Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment.

In the paracortex of the lymph node (LN), T zone fibroblastic reticular cells (TRCs) orchestrate an immune response by guiding lymphocyte migration both physically, by creating three-dimensional (3D) cell networks, and chemically, by secreting the chemokines CCL19 and CCL21 that direct interactions between CCR7-expressing cells, including mature dendritic cells and naive T cells. TRCs also enwrap matrix-based conduits that transport fluid from the subcapsular sinus to high endothelial venules, and fluid flow through the draining LN rapidly increases upon tissue injury or inflammation. To determine whether fluid flow affects TRC organization or function within a 3D network, we regenerated the 3D LN T zone stromal network by culturing murine TRC clones within a macroporous polyurethane scaffold containing type I collagen and Matrigel and applying slow interstitial flow (1-23 microm/min). We show that the 3D environment and slow interstitial flow are important regulators of TRC morphology, organization, and CCL21 secretion. Without flow, CCL21 expression could not be detected. Furthermore, when flow through the LN was blocked in mice in vivo, CCL21 gene expression was down-regulated within 2 h. These results highlight the importance of lymph flow as a homeostatic regulator of constitutive TRC activity and introduce the concept that increased lymph flow may act as an early inflammatory cue to enhance CCL21 expression by TRCs, thereby ensuring efficient immune cell trafficking, lymph sampling, and immune response induction.

Plasmacytoid dendritic cells and regulatory T cells in the tumor microenvironment: A dangerous liaison.

Tumor-infiltrating plasmacytoid dendritic cells (pDCs) have been associated with poor patient prognosis. We have recently uncovered the ability of pDCs to activate and expand a subset of tumor-infiltrating FOXP3(+) regulatory T cells that express inducible costimulator (ICOS), providing new insights into the mechanisms that govern the escape of cancer from immunosurveillance.

Role of the microenvironment on epithelial stem cell fate

Stratified epithelia of mammals contain adult stem/progenitor cells that are instrumental for renewal, regeneration and repair. We have recently demonstrated, using clonal and functional analysis, that all stratified epithelia contain clonogenic stem cells that can respond to skin morphogenetic signals, while cells obtained from simple or pseudo-stratified epithelia cannot. A genome-wide expression analysis favors multilineage priming rather than reprogramming. Collectively, these observations are reminiscent of epithelial metaplasia, a phenomenon in which a cell adopts the phenotype of another epithelial cell, often in response to repeated environmental stress, e.g. smoking, alcohol and micro-traumatisms. Furthermore, they support the notion that metaplasia results from the expression of an unseen potency, revealed by an environmental deficiency. The thymus supposedly contains only progenitor epithelial cells but no stem cells. We have demonstrated that the thymus also contains a small population of clonogenic cells that can function as bona fide multipotent hair follicle stem cells in response to an inductive skin microenvironment and a genome-wide expression analysis indicates that it correlates with robust changes in the expression of genes important for thymus identity. Hence, multilineage priming or reprogramming can account for the fate change of epithelial stem/progenitor cells in response to a varying microenvironment.

Targeting the intragraft microenvironment and the development of chronic allograft rejection.

In this review, we discuss a paradigm whereby changes in the intragraft microenvironment promote or sustain the development of chronic allograft rejection. A key feature of this model involves the microvasculature including (a) endothelial cell (EC) destruction, and (b) EC proliferation, both of which result from alloimmune leukocyte- and/or alloantibody-induced responses. These changes in the microvasculature likely create abnormal blood flow patterns and thus promote local tissue hypoxia. Another feature of the chronic rejection microenvironment involves the overexpression of vascular endothelial growth factor (VEGF). VEGF stimulates EC activation and proliferation and it has potential to sustain inflammation via direct interactions with leukocytes. In this manner, VEGF may promote ongoing tissue injury. Finally, we review how these events can be targeted therapeutically using mTOR inhibitors. EC activation and proliferation as well as VEGF-VEGFR interactions require PI-3K/Akt/mTOR intracellular signaling. Thus, agents that inhibit this signaling pathway within the graft may also target the progression of chronic rejection and thus promote long-term graft survival.

