Reciprocal Signaling Research Articles

Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the Caenorhabditis elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell-specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor-2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the Caenorhabditis elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell-specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor-2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

Abstract A002: A reciprocal signaling crosstalk between the AR and BCl2-AKT pathways induces castration resistance in castration sensitive prostate cancer

Abstract Introduction: A leading contributor to the significant mortality burden of prostate cancer (PC) is the resistance to androgen deprivation therapy (ADT). Although most PCs are initially sensitive to ADT, the duration of response is variable, and relapse invariably occurs in the transition to metastatic castration-resistant prostate cancer (mCRPC), the lethal form of the disease. Therefore, it is essential to discover and validate early resistance mechanisms that initially protect castration sensitive prostate cancer (CSPC) cells from ADT and to develop novel combination therapies that can block or delay the ADT-induced shift from CSPC to mCRPC. Overexpression of the hallmark antiapoptotic gene B-cell lymphoma 2 (BCL2) is a potential mechanism of resistance to ADT. Methods: To discover the molecular drivers of castration resistance, we analyzed the tumor transcriptomic profile of a cohort of localized PC patients (N=58) treated with neoadjuvant-intensive ADT (NCT00924469; Taplin et al, 2014, Sowalsky et al, 2018). Experimentally, we treated CSPC cells and genetically engineered mice (GEM) prostate tumors with ADT and analyzed the signaling pathways, which were enriched post-ADT treatment compared untreated samples. Results: We discovered ~10-fold increased mRNA expression of BCL2 (p<0.001) in ADT-treated localized PC patients compared to untreated (standard of care surgery) patients in NCT00924469 samples. Our experimental study showed that treatment with AR inhibitors strongly augments BCL2 expression in human CSPC cell lines and GEM PC model, indicating possible direct negative regulation of BCL2 by the AR-signaling pathway. Mechanistically, our preliminary data reveal a striking induction of cell metabolism pathways (fatty acid and xenobiotic metabolism), including activation of PI3K/AKT signaling in BCL2-overexpressing CSPC cells. We used siRNA-mediated BCL2 knockdown in LNCaP cells and showed that loss of BCL2 strongly suppressed ADT-induced AKT- phosphorylation. We also showed increased Bcl2 mRNA expression in PTEN-knockout mice-derived prostate organoids compared to the wild-type control. Furthermore, we observed that AKT kinase -inhibitor MK2206 strongly inhibits enzalutamide-induced BCL2 expression in LNCaP cells. Conclusion: Together our data indicate a non-canonical role of BCL2 in activating PI3K/AKT signaling in prostate cancer. For the first time, we demonstrate the crucial importance of AR-BCL2-AKT signaling pathway crosstalk in CSPC biology and reveal this complex signaling crosstalk may act as a key driver of CSPC transformation to lethal CRPC. Citation Format: Goutam Chakraborty, Rahim Hirani, Subhiksha Nandakumar, Nabila Zaman, Teja Muralidhar Kalidindi, Sai Harisha Rajanala, Gwo-Shu M. Lee, Konrad H. Stopsack, Naga Vara Kishore Pillarsetty, Lorelei A. Mucci, Daniel C. Danila, Philip W. Kantoff. A reciprocal signaling crosstalk between the AR and BCl2-AKT pathways induces castration resistance in castration sensitive prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A002.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

Macrophages and stem/progenitor cells interplay in adipose tissue and skeletal muscle: a review.

Like all immune cells, macrophages do not act autonomously but in unison with other immune cells, surrounding tissues, and the niche they occupy. Constant exchange of information between cellular and noncellular participants within a tissue allows for preserving homeostasis and defining responses in a pathologic environment. Although molecular mechanisms and pathways involved in reciprocal signaling between macrophages and other immune cells have been known for decades, much less is known about interactions between macrophages and stem/progenitor cells. Based on the time when stem cells form, there are two stem cell types: embryonic stem cells existing only in an early embryo, which are pluripotent and can differentiate into any cell type present in an adult, and somatic (adult) stem cells formed in fetus and persisting for whole adult life. Tissues and organs have their own (tissue-specific and organ-specific) adult stem cells, which serve as a reserve for tissue homeostasis and regeneration after injury. It is still uncertain whether organ- and tissue-specific stem cells are actual stem cells or just progenitor cells. The important question is how stem/progenitor cells can sculpt macrophage phenotype and functions. Even less is known if or how macrophages can shape stem/progenitor cell functions, their divisions, and fate. We describe here examples from recent studies of how stem/progenitor cells can affect macrophages and how macrophages can influence stem/progenitor cell properties, functions, and destiny.

