How circulating tumor cluster biology contributes to the metastatic cascade: from invasion to dissemination and dormancy.

Circulating tumor cells (CTCs) are known to be prognostic for metastatic relapse and are detected in patients as solitary cells or cell clusters. Circulating tumor cell clusters (CTC clusters) have been observed clinically for decades and are of significantly higher metastatic potential compared to solitary CTCs. Recent studies suggest distinct differences in CTC cluster biology regarding invasion and survival in circulation. However, differences regarding dissemination, dormancy, and reawakening require more investigations compared to solitary CTCs. Here, we review the current state of CTC cluster research and consider their clinical significance. In addition, we discuss the concept of collective invasion by CTC clusters and molecular evidence as to how cluster survival in circulation compares to that of solitary CTCs. Molecular differences between solitary and clustered CTCs during dormancy and reawakening programs will also be discussed. We also highlight future directions to advance our current understanding of CTC cluster biology.

Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Should I stay or should I go? Spatio-temporal control of cellular anchorage by hematopoietic factors orchestrates tumor metastatic cascade.

The term "metastatic cascade" defines a process whereby few tumor cells complete a sequence of steps to leave the primary tumor to reach one or more sites elsewhere in the body, usually through the bloodstream to develop one or several metastases. Due to the nature and plasticity of cancer, unfortunately no specific and functional anti-metastatic drugs are available. In this Commentary, we are highlighting how four essential factors are able to induce adhesion-to-suspension transition (herein referred to as AST) in human cancer cells and how this process may play a key role in tumor metastasis. We further underlined the potential role of hematopoietic transcriptional regulators in reprogramming anchorage dependency of cells, supporting the possible targeting of AST factors as promising therapeutic strategy to overcome metastasis in solid tumor cells.

Oscillatory Hypoxia Can Induce Senescence of Adipose-Derived Mesenchymal Stromal Cells Potentiating Invasive Transformation of Breast Epithelial Cells.

Obesity is strongly associated with occurrence, metastasis, and resistance to therapy in breast cancers, which also exhibit high adipose content in the tumor microenvironment. Adipose tissue-derived mesenchymal stromal cells (ASCs) are recruited to breast cancer by many mechanisms, including hypoxia, and contribute to metastatic transition of the cancer. Breast cancers are characterized by regions of hypoxia, which can be temporally unstable owing to a mismatch between oxygen supply and consumption. Using a high-sensitivity nanopatterned stromal invasion assay, we found that ASCs could promote stromal invasion of not only breast cancer cell lines but also MCF10A1, a cell line derived from untransformed breast epithelium. RNA sequencing of MCF10A1 cells conditioned with medium from ASCs revealed upregulation of genes associated with increased cell migration, chemotaxis, and metastasis. Furthermore, we found that fluctuating or oscillating hypoxia could induce senescence in ASCs, which could result in an increased invasive potential in the treated MCF10A1 cells. These findings highlight the complex interplay within the breast cancer microenvironment, hypoxia, and the role of ASCs in transforming even non-cancerous breast epithelium toward an invasive phenotype, providing insights into early metastatic events.

Primary Colorectal Tumor Displays Differential Genomic Expression Profiles Associated with Hepatic and Peritoneal Metastases.

BACKGROUND: Despite improvements in characterization of CRC heterogeneity, appropriate risk stratification tools are still lacking in clinical practice. This study aimed to elucidate the primary tumor transcriptomic signatures associated with distinct metastatic routes. METHODS: Primary tumor specimens obtained from CRC patients with either isolated LM (CRC-Liver) or PM (CRC-Peritoneum) were analyzed by transcriptomic mRNA sequencing, gene set enrichment analyses (GSEA) and immunohistochemistry. We further assessed the clinico-pathological associations and prognostic value of our signature in the COAD-TCGA independent cohort. RESULTS: We identified a significantly different distribution of Consensus Molecular Subtypes between CRC-Liver and CRC-peritoneum groups. A transcriptomic signature based on 61 genes discriminated between liver and peritoneal metastatic routes. GSEA showed a higher expression of immune response and epithelial invasion pathways in CRC-Peritoneum samples and activation of proliferation and metabolic pathways in CRC-Liver samples. The biological relevance of RNA-Seq results was validated by the immunohistochemical expression of three significantly differentially expressed genes (ACE2, CLDN18 and DUSP4) in our signature. In silico analysis of the COAD-TCGA showed that the CRC-Peritoneum signature was associated with negative prognostic factors and poor overall and disease-free survivals. CONCLUSIONS: CRC primary tumors spreading to the liver and peritoneum display significantly different transcriptomic profiles. The implementation of this signature in clinical practice could contribute to identify new therapeutic targets for stage IV CRC and to define individualized follow-up programs in stage II-III CRC.

