Non-invasive Cells Research Articles

Emerin deficiency drives MCF7 cells to an invasive phenotype

During metastasis, cancer cells traverse the vasculature by squeezing through very small gaps in the endothelium. Thus, nuclei in metastatic cancer cells must become more malleable to move through these gaps. Our lab showed invasive breast cancer cells have 50% less emerin protein resulting in smaller, misshapen nuclei, and higher metastasis rates than non-cancerous controls. Thus, emerin deficiency was predicted to cause increased nuclear compliance, cell migration, and metastasis. We tested this hypothesis by downregulating emerin in noninvasive MCF7 cells and found emerin knockdown causes smaller, dysmorphic nuclei, resulting in increased impeded cell migration. Emerin reduction in invasive breast cancer cells showed similar results. Supporting the clinical relevance of emerin reduction in cancer progression, our analysis of 192 breast cancer patient samples showed emerin expression inversely correlates with cancer invasiveness. We conclude emerin loss is an important driver of invasive transformation and has utility as a biomarker for tumor progression.

Emerin deficiency drives MCF7 cells to an invasive phenotype.

During metastasis, cancer cells traverse the vasculature by squeezing through very small gaps in the endothelium. Thus, nuclei in metastatic cancer cells must become more malleable to move through these gaps. Our lab showed invasive breast cancer cells have 50% less emerin protein resulting in smaller, misshapen nuclei, and higher metastasis rates than non-cancerous controls. Thus, emerin deficiency was predicted to cause increased nuclear compliance, cell migration, and metastasis. We tested this hypothesis by downregulating emerin in noninvasive MCF7 cells and found emerin knockdown causes smaller, dysmorphic nuclei, resulting in increased impeded cell migration. Emerin reduction in invasive breast cancer cells showed similar results. Supporting the clinical relevance of emerin reduction in cancer progression, our analysis of 192 breast cancer patient samples showed emerin expression inversely correlates with cancer invasiveness. We conclude emerin loss is an important driver of invasive transformation and has utility as a biomarker for tumor progression.

Ductal carcinoma in situ: Molecular and cellular basis of malignancy.

e12582 Background: The goal of our study is to improve the diagnosis and treatment of DCIS through the identification of the factors driving its invasion and metastasis. Methods: We utilized the Mouse INtraDuctal (MIND) model to investigate the mechanism(s) by which some DCIS become invasive while others remain indolent. This model closely replicates the human condition by injecting non-invasive epithelial cells from patients into mouse mammary ducts to form in situ lesions. In some cases, these cells will display invasive behavior by overcoming the natural barriers of myoepithelium. To compare these two processes, we performed single-cell (sc) RNA-sequencing analysis of 60,000 cells from the pre-transplant time-point representing 17 DCIS (11 became invasive, 6 remained non-invasive). We also performed scATAC/RNA sequencing of a DCIS with associated IDC. Results: Our analysis revealed that the proportion of endothelial cells was higher in cases that became invasive, while the proportion of plasma and T cells was higher in those that remained non-invasive (t-test; P<0.05). This finding suggested that invasive cells reprogrammed the immune microenvironment to exclude immune cells as a means of evading immune surveillance. Additionally, we performed cell-cell interaction (CCI) analysis by CellPhoneDB. This analysis revealed highly ranked CCIs in invasive cases, including luminal cell interactions with pericytes via Wnt5A-SFRP1. On the other hand, highly ranked CCIs in non-invasive cases included HR-positive luminal cell interactions with T cells via CD226-Nectin-2 and CD96-Nectin1. Notably, CD226 and CD96 are T cell receptors that enhance CD8+ T cell activity. These results support the potential role of T-cell interactions with DCIS luminal cells in preventing their invasive transition. We conducted additional analysis of sc-RNA-sequencing data using a stem cell-based gene signature algorithm called CytoTRACE. Our analysis revealed that invasive cells were enriched in cell clusters with significantly higher stem cell scores than non-invasive cells. In another analysis of a patient DCIS with associated IDC using sc-ATAC and RNA sequencing, we discovered that the open chromatin regions in the stem cell cluster were enriched for FOXA1 binding motifs. This cluster also had the highest expression of two pioneer factors (FOXA1 and GRHL2), NEAT1 (paraspeckle protein), ERBB2, 3,4, and ANKRD30A (cell surface protein). Our findings suggest that the pioneer factors FOXA1 and GRHL2 play a crucial role as transcriptional drivers of stemness. Additionally, the ANKRD30A could be used to isolate stem cell clusters to validate their self-renewal potential. Conclusions: DCIS progression is facilitated by interactions between stromal, immune, and epithelial cells, which lead to epigenetic and transcriptional changes. This results in the formation of stem-like cells that are characterized by their plasticity and invasive capacity.

