Micro-channels array device fabricated via two photon lithography for cell migration studies in Neuroblastoma metastatic dissemination

The Urokinase Receptor Promotes Cancer Metastasis Independently of Urokinase-Type Plasminogen Activator in Mice

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Metastasis is the leading cause of deaths among cancer patients. Metastatic dissemination, during which cancer cells from the primary tumor reach the secondary organ, consists of a cascade of events, starting with cancer cell invasion and migration through the surrounding tissue [1]–[3]. During cancer cells invasion, tumor cell movement is directed by multiple guiding cues present in the tissue at different concentrations [1, 4]. Guiding cues can be biophysical, such as aligned collagen fibers inducing contact guidance or biochemical, such as gradients of chemoattractants inducing chemotaxis. Following individual cues will lead to directed cell migration, where the cell velocity and cell persistence will be determined by the concentration of the cue and the presence of the cell receptors that are responsible for communication with the particular cue. However, in complex microenvironments, where multiple cues are present at different levels, distances and orientations, cues can compete with or synergize each other to guide the cell migration. In such conditions, analyzing the cell migration parameters (velocity, persistence, directionality etc.) is a challenging task.

Cell migration is central to physiological responses to injury and infection and in the design of biomaterial implants. The ability to tune the properties of adhesive materials and relate those properties in a quantitative way to the dynamics of intracellular processes remains a definite challenge in the manipulation of cell migration. Here, we propose the use of poly(vinylmethylsiloxane) (PVMS) networks as novel substrata for cell adhesion and migration. These materials offer the ability to tune independently chemical functionality and elastic modulus. Importantly, PVMS networks are compatible with total internal reflection fluorescence (TIRF) microscopy, which is ideal for interrogating the cell-substratum interface; this latter characteristic presents a distinct advantage over polyacrylamide gels and other materials that swell with water. To demonstrate these capabilities, adhesive peptides containing the arginyl-glycyl-aspartic acid (RGD) tripeptide motif were successfully grafted to the surface of PVMS network using a carboxyl-terminated thiol as a linker. Peptide-specific adhesion, spreading, and random migration of NIH 3T3 mouse fibroblasts were characterized. These experiments show that a peptide containing the synergy sequence of fibronectin (PHSRN) in addition to RGD promotes more productive cell migration without markedly enhancing cell adhesion strength. Using TIRF microscopy, the dynamics of signal transduction through the phosphoinositide 3-kinase pathway were monitored in cells as they migrated on peptide-grafted PVMS surfaces. This approach offers a promising avenue for studies of directed migration and mechanotransduction at the level of intracellular processes.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-β3 (PLC-β3) and calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-β3, or (CaMKKβ) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.

Ewing sarcoma (ES) is one of the most common and lethal pediatric cancers with a 5-year survival rate of less than 30% for those with metastatic disease. Approximately 25% of ES patients have metastatic disease at time of diagnosis. Current treatment for ES rests on an intensive regimen of five chemotherapeutic agents with a high level of toxicity. Recurrent ES is incurable. Hence, there is a desperate need for more effective and less toxic treatments. A major obstacle in developing new, more effective therapies against ES is the lack of animal models. Zebrafish offers a robust vertebrate model for investigating cancer and evaluating the efficacy of anti-cancer drugs. Many genes, signaling pathways, and mechanisms of carcinogenesis and metastasis are highly conserved between humans and zebrafish. As zebrafish do not develop an adaptive immune response until approximately four weeks of age, they present an ideal system for xenograft tolerance in an immunocompetent organism. In addition, the transparency of zebrafish embryos permits high-resolution optical imaging in vivo and allows for easy tracking of tumor cell migration and metastasis. Our lab has created a ES xenograft model in zebrafish by injecting mCherry-labeled ES cell lines into the transgenic strain Tg(fli:GFP). This strain marks the zebrafish vasculature green, allowing for effective tracking of the injected red cancer cells. Our lab transduced the human ES cell lines A673 and MHH-ES-1, both of which contain the EWSR1:FLI fusion and TP53 mutations, with the mCherry construct. The top 10% brightest cells were selected to allow for easy visualization within the zebrafish. Approximately 200 labeled cells were injected into the yolk at two days post fertilization (2 dpf). Cell growth and migration were tracked until 5 dpf. Using fluorescent microscopy, images of each injected fish were collected at one day post injection (1 dpi), corresponding to 3 dpf, and at 3dpi, corresponding to 5 dpf. Using ImageJ software, these images were carefully analyzed for tumor growth, cell migration, and metastasis. To quantify tumor invasion and metastatic dissemination, we used flow cytometry to compare total ES cell numbers between 1 dpi and 3 dpi within the zebrafish. Our results show that metastasis within the zebrafish, which is indicated by ES cell growth within the tail region, occurs in approximately 15% of injected fish. Data from fluorescent microscopy and flow cytometry demonstrate primary tumor growth, within the zebrafish yolk, occurs in nearly all injected fish. Flow cytometry work is ongoing to quantify the percent increase in tumor growth within injected fish at 5dpf. Large–scale drug screens can be easily performed in the zebrafish due to their small size and ease of drug delivery, which consists simply of immersing fish into drug solutions. Ongoing work includes testing our xenograft model using two known chemotherapeutic agents against ES, doxorubicin and ifosfamide, with the goal of providing a benchmark for novel drugs’ responses on cell migration and metastases. Citation Format: Rebecca A. Anderson, Usua Oyarbide Cuervas-Mon, Seth Corey. Patterns and quantitation of migration and metastasis in a zebrafish xenograft model of ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B007.

