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Shakhpazyan, Nikolay, Liudmila Mikhaleva, Arkady Bedzhanyan, et al. "Cellular and Molecular Mechanisms of the Tumor Stroma in Colorectal Cancer: Insights into Disease Progression and Therapeutic Targets." Biomedicines 11, no. 9 (2023): 2361. http://dx.doi.org/10.3390/biomedicines11092361.
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Colorectal cancer (CRC) is a major health burden worldwide and is the third most common type of cancer. The early detection and diagnosis of CRC is critical to improve patient outcomes. This review explores the intricate interplay between the tumor microenvironment, stromal interactions, and the progression and metastasis of colorectal cancer. The review begins by assessing the gut microbiome’s influence on CRC development, emphasizing its association with gut-associated lymphoid tissue (GALT). The role of the Wnt signaling pathway in CRC tumor stroma is scrutinized, elucidating its impact on disease progression. Tumor budding, its effect on tumor stroma, and the implications for patient prognosis are investigated. The review also identifies conserved oncogenic signatures (COS) within CRC stroma and explores their potential as therapeutic targets. Lastly, the seed and soil hypothesis is employed to contextualize metastasis, accentuating the significance of both tumor cells and the surrounding stroma in metastatic propensity. This review highlights the intricate interdependence between CRC cells and their microenvironment, providing valuable insights into prospective therapeutic approaches targeting tumor–stroma interactions.
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Matsumoto, Mitsuru, Kikue Iwamasa, Paul D. Rennert та ін. "Involvement of Distinct Cellular Compartments in the Abnormal Lymphoid Organogenesis in Lymphotoxin-α-Deficient Mice and Alymphoplasia (aly) Mice Defined by the Chimeric Analysis". Journal of Immunology 163, № 3 (1999): 1584–91. http://dx.doi.org/10.4049/jimmunol.163.3.1584.
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Abstract Both lymphotoxin-α (LTα)-deficient mice and alymphoplasia (aly) mice, a natural mutant strain, manifest a quite similar phenotype: lack of lymph nodes (LN) and Peyer’s patches (PP), with disturbed spleen architecture. The mechanisms underlying the defective lymphoid organogenesis in these mice were investigated by generating aggregation chimeras; ex vivo fused morulae were implanted into pseudo-pregnant host females and allowed to develop to term. Chimeric mice between LTα-deficient mice and wild-type mice restored LN and PP almost completely, suggesting that LTα expressed by circulating bone marrow-derived cells is essential for lymphoid organogenesis as well as for organization of spleen architecture. By contrast, chimeric mice between aly mice and wild-type mice showed only limited restoration of LN and PP. This suggests that the putative aly gene product does not act as a circulating ligand for lymphoid organogenesis, like LTα. Rather, abnormal development of lymphoid organs in aly mice seems most likely due to the defective development of the incipient stromal cells of the LN and PP. Supporting this hypothesis, up-regulation of VCAM-1 on aly mouse embryonic fibroblasts by signals through LTβR, which is exclusively expressed by nonlymphoid cells, was disturbed. These studies demonstrate that LTα and the putative aly gene product together control lymphoid organogenesis with a close mechanistic relationship in their biochemical pathways through governing the distinct cellular compartments, the former acting as a circulating ligand and the latter as a LTβR-signaling molecule expressed by the stroma of the lymphoid organs.
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Nitta, Takeshi. "Understanding the Thymic Microenvironment: the Cellular and Molecular Basis of T Cell Development." Central Asian Journal of Medical Sciences 2, no. 2 (2016): 111–26. http://dx.doi.org/10.24079/cajms.2016.02.002.
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Objectives: The thymus is a primary lymphoid organ that provides specialized microenvironment for T cell development. A variety of thymic stromal cells form the thymic tissue architecture and critically regulate the development and repertoire selection of T cells. Methods: We reviewed historical and recent studies on thymic stromal cells, especially focusing on the well-characterized functions of thymic epithelial cells (TECs) and the significance of as yet less characterized non-TEC thymic stromal cells and hematopoietic antigen-presenting cells in the regulation of T cell development. Results: Cortical TECs (cTECs) induce positive selection of diverse and functional T cells, while medullary TECs (mTECs) establish T cell tolerance via the negative selection of auto-reactive T cells and their conversion into regulatory T cells. These modes of T cell tolerance induction are also mediated by hematopoietic antigen-presenting cells such as dendritic cells and thymic B cells. Thymic mesenchymal cells, a prominent component of non-TEC thymic stromal cells, support the development and maintenance of TECs and thereby T cell production. Conclusion: Understanding the cellular and molecular basis for thymic stromal subsets will provide invaluable information toward in vivo reconstitution of the thymic microenvironment for future therapeutic applications.
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Shadlinskaya, S. V. "Cellular composition and microanatomy of lymphoid formations of the vestibule of the vagina in postnatal ontogenesis." Sechenov Medical Journal 10, no. 1 (2019): 57–62. http://dx.doi.org/10.47093/22187332.2019.1.57-62.
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Aim - presents data on the number of cells of the lymphoid series and the cellular composition of the lymphoid tissue in the mucosa of the vaginal vestibule at different age periods of ontogenesis. Materials and methods. The study material was preparations of the vaginal vestibule taken from 45 women of different ages (from newborns to 75 years). By age periods, the material was subdivided according to the standard scheme of age periodization. Cross sections of the organ wall were stained with hematoxylin - eosin and according to Van Gieson. In a number of cases, the silvering of Grimelius was performed. Obtained during the study of digital data were subjected to statistical processing. Results. Analysis of the results showed that lymphoid formations are located mainly near the glands. Glandular - lymphoid relationships are less impressed in newborns, to the maximum extent from early childhood to the 1st period of adulthood, in the elderly and senile age of cells. There are fewer lymphoid rows near the initial divisions and in the stroma of the glands. The lymphoid apparatus of the vaginal vestibule is represented by all the morphogenetic forms of the lymphoid tissue. The qualitative composition of lymphoid tissue is of the same type in all its morphogenetic forms, regardless of age. It has been established that there are close microsynthopic connections between immune structures and small glands of the vestibule. The intensity of these relationships has ontogenetic features. They are relatively weak in new - borns, are maximal in early childhood and weaken after the 1st period of adulthood. Conclusion. The study allowed us to identify previously unknown patterns of morphogenesis of lymphoid structures of the vaginal vestibule.
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Cakala-Jakimowicz, Marta, Paulina Kolodziej-Wojnar, and Monika Puzianowska-Kuznicka. "Aging-Related Cellular, Structural and Functional Changes in the Lymph Nodes: A Significant Component of Immunosenescence? An Overview." Cells 10, no. 11 (2021): 3148. http://dx.doi.org/10.3390/cells10113148.
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Aging affects all tissues and organs. Aging of the immune system results in the severe disruption of its functions, leading to an increased susceptibility to infections, an increase in autoimmune disorders and cancer incidence, and a decreased response to vaccines. Lymph nodes are precisely organized structures of the peripheral lymphoid organs and are the key sites coordinating innate and long-term adaptive immune responses to external antigens and vaccines. They are also involved in immune tolerance. The aging of lymph nodes results in decreased cell transport to and within the nodes, a disturbance in the structure and organization of nodal zones, incorrect location of individual immune cell types and impaired intercellular interactions, as well as changes in the production of adequate amounts of chemokines and cytokines necessary for immune cell proliferation, survival and function, impaired naïve T- and B-cell homeostasis, and a diminished long-term humoral response. Understanding the causes of these stromal and lymphoid microenvironment changes in the lymph nodes that cause the aging-related dysfunction of the immune system can help to improve long-term immune responses and the effectiveness of vaccines in the elderly.
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Jansen, Caroline S., BaoHan Vo, Ewelina Sobierajska, et al. "Abstract B030: Form and function in intratumoral immune organization: Understanding the cellular composition of TCF1+ CD8+ T cell niches in human cancer." Cancer Research 83, no. 16_Supplement (2023): B030. http://dx.doi.org/10.1158/1538-7445.kidney23-b030.
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Abstract Tumor infiltrating T cells are a positive prognosticator in many tumor types, and our prior work demonstrated that in renal cell carcinoma, patients with higher CD8 T cell infiltration have improved survival (Jansen Nature 2019). We described that this T cell response is supported by TCF1+ stem-like CD8 T cells, which reside within dense regions of closely clustered antigen presenting cells within the tumor. Interestingly, aggregations of immune cells have also been described in other settings and termed ‘tertiary lymphoid structures’ (TLS), raising an important question of whether the described immune niches are similar to these TLS or if they represent a distinct type of peripheral immune organization. In the work presented here, we use quantitative immunofluorescence image analysis to examine the cellular composition and organization of intratumoral immune niches, TLS, and secondary lymphoid tissue. The novel data presented here describes how immune niches are distinct from TLS and are more similar to the organization of T cell zones in secondary lymphoid tissue. Immune niches are T cell dominant structures, with T cell and antigen presenting cell compartments similar to T cell zones, whereas these niches profoundly lack the B cell presence seen in TLS and B cell zones of secondary lymphoid tissue. Immune niches are comparatively small in size (50-200um), while TLS are much larger (often >1mm), and, importantly, immune niches are found inside the tumor border, while TLS are located peripherally in tumor tissue. Notably, we find that immune niches have a marked presence of aSMA+ stromal cells, which are similar to those found in T cell zones but absent in B cell zones and TLS. When probed by flow cytometry and RNAseq, we find that aSMA+ stroma in immune niches are myofibroblast-like, similar those known to maintain the structure of secondary lymphoid tissues, and are distinct from those found in healthy kidney tissue. These data suggest these aSMA+ cells may play an important role in the organization of immune niches in tumor tissue. As we have shown that a robust T cell response is beneficial for patient outcomes, it is crucial to understand the mechanisms which support that response. Recently, our group defined that CD8 T cell activation in cancer is comprised of two phases, with an initial priming phase in the lymph node and a second phase of effector program acquisition in the tumor tissue (Prokhnevska Immunity 2023). We suggest that these immune niches represent an important hub for this development of T cell function, which is required for the robust, effective anti-tumor T cell response that supports improved patient outcomes and is the basis for the response to immunotherapy. Advancing our understanding of intratumoral immune organization will allow for enhanced biomarker discovery, improvement in existing therapies, and innovation in developing novel therapeutic strategies that will convert immunologically “cold” tumors to be immunologically “hot” and enhance anti-tumor immunity. Citation Format: Caroline S. Jansen, BaoHan Vo, Ewelina Sobierajska, Rachel Greenwald, Patrick Mullane, Nataliya Prokhnevska, Maria Cardenas, Mehmet Asim Bilen, Adeboye O. Osunkoya, Viraj A. Master, Haydn T. Kissick. Form and function in intratumoral immune organization: Understanding the cellular composition of TCF1+ CD8+ T cell niches in human cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B030.
