Untreated Stroma Research Articles

Abstract 3963: Mass spectrometry reveals a downregulation of calreticulin after neoadjuvant chemotherapy in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinomas are one of the deadliest cancer types with limited response to current therapies. Thus, this study examined changes in the proteome of epithelial cells in pancreatic cancer after neoadjuvant chemotherapy with gemcitabine, to identify new therapeutic targets. Methods: Upon receiving patient consent, fresh pancreatic cancer tissue was received from surgical tumor resections at the University Hospital of Bonn. We mechanically and enzymatically digested and purified the tissue to create single-cell suspensions. After staining the cells against CD45 und CD326, we purified epithelial cells, blood cells, and stroma through FACS. To analyze their proteome, the sorted cells were lysed in TEAB buffer and the protein lysates were measured by the mass spectrometry core facility at the University of Bonn. We then analyzed the data using DAVID and Shiny GO. Results: Gene enrichment analyses showed a significantly reduced expression of calreticulin after neoadjuvant chemotherapy in epithelial cells. A direct comparison between untreated and treated epithelium cancer cells using a heat map showed a decreased expression of actin and enzymes of glycolysis in treated cancer cells. Moreover, untreated epithelial cells showed an increase of proteins related to MHC class Ib protein complex assembly and antigen processing and presentation of endogenous peptide antigen via MHC class. In contrast, treated cells showed a higher presence of proteins for nutrient supply from the blood. Furthermore, untreated stroma cells exhibited an increased number of proteins responsible for mesenchymal migration and for actin filament fragmentation. Conclusion: This study shows that neoadjuvant chemotherapy with gemcitabine leads to distinct changes in the proteome of tumor cells in pancreatic cancers. In particular, a decreased expression of the “eat-me-signal” calreticulin impairs macrophage-mediated cancer cell phagocytosis, thereby indicating a mechanism by which cancer cells protect themselves after chemotherapy. Citation Format: Michael C. Stoffel, Elisabeth BH Dingendorf, Dahlia L. Kittel, Glen Kristiansen, Tristan Lerbs. Mass spectrometry reveals a downregulation of calreticulin after neoadjuvant chemotherapy in pancreatic cancer. [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 3963.

Genipin Delays Corneal Stromal Enzymatic Digestion.

PurposeTo evaluate the use of genipin in delaying enzymatic digestion of corneal stroma.MethodsHuman corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy.ResultsGenipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis.ConclusionsGenipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging.Translational RelevanceThis study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity.

Azacitidine Directly Acts on the Mesenchymal Stromal Cell Compartment in Myelodysplastic Syndromes to Alter Niche Function and Suppress Malignant Hematopoeitic Stem/Progenitor Cells

