Fibroblastic Stromal Cells Research Articles

From Graft-Versus-Host Disease to Lymphoma Pathogenesis: Emerging Roles for Stromal Notch Ligands in Hematology

Notch signaling is a highly conserved signaling pathway with multiple functions in health and disease. Notch ligands of the Delta-like and Jagged families interact with Notch1-4 on adjacent cells to trigger proteolytic activation of the Notch receptors, followed by context-dependent transcriptional activation of Notch target genes. In the hematopoietic system, Notch was first identified for its oncogenic role in T cell acute lymphoblastic leukemia (T-ALL). More recently, Notch has been recognized as a recurrent oncogenic pathway in multiple B cell lymphoproliferative disorders, including CLL/SLL, mantle cell lymphoma and marginal zone lymphoma. In parallel, essential functions of Notch signaling have been identified in early T cell development and B cell homeostasis, as well as in regulating T and B cell immune responses in secondary lymphoid organs. We have discovered a conserved pathogenic function of Notch signaling in the control of graft-versus-host disease (GVHD), both in mouse and non-human primate models of hematopoietic cell transplantation. Within days after allogeneic hematopoietic cell transplantation, donor-derived T cells interact with Delta-like Notch ligands expressed by specialized subsets of non-hematopoietic fibroblastic stromal cells in spleen and lymph nodes. In turn, Notch signaling programs alloreactive T cells to become pathogenic effector cells that mediate long-term damage in GVHD target tissues, while suppressing the expansion of regulatory T cells. Emerging evidence suggests the existence of an active crosstalk between T cells, B cells and well-defined specialized immunological stromal niches expressing Notch ligands in secondary lymphoid organs, both in GVHD and in other types of immune responses. Moreover, many B cell lymphomas demonstrate oncogenic Notch activation as a result of gain-of-function mutations that increase the persistence of intracellular Notch after proteolytic cleavage, thus requiring the presence of Notch ligands in the microenvironment to trigger oncogenic Notch signals. New evidence suggests that key Notch ligands in human lymphomas are present in the lymph node microenvironment, and that oncogenic Notch activation can happen even in the absence of mutations in a substantial fraction of lymphoma patients, including the majority of patients with CLL/SLL. In these cases, lymphoma cells appear to interact with microenvironmental Notch ligands in secondary lymphoid organs through unmutated Notch receptors, borrowing from the playbook of normal lymphocytes. Furthermore, the expression of Notch ligands by defined subsets of fibroblastic stromal cells illustrates a new paradigm through which microenvironmental signals can profoundly influence the differentiation and function of adjacent lymphoid cells. We speculate that better understanding the regulation of Notch signaling and fibroblastic stromal cells in secondary lymphoid organs will provide valuable new information with translational potential both in immunobiology and in malignant hematology. Disclosures Maillard: Genentech: Consultancy; Regeneron: Consultancy.

Microenvironmental Regulation of Long Noncoding RNA LINC01133 Promotes Cancer Stem Cell-Like Phenotypic Traits in Triple-Negative Breast Cancers

The fibrotic tumor microenvironment is a critical player in the pathogenesis of triple-negative breast cancers (TNBCs), with the presence of fibroblastic infiltrates particularly correlating with tumors that are clinically advanced. On this front, we previously demonstrated that TNBCs are highly enriched in fibroblastic stromal progenitor cells called mesenchymal stem/stromal cells (MSCs) and that such cells play critical roles in promoting TNBC initiation and progression. How TNBC cells respond to MSC stimulation, however, is not fully understood, and stands to reveal contextual signals used by TNBC cells during tumor development and provide biomarkers and therapeutic targets of pertinence to TNBC management. Here, we report that MSCs strongly induced the long noncoding RNA (lncRNA) LINC01133 in neighboring TNBC cells. Indeed, although lncRNAs have been tightly associated with cancer development, their contributions to breast cancer in general, and to TNBC pathogenesis in particular, have not been fully elucidated, and we set out to determine if LINC01133 regulated malignant traits in TNBC cells. We establish that LINC01133 is sufficient, on its own, in promoting phenotypic and growth characteristics of cancer stem cell-like cells, and that it is a direct mediator of the MSC-triggered miR-199a-FOXP2 pathway in TNBC models. Furthermore, we show that LINC01133 is a critical regulator of the pluripotency-determining gene Kruppel-Like Factor 4 (KLF4), and that it represents a biomarker and prognosticator of disease outcome in the clinic. Collectively, our findings introduce LINC01133 as a novel functional driver of malignancy and a potential theranostic in TNBC. Stem Cells 2019;37:1281-1292.