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Voermans, Carlijn, and Mette D. Hazenberg. "Cellular therapies for graft-versus-host disease: a tale of tissue repair and tolerance." Blood 136, no. 4 (2020): 410–17. http://dx.doi.org/10.1182/blood.2019000951.
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Abstract The success of allogeneic hematopoietic cell transplantation depends heavily on the delicate balance between the activity of the donor immune system against malignant and nonmalignant cells of the recipient. Abrogation of alloreactivity will lead to disease relapse, whereas untamed allo-immune responses will lead to lethal graft-versus-host disease (GVHD). A number of cell types have been identified that can be used to suppress alloreactive immune cells and prevent lethal GVHD in mice. Of those, mesenchymal stromal cells and, to a lesser extent, regulatory T cells have demonstrated efficacy in humans. Ideally, cellular therapy for GVHD will not affect alloreactive immune responses against tumor cells. The importance of tissue damage in the pathophysiology of GVHD rationalizes the development of cells that support tissue homeostasis and repair, such as innate lymphoid cells. We discuss recent developments in the field of cellular therapy to prevent and treat acute and chronic GVHD, in the context of GVHD pathophysiology.
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Baron, Chloé S., Serine Avagyan, Song Yang, Aaron McKenna, and Leonard Zon. "Abstract A27: Cellular barcoding of the leukemic niche reveals an apelin-mediated clonal expansion of niche endothelial and mesenchymal stromal cell clones." Blood Cancer Discovery 4, no. 3_Supplement (2023): A27. http://dx.doi.org/10.1158/2643-3249.aml23-a27.
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Abstract Hematopoietic stem and progenitor cells (HSPCs) reside in niches that provide regulatory signals for their function. Perturbations such as acute leukemia induce cellular and molecular insults to the niche to support disease progression. Mutation of the MYC oncogene family is a frequent event leading to leukemogenesis. We developed a new zebrafish model of acute erythroid leukemia (AEL) by overexpression of human CMYC under the blood specific promotor draculin (drl). Analyses of drl:CMYC marrows demonstrated a significant expansion of progenitors and a decrease of erythroid, lymphoid and myeloid mature cells (fc=4.8, -4.5, -3.3, -6.5; p<0.000001). RNA-Sequencing of drl:CMYC marrows revealed an upregulation of the erythroid master regulator gata1a (fc=1.4, p=0.01) and fetal hemoglobins hbbe1.1/2 (fc=4.7, 2.9; p=0.0004). Primary and secondary transplantation of drl:CMYC marrows resulted in engraftment and disease propagation (7/7; 17/18). We used the cellular barcoding GESTALT technique to uniquely barcode single cells using CRISPR-CAS9 during embryonic development. We induced a round of barcoding at the one-cell stage and another one at 28 hours post-fertilization, the time of HSC birth. We injected these GESTALT embryos with drl:CMYC to induce AEL, barcode HSPCs and their niche to perform clone tracing. The number of HSPC clones was decreased by half compared to controls (p=0.008) indicative of a clonal expansion of the disease. We performed barcode and single-cell transcriptome profiling of flk1:GFP+ niche endothelial cells and found a significant decrease in the number of endothelial cells clones (fc= -3.5, p<0.05) paired with the identification of a novel AEL venous endothelial population upregulating 99 genes (fc>1; p<0.05) suggestive of angiogenesis likely supporting leukemogenesis. We sorted cxcl12a:dsRed+ niche stromal cells and found that AEL marrows have significantly less stromal clones (fc= -2.1, p<0.01) that are selectively amplified (>20% of the stromal compartment). We hypothesized that AEL expands a subset of stromal cells to promote disease progression and scRNA-Seq of 3,263 cxcl12a:dsRed+ stromal cells revealed an increased fraction of lepr+ mesenchymal stromal cells (MSCs, 66 vs 24% in controls). We hypothesized that AEL progenitors secrete a signal that can specifically remodel the marrow niche and we mined our scRNA-Sequencing data for a candidate ligand significantly upregulated in AEL cells compared to control HSPCs (fc>1; p<0.05) and paired receptors expressed on more than 20% of AEL associated endothelial and/or stromal cells. We identified the peptide hormone apelin expressed by the leukemia and tested if apelin alone could remodel the normal marrow niche endothelial and stromal cells by overexpressing apelin under the drl promotor. Adult marrow analyses revealed a decrease in the absolute number of stromal cells per marrow (fc= -2.2, p<0.001). Together our data support a model in which leukemia induces a clonal expansion of HSPC niche clones via apelin signaling to promote disease progression. Citation Format: Chloé S Baron, Serine Avagyan, Song Yang, Aaron McKenna, Leonard Zon. Cellular barcoding of the leukemic niche reveals an apelin-mediated clonal expansion of niche endothelial and mesenchymal stromal cell clones [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A27.
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Aggarwal, Vaishali, Radhika Srinivasan, Amanjit Bal, et al. "Mutational Spectrum of Stromal Genes By Whole Exome Sequencing and Stromal-Cellular Interaction in Diffuse Large B-Cell Lymphoma." Blood 126, no. 23 (2015): 2649. http://dx.doi.org/10.1182/blood.v126.23.2649.2649.
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Abstract Introduction: Morphological sub-classification of DLBCL into germinal center B-cell-like (GCB) and activated B-cell like (ABC) lacks sufficient reproducibility for risk stratification and predicting survival based on International Prognostic Index (IPI). The importance of stromal cells and their cross-talk with lymphoma cells is important in lymphomagenesis and progression. We postulate that mutations in stromal genes may regulate the rate of tumor progression in DLBCL and understanding the stromal-cellular interaction in DLBCL may help evolve better markers of prognostication and identify novel therapeutic targets. Methods: The study was approved by the Institute ethics committee and patients were enrolled after an informed consent. Frozen samples from 6 cases which included 4 cases of de novo nodal DLBCL (2 cases each of GCB and ABC subtypes as per Han's algorithm) and two control cases of non-neoplastic reactive lymphoid hyperplasia were subjected to AmpliSeqExome - Whole Exome Sequencing(WES) using Ion TorrentTM and compared against human reference genome (hg19). Torrent suiteTM 4.4 version (Life Technologies) and Ion ReporterTM software was used to perform tumor vs normal analysis by choosing variants for missense, frameshift insertions/ deletions, stoploss and single nucleotide variations (SNP). Filtering for common SNPs were done by UCSC common SNPs followed by inclusion in dbSNP and COSMIC and further filtered by predictive functional scores of SIFT < 0.05 and Polyphen (0.15 - 1.0). Variants were visualized using Integrated Genome Viewer (IGV) software. Bioinformatics analysis was done using PANTHER, STRING and PCViz and data mined for mutations in extracellular matrix genes. Selected genes were validated in DLBCL cases using Sanger sequencing BigDye terminator v3.1 cycle sequencing kit (Life Technologies) and ABI PRISM 3130 analyzer (Life Technologies). Further, in-vitro experiments were carried out to evaluate the effect of the conditioned media of CD19- stromal cells on the CD19+ lymphoma cells. Fresh tissue from 5 cases of nodal DLBCL were collected in RPMI 1640, 20% FBS and 1% penicillin and streptomycin and subjected to primary culture at 37°C and 5% CO2 humidified atmosphere. After 24 hours, the cultured cells were sorted into CD19+ and CD19- cells (BD FACSAriaTM) which were re-cultured for 24 hours separately. After this, the conditioned media of CD19- cells (comprising stromal cells predominantly) was transferred onto CD19+ cells and incubated for further 48 hours. Thereafter, cytokine (IL-6, IL-10, TNF-α, IFN-γ, IL-2, IL-4, IL-17) analysis of conditioned media in comparison to mock controls was performed using BD THI/THII kit (560484). Results: Analysis of filtered mutated genes using Panther revealed thirty four genes coding for extracellular matrix mutated specifically in GCB subtype and 26 mutated genes in ABC DLBCL. These genes were then analyzed for association in STRING. STRING identified LAMC3, AGRN, LAMA2, LAMB2, COL5A2, COL13A1,FN1,TGFB3, LTBP1 andADAMTS16 in GCB phenotype whereas LAMB1,LAMA3,COL4A2, COL28A1,COL5A3, IBSP, FGA, MUC6, MUC2, MUC5B,SPARCL1, VWF, GAS6 and USH2A were mutated in ABC DLBCL (Figure 1A and B). Mutations of COL5A2, COL13A1, LAMB2, MUC2, MUC6, and MUC5B were further validated using Sanger sequencing in individual DLBCL cases. This advocated the role of collagen scaffolding and laminin cross-linking in DLBCL progression. In vitro experiments for effect of stromal cells on the secreted cytokine profile revealed increased IL-6, IL-10, TNF-α, IFN-γ, IL-4, IL-17 levels respectively in the media of CD19+ sorted lymphoma cells upon addition of conditioned media of CD19-stromal cells in comparison to controls. Conclusion: Mutational profile of COL5A2, COL13A1, LAMB2, MUC2, MUC6, and MUC5B stromal genes in DLBCL may help in refinement of risk stratification based on morphology. The cross-talk between neoplastic cells and interacting tumor microenvironment cells has implications for therapy in DLBCL. GCB Mutation Profile ABC Mutation Profile Figure 1. Stromal gene signature profile in ABC and GCB subsets of DLBCL. Figure 1. Stromal gene signature profile in ABC and GCB subsets of DLBCL. Figure 2. Association among Stromal genes in GCB and ABC subtypes of DLBCL using STRING. Figure 2. Association among Stromal genes in GCB and ABC subtypes of DLBCL using STRING. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.