Legumain Functions as a Transient TrkB Sheddase.

While primarily found in endo-lysosomal compartments, the cysteine protease legumain can also translocate to the cell surface if stabilized by the interaction with the RGD-dependent integrin receptor αVβ3. Previously, it has been shown that legumain expression is inversely related to BDNF-TrkB activity. Here we show that legumain can conversely act on TrkB-BDNF by processing the C-terminal linker region of the TrkB ectodomain in vitro. Importantly, when in complex with BDNF, TrkB was not cleaved by legumain. Legumain-processed TrkB was still able to bind BDNF, suggesting a potential scavenger function of soluble TrkB towards BDNF. The work thus presents another mechanistic link explaining the reciprocal TrkB signaling and δ-secretase activity of legumain, with relevance for neurodegeneration.

Initial employability development: introducing a conceptual model integrating signalling and social exchange mechanisms

ABSTRACT This article develops a conceptual understanding of initial employability development, specifically the transition into first career destinations. Substantial previous research focuses on the role of individuals in ensuring employment readiness and continued employability development. Studies also identify the importance of employers for sustained employability. However, the interdependence and interaction between employers and individuals are under-theorized. We integrate social exchange theory and signalling theory to conceptualize initial employability development. We propose a cyclical model that comprises interactive and reciprocal signalling and social exchange processes between employers and individuals. The model overcomes the limitations of linear conceptualizations and one-sided models that theorize employability as either the outcome of human capital resources deployed by individuals or as managed by employers in employability development processes. We extend the processual approach to employability by redirecting theoretical attention to the interaction of signalling and social exchange as fundamental to employability development. We conclude by discussing implications in relation to employability research and theory development.

‘Openness in Action’ Early Steps in Cosmic Phenomenology

‘Openness in Action’ Early Steps in Cosmic Phenomenology

Reciprocal FGF19-GLI2 signaling induces epithelial-to-mesenchymal transition to promote lung squamous cell carcinoma metastasis.

Metastatic lung squamous cell carcinoma (LUSC) is one of the most common causes of cancer death worldwide. As yet, however, the molecular mechanism underlying LUSC metastasis remains elusive. In this study, we report a novel mechanism involving signaling interactions between FGF19 and GLI2 that could drive the progression of LUSC. The expression of FGF19 in human LUSC samples was assessed by immunohistochemistry. The concentration of FGF19 in serum samples was assessed by ELISA. RNA sequencing, scratch wound-healing, trans-well, GO analysis, GSEA, luciferase reporter, Western blotting, immunofluorescence and immunohistochemistry assays, as well as an animal model were used to investigate the molecular mechanism underlying FGF19 driven LUSC progression. The therapeutic effect of a GLI2 inhibitor was determined using both in vitro cellular and in vivo animal experiments. We found that FGF19, a member of the fibroblast growth factor family, plays a crucial role in the invasion and metastasis of LUSC, and identified GLI2 as an important downstream effector of FGF19 involved in metastasis. Surprisingly, we found that FGF19 and GLI2 could reciprocally induce the expression of each other, and form a positive feedback loop to promote LUSC cell invasion and metastasis. These findings were corroborated by an association between a poor prognosis of LUSC patients and FGF19/GLI2 co-expression. In addition, we found that the GLI inhibitor GANT61 could effectively reduce FGF19-mediated LUSC invasion and metastasis. Our data suggest that FGF19 may serve as a novel biomarker for predicting metastatic LUSC. Intervening with the FGF19-GLI2 feedback loop may be a strategy for the treatment of FGF19-driven LUSC metastasis.

Higher immunoexpression of CK14 from the Wnt-1/β-catenin pathway in the development of odontomas.

