Engrailed-1 Promotes Pancreatic Cancer Metastasis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

A human initial lymphatic chip reveals distinct mechanisms of primary lymphatic valve dysfunction in acute and chronic inflammation.

Interstitial fluid uptake and retention by lymphatic vessels (LVs) play a role in maintaining interstitial fluid homeostasis. While it is well-established that intraluminal lymphatic valves in the collecting LVs prevent fluid backflow (secondary lymphatic valves), a separate valve system in the initial LVs that only permits interstitial fluid influx into the LVs, preventing fluid leakage back to the interstitium (primary lymphatic valves), remains incompletely understood. Although lymphatic dysfunction is commonly observed in inflammation and autoimmune diseases, how the primary lymphatic valves are affected by acute and chronic inflammation has scarcely been explored and even less so using in vitro lymphatic models. Here, we developed a human initial lymphatic vessel chip where interstitial fluid pressure and luminal fluid pressure are controlled to examine primary lymph valve function. In normal conditions, lymphatic drainage (fluid uptake) and permeability (fluid leakage) in engineered LVs were maintained high and low, respectively, which was consistent with our understanding of healthy primary lymph valves. Next, we examined the effects of acute and chronic inflammation. Under the acute inflammation condition with a TNF-α treatment (2 hours), degradation of fibrillin and impeded lymphatic drainage were observed, which were reversed by treatment with anti-inflammatory dexamethasone. Surprisingly, the chronic inflammation condition (repeated TNF-α treatments during 48 hours) deposited fibrillin to compensate for the fibrillin loss, showing no change in lymphatic drainage. Instead, the chronic inflammation condition led to cell death and disruption of lymphatic endothelial cell-cell junctions, increasing lymphatic permeability and fluid leakage. Our human lymphatic model shows two distinct mechanisms by which primary lymphatic valve dysfunction occurs in acute and chronic inflammation.

Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT.

Gastrointestinal stromal tumors (GISTs) frequently show KIT mutations, accompanied by overexpression and aberrant localization of mutant KIT (MT-KIT). As previously established by multiple studies, including ours, we confirmed that MT-KIT initiates downstream signaling in the Golgi complex. Basic leucine zipper nuclear factor 1 (BLZF1) was identified as a novel MT-KIT-binding partner that tethers MT-KIT to the Golgi complex. Sustained activation of activated transcription factor 6 (ATF6), which belongs to the unfolded protein response (UPR) family, alleviates endoplasmic reticulum (ER) stress by upregulating chaperone expression, including heat shock protein 90 (HSP90), which assists in MT-KIT folding. BLZF1 knockdown and ATF6 inhibition suppressed both imatinib-sensitive and -resistant GIST in vitro. ATF6 inhibitors further showed potent antitumor effects in GIST xenografts, and the effect was enhanced with ER stress-inducing drugs. ATF6 activation was frequently observed in 67% of patients with GIST (n = 42), and was significantly associated with poorer relapse-free survival (P = 0.033). Overall, GIST bypasses ER quality control (QC) and ER stress-mediated cell death via UPR activation and uses the QC-free Golgi to initiate signaling.

Microphysiological Systems for Cancer Immunotherapy Research and Development.

Cancer immunotherapy focuses on the use of patients' adaptive immune systems to combat cancer. In the past decade, FDA has approved many immunotherapy products for cancer patients who suffer from primary tumors, tumor relapse, and metastases. However, these immunotherapies still show resistance in many patients and often lead to inconsistent responses in patients due to variations in tumor genetic mutations and tumor immune microenvironment. Microfluidics-based organ-on-a-chip technologies or microphysiological systems have opened new ways that can provide relatively fast screening for personalized immunotherapy and help researchers and clinicians understand tumor-immune interactions in a patient-specific manner. They also have the potential to overcome the limitations of traditional drug screening and testing, given the models provide a more realistic 3D microenvironment with better controllability, reproducibility, and physiological relevance. This review focuses on the cutting-edge microphysiological organ-on-a-chip devices developed in recent years for studying cancer immunity and testing cancer immunotherapeutic agents, as well as some of the largest challenges of translating this technology to clinical applications in immunotherapy and personalized medicine.

