Ketogenic diet does not promote triple-negative and luminal mammary tumor growth and metastasis in experimental mice.

Ketogenic diets (KDs) can improve the well-being and quality of life of breast cancer patients. However, data on the effects of KDs on mammary tumors are inconclusive, and the influence of KDs on metastasis in general remains to be investigated. We therefore assessed the impact of a KD on growth and metastasis of triple negative murine 4T1 mammary tumors, and on the progression of luminal breast tumors in an autochthonous MMTV-PyMT mouse model. We found that KD did not influence the metastasis of 4T1 and MMTV-PyMT mammary tumors, but impaired 4T1 tumor cell proliferation in vivo, and also temporarily reduced 4T1 primary tumor growth. Notably, the ketogenic ratio (the mass of dietary fat in relation to the mass of dietary carbohydrates and protein) that is needed to induce robust ketosis was twice as high in mice as compared to humans. Surprisingly, only female but not male mice responded to KD with a sustained increase in blood ß-hydroxybutyrate levels. Together, our data show that ketosis does not foster primary tumor growth and metastasis, suggesting that KDs can be safely applied in the context of luminal breast cancer, and may even be advantageous for patients with triple negative tumors. Furthermore, our data indicate that when performing experiments with KDs in mice, the ketogenic ratio needed to induce ketosis must be verified, and the sex of the mice should also be taken into account.

Increased Circulating Osteopontin Levels Promote Primary Tumour Growth, but Do Not Induce Metastasis in Melanoma.

Osteopontin (OPN) is a phosphoprotein with diverse functions in various physiological and pathological processes. OPN expression is increased in multiple cancers, and OPN within tumour tissue has been shown to promote key stages of cancer development. OPN levels are also elevated in the circulation of cancer patients, which in some cases has been correlated with enhanced metastatic propensity and poor prognosis. However, the precise impact of circulating OPN (cOPN) on tumour growth and progression remains insufficiently understood. To examine the role of cOPN, we used a melanoma model, in which we stably increased the levels of cOPN through adeno-associated virus-mediated transduction. We found that increased cOPN promoted the growth of primary tumours, but did not significantly alter the spontaneous metastasis of melanoma cells to the lymph nodes or lungs, despite an increase in the expression of multiple factors linked to tumour progression. To assess whether cOPN has a role at later stages of metastasis formation, we employed an experimental metastasis model, but again could not detect any increase in pulmonary metastasis in animals with elevated levels of cOPN. These results demonstrate that increased levels of OPN in the circulation play distinct roles during different stages of melanoma progression.

CEMIP, a Promising Biomarker That Promotes the Progression and Metastasis of Colorectal and Other Types of Cancer.

Originally discovered as a hypothetical protein with unknown function, CEMIP (cell migration-inducing and hyaluronan-binding protein) has been implicated in the pathogenesis of numerous diseases, including deafness, arthritis, atherosclerosis, idiopathic pulmonary fibrosis, and cancer. Although a comprehensive definition of its molecular functions is still in progress, major functions ascribed to CEMIP include the depolymerization of the extracellular matrix component hyaluronic acid (HA) and the regulation of a number of signaling pathways. CEMIP is a promising biomarker for colorectal cancer. Its expression is associated with poor prognosis for patients suffering from colorectal and other types of cancer and functionally contributes to tumor progression and metastasis. Here, we review our current understanding of how CEMIP is able to foster the process of tumor growth and metastasis, focusing particularly on colorectal cancer. Studies in cancer cells suggest that CEMIP exerts its pro-tumorigenic and pro-metastatic activities through stimulating migration and invasion, suppressing cell death and promoting survival, degrading HA, regulating pro-metastatic signaling pathways, inducing the epithelial-mesenchymal transition (EMT) program, and contributing to the metabolic reprogramming and pre-metastatic conditioning of future metastatic microenvironments. There is also increasing evidence indicating that CEMIP may be expressed in cells within the tumor microenvironment that promote tumorigenesis and metastasis formation, although this remains in an early stage of investigation. CEMIP expression and activity can be therapeutically targeted at a number of levels, and preliminary findings in animal models show encouraging results in terms of reduced tumor growth and metastasis, as well as combating therapy resistance. Taken together, CEMIP represents an exciting new player in the progression of colorectal and other types of cancer that holds promise as a therapeutic target and biomarker.

