Mechanisms Of Cancer Metastasis Research Articles

PADI3 inhibits epithelial-mesenchymal transition by targeting CKS1-induced signal transduction in colon cancer.

Protein arginine deiminase 3 (PADI3) is involved in various biological processes of human disease. PADI3 has recently received increasing attention due to its role in tumorigenesis. In a previous study, we found that PADI3 plays a tumor suppressor role in colon cancer by inducing cell cycle arrest, but its critical role and mechanism in cancer metastasis remain obscure. In this study, we fully studied the role of PADI3 in colon cancer cell metastasis. The expression levels of related proteins were detected by Western blotting, and Transwell and wound healing assays were used to examine the cell migration ability. Flow cytometry was used to measure and exclude cell apoptosis-affected cell migration. Both overexpression and rescue experiments were employed to elucidate the molecular mechanism of CKS1 in colon cancer cells. The expression levels of PADI3 and CKS1 are negatively related, and PADI3 can promote CKS1 degradation in a ubiquitin-dependent manner. PADI3 can suppress colon cancer cell migration and reduce the wound healing speed by inhibiting CKS1 expression. The molecular mechanism showed that CKS1 can promote EMT by increasing Snail and N-cadherin expression and suppressing E-cadherin expression. PADI3, as a suppressor of CKS1, can block the process of EMT by impairing CKS1-induced Snail upregulation and E-cadherin downregulation; however, the expression of N-cadherin cannot be rescued. CKS1 promotes EMT in colon cancer by regulating Snail/E-cadherin expression, and this effect can be reversed by PADI3 via the promotion of CKS1 degradation in a ubiquitylation-dependent manner.

Endogenous mRNA-Driven "One-To-More" Signal Amplification of DNA Probe for Intracellular miR155 Sensing.

The detection of specific intracellular microRNAs could be potentially helpful in understanding the underlying mechanisms of cancer metastasis and invasion. MiRNAs are usually present in lower expression levels, especially in early stage of cancer. Here, we proposed a "one-to-more" amplification strategy for miRNA imaging, by virtue of DNA strand displacements with dual-amplification. This approach involves leveraging high-abundance endogenous mRNA as fuel strand to drive cascade reactions between DNA strands for amplification, enabling the monitoring of low-abundance intracellular microRNA155. Notably, in comparison to the traditional "one-to-one" signal triggering mode, our "one-to-more" amplification strategy led to a remarkable 11.8-fold increase in fluorescence signal. Our approach not only demonstrates a high sensitivity and specificity in detecting miR155, but also allows for discrimination of miR155 expression levels in different cell lines. With the advantages of intracellular signal amplification and reduced background signal, this approach holds substantial potential in the early diagnosis of cancer.

Exosome-mediated secretion of miR-127-3p regulated by RAB27A accelerates metastasis in renal cell carcinoma

BackgroundThe exosome-mediated extracellular secretion of miRNAs occurs in many cancers, and RAB27A is a potent regulator of exosome secretion. For metastatic renal cell carcinoma (RCC), this study examines the mechanisms of cancer metastasis via the RAB27A-regulated secretion of specific miRNAs.MethodsRAB27A knockdown (KD) and overexpressing (OE) RCC cells were used to examine cell migration and adhesion. The particle counts and sizes of exosomes in RAB27A OE cells were analyzed using Exoview, and those of intraluminal vesicles (ILV) and multivesicular bodies (MVB) were measured using an electron microscope. Analysis of RNA sequences, protein–protein interaction networks, and the competing endogenous RNA (ceRNA) network were used to identify representative downregulated miRNAs that are likely to undergo cargo-sorting into exosomes and subsequent secretion. A molecular beacon of miR-137-3p, one of the most representatively downregulated genes with a fold change of 339, was produced, and its secretion was analyzed using Exoview. RAB27A OE and control cells were incubated in an exosome-containing media to determine the uptake of tumor suppressor miRNAs that affect cancer cell metastasis.ResultsMigration and cell adhesion were higher in RAB27A OE cells than in RAB27A KD cells. Electron microscopy revealed that the numbers of multivesicular bodies and intraluminal vesicles per cell were higher in RAB27A OE cells than in control cells, suggesting their secretion. The finding revealed that miR-127-3p was sorted into exosomes and disposed of extracellularly. Protein–protein interaction analysis revealed MYCN to be the most significant hub for RAB27A-OE RCC cells. ceRNA network analysis revealed that MAPK4 interacted strongly with miR-127-3p.ConclusionThe disposal of miR-127-3p through exosome secretion in RAB27A overexpressing cells may not inhibit the MAPK pathway to gain metastatic potential by activating MYCN. The exosomes containing miRNAs are valuable therapeutic targets for cancer treatment.