Comprehensive characterization of distinct genetic alterations in metastatic breast cancer across various metastatic sites.

Metastasis is the major cause of death in breast cancer patients. Although previous large-scale analyses have identified frequently altered genes specific to metastatic breast cancer (MBC) compared with those in primary breast cancer (PBC), metastatic site-specific altered genes in MBC remain largely uncharacterized. Moreover, large-scale analyses are required owing to the low expected frequency of such alterations, likely caused by tumor heterogeneity and late dissemination of breast cancer. To clarify MBC-specific genetic alterations, we integrated publicly available clinical and mutation data of 261 genes, including MBC drivers, from 4268 MBC and 5217 PBC patients from eight different cohorts. We performed meta-analyses and logistic regression analyses to identify MBC-enriched genetic alterations relative to those in PBC across 15 different metastatic site sets. We identified 11 genes that were more frequently altered in MBC samples from pan-metastatic sites, including four genes (SMARCA4, TSC2, ATRX, and AURKA) which were not identified previously. ARID2 mutations were enriched in treatment-naïve de novo and post-treatment MBC samples, compared with that in treatment-naïve PBC samples. In metastatic site-specific analyses, associations of ESR1 with liver metastasis and RICTOR with bone metastasis were significant, regardless of intrinsic subtypes. Among the 15 metastatic site sets, ESR1 mutations were enriched in the liver and depleted in the lymph nodes, whereas TP53 mutations showed an opposite trend. Seven potential MBC driver mutations showed similar preferential enrichment in specific metastatic sites. This large-scale study identified new MBC genetic alterations according to various metastatic sites and highlights their potential role in breast cancer organotropism.

In vitro modeling of solid tumor interactions with perfused blood vessels.

Molecular crosstalk between intra-tumor blood vessels and tumor cells plays many critical roles in tumorigenesis and cancer metastasis. However, it has been very difficult to investigate the biochemical mechanisms underlying the overlapping, multifactorial processes that occur at the tumor-vascular interface using conventional murine models alone. Moreover, traditional two-dimensional (2D) culture models used in cancer research do not recapitulate aspects of the 3D tumor microenvironment. In the present study, we introduce a microfluidic model of the solid tumor-vascular interface composed of a human umbilical vein endothelial cell (HUVEC)-lined, perfusable, bioengineered blood vessel and tumor spheroids embedded in an extracellular matrix (ECM). We sought to optimize our model by varying the composition of the tumor spheroids (MDA-MB-231 breast tumor cells + mesenchymal stem cells (MSCs)/human lung fibroblasts (HLFs)/HUVECs) and the extracellular matrix (ECM: collagen, Matrigel, and fibrin gels with or without free HLFs) that we used. Our results indicate that culturing tumor spheroids containing MDA-MB-231 cells + HUVECs in an HLF-laden, fibrin-based ECM within our microfluidic device optimally (1) enhances the sprouting and migration of tumor spheroids, (2) promotes angiogenesis, (3) facilitates vascular invasion, and (4) preserves the structural integrity and functionality of HUVEC-lined microfluidic channels. This model may provide a platform for drug screening and mechanism studies on solid tumor interactions with functional blood vessels.

Rapid multilayer microfabrication for modeling organotropic metastasis in breast cancer.

Triple-negative breast cancer (TNBC) is one of the most insidious forms of breast cancer with high rates of metastasis, resulting in major mortalities in breast cancer patients. To better understand and treat TNBC metastasis, investigation of TNBC interactions with blood vasculatures is crucial. Among multiple metastatic processes, a step of TNBC exit from the blood vessels ('extravasation') in the pre-metastatic organs determines the final site of the metastasis. Here, we present a rapid multilayer microfabrication method of transferring a three-dimensional (3D) overhang pattern to a substrate with a sacrificial layer to reconstitute a 3D blood vessel surrounded by the extracellular matrix containing organ-specific parenchymal cells. Bones and lungs are the most common sites of breast cancer metastasis. We modeled organotropic bone and lung metastasis in TNBC by introducing subpopulations of TNBC metastases into a vessel lumen surrounded by osteoblasts, bone marrow derived mesenchymal stem cells, and lung fibroblasts. We found that bone-like microenviroment with osteoblasts and mesenchymal stem cells promoted extravasation of the bone-tropic TNBC cells, whereas the lung-like microenviroment promoted extravasation of the lung-tropic TNBC cells. Given that these organ-specific parenchymal cells do not impact vascular permeability, our results suggest that the parenchymal cells dictate selective extravasation of the bone-tropic or lung-tropic TNBC cells in our system.