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Belli, Carmen, Gabriele Antonarelli, Matteo Repetto, Luca Boscolo Bielo, Edoardo Crimini, and Giuseppe Curigliano. "Targeting Cellular Components of the Tumor Microenvironment in Solid Malignancies." Cancers 14, no. 17 (2022): 4278. http://dx.doi.org/10.3390/cancers14174278.
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Cancers are composed of transformed cells, characterized by aberrant growth and invasiveness, in close relationship with non-transformed healthy cells and stromal tissue. The latter two comprise the so-called tumor microenvironment (TME), which plays a key role in tumorigenesis, cancer progression, metastatic seeding, and therapy resistance. In these regards, cancer-TME interactions are complex and dynamic, with malignant cells actively imposing an immune-suppressive and tumor-promoting state on surrounding, non-transformed, cells. Immune cells (both lymphoid and myeloid) can be recruited from the circulation and/or bone marrow by means of chemotactic signals, and their functionality is hijacked upon arrival at tumor sites. Molecular characterization of tumor-TME interactions led to the introduction of novel anti-cancer therapies targeting specific components of the TME, such as immune checkpoint blockers (ICB) (i.e., anti-programmed death 1, anti-PD1; anti-Cytotoxic T-Lymphocyte Antigen 4, anti-CTLA4). However, ICB resistance often develops and, despite the introduction of newer technologies able to study the TME at the single-cell level, a detailed understanding of all tumor-TME connections is still largely lacking. In this work, we highlight the main cellular and extracellular components of the TME, discuss their dynamics and functionality, and provide an outlook on the most relevant clinical data obtained with novel TME-targeting agents, with a focus on T lymphocytes, macrophages, and cancer-associated fibroblasts.
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Reglero-Real, Natalia, Diego García-Weber, and Jaime Millán. "Cellular Barriers after Extravasation: Leukocyte Interactions with Polarized Epithelia in the Inflamed Tissue." Mediators of Inflammation 2016 (2016): 1–10. http://dx.doi.org/10.1155/2016/7650260.
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During the inflammatory response, immune cells egress from the circulation and follow a chemotactic and haptotactic gradient within the tissue, interacting with matrix components in the stroma and with parenchymal cells, which guide them towards the sites of inflammation. Polarized epithelial cells compartmentalize tissue cavities and are often exposed to inflammatory challenges such as toxics or infections in non-lymphoid tissues. Apicobasal polarity is critical to the specialized functions of these epithelia. Indeed, a common feature of epithelial dysfunction is the loss of polarity. Here we review evidence showing that apicobasal polarity regulates the inflammatory response: various polarized epithelia asymmetrically secrete chemotactic mediators and polarize adhesion receptors that dictate the route of leukocyte migration within the parenchyma. We also discuss recent findings showing that the loss of apicobasal polarity increases leukocyte adhesion to epithelial cells and the consequences that this could have for the inflammatory response towards damaged, infected or transformed epithelial cells.
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Szeifert, György T., Isabelle Salmon, Sandrine Rorive, et al. "Does gamma knife surgery stimulate cellular immune response to metastatic brain tumors? A histopathological and immunohistochemical study." Journal of Neurosurgery 102, Special_Supplement (2005): 180–84. http://dx.doi.org/10.3171/sup.2005.102.s_supplement.0180.
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Object. The aim of this study was to analyze the cellular immune response and histopathological changes in secondary brain tumors after gamma knife surgery (GKS). Methods. Two hundred ten patients with cerebral metastases underwent GKS. Seven patients underwent subsequent craniotomy for tumor removal between 1 and 33 months after GKS. Four of these patients had one tumor, two patients had two tumors, and one patient had three. Histological and immunohistochemical investigations were performed. In addition to routine H & E and Mallory trichrome staining, immunohistochemical reactions were conducted to characterize the phenotypic nature of the cell population contributing to the tissue immune response to neoplastic deposits after radiosurgery. Light microscopy revealed an intensive lymphocytic infiltration in the parenchyma and stroma of tumor samples obtained in patients in whom surgery was performed over 6 months after GKS. Contrary to this, extensive areas of tissue necrosis with either an absent or scanty lymphoid population were observed in the poorly controlled neoplastic specimens obtained in cases in which surgery was undertaken in patients less than 6 months after GKS. Immunohistochemical characterization demonstrated the predominance of CD3-positive T cells in the lymphoid infiltration. Conclusions. Histopathological findings of the present study are consistent with a cellular immune response of natural killer cells against metastatic brain tumors, presumably stimulated by the ionizing energy of focused radiation.
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Szeifert, György T., Isabelle Salmon, Sandrine Rorive, et al. "Does gamma knife surgery stimulate cellular immune response to metastatic brain tumors? A histopathological and immunohistochemical study." Journal of Neurosurgery 102 (January 2005): 180–84. http://dx.doi.org/10.3171/jns.2005.102.s_supplement.0180.
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Object.The aim of this study was to analyze the cellular immune response and histopathological changes in secondary brain tumors after gamma knife surgery (GKS).Methods.Two hundred ten patients with cerebral metastases underwent GKS. Seven patients underwent subsequent craniotomy for tumor removal between 1 and 33 months after GKS. Four of these patients had one tumor, two patients had two tumors, and one patient had three. Histological and immunohistochemical investigations were performed. In addition to routine H & E and Mallory trichrome staining, immunohistochemical reactions were conducted to characterize the phenotypic nature of the cell population contributing to the tissue immune response to neoplastic deposits after radiosurgery.Light microscopy revealed an intensive lymphocytic infiltration in the parenchyma and stroma of tumor samples obtained in patients in whom surgery was performed over 6 months after GKS. Contrary to this, extensive areas of tissue necrosis with either an absent or scanty lymphoid population were observed in the poorly controlled neoplastic specimens obtained in cases in which surgery was undertaken in patients less than 6 months after GKS. Immunohistochemical characterization demonstrated the predominance of CD3-positive T cells in the lymphoid infiltration.Conclusions.Histopathological findings of the present study are consistent with a cellular immune response of natural killer cells against metastatic brain tumors, presumably stimulated by the ionizing energy of focused radiation.
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Humby, Frances, Myles Lewis, Nandhini Ramamoorthi, et al. "Synovial cellular and molecular signatures stratify clinical response to csDMARD therapy and predict radiographic progression in early rheumatoid arthritis patients." Annals of the Rheumatic Diseases 78, no. 6 (2019): 761–72. http://dx.doi.org/10.1136/annrheumdis-2018-214539.
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ObjectivesTo unravel the hierarchy of cellular/molecular pathways in the disease tissue of early, treatment-naïve rheumatoid arthritis (RA) patients and determine their relationship with clinical phenotypes and treatment response/outcomes longitudinally.Methods144 consecutive treatment-naïve early RA patients (<12 months symptoms duration) underwent ultrasound-guided synovial biopsy before and 6 months after disease-modifying antirheumatic drug (DMARD) initiation. Synovial biopsies were analysed for cellular (immunohistology) and molecular (NanoString) characteristics and results compared with clinical and imaging outcomes. Differential gene expression analysis and logistic regression were applied to define variables correlating with treatment response and predicting radiographic progression.ResultsCellular and molecular analyses of synovial tissue demonstrated for the first time in early RA the presence of three pathology groups: (1) lympho-myeloid dominated by the presence of B cells in addition to myeloid cells; (2) diffuse-myeloid with myeloid lineage predominance but poor in B cells nd (3) pauci-immune characterised by scanty immune cells and prevalent stromal cells. Longitudinal correlation of molecular signatures demonstrated that elevation of myeloid- and lymphoid-associated gene expression strongly correlated with disease activity, acute phase reactants and DMARD response at 6 months. Furthermore, elevation of synovial lymphoid-associated genes correlated with autoantibody positivity and elevation of osteoclast-targeting genes predicting radiographic joint damage progression at 12 months. Patients with predominant pauci-immune pathology showed less severe disease activity and radiographic progression.ConclusionsWe demonstrate at disease presentation, prior to pathology modulation by therapy, the presence of specific cellular/molecular synovial signatures that delineate disease severity/progression and therapeutic response and may pave the way to more precise definition of RA taxonomy, therapeutic targeting and improved outcomes.
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Zhu, Jiang, Yi Zhang, Nacksung Kim, et al. "Osteoblasts Support Early B Lymphoiesis as well as Stem Cell Proliferation and Myelopoiesis: Identification of the Mammalian Cellular Analog of the Bursa of Fabricius." Blood 104, no. 11 (2004): 508. http://dx.doi.org/10.1182/blood.v104.11.508.508.