Effects of Garlic on Breast Tumor Cells with a Triple Negative Phenotype: Peculiar Subtype-Dependent Down-Modulation of Akt Signaling.

Breast cancer includes tumor subgroups with morphological, molecular, and clinical differences. Intrinsic heterogeneity especially characterizes breast tumors with a triple negative phenotype, often leading to the failure of even the most advanced therapeutic strategies. To improve breast cancer treatment, the use of natural agents to integrate conventional therapies is the subject of ever-increasing attention. In this context, garlic (Allium sativum) shows anti-cancerous potential, interfering with the proliferation, motility, and malignant progression of both non-invasive and invasive breast tumor cells. As heterogeneity could be at the basis of variable effects, the main objective of our study was to evaluate the anti-tumoral activity of a garlic extract in breast cancer cells with a triple negative phenotype. Established triple negative breast cancer (TNBC) cell lines from patient-derived xenografts (PDXs) were used, revealing subtype-dependent effects on morphology, cell cycle, and invasive potential, correlated with the peculiar down-modulation of Akt signaling, a crucial regulator in solid tumors. Our results first demonstrate that the effects of garlic on TNBC breast cancer are not unique and suggest that only more precise knowledge of the mechanisms activated by this natural compound in each tumor will allow for the inclusion of garlic in personalized therapeutic approaches to breast cancer.

Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding

Overexpression of the transmembrane matrix metalloproteinase MT1-MMP/MMP14 promotes cancer cell invasion. Here we show that MT1-MMP-positive cancer cells turn MT1-MMP-negative cells invasive by transferring a soluble catalytic ectodomain of MT1-MMP. Surprisingly, this effect depends on the presence of TKS4 and TKS5 in the donor cell, adaptor proteins previously implicated in invadopodia formation. In endosomes of the donor cell, TKS4/5 promote ADAM-mediated cleavage of MT1-MMP by bridging the two proteases, and cleavage is stimulated by the low intraluminal pH of endosomes. The bridging depends on the PX domains of TKS4/5, which coincidently interact with the cytosolic tail of MT1-MMP and endosomal phosphatidylinositol 3-phosphate. MT1-MMP recruits TKS4/5 into multivesicular endosomes for their subsequent co-secretion in extracellular vesicles, together with the enzymatically active ectodomain. The shed ectodomain converts non-invasive recipient cells into an invasive phenotype. Thus, TKS4/5 promote intercellular transfer of cancer cell invasiveness by facilitating ADAM-mediated shedding of MT1-MMP in acidic endosomes.

The dopamine receptor D1 inhibitor, SKF83566, suppresses GBM stemness and invasion through the DRD1-c-Myc-UHRF1 interactions.

Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.

Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)β1. We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFβ1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFβ1-induced fibroblast invasion and RHOA pathway activity. Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFβ1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-β) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-β signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-β biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-β signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV’s unique role, TGF-β signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-β. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-β signaling to nearly-normal levels. Otherwise, TGF-β signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-β signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-β signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-β signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-β biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-β signaling levels in breast cancer cells.

Expression pattern of osteopontin isoforms in malignant melanoma cell lines.