Targeting phosphoglycerate kinases by tatridin A, a natural sesquiterpenoid endowed with anti-cancer activity, using a proteomic platform.

Tatridin A (TatA) is a germacrane sesquiterpenoid containing one E-double bond and one Z-double bond in its 10-membered ring, which is fused to a 3-methylene-dihydrofuran-2-one moiety. Tatridin A bioactivity has been poorly investigated despite its interesting chemical structure. Here, a functional proteomic platform was adapted to disclose its most reliable targets in leukemia monocytic cells, and phosphoglycerate kinases were recognized as the most affine enzymes. Through a combination of limited proteolysis and molecular docking, it has been discovered that tatridin A interacts with the active domains of phosphoglycerate kinase 1, altering its hinge region, and it can be accountable for tatridin A inhibition potency on enzyme activity. A more detailed tatridin A biological profile showed that it is also fully active against gastric cancer cells, downregulating the mRNA levels of chemokine receptor 4 and ß-catenin and inhibiting the invasiveness of living KATO III cells as a direct consequence of phosphoglycerate kinase 1 antagonism.

Establishment of the SIS scaffold-based 3D model of human peritoneum for studying the dissemination of ovarian cancer.

Ovarian cancer is the second most common gynecological malignancy in women. More than 70% of the cases are diagnosed at the advanced stage, presenting as primary peritoneal metastasis, which results in a poor 5-year survival rate of around 40%. Mechanisms of peritoneal metastasis, including adhesion, migration, and invasion, are still not completely understood and therapeutic options are extremely limited. Therefore, there is a strong requirement for a 3D model mimicking the in vivo situation. In this study, we describe the establishment of a 3D tissue model of the human peritoneum based on decellularized porcine small intestinal submucosa (SIS) scaffold. The SIS scaffold was populated with human dermal fibroblasts, with LP-9 cells on the apical side representing the peritoneal mesothelium, while HUVEC cells on the basal side of the scaffold served to mimic the endothelial cell layer. Functional analyses of the transepithelial electrical resistance (TEER) and the FITC-dextran assay indicated the high barrier integrity of our model. The histological, immunohistochemical, and ultrastructural analyses showed the main characteristics of the site of adhesion. Initial experiments using the SKOV-3 cell line as representative for ovarian carcinoma demonstrated the usefulness of our models for studying tumor cell adhesion, as well as the effect of tumor cells on endothelial cell-to-cell contacts. Taken together, our data show that the novel peritoneal 3D tissue model is a promising tool for studying the peritoneal dissemination of ovarian cancer.