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Abstract The self-renewal, survival and differentiation of hematopoietic stem cells (HSC) are greatly influenced by the activities of neighboring osteoblasts and non-osteogenic bone marrow (BM) stromal cells such as fibroblasts, endothelial cells and adipocytes. Previously, we showed that osteoblasts from human long bones support the in vitro self-renewal as well as myeloid differentiation of human CD34+ cord blood cells. Recently, Li’s and Scadden’s groups provided in vivo evidence indicating a primary role of trabecular osteoblasts as a major component of HSC niche and of stromal osteoblastic cells in facilitating the self-renewal of HSCs. We have now asked whether osteoblasts contribute to early lymphopoiesis as well as myelopoiesis, by measuring the cellular outpout of purified HSCs on isolated osteoblasts alone, or with added non-osteoblast stromal cytokines as well. We prepared mature osteoblasts, as monitored and confirmed by homogeneous OPN and CD61 expression, by pretreating osteoblastic cells isolated from neonatal calvaria of C57BL/6 mice (CD45.2) with 1X10−7 M PTH. Purified OB were then co-cultured for 6 days with Lin− BM cells (CD45.1+) isolated from congenic B6 mice(CD45.1) and labeled with CFSE. Osteoblast coculture stimulated the proliferation of Lin− CD45.1+ BM cells 50-fold during culture, with most cells (87%) remaining tightly adherent to the osteoblast monolayer; no live cells were recovered from Lin− BM cell culture without osteoblasts. In addition to mature granulocytes/monocytes, a substantial amount of CD45.1+B220+ B lymphocytes (about 10% of small size cells gated by forward and side scatter), were detected. In contrast, very few CD45.1+Lin-Sca-1+c-Kit+ (LSK) cells or CD45.1+Lin−Sca-1−c-Kit+ (CMP) cells were detected under these conditions. Most B220+ cells attached to osteoblasts were found to be CD43+CD24+ pre-B cells undergoing division. In contrast to the cells recovered attached to the osteoblasts, the pre-B lymphocytes found in suspension were more mature with phenotype of B220+CD43−CD24+. Prevention of direct contact of Lin− BM cells with osteoblasts by Transwell co-culture abrogated the production of pre-B cells in both adherent and suspension compartments, indicating that physical contact is required for the interaction. Interestingly, when 20ng/ml of SCF, 6ng/ml of IL-3, 10ng/ml of IL-6 and 25ng/ml TPO were added to osteoblast/Lin− cell co-culture, B lymphpoiesis was repressed, while the production of CD45.1+LSK HSCs and CMPs was significantly enhanced. These data demonstrate a direct role of osteoblasts in inducing and supporting the early development of B lymphocytes from HSCs or/and common lymphoid progenitors. Additional cytokines, perhaps provided in specific in vivo niches by non-osteogenic stromal cells, cooperate with the stimulatory signals from osteoblasts to promote the survival and expansion of HSCs. Taken together, these results suggest that osteoblasts may be the mammalian analog of the avian Bursa of Fabricius, and that their local degree of proximity to non-osteogenic stromal cells may define specific microniches for stem cell survival, myelopoiesis and/or B lymphopoiesis.
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Ho, Anthony D., Patrick Horn, Marco Hennrich, et al. "Proteomics Analysis of Cellular Network in Human Bone Marrow Reveals Lineage Skewing Towards Megakaryocytes and Decrease in Lymphoid Development upon Aging." Blood 128, no. 22 (2016): 2658. http://dx.doi.org/10.1182/blood.v128.22.2658.2658.
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Abstract Background: Whereas proteomics analyses at a cellular level have been performed in murine HPC, most proteomics studies on human material have thus far focused on examining human tissues with a mixture of cell types and not at a specific cellular level. Whole proteomics analyses might reveal genuine molecular alterations und mechanisms of aging on human hematopoietic cells. Aim: To identify the age-related molecular changes in human hematopoietic progenitor cells (HPC) as well as in the cellular elements of the human bone marrow niche, we have applied mass spectrometry-based platform for proteomics analysis of this network of bone marrow (BM) cells at a cellular level. The overarching goal is to acquire a systems understanding of the molecular mechanisms involved in aging of human HPC as well as other cellular constituents of the marrow niche, and to combine the cell biological, proteomic, and genetic studies for a better understanding of age-related diseases such as MDS. Methods: In this study, we have simultaneously characterized at the proteomics level HPC as well as the cellular microenvironment in BM from each individual human subject. Cells were harvested from the BM of 69 healthy human subjects [Age Group (AG) 20-29 years: n=19; AG 30-39: n=16; AG 40-49: n=9; AG 50+: N=15]. BM cells were separated for nucleated cells using Ficoll, followed by sorting with specific marker constellations, i.e. HPC (CD34+), lymphoid cells (CD34-/CD45+/SSClow), myeloid precursors (CD34-/CD45med/SSChigh), monocyte-macrophages (CD34-/CD45+/CD14+), erythroid precursors (CD34-/CD45-), and mesenchymal stromal cells (MSC) based on plastic adherence upon culture. Results: In 60 of these 69 healthy subjects we were able to harvest an adequate amount of material from 5 to 6 the aforementioned subsets from the respective individual, yielding altogether 342 samples for proteomics analysis. After digestion (trypsinization), isobaric labelling of peptides (TMT) of appropriate quality was successfully performed in 270 of these 342 samples derived from 69 healthy human subjects. These 270 samples underwent tandem mass-spectrometry analyses. The number of proteins identified were >12,000, covering 65% of the estimated human proteome (HPC: >7,500; Lymphoid cells: >8,500; Myeloid Precursors: >7,500; Mono/Macro: >8,500; Erythroid Precursors: >6,500; and in MSC: >9,000 proteins). We have then focused on the molecular mechanisms, specifically proteomic alterations involved in aging of these 6 cell types. Gene Set Enrichment Analysis (GSEA) using previously defined gene sets specific for common lymphoid progenitors (CLP), bi-potent granulocyte/macrophage progenitors (GMP), and megakaryocyte progenitors (MKP) revealed significant down-regulation of CLP related proteins and significant up-regulation of MKP related proteins in HPC upon aging. The same down-regulation of CLP proteins and up-regulation of MKP proteins were also found in the BM lymphoid cells. Further significant alterations of note are a decrease in cell cycle related proteins upon aging in HPC, lymphoid cells, as well as in MSC, and an increase in GMP related proteins upon aging in lymphoid cells, erythroid precursors and in MSC. Extensive pathway and network analyses are underway. Conclusions: We have for the first time accomplished a multiplex and comprehensive analysis of a community of cells in human BM, comprising HPC and 5 other cell types that constitute the niche and have identified >12,000 proteins in this cellular network. Proteomics analyses have demonstrated a significant down-regulation of CLP related proteins in human HPC and in marrow lymphoid cells, as well as an up-regulation of MKP in all the cell types tested upon aging. Of note is also a decrease in cell cycle related proteins with age. This study has thus provided evidence that lineage skewing towards megakaryocytic and decrease in lymphoid development upon aging occur in human HPC as well as in other human BM cells. Disclosures No relevant conflicts of interest to declare.
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Vorontsova, Z. A., and E. F. Kudaeva. "Cellular reactions of the mucous membrane of the intestinal system after uranium incorporation." Morphological newsletter 28, no. 1 (2020): 9–15. http://dx.doi.org/10.20340/mv-mn.2020.28(1):9-15.
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A pilot study, which was performed on white laboratory male rats and included a morphological study of the mucous membrane of the jejunum and colon, revealed the long-term effect of a single oral exposure to an aqueous solution of depleted uranium oxides; this supported dynamic changes in the studied criteria determining metabolic, regenerative, and local regulatory processes in one, three and six months. A lymphoid component appeared to be the criterion of high radio sensitivity and a marker of nonspecific protection indicating deterioration of homeostasis. In the time dynamics of long-term periods, a change in all the parameters was detected when using immunohistochemical, histochemical, and specific methods at the level of the epithelium lining the relief formations of the mucous membranes of the intestinal system organs. The oxidative stress resulting in three months after exposure to depleted uranium, which was evidenced by a significant decrease in the content of anti-apoptotic Bcl-2 protein in the epithelial cells of the jejunely mucosa. Signs of its delayed effect after six months in the colon induced the pronounced nature of the reactions of stromal cell components that determined patterns of protective mechanisms of the intestinal-associated immune system with a greater expressiveness in the jejunum. The factual material cited in the work suggests the existence of a homeostatic immune response in the mucous membrane of the small and large intestines that controls cell proliferation, as a special form of immune surveillance of the state of cytodifferentiation.
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Shanmugam, Vignesh, Neriman Tokcan, Daniel Chafamo, et al. "Genome-Scale High-Resolution Spatial Mapping of the Pro-Tumorigenic Cellular Niche in Classic Hodgkin Lymphoma." Blood 142, Supplement 1 (2023): 175. http://dx.doi.org/10.1182/blood-2023-185522.
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Introduction: A fundamental hallmark of cancer is that tumor cells repurpose the tissue microenvironment to promote their own survival. An increased understanding of these mechanisms may lead to improved microenvironment-directed therapies, particularly in lymphoid malignancies. In classic Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed Sternberg (HRS) cells are surrounded by a CD4+ T-cell and macrophage-rich inflammatory infiltrate. Recent multiplexed immunofluorescence studies suggest that the micron-scale niche around HRS cells is composed of distinct populations of PD-L1+ macrophages and CD4+ T cells, including regulatory CTLA4+ and LAG3+ subsets (Carey et al. Blood 2017, Patel et al. Blood 2019 and Aoki et al. Cancer Discov 2020). However, the topography of the intact tumor microenvironment of cHL requires further definition. Recent single-cell RNA sequencing studies have led to important insights into the biology of cHL; however, they do not adequately capture myeloid cells, fibroblasts, and HRS cells, likely due to the relative fragility of these cells in conventional tissue dissociation protocols. In this study, we use tandem single nucleus and spatially resolved RNA sequencing to systematically dissect the pro-tumorigenic cellular niche of cHL to define potentially targetable microenvironmental dependencies. Methods: Using single nucleus (10X Chromium V3.1 gene expression) and spatially resolved RNA sequencing (Slide-seqV2 and 10X Visium), we generated a dataset of 12 newly diagnosed cHL and 7 reactive lymphoid tissues comprising over 324,855 nuclei and 2.5 million spatially resolved gene expression profiles to construct a genome-scale single cell and spatially resolved atlas of cHL. To validate the expression of ligands/receptors on proximal cell types within the HRS cell niche at single-cell resolution, we also generated an orthogonal 1000-plex image-based spatial transcriptomic dataset (CosMx Spatial Molecular Imager). Additionally, we developed a novel Bayesian consensus tensor factorization approach (C-ZIPTF) to infer shared cancer-associated immune signatures and ligand-receptor interactions. Results: Single nucleus RNA sequencing captures a diversity of cell types and cell states in cHL, including distinct myeloid and stromal cell subsets and HRS cells. Copy number inference analysis identifies the HRS cells, and unsupervised consensus tensor factorization reveals recurrent HRS cell transcriptional states, including aberrant myeloid, stromal, neuronal, and secretory/cytokine programs. Of all clinical and histological parameters, EBV infection status showed the strongest association with HRS cell transcriptional state. Regulatory T cells, macrophages, and fibroblasts are quantitatively increased in cHL compared to reactive lymphoid tissues and additionally exhibit qualitatively altered expression programs. Using C-ZIPTF, we identify disease-defining and prognostically relevant immunosuppressive regulatory T cell and myeloid cell programs and an activated pro-fibrotic myofibroblast-like fibroblast signature. Spatial analysis provides strong evidence for a “niche” model, i.e., the spatial organization of cell types and states around HRS cells. Specifically, CD4+ T cells and myeloid cells are spatially enriched in the immediate proximity of HRS cells and, plasma cells are depleted. Moreover, the CD4+ T cells and myeloid cells exhibit a specialized pro-tumorigenic gene expression program with increased expression of multiple immunosuppressive cytokines, checkpoint molecules, and putative growth factors for HRS cells. Spatially-aware ligand-receptor interaction analysis enables systematic identification and prioritization of novel growth factors for HRS cells. Functional testing of these putative growth factors using an ex vivo 3D cell culture system confirms their growth-promoting effect on primary HRS cells. Conclusions: The HRS cells in cHL are surrounded by a distinctive pro-tumorigenic cellular niche that not only mediates immune evasion but may also directly promote tumor cell growth. These observations provide a rich set of hypotheses for preclinical model development and microenvironment-directed therapies. More broadly, our study demonstrates the power of spatial genomic approaches to deconvolute the molecular architecture of pro-tumorigenic cellular niches in tumor ecosystems.