A biomimetic pancreatic cancer on-chip reveals endothelial ablation via ALK7 signaling.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, lethal malignancy that invades adjacent vasculatures and spreads to distant sites before clinical detection. Although invasion into the peripancreatic vasculature is one of the hallmarks of PDAC, paradoxically, PDAC tumors also exhibit hypovascularity. How PDAC tumors become hypovascular is poorly understood. We describe an organotypic PDAC-on-a-chip culture model that emulates vascular invasion and tumor-blood vessel interactions to better understand PDAC-vascular interactions. The model features a 3D matrix containing juxtaposed PDAC and perfusable endothelial lumens. PDAC cells invaded through intervening matrix, into vessel lumen, and ablated the endothelial cells, leaving behind tumor-filled luminal structures. Endothelial ablation was also observed in in vivo PDAC models. We also identified the activin-ALK7 pathway as a mediator of endothelial ablation by PDAC. This tumor-on-a-chip model provides an important in vitro platform for investigating the process of PDAC-driven endothelial ablation and may provide a mechanism for tumor hypovascularity.

Biomimetic peptide display from a polymeric nanoparticle surface for targeting and antitumor activity to human triple-negative breast cancer cells.

While poly(lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) can encapsulate drug cargos and prolong circulation times, they show nonspecific accumulation in off-target tissues. Targeted delivery of drugs to tumor tissue and tumor vasculature is a promising approach for treating solid tumors while enhancing specificity and reducing systemic toxicity. AXT050, a collagen-IV derived peptide with both antitumor and antiangiogenic properties, is shown to bind to tumor-associated integrins with high affinity, which leads to targeted accumulation in tumor tissue. AXT050 conjugated to PLGA-PEG NPs at precisely controlled surface density functions both as a targeting agent to human tumor cells and demonstrates potential for simultaneous antitumorigenic and antiangiogenic activity. These targeted NPs cause inhibition of adhesion and proliferation in vitro when added to human triple-negative breast cancer cells and microvascular endothelial cells through binding to integrin αV ß3 . Furthermore, we find an in vivo biphasic relationship between tumor targeting and surface coating density of NPs coated with AXT050. NPs with an intermediate level of 10% peptide surface coating show approximately twofold greater accumulation in tumors and lower accumulation in the liver compared to nontargeted PLGA-PEG NPs in a murine biodistribution model. Display of biomimetic peptides from NP surfaces to both target and inhibit cancer cells has the potential to enhance the activity of cancer nanomedicines. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1753-1764, 2018.

Human Organ Chip Models Recapitulate Orthotopic Lung Cancer Growth, Therapeutic Responses, and Tumor Dormancy In Vitro.

Here, we show that microfluidic organ-on-a-chip (organ chip) cell culture technology can be used to create in vitro human orthotopic models of non-small-cell lung cancer (NSCLC) that recapitulate organ microenvironment-specific cancer growth, tumor dormancy, and responses to tyrosine kinase inhibitor (TKI) therapy observed in human patients in vivo. Use of the mechanical actuation functionalities of this technology revealed a previously unknown sensitivity of lung cancer cell growth, invasion, and TKI therapeutic responses to physical cues associated with breathing motions, which appear to be mediated by changes in signaling through epidermal growth factor receptor (EGFR) and MET protein kinase. These findings might help to explain the high level of resistance to therapy in cancer patients with minimal residual disease in regions of the lung that remain functionally aerated and mobile, in addition to providing an experimental model to study cancer persister cells and mechanisms of tumor dormancy in vitro.