Characterization and clinical evaluation of CD10+ stroma cells in the breast cancer microenvironment.

PURPOSE: There is growing evidence that interaction between stromal and tumor cells is pivotal in breast cancer progression and response to therapy. Based on earlier research suggesting that during breast cancer progression, striking changes occur in CD10(+) stromal cells, we aimed to better characterize this cell population and its clinical relevance. EXPERIMENTAL DESIGN: We developed a CD10(+) stroma gene expression signature (using HG U133 Plus 2.0) on the basis of the comparison of CD10 cells isolated from tumoral (n = 28) and normal (n = 3) breast tissue. We further characterized the CD10(+) cells by coculture experiments of representative breast cancer cell lines with the different CD10(+) stromal cell types (fibroblasts, myoepithelial, and mesenchymal stem cells). We then evaluated its clinical relevance in terms of in situ to invasive progression, invasive breast cancer prognosis, and prediction of efficacy of chemotherapy using publicly available data sets. RESULTS: This 12-gene CD10(+) stroma signature includes, among others, genes involved in matrix remodeling (MMP11, MMP13, and COL10A1) and genes related to osteoblast differentiation (periostin). The coculture experiments showed that all 3 CD10(+) cell types contribute to the CD10(+) stroma signature, although mesenchymal stem cells have the highest CD10(+) stroma signature score. Of interest, this signature showed an important role in differentiating in situ from invasive breast cancer, in prognosis of the HER2(+) subpopulation of breast cancer only, and potentially in nonresponse to chemotherapy for those patients. CONCLUSIONS: Our results highlight the importance of CD10(+) cells in breast cancer prognosis and efficacy of chemotherapy, particularly within the HER2(+) breast cancer disease.

Tumor microenvironment and immune effects of antineoplastic therapy in lymphoproliferative syndromes.

Lymphomas represent a wide group of heterogenic diseases with different biological and clinical behavior. The underlying microenvironment-specific composition seems to play an essential role in this scenario, harboring the ability to develop successful immune responses or, on the contrary, leading to immune evasion and even promotion of tumor growth. Depending on surrounding lymphoid infiltrates, lymphomas may have different prognosis. Moreover, recent evidences have emerged that confer a significant impact of main lymphoma's treatment over microenvironment, with clinical consequences. In this review, we summarize these concepts from a pathological and clinical perspective. Also, the state of the art of lymphoma's anti-idiotype vaccine development is revised, highlighting the situations where this strategy has proven to be successful and eventual clues to obtain better results in the future.

Immune Microenvironment in Colorectal Cancer: A New Hallmark to Change Old Paradigms

Impact of immune microenvironment in prognosis of solid tumors has been extensively studied in the last few years. Specifically in colorectal carcinoma, increased knowledge of the immune events around these tumors and their relation with clinical outcomes have led to consider immune microenvironment as one of the most important prognostic factors in this disease. In this review we will summarize and update the current knowledge with respect to this intriguing and complex new hallmark of cancer, paying special attention to infiltration by T-infiltrating lymphocytes and their subtypes in colorectal cancer, as well as its eventual clinical translation in terms of long-term prognosis. Finally, we suggest some possible investigational approaches based on combinatorial strategies to trigger and boost immune reaction against tumor cells.

Analysis of selectin ligands supporting hematologic malignancy cell interactions with their microenvironment : focus on leukemia and multiple myeloma