Osteopontin (OPN) is a secreted integrin-binding protein that plays a role in inflammation, cellular viability, cell adhesion and migration, cancer development, and diabetes through different mechanisms. The splice variants of OPN can play essential roles in cancer development, progression, and metastasis formation; however, limited data are available about the role of OPN isoforms in human malignant melanoma. Our goal was to define the gene expression patterns of five OPN variants (OPN4, OPN5, OPNa, OPNb, and OPNc), integrin, and CD44 receptor genes in primary and metastatic melanoma-originated cell lines (n = 19), and to explore the association of the expression patterns with clinicopathological parameters. We evaluated the invasive property of the cell lines and investigated the potential association between the invasion and gene expression of OPN isoforms. We found a significant rise in the expression of OPNc in the invasive cell lines compared to the noninvasive cells and detected significantly higher expression of the OPN splice variants in melanoma cell lines originating from more advanced stages tumors than cell lines originating from early-stage melanomas. The correlation analysis revealed that all five OPN variants positively correlated with ITGB3 and ITGA9, whereas OPN5 positively correlated with ITGB1, ITGAV, ITGA6, and CD44. OPN can activate extracellular signal-regulated kinase signaling through binding to α9β1 integrin, promoting melanoma tumor cell migration. It is possible that such associations between OPN splice variants and integrin receptors may play a role in melanoma progression. In conclusion, our findings suggest that high expression of OPNc correlates with the invasive behavior of melanoma cells.

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans.

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch in C. elegans

A growing body of evidence suggests that cell division and basement membrane invasion are mutually exclusive cellular behaviors. How cells switch between proliferative and invasive states is not well understood. Here, we investigated this dichotomy in vivo by examining two cell types in the developing Caenorhabditis elegans somatic gonad that derive from equipotent progenitors, but exhibit distinct cell behaviors: the post-mitotic, invasive anchor cell and the neighboring proliferative, non-invasive ventral uterine (VU) cells. We show that the fates of these cells post-specification are more plastic than previously appreciated and that levels of NHR-67 are important for discriminating between invasive and proliferative behavior. Transcription of NHR-67 is downregulated following post-translational degradation of its direct upstream regulator, HLH-2 (E/Daughterless) in VU cells. In the nuclei of VU cells, residual NHR-67 protein is compartmentalized into discrete punctae that are dynamic over the cell cycle and exhibit liquid-like properties. By screening for proteins that colocalize with NHR-67 punctae, we identified new regulators of uterine cell fate maintenance: homologs of the transcriptional co-repressor Groucho (UNC-37 and LSY-22), as well as the TCF/LEF homolog POP-1. We propose a model in which the association of NHR-67 with the Groucho/TCF complex suppresses the default invasive state in non-invasive cells, which complements transcriptional regulation to add robustness to the proliferative-invasive cellular switch in vivo.

Targeting tumor microenvironment of metastatic triple negative breast cancer cells via exosomes derived from non-invasive breast cancer cells for multi-drug resistance inhibition and enhancing drug susceptibility

Targeting tumor microenvironment of metastatic triple negative breast cancer cells via exosomes derived from non-invasive breast cancer cells for multi-drug resistance inhibition and enhancing drug susceptibility

Cluster of differentiation 147 (CD147) as potential membrane protein biomarker for bladder cancer cells

Studies reveal that alterations in membrane protein (MP) patterns are associated with underlying drug resistance to chemotherapy. Therefore, the tryptic-digested MPs from the bladder cancer cell line were subjected to global proteomics using LC-MS/MS to identify the highly expressed potential MPs in bladder cancer cells. Our findings revealed the identification of MP biomarkers, CD147, and caveolin-1. Immunocytochemistry analysis confirmed the presence of CD147 on the cell membrane, while caveolin-1 showed positive signals without apparent staining on the membrane, suggesting its existence in multiple locations. Western blot analysis confirmed the higher expression of CD147 in non-invasive (RT 112) and metastatic (UM-UC-13) bladder cancer cells compared to invasive bladder cancer cells (5637 and J82), suggesting its potential as an MP biomarker for both of the former subtypes. The identified MPs could be used as drug therapy targets aimed at improving drug sensitivity and enhancing treatment outcomes in bladder cancer patients. SignificanceIdentification of the membrane proteins associated with bladder cancer recurrence is crucial to understanding the mechanisms underlying the drug resistance to chemotherapy.

