Neighboring Stroma Research Articles

Tumor-associated tissue eosinophilia promotes angiogenesis and metastasis in head and neck squamous cell carcinoma

Eosinophils are terminally differentiated leukocytes that participate in the process of chronic inflammation and allergy and are able to release multiple cytokines into the surrounding tissue environment. Tumor-associated tissue eosinophilia (TATE) is the presence of eosinophils in the tumor or in the neighboring stroma and has been observed in various types of cancer. In head and neck squamous cell carcinoma (HNSCC), the clinical relevance of TATE has not been concluded yet because of the inconsistent results in different studies. In our study, we focus on the prognostic effects of TATE on HNSCC and how TATE can influence tumor behavior and tumor microenvironment. We first showed that in both the TCGA-HNSC cohort and our cohort of patients with HNSCC who had received curative surgery, TATE is correlated with worse overall survival. To investigate the underlying mechanism of how TATE leads to poor clinical outcomes, we showed that activated eosinophils produce a variety of cytokines and chemokines, and activated TATE-derived culture medium promotes tumor migration mainly through CCL2. We also showed that eosinophils are capable of inducing angiogenesis and that HNSCC samples enriched with TATE are highly correlated with tumor angiogenesis. Furthermore, HNSCC enriched with TATE had more aggressive pathological features, including regional lymph node metastasis, perineural invasion, lymphovascular invasion, and tumor growth. Lastly, we showed that HNSCC enriched with TATE is associated with immunosuppressive tumor microenvironment. Taken together, our results suggest that TATE promotes cancer metastasis and angiogenesis which results in a poor clinical outcomes in HNSCC.

Abstract 2141: Stromal SPARC deficiency skews prostate cancer toward neuroendocrine differentiation

Abstract Tumor progression is a multifaceted process in which, complex interactions between tumor and different types of stromal cells and extracellular matrix components, actively contribute to its phenotypic heterogeneity. Among extracellular matrix proteins, secreted protein acidic and rich in cysteine (SPARC) has been deeply studied since conflicting reports have described its expression to be either increased or decreased in different cancer settings, also depending on whether it is produced by the neoplasm or by the neighboring stroma. Nevertheless, the different contribution of tumor- or stromal-derived SPARC in prostate tumor microenvironment has not been addressed at least for tumor tissue histotype. Given this evidence, we aimed at providing new insights into the mechanism by which SPARC modulation influences prostate cancer development and progression. We modeled human disease using TRAMP mice, which spontaneously develop autochthonous prostate tumors following the onset of puberty. Crossing TRAMP mice with Sparc-/- mice, we found the appearance of focal areas of neuroendocrine differentiation within adenocarcinoma. In patients this phenomenon commonly results after androgen ablation therapy and correlates with poor prognosis. Interestingly, areas of neuroendocrine differentiation in Sparc-/- TRAMP mice were both positive for cytokeratin 8 and synaptophysin (usually expressed by luminal proliferating cells within adenocarcinoma or neuroendocrine cells, respectively), further suggesting a differentiation of adenocarcinoma cells to a neuroendocrine-like phenotype. Moreover, immunohistochemistry showed SPARC positivity not only in scattered tumor cells but also in fibroblasts and myeloid cells infiltrating TRAMP prostate. Accordingly, in vitro experiments suggested that stromal-derived SPARC limits neuroendocrine differentiation of prostate cancer cells, while they excluded a role of endogenous SPARC in this phenomenon. Indeed, prostate cancer cell lines co-cultured in presence of Sparc-deficient fibroblasts increased or acquired neuroendocrine features. This likely occurs through the effect of IL-6, a cytokine recently discovered to induce neuroendocrine differentiation, and that we found to be released by Sparc-deficient, but not sufficient, fibroblasts. Data collected so far indicate that stromal SPARC deficiency skews prostate carcinogenesis toward neuroendocrine differentiation. A deeper understanding of the molecular mechanisms governing the balance between prostate adenocarcinoma and neuroendocrine tumors according to extracellular matrix composition will provide important insights for the development of new prognostic and therapeutic strategies. Citation Format: Claudia Enriquez, Valeria Cancila, Ivano Arioli, Claudio Tripodo, Mario Paolo Colombo, Elena Jachetti. Stromal SPARC deficiency skews prostate cancer toward neuroendocrine differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2141.