Laminar flow downregulates Notch activity to promote lymphatic sprouting.

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow-induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow-induced lymphatic sprouting.

The Angiogenic Secretome in VEGF overexpressing Breast Cancer Xenografts.

The plasticity of cancer cells and the fluidity of the tumor microenvironment continue to present major challenges in the comprehensive understanding of cancer that is essential to design effective treatments. The tumor interstitial fluid (TIF) encompasses the secretome and holds the key to several of the phenotypic characteristics of cancer. Difficulties in sampling this fluid have resulted in limited characterization of its components. Here we have sampled TIF from triple negative and estrogen receptor (ER)-positive human breast tumor xenografts with or without VEGF overexpression. Angiogenesis-related factors were characterized in the TIF and plasma, to understand the relationship between the TIF and plasma secretomes. Clear differences were observed between the TIF and plasma angiogenic secretomes in triple negative MDA-MB-231 breast cancer xenografts compared to ER-positive MCF-7 xenografts with or without VEGF overexpression that provide new insights into TIF components and the role of VEGF in modifying the angiogenic secretome.

Biomimetic on-a-chip platforms for studying cancer metastasis.

Cancer metastasis is a multi-step, secondary tumor formation that is responsible for the vast majority of deaths in cancer patients. Animal models have served as one of the major tools for studying metastatic diseases. However, these metastasis models inherently lack the ability to decouple many of the key parameters that might contribute to cancer progression, and therefore ultimately limit detailed, mechanistic investigation of metastasis. Recently, organ-on-a-chip model systems have been developed for various tissue types with the potential to recapitulate major components of metastasis. Here, we discuss recent advances in in vitro biomimetic on-a-chip models for cancer metastasis.

Analysis of gene expression of secreted factors associated with breast cancer metastases in breast cancer subtypes.

Breast cancer is a heterogeneous disease, having multiple subtypes with different malignant phenotypes. The triple-negative breast cancer, or basal breast cancer, is highly aggressive, metastatic, and difficult to treat. Previously, we identified that key molecules (IL6, CSF2, CCL5, VEGFA, and VEGFC) secreted by tumor cells and stromal cells in basal breast cancer can promote metastasis. It remains to assess whether these molecules function similarly in other subtypes of breast cancer. Here, we characterize the relative gene expression of the five secreted molecules and their associated receptors (GP130, GMRA, GMRB, CCR5, VEGFR2, NRP1, VEGFR3, NRP2) in the basal, HER2 (human epidermal growth factor receptor 2) positive, luminal A, and luminal B subtypes using high throughput data from tumor samples in The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). IL6 and CCL5 gene expression are basal breast cancer specific, whereas high gene expression of GP130 was observed in luminal A/B. VEGFA/C and CSF2 mRNA are overexpressed in HER2 positive breast cancer, with VEGFA and CSF2 also overexpressed in basal breast cancer. Further study of the specific protein function of these factors within their associated cancer subtypes may yield personalized biomarkers and treatment modalities.

Crosstalk between cancer cells and blood endothelial and lymphatic endothelial cells in tumour and organ microenvironment.

Tumour and organ microenvironments are crucial for cancer progression and metastasis. Crosstalk between multiple non-malignant cell types in the microenvironments and cancer cells promotes tumour growth and metastasis. Blood and lymphatic endothelial cells (BEC and LEC) are two of the components in the microenvironments. Tumour blood vessels (BV), comprising BEC, serve as conduits for blood supply into the tumour, and are important for tumour growth as well as haematogenous tumour dissemination. Lymphatic vessels (LV), comprising LEC, which are relatively leaky compared with BV, are essential for lymphogenous tumour dissemination. In addition to describing the conventional roles of the BV and LV, we also discuss newly emerging roles of these endothelial cells: their crosstalk with cancer cells via molecules secreted by the BEC and LEC (also called angiocrine and lymphangiocrine factors). This review suggests that BEC and LEC in various microenvironments can be orchestrators of tumour progression and proposes new mechanism-based strategies to discover new therapies to supplement conventional anti-angiogenic and anti-lymphangiogenic therapies.