CBD Inhibits In Vivo Development of Human Breast Cancer Tumors.

Inflammation is a critical component of cancer development. Previously, we showed in vitro that IL-1β treatment of non-invasive human breast cancer MCF-7 cells promoted their transition to a malignant phenotype (6D cells). This epithelial-mesenchymal transition was reverted by exposure to cannabidiol (CBD). We show in a murine model that subcutaneous inoculation of 6D cells induced formation and development of tumors, the cells of which keep traits of malignancy. These processes were interrupted by administration of CBD under two schemes: therapeutic and prophylactic. In the therapeutic scheme, 6D cells inoculated mice developed tumors that reached a mean volume of 540 mm3 at 45 days, while 50% of CBD-treated mice showed gradual resorption of tumors. In the prophylactic scheme, mice were pre-treated for 15 days with CBD before cells inoculation. The tumors formed remained small and were eliminated under continuous CBD treatment in 66% of the animals. Histological and molecular characterization of tumors, from both schemes, revealed that CBD-treated cells decreased the expression of malignancy markers and show traits related with apoptosis. These results confirm that in vivo CBD blocks development of breast cancer tumors formed by cells induced to malignancy by IL-1β, endorsing its therapeutic potential for cancer treatment.

Cell contractility drives mechanical memory of oral squamous cell carcinoma.

Matrix stiffening is ubiquitous in solid tumors and can direct epithelial-mesenchymal transition (EMT) and cancer cell migration. Stiffened niche can even cause poorly invasive oral squamous cell carcinoma (OSCC) cell lines to acquire a less adherent, more migratory phenotype, but mechanisms and durability of this acquired "mechanical memory" are unclear. Here, we observed that contractility and its downstream signals could underlie memory acquisition; invasive SSC25 cells overexpress myosin II (vs. noninvasive Cal27 cells) consistent with OSCC. However, prolonged exposure of Cal27 cells to a stiff niche or contractile agonists up-regulated myosin and EMT markers and enabled them to migrate as fast as SCC25 cells, which persisted even when the niche softened and indicated "memory" of their prior niche. Stiffness-mediated mesenchymal phenotype acquisition required AKT signaling and was also observed in patient samples, whereas phenotype recall on soft substrates required focal adhesion kinase (FAK) activity. Phenotype durability was further observed in transcriptomic differences between preconditioned Cal27 cells cultured without or with FAK or AKT antagonists, and such transcriptional differences corresponded to discrepant patient outcomes. These data suggest that mechanical memory, mediated by contractility via distinct kinase signaling, may be necessary for OSCC to disseminate.

MicroRNA-188-5p inhibits hepatocellular carcinoma proliferation and migration by targeting forkhead box N2

BackgroundThis study aimed to identify the biological functions, expression modes, and possible mechanisms underlying the relationship between metastatic human hepatocellular carcinoma (HCC) and MicroRNA-188-5p (miR-188) dysregulation using cell lines.MethodsA decrease in miR-188 was detected in low and high metastatic HCC cells compared to that in normal hepatic cells and non-invasive cell lines. Gain- and loss-of-function experiments were performed in vitro to investigate the role of miR-188 in cancer cell (Hep3B, HepG2, HLF, and LM3) proliferation and migration.ResultsmiR-188 mimic transfection inhibited the proliferation of metastatic HLF and LM3 cells but not non-invasive HepG2 and Hep3B cells; nonetheless, miR-188 suppression promoted the growth of HLF and LM3 cells. miR-188 upregulation inhibited the migratory rate and invasive capacity of HLF and LM3, rather than HepG2 and Hep3B cells, whereas transfection of a miR-188 inhibitor in HLF and LM3 cells had the opposite effects. Dual-luciferase reporter assays and bioinformatics prediction confirmed that miR-188 could directly target forkhead box N2 (FOXN2) in HLF and LM3 cells. Transfection of miR-188 mimics reduced FOXN2 levels, whereas miR-188 inhibition resulted in the opposite result, in HLF and LM3 cells. Overexpression of FOXN2 in HLF and LM3 cells abrogated miR-188 mimic-induced downregulation of proliferation, migration, and invasion. In addition, we found that miR-188 upregulation impaired tumor growth in vivo.ConclusionsIn summary, this study showed thatmiR-188 inhibits the proliferation and migration of metastatic HCC cells by targeting FOXN2.