Metastatic Transition of Pancreatic Ductal Cell Adenocarcinoma Is Accompanied by the Emergence of Pro-Invasive Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) are now appreciated as key regulators of cancer metastasis, particularly in cancers with high stromal content, e.g., pancreatic ductal cell carcinoma (PDAC). However, it is not yet well understood if fibroblasts are always primed to be cooperative in PDAC transition to metastasis, if they undergo transformation which ensures their cooperativity, and if such transformations are cancer-driven or intrinsic to fibroblasts. We performed a fibroblast-centric analysis of PDAC cancer, as it transitioned from the primary site to trespass stromal compartment reaching the lymph node using published single-cell RNA sequencing data by Peng et al. We have characterized the change in fibroblast response to cancer from a normal wound healing response in the initial stages to the emergence of subclasses with myofibroblast and inflammatory fibroblasts such as signatures. We have previously posited "Evolved Levels of Invasibility (ELI)", a framework describing the evolution of stromal invasability as a selected phenotype, which explains the large and correlated reduction in stromal invasion by placental trophoblasts and cancer cells in certain mammals. Within PDAC samples, we found large changes in fibroblast subclasses at succeeding stages of PDAC progression, with the emergence of specific subclasses when cancer trespasses stroma to metastasize to proximal lymph nodes (stage IIA to IIB). Surprisingly, we found that the initial metastatic transition is accompanied by downregulation of ELI-predicted pro-resistive genes, and the emergence of a subclass of fibroblasts with ELI-predicted increased invasibility. Interestingly, this trend was also observed in stellate cells. Using a larger cohort of bulk RNAseq data from The Cancer Genome Atlas for PDAC cancers, we confirmed that genes describing this emergent fibroblast subclass are also correlated with lymph node metastasis of cancer cells. Experimental testing of selected genes characterizing pro-resistive and pro-invasive fibroblast clusters confirmed their contribution in regulating stromal invasability as a phenotype. Our data confirm that the complexity of stromal response to cancer is really a function of stage-wise emergence of distinct fibroblast clusters, characterized by distinct gene sets which confer initially a predominantly pro-resistive and then a pro-invasive property to the stroma. Stromal response therefore transitions from being tumor-limiting to a pro-metastatic state, facilitating stromal trespass and the onset of metastasis.

Evolved Resistance to Placental Invasion Secondarily Confers Increased Survival in Melanoma Patients.

Mammals exhibit large differences in rates of cancer malignancy, even though the tumor formation rates may be similar. In placental mammals, rates of malignancy correlate with the extent of placental invasion. Our Evolved Levels of Invasibility (ELI) framework links these two phenomena identifying genes that potentially confer resistance in stromal fibroblasts to limit invasion, from trophoblasts in the endometrium, and from disseminating melanoma in the skin. Herein, using patient data from The Cancer Genome Atlas (TCGA), we report that these anti-invasive genes may be crucial in melanoma progression in human patients, and that their loss is correlated with increased cancer spread and lowered survival. Our results suggest that, surprisingly, these anti-invasive genes, which have lower expression in humans compared to species with non-invasive placentation, may potentially prevent stromal invasion, while a further reduction in their levels increases the malignancy and lethality of melanoma. Our work links evolution, comparative biology, and cancer progression across tissues, indicating new avenues for using evolutionary medicine to prognosticate and treat human cancers.

Signaling network involved in the GPC3-induced inhibition of breast cancer progression: role of canonical Wnt pathway.

PURPOSE: We have shown that GPC3 overexpression in breast cancer cells inhibits in vivo tumor progression, by acting as a metastatic suppressor. GPC3-overexpressing cells are less clonogenic, viable and motile, while their homotypic adhesion is increased. We have presented evidences indicating that GPC3 inhibits canonical Wnt and Akt pathways, while non-canonical Wnt and p38MAPK cascades are activated. In this study, we aimed to investigate whether GPC3-induced Wnt signaling inhibition modulates breast cancer cell properties as well as to describe the interactions among pathways modulated by GPC3. METHODS: Fluorescence microscopy, qRT-PCR microarray, gene reporter assay and Western blotting were performed to determine gene expression levels, signaling pathway activities and molecule localization. Lithium was employed to activate canonical Wnt pathway and treated LM3-GPC3 cell viability, migration, cytoskeleton organization and homotypic adhesion were assessed using MTS, wound healing, phalloidin staining and suspension growth assays, respectively. RESULTS: We provide new data demonstrating that GPC3 blocks-also at a transcriptional level-both autocrine and paracrine canonical Wnt activities, and that this inhibition is required for GPC3 to modulate migration and homotypic adhesion. Our results indicate that GPC3 is secreted into the extracellular media, suggesting that secreted GPC3 competes with Wnt factors or interacts with them and thus prevents Wnt binding to Fz receptors. We also describe the complex network of interactions among GPC3-modulated signaling pathways. CONCLUSION: GPC3 is operating through an intricate molecular signaling network. From the balance of these interactions, the inhibition of breast metastatic spread induced by GPC3 emerges.

Regulation of the prometastatic neuregulin-MMP13 axis by SRC family kinases: therapeutic implications.