Canonical NF\u03baB signaling in myeloid cells is required for the glioblastoma growth

Tumor development and therapeutic resistance are linked with tumor-associated macrophage (TAM) and myeloid-derived suppressor cell (MDSC) infiltration in tumors via chemokine axis. Chemokine expression, which determines the pro or anti-inflammatory status of myeloid cells, are partly regulated by the nuclear factor-kappa B (NF-κB) pathway. Here, we identified that conditional deletion of canonical NF-κB signaling (p65) in myeloid cells inhibited syngeneic glioblastoma (GBM) through decreased CD45 infiltration in tumors, as characterized by decreased TAMs (CD206+) and MDSCs (Gr1+ CD11b+), increased dendritic cells (CD86+) and cytotoxic T cells (CD8+) in the p65 knockout (KO) mice. Proinflammatory cytokines (IFNγ, MCP1, MIP1α, and TNFα) and myeloid differentiation factor (Endoglin) were increased in myeloid cells from p65 KO tumor, which demonstrated an influence on CD8+T cell proliferation. In contrast, p65KO athymic chimeric mice with human GBM, failed to inhibit tumor growth, confirming the contribution of T cells in an immune competent model. The analysis of human datasets and GBM tumors revealed higher expression of p65 in GBM-associated CD68+ macrophages compared to neighboring stroma. Thus, canonical NF-κB signaling has an anti-inflammatory role and is required for macrophage polarization, immune suppression, and GBM growth. Combining an NF-κB inhibitor with standard therapy could improve antitumor immunity in GBM.

Mitochondrial and glycolytic metabolic compartmentalization in diffuse large B-cell lymphoma

Metabolic heterogeneity between neoplastic cells and surrounding stroma has been described in several epithelial malignancies; however, the metabolic phenotypes of neoplastic lymphocytes and neighboring stroma in diffuse large B-cell lymphoma (DLBCL) is unknown. We investigated the metabolic phenotypes of human DLBCL tumors by using immunohistochemical markers of glycolytic and mitochondrial oxidative phosphorylation (OXPHOS) metabolism. The lactate importer MCT4 is a marker of glycolysis, whereas the lactate importer MCT1 and TOMM20 are markers of OXPHOS metabolism. Staining patterns were assessed in 33 DLBCL samples as well as 18 control samples (non-neoplastic lymph nodes). TOMM20 and MCT1 were highly expressed in neoplastic lymphocytes, indicating an OXPHOS phenotype, whereas non-neoplastic lymphocytes in the control samples did not express these markers. Stromal cells in DLBCL samples strongly expressed MCT4, displaying a glycolytic phenotype, a feature not seen in stromal elements of non-neoplastic lymphatic tissue. Furthermore, the differential expression of lactate exporters (MCT4) on tumor-associated stroma and lactate importers (MCT1) on neoplastic lymphocytes support the hypothesis that neoplastic cells are metabolically linked to the stroma likely via mutually beneficial reprogramming. MCT4 is a marker of tumor-associated stroma in neoplastic tissue. Our findings suggest that disruption of neoplastic-stromal cell metabolic heterogeneity including MCT1 and MCT4 blockade should be studied to determine if it could represent a novel treatment target in DLBCL.

The Epithelial-to-Mesenchymal Transition-Like Process in Glioblastoma: An Updated Systematic Review and In Silico Investigation.

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer due to its highly invasive nature that impedes the surgical removal of all tumor cells, making relapse inevitable. However, the mechanisms used by glioma cells to invade the surrounding tissue are still unclear. In this context, epithelial-to-mesenchymal transition (EMT) has emerged as a key regulator of this invasive state and although the real relevance of this program in malignant glioma is still controversial, it has been strongly associated with GBM malignancy. EMT is a very complex process regulated by several families of transcriptional factors through many signaling pathways that form a network that allows cancer cells to acquire invasive properties and penetrate the neighboring stroma, resulting in the formation of an advantageous microenvironment for cancer progression and metastasis. In this systematic review, we focus on the molecular mechanisms of EMT including EMT-factors, drug resistance, miRNA, and new therapeutic strategies. In addition, we address controversial questions about mesenchymal shift in GBMs with a bioinformatics analysis to show that in terms of epithelial and mesenchymal phenotype, the majority of GBMs samples analyzed have a profile more mesenchymal than epithelial. If induced, this phenotype can be shifted toward an even more mesenchymal phenotype in an EMT-like process in glioma cells. A better understanding of the molecular regulation of the EMT during tumor spreading will help to provide potential therapeutic interventions to target this program when treating GBM.

