WT Stroma Research Articles

Lyn Kinase Contributes to the Reprogramming of Fibroblasts Promoting Chronic Lymphocytic Leukemia Progression

Growth of chronic lymphocytic leukemia cells strongly depends on a nurturing microenvironmental niche that is specifically primed by diverse, bi-directional interactions to promote leukemic homing, proliferation and progression. The Src-family kinase Lyn was previously identified by our group as a key factor for the formation of this pro-leukemic niche and for the expansion of CLL cells, using the Eµ-TCL1 mouse model. In order to attribute the pro-leukemic function of Lyn to a specific cell type, chimeric mice with lineage-specific defects of Lyn within hematopoietic or non-hematopoietic compartments were generated by irradiating BL6J-mice lethally and restoring their hematopoietic system with Lyn-WT or Lyn-KO stem cells. Consecutively mice were xenotransplanted with TCL1+-malignant cells. Lyn deficiency within the non-hematopoietic compartment decelerated leukemic expansion to a higher degree than did Lyn deficiency within the hematopoietic compartment. Completely Lyn deficient mice showed a more prominent retardation of leukemic expansion compared with both lineage specific Lyn deficient mouse strains, suggesting an additive effect of the two distinct compartments for leukemic expansion. In focusing on the non-hematopoietic fibroblastic bystander cells, primary human CLL cells were cocultured in vitro with Lyn-deficient mouse embryonic fibroblasts as well as Lyn-KO human HS5 cells, generated via the CRISPR-Cas9 system, and leukemic cell survival was assessed over time. All Lyn-deficient fibroblasts showed a significantly reduced feeding capacity for CLL cells compared to WT stroma, indicating the functional relevance of Lyn in leukemia-associated fibroblasts. Subsequently, transcriptomic, proteomic and phosphoproteomic alterations related to Lyn-KO in HS5 cells were comprehensively analyzed, revealing a surprisingly extensive change in gene and protein expression pattern that appeared to be regulated mainly at the transcriptional level. The differentially expressed genes were remarkably often extracellular matrix (ECM)-, cytoskeleton- or cytokine-associated. GO-term enrichment analysis additionally suggested a correlation with ECM processes. Therefore, we hypothesized that Lyn-deficiency might induce transcriptional changes of the cancer-associated fibroblast (CAF)-like phenotype, thus leading to a reduction in leukemic feeding capacity. The diminished expression of several CAF-makers congruent with this reduced activation status was validated in Lyn-KO fibroblasts, as well as the transcriptionally regulated differential expression of chosen target genes. Amongst those, the deubiquitinating enzyme UCHL1 was most abundantly reduced in Lyn-KO HS5 cells, showing an almost complete loss of mRNA and protein expression. Application of a specific UCHL1-inhibitor -in a dose without toxic effects on CLL cells - to CLL-stroma coculture resulted in a significantly hampered feeder effect and reduced CLL cell survival, implying a functional relevance of microenvironmental UCHL1 for stromal support in our system. Additionally, stroma cell death induced by higher drug concentrations in WT cells was completely prohibited in Lyn-KO stroma, illustrating the importance of Lyn for regulating UCHL1 expression and function. In summary, we propose that the Lyn kinase contributes to the formation of a supportive microenvironment via the transcriptional reprogramming of stroma fibroblasts into a "CAF-like" phenotype, which echances viability of CLL cells. In addition, UCHL1 might be a potentially druggable mediator of this activation process. Disclosures Hallek: Roche, Gilead Sciences, Inc., Mundipharma, Janssen, Celgene, Pharmacyclics, AbbVie: Honoraria, Research Funding, Speakers Bureau.