Metastatic dissemination of tumor cells is responsible for the fatal outcome of breast cancer. Therefore, understanding the mechanisms involved in dissemination is essential for the development of new therapeutic strategies to prevent metastasis. One mechanism involved in metastatic dissemination of breast cancer cells is dependent on control of the production of matrix metalloproteinases by the neuregulins (NRGs). The NRGs are polypeptide factors that act by binding to the ErbB/HER subfamily of receptor tyrosine kinases. NRG-mediated activation of HER receptors causes an increase in the production of metalloprotease 13 (MMP13, also termed collagenase-3), which facilitates metastatic dissemination of breast tumors. In this context, we aimed to explore whether the clinically approved tyrosine kinase inhibitor dasatinib was able to neutralize this mechanism of metastatic dissemination. Here, we show that dasatinib restricted NRG-induced MMP13 upregulation, both in vitro and in vivo, and in vivo metastatic dissemination of breast cancer cells. Chemical proteomics studies showed that the main cellular targets of dasatinib were SRC family kinases (SFKs). Moreover, genetic studies showed that knockdown of SRC or YES strongly inhibited NRG-induced MMP13 upregulation in vitro. Mechanistically, dasatinib treatment or knockdown of SRC also inhibited ERK1/2 kinases in vitro, which were required for NRG-induced MMP13 upregulation. These results open the possibility of clinically exploring the antitumoral action of dasatinib in those tumors in which the NRG-MMP13 signaling axis may play a relevant role in the control of tumor cell dissemination.

Chorioallantoic Membrane Microtumor Model to Study the Mechanisms of Tumor Angiogenesis, Vascular Permeability, and Tumor Cell Intravasation.

The mechanisms governing the development of angiogenic blood vessels, which not only deliver the nutrients to growing tumors but also provide the conduits for tumor cell dissemination, are still not fully resolved. The model systems based on the grafting of human tumor cells onto the chorioallantoic membrane (CAM) of the chick embryo offer several advantages to study complex processes underlying tumor angiogenesis and tumor cell dissemination. In particular, the CAM model described here allows for investigation of multiple microtumors as independent entities, thereby greatly facilitating quantification and statistical analyses of tumor neovascularization and cancer spreading. This CAM microtumor system was designed specifically to measure the level of tumor cell intravasation in combination with quantitative analyses of the microarchitecture and permeability of the intratumoral angiogenic blood vessels. By using this newly established microtumor model we have demonstrated the functional involvement of tumor matrix metalloproteinase-1 (MMP-1) and epidermal growth factor receptor (EGFR) in regulating the development of a distinct angiogenic vasculature capable of sustaining tumor cell intravasation and metastasis.

Circulating galectin-3 in the bloodstream: An emerging promoter of cancer metastasis.

Increased concentrations of free circulating galectin-3 are commonly seen in the blood circulation of patients with many types of cancers including colorectal cancer. Recent studies have shown that changes in circulating galectin-3 levels in cancer patients may contribute significantly to the metastatic spread of disseminating cancer cells by enhancing their ability to adhere to blood vessel endothelium and by helping their avoidance of immune surveillance. Thus, targeting the galectin-3 actions in the circulation may hold significant promise for future development of novel therapeutic agents to prevent metastasis and reduce cancer-associated fatality.

Pancreatic cancer--low survival rates.

INTRODUCTION: Cancers of the pancreas are identified in 11 800 to 13 500 patients each year in Germany. Epidemiological studies prove smoking and chronic alcohol consumption as causes of about 30% of pancreatic cancers. METHODS: Selective literature review. RESULTS: Only patients within TNM stage I and II have after oncologic tumor extirpation a chance for long term survival. Controlled prospective clinical trials demonstrated adjuvant chemotherapy yielding an additional significant survival benefit. The 3- and 5-year-survival after R0-resection and adjuvant chemotherapy are about 30% and below 15% respectively. Using the criteria of observed 5-year-survival less than 2% of all pancreatic cancer patients are alive. After R0-resection the median survival time is between 17 and 28 months, after R1/2-resection between 8 and 22 months. DISCUSSION: Pancreatic cancer is even today for more than 95% of the patients incurable. Strategies to prevent pancreatic cancer are intended to stop smoking and chronic alcohol consumption and early surgical extirpation of cystic neoplastic lesions. For patients with established pancreatic cancer risk a follow-up protocol is discussed.