CONTEXTE: Les sélectines sont une famille de trois protéines qui règlent la capture et le roulement des leucocytes et qui initient la cascade d'adhésion. Elles contrôlent également la migration des leucocytes en réponse à un stimulus physiologique ou inflammatoire pour atteindre un organe cible. Le rôle des sélectines et des leurs ligands est bien connu dans l'adhésion des leucocytes normaux à l'endothélium; en revanche, la nature des ligands des sélectines exprimés par les cellules leucémiques et le myélome multiple est peu connue. La récente découverte que la E- et la P-sélectine sont exprimées par les cellules endothéliales et du stroma de la moelle osseuse, nous a incité à examiner leur rôle dans les interactions des cellules malignes avec leur environnement médullaire. RÉSULTATS: Les analyses ont été conduites sur les cellules du sang ou de la moelle osseuse prélevées à des patients atteints de leucémie aiguë ou de myélome multiple et sur des lignées cellulaires. Les ligands des sélectines qui ont été identifiés sur les blastes leucémiques ou les plasmocytes, sont « P-selectin glycoprotein ligand-1 » (PSGL-1), CD44, CD43 et l'endoglycan (EGC), ainsi que les saccharides fucosylés sLex et CLA. Nous avons vérifié dans des expériences d'adhésion cellulaire effectuées dans des conditions de flux que ces ligands sont fonctionnels, étant porteurs des sucres mentionnés, et qu'ils sont capables de supporter le roulement cellulaire dépendant des sélectines. De plus, nous avons montré que la liaison de ces ligands génère des signaux intracellulaires favorisant la prolifération et la survie des cellules de myélome. CONCLUSION. Les données présentées ici montrent que la E- et la P- sélectine du microenvironnement médullaire interagissent avec les cellules leucémiques et de myélome multiple, et que ces interactions activent des voies de signalisation contrôlant la prolifération et la survie cellulaire. Ces effets protecteurs sont impliqués dans la persistance de clones cellulaires malins résistant aux traitements et peuvent conduire à la récidive de la maladie. L'inhibition de ces interactions pourrait fournir de nouvelles options thérapeutiques pour le traitement de ces maladies de mauvais pronostic. - BACKGROUND: Selectins are a family of glycoproteins involved in the first steps of the adhesion cascade, tethering and rolling, during which leukocytes sense tissue specific signals and commit the cells to enter in a particular organ or inflammation site. While the role of selectins and their ligands is well established in supporting normal leukocyte adhesion to vascular endothelium, our knowledge of selectin ligands in two hematological malignancies, acute leukemia and multiple myeloma, is incomplete. The recent discovery that E- and P- selectin are also expressed on bone marrow (BM) endothelial and stromal cells, prompted us to investigate a potential role in selectin-mediated interaction of malignant cells with its protective BM microenvironment. RESULTS. Using cells obtained from blood or BM of patients affected by acute myeloid or lymphoblastic leukemia, or multiple myeloma, as well as cell lines, we characterized the expression of selectin ligands on blasts and plasma cells and identified P-selectin glycoprotein ligand-1 (PSGL-1), CD44, CD43 and endoglycan (EGC), as well as sLex/CLA determinants. Rolling assays under flow conditions allowed us to verify that these ligands are functional, i.e. correctly glycosylated and able to support selectin-mediated rolling. Moreover, we demonstrated that these ligands trigger proliferation and pro-survival signals upon engagement on myeloma cells. CONCLUSIONS. Data presented here demonstrate that E- and P-selectin in the BM microenvironment interact with leukemia and myeloma cells, and suggest that they have an impact on proliferation and survival of malignant plasma cells. These protective effects may induce drug resistance in malignant clones, leading to disease relapse. Interfering with these interactions could provide new therapeutic options. - Le corps humain dépend du système immunitaire pour sa protection face aux agressions, notamment des bactéries ou des virus, ou face à une dysfonction de l'organisme. Ce système est composé de plusieurs types cellulaires, regroupés sous le nom de leucocytes, qui participent à son fonctionnement. Ces cellules se développent à partir d'une cellule souche hématopo'iétique commune qui réside dans la moelle osseuse. Comme c'est le cas dans les autres tissus, les cellules du système immunitaire peuvent aussi développer des cancers, appelés tumeurs hématopoïétiques ou tumeurs du sang. Bien que ces maladies puissent être traitées avec succès grâce à de fortes doses de chimiothérapies ou à d'autres moyens comme les greffes, les patients connaissent un fort taux de rechute. La raison de ces récidives est la survie d'une partie des cellules malignes dans la moelle osseuse, où elles reçoivent une protection au traitement par le biais de l'interaction avec d'autres cellules. Les sélectines (E-, P- et L-sélectine) régulent l'interaction des leucocytes avec l'endothélium (la paroi des vaisseaux sanguins), d'autres leucocytes et les plaquettes ; ces interactions surviennent quand les leucocytes atteignent un site d'inflammation ou un organe cible. Dans la moelle osseuse, la E- et la P-sélectine se trouvent sur les cellules de l'endothélium et sur les macrophages, qui sont d'autres leucocytes faisant partie du stroma de la moelle. Elles pourraient être impliquées dans la protection des cellules cancéreuses évoquée plus haut. Les molécules d'adhésion avec lesquelles les sélectines s'associent, autrement dit les ligands des sélectines, sont des glycoprotéines. Ces protéines ont besoin de sucres spécifiques pour acquérir une telle capacité d'adhésion. Dans le cadre de cette thèse, nous avons étudié deux types de cellules extraites du sang et de la moelle osseuse des patients atteints d'une leucémie aiguë (les blastes) ou de myélome multiple (les plasmocytes), et leur capacité à se lier aux sélectines. Nous avons démontré une interaction entre ces cellules malignes et la E- et/ou la P-sélectine, à condition que les ligands soient correctement décorés. De plus, lors que les plasmocytes se lient aux sélectines, une cascade de signaux à l'intérieur des cellules stimule leur prolifération et leur survie. L'ensemble de ces résultats permet l'identification de nouvelles cibles thérapeutiques potentielles de ces hémopathies de mauvais pronostic.