Tooth development depends on a series of reciprocal signaling interactions between the oral epithelium and ectomesenchyme. This study aimed to investigate the role of CK14, a protein involved in Wnt-1/β-catenin signaling, in odontogenesis and the development of odontomas. This cross-sectional, retrospective, immunohistochemical study analyzed 30 compound odontomas, 30 complex odontomas, and 17 tooth germs. Higher immunoexpression of CK14 was observed in odontogenic epithelial cells of tooth germs (p < 0.001) and odontogenic epithelial cells of odontomas (p < 0.001). There was higher immunoexpression of Wnt-1 and β-catenin proteins in epithelial cells of tooth germs (p = 0.002 and p < 0.001, respectively), as well as in the ectomesenchyme of odontomas (p = 0.003 and p < 0.001, respectively). β-Catenin was moderately and significantly correlated with CK14 in the membrane of reduced enamel epithelial cells in odontomas (p = 0.007). Higher immunoexpression of CK14 was observed in the odontogenic epithelium during the bud and cap stages and lower immunoexpression in the internal enamel epithelium during the bell stage. In odontomas, lower expression of Wnt-1/β-catenin and higher immunoexpression of CK14 were found in odontogenic epithelial cells, especially adjacent to the mineralized material resembling the tooth formed in these lesions.

Multiple congenital malformations arise from somatic mosaicism for constitutively active Pik3ca signaling.

Recurrent missense mutations of the PIK3CA oncogene are among the most frequent drivers of human cancers. These often lead to constitutive activation of its product p110α, a phosphatidylinositol 3-kinase (PI3K) catalytic subunit. In addition to causing a broad range of cancers, the H1047R mutation is also found in affected tissues of a distinct set of congenital tumors and malformations. Collectively termed PIK3CA-related disorders (PRDs), these lead to overgrowth of brain, adipose, connective and musculoskeletal tissues and/or blood and lymphatic vessel components. Vascular malformations are frequently observed in PRD, due to cell-autonomous activation of PI3K signaling within endothelial cells. These, like most muscle, connective tissue and bone, are derived from the embryonic mesoderm. However, important organ systems affected in PRDs are neuroectodermal derivatives. To further examine their development, we drove the most common post-zygotic activating mutation of Pik3ca in neural crest and related embryonic lineages. Outcomes included macrocephaly, cleft secondary palate and more subtle skull anomalies. Surprisingly, Pik3ca-mutant subpopulations of neural crest origin were also associated with widespread cephalic vascular anomalies. Mesectodermal neural crest is a major source of non-endothelial connective tissue in the head, but not the body. To examine the response of vascular connective tissues of the body to constitutive Pik3ca activity during development, we expressed the mutation by way of an Egr2 (Krox20) Cre driver. Lineage tracing led us to observe new lineages that had normally once expressed Krox20 and that may be co-opted in pathogenesis, including vascular pericytes and perimysial fibroblasts. Finally, Schwann cell precursors having transcribed either Krox20 or Sox10 and induced to express constitutively active PI3K were associated with vascular and other tumors. These murine phenotypes may aid discovery of new candidate human PRDs affecting craniofacial and vascular smooth muscle development as well as the reciprocal paracrine signaling mechanisms leading to tissue overgrowth.

Indole pyruvate decarboxylase gene regulates the auxin synthesis pathway in rice by interacting with the indole-3-acetic acid-amido synthetase gene, promoting root hair development under cadmium stress.

This research focused on cadmium (Cd), which negatively affects plant growth and auxin hemostasis. In plants, many processes are indirectly controlled through the expression of certain genes due to the secretion of bacterial auxin, as indole-3-acetic acid (IAA) acts as a reciprocal signaling molecule in plant-microbe interaction. The aim of current studies was to investigate responsible genes in rice for plant-microbe interaction and lateral root development due to the involvement of several metabolic pathways. Studies revealed that GH3-2 interacts with endogenous IAA in a homeostasis manner without directly providing IAA. In rice, indole-3-pyruvate decarboxylase (IPDC) transgenic lines showed a 40% increase in lateral roots. Auxin levels and YUCCA (auxin biosynthesis gene) expression were monitored in osaux1 mutant lines inoculated with Bacillus cereus exposed to Cd. The results showed an increase in root hairs (RHs) and lateral root density, changes in auxin levels, and expression of the YUCCA gene. B. cereus normalizes the oxidative stress caused by Cd due to the accumulation of and H2O2 in osaux1 mutant lines. Furthermore, the inoculation of B. cereus increases DR5:GUS expression, indicating that bacterial species have a positive role in auxin regulation. Thus, the current study suggests that B. cereus and IPDC transgenic lines increase the RH development in rice by interacting with IAA synthetase genes in the host plant, alleviating Cd toxicity and enhancing plant defense mechanisms.