Bone Marrow Niche - Multiple Myeloma Cross-Talk Generates Bortezomib Resistance

Bortezomib (BTZ), a proteasome inhibitor that induces accumulation of misfolded proteins and apoptosis in highly secretory cells, has improved survival in multiple myeloma (MM) but fails to achieve cure. It has been postulated that a population of MM cells, phenotypically similar to B-cells, are intrinsically resistant to BTZ, and contribute to minimal residual disease (MRD) and relapse (Matsui et al., 2008).Bone marrow (BM) stroma expresses CYP26 enzymes and creates a retinoic acid (RA)-low environment that prevents differentiation of normal and malignant cells (Meng et al., 2015). Since RA promotes plasma cell differentiation, Ig secretion (Ertesvag et al., 2007) and thus, ER-stress (Xu et al., 2007), we tested if the BM niche induces BTZ resistance by promoting a B-cell program in MM. Consistent with this, MM cells (H929 and MM1S) cultured under low RA conditions (co-cultured with BM stroma or treated with AGN194310, a pan-RA receptor inhibitor) up regulated B-cell markers (BCL6), and down regulated genes associated with plasma cell differentiation and BTZ sensitivity (XBP1s, Blimp, CHOP) (Fig 1A). In addition, inhibition of stromal CYP26 via R115866 reversed these gene expression changes in stroma co-culture conditions (Fig 1A).To verify whether a low-RA environment promotes BTZ resistance, we incubated MM cells with BM mesenchymal cells for 5 days. Then, MM cells were separated from stroma, and treated with BTZ. Incubation with BM stroma induced BTZ resistance of MM cells, which was completely overcome by CYP26 inhibition or by the CYP26 resistant retinoid IRX5183 (Fig 1B). Moreover, MM cells pretreated with AGN for 5 days were also resistant to BTZ (Fig 1B). Interestingly, this BTZ resistant phenotype was preserved for up to 48h upon removal of MM cells from stromal co-culture (Fig 1C).Recent studies suggest that malignant cells remodel their microenvironments to build a more protective niche (Schepers et al., 2013). Consistent with this, we found that stromal CYP26A1 was highly upregulated after co-culture with MM cells. Supernatant from MM cells had similar effects, implicating a soluble factor on this interaction. Since secretion of SHH was implicated in chemotherapy resistance in MM (Liu et al., 2014), we tested if MM-induced CYP26 up-regulation was dependent on SHH signaling. As hypothesized, treatment with the Smo antagonist cyclopamine, or the use of SMO knockout (KO) stromal cells, overcame CYP26A1 up regulation by MM cells (Fig 1D)To test if cell extrinsic SHH signaling in stromal niche contributes to BTZ resistance by modulating RA pathway in MM cells, we modeled MM-BM stroma interactions in a xenograft setting. Each mouse carried two subcutaneous tumors consisting of Luciferase expressing MM1S cells and SmoFl/Fl BM stroma cells, transduced with either a control vector (WT stroma) or Cre-recombinase (Smo KO stroma) (Fig E). Mice were treated with IRX5183, BTZ, or the combination. Consistent with our in vitro data, tumors with Smo KO stroma showed a significant response to BTZ, while those with WT stroma were refractory, as determined by exponential increase in bioluminescence. However, the combination of IRX+BTZ resulted in a dramatic and equivalent response regardless of the phenotype of the stromal compartment (Fig 2A, 2B). Moreover, tumors with Smo KO stroma, or treated with IRX, showed decrease expression of B-cell markers, and up-regulation of genes associated with plasma cell differentiation and BTZ sensitivity.In conclusion, we show that the BM niche promotes a B-cell program in MM, characterized by a low ER-stress and BTZ resistance. This niche-induced program is maintained even after displacement from the BM, which may have important implications for the use of mobilization-sensitization strategies. Finally, we propose the existence of a bi-directional crosstalk between MM cells and their respective niches. In our model, MM cells via secretion of SHH modify BM stroma to create RA-low environments, induce BTZ resistance and contribute to MRD. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Effects of the loss of conjunctival Muc16 on corneal epithelium and stroma in mice.