Angiogenesis is an essential process for disease progression in many solid tumors. There are several major cascade events in the angiogenic process that can be targeted to inhibit new blood vessel formation in the tumor tissue. The purpose of this work is to evaluate the inhibitory effect of paclitaxel (PTX) and rapamycin (RAP) as individual and in dual drug-loaded poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles on the angiogenic cascade processes of proliferation, migration, and tube formation. PEG-b-PLA PTX and/or RAP micelles were formed and characterized for size and drug loading. Sizes of individual and dual drug micelles were below 40 nm. PEG-b-PLA micelles significantly enhanced the aqueous solubility of PTX 1.80 mg/mL and RAP 1.60 mg/mL. The PTX-RAP dual drug PEG-b-PLA micelles were able to load PTX and RAP at 1.60 mg/mL for both drugs. Cell proliferation, apoptosis, tubule formation, and migration studies were performed in human umbilical vein endothelial cells (HUVEC). PTX and RAP in DMSO inhibited HUVEC proliferation with IC50 values of 0.82 ± 0.02 and 13 829 ± 681 nM, respectively, while the combination of both drugs in DMSO produced synergistic inhibition. PTX and RAP individual micelles had IC50 values of 6.3 ± 1.1 and 14 051 ± 821 nM, respectively. PTX and dual drug micelles had a synergistic inhibition effect on HUVEC proliferation through the induction of apoptosis via caspase 3/7 activity. In vitro tube formation assay demonstrated significant inhibition of tube formation upon treatment with dual drug micelles as compared to individual PTX or RAP micelles. Migration studies in HUVEC have shown that individual PTX micelles inhibited cell migration at 1 nM, while RAP micelles did not show any inhibitory effect on cell migration. Interestingly, the presence of RAP in the dual drug micelles was able to initiate the inhibition of the migration of HUVEC at 0.1 nM concentration of PTX. These results indicate that PTX-RAP dual drug micelles have antiangiogenic effects in vitro mediated through three major events in the angiogenic process and have strong potential for further development as antiangiogenic chemotherapy.

lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

All-trans retinoic acid (RA), the primary metabolite of vitamin A, controls the development and homeostasis of organisms and tissues. RA and its natural and synthetic derivatives, both known as retinoids, are promising agents in treating and chemopreventing different neoplasias, including breast cancer (BC). Focal adhesion kinase (FAK) is a crucial regulator of cell migration, and its overexpression is associated with tumor metastatic behavior. Thus, pharmaceutical FAK inhibitors (FAKi) have been developed to counter its action. In this work, we hypothesize that the RA plus FAKi (RA + FAKi) approach could improve the inhibition of tumor progression. By in silico analysis and its subsequent validation by qPCR, we confirmed RARA, SRC, and PTK2 (encoding RARα, Src, and FAK, respectively) overexpression in all breast cells tested. We also showed a different pattern of genes up/down-regulated between RA-resistant and RA-sensitive BC cells. In addition, we demonstrated that both RA-resistant BC cells (MDA-MB-231 and MDA-MB-468) display the same behavior after RA treatment, modulating the expression of genes involved in Src-FAK signaling. Furthermore, we demonstrated that although RA and FAKi administered separately decrease viability, adhesion, and migration in mammary adenocarcinoma LM3 cells, their combination exerts a higher effect. Additionally, we show that both drugs individually, as well as in combination, induce the expression of apoptosis markers such as active-caspase-3 and cleaved-PARP1. We also provided evidence that RA effects are extrapolated to other cancer cells, including T-47D BC and the human cervical carcinoma HeLa cells. In an orthotopic assay of LM3 tumor growth, whereas RA and FAKi administered separately reduced tumor growth, the combined treatment induced a more potent inhibition increasing mice survival. Moreover, in an experimental metastatic assay, RA significantly reduced metastatic lung dissemination of LM3 cells. Overall, these results indicate that RA resistance could reflect deregulation of most RA-target genes, including genes encoding components of the Src-FAK pathway. Our study demonstrates that RA plays an essential role in disrupting BC tumor growth and metastatic dissemination in vitro and in vivo by controlling FAK expression and localization. RA plus FAKi exacerbate these effects, thus suggesting that the sensitivity to RA therapies could be increased with FAKi coadministration in BC tumors.

Pancreatic ductal adenocarcinoma (PDAC) has a dire prognosis due to late diagnosis and ineffective treatments. Myoferlin, a transmembrane protein, is overexpressed in PDAC and correlates with poor patient survival, making it a potential therapeutic target. Several reports indicated that myoferlin depletion in cancer cells reduced migration or metastatic dissemination, but they failed to provide a consistent mechanism. Understanding the biology behind the dissemination of PDAC is of outmost importance, as this disease is often diagnosed at an advanced stage. This study aims to further elucidate myoferlin’s role in cell migration. Myoferlin expression in PDAC patients correlated with gene sets related to the actin cytoskeleton, and myoferlin knockdown in PDAC cell lines similarly affected these gene sets. Electron microscopy revealed that myoferlin depletion altered cytoskeleton ultrastructure. Immunofluorescence confirmed that myoferlin knockdown disorganized microfilaments, despite not altering β-actin abundance. Functionally, myoferlin depletion significantly reduced PDAC cell migration. While core machinery for actin polymerization/depolymerization and EMT markers remained unaffected, myoferlin knockdown increased the abundance of focal adhesion components. However, immunofluorescence showed a decrease in the number of functional focal adhesions, with paxillin accumulating in the cytosol. Cell migration relies on the dynamics of focal adhesions. The reduction in functional focal adhesions indicates that the cell has fewer points of attachment for migration. We suggest this impairment was linked to a significant decrease in clathrin heavy chain abundance and pointed to a disrupted clathrin-mediated endocytosis. This study highlights a novel role for myoferlin in focal adhesion recycling in PDAC and provides a consistent mechanism explaining how myoferlin contributes to PDAC cell migration, a significant event in metastatic dissemination, and reinforce its potential as a therapeutic target.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-27134-2.