Vasculature-specific MRI reveals differential anti-angiogenic effects of a biomimetic peptide in an orthotopic breast cancer model.

Translational vasculature-specific MRI biomarkers were used to measure the effects of a novel anti-angiogenic biomimetic peptide in an orthotopic MDA-MB-231 human triple-negative breast cancer model at an early growth stage. In vivo diffusion-weighted and steady-state susceptibility contrast (SSC) MRI was performed pre-treatment and 2 weeks post-treatment in tumor volume-matched treatment and control groups (n = 5/group). Treatment response was measured by changes in tumor volume; baseline transverse relaxation time (T2); apparent diffusion coefficient (ADC); and SSC-MRI metrics of blood volume, vessel size, and vessel density. These vasculature-specific SSC-MRI biomarkers were compared to the more conventional, non-vascular biomarkers (tumor growth, ADC, and T2) in terms of their sensitivity to anti-angiogenic treatment response. After 2 weeks of peptide treatment, tumor growth inhibition was evident but not yet significant, and the changes in ADC or T2 were not significantly different between treated and control groups. In contrast, the vascular MRI biomarkers revealed a significant anti-angiogenic response to the peptide after 2 weeks­blood volume and vessel size decreased, and vessel density increased in treated tumors; the opposite was seen in control tumors. The MRI results were validated with histology­H&E staining showed no difference in tumor viability between groups, while peptide-treated tumors exhibited decreased vascularity. These results indicate that translational SSC-MRI biomarkers are able to detect the differential effects of anti-angiogenic therapy on the tumor vasculature before significant tumor growth inhibition or changes in tumor viability.

A biomimetic collagen derived peptide exhibits anti-angiogenic activity in triple negative breast cancer.

We investigated the application of a mimetic 20 amino acid peptide derived from type IV collagen for treatment of breast cancer. We showed that the peptide induced a decrease of proliferation, adhesion, and migration of endothelial and tumor cells in vitro. We also observed an inhibition of triple negative MDA-MB-231 xenograft growth by 75% relative to control when administered intraperitoneally for 27 days at 10 mg/kg. We monitored in vivo the changes in vascular properties throughout the treatment using MRI and found that the vascular volume and permeability surface area product decreased significantly. The treatment also resulted in an increase of caspase-3 activity and in a reduction of microvascular density. The multiple mode of action of this peptide, i.e., anti-angiogenic, and anti-tumorigenic, makes it a viable candidate as a therapeutic agent as a monotherapy or in combination with other compounds.

Inhibition of breast cancer growth and metastasis by a biomimetic peptide.

Metastasis is the main cause of mortality in cancer patients. Though there are many anti-cancer drugs targeting primary tumor growth, anti-metastatic agents are rarely developed. Angiogenesis and lymphangiogenesis are crucial for cancer progression, particularly, lymphangiogenesis is pivotal for metastasis in breast cancer. Here we report that a novel collagen IV derived biomimetic peptide inhibits breast cancer growth and metastasis by blocking angiogenesis and lymphangiogenesis. The peptide inhibits blood and lymphatic endothelial cell viability, migration, adhesion, and tube formation by targeting IGF1R and Met signals. The peptide blocks MDA-MB-231 tumor growth by inhibiting tumor angiogenesis in vivo. Moreover, the peptide inhibits lymphangiogenesis in primary tumors. MDA-MB-231 tumor conditioned media (TCM) was employed to accelerate spontaneous metastasis in tumor xenografts, and the anti-metastatic activity of the peptide was tested in this model. The peptide prevents metastasis to the lungs and lymph nodes by inhibiting TCM-induced lymphangiogenesis and angiogenesis in the pre-metastatic organs. In summary, a novel biomimetic peptide inhibits breast cancer growth and metastasis by blocking angiogenesis and lymphangiogenesis in the pre-metastatic organs as well as primary tumors.