Abstract 2757: Investigating regulatory drivers of prostate cancer metastasis in circulating tumor cells

Abstract Prostate cancer is the most common cancer in men and the second-leading cause of death. Most prostate cancer deaths are due to metastasis, particularly to the bone. Metastasis occurs when cells from the primary tumor travel through the blood as circulating tumor cells (CTCs) and settle in metastatic sites. CTCs are difficult to collect from patients, and understanding the relationships between CTCs and the tumor phenotypes of origin could reveal mechanisms of cancer metastasis. Hence, we utilized several genetically engineered mouse models (GEMMs) of metastatic prostate cancer to evaluate tumor phenotypes and CTCs. We compared the tumor phenotypes of several GEMMs of metastatic prostate cancer and identified a level of heterogeneity among tumors, particularly focusing on a GEMM that demonstrated severe metastasis, especially to the bone. Next, we analyzed CTCs from this GEMM and focused our analysis on transcriptional states that are regulated by proteins called master regulators (MRs). MRs control the expression of groups of genes called regulons, and pharmacologically inhibiting these MRs may lead to reversal of cancer phenotypes. We hypothesized that these MRs may be mechanistic drivers of CTC cell state in prostate cancer metastasis. We investigated MRs enriched in CTCs from mice and screened and validated Lap2, the lamina-associated polypeptide 2 protein, as a regulatory driver in mice with metastatic prostate cancer. Analysis of bulk RNA-seq profiles from primary and metastatic prostate samples in The Cancer Genome Atlas and Stand Up to Cancer cohorts, respectively, revealed that the regulon-based activity of Lap2 was significantly higher in the latter group. Moreover, analysis of single-cell RNA-seq profiles of CTCs isolated from prostate cancer patients confirmed aberrant Lap2 activity. Finally, the role of Lap2 was assessed in human tissue from an engineered multi-organ-on-a-chip model of metastasis. Silencing of Lap2 production in cells dissociated from mouse CTC-derived organoids inhibited extravasation to the bone in the model, suggesting a critical role for Lap2 in metastasis. Our findings illustrate the unique transcriptional profile of prostate cancer CTCs associated with their metastatic potential. We identified Lap2 as a novel driver of CTC dissemination and metastasis in mice and validated its activity in human prostate cancer, which can aid future therapies to prevent metastatic progression in patients. Citation Format: Aunika Zheng, Aleksandar Obradovic, Kacey Ronaldson-Bouchard, Gordana Vunjak-Novakovic, Andrea Califano, Peter A. Sims, Cory Abate-Shen, Arianna Giacobbe. Investigating regulatory drivers of prostate cancer metastasis in circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2757.

Abstract 1449: Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models

Abstract Breast cancer brain metastasis is one of the most common forms of breast cancer metastasis and a major cause of morbidity and mortality. Patients who develop brain metastases tend to be associated with progressive neurologic deficits and short overall survival, which represents an unmet medical need. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information gained as to how cancer cells extravasate and colonize to the brain. Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in cholesterol biosynthesis, plays a critical role in the process of breast cancer metastasis to the brain. Thus, toward a better understanding of the underlying molecular mechanisms and applications of molecular target(s) for brain metastasis therapy, murine models were employed for investigation of brain extravasation and colonization with intracardiac or direct intracranial injections of GFP/FLuc-labeled human MDA-MB-231 metastatic breast carcinoma cells. The MDA-MB-231 cells were found to metastasize to the brain and other organs following intracardiac implantation into nude mice and were further isolated from each tissue. Interestingly, the isolated brain-metastasized tumor cells (MDA-MB-231-BrM) exhibited a significant upregulation of SQLE expression when compared with those that metastasized to other distal sites. The essential role of SQLE in the specific steps of the breast cancer-to-brain metastatic process was evaluated by ex vivo immunofluorescence analysis and immunohistochemistry staining of brain slices from the study subjects. Our data demonstrate that high SQLE expression is essential for MDA-MB-231-BrM to extravasate into the parenchyma, as well as the formation of micro- and macro-metastases in the brain. Interestingly, we found that blood vessel co-option and the surrounding neuronal cells play key roles in supporting MDA-MB-231-BrM to develop brain macro-metastases. In vitro blood-brain barrier (BBB) models further demonstrated the critical role of SQLE in promoting tumor cell invasion and penetration through the BBB. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast cancer brain metastasis, we evaluated the inhibitory effects of terbinafine (a SQLE inhibitor) in both the MDA-MB-231-BrM orthotopic and intracardiac mouse models. Pharmacologic inhibition of SQLE by terbinafine suppressed MDA-MB-231-BrM tumor growth at the mammary fat pad and distal metastases to the brain, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer brain metastasis. Citation Format: Pony Yu-Ling Lee, Kun-Yuan Lin, Chao-Di Chang, Shan-Yun Cheng, Ya-Wen Hung, Chih-Chieh Yang, Yu-Hsien Chang, Chien-Chang Shen, Jia-Yun Yeh, Yung-Lung Yu, Shu-Ping Wang. Targeting squalene epoxidase for breast cancer brain metastasis: The evaluation of a new therapeutic target for brain extravasation and colonization using animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1449.