Ovarian Fibrothecoma

A postmenopausal woman presented with abdominal pain and increased urinary frequency. A unilateral solid ovarian mass was identified and excised. What is the best diagnosis for the lesion?a.Adult granulosa cell tumorb.Leiomyomac.Fibrothecomad.Clear cell carcinoma Answer: c. Fibrothecoma Fibroma/thecoma/fibrothecoma is a spectrum of benign sex cord–stromal tumors composed of fibroblastic stromal cells and/or luteinized theca-like cells. When both cell types are present in a tumor, it is typically classified as fibrothecoma. These tumors can occur at any age, but most commonly arise in middle-aged to postmenopausal women.1Young R.H. Ovarian sex cord-stromal tumours and their mimics.Pathology. 2018; 50: 5-15Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar

Contribution of fibroblasts to tunnel formation and inflammation in hidradenitis suppurativa/ acne inversa.

The precise pathogenic mechanisms in the development, persistence and worsening of hidradenitis suppurativa (HS) remain ill-defined. This chronic inflammatory dermatosis displays a strong Th1 and Th17 inflammatory signature with elevated levels of TNF-α, IL-1β, IL-17 and IFNγ in lesional and perilesional tissue. HS significantly differs to other chronic inflammatory dermatoses due to the development of hypertrophic scarring and dermal tunnels. The development of scarring and tunnels suggests that fibroblastic stromal cells (including myofibroblasts, fibroblasts, pericytes etc) may be involved in the development and progression of disease. Heterogeneous populations of fibroblasts have been identified in other inflammatory disorders and malignancy which contribute to inflammation and present novel therapeutic targets for fibrotic disorders. Findings in HS are consistent with these fibroblast subpopulations and may contribute to tunnel formation, aggressive squamous cell carcinoma and the phenotypic presentation of familial HS variants. We describe the existing knowledge regarding these mechanistic pathways and methods to confirm their involvement in the pathogenesis of HS.

Abstract 1143: The circulating tumor cell (CTC) microenvironment visualized by color coded imaging

Abstract Background/Aim: An important question is whether cancer cells circulate with stromal cells. A color-coded mouse model of metastatic lymphoma was utilized to investigate the microenvironment of CTC clusters using fluorescent-protein imaging. Materials and Methods: EL-4 mouse lymphoma cells expressing red fluorescent protein (RFP) were injected into the spleen of transgenic C57BL6-green fluorescent protein (GFP) mice. The cellular composition of CTC clusters both in heart blood and portal blood was imaged with confocal microscopy. Results: CTC clusters comprised 8.8% of CTCs determined by color-coded imaging. Heterotypic CTC clusters containing other types of cells were distinguishable from homotypic CTCs. Heterotypic CTC clusters comprising cancer cells and fibroblasts were more rare than homotypic ones. Heterotypic CTC clusters with fibroblasts were observed only in portal blood, not in heart blood. Conclusion: CTCs can form clusters, which may contain fibroblast or other stromal cells that may play a role in promoting CTC metastasis. Citation Format: Atsushi Suetsugu, Tomoyuki Satake, Miki Nakamura, Masahito Shimizu, Shigetoyo Saji, Hisataka Moriwaki, Robert M. Hoffman. The circulating tumor cell (CTC) microenvironment visualized by color coded imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1143.

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6+ macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression oftwo rate-limiting enzymes in retinol metabolism. Depletion of Wt1+ stromal cellsreduced the frequency of GATA6+ macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1+mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.

