In solid tumors, vascular structure and function varies from the core to the periphery. This structural heterogeneity has been proposed to influence the mechanisms by which tumor cells enter the ...circulation. Blood vessels exhibit regional defects in endothelial coverage, which can result in cancer cells directly exposed to flow and potentially promoting intravasation. Consistent with prior reports, we observed in human breast tumors and in a mouse model of breast cancer that approximately 6% of vessels consisted of both endothelial cells and tumor cells, so-called mosaic vessels. Due, in part, to the challenges associated with observing tumor-vessel interactions deep within tumors in real-time, the mechanisms by which mosaic vessels form remain incompletely understood. We developed a tissue-engineered model containing a physiologically realistic microvessel in coculture with mammary tumor organoids. This approach allows real-time and quantitative assessment of tumor-vessel interactions under conditions that recapitulate many
features. Imaging revealed that tumor organoids integrate into the endothelial cell lining, resulting in mosaic vessels with gaps in the basement membrane. While mosaic vessel formation was the most frequently observed interaction, tumor organoids also actively constricted and displaced vessels. Furthermore, intravasation of cancer cell clusters was observed following the formation of a mosaic vessel. Taken together, our data reveal that cancer cells can rapidly reshape, destroy, or integrate into existing blood vessels, thereby affecting oxygenation, perfusion, and systemic dissemination. Our novel assay also enables future studies to identify targetable mechanisms of vascular recruitment and intravasation. SIGNIFICANCE: A tissue-engineered microdevice that recapitulates the tumor-vascular microenvironment enables real-time imaging of the cellular mechanisms of mosaic vessel formation and vascular defect generation.
Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct ...clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14⁺ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14⁺ epithelial tumor cell clusters disseminate collectively to colonize distant organs.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe elimination ...(clearance), focusing on the intramural periarterial drainage (IPAD) pathway.
Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min to 48 h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD-vascular matrix analyzed in 11,668 metastases.
In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85-93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways.
Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.
The mammary epithelium elaborates through hormonally regulated changes in proliferation, migration and differentiation. Non-muscle myosin II (NMII) functions at the interface between contractility, ...adhesion and signal transduction. It is therefore a plausible regulator of mammary morphogenesis. We tested the genetic requirement for NMIIA and NMIIB in mammary morphogenesis through deletion of the three NMII heavy chain-encoding genes (
,
and
; also known as
,
and
, respectively) that confer specificity to the complex. Surprisingly, mosaic loss, but not ubiquitous loss, of
and
induced high levels of proliferation in 3D culture. This phenotype was observed even when cells were cultured in basal medium, which does not support tissue level growth of wild-type epithelium. Mosaic loss of NMIIA and NMIIB combined with FGF signaling to induce hyperplasia. Mosaic analysis revealed that the cells that were null for both NMIIA and NMIIB, as well as wild-type cells, proliferated, indicating that the regulation of proliferation is both cell autonomous and non-autonomous within epithelial tissues. This phenotype appears to be mediated by cell-cell contact, as co-culture did not induce proliferation. Mosaic loss of NMIIA and NMIIB also induced excess proliferation
Our data therefore reveal a role for NMIIA and NMIIB as negative regulators of proliferation in the mammary epithelium.
The bifunctional alkylating agents epichlorohydrin (ECH) and diepoxybutane (DEB) have been linked to increased cancer risks in industrial workers. These compounds react with DNA and proteins, leading ...to genotoxic effects. We used the comet assay to monitor formation of cross-links in HL-60 cells treated with ECH, DEB, and the structurally related anti-cancer drug mechlorethamine (HN2). We report a time- and dose-dependent cytotoxicity that correlated with cross-linking activity, following the order HN2 > DEB > ECH. The rate of cross-link repair also varied with drug, with ECH-induced lesions the fastest to repair. High drug doses led to the formation of saturating amounts of HN2 cross-links that were repaired inefficiently. DEB and ECH produced fewer overall cross-links, but some were also resistant to repair. These persistent cross-links may activate cell-cycle arrest to allow repair of damage, with prolonged arrest triggering apoptosis. Quantitative reverse transcription polymerase chain reaction experiments revealed that treatment of HL-60 cells with DEB and ECH results in up-regulation of several genes involved in the intrinsic (mitochondrial) apoptosis pathway, including BAX, BAK1, CASP-9, APAF-1, and BCL-2. These findings contribute to our understanding of the principles underlying the carcinogenic potentials of these xenobiotics.
