Comparison of Decellularized Human Dermal Scaffolds versus Bovine Collagen/Elastin Matrices for Engineering of Soft-Tissue Flaps.

BACKGROUND: Free flap-based soft-tissue reconstruction comes at the price of donor-site morbidity. The arteriovenous loop (AVL) technique can overcome this issue by allowing for the de novo generation of axially vascularized soft-tissue flaps from vein grafts embedded into different matrices. Application of the AVL technique has been limited by insufficient long-term volume retention and poor tissue stability. The authors investigated the suitability of a novel human dermal scaffold to improve volume retention and tissue stability. METHODS: AVLs were created in 28 immunocompetent rats and embedded in either decellularized human dermal scaffolds (experimental group, n = 14) (Epiflex) or bovine collagen/elastin matrices (control group, n = 14) (MatriDerm) in subcutaneous polytetrafluoroethylene chambers. The weight and volume of engineered tissues, the extent of angiogenesis, and the proportion of proliferating cells were compared between groups on postoperative days (PODs) 21 and 28 by means of immunohistochemistry and micro-computed tomography. RESULTS: On POD 28, both groups displayed homogeneous microvascular networks on histopathology and micro-computed tomography. Mean microvessel counts and surface areas and the percentage of proliferating cells did not differ between the groups. However, the experimental human scaffold group displayed significantly smaller volume loss and significantly less tissue degradation compared with bovine matrix controls (volume retention, 102% ± 5% versus 27% ± 7% on POD 21, and 79% ± 12% versus 12% ± 7% on POD 28, respectively; P < 0.0001). CONCLUSION: Compared with bovine matrices, decellularized human scaffolds allow for superior volume retention and tissue stability of de novo engineered soft-tissue AVL flaps in rats. CLINICAL RELEVANCE STATEMENT: AVLs allow for the de novo generation of vascularized soft-tissue flaps. However, insufficient long-term volume retention is still an issue. The authors' study shows that decellularized human matrices guarantee superior volume stability of de novo grown soft-tissue flaps in rats.

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.

The role of host response to chemotherapy: resistance, metastasis and clinical implications.

Chemotherapy remains the primary treatment for most metastatic cancers. However, the response to chemotherapy and targeted agents is often transient, and concurrent development of resistance is the primary impediment to effective cancer therapy. Strategies to overcome resistance to treatment have focused on cancer cell intrinsic factors and the tumor microenvironment (TME). Recent evidence indicates that systemic chemotherapy has a significant impact on the host that either facilitates tumor growth, allowing metastatic spread, or renders treatment ineffective. These host responses include the release of bone marrow-derived cells, activation of stromal cells in the TME, and induction of different molecular effectors. Here, we provide an overview of chemotherapy-induced systemic host responses that support tumor aggressiveness and metastasis, and which contribute to therapy resistance. Studying host responses to chemotherapy provides a solid basis for the development of adjuvant strategies to improve treatment outcomes and delay resistance to chemotherapy. This review discusses the emerging field of host response to cancer therapy, and its preclinical and potential clinical implications, explaining how under certain circumstances, these host effects contribute to metastasis and resistance to chemotherapy.

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.

Detection of Cellular Senescence Reveals the Existence of Senescent Tumor Cells within Invasive Breast Carcinomas and Related Metastases.

Oncogene-induced senescence is thought to constitute a barrier to carcinogenesis by arresting cells at risk of malignant transformation. However, numerous findings suggest that senescent cells may conversely promote tumor growth and metastatic progression, for example, through the senescence-associated secretory phenotype (SASP) they produce. Here, we investigated the degree to which senescent tumor cells exist within untreated human primary breast carcinomas and whether the presence of senescent cancer cells in primary tumors is recapitulated in their matched lymph node metastases. For the detection of senescence, we used SA-ß-galactosidase (SA-ß-gal) staining and other senescence markers such as Ki67, p21, p53, and p16. In patients with invasive luminal A and B breast carcinomas, we found broad similarities in the appearance of cancer cells between primary tumors and their corresponding metastases. Analysis of lymph nodes from patients with other breast cancer subtypes also revealed senescent tumor cells within metastatic lesions. Collectively, our findings show that senescent tumor cells exist within primary breast carcinomas and metastatic lesions. These results suggest a potential role for senescent breast tumor cells during metastatic progression and raise the question as to whether the targeting of senescent tumor cells with anti-senescent drugs might represent a novel avenue for improved treatment of breast and other cancers.