PS913 ROLE OF CHOROID PLEXUS STROMA IN TUMOUR CHEMORESISTANCE: A SANCTUARY FOR LEUKAEMIA CELLS IN THE CENTRAL NERVOUS SYSTEM

Background:Central Nervous System (CNS) relapses are still one of the main causes of morbimortality in childhood acute lymphoblastic leukaemia (ALL) patients despite the reduction achieved by CNS‐directed prophylactic chemotherapy. Although CNS colonization by ALL cells has been already associated to both increased survival and enhanced chemoresistance of blasts to intrathecal therapy, the mechanisms of protection and tumour cell maintenance in this compartment, as well as the interactions established between ALL and CNS cells, still need to be better understood.Aims:We propose in vivo and in vitro studies aimed at determining the location of leukaemia cells in the choroid plexus (CP) and the relevance of their interaction with stromal fibroblastic cells to create a tumour supportive microenvironment. Also, we explore the contribution of stromal cells to the ALL chemoresistance.Methods:Brain histopathology evaluation of immunodeficient NSG mice transplanted with human primary ALL cells or the Nalm‐6 cell line was performed by using electron and fluorescence microscopy. Leukemic co‐cultures with human CP cells in the presence or absence of different concentrations of chemotherapeutic drugs were used for viability assays. Changes in CP microenvironment were further tested by RT‐qPCR, flow cytometry, ELISA and CBA assays.Results:The histological analysis of a xenograft model of ALL revealed the presence of leukaemic cells not only in the cerebral ventricles floating in the cerebrospinal fluid, but also interacting with the CP epithelium and within the CP stroma of those animals with CNS infiltration. The proximity of ALL cells to CP capillaries suggests they may easily enter through this fenestrated endothelium. Evaluation of interactions between leukaemic cells and stromal cells showed an upregulated expression of very late antigen‐4 (VLA‐4) in ALL cells and vascular cell adhesion molecule (VCAM1) in CP fibroblasts. Furthermore, in these co‐cultures, ALL cells exhibited a reduced cell proliferation and an enhanced cell viability following treatment with chemotherapeutic agents. However, the blockade of VCAM1‐VLA‐4 interaction was found to impair those effects suggesting that VCAM1‐VLA‐4 interactions are, at least in part, responsible of protecting leukemic cells from cell death.Summary/Conclusion:Choroid plexus is proposed as a possible sanctuary for leukemic cells. There, ALL cells may remain attached to fibroblasts in a dormant state and protected from intrathecal chemotherapy treatment, thus favouring later relapses.

PS914 CONDITIONAL MEF2C EXPRESSION IN THE MOUSE THYMUS DRIVES DEVELOPMENT OF T/B-BIPHENOTYPIC LYMPHOID LEUKEMIA/LYMPHOMA

Background: Central Nervous System (CNS) relapses are still one of the main causes of morbimortality in childhood acute lymphoblastic leukaemia (ALL) patients despite the reduction achieved by CNS-directed prophylactic chemotherapy. Although CNS colonization by ALL cells has been already associated to both increased survival and enhanced chemoresistance of blasts to intrathecal therapy, the mechanisms of protection and tumour cell maintenance in this compartment, as well as the interactions established between ALL and CNS cells, still need to be better understood. Aims: We propose in vivo and in vitro studies aimed at determining the location of leukaemia cells in the choroid plexus (CP) and the relevance of their interaction with stromal fibroblastic cells to create a tumour supportive microenvironment. Also, we explore the contribution of stromal cells to the ALL chemoresistance. Methods: Brain histopathology evaluation of immunodeficient NSG mice transplanted with human primary ALL cells or the Nalm-6 cell line was performed by using electron and fluorescence microscopy. Leukemic co-cultures with human CP cells in the presence or absence of different concentrations of chemotherapeutic drugs were used for viability assays. Changes in CP microenvironment were further tested by RT-qPCR, flow cytometry, ELISA and CBA assays. Results: The histological analysis of a xenograft model of ALL revealed the presence of leukaemic cells not only in the cerebral ventricles floating in the cerebrospinal fluid, but also interacting with the CP epithelium and within the CP stroma of those animals with CNS infiltration. The proximity of ALL cells to CP capillaries suggests they may easily enter through this fenestrated endothelium. Evaluation of interactions between leukaemic cells and stromal cells showed an upregulated expression of very late antigen-4 (VLA-4) in ALL cells and vascular cell adhesion molecule (VCAM1) in CP fibroblasts. Furthermore, in these co-cultures, ALL cells exhibited a reduced cell proliferation and an enhanced cell viability following treatment with chemotherapeutic agents. However, the blockade of VCAM1-VLA-4 interaction was found to impair those effects suggesting that VCAM1-VLA-4 interactions are, at least in part, responsible of protecting leukemic cells from cell death. Summary/Conclusion: Choroid plexus is proposed as a possible sanctuary for leukemic cells. There, ALL cells may remain attached to fibroblasts in a dormant state and protected from intrathecal chemotherapy treatment, thus favouring later relapses.