To examine the role of conjunctival Muc16 in the homeostasis of the ocular surface epithelium and stroma using Muc16-null knockout (KO) mice. We used KO mice (n = 58) and C57/BL6 (WT) mice (n = 58). Histology and immunohistochemistry were employed to analyze the phenotypes in the ocular surface epithelium. The expression of phospho-Stat3, AP-1 components, interleukin 6 (IL-6), and tumor necrosis factor-α (TNFα) in the cornea and conjunctiva was examined. The shape of the nuclei of corneal epithelial cells was examined to evaluate intraepithelial cell differentiation. Epithelial cell proliferation was studied using bromo-deoxyuridine labeling. Finally, the wound healing of a round defect (2-mm diameter) in the corneal epithelium was measured. The keratocyte phenotype and macrophage invasion in the stroma were evaluated after epithelial repair. The loss of Muc16 activated Stat3 signal, affected JunB signal, and upregulated the expression of IL-6 in the conjunctiva. Basal-like cells were observed in the suprabasal layer of the corneal epithelium with an increase in proliferation. The loss of Muc16 accelerated the wound healing of the corneal epithelium. The incidence of myofibroblast appearance and macrophage invasion were more marked in KO stroma than in WT stroma after epithelial repair. The loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma. The mechanism might include the upregulation of the inflammatory signaling cascade (i.e., Stat3 signal, and IL-6 expression in the KO conjunctiva). Current data provides insight into the research of the pathophysiology of dry eye syndrome.

P190-B RhoGAP Is Critical for Hematopoietic Stem Cell Niche Regulation

Abstract 221Hematopoiesis is regulated by components of the stromal microenvironment, so-called niche. Although the concept of hematopoietic stem and progenitor cell (HSC/P) niche is well known, its molecular regulation remains ill-defined. Here, we provide evidence that p190-B GTPase Activating Protein (p190-B), a negative regulator of Rho activity, is a regulator of mesenchymal/stromal cell functions necessary for normal hematopoiesis during fetal development. Mice lacking p190-B die before birth. At day 14.5 post coitum, p190-B−/− embryos are paler than WT embryos with a 40% lower hematocrit. Cellularity and numbers of burst forming-unit (BFU) erythroid (E), colony forming-unit (CFU)-granulo-monocytic and CFU-erythroid per p190-B−/− fetal livers (FL) were 50% lower than WT. In addition, the frequency of LongTerm-HSC (LinnegScaposKitposCD150posCD48neg), ShortTerm-HSC (LSK-CD150posCD48pos), and progenitors (LK) appeared significantly reduced in the bone marrow of e17.5/18 embryos. These data suggest a defect in fetal hematopoiesis. However, p190-B−/− FL cells were able to fully reconstitute hematopoiesis of adult irradiated recipients (Xu, Blood 2009). Mice reconstituted with p190-B−/− FL cells exhibited bone marrow content, white blood count, red blood count and hematocrit similar to those reconstituted with WT cells. Furthermore, hematopoietic recovery of p190-B−/− reconstituted animals following 5-Fluorouracil or phenylhydrazine-induced stress was comparable to that of WT. Therefore, FL HSC/P retain hematopoietic potential, which suggests a non-cell autonomous defect in p190-B−/− embryos. In the present study, we examined in more detail this possibility. The numbers of CFU-fibroblast (CFU-F) were 2-fold lower in p190-B−/− FL compared to WT FL. We next examined the hematopoietic supportive capacity of p190-B−/− FL microenvironment using stromal cell cultures derived from e14.5 FL. No noticeable differences were observed during the establishment of the stromal cell cultures. p190-B−/− stromal cells exhibited a spindle-like shape and expressed CD90, CD44 and Sca-1 but not CD45, CD31 or CD11b similarly to WT stromal cells. Cobblestone Area Forming Cell assays (CAFC) was performed in co-culture between various number of WT BM cells and p190-B−/− and WT stromal cells. We examined 5 independent stromal cell cultures from both genotypes. Frequency of CAFC was 75% lower on p190-B−/− stroma than WT stroma at both 1 week (1 in 5 × 10 3 versus 1 in 21 × 10 3, n = 5 p < 0.01) and 2 weeks (1 in 21 × 10 3 versus 1 in 87 × 10 3, n = 4 p < 0.01) in culture. CFU with cells recovered from one week coculture was also performed. Cocultures with p190-B−/− stroma gave rise to 2-fold less CFU than with WT stroma (538+90 versus 255+81, p < 0.01, one representative experiment of 4). Using competitive repopulation assay, we then assessed the in vivo repopulation ability of CD45.2+ hematopoietic cells co-cultured for one week on each stroma. Fifteen weeks following transplantation, the frequency of CD45.2+ cells in the peripheral blood of mice that received cells cocultured on p190-B−/− stroma was dramatically reduced compared to mice that received cells cocultured on WT stroma (0.43% + 0.37 versus 32.4% + 7.5, n = 6, p < 0.01), equivalent to a 100-fold difference in calculated competitive repopulation unit. Therefore, p190-B−/− stroma exhibited defective hematopoietic supportive activity. Interestingly, p190-B−/− stromal cells, like WT, showed characteristic of mesenchymal stem cells (MSC), i.e. ability to give rise to CFU-F and to differentiate to adipocytes and osteoblasts. They express relatively high level of nestin and osteopontin. At a mechanistic level, gene expression analysis of molecules known to play a critical role in HSC maintenance in the niche indicated 200-fold downregulation of Kit-ligand and Wnt3a in p190-B−/− stroma compared to WT stroma (p<0.05). Surprisingly, BMP4, angiopoietin and CXCL12 were upregulated in p190-B−/− stroma, 35-fold, 14-fold and 16-fold, respectively (p<0.05). Hence, p190-B expression in MSC/stromal cell microenvironment fine-tunes niche regulatory pathways to maintain normal hematopoiesis. Therefore, p190-B may be a critical regulator of the mesenchymal stem/hematopoietic stem and progenitor niche. Disclosures:No relevant conflicts of interest to declare.