Sulfated hyaluronic acid inhibits the hyaluronidase CEMIP and regulates the HA metabolism, proliferation and differentiation of fibroblasts.

Hyaluronan (HA) is an extracellular matrix component that regulates a variety of physiological and pathological processes. The function of HA depends both on its overall amount and on its size, properties that are controlled by HA synthesizing and degrading enzymes. The lack of inhibitors that can specifically block individual HA degrading enzymes has hampered attempts to understand the contribution of individual hyaluronidases to different physiological and pathological processes. CEMIP is a recently discovered hyaluronidase that cleaves HA through mechanisms and under conditions that are distinct from those of other hyaluronidases such as HYAL1 or HYAL2. The role of its hyaluronidase activity in physiology and disease is poorly understood. Here, we characterized a series of sulfated HA derivatives (sHA) with different sizes and degrees of sulfation for their ability to inhibit specific hyaluronidases. We found that highly sulfated sHA derivatives potently inhibited CEMIP hyaluronidase activity. One of these compounds, designated here as sHA3.7, was characterized further and shown to inhibit CEMIP with considerable selectivity over other hyaluronidases. Inhibition of CEMIP with sHA3.7 in fibroblasts, which are the main producers of HA in the interstitial matrix, increased the cellular levels of total and high molecular weight HA, while decreasing the fraction of low molecular weight HA fragments. Genetic deletion of CEMIP in mouse embryonic fibroblasts (MEFs) produced analogous results and confirmed that the effects of sHA3.7 on HA levels were mediated by CEMIP inhibition. Importantly, both CEMIP deletion and its inhibition by sHA3.7 suppressed fibroblast proliferation, while promoting differentiation into myofibroblasts, as reflected in a lack of CEMIP in myofibroblasts within skin wounds in experimental mice. By contrast, adipogenic and osteogenic differentiation were attenuated upon CEMIP loss or inhibition. Our results demonstrate the importance of CEMIP for the HA metabolism, proliferation and differentiation of fibroblasts, and suggest that inhibition of CEMIP with sulfated HA derivatives such as sHA3.7 has potential utility in pathological conditions that are dependent on CEMIP function.

Loss of ASAP1 in the MMTV-PyMT model of luminal breast cancer activates AKT, accelerates tumorigenesis, and promotes metastasis.

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis in a variety of cancers, and can promote cell migration, invasion and metastasis. Although amplification and expression of ASAP1 has been associated with poor survival in breast cancer, we found that in the autochthonous MMTV-PyMT model of luminal breast cancer, ablation of ASAP1 resulted in an earlier onset of tumor initiation and increased metastasis. This was due to tumor cell-intrinsic effects of ASAP1 deletion, as ASAP1 deficiency in tumor, but not in stromal cells was sufficient to replicate the enhanced tumorigenicity and metastasis observed in the ASAP1-null MMTV-PyMT mice. Loss of ASAP1 in MMTV-PyMT mice had no effect on proliferation, apoptosis, angiogenesis or immune cell infiltration, but enhanced mammary gland hyperplasia and tumor cell invasion, indicating that ASAP1 can accelerate tumor initiation and promote dissemination. Mechanistically, these effects were associated with a potent activation of AKT. Importantly, lower ASAP1 levels correlated with poor prognosis and enhanced AKT activation in human ER+/luminal breast tumors, validating our findings in the MMTV-PyMT mouse model for this subtype of breast cancer. Taken together, our findings reveal that ASAP1 can have distinct functions in different tumor types and demonstrate a tumor suppressive activity for ASAP1 in luminal breast cancer.