Co-Detection of miR-21 and TNF-α mRNA in Budding Cancer Cells in Colorectal Cancer.

MicroRNA-21 (miR-21) is upregulated in many cancers including colon cancers and is a prognostic indicator of recurrence and poor prognosis. In colon cancers, miR-21 is highly expressed in stromal fibroblastic cells and more weakly in a subset of cancer cells, particularly budding cancer cells. Exploration of the expression of inflammatory markers in colon cancers revealed tumor necrosis factor alpha (TNF-α) mRNA expression at the invasive front of colon cancers. Surprisingly, a majority of the TNF-α mRNA expressing cells were found to be cancer cells and not inflammatory cells. Because miR-21 is positively involved in cell survival and TNF-α promotes necrosis, we found it interesting to analyze the presence of miR-21 in areas of TNF-α mRNA expression at the invasive front of colon cancers. For this purpose, we developed an automated procedure for the co-staining of miR-21, TNF-α mRNA and the cancer cell marker cytokeratin based on analysis of frozen colon cancer tissue samples (n = 4) with evident cancer cell budding. In all four cases, TNF-α mRNA was seen in a small subset of cancer cells at the invasive front. Evaluation of miR-21 and TNF-α mRNA expression was performed on digital slides obtained by confocal slide scanning microscopy. Both co-expression and lack of co-expression with miR-21 in the budding cancer cells was noted, suggesting non-correlated expression. miR-21 was more often seen in cancer cells than TNF-α mRNA. In conclusion, we report that miR-21 is not linked to expression of the pro-inflammatory cytokine TNF-α mRNA, but that miR-21 and TNF-α both take part in the cancer expansion at the invasive front of colon cancers. We hypothesize that miR-21 may protect both fibroblasts and cancer cells from cell death directed by TNF-α paracrine and autocrine activity.

Non-canonical Inflammasome-Mediated IL-1β Production by Primary Endometrial Epithelial and Stromal Fibroblast Cells Is NLRP3 and Caspase-4 Dependent.

Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1β early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1β expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1β expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1β was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1β protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1β production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1β expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1β. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1β protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1β production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1β production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle.

Essential Role of Canonical NF-κB Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs.

Organized tissue structure in the secondary lymphoid organs (SLOs) tightly depends on the development of fibroblastic stromal cells (FSCs) of mesenchymal origin; however, the mechanisms of this relationship are poorly understood. In this study, we specifically inactivated the canonical NF-κB pathway in FSCs in vivo by conditionally inducing IκBα mutant in a Ccl19-IκBSR mouse system in which NF-κB activity is likely to be suppressed in fetal FSC progenitors. Given that NF-κB activation in fetal FSCs is essential for SLO development, the animals were expected to lack SLOs. However, all SLOs were preserved in Ccl19-IκBSR mice. Instead, the T cell area was severely disturbed by the lack of CCL21-expressing FSCs, whereas the follicles and associated FSC networks were formed. Fate mapping revealed that IκBSR-expressing cells constituted only a small fraction of stromal compartment outside the follicles. Taken together, our findings indicate an essential role of the canonical NF-κB pathway activity in the development of three FSC subsets common to SLOs and suggest transient or stochastic CCL19 expression in FSC progenitors and a compensatory differentiation program of follicular FSCs.

Donor T Cells Require Notch Signals but Not Alloantigen Presentation from Specialized Secondary Lymphoid Organ Fibroblasts to Drive Graft-Versus-Host Disease