Alternative mammalian strategies leading towards gastrulation: losing polar trophoblast (Rauber's layer) or gaining an epiblast cavity.

Using embryological data from 14 mammalian orders, the hypothesis is presented that in placental mammals, epiblast cavitation and polar trophoblast loss are alternative developmental solutions to shield the central epiblast from extraembryonic signalling. It is argued that such reciprocal signalling between the edge of the epiblast and the adjoining polar trophoblast or edge of the mural trophoblast or with the amniotic ectoderm is necessary for the induction of gastrulation. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Reciprocal signaling between adipose tissue depots and the central nervous system.

In humans, various dietary and social factors led to the development of increased brain sizes alongside large adipose tissue stores. Complex reciprocal signaling mechanisms allow for a fine-tuned interaction between the two organs to regulate energy homeostasis of the organism. As an endocrine organ, adipose tissue secretes various hormones, cytokines, and metabolites that signal energy availability to the central nervous system (CNS). Vice versa, the CNS is a critical regulator of adipose tissue function through neural networks that integrate information from the periphery and regulate sympathetic nerve outflow. This review discusses the various reciprocal signaling mechanisms in the CNS and adipose tissue to maintain organismal energy homeostasis. We are focusing on the integration of afferent signals from the periphery in neuronal populations of the mediobasal hypothalamus as well as the efferent signals from the CNS to adipose tissue and its implications for adipose tissue function. Furthermore, we are discussing central mechanisms that fine-tune the immune system in adipose tissue depots and contribute to organ homeostasis. Elucidating this complex signaling network that integrates peripheral signals to generate physiological outputs to maintain the optimal energy balance of the organism is crucial for understanding the pathophysiology of obesity and metabolic diseases such as type 2 diabetes.

A reciprocal learning strategy for semisupervised medical image segmentation.

Semisupervised strategy has been utilized to alleviate issues from segmentation applications due to challenges in collecting abundant annotated segmentation masks, which is an essential prerequisite for training high-performance 3D convolutional neural networks (CNNs) . Existing semisupervised segmentation methods are mainly concerned with how to generate the pseudo labels with regularization but not evaluate the quality of the pseudo labels explicitly. To alleviate this problem, we offer a simple yet effective reciprocal learning strategy for semisupervised volumetric medical image segmentation, which generates more reliable pseudo labels for the unannotateddata. Our proposed reciprocal learning is achieved through a pair of networks, one as a teacher network and the other as a student network. The student network learns from pseudo labels generated by the teacher network. In addition, the teacher network autonomously optimizes its parameters based on the reciprocal feedback signals from the student's performance on the annotated images. The efficacy of the proposed method is evaluated on three medical image data sets, including 82 pancreas computed tomography (CT) scans (training/testing: 62/20), 100 left atrium gadolinium-enhanced magnetic resonance (MR) scans (training/testing: 80/20), and 200 breast cancer MR scans (training/testing: 68/132). The comparison methods include mean teacher (MT) model, uncertainty-aware MT (UA-MT) model, shape-aware adversarial network (SASSNet), and transformation-consistent self-ensembling model (TCSM). The evaluation metrics are Dice similarity coefficient (Dice), Jaccard index (Jaccard), 95% Hausdorff distance (95HD), and average surface distance (ASD). The Wilcoxon signed-rank test is used to conduct the statisticalanalyses. By utilizing 20% labeled data and 80% unlabeled data for training, our proposed method achieves an average Dice of 84.77%/90.46%/78.53%, Jaccard of 73.71%/82.67%/69.00%, ASD of 1.58/1.90/0.57, and 95HD of 6.24/5.97/4.34 on pancreas/left atrium/breast data sets, respectively. These results outperform several cutting-edge semisupervised approaches, showing the feasibility of our method for the challenging semisupervised segmentationapplications. The proposed reciprocal learning strategy is a general semisupervised solution and has the potential to be applied for other 3D segmentationtasks.

Directed differentiation of human pluripotent stem cells into diverse organ-specific mesenchyme of the digestive and respiratory systems.

Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the endoderm and adjacent mesoderm cells in the fetal foregut. Although the recent successes in recapitulating developmental signaling in vitro has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe a protocol to differentiate hPSCs into different types of organ-specific mesoderm, leveraging signaling networks and molecular markers elucidated from single-cell transcriptomics of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm treated with either retinoic acid (RA) or RA together with a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm, respectively, after 4-d cultures. These are directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4 to 7 in cultures. By day 7, the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60-90% pure liver septum transversum/mesothelium-like, 70-80% pure liver-like fibroblasts and populations of ~35% respiratory-like mesoderm, gastric-like mesoderm or esophageal-like mesoderm. This protocol can be performed by anyone with moderate experience differentiating hPSCs, provides a novel platform to study human mesoderm development and can be used to engineer more complex foregut tissue for disease modeling and regenerative medicine.

Breast Cancer-Stromal Interactions: Adipose-Derived Stromal/Stem Cell Age and Cancer Subtype Mediated Remodeling.

Adipose tissue is characterized as an endocrine organ that acts as a source of hormones and paracrine factors. In diseases such as cancer, endocrine and paracrine signals from adipose tissue contribute to cancer progression. Young individuals with estrogen receptor-alpha positive (ER-α+) breast cancer (BC) have an increased resistance to endocrine therapies, suggesting that alternative estrogen signaling is activated within these cells. Despite this, the effects of stromal age on the endocrine response in BC are not well defined. To identify differences between young and aged ER-α+ breast tumors, RNA sequencing data were obtained from The Cancer Genome Atlas. Analysis revealed enrichment of matrix and paracrine factors in young (≤40 years old) patients compared to aged (≥65 years old) tumor samples. Adipose-derived stromal/stem cells (ASCs) from noncancerous lipoaspirate of young and aged donors were evaluated for alterations in matrix production and paracrine secreted factors to determine if the tumor stroma could alter estrogen signaling. Young and aged ASCs demonstrated comparable proliferation, differentiation, and matrix production, but exhibited differences in the expression levels of inflammatory cytokines (Interferon gamma, interleukin [IL]-8, IL-10, Tumor necrosis factor alpha, IL-2, and IL-6). Conditioned media (CM)-based experiments showed that young ASC donor age elevated endocrine response in ER-α+ BC cell lines. MCF-7 ER-α+ BC cell line treated with secreted factors from young ASCs had enhanced ER-α regulated genes (PGR and SDF-1) compared to MCF-7 cells treated with aged ASC CM. Western blot analysis demonstrated increased activation levels of p-ER ser-167 in the MCF-7 cell line treated with young ASC secreted factors. To determine if ER-α+ BC cells heightened the cytokine release in ASCs, ASCs were stimulated with MCF-7-derived CM. Results demonstrated no change in growth factors or cytokines when treated with the ER-α+ secretome. In contrast to ER-α+ CM, the ER-α negative MDA-MB-231 derived CM demonstrated increased stimulation of pro-inflammatory cytokines in ASCs. While there was no observed change in the release of selected paracrine factors, MCF-7 cells did induce matrix production and a pro-adipogenic lineage commitment. The adipogenesis was evident by increased collagen content through Sirius Red/Fast Green Collagen stain, lipid accumulation evident by Oil Red O stain, and significantly increased expression in PPARγ mRNA expression. The data from this study provide evidence suggesting more of a subtype-dependent than an age-dependent difference in stromal response to BC, suggesting that this signaling is not heightened by reciprocal signals from ER-α+ BC cell lines. These results are important in understanding the mechanisms of estrogen signaling and the dynamic and reciprocal nature of cancer cell-stromal cell crosstalk that can lead to tumor heterogeneity and variance in response to therapy.

R-SPONDIN2+ mesenchymal cells form the bud tip progenitor niche during human lung development.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

Task-Dependent Effective Connectivity of the Reward Network During Food Cue-Reactivity: A Dynamic Causal Modeling Investigation.