Abstract 7503: Non-clinical study of circulating tumor cells in humanized mice: Post-xenograft effects in immunodeficient mice

Abstract Circulating tumor cells (CTCs) possess the potential to serve as innovative markers in clinical trials, contributing to the understanding of cancer metastasis mechanisms, diagnosis, prognosis, the development of personalized treatment strategies, and enabling the monitoring of treatment efficacy. NSG mice, which are a strain of immunodeficient mice frequently utilized as a non-clinical model for cancer research, exhibit diminished levels of natural killer (NK) cells and abnormal monocytes in their bloodstream. In a previous study, a positive correlation was observed between the duration of injection and the levels of white blood cells (WBC) in mice that were injected with various cancer cell lines derived from humans. To evaluate the correlation among the primary tumor and circulating tumor cells (CTCs), as well as the impact on immune cells, we assessed changes in immune cell composition, primary tumor size, and enumeration of CTCs in xenografted mice over a period of time. This investigation was carried out using CytoGen's Smart Biopsy™ systems. The results, which were obtained using equipment including CytoGen's Smart Biopsy™ Systems, showed that the distribution of white blood cell (WBC) components exhibited variations based on age, primary tumor burden, and circulating tumor cell (CTC) count following injection. A notable association was observed between the augmentation of immune cells and the quantity of circulating tumor cells (CTCs) present in the bloodstream. Therefore, alterations in immune cells may potentially impact the potential to affect the ability to influence the efficacy of cancer immunotherapy, metabolic therapy, and anti-metastatic therapy. When conducting non-clinical investigations with the objective of developing cancer treatment protocols, it is crucial to meticulously select humanized or immunodeficient mice that are appropriate for the study. Therefore, it is advisable to perform regular monitoring of murine blood samples in order to observe any variations in the counts of circulating tumor cells (CTCs), while considering a standardized blood volume per individual. In the immunodeficient mouse model, the existence of circulating tumor cells (CTCs) results in an increase in the quantity of cells present in the bloodstream. The observed rise in circulating tumor cells (CTCs) is found to be concurrent with the progression and development of the primary tumor. The consideration of this aspect is of utmost importance in the formulation of non-clinical studies aimed at the development of cancer treatment protocols. Citation Format: Sehyung Pak, Hajin Pak, Hee Jin Yoo, Sra Min, Hui Ji Kim, JaeYoon Lee, Min-Ji Song, Jaesung Choi, Aejin Jeong, Hanbyeol Kim, Jung Won Kim, Hai-Chon Lee. Non-clinical study of circulating tumor cells in humanized mice: Post-xenograft effects in immunodeficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7503.

A glimpse into novel acylations and their emerging role in regulating cancer metastasis.

Metastatic cancer is a major cause of cancer-related mortality; however, the complex regulation process remains to be further elucidated. A large amount of preliminary investigations focus on the role of epigenetic mechanisms in cancer metastasis. Notably, the posttranslational modifications were found to be critically involved in malignancy, thus attracting considerable attention. Beyond acetylation, novel forms of acylation have been recently identified following advances in mass spectrometry, proteomics technologies, and bioinformatics, such as propionylation, butyrylation, malonylation, succinylation, crotonylation, 2-hydroxyisobutyrylation, lactylation, among others. These novel acylations play pivotal roles in regulating different aspects of energy mechanism and mediating signal transduction by covalently modifying histone or nonhistone proteins. Furthermore, these acylations and their modifying enzymes show promise regarding the diagnosis and treatment of tumors, especially tumor metastasis. Here, we comprehensively review the identification and characterization of 11 novel acylations, and the corresponding modifying enzymes, highlighting their significance for tumor metastasis. We also focus on their potential application as clinical therapeutic targets and diagnostic predictors, discussing the current obstacles and future research prospects.