Notch signals drive pathogenic T cell alloreactivity in mouse models of allogeneic bone marrow transplantation (allo-BMT) leading to lethal graft-versus-host disease (GVHD). In a mouse model of MHC-mismatched allo-BMT with high intensity whole body conditioning (11 Gy), residual recipient or donor hematopoietic cells were dispensable sources of Notch signals to drive T cell alloreactivity. Instead, abrogating the expression of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands in Ccl19-Cre+ secondary lymphoid organ (SLO) fibroblastic stromal cells protected mice from GVHD (Chung et al., JCI 2017). In this study we sought to 1) understand the expression and regulation of Notch ligands in fibroblastic stromal cells during allo-BMT; 2) test whether fibroblastic stromal cells are still critical sources of Notch signals in models of allotransplantation with reduced or no conditioning (when other radiation-sensitive sources of Notch ligands are better preserved); and 3) assess whether SLO fibroblastic stromal also present alloantigens to T cells during GVHD.To characterize stromal sources of Notch ligands early after allo-BMT, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele in Dll4-mCherry BAC reporter mice. We observed combined Ccl19-Cre activity and Dll4-mCherry reporter expression in CD157hi fibroblastic reticular cells, MAdCAM1+ marginal reticular cells, CD21+ follicular dendritic cells, and a subset of MAdCAM1+ lymphatic endothelial cells. 12 hours after high intensity conditioning and allo-BMT, Dll4 mRNA and cell surface expression increased in fibroblastic but not in hematopoietic or endothelial cellular compartments. Dll4 upregulation required transfer of alloreactive T cells and was potentiated by the intensity of myeloablative conditioning, suggesting that fibroblastic stromal cells have increased availability of Notch ligands to drive GVHD after high-intensity conditioning.To assess the functional impact of conditioning on the cellular sources of Notch ligands, we studied a parent-to-F1 allo-BMT model in which recipients are tolerant to donor-derived T cells, and the conditioning intensity can be titrated (C57BL/6 [H-2b] or BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b]). Cell-specific genetic loss of function systems combining floxed alleles for Dll1 and Dll4 with Ccl19-Cre+ and Mx-Cre+ transgenes allowed conditional deletion in SLO fibroblastic stromal cells or hematopoietic cells, respectively. Across all conditioning intensities tested, upregulation of core-2 O-glycosylation machinery in alloreactive T cells was critically regulated by Notch signals. Thus, induction of the core-2 glycoform of CD43 could be used as a sensitive surrogate readout of Notch signals received by alloreactive T cells. In models of allotransplantation with high-intensity (11 Gy), low-intensity (3 Gy), and no conditioning, fibroblastic stromal cells remained essential and hematopoietic cells were dispensable as sources of Notch signals driving both T cell core-2 O-glycosylation and lethal alloimmunity.Because previous studies indicated that non-hematopoietic cells can serve as antigen-presenting cells in CD4+ T cell mediated GVHD, we tested if fibroblastic stromal cells could present MHC class II alloantigens in addition to Notch ligands. We used Ccl19-Cre+ crossed to mice with a floxed I-A allele to delete the cell-intrinsic ability of fibroblastic stromal cells to present MHC class II alloantigens. In multiple models of high-intensity conditioned CD4+ T cell mediated GVHD (BALB/c [H-2d] into BALB/c x C57BL/6 F1 [H-2d/b] and TEa TCR Tg into BALB/c x C57BL/6 F1 [H-2d/b]), fibroblastic stromal cells were dispensable as sources of alloantigen to drive T cell alloreactivity and lethal GVHD. Together, these results indicate that across varying conditioning intensities, specialized subsets of SLO fibroblastic stromal cells critically regulate T cell alloreactivity through presentation of Notch ligands independently of antigen presentation. Selective targeting of Delta-like Notch ligand expression on SLO fibroblastic cells may prevent T cell immunopathology while preserving other beneficial antigen-dependent T cell responses after both high-intensity conditioned allo-BMT and allotransplantation with reduced intensity conditioning. DisclosuresBlazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Siebel:Genentech: Employment.

MiR-21 expression analysis in budding colon cancer cells by confocal slide scanning microscopy

MicroRNA-21 (miR-21) expression in stromal fibroblastic cells in colorectal cancer is well-documented, whereas miR-21 expression in tumor budding cells (TBCs) is poorly described. TBCs are locally invasive carcinoma cells with increased metastatic properties and characteristics of epithelial to mesenchymal transition. This study was conducted to better characterize the expression of miR-21 in TBCs. First, chromogenic miR-21 in situ hybridization (ISH) staining was performed in 58 colon adenocarcinomas with evident TBCs. Then, to obtain unambiguous identification of miR-21 in the TBCs, twenty cases were selected for an additional multiplex fluorescence analysis combining miR-21 ISH with cytokeratin and laminin-5γ2 immunofluorescence. Employing confocal slide scanning microscopy, comprehensive digital images of the invasive front (10–40 mm2) were obtained from 16 of the 20 cases, and miR-21 expression was evaluated in cytokeratin-positive TBCs. The high resolution of the confocal digital slide images allowed a detailed examination of the confocal stacks of the multiplex-stained tissue sections. The cases with the highest fraction of miR-21 positive TBCs were all stage III cancers defined by the presence of regional lymph node metastasis. Some of the miR-21 positive TBCs were also laminin-5γ2 positive. The confocal image stacks also revealed that some TBCs were actually directly connected to malignant glands. In conclusion, miR-21 expression was unambiguously identified in TBCs by evaluation of digital slides obtained by confocal slide scanning microscopy. In addition, the digital confocal slides provided a more detailed understanding of local cancer cell invasion by allowing evaluation of the cell structures in three dimensions.