Quantitative Detection of Disseminated Melanoma Cells by Trp-1 Transcript Analysis Reveals Stochastic Distribution of Pulmonary Metastases.

A better understanding of the process of melanoma metastasis is required to underpin the development of novel therapies that will improve patient outcomes. The use of appropriate animal models is indispensable for investigating the mechanisms of melanoma metastasis. However, reliable and practicable quantification of metastases in experimental mice remains a challenge, particularly if the metastatic burden is low. Here, we describe a qRT-PCR-based protocol that employs the melanocytic marker Trp-1 for the sensitive quantification of melanoma metastases in the murine lung. Using this protocol, we were able to detect the presence of as few as 100 disseminated melanoma cells in lung tissue. This allowed us to quantify metastatic burden in a spontaneous syngeneic B16-F10 metastasis model, even in the absence of visible metastases, as well as in the autochthonous Tg(Grm1)/Cyld-/- melanoma model. Importantly, we also observed an uneven distribution of disseminated melanoma cells amongst the five lobes of the murine lung, which varied considerably from animal to animal. Together, our findings demonstrate that the qRT-PCR-based detection of Trp-1 allows the quantification of low pulmonary metastatic burden in both transplantable and autochthonous murine melanoma models, and show that the analysis of lung metastasis in such models needs to take into account the stochastic distribution of metastatic lesions amongst the lung lobes.

Application of ethyl cinnamate based optical tissue clearing and expansion microscopy combined with retrograde perfusion for 3D lung imaging.

PURPOSE: 3 D imaging of the lung is not a trivial undertaking as during preparation the lung may collapse. Also serial sections and scans followed by 3 D reconstruction may lead to artifacts. The present study aims to figure out the best way to perform 3 D imaging in lung research. MATERIALS AND METHODS: We applied an optical tissue clearing (OTC) method, which uses ethyl cinnamate (ECi) as a fast, nontoxic and cheap clearing solvent, for 3 D imaging of retrograde perfused lungs by laser confocal fluorescence microscopy and light sheet fluorescence microscopy. We also introduced expansion microscopy (ExM), a cutting-edge technique, in 3 D imaging of lungs. We examined and compared the usefulness of these techniques for 3 D lung imaging. The ExM protocol was further extended to paraffin-embedded lung metastases blocks. RESULTS: The MHI148-PEI labeled lung vasculature was visualized by retrograde perfusion combined with trachea ligation and ECi based OTC. As compared with trans-cardiac perfusion, the retrograde perfusion results in a better maintenance of lung morphology. 3 D structure of alveoli, vascular branches and cilia in lung were revealed by immunofluorescence staining after ExM. 3 D distribution of microvasculature and neutrophil cells in 10 years old paraffin-embedded lung metastases were analyzed by ExM. CONCLUSIONS: The retrograde perfusion combined with trachea ligation technique could be applied in the lung research in mice. 3 D structure of lung vasculature can be visualized by MHI148-PEI perfusion and ECi based OTC in an efficient way. ExM and immunofluorescence staining protocol is highly recommended to perform 3 D imaging of fresh fixed lung as well as paraffin-embedded lung blocks.

Author Correction: Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells.

BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.

TGFß counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides.

During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4-25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFß in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes. KEY MESSAGES : sHA promotes lymphangiogenesis primarily through increased LEC proliferation sHA induces proliferation in a narrow concentration window due to upregulated TGFß Smaller HA oligosaccharides more potently induce proliferation than larger ones VEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHA Sialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC.

TGF-ß1 Is Present at High Levels in Wound Fluid from Breast Cancer Patients Immediately Post-Surgery, and Is Not Increased by Intraoperative Radiation Therapy (IORT).