Fat Grafts Show Higher Hypoxia, Angiogenesis, Adipocyte Proliferation, and Macrophage Infiltration than Flaps in a Pilot Mouse Study.

BACKGROUND: Over 137,000 breast reconstructions are performed annually by American Society of Plastic Surgeons (ASPS) members. Vascularized flaps and avascular lipofilling each account for over 33,000 autologous reconstructions. Although clinical and experimental observations suggest biologic differences with diverging effects on locoregional tumor control, comparative animal models are lacking. The authors standardized existing techniques in immunocompetent mice, laying the foundation for in vivo models of autologous breast reconstruction combinable with orthotopic tumor implantations. METHODS: Twenty-five groin flaps and 39 fat grafts were transferred in female BALB/c-mice. Adipocytes were tracked via Hoechst-Calcein-DiI staining ( n = 2 per group), and postoperative volume retentions were compared via magnetic resonance imaging ( n = 3 per group) on days 1, 11, 21, and 31. Proliferation indices, microvessel densities, tissue hypoxia, and macrophage infiltrates were compared via Ki67, CD31, pimonidazole, and hematoxylin-eosin staining on days 5, 10, 15, 20, and 30 ( n = 4 per group). RESULTS: Viable adipocytes were present in both groups. Graft volumes plateaued at 42.7 ± 1.2% versus 81.8 ± 4.0% of flaps ( P < 0.001). Initially, grafts contained more hypoxic cells (day 5: 15.192 ± 1.249 versus 1.157 ± 192; P < 0.001), followed by higher proliferation (day 15: 25.2 ± 1.0% versus 0.0 ± 0.0%; P < 0.001), higher microvessel numbers (day 30: 307.0 ± 13.2 versus 178.0 ± 10.6; P < 0.001), and more pronounced macrophage infiltrates (graded 3 versus 2; P < 0.01). CONCLUSION: This comparative murine pilot study of vascularized flaps versus avascular lipofilling suggests differences in volume retention, proliferation, angiogenesis, hypoxia, and inflammation. CLINICAL RELEVANCE STATEMENT: The biological differences of fat grafting versus flap transfer are not fully understood because no single comparative experimental model has been established to date. The authors present the first comparative small animal model of both techniques, which will allow the gaining of deeper insights into their biological effects.

CEMIP (HYBID, KIAA1199): structure, function and expression in health and disease.

CEMIP (cell migration-inducing protein), also known as KIAA1199 or HYBID, is a protein involved in the depolymerisation of hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix. CEMIP was originally described in patients affected by nonsyndromic hearing loss and has subsequently been shown to play a key role in tumour initiation and progression, as well as arthritis, atherosclerosis and idiopathic pulmonary fibrosis. Despite the vast literature associating CEMIP with these diseases, its biology remains elusive. The present review article summarises all the major scientific evidence regarding its structure, function, role and expression, and attempts to cast light on a protein that modulates EMT, fibrosis and tissue inflammation, an unmet key aspect in several inflammatory disease conditions.

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.

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.

Cancer microenvironment and genomics: evolution in process.

Cancer heterogeneity is a result of genetic mutations within the cancer cells. Their proliferation is not only driven by autocrine functions but also under the influence of cancer microenvironment, which consists of normal stromal cells such as infiltrating immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, vascular and lymphatic channels. The relationship between cancer cells and cancer microenvironment is a critical one and we are just on the verge to understand it on a molecular level. Cancer microenvironment may serve as a selective force to modulate cancer cells to allow them to evolve into more aggressive clones with ability to invade the lymphatic or vascular channels to spread to regional lymph nodes and distant sites. It is important to understand these steps of cancer evolution within the cancer microenvironment towards invasion so that therapeutic strategies can be developed to control or stop these processes.