Relaxin-Induced Proliferation of Epithelial and Stromal Cells in the Mouse Cervix Requires Estrogen Receptor-Alpha Within Stromal Cells Binding to Estrogen Response Elements.

Relaxin is secreted by the corpora lutea during the second half of pregnancy in mice, rats and pigs. Relaxin stimulates marked growth and extensibility of the lower reproductive tract and thereby facilitates birth in these species. Relaxin-dependent growth of the cervix and vagina is accompanied by increases in both stromal and epithelial cells. Recently, we reported that relaxin receptor-expressing cells in the stroma are necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells of mouse cervix and vagina. Estrogen is secreted in increasing amounts by developing follicles as pregnancy progresses in rats and mice. An understanding of relaxin's growth-promoting actions on the lower reproductive tract in rodents requires knowledge of the cellular and molecular actions of estrogen, because relaxin-induced growth of the cervix and vagina is obligatorily dependent upon estrogen signaling through estrogen receptor alpha (ERα). There were two objectives in this study. The objective of experiment 1 was to determine whether stromal and/or epithelial ERα is required for relaxin to promote proliferation of stromal and epithelial cells in the mouse cervix. Tissue recombinants were prepared with stroma (St) and epithelium (Ep) from wild type (wt) and ERα knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of intact syngeneic female mice. Following 3 weeks of transplant growth, hosts were ovariectomized and fitted with silicon implants containing estradiol-17β (designated day 1 of treatment). Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on day 8 through 0600 h on day 10 of treatment. To enable morphometric immunohistochemical analysis of cell proliferation, 5-bromo-2'-deoxyuridine (BrdU) was injected sc 10 h before cervices were collected at 1000 h on day 10. Relaxin markedly increased proliferation of epithelial and stromal cells in tissue recombinants containing wt stroma (P< 0.001), but not in tissue recombinants prepared with ko stroma, regardless of whether epithelium was derived from wt or ko mice. The objective of experiment 2 was to determine if ERα enables relaxin's proliferative effects on the cervix by acting through the classical signal transduction pathway in which the estrogen-ERα complex binds to specific estrogen response elements (ERE) in regulatory regions of target genes or through a non-classical pathway in which ERα binding to an ERE is not required. Wild type mice (ERERα+/+), mutant ERα DNA binding domain knock-in (NERKI) mice (ERα+/AA & ERα-/AA) and ERα null mice (ERα-/-) were ovariectomized at 4 weeks of age and treated with estrogen or estrogen plus relaxin as described for experiment 1. Following the administration of relaxin, the rate of cell proliferation increased dramatically in the two groups that contained wt ERα (ERα+/+ and ERα+/AA) but not in groups ERα-/AA and ER-/-. that lack a classical ER signaling pathway. In conclusion, this study demonstrates that estradiol-17β acting through ERα-expressing cells in the stroma, using a classical signaling pathway, is necessary for relaxin to promote marked proliferation in both stromal and epithelial cells of the mouse cervix. (platform)