A SERS-assisted 3D organotypic microfluidic chip for in-situ visualization and monitoring breast cancer extravasation process

Extravasation, as one of the key steps in cancer metastasis, refers to the process where tumor cells escape the bloodstream by crossing the vascular endothelium and invade the targeted tissue, which accounts for the low five-year survival rate of cancer patients. Understanding the mechanism of cancer metastasis and inhibiting extravasation are crucial to improve patient prognosis. Here, a 3D organotypic microfluidic chip combined with SERS-based protein imprinted nanomaterials (SPINs) was proposed to study the extravasation process in vitro. The chip consists of a collagen gel channel and a vascular channel where human vein endothelial cells (HUVECs) and breast cancer cells are injected sequentially to induce extravasation. By comparing two subtypes of breast cancer cells (MCF-7 and MDA-MB-231), we successfully observed the difference in extravasation capabilities between two kinds of cells through fluorescence imaging. Meanwhile, thanks to the high specificity of molecular imprinting technology and the high sensitivity of surface enhanced Raman scattering (SERS), SPINs were utilized to analyze the concentration of several cancer secretions (interleukin-6 and interleukin-8) in complex biological fluid in real-time. Further, our model showed that downregulation of secretions by therapeutic drugs can inhibit the extravasation of breast cancers. This microfluidic model may pave the way for the fundamental research of the cancer metastasis and evaluating the therapeutic efficacy of potential drugs.

The influence of silver nanoparticles on the process of epithelial transition in the context of cancer metastases.

Exposure to nanoparticles (NPs) can occur in a variety of occupational situations. Ultrafine particles of natural and anthropological origin toxicity has been described in epidemiological studies. Meanwhile, the risks associated with NPs exposure are not comprehensively assessed. A wide spectrum of NPs toxicity has been demonstrated, mainly through the induction of oxidative stress and inflammatory mediators. Among the newly described mechanisms of NPs toxicity is the induction of fibrosis via the epithelial-mesenchymal transition (EMT), which is also a key mechanism of cancer metastasis. The effect of NPs on EMT in the context of metastasis has not been sufficiently described so far, and the results of studies do not allow for the formulation of unambiguous conclusions. Therefore, the aim of the work was to determine the biological activity of silver NPs against MDA-MB-436 triple-negative breast cancer cells. Exposure to nanoparticles (NPs) can occur in a variety of occupational situations. Ultrafine particles of natural and anthropological origin toxicity has been described in epidemiological studies. Meanwhile, the risks associated with NPs exposure are not comprehensively assessed. A wide spectrum of NPs toxicity has been demonstrated, mainly through the induction of oxidative stress and inflammatory mediators. Among the newly described mechanisms of NPs toxicity is the induction of fibrosis via the epithelial-mesenchymal transition (EMT), which is also a key mechanism of cancer metastasis. The effect of NPs on EMT in the context of metastasis has not been sufficiently described so far, and the results of studies do not allow for the formulation of unambiguous conclusions. Therefore, the aim of the work was to determine the biological activity of silver NPs against MDA-MB-436 triple-negative breast cancer cells. Silver nanoparticles (AgNPs) cause a statistically significant increase in relative expression of all tested mesenchymal EMT markers - cadherin 2, vimentin, matrix metalloproteinase 2 and matrix metalloproteinase 9. At the same time, reduction of epithelial cadherin 1 expression was observed. The level of MDA-MB-436 migration and TGF-beta 1 secretion was slighty increased in AgNPs-treated cells, with no influence on invasion potential. Potentially prometastatic effect of AgNPs encourages further work on the safety of nanomaterials. Med Pr Work Health Saf. 2023;74(6):541-8.

KCTD4 interacts with CLIC1 to disrupt calcium homeostasis and promote metastasis in esophageal cancer.