A Distinct Subset of Fibroblastic Stromal Cells Constitutes the Cortex-Medulla Boundary Subcompartment of the Lymph Node

The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRβ, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

Microfluidic Formation of Coculture Tumor Spheroids with Stromal Cells As a Novel 3D Tumor Model for Drug Testing.

Three-dimensional (3D) tumor spheroids offer unprecedented capability for drug screening because of their unique features such as spatial 3D structure, relevant physiological responses, more complex intercellular network, and stroma-cancer cell interactions. Microfluidic technology provides a facile strategy to make uniform tumor spheroids with potential of high-throughput production. In this article, we develop a microfluidic approach to produce core-shell alginate particles, which allows the separate confinement of different cells in the core and shell structure. To reconstitute the complex tumor structure, we encapsulated tumor cells in the core and stromal fibroblast cells in the shell. These coculture tumor spheroids were applied for drug evaluation showing similar drug resistance as those prepared using conventional methods in well plates. These results demonstrated that our microfluidic approach are facile and versatile for making various tumor spheroids with uniform size but different components to better mimic tumor microenvironment. Moreover, the production rate of around 200 spheroids/min indicates the great potential of this approach for high-throughput drug screening.

Neonatally imprinted stromal cell subsets induce tolerogenic dendritic cells in mesenteric lymph nodes

Gut-draining mesenteric lymph nodes (mLNs) are important for inducing peripheral tolerance towards food and commensal antigens by providing an optimal microenvironment for de novo generation of Foxp3+ regulatory T cells (Tregs). We previously identified microbiota-imprinted mLN stromal cells as a critical component in tolerance induction. Here we show that this imprinting process already takes place in the neonatal phase, and renders the mLN stromal cell compartment resistant to inflammatory perturbations later in life. LN transplantation and single-cell RNA-seq uncover stably imprinted expression signatures in mLN fibroblastic stromal cells. Subsetting common stromal cells across gut-draining mLNs and skin-draining LNs further refine their location-specific immunomodulatory functions, such as subset-specific expression of Aldh1a2/3. Finally, we demonstrate that mLN stromal cells shape resident dendritic cells to attain high Treg-inducing capacity in a Bmp2-dependent manner. Thus, crosstalk between mLN stromal and resident dendritic cells provides a robust regulatory mechanism for the maintenance of intestinal tolerance.

Abstract 4162: Lentivirally delivered shRNA knockdown of CXCL12 is effective at preventing radiation fibrosis in normal tissues