In patients with low-risk breast cancer, intraoperative radiotherapy (IORT) during breast-conserving surgery is a novel and convenient treatment option for delivering a single high dose of irradiation directly to the tumour bed. However, edema and fibrosis can develop after surgery and radiotherapy, which can subsequently impair quality of life. TGF- ß is a strong inducer of the extracellular matrix component hyaluronan (HA). TGF-ß expression and HA metabolism can be modulated by irradiation experimentally, and are involved in edema and fibrosis. We therefore hypothesized that IORT may regulate these factors.Wound fluid (WF) draining from breast lumpectomy sites was collected and levels of TGF-ß1 and HA were determined by ELISA. Proliferation and marker expression was analyzed in primary lymphatic endothelial cells (LECs) treated with recombinant TGF-ß or WF. Our results show that IORT does not change TGF-ß1 or HA levels in wound fluid draining from breast lumpectomy sites, and does not lead to accumulation of sHA oligosaccharides. Nevertheless, concentrations of TGF-ß1 were high in WF from patients regardless of IORT, at concentrations well above those associated with fibrosis and the suppression of LEC identity. Consistently, we found that TGF-ß in WF is active and inhibits LEC proliferation. Furthermore, all three TGF-ß isoforms inhibited LEC proliferation and suppressed LEC marker expression at pathophysiologically relevant concentrations. Given that TGF-ß contributes to edema and plays a role in the regulation of LEC identity, we suggest that inhibition of TGF-ß directly after surgery might prevent the development of side effects such as edema and fibrosis.

Hyaluronidase-1 expression promotes lung metastasis in syngeneic mouse tumor models without affecting accumulation of small hyaluronan oligosaccharides in tumor interstitial fluid.

Enhanced levels in tumors of hyaluronan, a glycosaminoglycan component of the extracellular matrix, and hyaluronidases such as hyaluronidase-1 (Hyal1) that degrade hyaluronan have both been linked to poor prognosis and metastasis, suggesting that the turnover of hyaluronan might contribute to tumor progression. Small hyaluronan oligosaccharides (sHA) can accumulate in tumor interstitial fluid (TIF), and have been implicated in a number of processes that drive tumor progression, including MMP expression and angiogenesis. The properties of Hyal1 suggest that it might contribute to the degradation of hyaluronan in tumors and the subsequent accumulation of sHA. Accumulation of Hyal1-produced sHA may therefore account for the association between Hyal1 and metastasis. Here we have investigated this hypothesis using mouse syngeneic breast tumor models. Specifically, we modulated Hyal1 expression and activity either in the tumor cells themselves, or in the stromal compartment by using Hyal1 knockout (KO) mice. These approaches did not change sHA levels in TIF, but nevertheless fostered metastasis to the lung in some of the models used in the study. Together, these data suggest that Hyal1 can promote lung metastasis in a manner that is not dependent on altered accumulation of sHA in TIF.

Sugars in the microenvironment: the sticky problem of HA turnover in tumors.

The properties and behavior of tumor cells are closely regulated by their microenvironment. Accordingly, stromal cells and extracellular matrix components can have a pronounced effect on cancer initiation, growth, and progression. The linear glycosaminoglycan hyaluronan (HA) is a major component of the extracellular matrix. Altered synthesis and degradation of HA in the tumor context has been implicated in many aspects of tumor biology. In particular, the accumulation of small HA oligosaccharides (sHA) in the tumor interstitial space may play a decisive role, due to the ability of sHA to activate a number of biological processes that are not modulated by high molecular weight (HMW)-HA. In this article, we review the normal physiological role and metabolism of HA and then survey the evidence implicating HA in tumor growth and progression, focusing in particular on the potential contribution of sHA to these processes.

Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways.