Microclaw Array Fabricated by Single Exposure of Femtosecond Airy Beam and Self-Assembly for Regulating Cell Migratory Plasticity.

The highly aligned extracellular matrix of metastatic breast cancer cells is considered to be the "highway" of cancer invasion, which strongly promotes the directional migration of cancer cells to break through the basement membrane. However, how the reorganized extracellular matrix regulates cancer cell migration remains unknown. Here, a single exposure of a femtosecond Airy beam followed by a capillary-assisted self-assembly process was used to fabricate a microclaw-array, which was used to mimic the highly oriented extracellular matrix of tumor cells and the pores in the matrix or basement membrane during cell invasion. Through the experiment, we found that metastatic breast cancer MDA-MB-231 cells and normal breast epithelial MCF-10A cells exhibit three major migration phenotypes on microclaw-array assembled with different lateral spacings: guidance, impasse, and penetration, whereas guided and penetrating migration are almost completely arrested in noninvasive MCF-7 cells. In addition, different mammary breast epithelial cells differ in their ability to spontaneously perceive and respond to the topology of the extracellular matrix at the subcellular and molecular levels, which ultimately affects the cell migratory phenotype and pathfinding. Altogether, we fabricated a microclaw-array as a flexible and high-throughput tool to mimic the extracellular matrix during invasion to study the migratory plasticity of cancer cells.

Analysis of EMT induction in a non-invasive breast cancer cell line by mesenchymal stem cell supernatant: Study of 2D and 3D microfluidic based aggregate formation and migration ability, and cytoskeleton remodeling.

The process of Epithelial-to-mesenchymal transition (EMT) as a phenotypic invasive shift and the factors affecting it, are under extensive research. Application of supernatants of human adipose-derived mesenchymal stem cells (hADMSCs) on non-invasive cancer cells is a well known method of in vitro induction of EMT like process. While previous researches have focused on the effects of hADMSCs supernatant on the biochemical signaling pathways of the cells through expression of different proteins and genes, we investigated pro-carcinogic alterations of physico-mechanical cues in terms of changes in cell motility and aggregated formation in 3D microenvironments, and cytoskeletal actin-myosin content and fiber arrangement. MCF-7 cancer cells were treated by the supernatant from 48hour-starved hADMSCs, and their vimentin/E-cadherin expressions were evaluated. The invasive potential of treated and non-treated cells was measured and compared through aggregate formation and migration capability. Furthermore, alterations in cell and nucleus morphologies were studied, and F-actin and myosin-II alterations in terms of content and arrangement were investigated. Results indicated that application of hADMSCs supernatant enhanced vimentin expression as the biomarker of EMT, and induced pro-carcinogenic effects on non-invasive cancer cells through increased invasive potential by higher cell motility and reduced aggregate formation, rearrangement of actin structure and generation of more stress fibers, together with increased myosin II that lead to enhanced cell motility and traction force. Our results indicated that in vitro induction of EMT through mesenchymal supernatant influenced biophysical features of cancer cells through cytoskeletal remodeling that emphasizes the interconnection of chemical and physical signaling pathways during cancer progress and invasion. Results give a better insight to EMT as a biological process and the synergy between biochemical and biophysical parameters that contribute to this process, and eventually assist in improving cancer treatment strategies.