Neural reactivity to food cues may play a central role in overeating and excess weight gain. Functional magnetic resonance imaging (fMRI) studies have implicated regions of the reward network in dysfunctional food cue-reactivity, but neural interactions underlying observed patterns of signal change remain poorly understood. Fifty overweight and obese participants with self-reported cue-induced food craving viewed food and neutral cues during fMRI scanning. Regions of the reward network with significantly greater food versus neutral cue-reactivity were used to specify plausible models of task-related neural interactions underlying the observed blood oxygenation level-dependent (BOLD) signal, and a bi-hemispheric winning model was identified in a dynamic causal modeling (DCM) framework. Neuro-behavioral correlations are investigated with group factor analysis (GFA) and Pearson’s correlation tests. The ventral tegmental area (VTA), amygdalae, and orbitofrontal cortices (OFC) showed significant food cue-reactivity. DCM suggests these activations are produced by largely reciprocal dynamic signaling between these regions, with food cues causing regional disinhibition and an apparent shifting of activity to the right amygdala. Intrinsic self-inhibition in the VTA and right amygdala is negatively correlated with measures of food craving and hunger and right-amygdalar disinhibition by food cues is associated with the intensity of cue-induced food craving, but no robust cross-unit latent factors were identified between the neural group and behavioral or demographic variable groups. Our results suggest a rich array of dynamic signals drive reward network cue-reactivity, with the amygdalae mediating much of the dynamic signaling between the VTA and OFCs. Neuro-behavioral correlations suggest particularly crucial roles for the VTA, right amygdala, and the right OFC-amygdala connection but the more robust GFA identified no cross-unit factors, so these correlations should be interpreted with caution. This investigation provides novel insights into dynamic circuit mechanisms with etiologic relevance to obesity, suggesting pathways in biomarker development and intervention.

Abstract 6383: Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis

Abstract Cancer stem-like cells (CSCs) and the differentiated bulk cells mainly contribute to cellular heterogeneity within a tumor. The dichotomous division pattern of the CSCs sitting atop the tumor hierarchy allow them to self-renew, and differentiate to initiate and repopulate aggressive tumors. These CSC-differentiation-derived bulk tumor cells reprogram to stem-like states, accounting for high plasticity within a tumor; a phenomenon governed by the distinct epigenetic profiles of the two cell populations. Reciprocal signaling between the tumor and its complex microenvironment is a vital driver of plasticity and heterogeneity in cancer. The indispensable role of CSCs in cellular cross-talk in the tumor niche as critical drivers of tumorigenesis is well-studied. However, the massive outnumbering of the CSCs by the differentiated cells raises several vital questions regarding the role of the latter in tumor growth; whether they contribute to the complex signaling within the tumor microenvironment (TME) to drive tumor growth remains unexplored. In this study, we aimed to mechanistically delineate the distinct roles of the cancer stem-like and differentiated cells by investigating their interactions with other stromal cells in the TME.In our model system, Glioblastoma (GBM), a highly aggressive brain cancer, we adopted a quantitative, label-free, high-throughput proteomics-based approach to identify the differentially abundant secreted proteins between the glioma stem-like cell (GSC) and differentiated glioma cell (DGC). This analysis showed a significantly higher abundance of the Fibromodulin (FMOD), a collagen-binding proteoglycan, in the conditioned medium of the DGCs compared with that of the GSCs. Our investigation revealed that an activated TGF-β signaling leads to p-SMAD2 occupancy on the FMOD promoter region, leading to increased FMOD secretion by the DGCs. DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth in a subcutaneous mouse model. We also observed that FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. Moreover, DGC-secreted FMOD activates integrin-dependent Notch signaling in endothelial cells, promoting angiogenesis. FMOD-silencing also inhibits the capability of the transdifferentiated endothelial cells (derived from the DGCs) to form angiogenic networks. Conditional silencing of FMOD in the de novo formed DGCs in an orthotopic intracranial C57BL/6 mouse model inhibited the growth of GSC-initiated tumors due to poorly developed vasculature, thereby increasing mouse survival. The same observation was recapitulated using patient-derived GSCs in an immunocompromised intracranial mouse model, thereby highlighting the human relevance of our findings. Collectively, our findings demonstrate how paracrine signaling between DGC-secreted FMOD and the stromal cells promotes tumor growth by enhancing angiogenesis, thereby highlighting a previously less studied supportive role of DGCs in tumor growth, albeit the GSCs retaining the tumor-initiating capacity. Thus our study identifies DGC-specific FMOD as a potential therapeutic target and emphasizes that targeting both cancer stem cells and differentiated cancer bulk cells is vital to achieving a durable response in GBM. Citation Format: Shreoshi Sengupta, Kumaravel Somasundaram, Dinorah Friedmann Morvinski, Philippe Marin, Prasasvi Reddy, Arani Mukherjee, Prerna Magod, Mainak Mondal, Serge Urbach. Paracrine signaling between differentiated tumor cell-secreted fibromodulin and the stroma promotes tumor growth by enhancing angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6383.