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Capron, C., K. Jondeau, L. Casetti, et al. "Viability and stress protection of chronic lymphoid leukemia cells involves overactivation of mitochondrial phosphoSTAT3Ser727." Cell Death & Disease 5, no. 10 (2014): e1451-e1451. http://dx.doi.org/10.1038/cddis.2014.393.
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Abstract Chronic lymphoid leukemia (CLL) is characterized by the accumulation of functionally defective CD5-positive B lymphocytes. The clinical course of CLL is highly variable, ranging from a long-lasting indolent disease to an unpredictable and rapidly progressing leukemia requiring treatment. It is thus important to identify novel factors that reflect disease progression or contribute to its assessment. Here, we report on a novel STAT3-mediated pathway that characterizes CLL B cells-extended viability and oxidative stress control. We observed that leukemic but not normal B cells from CLL patients exhibit constitutive activation of an atypical form of the STAT3 signaling factor, phosphorylated on serine 727 (Ser727) in the absence of detectable canonical tyrosine 705 (Tyr705)-dependent activation in vivo. The Ser727-phosphorylated STAT3 molecule (pSTAT3Ser727) is localized to the mitochondria and associates with complex I of the respiratory chain. This pSer727 modification is further controlled by glutathione-dependent antioxidant pathway(s) that mediate stromal protection of the leukemic B cells and regulate their viability. Importantly, pSTAT3Ser727, but neither Tyr705-phosphorylated STAT3 nor total STAT3, levels correlate with prolonged in vivo CLL B cells survival. Furthermore, STAT3 activity contributes to the resistance to apoptosis of CLL, but not normal B cells, in vitro. These data reveal that mitochondrial (Mt) pSTAT3Ser727 overactivity is part of the antioxidant defense pathway of CLL B cells that regulates their viability. Mt pSTAT3Ser727 appears to be a newly identified cell-protective signal involved in CLL cells survival. Targeting pSTAT3Ser727 could be a promising new therapeutic approach.
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Song, Xiaofei, Xiaoqing Yu, Carlos M. Moran-Segura, G. Daniel Grass, Roger Li, and Xuefeng Wang. "Abstract 6245: Missed opportunity at the subcellular level: Enhancing the utility of cellular imaging modality in spatial transcriptomic profiling of tumor tissues." Cancer Research 84, no. 6_Supplement (2024): 6245. http://dx.doi.org/10.1158/1538-7445.am2024-6245.
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Abstract Mini-bulk or single-spot spatial transcriptomic technologies, such as GeoMX Digital Spatial Profiler (DSP), have revolutionized our ability to probe spatial heterogeneity and examine transcripts at a subcellular level. In cancer research, this level of granularity is crucial for uncovering the distinct gene expression signatures within specific compartments or structures of interest such as tumor, immune, stroma, and tertiary lymphoid structures. However, in most GeoMX DSP studies, the spatial information obtained from multiplex immunofluorescence imaging has been primarily used for identifying the regions of interest (ROI), rather than as an integral part of the downstream transcriptomic data interpretation. To fix this missed opportunity, we developed a machine learning based analytical framework to fully leverage the rich spatial context provided by the in situ imaging modality. The framework has two main functionalities. (1) ROI cropping and cell segmentation: our framework enables the automated cropping of ROI images, followed by a cell segmentation process utilizing a multi-color-optimized deep learning model pre-trained based on the TissueNet. (2) Cell typing and integrative spatial analysis: the pipeline extracts cell-level morphological features (e.g. cell size and circularity), and AI-driven characteristics. These features are subsequently employed for cell typing and the quantification of cell mixture. The framework is equipped to perform a series of advanced spatial analyses, such as spatial metrics calculation and consensus clustering analysis with the matched gene expression data. To validate the framework, we conducted GeoMX DSP analyses on samples obtained from bladder cancer and upper tract urothelial carcinoma, encompassing 56 ROIs annotated by pathologists. Our results demonstrated high accuracy, with the calculated tumor-immune proportions aligning precisely with the original annotations. We further compared the cell deconvolution results of our framework with those obtained using SpatialDecon. Consistency was noted across both methods, with a high correlation reaching 0.90. However, we observed that SpatialDecon tends to underestimate tumor purity in tumor core regions, with discrepancies as large as 0.30, and overestimating tumor purity in stromal regions. Additionally, analysis of ROIs in tumor margins revealed that SpatialDecon consistently overestimates the overall proportions of immune cells. In conclusion, our results underscore the importance of integrating in situ imaging with subcellular-level spatial transcriptomics for a more accurate and reliable analysis of tumor tissues. Our approach provides critical insights into the tumor microenvironment and cellular interactions, with significant implications for both research and clinical applications in oncology. Citation Format: Xiaofei Song, Xiaoqing Yu, Carlos M. Moran-Segura, G Daniel Grass, Roger Li, Xuefeng Wang. Missed opportunity at the subcellular level: Enhancing the utility of cellular imaging modality in spatial transcriptomic profiling of tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6245.
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Damle, Rajendra N., Taraneh Banapour, Sonal Temburni, et al. "Remarkable Differences in Cellular Activation State and Migratory and Proliferative Potential among Clonal Cells Derived from Different Tissues of Chronic Lymphocytic Leukemia Patients." Blood 108, no. 11 (2006): 2817. http://dx.doi.org/10.1182/blood.v108.11.2817.2817.
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Abstract An antigen-independent process that takes place in the bone marrow (BM) leads to the birth of B cells from bone marrow precursors. Cross-talk between components of the microenvironment of BM or secondary lymphoid compartment(s), eg. spleen (SP), nurtures the subsequent evolution of B cells and governs in large part the natural history of normal B cells and B cell malignancies including chronic lymphocytic leukemia (B-CLL). Interaction of B cells with stromal elements confers upon them features enabling their transient sequestration, proliferation and extended survival. In this report we have compared the characteristics of clonal B-CLL cells obtained from paired specimens (peripheral blood, PB with corresponding BM or SP obtained within an interval of less than 1 month of each other) from 17 individual untreated B-CLL patients. These cells were tested by surface immunofluorescence and flow cytometry for their expression of a panel of chemokine receptors (CCR −1, −2, −4, and −7 and CXCR−1, −2, −3, and −4) and markers of cellular activation (CD23, CD62L, CD69, CD71 and HLA-DR). The relative age of the B cells (telomere length) and their ability to maintain telomere length (telomerase activity) were studied in paired BM/SP and PB samples from 15 of these cases. Although PB-, BM- and SP-derived B cells expressed activation markers, specifically, the percentages of cells expressing ZAP-70 and Ki-67 were significantly higher in BM- and SP-derived B cells than those expressed by corresponding PB-derived B cells (p<0.01). Increase in extent of CD38-positivity among members of the clone correlates with poor prognosis in B-CLL. Interestingly, in cases with low CD38 expression (<30% cells expressing CD38), the percentage of B-CLL cells expressing CXCR3 and CCR7 was significantly higher among PB-B cells, than BM/SP-derived B cells. No such differences existed in cases with high CD38 expression. This suggests a role for these receptors and their ligands in maintaining homeostasis of B-CLL cells in this subset of cases (that does not show remarkable progression of disease). In each of the 15 cases studied BM and SP-B cells had significantly higher telomerase activity (p<0.01) than those in PB, although telomere lengths of B cells from both sources were comparable. These findings highlight important differences in cellular kinetics among lymphoid compartments in B-CLL.
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Subramanian, Ajay, Neda Nemat-Gorgani, Timothy J. Ellis-Caleo, et al. "Abstract 5960: Identification and validation of sarcoma cellular ecosystems associated with prognosis and predictive of immunotherapy response." Cancer Research 83, no. 7_Supplement (2023): 5960. http://dx.doi.org/10.1158/1538-7445.am2023-5960.