Abstract 3348: Unraveling new strategies for early detection of invasive cancer cells: A special focus on the nuclear and cytoskeletal architecture

Abstract Introduction: Cancer invasion is a hallmark of cancer progression and the leading cause of cancer mortality worldwide. However, the identification of invasive cancer cells at an early stage remains challenging. Therefore, there is an urgent need to develop novel biomarkers and innovative strategies to successfully tackle cancer invasion. It is well established that the structure and organization of the nucleus and the cytoskeleton are dynamically orchestrated during many cellular processes, including cancer invasion. Thus, in this study, we investigated fine-tuned nuclear and cytoskeletal patterns associated with invasion, taking into consideration the cell-extracellular matrix (ECM) interaction. Methods: In this study, we took advantage of an ECM Array platform (MicroMatrix 36, MicroStem) and a panel of invasive and non-invasive gastric cancer cells. For validation purposes, we assessed cells transfected with wild type or mutant E-cadherin, which lead to distinct adhesion and invasion phenotypes. A comprehensive analysis of nuclei and cytoskeletal architectural features was performed. Nuclei area, intensity, perimeter, eccentricity, circularity, solidity and entropy were evaluated. Notably, a new computational pipeline was developed to characterize cytoskeletal structures, namely orientation, compactness, radiality, bundling, parallelness, morphology, among others. Results and Discussion: Our results have shown that the invasive abilities of cancer cells are associated with unique nuclear and cytoskeletal modifications. Moreover, we verified that invasive cells adhere preferentially to specific ECM components. Interestingly, we could observe an association between nuclear phenotypes and increased attachment abilities, suggesting that the ECM may modulate nuclear morphometrics thus affecting the invasive behavior of cancer cells. Of note, cell lines harboring E-cadherin mutations corroborated the existence of a nuclear signature associated with invasive potential. Remarkably, the proposed computational framework has shown that the microtubules of cells with disrupted E-cadherin are shorter, have uniform length patterns and are more compactly distributed as compared with cells with wild-type E-cadherin. Conclusion: Overall, this study demonstrates that the use of nuclear and cytoskeletal architectural features could provide an efficient strategy to identify cells with invasive potential. Further investigation and integration of this approach with other cellular properties could have an impact in diagnosis, prognosis and therapeutic strategies. Citation Format: Maria Sofia Fernandes, Joana Figueiredo, Diogo Vieira, Soraia Melo, Ana Margarida Moreira, João Miguel Sanches, Raquel Seruca. Unraveling new strategies for early detection of invasive cancer cells: A special focus on the nuclear and cytoskeletal architecture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3348.

The location of estrogen receptor variant ER-α36 is associated with the invasion of glioblastoma

Glioblastoma (GBM) is the most common central nervous system tumor and is associated with poor outcomes. There have been no significant improvements in GBM mortality in recent decades. ER-α36 is a variant of ER-α66 that may be involved in carcinoma growth and proliferation via genomic and nongenomic mechanisms. This variant might play an essential role in tamoxifen resistance of several tumors. Previously, our laboratory found that ER-α36 is expressed in GBM and participates in proliferation; nevertheless, the role of ER-α36 in GBM invasion remains unknown. This study aimed to determine the effects of the ER-α36 modulator SNG162 on GBM growth and invasion. U251 cells, U87cells, and U87-36KD cells with knockdown of ER-α36 expression were cultured under the two-dimensional and the three-dimensional (3D) environments. GBM cells growth was examined by cell counting, flow cytometry, western blot, and MTT assays. Invasiveness was measured using confocal microscopy in the 3D environment. Growth of U87 cells with downregulated EGFR and ER-α36 expression was significantly reduced after treatment with 1 µM, 3 µM, and 5 µM of SNG162; growth inhibition in U251 cells was more potent than in U87 cells, although the expression level of ER-α36 in U251 cells was lower than in U87 cells. We found that 1 μM SNG162 suppressed E2-induced MAPK/ERK pathway activation in U87 cells. We also showed that SNG162 inhibited U87 cells invasion; however, it did not significantly affect U251 and U87-36KD cells invasion using the 3D culture method. Finally, we determined that ER-α36 was expressed in the nucleus of invading GBM cells, and SNG162 significantly inhibited the expression of ER-α36 in these cells. SNG162 inhibited the expression of EGFR on cell membranes of non-invasive GBM cells. These results suggest that SNG162 could be a therapeutic agent for GBM by targeting ER-α36.