•Cytotoxicity paralleled cross-linking in HL-60 cells for the drugs of interest.•This is the first report of epichlorohydrin (ECH) interstrand cross-links in cells.•At low drug doses, the cross-links had differential rates of repair.•Higher drug doses led to persistent cross-links that were resistant to repair.•ECH and diepoxybutane activated the intrinsic apoptosis pathway in HL-60 cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Real time quantitative reverse transcription PCR was used to monitor changes in apoptotic gene expression after treating cells with the DNA cross-linkers epichlorohydrin (ECH) and diepoxybutane ...(DEB). This article presents the data obtained from application of the comparative CT method to the amplification of twelve apoptotic genes in human MCF10-A cells and eight genes in HUVEC cells. Further insight regarding the significance of these data can be found in “Cross-linking by epichlorohydrin and diepoxybutane correlates with cytotoxicity and leads to apoptosis in human leukemia (HL-60) cells” (Le et al., 2018) 1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Leptomeningeal metastases (LM), characterized by cancer cell growth in the linings of the brain and spinal cord, and the cerebrospinal fluid (CSF) compartment, have a dire prognosis ...represent 5-15% of central nervous system metastases. LM occur in all subclasses of breast cancer and are currently treated with intrathecal methotrexate or liposomal cytarabine or radiation therapy. Despite initial response to current treatments, median survival remains poor. Relatively little is known on the development of LM, due to the lack of adequate mouse models. This study aimed to develop and validate in vivo models of LM and to use them to credential potential therapies. Three breast cancer models were established using intracisternal injections of tumor cells covering the HER2+, ER+ lobular and triple negative breast cancer subtypes. The HER2+ model was produced from the JIMT-1-BR cell line that previously was enriched for metastasis to the brain parenchyma, reflecting clinical observations of concurrent parenchymal and LM spread. Multiple endpoints were implemented to assess disease progression: bioluminescence imaging, histopathology, MRI, tumor cells in CSF. Bulk RNA sequencing on all LM-tropic cell lines revealed significant alterations in cytokine-cytokine receptor interactions, calcium signaling pathways, cell adhesion molecules, and TNF signaling pathways across all models. Notably, several genes including PORCN, PGBD5, FAM43A and FHOD3 were differentially expressed and consistently alter across all models. Validation of these key genes is underway. For the JIMT-1-BR HER2+ model, efficacy studies compared standard therapy (trastuzumab + paclitaxel) with the antibody-drug conjugate trastuzumab deruxtecan (T-DXd). T-DXd (10 mg/kg, iv q3w) demonstrated superior efficacy with extended survival and reduced extent of edema on MRI. This study established and validated three in vivo models of LM colonization. These models, incorporating diverse endpoints, and drug testing capabilities, offer a valuable platform for identifying novel therapeutic strategies to combat this aggressive form of breast cancer progression.
Abstract
Metastasis is responsible for the majority of cancer related deaths; however, many of the biological and physical details surrounding the critical steps (e.g. invasion and intravasation) are ...largely unknown, in part due to the difficulty in recapitulating and visualizing these dynamic processes. To elucidate these mechanisms, we have developed an in vitro model of invasion and intravasation that comprises tumor cells embedded within an extracellular matrix (ECM) surrounding an engineered microvessel. The microvessel is cylindrical, 150 µm in diameter, and lined with endothelial cells forming a functional barrier that is maintained under constant perfusion at a shear stress of 2-8 dyne cm-2. Using this biomimetic tumor-microvessel platform, we have previously visualized invasion and intravasation of single MDA-MB-231 breast cancer cells into the microvessel and escape into flow. Here, we present mechanistic details of intravasation from both single tumor cells (MDA-MB-231) and mouse mammary tumor organoids (MMTV-PyMT). For single cells, we observe a mitosis-mediated mechanism of intravasation where tumor cell entry into flow is prefaced by cell division at the ECM-vessel interface. For tumor organoids, we see vascular deformation and destabilization from growing tumors that impinge upon proximal vessels, and that bulk intravasation is mediated by shear stress and tumor cell adhesion. As we characterize the various ways in which tumor cells interact with the vessel endothelium and intravasate, we can explore strategies involving the tumor vasculature and clinically relevant drugs to inhibit critical steps in the metastatic cascade.
Citation Format: Andrew D. Wong, Vanesa Silvestri, Anderw J. Ewald, Peter C. Searson. Visualization of the mechanisms of metastasis within a biomimetic engineered tumor microenvironment encompassing a perfusable cylindrical 3D microvessel abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5017. doi:10.1158/1538-7445.AM2017-5017
Abstract
Metastasis is responsible for the majority of cancer related deaths; however, many of the biological and physical details surrounding the critical steps (e.g. invasion and intravasation) are ...largely unknown, in part due to the difficulty in recapitulating and visualizing these dynamic processes. To elucidate these mechanisms, we have developed an in vitro model of invasion and intravasation that comprises tumor cells embedded within an extracellular matrix (ECM) surrounding an engineered microvessel. The microvessel is cylindrical, 150 μm in diameter, and lined with endothelial cells forming a functional barrier that is maintained under constant perfusion at a shear stress of 2-8 dyne cm-2. Using this biomimetic tumor-microvessel platform, we have previously visualized invasion and intravasation of single MDA-MB-231 breast cancer cells into the microvessel and escape into flow. Here, we present mechanistic details of intravasation from both single tumor cells (MDA-MB-231) and mouse mammary tumor organoids (MMTV-PyMT). For single cells, we observe a mitosis-mediated mechanism of intravasation where tumor cell entry into flow is prefaced by cell division at the ECM-vessel interface. For tumor organoids, we see vascular deformation and destabilization from growing tumors that impinge upon proximal vessels, and that bulk intravasation is mediated by shear stress and tumor cell adhesion. As we characterize the various ways in which tumor cells interact with the vessel endothelium and intravasate, we can explore strategies involving the tumor vasculature and clinically relevant drugs to inhibit critical steps in the metastatic cascade.
Citation Format: Andrew D. Wong, Vanesa Silvestri, Andrew J. Ewald, Peter C. Searson. Visualization of the mechanisms of metastasis within a biomimetic engineered tumor microenvironment encompassing a perfusable cylindrical 3D microvessel. abstract. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B07.
Abstract The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe ...elimination (clearance), focusing on the intramural periarterial drainage (IPAD) pathway. Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min to 48 h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD-vascular matrix analyzed in 11,668 metastases. In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85–93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways. Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.
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