Background: Hematopoietic stem/progenitor cells (HSPC) are highly dependent on interaction with the specific bone marrow microenvironment (niche) which supports their survival and directs their differentiation. Increasing evidence suggests that the niche plays a central role in the development of clonal stem cell disorders such as myelodysplastic syndromes (MDS). Malignant clonal HSPC in MDS have been shown to reprogram mesenchymal stromal cells (MSC) to promote disease progression. In turn, the altered stromal microenvironment in MDS may directly impair healthy hematopoiesis. Accordingly, MSC isolated from MDS patients have shown phenotypical and functional abnormalities, which were not observed in patients successfully treated with azacitidine (AZA), currently the standard of care for higher-risk MDS patients. Although AZA is a potent inhibitor of DNA methyltransferases and is thus considered a hypomethylating agent, the exact anti-leukemic mechanisms are not understood and a clear correlation between DNA demethylation and response to AZA has not been demonstrated thus far. Hence, we looked beyond the current focus of AZA effects on clonal hematopoiesis and hypothesized that AZA may also act through direct modulation of the MSC compartment. To answer this question, we examined primary MSC treated in vitro with AZA as well as the functional interaction between MSC and HSPC after AZA treatment.Methods: Primary MSC were isolated from bone marrow of untreated MDS patients or age-matched healthy controls, expanded by plastic adherence, cultured until confluency under defined conditions and used for up to 4 passages. Identity of MSC was confirmed by presence of characteristic cell surface markers CD73, CD271, CD105 and CD90 and absence of CD45 and CD34 by flow cytometry. MSC were differentiated into the adipogenic or osteogenic lineage after in vitro treatment with AZA (10 uM). Co-culture experiments with CD34+ HSPC from MDS patients or healthy controls were performed on AZA-treated or untreated MDS-MSC or healthy MSC, respectively. After four days, CD34+ cells were harvested and assessed by colony-forming unit assays in methylcellulose. Single colonies were genotyped for MDS-associated mutations known to be present in the bone marrow sample. Each colony was genotyped for one or two known MDS-associated mutations and an input patient specimen was used as a control. Finally, RNA sequencing was performed on a mesenchymal stromal cell line (EL08-1D2), either untreated or treated with AZA for 4 days in vitro to uncover pathways affected by AZA in stromal cells.Results: MDS-MSC showed a decreased proliferative capacity as well as markedly impaired differentiation into the osteoblastic lineage compared to healthy MSC. Treatment with AZA had no effect on cell surface marker expression or viability of either healthy or MDS-MSC. AZA treatment significantly reduced the ability of MDS-MSC to differentiate towards the adipogenic lineage while osteogenic differentiation was severely impaired with or without AZA treatment. In contrast, osteogenic differentiation of healthy MSC was enhanced after AZA treatment. In co-culture experiments pretreatment with AZA significantly improved stromal support of healthy CD34+ HSPC while malignant MDS-HSPC were suppressed. RNA sequencing analysis revealed differential gene expression in AZA-treated vs untreated stroma with significantly more genes upregulated in AZA-treated samples. Specific changes in pathways involved in ECM-receptor interaction, cell adhesion molecules (CAMs), cytokine-cytokine receptor interaction and metabolic pathways in AZA treated EL08-1D2 stromal cells were determined and validated in primary MDS MSC samples.Conclusion: We show here that AZA directly modulates altered MSC function in MDS, thus influencing interaction with HSPC and leading to suppression of malignant hematopoiesis. Our data suggest an additional mode of action by which AZA exerts its therapeutic activity. To our knowledge this analysis is the first to examine the direct effect of AZA treatment on MSC. DisclosuresGötze:BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Abbvie: Honoraria.

Quantization of collagen organization in the stroma with a new order coefficient.

Many optical and biomechanical properties of the cornea, specifically the transparency of the stroma and its stiffness, can be traced to the degree of order and direction of the constituent collagen fibers. To measure the degree of order inside the cornea, a new metric, the order coefficient, was introduced to quantify the organization of the collagen fibers from images of the stroma produced with a custom-developed second harmonic generation microscope. The order coefficient method gave a quantitative assessment of the differences in stromal collagen arrangement across the cornea depths and between untreated stroma and cross-linked stroma.

A Novel Platform For The Identification Of Stromal-Derived Factors That Enhance Acute Lymphoblastic Leukemia Cell Survival In The Context Of Maintenance Chemotherapy