Abstract Late Adverse Effects (LAEs) following adjuvant radiotherapy (RT) are common sequelae in free flaps used for breast reconstruction and result in ongoing healthcare burdens during the phase of cancer survivorship. Intra-arterial delivery of gene therapy to an isolated vascular region permits localized, targeted genetic modulation of reconstructed tissue, avoiding potential off-target effects in microscopic residual disease. We studied the immunological component of LAE development in a validated accelerated rodent model recapitulating autologous flap reconstruction, across 90 days after RT (50 Gy/3). CXCL12 was significantly up-regulated from 2-21 days after RT, peaking at 7 days (p<0.001), and corresponded to an increase in CXCR4 expression (p=0.047). Multiplexed confocal imaging co-localised CXCL12 and γH2AX in fibroblast stromal cells after RT, confirming that CXCL12 was a flap-originating signal suitable for our therapy modality. This was associated with increased macrophage ingress and fibroblast activation as characterized by immunohistochemical (p=0.002) and flow cytometric end-points (p=0.035). In vitro work corroborated the relevance of the CXCL12 axis in the development of LAEs, demonstrating significant increase in CXCL12's transcriptome expression (p=0.013) and protein secretion (p=0.03) in primary rat fibroblasts stimulated with RT, high expression of CXCL12's receptors, CXCR4 and CXCR7 on M1 and M2 rat macrophages, and increased fibroblast activation, assayed with αSMA expression, with co-stimulation of CXCL12 and TGFβ. We hypothesized that LAEs in animals with flap-targeted knockdown of CXCL12 would be reduced. Flaps were infected with vascularly-delivered, lentiviral particles encoding shRNA against CXCL12 (LVshCXCL12), scrambled controls (LVSCR) alongside sham (phosphate-buffered saline (PBS)) and un-irradiated controls. Flaps infected with LVshCXCL12 exhibited reversal of LAE-mediated flap contracture and were not significantly different from un-irradiated flaps at 90 days post-RT, but were significantly greater in area compared to sham flaps or those infected with LVSCR (p<0.0001). Radiation Therapy Oncology Group (RTOG) LAE scores were also significantly improved compared with LvSCR (p<0.0001) and PBS groups (p=0.0011). Endpoint assays demonstrated significant vascular preservation (p=0.049) and reduced tissue collagen deposition (p=0.019). In conclusion, targeted knockdown of CXCL12 is effective at preventing the development of LAEs in flap tissue exposed to RT and is a promising strategy for clinical translation. Citation Format: James T. Paget, Martin McLaughlin, Joan N. Kyula, David Mansfield, Henry G. Smith, Victoria Roulstone, Paul A. Harris, Alan A. Melcher, Kevin J. Harrington, Aadil A. Khan. Lentivirally delivered shRNA knockdown of CXCL12 is effective at preventing radiation fibrosis in normal tissues [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 4162.

PO-121 Lentivirally-delivered ShRNA knockdown of CXCL12 prevents fibrosis in a rodent model of radiation late adverse effects

IntroductionLate Adverse Effects (LAEs) following adjuvant radiotherapy (RT) are common sequelae in free flaps used for breast reconstruction. Intra-arterial delivery of gene therapy to an isolated vascular region permits localised, targeted genetic modulation of reconstructed tissue, avoiding potential off-target effects in microscopic residual disease. We hypothesised that targeted knockdown of CXCL12 would reduce LAEs.Material and methodsLAE development was studied across 8 time points to 90 days after 50 Gy/3 in 24 male Fischer 344 rats in a validated LAE flap model. RT and control human skin and subcuticular tissue was obtained for validation from the PRADA study.Efficacy of ShCXCL12 against LAEs was assessed in 72 animals. 1 × 108 lentiviral particles encoding ShRNA against CXCL12 (ShCXCL12), RNA overexpressing CXCL12 (OeCXCL12), or scrambled control (SCR), or sham infection with PBS were delivered intra-arterially to the flap tissue 30 days before RT. Acute and Late toxicities were assessed using Radiation Therapy Oncology Group (RTOG) scores with tissue endpoints at 7, 21 and 90 days after RT.Results and discussionsCXCL12 and CXCR4 were up-regulated after RT, peaking at 7 days (p=0.001 and p=0.047 respectively). Confocal imaging co-localised CXCL12 and γH2AX in fibroblast stromal cells after RT, confirming that CXCL12 was flap-originating and suitable for our therapy. This was associated with increased macrophage ingress and fibroblast activation characterised by immunohistochemical and flow cytometric end-points (p=0.035). We corroborated these findings in human RT tissue, demonstrating increased CXCL12 (p=0.035) and CD68 (p=0.027) expression compared to paired controls.ShCXCL12 animals had reduced acute toxicities compared to LVSCR or PBS groups. OeCXCL12 animals had significantly worse toxicity. ShCXCL12 animals exhibited reversal of flap contracture and retained significantly greater flap area overall. RTOG late toxicity scores were significantly improved (p<0.0001).At day 7 and day 21 ShCXCL12 tissue contained fewer macrophages. This was not seen by day 90. Fibroblasts did not differ at day 7, but were reduced by day 21 and at day 90. Endpoint assays confirmed amelioration of the LAE phenotype with reduced tissue collagen deposition and reduced Acta2 expression.ConclusionCXCL12 over-expression post-RT signals an innate immune response mediated primarily by macrophages. Targeted ShCXCL12 can prevent the development of LAEs. Further work aims to understand the mechanism of immune-fibroblast cross-talk.