New insights into the role of the immune microenvironment in breast carcinoma.

Recently, immune edition has been recognized as a new hallmark of cancer. In this respect, some clinical trials in breast cancer have reported imppressive outcomes related to laboratory immune findings, especially in the neoadjuvant and metastatic setting. Infiltration by tumor infiltrating lymphocytes (TIL) and their subtypes, tumor-associated macrophages (TAM) and myeloid-derived suppressive cells (MDSC) seem bona fide prognostic and even predictive biomarkers, that will eventually be incorporated into diagnostic and therapeutic algorithms of breast cancer. In addition, the complex interaction of costimulatory and coinhibitory molecules on the immune synapse and the different signals that they may exert represent another exciting field to explore. In this review we try to summarize and elucidate these new concepts and knowledge from a translational perspective focusing on breast cancer, paying special attention to those aspects that might have more significance in clinical practice and could be useful to design successful therapeutic strategies in the future.

Characterization of the effects of radiation therapy on the tumour microenvironment and their consequences on cancer progression

RESUME La radiothérapie est utilisée avec succès pour le traitement d'un grand nombre de pathologies tumorales (1). Cependant, les récidives post-actiniques sont associées à un risque accru de développer des métastases régionales et à distance (2, 3). La prise en charge de ce type de patients demeure insatisfaisante à l'heure actuelle, principalement parce que les mécanismes physio-pathologiques sous- sous-jacents restent mal compris. Etant donné le rôle primordial du stroma dans la progression tumorale (4) et l'importance des effets de la radiothérapie sur le micro-environnement des tumeurs (5), nous avons émis l'hypothèse que la radiothérapie pouvait engendrer des modifications stromales susceptibles de contribuer à l'émergence d'un phénotype tumoral plus agressif. Nous avons observé que l'exposition préalable d'un environnement tumoral à des radiations ionisantes engendre une inhibition locale et à long terme de l'angiogenèse. Cette inhibition conduit à la création d'un environnement tumoral hypoxique favorisant l'invasion et la métastatisation tumorale. Les mécanismes sous-jacents impliquent l'activation de gènes prométastatiques sous le contrôle du facteur de transcription HIF-1, ainsi que la sélection hypoxique de cellules hautement invasives et métastatiques. Par des analyses de profile d'expression génétique ainsi que par des analyses fonctionnelles, nous avons identifié la protéine matri-cellulaire CYR61 ainsi que ses partenaires d'interaction, les intégrines aVb5/aVb3, comme médiateurs importants de ces effets. De plus, une corrélation significative a également été trouvée entre le niveau d'expression de CYR61 et le taux d'hypoxie dans un grand nombre de carcinomes mammaires chez l'humain. Une association a aussi été observée entre le niveau d'expression de CYR61 et le pronostic de patientes souffrant d'un cancer du sein traité par chimiothérapie adjuvante. Globalement ces résultats identifient l'interaction entre la protéine CYR61 et ses récepteurs aVb5/aVb3 comme un mécanisme important du processus de métastatisation et en font une cible thérapeutique potentielle pour le traitement de patients souffrant d'une récidive tumorale après un traitement de radiothérapie. Finalement, bien que l'inhibition de l'angiogenèse soit locale dans ce cas particulier, nos résultats justifient une surveillance particulière des patients souffrant d'une pathologie tumorale et étant au bénéfice d'un traitement inhibiteur de l'angiogenèse. SUMMARY Radiotherapy is successfully used to treat a large variety of tumours (1 ). However, cancer patients experiencing local recurrent disease after radiation therapy are at increased risk of developing regional and distant metastasis (2, 3). The clinical management of this condition represents a difficult and challenging issue, mainly because the underlying physio-pathological mechanisms remain poorly understood. Given the well established role of the tumour stroma in promoting cancer progression (4) and since radiotherapy is known to persistently alter the tumour microenvironment (5), we hypothesized that ionising radiations may generate stromal