Reasons to reconsider interpretation of densitometry for corneal transparency

To assess the effect of light scattering in the corneal epithelium on densitometric brightness of the stroma through mathematical modeling of the interaction between a light beam and the two-layer epithelium-stroma system. In order to study the scattering behavior of a plane-parallel non-coherent beam at the epithelium-stroma interface, a multi-age group was formed (87 patients, 174 eyes) that comprised two subgroups with equal number of assign participants: healthy patients with no systemic changes and mixed patients with undisturbed corneal transparency that, nevertheless, were under instillation therapy. In the first subgroup, the assessment of light scattering was done at random times, while in the second subgroup - within the first 200 seconds after the instillation in order to avoid a reaction of the ocular surface structures, including epithelium (rapid response of epithelial cells to instillations). Light scattering intensity was measured with Pentacam (USA). The densitometric curve and brightness maxima of the epithelium and neighboring stroma as well as the mid and upper stroma were recorded for each patient. A physico-mathematical model of the interaction between a light beam and the two-layer epithelium-stroma system was created. The study confirmed our theoretical CONCLUSION: on the effect size of light scattering intensity on the apparent densitometric brightness of the stroma. Direct densitometry of multilayered spherical objects, of which each layer possesses its own optical properties, has been proved inaccurate. Complex optical interactions in a multilayered model cause a false increase in brightness of more transparent layers that lie behind those with pronounced light scattering properties. The function that describes the false increase in scattering ability of the layer that is more transparent rises sharply at the border between the layers.

Stromal Expression of CD34, α-Smooth Muscle Actin and CD26/DPPIV in Squamous Cell Carcinoma of the Skin: A Comparative Immunohistochemical Study

Invasion pathogenesis is one of the most complicated issues in the literature. There are numerous studies concerning the tumor markers implicated in the preinvasive-invasive tumor sequence. Despite ample studies on the invasion pathogenesis of cutaneous melanomas, there is limited and dispersed work presently available on non-melanoma skin cancer. The vast knowledge in the literature concerning this issue in squamous cell carcinoma comes mostly from the studies of the oral cavity, esophagus, larynx, and cervix. In this study, we investigated tumor-free neighboring stroma and tumor stroma in squamous cell carcinomas (SCCs) of the skin as well as keratoacanthomas (KAs) with respect to the presence of stromal CD34-positive (CD34+) fibrocytes and α-smooth muscle actin-positive (α-SMA+) myofibroblasts using seborrheic keratosis (SKs) and non-tumoral skin samples as controls. We also evaluated the stromal expression pattern of CD26/DPPIV (CD26), a tumor suppressor gene product that also has immunoregulatory properties. Immunohistochemistry was performed on samples of 31 SCC, 8 KA, 15 SK and 10 non-tumoral skin samples. Peri-tumoral stroma from resection margins was also evaluated. We found that CD34 and α-SMA demonstrated significantly different staining between benign and malignant squamous skin lesions consisting of a loss of CD34+ fibrocytes paralleled by a gain of α-SMA+ myofibroblasts in malignant tumor stroma. Additionally, it was shown that CD26 expression was lower in tumor stroma when compared to that of tumor neighboring stroma. However, we concluded that this finding may be attributable to the solar elastosis areas in the peritumoral tissue, which shows diffuse strong positivity for this marker.

Improved Response to nab-Paclitaxel Compared with Cremophor-Solubilized Paclitaxel is Independent of Secreted Protein Acidic and Rich in Cysteine Expression in Non-Small Cell Lung Cancer