Increasing evidences suggest the important role of calcium homeostasis in hallmarks of cancer, but its function and regulatory network in metastasis remain unclear. A comprehensive investigation of key regulators in cancer metastasis is urgently needed. Transcriptome sequencing (RNA-seq) of primary esophageal squamous cell carcinoma (ESCC) and matched metastatic tissues and a series of gain/loss-of-function experiments identified potassium channel tetramerization domain containing 4 (KCTD4) as a driver of cancer metastasis. KCTD4 expression was found upregulated in metastatic ESCC. High KCTD4 expression is associated with poor prognosis in patients with ESCC and contributes to cancer metastasis invitro and invivo. Mechanistically, KCTD4 binds to CLIC1 and disrupts its dimerization, thus increasing intracellular Ca2+ level to enhance NFATc1-dependent fibronectin transcription. KCTD4-induced fibronectin secretion activates fibroblasts in a paracrine manner, which in turn promotes cancer cell invasion via MMP24 signaling as positive feedback. Furthermore, a lead compound K279-0738 significantly suppresses cancer metastasis by targeting the KCTD4‒CLIC1 interaction, providing a potential therapeutic strategy. Taken together, our study not only uncovers KCTD4 as a regulator of calcium homeostasis, but also reveals KCTD4/CLIC1-Ca2+-NFATc1-fibronectin signaling as a novel mechanism of cancer metastasis. These findings validate KCTD4 as a potential prognostic biomarker and therapeutic target for ESCC.

Targeting squalene epoxidase in breast-to-brain metastasis: A new therapeutic target for brain extravasation and colonization using animal models.

1084 Background: Brain metastasis is the most common malignancy of the central nervous system which causes severe morbidity and mortality in multiple cancer types of patients and represents an unmet medical need. Several critical steps are required for a successful brain metastasis, including local invasion, intravasation, dissemination, extravasation, and colonization. Extensive research has been conducted to elucidate the mechanism of cancer metastasis with limited information toward how cancer cells extravasate and colonize. Methods: To understand the underlying molecular mechanisms and applications of molecular targets for brain metastasis therapy, murine models are employed for investigation of brain extravasation and colonization. To investigate the roles of SQLE in breast-to-brain metastasis in vivo, we silenced SQLE expression directly with lentiviral shRNA in the brain metastatic MDA-MB-231-BR cell line (231-BR/shSQLE) and used 231-BR cells expressing a scramble shRNA (231-BR/shScr) as the control. Brain metastases were induced by intracardiac, orthotopic, or direct intracranial injections of 231-BR/shSQLE or 231-BR/shScr cells into immune deficient mice. The essential roles of SQLE in the specific step(s) of breast-to-brain metastatic process were evaluated by ex vivo immunofluorescence analysis of brain slices from the animals. To verify SQLE as an oncogenic factor that can be selected as a potential therapeutic target in suppressing breast-to-brain metastasis, we evaluated the inhibitory effects of NB-598 (a SQLE inhibitor) in both the 231-BR orthotopic and intracardiac mouse models. Results: Recently, we identified a novel mechanism by which squalene epoxidase (SQLE), the second rate-limiting enzyme in the cholesterol biosynthesis, plays a critical role in the processes of breast cancer metastasized to the brain, especially in brain extravasation and colonization. Interestingly, the pharmacologic inhibition of SQLE has been widely used against fungal infections, and the next-generation SQLE inhibitors have been recently shown to exert an anticancer effect. Our data demonstrated that SQLE is essential for 231-BR cells to extravasate into the parenchyma as well as the formation of micro and macro-metastases in the brain. Interestingly, we found that astrocytes play a key role in supporting 231-BR-developed brain macro-metastasis. In vitro blood-brain barrier (BBB) models further demonstrated the critical roles of SQLE in promoting 231-BR cell invasion and penetration through BBB. Inhibition of SQLE by NB-598 demonstrated anti-tumor proliferation and anti-metastasis. Conclusions: Pharmacologic inhibition of SQLE by NB-598 suppressed 231-BR tumor growth at the mammary fat pad and distal metastases to organs, suggesting that targeting SQLE represents a therapeutic opportunity for breast cancer metastases.

Abstract 5598: Microfluidic isolation and capture of circulating tumor cells and clusters from mouse blood