Relaxin Acts on Stromal Cells to Promote Epithelial and Stromal Proliferation and Inhibit Apoptosis in the Mouse Cervix and Vagina

The objective of this study was to determine whether stromal and/or epithelial relaxin receptor (LGR7) is required for relaxin to promote proliferation and inhibit apoptosis of stromal and epithelial cells in the mouse cervix and vagina. Tissue recombinants were prepared with stroma (St) and epithelium (Ep) from wild-type (wt) and LGR7 knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep, and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of intact syngeneic female mice. After 3 wk of transplant growth, hosts were ovariectomized and fitted with silicon implants containing progesterone and estradiol-17beta (designated d 1 of treatment). Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on d 8 through 0600 h on d 10 of treatment. To evaluate cell proliferation, 5-bromo-2'-deoxyuridine was injected sc 10 h before cervices and vaginas were collected at 1000 h on d 10. Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick end labeling was used to quantify apoptosis. Relaxin markedly increased proliferation and decreased apoptosis of epithelial and stromal cells in tissue recombinants containing wt stroma (P < 0.01) but had no effect on tissue recombinants prepared with ko stroma, regardless of whether epithelium was derived from wt or ko mice. In conclusion, this study shows that LGR7-expressing cells in the stroma are both necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells of cervix and vagina, whereas epithelial LGR7 does not affect these processes.

β-Catenin Expression in Cultured Bone Marrow Stromal Cells Is Required To Maintain Production of Osteoblasts and Hematopoietic Progenitor Cells.

The canonical Wnt signaling pathway is defined by Wnt ligand-mediated stabilization and nuclear translocation of β-catenin to induce target gene expression. This pathway has been demonstrated to regulate differentiation of mesenchymal tissue, which includes the cell types (e.g. osteoblasts, myofibroblasts, adipocytes) that comprise the stromal cells of the hematopoietic microenvironment. We hypothesized that loss of canonical Wnt signaling would result in disruption of the ability of stromal cells to support hematopoiesis. To test this hypothesis, we generated transgenic mice that expressed conditonal loss of function β-catenin alleles along with Cre-recombinase under the control of the inducible Mx1 promoter, which is active in bone marrow. We induced excision of β-catenin by injecting β-cateninlox/lox Mx-cre+/cre mice with 10 doses of 300 mg/ml pIpC. Whole bone marrow from treated (KO) and untreated (WT) animals was used to establish Dexter stromal cultures with an input of 1 × 106 cells/cm2 culture surface area. PCR performed on DNA isolated from KO stromal cells showed that nearly 100% deletion of β-catenin occurred with this regimen. To determine the ability of KO stroma to support hematopoiesis, irradiated KO and WT stromal cultures were seeded with 4 × 104 normal lin− cells/cm2. There were no differences in cell expansion, cell cycle activity, or apoptosis between hematopoietic cells cultured on WT vs. KO stroma. We determined the capacity of β-catenin deficient stroma to maintain hematopoietic progenitors by measuring myeloid CFU formation after 1, 2, and 3 weeks in culture. After 1 week, hematopoietic cells cultured on WT stroma contained 5-fold more CFU-GM (151.7 ± 21.4 CFU-GM/1×104 cells) than cells cultured on KO stroma (28.7 ± 4.9; n = 6, p < .001). Similar differences in CFU-GM formation were observed after 2 weeks (WT 46.5 ± 8.0 vs. KO 10.3 ± 1.7; n = 6, p< .001) and 3 weeks (WT 16.5 ± 2.8 vs. KO 2.6 ± 1.5; n = 6, p < .001) in culture. This decrease in the production of hematopoietic progenitor cells was not due to decreased numbers of stromal cells as the average number of KO stromal cells (4.8 ± 0.07 × 104/cm2) was greater than WT (3.7 ± 0.7 × 104/cm2; n = 3, p = .05). We also determined the ability of WT and KO mesenchymal progenitors to generate fibroblast colonies (CFU-F) and found no difference between WT (17 ± 1.8 CFU-F/1 × 106 bone marrow cells) and KO (15.8 ± 3.5; n = 4, p = .54). Canonical Wnt signaling has been proposed to regulate the differentiation of mesenchymal stem cells into osteoblasts. Since osteoblasts contribute to the proper regulation of hematopoiesis, we hypothesized that the depletion of hematopoietic progentiors in KO stromal cultures is due to a reduction in the number of osteoblasts. To detect osteoblasts in vitro, we performed histochemical staining to detect alkaline phosphatase (ALP) activity in WT and KO stromal cultures and scored the positive cells. We observed a significant 50% reduction in the percentage of ALP+ cells in KO stroma (13.2 ± 4.8%) compared to WT (28.0 ± 7.9%) (n = 3, p = .05). In summary, these data indicate that loss of canonical Wnt signaling results in decreased support of hematopoietic progenitors and osteoblasts. From these data, we propose a model in which canonical Wnt signaling is necessary to maintain normal numbers of osteoblasts within the bone marrow stroma and that loss of β-catenin leads to a decrease in the number of osteoblasts and a subsequent reduction in the ability of the stroma to support hematopoiesis.