modifications contributing to the metastatic spread of relapsing tumours. Here, we report that irradiation of the prospective tumour microenvironment promotes tumour invasion and metastasis through a mechanism of local and sustained impairment of angiogenesis leading to both HIF-1 dependent activation of pro-metastatic genes and hypoxia-mediated selection of highly metastatic tumour cell variants. Through gene expression profiling and functional experiments, we identified the matricellular signalling protein CYR61 and its interaction partners aVb5/ aVb3 integrins as critical mediators of these effects. Furthermore, we found a significant correlation between CYR61 expression and the hypoxic status of a large number of human mammary carcinomas. A positive correlation between increased levels of CYR61 expression and shorter relapse free survival was also identified in breast cancer patients treated with adjuvant chemotherapy. Together, these results identify CYR61 and aVb5/aVb3 integrins as critical mediators of metastasis and potential therapeutic targets to improve outcome in patients with post-radiation tumour recurrences. Finally, although inhibition of angiogenesis is local in this setting, our data warrant close monitoring of tumour progression in patients under anti-angiogenic therapy.

The microenvironment in T-cell lymphomas: Emerging themes

Peripheral T-cell lymphomas (PTCLs) are heterogeneous and uncommon malignancies characterized by an aggressive clinical course and a mostly poor outcome with current treatment strategies. Despite novel insights into their pathobiology provided by recent genome-wide molecular studies, several entities remain poorly characterized. In addition to the neoplastic cell population, PTCLs have a microenvironment component, composed of non-tumor cells and stroma, which is quantitatively and qualitatively variable, and which may have an effect on their pathological and clinical features. The best example is provided by angioimmunoblastic T-cell lymphoma (AITL), a designation reflecting the typical vascularization and reactive immunoblastic content of the tumor tissues. In this disease, a complex network of interactions between the lymphoma cells and the microenvironment exists, presumably mediated by the neoplastic T cells with follicular helper T-cell properties. A better understanding of the crosstalk between neoplastic T or NK cells and their microenvironment may have important implications for guiding the development of novel therapies.

The role of population origin and microenvironment in seedling emergence and early survival in Mediterranean maritime pine (Pinus pinaster Aiton).

Understanding tree recruitment is needed to forecast future forest distribution. Many studies have reported the relevant ecological factors that affect recruitment success in trees, but the potential for genetic-based differences in recruitment has often been neglected. In this study, we established a semi-natural reciprocal sowing experiment to test for local adaptation and microenvironment effects (evaluated here by canopy cover) in the emergence and early survival of maritime pine (Pinus pinaster Aiton), an emblematic Mediterranean forest tree. A novel application of molecular markers was also developed to test for family selection and, thus, for potential genetic change over generations. Overall, we did not find evidence to support local adaptation at the recruitment stage in our semi-natural experiment. Moreover, only weak family selection (if any) was found, suggesting that in stressful environments with low survival, stochastic processes and among-year climate variability may drive recruitment. Nevertheless, our study revealed that, at early stages of recruitment, microenvironments may favor the population with the best adapted life strategy, irrespectively of its (local or non-local) origin. We also found that emergence time is a key factor for seedling survival in stressful Mediterranean environments. Our study highlights the complexity of the factors influencing the early stages of establishment of maritime pine and provides insights into possible management actions aimed at environmental change impact mitigation. In particular, we found that the high stochasticity of the recruitment process in stressful environments and the differences in population-specific adaptive strategies may difficult assisted migration schemes.