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Abstract The tumor microenvironment contributes to tumorigenesis, disease progression, and response to therapy in soft tissue sarcomas (STSs). However, characterizing the malignant and stromal cells that make up STSs and correlating their abundance with clinical outcomes has proven difficult in fixed clinical specimens. We employed a machine learning framework called EcoTyper (Luca et al. Cell 2021) to identify cell type-specific transcriptional states and define tumor ecotypes consisting of co-occurring cell states from bulk transcriptomes. Analyzing 292 previously published STSs profiled by bulk RNA-sequencing (RNA-Seq), we identified 23 transcriptionally-defined sarcoma cell states in malignant, immune, and other stromal cells and validated the majority of these cell states using single cell RNA-Seq. Although sarcomas originate from mesenchymal tissues, we identified four epithelial-like sarcoma cell states and observed epithelial differentiation and expression of epithelial markers within malignant sarcoma cells across histologies. Cell states reflected known and novel cell phenotypes and many were strongly associated with patient outcomes in our RNA-Seq training cohort and an independent validation cohort of STSs profiled by microarray (n=309). By identifying co-occurring cell states across STSs, we discovered 3 sarcoma ecotypes associated with underlying genomic alterations and distinct clinical outcomes. On spatial transcriptomic analysis, we observed co-localization of cell states within the same sarcoma ecotypes and spatial aggregation of ecotypes within STSs, suggesting that sarcoma ecotypes represent distinct functional units within human sarcomas. One ecotype (sarcoma ecotype 3: SE3) defined by CLEC5A/SPP1+ M2-like immunosuppressive macrophages and MYC/MTORC1-activated epithelial-like malignant cells was associated with inferior progression-free survival (PFS) in the RNA-Seq training cohort (P=0.01) and inferior metastasis-free survival in the microarray validation cohort (P=0.002). Remarkably, SE3 was associated with improved PFS in patients with metastatic STSs treated with ipilimumab and nivolumab (n=38, P=0.003) but not patients treated with chemotherapy. Furthermore, SE3 outperformed previously reported predictors of immunotherapy response in STSs, PD-L1 expression and tertiary lymphoid structures (TLS), for predicting response to immunotherapy (AUCs: SE3 0.87; PD-L1 0.81; TLS 0.62). Finally, SE3 similarly predicted response to immunotherapy in an independent validation cohort (n=29, AUC 0.89). In summary, our findings provide a high-resolution cell atlas of STSs to guide the development of novel therapeutic strategies. In addition, we have identified a predictive biomarker of response to immune checkpoint inhibition that may enable personalization of systemic therapy in patients with advanced STSs. Citation Format: Ajay Subramanian, Neda Nemat-Gorgani, Timothy J. Ellis-Caleo, David G.P. van IJzendoorn, Timothy J. Sears, Anish Somani, Bogdan A. Luca, Maggie Y. Zhou, Martina Bradic, Ileana A. Torres, Eniola Oladipo, Christin New, Deborah E. Kenney, Raffi S. Avedian, Robert J. Steffner, Michael S. Binkley, David G. Mohler, William D. Tap, Sandra P. D’Angelo, Matt van de Rijn, Kristen N. Ganjoo, Nam Q. Bui, Gregory W. Charville, Aaron M. Newman, Everett J. Moding. Identification and validation of sarcoma cellular ecosystems associated with prognosis and predictive of immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5960.
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Steen, Chloe B., Bogdan A. Luca, Mohammad Shahrokh Esfahani, et al. "An Atlas of Clinically-Distinct Tumor Cellular Ecosystems in Diffuse Large B Cell Lymphoma." Blood 134, Supplement_1 (2019): 655. http://dx.doi.org/10.1182/blood-2019-129461.
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Background: Diffuse large B cell lymphoma (DLBCL) exhibits significant clinical and biological heterogeneity, in part due to cell-of-origin subtypes, somatic alterations, and diverse stromal constituents within the tumor microenvironment (TME). Several immunologically-active lymphoma therapies are known to rely on innate and adaptive anti-tumor responses occurring within this dynamic TME, including agents that are approved (e.g., rituximab, lenalidomide, CART19, ibrutinib) or emerging (e.g., anti-CD47, checkpoint inhibitors). We hypothesized that a large-scale characterization of the cellular heterogeneity in DLBCL might reveal previously unknown biological variation in the TME linked to tumor subtypes and genotypes, therapeutic responses and clinical outcomes, with implications for future personalization of immunotherapy. Methods: Using a combination of lymphoma single-cell RNA sequencing (scRNA-seq) and bulk tumor transcriptome deconvolution (CIBERSORTx; Newman et al., Nat Biotech, 2019), we developed a new machine learning framework for identifying cellular states and ecosystems that reflect fundamental TME subtypes and distinctions in tumor biology (Fig. 1). Specifically, using CIBERSORTx, we purified the transcriptomes of B cells and 12 different TME cell types, including immune and stromal subsets, from 1,279 DLBCL tumor biopsies profiled in 3 prior studies (Reddy et al., Cell 2017; Schmitz et al., NEJM 2018; Chapuy et al., Nat Med 2018). Then, we defined distinct transcriptional states for each of the 13 cell types, which we validated at single-cell resolution, using a combination of two scRNA-seq techniques (Smart-Seq2 and 10x Chromium 5' GEP, BCR and TCR) to profile primary DLBCL, FL, and human tonsils, as well as leveraging multiple scRNA-seq datasets from previous studies. We identified robust co-associations between cell states that form tumor cellular ecosystems, which we validated in independent datasets of bulk DLBCL tumor gene expression profiles. Finally, we related TME ecosystems to defined tumor subtypes, including genotype classes, and to clinical outcomes. Results: By systematically characterizing the landscape of cellular heterogeneity in nearly 1,300 DLBCL tumors, we defined an atlas of 49 distinct transcriptional states across 13 major cell types. These novel cell states spanned diverse innate and adaptive immune effector cells of the lymphoid and myeloid lineages, as well as tumor-associated fibroblasts. Remarkably, 94% of these states (46 of 49) could be validated in a compendium of ~200,000 single-cell transcriptomes derived from lymphomas, healthy control tonsils, and other tissue types. Moreover, single cells from DLBCL, FL and tonsils best mirrored these newly discovered cell states. We next characterized the biology and potential clinical utility of each cell state. We observed clear distinctions in the transcriptional programs of immune and stromal elements between germinal center and activated B cell DLBCL, as well as between known mutational subtypes. Importantly, many cell states reflected novel phenotypic groupings, and the majority were significantly associated with overall survival (P&lt;0.05). These findings were highly concordant both within and across 3 independent DLBCL cohorts. By identifying groups of DLBCL patients with similar communities of cellular states, we defined cohesive cellular ecosystems that collectively capture the landscape of transcriptional heterogeneity in DLBCL tumors. Patients whose tumors were assigned to these ecosystems exhibited striking variation in overall survival. Importantly, the ecosystems defined distinct subgroups that could not be fully recapitulated by known transcriptional and genetic subtypes. Moreover, several TME classes showed significant enrichments in canonical or novel tumor genotypes, suggesting an evolutionary interplay between the tumor and host microenvironment. Conclusion: We describe a novel computational framework to digitally dissect the DLBCL TME and an atlas of novel states for diverse cell types in these tumors. We show how cellular states form cohesive tumor ecosystems, which exhibit distinct clinical outcomes and novel somatic alterations. These results expand our understanding of cellular heterogeneity in DLBCL, with implications for the development of individualized immunotherapies. Disclosures Kurtz: Roche: Consultancy. Advani:Kura: Research Funding; Merck: Research Funding; Millennium: Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cell Medica, Ltd: Consultancy; Kyowa Kirin Pharmaceutical Developments, Inc.: Consultancy; Stanford University: Employment, Equity Ownership; Janssen: Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Research Funding; Infinity Pharma: Research Funding; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Celmed: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forty-Seven: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc./Kite Pharma, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agensys: Research Funding. Diehn:Roche: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; BioNTech: Consultancy; Quanticell: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.
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Ruggeri, Loredana, Elena Urbani, Davide Chiasserini, et al. "Alloreactive Natural Killer Cells Initiate a Unique Cellular and Molecular Pathway That Greatly Accelerates Immune Reconstitution after Allogeneic Bone Marrow Transplantation." Blood 128, no. 22 (2016): 548. http://dx.doi.org/10.1182/blood.v128.22.548.548.
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Abstract One outstanding issue in allogeneic hematopoietic transplantation is impaired immune reconstitution. As the primary site of T cell development, the thymus plays a key role in the generation of a strong yet self-tolerant adaptive immune response, essential in the face of the potential threat from pathogens or neoplasia. Allogeneic hematopoietic transplantation may acutely damage the thymus through the chemo or radiotherapy, antibody therapy of the conditioning regime, infections acquired by the immunosuppressed patient, and thymic graft versus host disease. To date, attempts to improve thymic reconstitution have been disappointing. Pre-clinical experiments and pilot clinical trials tried to assess the role of a variety of therapeutic approaches, such as transfer of lymphoid progenitor cells, thymic grafts, or enhancement of thymopoiesis by administration of hormonal or cytokine/growth factor-based therapies, such as sex-steroid blockade, and IL-7, IL-22, KGF, or Flt-3 ligand administration (reviewed in Chaudhry et al., Immunol Rev. 2016). In mouse MHC mismatched transplantation models (F1 H-2d/b→parent H-2b), we previously found that infusion of donor versus recipient alloreactive NK cells eradicated recipient-type lympho-hematopoietic lineage cells, thereby enhancing engraftment, protecting from GvHD and eradicating leukemia (Ruggeri et al., Science 2002). Here, in the same models we show that infusion of alloreactive NK cells greatly accelerates the post-hematopoietic transplant recovery of donor-type immune cells, i.e., dendritic cells (DCs) (p<0.001), B lineage cells (p<0.001) and thymocytes (p<0.001) and maturation to B (p<0.001) and T cells (p<0.001). By the use of recipient chimeric mice displaying different tissue (i.e., hematopoietic vs non-hematopoietic) susceptibility to donor alloreactive NK cell killing, we show that a specific interaction between donor alloreactive NK cells and recipient DCs is responsible for the accelerated immune rebuilding. We find that donor-versus-recipient alloreactive NK cells trigger recipient DCs to synthesize a protein factor in a DNA translation-depended fashion (i.e., blocking DNA transcription in DCs abrogated the DC ability to produce the factor), and release it. Infusion of NK/DC co-culture supernatants containing this factor induced bone marrow and thymic stromal cells to produce IL-7 (p<0.001) and c-Kit ligand (p<0.001) and, thereby, the extraordinarily accelerated maturation of donor DCs, B- and T-cell precursors. Interestingly, in vitro experiments with human thymic stromal cells that support human thymocyte proliferation and differentiation demonstrated the exact same mechanisms. Supernatants from human alloreactive NK cell clones and human (HLA-class I KIR ligand mismatched) allogeneic DCs induced IL-7 production by human thymic stromal cells which in turn supported accelerated proliferation and maturation of human thymocytes (p<0.001). The murine and the human "immune rebuilding" factors displayed biochemical similarities as they both are highly hydrophobic 12KDa molecular weight proteins. Mass spectrometry analysis by stable isotope labeling with amino acids in cell culture (SILAC) identified Beta-2 Microglobulin (B2M) as the newly synthesized protein sharing the above biochemical features and present both in murine and human samples. B2M-KO mice used as recipients of MHC mismatched bone marrow transplant and given donor versus recipient alloreactive NK cells were unable to undergo accelerated immune rebuilding. However, their defect was repaired and accelerated rebuilding of donor-type DCs (p<0.001), B lineage cells (p<0.001) and thymocytes (p<0.001) was restored by the administration of culture supernatants obtained from alloreactive NK cells and wild-type (non-KO) MHC mismatched DCs. Finally, RNA interference experiments that silenced the B2M gene in human DCs resulted in loss of biological activity of supernatants obtained from alloreactive NK cells and B2M-silenced DCs. B2M plays a key role in the immune system as it is known to be part of MHC class I molecules. However, its role in signaling for immune precursor cell development has never been recognized. Here we report the discovery of a novel cellular and molecular pathway initiated by alloreactive NK cells and mediated by B2M that leads to greatly accelerated rebuilding of B and T cells after hematopoietic transplantation. Disclosures No relevant conflicts of interest to declare.