Collective cell migration is the movement of groups of cells in a coordinated and connected manner. It is essential in development, and has recently been connected to the spread of cancer metastasis. Despite the fact that collective cell migration is essential, most studies of cell migration are looking at single cell migration events. While many of the same processes that regulate single cell migration likely regulate collective cell migration, collective cell migration presents its own individual challenges. For this reason, it is important to study genes and signaling molecules known to regulate single cell migration in the collective cell context. One regulator of single cell migration that has not been looked at in the context of collective cell migration is Prostaglandin Signaling. Prostaglandins are short range, lipid signaling molecules regulated by release of arachidonic acid and by the conversion of that arachidonic acid into the prostaglandin precursor by cyclooxygenases. There are two cyclooxygenase enzymes in humans (COX1 and COX2), and these enzymes are the targets of many non-steroidal anti-inflammatory drugs like aspirin. Studies in both mice, zebrafish, and humans all show that these enzymes are essential to development and can regulate cancer cell metastasis. This thesis focuses on prostaglandins in collective cell migration, and how they regulate this process. Using Drosophila melanogaster oogenesis, we examine the role of the Drosophila Cox like enzyme Pxt in border cell migration. Border cell migration occurs during Stage 9 of oogenesis and consists of a set of 8-10 somatic cells which delaminate from the anterior tip of the follicle and migrate collectively between the much larger nurse cells to the nurse cell-oocyte border. This migration is essential to the fertility of the female fly, and is a common model for examining collective cell migration. Here I make use of this model to examine the effects of pxt both within the migratory cells, and within the nurse cells which represent the microenvironment of the border cell cluster. To do this however, I first had to develop a new method of migration quantification. Chapter 2 outlines the method I developed to quantify border cell migration using fixed imaging analysis. Typically border cell migration is assessed at Stage 10 to look for completion, or during Stage 9 using live imaging. As live imaging proved impossible in these mutants, I assessed border cell migration using a fixed imaging analysis that quantifies the amount the border cell cluster is ahead of or behind the position of the outer follicle cells. As the outer follicle cell position is not altered in any known border cell mutants, we are able to take advantage of this fact to assess border cell migration in a highly quantitative method. In Chapter 3, I present the main body of my thesis and show that Pxt is essential to proper border cell migration. Here I show that loss of pxt results in delayed border cell migration and apparent cluster morphology defects. I also show that somatic loss of Pxt results in delayed migration but without apparent cluster morphology defects. From the results presented in this chapter I propose that border cell migration is regulated both within the migratory cells, and by the microenvironment of these cells. In conclusion, prostaglandins are essential in collective, invasive cell migration and act both within the migratory cluster and in the surrounding microenvironment in different ways to promote migration. These functions, which are likely conserved across species, are essential to proper migration and make excellent targets for better understanding of their role in development and in disease. It is likely that the work presented in this thesis lays the groundwork for future experiments which will better understand the targets of prostaglandin signaling in both the microenvironment and the migratory cells. Furthermore, it further establishes Drosophila as an excellent model for understanding the mechanisms behind prostaglandin signaling.

Migration of Cells in a Social Context

The Bcl-xL apoptosis inhibitor plays a major role in vertebrate development. In addition to its effect on apoptosis, Bcl-xL is also involved in cell migration and mitochondrial metabolism. These effects may favour the onset and dissemination of metastasis. However, the underlying molecular mechanisms remain to be fully understood. Here we focus on the control of cell migration by Bcl-xL in the context of breast cancer cells. We show that Bcl-xL silencing led to migration defects in Hs578T and MDA-MB231 cells. These defects were rescued by re-expressing mitochondria-addressed, but not endoplasmic reticulum-addressed, Bcl-xL. The use of BH3 mimetics, such as ABT-737 and WEHI-539 confirmed that the effect of Bcl-xL on migration did not depend on interactions with BH3-containing death accelerators such as Bax or BH3-only proteins. In contrast, the use of a BH4 peptide that disrupts the Bcl-xL/VDAC1 complex supports that Bcl-xL by acting on VDAC1 permeability contributes to cell migration through the promotion of reactive oxygen species production by the electron transport chain. Collectively our data highlight the key role of Bcl-xL at the interface between cell metabolism, cell death, and cell migration, thus exposing the VDAC1/Bcl-xL interaction as a promising target for anti-tumour therapy in the context of metastatic breast cancer.

Normal brain development relies on precise genetic and epigenetic spatiotemporal regulation of gene expression. How dysregulation of neurodevelopment relates to medulloblastoma, the most common pediatric brain tumor, remains elusive. Here, we uncovered a novel neurodevelopmental epigenomic program that regulates Purkinje cell migration in developing cerebellum is hijacked to induce tumor metastatic dissemination in medulloblastoma. Integrating publicly available datasets with our in-house data, unsupervised analyses revealed that BAF60C/SMARCD3, a subunit of SWI/SNF chromatin remodeling complex, promotes tumor cell migration in vitro and metastasis in vivo. Based on analyzing the single-cell RNAseq data of cerebellum developmental trajectory in mice and humans, aligning with the medulloblastoma patients’ datasets, we found that BAF60C/SMARCD3 regulated DAB1-mediated Reelin signaling is involved in Purkinje cell positioning during cerebellum development and medulloblastoma metastasis by orchestrating the cis-regulatory elements (CREs) at the DAB1 gene locus. Analysis of spatiotemporal gene expression and chromatin architecture in the human and mouse cerebellum demonstrated that transcription activity of the BAF60C/SMARCD3-DAB1 circuit is downregulated in a mature state of cerebellar development, however, is upregulated in metastatic medulloblastoma. We further identified that a core set of transcription factors, enhancer of zeste homolog 2 (EZH2) and nuclear factor I X (NFIX), bi-directionally control BAF60C/SMARCD3 transcriptional regulation by coordinating with the CREs at the BAF60C/SMARCD3 gene locus to form a chromatin hub during developing cerebellar development and medulloblastoma metastatic dissemination. Highly expressed BAF60C/SMARCD3 activates the Reelin/DAB1 signaling pathway downstream Src kinase, which was validated in the pair-wised primary and metastatic tumors from medulloblastoma patients. Preclinical medulloblastoma mouse models revealed that inhibiting Src activity reduces tumor cell migration and metastatic dissemination at a lower and safe dose. Together, these data deepen our understanding of how the developmental program influences disease progression and provide an opportunity for the development of therapeutics for this devastating brain cancer in children.