Abstract Background. Circulating tumor cells (CTCs) and their clusters have been regarded as potential targets for therapies and subjects for identifying certain mechanisms of cancer metastasis. Mouse models have been especially useful tools to investigate the process of metastasis, since they are easier to control and are less variable compared to clinical patient samples. Therefore, it is of interest to extract and study CTCs and CTC clusters from such mouse models. However, most isolation systems are designed and tested for human blood and cell lines. Sized-based methods of CTC isolation have been optimized for human cell lines, but mouse CTCs are generally smaller in size (i.e., ~14 µm EO771 breast cancer cells). Therefore, mouse cell separation necessitates a varied protocol for CTC isolation. We used mouse blood and EO771, a mouse breast cancer cell line, to demonstrate the isolation of mouse CTCs from mouse blood using a sized-based microfluidic device. We also validated the presence of endogenous CTCs and CTC-neutrophil clusters in a tumor bearing mouse model. Methods. Our microfluidic device employs inertial migration in a straight channel segment (150 µm × 50 µm × 24 mm) to isolate CTCs from blood samples. We spiked 100 and 1000 ZsGreen-expressing EO771 cells in 5 × diluted blood containing tdTomato-expressing neutrophils, easily distinguishing CTCs from the red-colored neutrophils. To enumerate the recovered cells, we used Cytospin to concentrate the cells into a glass slide. Lastly, we used hydrodynamic traps to capture CTC clusters from tumor-bearing mouse blood. The blood was extracted from the posterior vena cava of the mice at the endpoint experiment. Results. We achieved an overall recovery rate of 22-29%, which combines the recovery of the isolation device and the Cytospin. Here, we enumerate cells only after their extraction from the collection tube outside of the isolation device. Other methods with higher capture efficiency enumerate the recovered CTCs before they are harvested from the isolation device which is not always representative of the number of cells that are analyzed. We were also able to discover endogenous CTC-neutrophil clusters from tumor-bearing mice which we distinguished apart with anti-EpCAM staining of CTCs (green) and anti-Ly6G (red) staining of neutrophils. Conclusion. We demonstrated and optimized a procedure for isolating mouse-derived CTCs from naïve mice blood and CTC-neutrophil clusters from tumor-bearing mice with our microfluidic isolation devices, as opposed to the isolation of human CTCs as in other sized-based isolation systems. In future studies, we aim to continue to use our microfluidic devices to capture and study CTC and CTC clusters. Citation Format: Celine Macaraniag, Jian Zhou, Ian Papautsky, Jing Li, William Putzbach, Nissim Hay. Microfluidic isolation and capture of circulating tumor cells and clusters from mouse blood. [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 5598.

Cell scientist to watch – Guillaume Jacquemet

ABSTRACT Guillaume Jacquemet studied biology at the Université de Reims Champagne-Ardenne in France, before joining the lab of Martin Humphries at the University of Manchester in 2008 as part of a Wellcome-funded four-year PhD studentship. There, he studied cell–matrix interactions and the integrin-mediated regulation of small GTPases. In 2014, Guillaume moved to Turku, Finland for his postdoc with Johanna Ivaska to work on the role of filopodia in cell migration and cancer cell invasion. He set up his own research group, the Cell Migration Lab, at Åbo Akademi University, Turku, in 2019, where in addition to investigating the molecular mechanisms of cancer metastasis and cell migration, he also develops deep-learning-based image analysis tools.

All-in-one microfluidic design to integrate vascularized tumor spheroid into high-throughput platform.

The development of a scalable and highly reproducible in vitro tumor microenvironment (TME) platform still sheds light on new insights into cancer metastasis mechanisms and anticancer therapeutic strategies. Here, we present an all-in-one injection molded plastic array three-dimensional culture platform (All-in-One-IMPACT) that integrates vascularized tumor spheroids for highly reproducible, high-throughput experimentation. This device allows the formation of self-assembled cell spheroids on a chip by applying the hanging drop method to the cell culture channel. Then, when the hydrogel containing endothelial cells and fibroblasts is injected, the spheroid inside the droplet can be patterned together in three dimensions along the culture channel. In just two steps above, we can build a vascularized TME within a defined area. This process does not require specialized user skill and minimizes error-inducing steps, enabling both reproducibility and high throughput of the experiment. We have successfully demonstrated the process, from spheroid formation to tumor vascularization, using patient-derived cancer cells (PDCs) as well as various cancer cell lines. Furthermore, we performed combination therapies with Taxol (paclitaxel) and Avastin (bevacizumab), which are used in standard care for metastatic cancer. The All-in-One IMPACT is a powerful tool for establishing various anticancer treatment strategies through the development of a complex TME for use in high-throughput experiments.