The tumor microenvironment and its contribution to tumor evolution toward metastasis.

Cancer cells acquire cell-autonomous capacities to undergo limitless proliferation and survival through the activation of oncogenes and inactivation of tumor suppressor genes. Nevertheless, the formation of a clinically relevant tumor requires support from the surrounding normal stroma, also referred to as the tumor microenvironment. Carcinoma-associated fibroblasts, leukocytes, bone marrow-derived cells, blood and lymphatic vascular endothelial cells present within the tumor microenvironment contribute to tumor progression. Recent evidence indicates that the microenvironment provides essential cues to the maintenance of cancer stem cells/cancer initiating cells and to promote the seeding of cancer cells at metastatic sites. Furthermore, inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes impacting tumor progression. A growing number of studies demonstrate a positive correlation between angiogenesis, carcinoma-associated fibroblasts, and inflammatory infiltrating cells and poor outcome, thereby emphasizing the clinical relevance of the tumor microenvironment to aggressive tumor progression. Thus, the dynamic and reciprocal interactions between tumor cells and cells of the tumor microenvironment orchestrate events critical to tumor evolution toward metastasis, and many cellular and molecular elements of the microenvironment are emerging as attractive targets for therapeutic strategies.

Radiation-induced modifications of the tumor microenvironment promote metastasis.

Radiotherapy is successfully used to treat cancer. Emerging evidence, however, indicates that recurrences after radiotherapy are associated with increased local invasion, metastatic spreading and poor prognosis. Radiation-induced modifications of the tumor microenvironment have been proposed to contribute to increased aggressive tumor behavior, an effect also referred to as tumor bed effect, but the putative mechanisms involved have remained largely elusive. We have recently demonstrated that irradiation of the prospective tumor stroma impairs de novo angiogenesis through sustained inhibition of proliferation, migration and sprouting of endothelial cells. Experimental tumors growing within a pre-irradiated field have reduced tumor angiogenesis and tumor growth, increased hypoxia, necrosis, local invasion and distant metastasis. Mechanisms of progression involve adaptation of tumor cells to local hypoxic conditions as well as selection of cells with invasive and metastatic capacities. The matricellular protein CYR61 and integrin αVβ5 emerged as molecules that cooperate to mediate lung metastasis. Cilengitide, a small molecular inhibitor of αV integrins prevented lung metastasis formation. These results represent a conceptual advance to the understanding of the tumor bed effect and indicate that αV integrin inhibition might be a potential therapeutic approach for preventing metastasis in patients at risk for post-radiation recurrences.

Molecular pathways: emerging pathways mediating growth, invasion, and metastasis of tumors progressing in an irradiated microenvironment.

Radiotherapy is a well-established therapeutic modality in oncology. It provides survival benefits in several different cancer types. However, cancers relapsing after radiotherapy often develop into more aggressive conditions that are difficult to treat and are associated with poor prognosis. Cumulative experimental evidence indicates that the irradiated tumor bed contributes to such aggressive behavior. The involved mechanisms have for long remained elusive. Recent progress in the field revealed previously unrecognized cellular and molecular events promoting growth, invasion, and metastasis of tumors progressing in an irradiated microenvironment. Cellular mechanisms include inhibition of sprouting angiogenesis, formation of hypoxia, activation and differentiation of stromal cells, and recruitment of bone marrow-derived cells with vasculogenic and prometastatic activities. Identified pathways include TGF-β/ALK5, CXCL12/CXCR4, KITL/KIT, and CYR61/αVβ5 integrin. The availability of pharmacologic inhibitors impinging on these pathways opens novel opportunities for translational and clinical studies. These experimental results and ongoing work highlight the importance of the irradiated microenvironment in modulating the tumor response to radiotherapy and open new opportunities for the development of novel therapeutic strategies for patients with cancer who relapse after radiotherapy. Here, we review and discuss recent advances in the field and their translational and therapeutic implications to human cancer treatment.