The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is produced by tumor and/or neighboring stroma. SPARC expression is thought to facilitate the intracellular accumulation of nanoparticle albumin-bound paclitaxel (nab-paclitaxel, abraxane [ABX]). Gene hypermethylation is a common mechanism for loss of SPARC expression in non-small cell lung cancer (NSCLC). We aim to demonstrate the role of SPARC expression as biomarker for treatment selection using ABX in NSCLC and to evaluate the presence of synergistic antitumor effect when a demethylating agent is combined with ABX. We analyzed the SPARC messenger RNA expression and SPARC gene methylation status in 13 NSCLC cell lines and 22 minimally passaged patient-derived (PD) NSCLC tumors using real-time (RT) polymerase chain reaction. The effect of ABX on tumor growth was compared with cremophor-solubilized paclitaxel (taxol) in severe combined immunodeficiency mice bearing SPARC-positive PD xenografts. The effect of pretreatment with a demethylating agent, 5-Aza-2'-deoxycytidine (DEC) in SPARC-negative tumors was assessed. SPARC expression was weak to absent in 62% of established NSCLC cell lines and 68% of PD NSCLC tumor xenografts. SPARC expression could be up-regulated/restored by DEC treatment in both SPARC-negative cell lines and PD xenografts in vitro and in vivo. ABX demonstrated better antitumor efficacy than equitoxic dose of taxol in SPARC-expressing xenografts and some SPARC-negative xenografts. At equimolar doses in vitro, there was similar increased cytotoxicity on DEC pretreatment with either ABX or taxol in SPARC-negative cell lines. At equitoxic doses, there was similar additive antitumor activity of DEC with either ABX or taxol in SPARC-negative PD xenografts. Endogenous SPARC status is somewhat uncorrelated with response to ABX in NSCLC. The greater antitumor effect of ABX compared with equitoxic dose of taxol observed in SPARC-expressing NSCLC tumors can also be seen in some SPARC-negative tumors. DEC pretreatment similarly enhanced antitumor activity with either ABX or taxol in SPARC-negative tumors.

The role of SPARC in the TRAMP model of prostate carcinogenesis and progression

SPARC (Secreted Protein Acidic and Rich in Cysteine), is a matricellular glycoprotein that is produced by tumor and/or neighboring stroma. In human prostate cancer, SPARC immunoreactivity is highest in metastatic lesions but distinct contributions of tumoral and stromal SPARC to tumorigenesis and progression are unclear. To determine the role of SPARC in primary prostate tumorigenesis, we crossed SPARC-null (SP(-/-)) with TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. TRAMP(+)/SP(-/-) mice exhibited accelerated cancer development and progression. Compared to their TRAMP(+)/SP(-/-) counterparts, TRAMP(+)/SP(+/+) tumors had fewer proliferating cells, and decreased cyclins A and D1 with increased p21(Cip) and p27(Kip). Similar effects on proliferation and cell-cycle regulators were observed in human prostate cancer cell lines, transiently transfected with pSPARC. TRAMP(+)/SP(-/-) tumors exhibited decreased stromal collagen, enhanced matrix metalloproteinase activity and increased vascular endothelial growth factor, proinflammatory cytokines. To determine the contribution of stromal SPARC, we evaluated subcutaneous tumor growth of TRAMP cell lines in syngeneic SP(+/+) and SP(-/-) mice. Enhanced growth, decreased stromal collagen and increased proteolysis were noted in SP(-/-) mice. Our findings demonstrate that both tumor and stromal SPARC are limiting for primary prostate tumorigenesis and progression, through effects on the cell cycle and the creation of a less favorable tumor microenvironment.

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation. In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions. In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling (1). Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-microm gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