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Reimann, Christian, Andrea Schiavo, Julien Rouiller, et al. "Further Characterization of T-Cellular Precursors Generated From CD34+ Progenitors by Exposure to Immobilized Notch Ligand Delta-Like 4 In Vitro." Blood 116, no. 21 (2010): 3712. http://dx.doi.org/10.1182/blood.v116.21.3712.3712.
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Abstract Abstract 3712 Injection of donor derived T-cellular precursors has been proposed as a novel strategy to shorten delayed reconstitution of the T-lymphoid compartment following HSCT. In the past years, several research groups have successfully generated murine and human T-cellular precursors in vitro using Notchligand-based coculture systems such as OP9-DL1 or Tst-DL4. Murine T-cellular precursors generated in vitro, promoted reconstitution of the T-cellular compartment when applied in murine HSCT-models. In consistency, transfer of human T-cellular precursors, generated in vitro in coculture with OP9-DL1 or Tst-DL4 resulted in enhanced thymic repopulation in NOD/SCID/gc−/− mice. Yet, positive effects on peripheral T-cell reconstitution have not been reported. Moreover, clinical application of OP9-DL1 or Tst-DL4 coculture systems is limited, since they consist of murine stromal cells transduced with either DL1 or DL4. It has been described that exposure of CD34+ cells to immobilized DL4 induces T-cell differentiation in vitro and allows expansion human T-cellular precursors even in absence of stromal cell support. However, the hypothesis that DL4 alone can drive hematopoietic progenitors towards a T-cell fate in vitro, requires more evidence. Here, we further characterized the in vitro and in vivo potential of T-cellular precursors generated by single exposure to DL4. We exposed human CD34+ progenitors to immobilized DL4 in the presence of different cytokine combinations implicated in human haematopoiesis. Within 7 days, CD34+CD7+ and CD34−CD7++ T-cellular precursors emerged in the presence of DL4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while the CD34−CD7++ population further expanded. Two distinct progenitor subsets, CD5+ and CD5-, emerged within the CD34−CD7++ population. The CD34−CD7++CD5+ subset partially acquired CD1a, corresponding to a developmental stage between the early thymic progenitor (ETP) and the prethymocyte (pre-T) stage. Conversely to what observed in the OP9-DL1 system, T-cell development did not progress beyond the pre-T-stage. Indeed, we neither observed more advanced stages of T-cell development, such as immature single positive CD4+ cells, nor complete TCR-rearrangements. 7-day exposure to immobilized DL4 induced a 90-fold increase of T-precursor frequency in CD34+ progenitors (1/8800 before culture vs. 1/90 after culture) as confirmed by limiting dilution assays on OP9-DL1. All T-cellular precursor activity was restricted to cells expressing CD34, CD7 or both (frequency: 1/9). In particular, elevated T-cellular precursor levels were found in the subsets expressing CD7 (CD34+/CD7+ and CD34−/CD7+), while the T-cellular precursor frequency in the CD34+/CD7− subset was equal to that seen in non-cultured CD34+ progenitors. In consistency the CD34−CD7− population did not contain any detectable T-cellular precursors. After 7 day exposure to DL4, cells phenotypically corresponding to T-cellular precursors were transferred into NOD/SCID/gc−/− mice. Within 2 months following HSCT, cells exposed to DL4 were able to reconstitute the recipients' thymus and partially gave rise to peripheral T-cells. When injecting non-cultured CD34+ progenitors, thymic reconstitution was likewise seen 2 months after HSCT. However, intrathymic T-cell development was less advanced and peripheral T-cells were absent. In contrast, cells cultured in presence of a control peptide did not retain any potential to repopulate the recipients' thymus. Our experiments provide further evidence that exposure DL4 induces early human T-cell development and allows generation of large numbers of T-cellular precursors in vitro. These precursors feature phenotypical and molecular properties corresponding to early precursors found in the human thymus. Furthermore, they have an increased potential to further differentiate into mature T-cells in vitro and when transferred into immunodeficient mice. Our preliminary data suggest, that injection of T-cellular precursors accelerates T-cell reconstitution after HSCT and provides further evidence for the feasibility of this novel strategy of immunotherapy. Disclosures: No relevant conflicts of interest to declare.
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Grenier, Julien, Barbara Peyrard, Auria Godard, et al. "New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model." Blood 142, Supplement 1 (2023): 3855. http://dx.doi.org/10.1182/blood-2023-181742.
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Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.
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Palshikar, Mukta G., Maxwell Spurrell, Jack Demaray, et al. "Abstract 5492: Spatial organization and cellular composition of immunity hubs in human colorectal cancer." Cancer Research 84, no. 6_Supplement (2024): 5492. http://dx.doi.org/10.1158/1538-7445.am2024-5492.
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Abstract Background: Among colorectal cancer (CRC) tumors, mismatch repair deficient (MMRd) tumors have more immunogenic neo-antigens, hence more cytotoxic T-cell mediated anti-tumor immunity than mismatch repair proficient (MMRp) tumors. Previously1, we transcriptionally profiled over 300,000 cells from 62 MMRd and MMRp CRC tumors and found an MMRd-unique network of immunologically active transcriptional states enriched in interferon-stimulated genes (ISGs). With 4-color imaging, we showed that this program arises from spatially organized hubs of at least 3 cell types: IFNã-secreting T and interferon-responsive CXCL9/10/11+ myeloid and tumor cells. To characterize the breadth of cells involved in tumor immunity hubs, we must simultaneously measure the expression of 100s of genes in situ. Methods: To describe the cellular composition, structural organization and intercellular spatial interactions of these immunity hubs, we profiled primary untreated resection specimens from 17 CRC donors (n=12 MMRd, 5 MMRp) using multiplexed error-robust FISH (MERFISH) mRNA microscopy. Efforts were made to sample both the tumor body and the invasive border of the tumor. We designed a panel of 479 genes to capture key transcriptional programs from our single-cell atlas1. We wrote analytical pipelines to segment transcripts into cells, select high-quality cells, and organize cells into distinct zones in tissue. To leverage the rich information in the scRNA-seq atlas1, we designed a robust label transfer analysis to assign cell state labels to MERFISH cells by comparison to scRNAseq cells. Results: We profiled 7.5 million high quality MERFISH cells across 21 CRC tissue sections and labeled the cells into 7 major lineages: 63.19% epithelial, 22.52% stromal, 7.75% myeloid, 3.72% T/NK/ILCs, 1.45% plasma, 1.2% B, and 0.17% mast cells. We visualized the spatial location of these cell types. T cells aggregated into 3 distinct regions of tissue: tertiary lymphoid structures (TLS) outside the tumor, and smaller clusters near the invasive border and in the tumor center. We identified immunity hubs defined by colocalized CXCL9/10/11 expressing cells and T cells near the invasive border and in the center of MMRd tumors. As expected, we found enrichment of CXCL13, IFNG, and ISGs such as CD74 and MHCII genes at these locations. In contrast, we found evidence of CCL17+CCL19+LAMP3+ activated dendritic cells and B cells enriched in the TLS. These results support a distinct composition and localization of immunity hubs in tumors, in contrast to TLS outside tumors. Conclusions: Our study defines the composition, spatial organization, and intercellular interactions of immunity hubs, shedding light on the anti-tumor immune response in CRC. Ethics Approval: Approved by the DF/HCC Institutional Review Board (protocol #02-240).(1) Pelka, K. et al. Spatially organized multicellular immune hubs in human colorectal cancer. Cell 184, 4734-4752.e20 (2021) Citation Format: Mukta G. Palshikar, Maxwell Spurrell, Jack Demaray, Han Chen, Scott Engels, Roopa Madhu, Milan Parikh, Izabella Gazmora, Arnav Mehta, Nicolas Fernandez, Colles Price, Jiang He, Sonia Cohen, Karin Pelka, Nir Hacohen, Jonathan Chen, Ilya Korsunsky. Spatial organization and cellular composition of immunity hubs in human colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5492.
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Savino, Wilson, and Ailin Lepletier. "Thymus-derived hormonal and cellular control of cancer." Frontiers in Endocrinology 14 (July 17, 2023). http://dx.doi.org/10.3389/fendo.2023.1168186.
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The thymus gland is a central lymphoid organ in which developing T cell precursors, known as thymocytes, undergo differentiation into distinct type of mature T cells, ultimately migrating to the periphery where they exert specialized effector functions and orchestrate the immune responses against tumor cells, pathogens and self-antigens. The mechanisms supporting intrathymic T cell differentiation are pleiotropically regulated by thymic peptide hormones and cytokines produced by stromal cells in the thymic microenvironment and developing thymocytes. Interestingly, in the same way as T cells, thymic hormones (herein exemplified by thymosin, thymulin and thymopoietin), can circulate to impact immune cells and other cellular components in the periphery. Evidence on how thymic function influences tumor cell biology and response of patients with cancer to therapies remains unsatisfactory, although there has been some improvement in the knowledge provided by recent studies. Herein, we summarize research progression in the field of thymus-mediated immunoendocrine control of cancer, providing insights into how manipulation of the thymic microenvironment can influence treatment outcomes, including clinical responses and adverse effects of therapies. We review data obtained from clinical and preclinical cancer research to evidence the complexity of immunoendocrine interactions underpinning anti-tumor immunity.