Abstract 475: Characterization of miR-21 expression in budding colon cancer cells using digital images of multiplex fluorescence stained slides obtained by confocal scanning microscopy

Abstract This study was undertaken to analyze the presence and prevalence of microRNA-21 (miR-21) positive budding cancer cells in colon cancer using confocal scanning microscopy. Budding cancer cells are locally invasive cancer cells with increased metastatic properties and characteristics of epithelial to mesenchymal transition (EMT). Expression of miR-21 in stromal fibroblastic cells in colorectal cancers is well documented (1,2), whereas miR-21 expression in budding cells is poorly described. In order to characterize miR-21 positive budding cells, we first developed a multiplex fluorescence staining method by combining miR-21 in situ hybridization with immunohistochemical staining for cytokeratin and budding cell marker laminin-γ2, and stained 16 colon cancer cases (stage II, n=5, stage III, n=11). We then employed a confocal scanning microscope to obtain 15-55 GB digital images covering areas of 10-40 mm2 of the invasive front. The high resolution of the confocal digital images allowed detailed examination of the 4-fluorophore-stained slides using extended focus and z-stack images, e.g. in the discrimination of epithelial cells from adjacent stromal cells. All cytokeratin-positive budding cells were evaluated for the presence of miR-21 and laminin-γ2. Five out of the 16 cases had more than 10% miR-21 positive budding cells, a few of which were also laminin-γ2 positive. All five cases were stage III cancers. The presence of miR-21 in the budding cancer cells was not associated with the level of tumor budding. These observations suggest that the miR-21 expression in tumor budding cells increases with the progression of the cancer and is independent of laminin-γ2.

PO-175 The secretary miR-141 facilitates ovarian cancer metastasis through reprogramming stromal fibroblast cells in pre-metastatic niches formation

IntroductionRegardless of the modern advances in cancer therapeutics, cancer metastasis is still a major obstacle in the clinical management of ovarian cancer. Emerging evidence discloses exosomal miRNAs act as critical roles in cancer development. One of the possibility is the secretary miRNAs mediating communications between tumour cells and their tumour microenvironment. However, the functions and mechanisms of miRNAs in regulating cancer metastasis are not fully understood. We have previously identified that Hsa-miR-141 (miR-141) is not only aberrantly expressed in aggressive ovarian cancer but also enhances anoikis resistance in metastatic progression of ovarian cancer through targeting KLF12/SP1/Survivin axis. Here, we report that miR-141 is also a tumour secretary miRNA which can remodel the stromal cells to facilitate the formation of pre-metastatic niches for ovarian cancer metastasis.Material and methodsQPCR analysis was used to evaluate the level of exosomal miR-141 in the conditioned medium of ovarian cancer cells. T-HESCs and WPMY-1 were used for miR-141 mediated reprogramming stromal cells. Cytokine array, ELISA, QPCR and Western blot analyses were used for measuring the levels of EMMPRIN and GRO-α. LC-MS/MS and proteomic analyses to identify miR-141-mediated target, Yes Associated Protein 1(YAP1).Results and discussionsmiR-141 was frequently overexpressed in ovarian cancer cells and also secreted to the surroundings through exosomal pathway. Functional analyses revealed miR-141 enables to reprogram the stromal cells because miR-141-expressing stromal cells showed an escalating secretion of cytokines GRO-α and EMMPRIN in cultured media. By co-culture with ovarian cancer cells with above conditioned media, or the recombinant proteins of GRO-α and EMMPRIN, ovarian cancer cells exhibited increased cell proliferation. Proteomic profiling of miR-141 reprogrammed stromal cells revealed YAP1, a key downstream effector of the Hippo pathway was suppressed. Depletion of YAP1 in stromal cells also increased the expression of GRO-α and EMMPRIN in the conditioned media. On the contrary, restoration of YAP1 attenuated the escalated secretion of GRO-α and EMMPRIN, indicating the reduction of YAP1 might be required for increased YAP/TAZ –mediated transcriptional activities of GRO-α and EMMPRIN.ConclusionOur preliminary findings suggest the exosomal miR-141 could reprogram stromal cells through altering Hippo/YAP1 signalling in production of GRO-α and EMMPRIN, to facilitate metastatic colonisation of ovarian cancer.