Paracrine signaling through the epithelial estrogen receptor alpha is required for proliferation and morphogenesis in the mammary gland.

Estradiol is a major regulator of postnatal mammary gland development and thought to exert its effects through estrogen receptor alpha (ERalpha) expressed in the mammary gland stroma and epithelium. Previous studies, however, were confounded by the use of an ERalpha mutant strain that retains some of the protein with transactivation activity. Here, we use an ERalpha-/- mouse strain in which no ERalpha transcript can be detected to analyze mammary gland development in the complete absence of ERalpha signaling. The ERalpha-/- females show no development beyond a rudimentary ductal system. By grafting ERalpha-/- epithelium or stroma in combination with ERalpha WT stroma or epithelium, we show that the primary target for estradiol is the mammary epithelium, whereas a direct response of the mammary stroma is not required for mammary gland development to proceed normally. Mammary glands reconstituted with ERalpha-/- mammary epithelium exposed to pregnancy hormones show increased transcription of milk protein genes, indicating that ERalpha signaling is not an absolute requirement for a transcriptional response to pregnancy hormones. When ERalpha-/- mammary epithelial cells are in close vicinity to ERalpha WT cells, they proliferate and contribute to all aspects of mammary gland development, indicating that estradiol, like progesterone, orchestrates proliferation and morphogenesis by a paracrine mechanism, affecting nearby cells in the mammary epithelium.

Calcium in the toad ( Bufo marinus) cornea: Binding by the stroma

1. 1. The Ca concentration in the toad ( Bufo marinus) cornea was 2.6 mmol/kg wet wt compared at 1.0 mmol/l in the bathing aqueous humor and 2.8 mmol/kg wet wt in the separated corneal stromal layer. Cell Ca content was calculated to be about 1.8 mmol/kg wet wt. 2. 2. About 80% of the total Ca appears to be sequestered or bound to tissue components most of which (68% of the total) is associated with the stroma (2.2 mmol/kg wet wt stroma). 3. 3. About 85–90% of the Ca in the stroma is readily exchangeable with external 45Ca. 4. 4. The loss of accumulated 45Ca from the stroma was measured in vitro. This efflux of the isotope was enhanced by multivalent ions and was greatest when Ca 2+ or La 3+ was present in the external media. Other alkaline earth metal ions were not as effective. The relative effectiveness of this displacement of 45Ca was Ca = La > Sr > Ba > Mg. 5. 5. The results suggest that the Ca 2+ is bound by the amphibian stroma at sites that have a preference or specificity for this divalent ion as compared to the other alkaline earth metals. 6. 6. The possible functional role of this bound Ca is discussed.