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Stamatis, Pavlos, Carl Turesson, Despina Michailidou, and Aladdin J. Mohammad. "Pathogenesis of giant cell arteritis with focus on cellular populations." Frontiers in Medicine 9 (November 17, 2022). http://dx.doi.org/10.3389/fmed.2022.1058600.
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Giant cell arteritis (GCA), the most common non-infectious vasculitis, mainly affects elderly individuals. The disease usually affects the aorta and its main supra-aortic branches causing both general symptoms of inflammation and specific ischemic symptoms because of the limited blood flow due to arterial structural changes in the inflamed arteries. The pathogenesis of the GCA is complex and includes a dysregulated immune response that affects both the innate and the adaptive immunity. During the last two decades several studies have investigated interactions among antigen-presenting cells and lymphocytes, which contribute to the formation of the inflammatory infiltrate in the affected arteries. Toll-like receptor signaling and interactions through the VEGF-Notch-Jagged1 pathway are emerging as crucial events of the aberrant inflammatory response, facilitating among others the migration of inflammatory cells to the inflamed arteries and their interactions with the local stromal milieu. The increased use of checkpoint inhibitors in cancer immunotherapy and their immune-related adverse events has fed interest in the role of checkpoint dysfunction in GCA, and recent studies suggest a dysregulated check point system which is unable to suppress the inflammation in the previously immune-privileged arteries, leading to vasculitis. The role of B-cells is currently reevaluated because of new reports of considerable numbers of plasma cells in inflamed arteries as well as the formation of artery tertiary lymphoid organs. There is emerging evidence on previously less studied cell populations, such as the neutrophils, CD8+ T-cells, T regulatory cells and tissue residing memory cells as well as for stromal cells which were previously considered as innocent bystanders. The aim of this review is to summarize the evidence in the literature regarding the cell populations involved in the pathogenesis of GCA and especially in the context of an aged, immune system.
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Thieme, Elana, Tingting Liu, Nur Bruss, et al. "Dual BTK/SYK inhibition with CG-806 (luxeptinib) disrupts B-cell receptor and Bcl-2 signaling networks in mantle cell lymphoma." Cell Death & Disease 13, no. 3 (2022). http://dx.doi.org/10.1038/s41419-022-04684-1.
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AbstractAberrant B-cell receptor (BCR) signaling is a key driver in lymphoid malignancies. Bruton tyrosine kinase (BTK) inhibitors that disrupt BCR signaling have received regulatory approvals in therapy of mantle cell lymphoma (MCL). However, responses are incomplete and patients who experience BTK inhibitor therapy failure have dire outcomes. CG-806 (luxeptinib) is a dual BTK/SYK inhibitor in clinical development in hematologic malignancies. Here we investigated the pre-clinical activity of CG-806 in MCL. In vitro treatment with CG-806 thwarted survival of MCL cell lines and patient-derived MCL cells in a dose-dependent manner. CG-806 blocked BTK and SYK activation and abrogated BCR signaling. Contrary to ibrutinib, CG-806 downmodulated the anti-apoptotic proteins Mcl-1 and Bcl-xL, abrogated survival of ibrutinib-resistant MCL cell lines, and partially reversed the pro-survival effects of stromal microenvironment-mimicking conditions in primary MCL cells. Dual BTK/SYK inhibition led to mitochondrial membrane depolarization accompanied by mitophagy and metabolic reprogramming toward glycolysis. In vivo studies of CG-806 demonstrated improved survival in one of the two tested aggressive MCL PDX models. While suppression of the anti-apoptotic Bcl-2 family proteins and NFκB signaling correlated with in vivo drug sensitivity, OxPhos and MYC transcriptional programs were upregulated in the resistant model following treatment with CG-806. BAX and NFKBIA were implicated in susceptibility to CG-806 in a whole-genome CRISPR-Cas9 library screen (in a diffuse large B-cell lymphoma cell line). A high-throughput in vitro functional drug screen demonstrated synergy between CG-806 and Bcl-2 inhibitors. In sum, dual BTK/SYK inhibitor CG-806 disrupts BCR signaling and induces metabolic reprogramming and apoptosis in MCL. The Bcl-2 network is a key mediator of sensitivity to CG-806 and combined targeting of Bcl-2 demonstrates synergy with CG-806 warranting continued exploration in lymphoid malignancies.
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Santamaria Costa, X., R. Beatriz, K. Nanda, et al. "O-218 Single-cell RNA sequencing reveals that Ashermańs syndrome is caused by chronic inflammation that induces differential molecular and cellular cartography in the human endometrium." Human Reproduction 37, Supplement_1 (2022). http://dx.doi.org/10.1093/humrep/deac105.137.
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Abstract Study question What is the differential cellular, transcriptomic and immunological differences of the human endometrium in AS versus healthy patients at single cell resolution? Summary answer The epithelial fraction is decreased, the myeloid and lymphoid cell lineages increased with an altered inflammation, inhibition of angiogenesis and EM abnormal remodeling. What is known already Asherman’s Syndrome (AS) is an acquired pathological condition, defined by the presence of intrauterine adhesions (IUAs) causing the uterine walls to adhere to one another resulting in menstrual abnormalities, pelvic pain, infertility, recurrent miscarriage, and abnormal placentation. However, the underlaying cellular, transcriptomic and immunological mechanisms at the single-cell level that occur in AS have not been investigated. Study design, size, duration Single cell RNA-seq (scRNA-seq) was performed on 41,854 cells corresponding to endometrial biopsies from a total of 7 individuals with severe AS (AFS classification 1998). These patients were involved in a phase I/II, prospective, non-randomized, uncontrolled, multicenter, interventional clinical trial authorized by the Spanish Medicines Agency (AEMPS)(2016-003973-23). Control healthy endometrium was represented by 68,026 cell transcriptomes from our previous work (Wang et al. 2020). Participants/materials, setting, methods Seven patients were included. Ultrasound and hysteroscopies were performed in mid secretory phase. Endometrial specimens were digested with collagenase and filtered. Epithelial cells were digested with trypsin and red blood cells removed. After MACS live enrichment, cells were loaded onto ChromiumNext GEM (10xGenomics). Libraries were sequenced in a Novaseq, and reads processed with CellRanger. Quality filtering, normalization, clustering and differential expression analysis were applied from ‘Seurat’ package. Functional enrichment analysis was computed using ‘escape’ package. Main results and the role of chance In total 109,880 cell transcriptomes were compared and found changes in cell population ratios in two specific cell types. First, the epithelial fraction was decreased in AS compared to healthy condition (26.53% vs 45.7%, respectively) specifically the epithelium representing the opening of the window of implantation (WOI) (0.25% vs 2.01%respectively), and the ciliated epithelium (0.84% vs 6.12%, respectively). Second, the myeloid and lymphoid cell lineages, which are much more abundant in AS samples. Macrophages (1.97% vs 0.24%, respectively), CD8+ T cells (3.71% vs 1.34%, respectively), and CD8- T cells (2.28% vs 0.55%, respectively). In addition, there was a different transcriptomic composition represented by three differential linked clusters related to AS condition. First, a unique stromal cluster labelled as stromal_ACTA2 that express genes related to contractile functions (ACTA2, MYH11, DES). Second, a specific AS epithelium cluster closely related to antigen processing and presentation of HLA class II family genes. Third, a KRT8 ACTA2 cluster composed by genes related to collagen (COL3A1 and COL1A1) and IGFBP5. Enrichment analysis performed with ssGSEA revealed the functional impact of the AS condition identifying an increase in different GO terms related to tissue damage, pro-inflammatory processes, inhibition of angiogenesis. Limitations, reasons for caution Despite these promising results, this is an study in progress to be completed with 10 patients Wider implications of the findings These findings describe for the first time the pathophysiology of AS at single cell level with the functional involvement of inflammation, fibrosis, and defective angiogenesis in this pathological condition. Trial registration number Eudra CT 2016-003975-23
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García-Ceca, Javier, Sara Montero-Herradón, Ana González, Rosa Plaza, and Agustín G. Zapata. "Altered thymocyte development observed in EphA4-deficient mice courses with changes in both thymic epithelial and extracellular matrix organization." Cellular and Molecular Life Sciences 79, no. 11 (2022). http://dx.doi.org/10.1007/s00018-022-04610-w.
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AbstractEph receptors and their ligands, Ephrins, are involved in the thymocyte-thymic epithelial cell (TEC) interactions, key for the functional maturation of both thymocytes and thymic epithelium. Several years ago, we reported that the lack of EphA4, a Eph of the subfamily A, coursed with reduced proportions of double positive (DP) thymocytes apparently due to an altered thymic epithelial stroma [Munoz et al. in J Immunol 177:804–813, 2006]. In the present study, we reevaluate the lymphoid, epithelial, and extracellular matrix (ECM) phenotype of EphA4−/− mice grouped into three categories with respect to their proportions of DP thymocytes. Our results demonstrate a profound hypocellularity, specific alterations of T cell differentiation that affected not only DP thymocytes, but also double negative and single positive T cell subsets, as well as the proportions of positively and negatively selected thymocytes. In correlation, thymic histological organization changed markedly, especially in the cortex, as well as the proportions of both Ly51+UEA-1− cortical TECs and Ly51−UEA-1+ medullary TECs. The alterations observed in the expression of ECM components (Fibronectin, Laminin, Collagen IV), integrin receptors (VLA-4, VLA-6), chemokines (CXCL12, CCL25, CCL21) and their receptors (CXCR4, CCR7, CCR9) and in vitro transwell assays on the capacity of migration of WT and mutant thymocytes suggest that the lack of EphA4 alters T-cell differentiation by presumably affecting cell adhesion between TECs and T-TEC interactions rather than by thymocyte migration.