Abstract LB540: Potential role of the GPI anchor of Thy-1 in expanding ovarian cancer stem cells

Abstract Introduction: Ovarian cancer is the deadliest gynecologic cancer, and it is often detected at a late stage. Understanding the mechanisms of cancer metastasis can allow us to develop methods to detect ovarian cancers sooner. Screening for specific biomarkers present in at-risk patients may facilitate detection at earlier stages and improve prognoses. A hypothesis currently being developed involves tumor spheroids that infiltrate the abdominal cavity to drive metastasis. These spheroids have increased expression of proteins with a glycosylphosphatidylinositol anchor (GPI anchor). Increased expression of GPI anchored proteins has been previously linked to cancer cells when compared to non-cancerous cells. Thy-1 (CD90) is a cell surface glycoprotein containing a GPI anchor linkage is thought to have a role in forming and increasing proliferation of these cancer spheroids. Methods and Results: In this project, cells were cultured from two ovarian cancer cell lines (A1847 and C30) to determine if the GPI anchor was granting the spheroid proliferation capability as part of the CD90 protein structure. The A1847 and C30 ovarian cancer cell lines were transfected with a lentivirus to express three different versions of the GPI anchor + Thy1 amino acids. Construct I contain truncated Thy-1 protein with the C-terminal 30 amino acids with the GPI anchor, Construct II has truncated Thy-1 (the same C-terminal amino acids as construct I) without the GPI anchor, and Construct III has the full Thy-1 protein and the GPI anchor. The cell lines were grown on non-adherent plates to allow formation of spheroids. Spheroid formation is dependent on the number of cancer stem cells. The size and number of spheroids were measured using microscopy. Spheroids were defined as an aggregate of five or more cells. Spheroid density was calculated using ImageJ from five random fields for each cell line at 2, 24, 48, and 72 hours after seeding onto non-adherent plates. Analysis shows that construct I and construct III have a similar number and density of spheroids indicating that the GPI anchor and the 30 amino acids preceding the anchor are sufficient for promoting spheroid formation. This implies that this may be the region of interest of the Thy-1 protein that can be targeted for diagnosis and treatment. Conclusion: Determining the role of the GPI anchor and the amino acid components of Thy-1 for the self-renewal function of ovarian cancer stem cells is the first step in investigating possible roles for this glycan structure in ovarian cancer metastasis. The results of these studies can lead to future development of diagnostic and therapeutic techniques for earlier detection and treatment of ovarian cancer. Citation Format: Veronica Alvarez, Karen L. Abbott. Potential role of the GPI anchor of Thy-1 in expanding ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB540.

High-Metastatic Melanoma Cells Promote the Metastatic Capability of Low-Metastatic Melanoma Cells via Exosomal Transfer of miR-411-5p.

Melanoma is characterized by high rate of metastasis and mortality. Effective management of metastatic melanoma depends on renewed mechanistic understanding underlying melanoma progression and metastasis. The role of exosomes in mediating the interactions between cancer cells and the metastatic microenvironment is at the forefront of cancer research. Previous researches on the function of exosomes in metastasis have been primarily focused on tumor cell-derived exosomes in modifying the biological functions of stromal cells. Whether the cancer cells at the involved organ can modify the metastatic capability of each other has not been demonstrated. In this study, a paired M14 melanoma derivative cell line, i.e., M14-OL and POL, that we established and characterized were employed. Oligo-metastatic (M14-OL) and poly-metastatic (M14-POL) cell line were generated from three consecutive rounds of in vivo selection and passage. They exhibit high (POL cells) and low (OL cells) metastatic colonization efficiency in vivo, respectively. We show that exosomal crosstalk between metastatic cancer cells is a new mechanism of cancer metastasis. High-metastatic melanoma cells (POL) can augment the metastatic colonization capability of the low-metastatic melanoma cells (OL). POL achieves this goal by utilizing its exosomes to deliver functional miRNAs, in this case, miR-411-5p, to the OL cell. Upon entering OL cells, exosomal miR-411-5p enhance metastatic colonization efficiency by activation of the ERK signaling pathway. Moreover, miR-411-5p expression is higher in cancer tissues of other cancer types (colon, lung, rectum) compared with that of respective normal tissues. The clinical relevance of the present finding merits future investigations.

All-Ion Monitoring-Directed Low-Abundance Protein Quantification Reveals CALB2 as a Key Promoter in Hepatocellular Carcinoma Metastasis.

Because of the wide abundance range of the proteome, achieving high-coverage quantification of low-abundance proteins is always a major challenge. In this study, a complete pipeline focused on all-ion monitoring (AIM) is first constructed with the concept of untargeted parallel-reaction monitoring, including the seamless connection of protein sample preparation, liquid chromatography mass spectrometry (LC-MS) acquisition, and algorithm development to enable the in-depth quantitative analysis of low-abundance proteins. This pipeline significantly improves the reproducibility and sensitivity of sample preparation and LC-MS acquisition for low-abundance proteins, enabling all the precursors ions fragmented and collected. Contributed by the advantages of the AIM method with all the target precursor acquisition by the data-dependent acquisition (DDA) approach, together with the ability of data-independent acquisition to fragment all precursor ions, the quantitative accuracy and precision of low-abundance proteins are greatly enhanced. As a proof of concept, this pipeline is employed to discover the key differential proteins in the mechanism of hepatocellular carcinoma (HCC) metastasis. On the basis of the superiority of AIM, an extremely low-abundance protein, CALB2, is proposed to promote HCC metastasis in vitro and in vivo. We also reveal that CALB2 activates the TRPV2-Ca2+-ERK1/2 signaling pathway to induce HCC cell metastasis. In summary, we provide a universal AIM pipeline for the high-coverage quantification of low-abundance functional proteins to seek novel insights into the mechanisms of cancer metastasis.