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.

RASSF1A-Mediated Suppression of Estrogen Receptor Alpha (ERα)-Driven Breast Cancer Cell Growth Depends on the Hippo-Kinases LATS1 and 2.

Around 70% of breast cancers express the estrogen receptor alpha (ERα). This receptor is of central importance for breast cancer development and estrogen-dependent tumor growth. However, the molecular mechanisms that are responsible for the control of ERα expression and function in the context of breast carcinogenesis are complex and not fully understood. In previous work, we have demonstrated that the tumor suppressor RASSF1A suppresses estrogen-dependent growth of breast cancer cells through a complex network that keeps ERα expression and function under control. We observed that RASSF1A mediates the suppression of ERα expression through modulation of the Hippo effector Yes-associated protein 1 (YAP1) activity. Here we report that RASSF1A-mediated alteration of YAP1 depends on the Hippo-kinases LATS1 and LATS2. Based on these results, we conclude that inactivation of RASSF1A causes changes in the function of the Hippo signaling pathway and altered activation of YAP1, and as a consequence, increased expression and function of ERα. Thus, the inactivation of RASSF1A might constitute a fundamental event that supports the initiation of ERα-dependent breast cancer. Furthermore, our results support the notion that the Hippo pathway is important for the suppression of luminal breast cancers, and that the tumor-suppressor function of RASSF1A depends on LATS1 and LATS2.

IER2-induced senescence drives melanoma invasion through osteopontin.

Expression of the immediate-early response gene IER2 has been associated with the progression of several types of cancer, but its functional role is poorly understood. We found that increased IER2 expression in human melanoma is associated with shorter overall survival, and subsequently investigated the mechanisms through which IER2 exerts this effect. In experimental melanoma models, sustained expression of IER2 induced senescence in a subset of melanoma cells in a p53/MAPK/AKT-dependent manner. The senescent cells produced a characteristic secretome that included high levels of the extracellular phosphoglycoprotein osteopontin. Nuclear localization of the IER2 protein was critical for both the induction of senescence and osteopontin secretion. Osteopontin secreted by IER2-expressing senescent cells strongly stimulated the migration and invasion of non-senescent melanoma cells. Consistently, we observed coordinate expression of IER2, p53/p21, and osteopontin in primary human melanomas and metastases, highlighting the pathophysiological relevance of IER2-mediated senescence in melanoma progression. Together, our study reveals that sustained IER2 expression drives melanoma invasion and progression through stimulating osteopontin secretion via the stochastic induction of senescence.

Functional Characterization of Circulating Tumor Cells (CTCs) from Metastatic ER+/HER2- Breast Cancer Reveals Dependence on HER2 and FOXM1 for Endocrine Therapy Resistance and Tumor Cell Survival: Implications for Treatment of ER+/HER2- Breast Cancer.

Mechanisms of acquired endocrine resistance and late recurrence in patients with ER+/HER2- breast cancer are complex and not fully understood. Here, we evaluated mechanisms of acquired resistance in circulating tumor cells (CTCs) from an ER+/HER2- breast cancer patient who initially responded but later progressed under endocrine treatment. We found a switch from ERα-dependent to HER2-dependent and ERα-independent expression of FOXM1, which may enable disseminated ER+/HER2- cells to re-initiate tumor cell growth and metastasis formation in the presence of endocrine treatment. Our results also suggest a role for HER2 in resistance, even in ER+ breast cancer cells that have neither HER2 amplification nor activating HER2 mutations. We found that NFkB signaling sustains HER2 and FOXM1 expression in CTCs in the presence of ERα inhibitors. Inhibition of NFkB signaling blocked expression of HER2 and FOXM1 in the CTCs, and induced apoptosis. Thus, targeting of NFkB and FOXM1 might be an efficient therapeutic approach to prevent late recurrence and to treat endocrine resistance. Collectively our data show that CTCs from patients with endocrine resistance allow mechanisms of acquired endocrine resistance to be delineated, and can be used to test potential drug regimens for combatting resistance.

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.