Watching Immune Cells in Action

In contrast to most adult tissues, a unique feature f the vertebrate immune system is the motility of the mmune cells. Both innate and adaptive immune reponses are critically dependent on the migratory caacity of myeloid and lymphoid cells. For example, ffector cells of the innate immune system rapidly nter infected tissues, whereas sentinel dendritic cells DCs) in these sites mobilize and transit to lymph odes [1-5]. Lymphoid and myeloid cells also enter eripheral tissues in response to local signals such as hemokines and adhesion molecules present on the urface of endothelial cells [3]. In all of these sites, ells of the innate and adaptive immune systems comunicate extensively [6,7], each guiding the differeniation of the other, leading to changes in their paterns of gene expression, alterations in their repertoire f surface proteins, and production of effector moleules. These orchestrated series of events ensure the roper quality and magnitude of adaptive humoral and ell-mediated immune responses to infectious agents 8,9]. Equally important, the immune cells also interct with cells of the neighboring stroma, which has an mportant contribution to the eventual outcome of the mmune response. Until recently, support for this description of the mmune system comes from more than a century of xperimental analyses, with conclusions about in vivo ell dynamics inferred largely from serial static analsis of tissue sections (at relatively long intervals comared with the rate at which the underlying events ctually transpired) and dynamic studies of dissociated ells from in vitro cultures (in the absence of intact tromal elements normally present in situ) [10]. Even ith improvements in staining reagents, flow cytoetric equipment [11], and microscopy tools [12-14], hese early dynamic studies of immune cell behavior ave been limited to in vitro imaging of cultured cells, hich was used to clarify the molecular reorganization hat occurs during cell-cell contacts [15-17] and to ingle-color analysis of leukocyte behavior in small essels [18], which helped to confirm the rolling, stopd ing, and transmigration model of cell extravasations rom the blood to the tissues in situ [19]. However, ecent advances with in vivo imaging modalities have egun to afford direct visualization of these events in iving animals and tissue explants, allowing a renew ppreciation of the dynamics of immune cell behavior n situ. Positron emission tomography, magnetic resnance imaging, and luminescence imaging [20,21] ave been used to track immune cells in vivo. Alhough useful in tracking bulk population behavior, hese methods failed to offer the spatial resolution and ime scale required for single-cell analysis during an mmune response. Fortunately, advances in confocal nd, most recently, 2-photon laser scanning microsopy tools, combined with new intravital surgical aproaches that preserve the structural integrity, nervous nnervations, and blood and lymphatic circulation of the issues studied, have made high spatialand temporalesolution imaging of immune cell interaction in any tissues possible and have revolutionized our bility to probe more directly how immune cells oprate in vivo [22]. Two-photon microscopy was espeially attractive as a tool for intravital imaging because f the increased depth of tissue penetration with high patial resolution, high signal-to-noise ratio, and dereased photobleaching of out-of-focus planes [22,23], hus allowing prolonged periods of high-resolution ata collection on events that occur deep in intact tissues f living animals or intact tissue explants (Table 1). To date, data generated by 2-photon microscopy ethods have provided new perspectives and insights nto our understanding of how, where, and when cells nteract during the initiation of an immune response nd during the delivery of effector function at periphral sites [24-31]. Some initial studies have tracked the ovements of naive B and T cells after adoptive transer in the absence of antigenic stimulation, revealing he unexpectedly rapid pace of lymphocyte migration n secondary lymphoid tissues [25,26,31-38], a finding hat had important implications for our current modls of how rare antigen-specific lymphocytes and anigen-presenting cells (APCs) find one another to prouce an effective response. When a migrating T cell

CD44 distribution in sweat gland tumors suggests it has different functional roles in the various cell types.

CD44 is a polymorphic group of membrane glycoproteins with multiple functions that include cell adhesion. Since on normal sweat glands CD44 is expressed only in eccrine coil secretory cells, it has been considered as a possible marker of this type of differentiation. We have immunohistochemically investigated the distribution of CD44 in paraffin-embedded samples of 41 benign and malignant sweat gland tumors by using a monoclonal antibody directed against the standard isoform of CD44. CD44 was strongly expressed in epithelial cells at the peripheral row of syringomas and in cuticular areas of eccrine poromas. Apocrine tumors such as apocrine hidrocystoma, syringocystadenoma papilliferum, or hidradenoma papilliferum showed intense CD44 positivity in the portion of cells in contact with the neighboring stroma and focally on the luminal side of cells with apocrine secretion. Cylindromas and spiradenomas presented focal CD44 positivity, virtually limited to clear cells. Malignant neoplasms exhibited irregular CD44 staining, which was more intense in the less differentiated zones and tumors. Our results indicate that CD44 is not a useful marker for a specific form of sweat gland differentiation. Nevertheless, its characteristic patterns of distribution might reflect the variety of functional roles assumed by the different CD44 isoforms in each epithelial cell.

Active cell death in hormone-dependent tissues.

Active cell death (ACD) in hormone-dependent tissues such as the prostate and mammary gland is readily induced by hormone ablation and by treatment with anti-androgens or anti-estrogens, calcium channel agonists and TGF beta. These agents induce a variety of genes within the hormone-dependent epithelial cells including TRPM-2, transglutaminase, poly(ADP-ribose) polymerase, Hsp27 and several other unidentified genes. Not all epithelial cells in the glands are equally sensitive to the induction of ACD. In the prostate, the secretory epithelial cells that are sensitive to hormone ablation are localized in the distal region of the prostatic ducts, and are in direct contact with the neighboring stroma. In contrast, the epithelial cells in the proximal regions of the ducts are more resistant to hormone ablation, probably because the permissive effects of the stroma are attenuated by the presence of the basal epithelial cells, which are intercalated between the epithelium and stroma. The underlying biology of ACD in prostate and mammary glands, and its relevance to hormone resistance, is discussed in this review.