INTRODUCTION: Intervertebral disc (IVD) degeneration is characterized by extracellular matrix breakdown and is considered to be a primary cause of discogenic back pain. Although increases in pro-inflammatory cytokine levels within degenerating discs are associated with discogenic back pain, the mechanisms leading to their overproduction have not yet been elucidated. As fragmentation of matrix components occurs during IVD degeneration, we assessed the potential involvement of hyaluronic acid fragments (fHAs) in the induction of inflammatory and catabolic mediators. METHODS: Human IVD cells isolated from patient biopsies were stimulated with fHAs (6 to 12 disaccharides) and their effect on cytokine and matrix degrading enzyme production was assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The involvement of specific cell surface receptors and signal transduction pathways in mediating the effects of fHAs was tested using small interfering RNA (siRNA) approaches and kinase inhibition assays. RESULTS: Treatment of IVD cells with fHAs significantly increased mRNA expression levels of interleukin (IL)-1ß, IL-6, IL-8, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1 and -13. The stimulatory effects of fHAs on IL-6 protein production were significantly impaired when added to IVD cells in combination with either Toll-like receptor (TLR)-2 siRNA or a TLR2 neutralizing antibody. Furthermore, the ability of fHAs to enhance IL-6 and MMP-3 protein production was found to be dependent on the mitogen-activated protein (MAP) kinase signaling pathway. CONCLUSIONS: These findings suggest that fHAs may have the potential to mediate IVD degeneration and discogenic back pain through activation of the TLR2 signaling pathway in resident IVD cells.

Delphinidin is a novel inhibitor of lymphangiogenesis but promotes mammary tumor growth and metastasis formation in syngeneic experimental rats.

We have recently demonstrated that the anthocyanidin delphinidin (DEL), one of the most abundant dietary flavonoids, inhibits activation of ErbB and vascular endothelial growth factor receptor family members. These receptors play crucial roles in the context of tumor progression and the outgrowth of blood and lymphatic vessels. Here, we have developed an improved chemical synthesis for DEL in order to study the effects of the aglycon and its degradation product gallic acid (GA) on endothelial and tumor cells in vitro and in vivo. We found that DEL blocked the proliferation in vitro of primary human blood and lymphatic endothelial cells as well as human HT29 colon and rat MT-450 mammary carcinoma cells in a dose-dependent manner. In contrast, its degradation product GA had little effect. At higher concentrations, DEL induced apoptosis of endothelial and tumor cells. Furthermore, DEL potently blocked the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In the MT-450 rat syngeneic breast tumor model, it also significantly reduced angiogenesis and tumor-induced lymphangiogenesis when administered in vivo. These data reveal DEL to be a novel antilymphangiogenesis reagent. Surprisingly, however, the application of DEL unexpectedly promoted tumor growth and metastasis in the MT-450 tumor model, suggesting that the antiproliferative effect of DEL on cultured cells does not necessarily reflect the response of tumors to this anthocyanidin in vivo. Furthermore, while DEL may have utility as a cancer chemopreventative agent, its ability to promote tumor growth once a neoplasm develops also needs to be taken into consideration.

Spatio-temporal patterns of pancreatic cancer cells expressing CD44 isoforms on supported membranes displaying hyaluronic acid oligomers arrays.

In this paper, we designed a quantitative model of biological membranes by the deposition of planar lipid membranes on solid substrates (called supported membranes), and immobilized biotinylated oligomers of hyaluronic acid (oligo-HA, 6-8 disaccharide units in length) to the membrane surface via neutravidin cross-linkers. By controlling the self-assembly of biotinylated lipid anchors, the mean distance between the oligo-HA molecules on the membrane could be controlled to nm accuracy. The adhesion and motility of pancreatic adenocarcinoma cells expressing different splice variants of the HA-binding cell surface receptor CD44 on these surfaces were investigated quantitatively. The combination of label-free, time-lapse imaging of living cells and statistical analysis suggests that the static morphology (global shape and cytoskeleton remodeling) of cells, their stochastic morphological dynamics, and the probability of directed motion reflect the metastatic behaviour of the cancer cells.