the microenvironment and the tumor cells may lead to therapies that eliminate disseminated metastatic cells early on, and thus reduce distant disease relapse which is the leading cause of death for patients who are diagnosed with this illness.

Human-specific approaches to brain research for the 21st century: a South American perspective.

The 21st century paradigm in toxicology, which emphasizes mechanistic understanding and species-relevant modeling of human biology and pathophysiology, is gaining traction in the wider biosciences through a global workshop series organized by the BioMed21 Collaboration. The second of this series, entitled Emerging Technology Toward Pathway-Based Human Brain Research, was held in Brazil in 2017, bringing together leading South American and international scientists, research funders and other stakeholders. The aims were to foster strategic scientific dialogue and identify actionable consensus recommendations as a first step toward a roadmap for 21st century, human-specific health research and funding in the region.

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells.

Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with ß1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα+ vesicles are present within human breast tissues, and colocalization with ß1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.

Organoids: A historical perspective of thinking in three dimensions.

In the last ten years, there has been a dramatic surge in the number of publications where single or groups of cells are grown in substrata that have elements of basement membrane leading to the formation of tissue-like structures referred to as organoids. However, this field of research began many decades ago, when the pioneers of cell culture began to ask questions we still ask today: How does organogenesis occur? How do signals integrate to make such vastly different tissues and organs given that the sequence of the genome in our trillions of cells is identical? Here, we summarize how work over the past century generated the conceptual framework that has allowed us to make progress in the understanding of tissue-specific morphogenetic programs. The development of cell culture systems that provide accurate and physiologically relevant models are proving to be key in establishing appropriate platforms for the development of new therapeutic strategies.

Laminin Modulates the Stem Cell Population in LM05-E Murine Breast Cancer Cells through the Activation of the MAPK/ERK Pathway.

PURPOSE: We investigated the effects of laminin on the fraction of cells with self-renewing capacity in the estrogen-dependent, tamoxifen-sensitive LM05-E breast cancer cell line. We also determined whether laminin affected the response to tamoxifen. MATERIALS AND METHODS: The LM05-E breast cancer cell line was used as a model for all experiments. Aldehyde dehydrogenase (ALDH) activity, clonogenic and mammosphere assays were performed to measure the effects of laminin on modulation of the stem cell subpopulation. Pluripotent gene expression was analyzed by reverse transcriptase-polymerase chain reaction. The involvement of the mitogen-activated protein kinase (MAPK)/ERK pathway was determined using specific inhibitors. The effects of laminin on the response to tamoxifenwere determined and the involvement of α6 integrin was investigated. RESULTS: We found that pretreatment with laminin leads to a decrease in cells with the ability to form mammospheres that was accompanied by a decrease in ALDH activity. Moreover, exposure of mammospheres to laminin reduced the capacity to form secondary mammospheres and decreased the expression of Sox-2, Nanog, and Oct-4. We previously reported that 4-OH-tamoxifen leads to an increase in the expression of these genes in LM05-E cells. Treatment with signaling pathway inhibitors revealed that the MAPK/ERK pathway mediates the effects of laminin. Finally, laminin induced tamoxifen resistance in LM05-E cells through α6 integrin. CONCLUSION: Our results suggest that the final number of cells with self-renewing capacity in estrogen-dependent breast tumors may result from the combined effects of endocrine treatment and microenvironmental cues.