The Lymph Node Microenvironment May Invigorate Cancer Cells With Enhanced Metastatic Capacities.

Cancer metastasis, a typical malignant biological behavior involving the distant migration of tumor cells from the primary site to other organs, contributed majorly to cancer-related deaths of patients. Although constant efforts have been paid by researchers to elucidate the mechanisms of cancer metastasis, we are still far away from the definite answer. Recently, emerging evidence demonstrated that cancer metastasis is a continuous coevolutionary process mediated by the interactions between tumor cells and the host organ microenvironment, and epigenetic reprogramming of metastatic cancer cells may confer them with stronger metastatic capacities. The lymph node served as the first metastatic niche for many types of cancer, and the appearance of lymph node metastasis predicted poor prognosis. Importantly, multiple immune cells and stromal cells station and linger in the lymph nodes, which constitutes the complexity of the lymph node microenvironment. The active cross talk between cancer cells and immune cells could happen unceasingly within the metastatic environment of lymph nodes. Of note, diverse immune cells have been found to participate in the formation of malignant properties of tumor, including stemness and immune escape. Based on these available evidence and data, we hypothesize that the metastatic microenvironment of lymph nodes could drive cancer cells to metastasize to further organs through epigenetic mechanisms.

CRISPR/Cas9 Mediated Knockout of Cyclooxygenase-2 Gene Inhibits Invasiveness in A2058 Melanoma Cells.

The inducible isoenzyme cyclooxygenase-2 (COX-2) is an important hub in cellular signaling, which contributes to tumor progression by modulating and enhancing a pro-inflammatory tumor microenvironment, tumor growth, apoptosis resistance, angiogenesis and metastasis. In order to understand the role of COX-2 expression in melanoma, we investigated the functional knockout effect of COX-2 in A2058 human melanoma cells. COX-2 knockout was validated by Western blot and flow cytometry analysis. When comparing COX-2 knockout cells to controls, we observed significantly reduced invasion, colony and spheroid formation potential in cell monolayers and three-dimensional models in vitro, and significantly reduced tumor development in xenograft mouse models in vivo. Moreover, COX-2 knockout alters the metabolic activity of cells under normoxia and experimental hypoxia as demonstrated by using the radiotracers [18F]FDG and [18F]FMISO. Finally, a pilot protein array analysis in COX-2 knockout cells verified significantly altered downstream signaling pathways that can be linked to cellular and molecular mechanisms of cancer metastasis closely related to the enzyme. Given the complexity of the signaling pathways and the multifaceted role of COX-2, targeted suppression of COX-2 in melanoma cells, in combination with modulation of related signaling pathways, appears to be a promising therapeutic approach.

Towards targeting of shared mechanisms of cancer metastasis and therapy resistance.

Resistance to therapeutic treatment and metastatic progression jointly determine a fatal outcome of cancer. Cancer metastasis and therapeutic resistance are traditionally studied as separate fields using non-overlapping strategies. However, emerging evidence, including from in vivo imaging and in vitro organotypic culture, now suggests that both programmes cooperate and reinforce each other in the invasion niche and persist upon metastatic evasion. As a consequence, cancer cell subpopulations exhibiting metastatic invasion undergo multistep reprogramming that - beyond migration signalling - supports repair programmes, anti-apoptosis processes, metabolic adaptation, stemness and survival. Shared metastasis and therapy resistance signalling are mediated by multiple mechanisms, such as engagement of integrins and other context receptors, cell-cell communication, stress responses and metabolic reprogramming, which cooperate with effects elicited by autocrine and paracrine chemokine and growth factor cues present in the activated tumour microenvironment. These signals empower metastatic cells to cope with therapeutic assault and survive. Identifying nodes shared in metastasis and therapy resistance signalling networks should offer new opportunities to improve anticancer therapy beyond current strategies, to eliminate both nodular lesions and cells in metastatic transit.