The impairment of cyclic guanosine monophosphate (cGMP) signaling by overexpression of PDE5 isoform has been recently described in multiple human carcinomas. In addition, accumulating evidences indicate that PDE5 inhibitors could have direct anti-cancer activities as well as they may enhance the sensitivity of certain types of cancer to standard chemotherapeutic drugs. However, despite these studies, neither the expression of PDE5 in breast cancer subtypes nor the underlying regulatory molecular mechanisms by which PDE5 expression may contribute to breast cancer progression have been deeply studied. We demonstrated that PDE5 was expressed in different subtypes of breast cancer cell lines at higher levels than in non tumorogenic human epithelial breast cell lines. Increased levels were detected in more aggressive endocrine non responsive basal-like breast cancer cells. Interestingly, PDE5 was expressed at very low levels in luminal A-type breast cancer cell lines, which display low ki67 expression, weak invasive behavior and endocrine responsiveness (MCF-7 and T47D cells) compared to luminal B-like cells (such as ZR-75 cells). These results well correlated with data obtained in immunohistochemistry analyses of human breast cancer tissues, showing PDE5 expression in 30 of 35 tumor entities analyzed, with the highest intensity staining in high-grade tumors. Concomitantly, no cytoplasmic PDE5 staining was observed in non neoplastic tissues examined (n=5). In addition, retrospective analyses (n=1959, median follow-up time: 25 years) showed that high PDE5 expression in breast cancer patients was correlated with a statistically significant poorer survival compared to low PDE5-expressing patients. A more relevant discrimination is achieved in lymphnode-negative patients, suggesting a role of PDE5 for identifying early patients at high risk of rapid progression. In order to better ascertain the role of PDE5 in breast tumorogenesis, we selected a breast tumor cell line that express low levels of this enzyme, MCF-7 and engineered stable clones for overexpression studies. Both vector- and PDE5-stable MCF-7 clones demonstrated comparable proliferation rates; whereas, cell motility and invasion were dramatically increased in PDE5-overexpressing cells. RNA sequencing to compare the transcriptomes of vector- and PDE5-overexpressing MCF-7 cells identified differential expression of genes involved in cell migration and invasion. Particularly, based on pathway analysis we found marked changes in the expression of Rho GTPase family members, proteins involved in cell cytoskeleton organization, migration, and metastasis dissemination (Rho A, cdc42 and Rac signaling, activation score= 1.9, 1.342, and 0.302, respectively). Indeed, Rho and cdc42 pull-down assays revealed increased Rho GTPase activity in cells overexpressing PDE5. Moreover, the selective ROCK inhibitor Y-27632 as well as the PDE5 inhibitor sildenafil were able to significantly reduce both migration and invasion of PDE5 clones. Our data reveal that PDE5 expression enhances motility and invasiveness of breast cancer cells through the activation of the Rho family of GTPases, and highlight, for the first time, a novel role for PDE5 as a marker of poor outcome in breast cancer patients. Citation Format: Barone I, Campana A, Giordano C, Tarallo R, Rinaldi A, Bruno G, Gyorffy B, Lanzino M, Bonofiglio D, Catalano S, Ando' S. Phosphodiesterase type 5 promotes the invasive potential of breast cancer cells through Rho GTPase activation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-10.