IntroductionChildhood B-lineage acute lymphoblastic leukemia (ALL) is the most common form of malignancy in children. ALL is highly responsive to intensive, multi-agent chemotherapy, and most children enter remission; 25% of children relapse. Furthermore, cure requires an additional two years of low intensity maintenance chemotherapy with 6-mercaptopurine (6-MP) and methotrexate. It is known that serum levels of maintenance chemotherapy drugs are not highly cytotoxic for ALL cells, and it is thought that bone marrow stromal cells (BM-SC) provide trophic signals to leukemia cells that result in increased resistance to standard therapy. If anti-apoptotic signals from stromal cells to leukemia cells were known, new molecular targets for leukemia therapy could be developed. We are developing a conceptually simple screening system that will identify stromal cell-derived molecules that support ALL cell survival in the setting of 6MP and methotrexate. Our hypothesis is based on the observation that in serum-free conditions, BMSC prevent apoptosis of primary ALL cells. We reason that if we interfere with the production of key BMSC-derived trophic factors, an increase in ALL cell apoptosis in vitro will be seen. MethodsThe system has 3 components: (1) human marrow stromal cells; (2) primary ALL cells (not established cell lines); and (3) siRNAs to knockdown candidate stromal genes. We employ a human mesenchymal stromal cell (MSC) line immortalized with a human TERT gene that has been shown to be representative of primary human stroma. 20,000 BMSC are placed in wells of a 96-well plate and treated with siRNA. 48 hours later, 30,000 primary human ALL cells are added in serum-free media. After 5 days, viable ALL cells are measured by flow cytometry. Results(1) The addition of pharmacological concentrations of 6-MP to BMSC/ALL co-cultures does not significantly increase primary ALL apoptosis. Co-cultures were established as above and 6-MP was added at 1 micromolar. ALL survival was measured at 5 days. No increase in ALL survival was seen in the 6-MP containing cultures. On unmanipulated stroma cultured without 6-MP, 1948.8 (± 397.0) viable ALL cells were recovered; on manipulated stroma in the presence of 6-MP, 1698.5 (±299.3) (p value = 0.12). (2) Interference with stromal cell protein synthesis significantly increases ALL cell apoptosis. We hypothesize that interference with new protein synthesis might reduce stromal support of ALL cells. To test this we treated BMSC for 6 hours with 25 micrograms of G418 which irreversibly blocks polypeptide synthesis. Wells were then washed with serum-free medium. Stromal cells remained viable for up to 1 week. However, ALL cells apoptosis was much higher on such treated stromal cells (32.7±18.5 viable ALL cells on G418-treated stroma versus 1948.8 ± 397.0 on unmanipulated stroma, p value< 0.001)(Figure 1). (3) Interference with global stromal cell RNA transcription significantly increased ALL cell apoptosis. Triptolide is a molecule that irreversibly inhibits RNA transcription. BMSC were treated for 6 hr with 1 microgram triptolide and washed with serum-free medium. Stromal cells remain viable for up to 1 week. ALL cell apoptosis was significantly reduced by triptolide treatment of stroma (not shown in Figure 1). The number of viable ALL cells recovered from triptolide-treated stroma was 252.3±113.2, compared to 996.6±239.2 on unmanipulated stroma (p< 0.001). (4) Knockdown of CXCL12 gene expression in stromal cells signficantly increase ALL cell apoptosis. For proof of principle, we have started with CXCL12, a gene shown in CLL to contribute to leukemia cell survival. BMSC were treated with 0.3 picomolar anti-CXCL12 siRNA for 24 hr. As measured by quantitative RT-PCR, CXCL12 gene transcription was reduced by 80%. Moreover, there was a 53.5% reduction in ALL cell viability in ALL cells co-cultured with CXCL12 siRNA-treated stroma compared to ALL cells co-cultured with untreated stroma (p value< 0.001) (Figure 1). [Display omitted] ConclusionsThis BMSC cell/primary ALL cell system can be used to identify stromal cell genes that prevent apoptosis of primary ALL cells. The simple system can be scaled up to allow high throughput screening of candidate genes that have antagonistic or additive effects to maintenance chemotherapy drugs. Disclosures:No relevant conflicts of interest to declare.

Bone Marrow (BM) Stromal Expression Of Cytochrome P450 (CYP) Enzymes Protects Acute Myeloid Leukemia (AML) From All-Trans Retinoic Acid (atRA)

Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved the outcome of acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of atRA resistance have been previously reported (i.e., over-expression of PRAME or up-regulation of Tal1 pathway), yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, although atRA induces complete remissions (CRs) in &gt;80% of cases, most patients eventually develop disease recurrence. We have recently showed that BM stromal control of RA signaling via expression of CYP26 is important in determining normal hematopoietic stem cell fate. Thus, here we hypothesize that the BM microenvironment is responsible for the difference between the in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. It is well-recognized that pharmacological concentrations of atRA (10-7M x 3 days) differentiates NB-4 cells [a t(15;17) APL line] as demonstrated by decreased cellular expansion (32.9±1 vs. 3.5±1.3, mean±SD of cellular expansion folds of control vs. atRA-treated cultures, n=3, p&lt;0.01) induction of cell cycle arrest (52.7% vs. 86.2% Go/G1, control vs. atRA), decreased clonogenic activity (61±12% of control in atRA cultures, n=3, p&lt;0.01), increased expression of differentiation markers (47.4±7% vs. 84.5±13% CD11b positive cells, control vs. atRA, n=3, p=0.01), disappearance of blasts (44.5±7% vs 24.3±4% blasts, control vs. atRA, n=4, p&lt;0.01) and emergence of morphological neutrophils. We observed similar pro-differentiation effects of atRA on other AML lines including HL-60 [non-t(15:17) APL line], KG-1 (primitive line arising from MDS), Kasumi-1 [t(8;21)] and OCI-AML3 (NPM1 mutated). When the incubations were repeated with the 5 AML lines in the presence of BM stroma, atRA activity (i.e., upregulation of differentiation markers, inhibition of clonogenic growth) was blocked. Inhibition of stromal CYP26 by the competitive inhibitor R115866, rescued the AML cell sensitivity to atRA: clonogenic recovery of NB4 cells was 92±17% on stroma with atRA compared to 56±15.9% when R115866 was added) (mean±SD of clonogenic activity of untreated stroma cultures, n=3, p=0.03) and 67.5±8.9% cells expressed the differentiation marker CD11b when treated with atRA on stroma compared to 95.1±4.9% with the combination of atRA+R115866 (n=3, p=0.03). Similar results were observed using HL-60, KG-1, Kasumi-1 and OCI-AML3 cells. CD34+ AML blasts from the BM of newly-diagnosed patients with t(8;21) AML were cultured as above. When isolated from the BM, the blasts showed only low-level expression of mature myelomonocytic markers (n=3). However, the expression of differentiation markers was significantly increased when cultured in serum (which contains about 10-9M atRA) and even further with the addition of 10-7M atRA (Figure). The presence of stroma during culture was associated with maintenance of an immature leukemic phenotype; moreover, as seen above, inhibition of CYP26 by R115866 restored AML sensitivity to atRA (Figure). CYP26 inhibitor had no effect on AML cells in stroma independent cultures. Our data suggest that CYP26 activity in the BM microenvironment creates retinoid low sanctuaries that protect AML cells from systemic atRA therapy. Thus, inhibition of CYP26 activity provides a new opportunity to expand the clinical activity of atRA in both APL and non-APL AML. It is also tempting to hypothesize that the P450-mediated detoxification of drugs by the stroma is not a retinoid-specific phenomenon but rather a more general, cell-extrinsic mechanism of drug resistance. Bone marrow stroma protects primary AML blasts from atRA induced differentiation Flow cytometry analysis of CD15 expression of CD34+ blasts isolated from the BM of a patient with t(8;21) AML. On day 0, these cells express no CD15. Culture in the presence of serum results in rapid acquisition of CD15 (liquid control). This is further enhanced by treatment with 10-7M atRA. Co-culture with BM stroma inhibits CD15 expression and atRA has only minimal effect in the presence of stroma. Inhibition of stromal CYP26 overcomes stroma’s inhibition of atRA-mediated up-regulation of CD15. Disclosures: No relevant conflicts of interest to declare.

Balanced Wnt5a-Mediated Signaling Is Necessary for Normal Proliferation of Primitive Hematopoietic Cells.

Abstract 2533Poster Board II-510Multiple members of the Wnt family of ligands have been implicated in the regulation of self-renewal and proliferation of hematopoietic stem cells (HSCs). Previously, we have observed that ex vivo expansion of HSCs in the presence of recombinant murine Wnt5a (rmWnt5a) resulted in increased hematopoietic repopulation. Based on these data, we hypothesized that Wnt5a is necessary for normal function of HSCs and hematopoietic progenitors (HPCs). Since Wnt5a deficiency (Wnt5a−/−) is perinatal lethal in vivo, we tested this hypothesis using in vitro Dexter stroma cultures established using whole bone marrow. To determine the ability of Wnt5a to support hematopoiesis in the context of the adult hematopoietic microenvironment, we cultured lineage-negative (lin−) HPCs on irradiated bone marrow stroma in the presence of 5 μg/ml Wnt5a-neutralizing antibody (Wnt5a-Ab). After two weeks, we observed that hematopoietic cells cultured on untreated stroma contained 4.8-fold more myeloid CFU (33.1 ± 12.3 CFU/104 cells) than cells cultured on Wnt5a-Ab stroma (6.9 ± 0.7 CFU; n = 3, p < .01). A similar difference was observed after 4 weeks (control: 16.0 ± 7.2 CFU/104 cells; Wnt5a-Ab: 1.3 ± 2.3 CFU; n = 3; p = .03). In the converse experiment, lin− HPCs were cultured on stroma in the presence of Wnt5a conditioned medium (Wnt5a-CM). We observed after two weeks that hematopoietic cells cultured on stroma with control-CM contained 4.4-fold more myeloid CFU (29.8 ± 13.5 CFU/104 cells) than cells cultured on Wnt5a-CM stroma (6.8 ± 2.2 CFU; n = 4, p = .02). Together, these data indicate that Wnt5a-mediated signaling must be balanced in order for normal hematopoiesis to occur. To determine if the effects of Wnt5a required the presence of developmental stage-specific factors, we established stroma cultures from fetal spleens harvested from E17.5 Wnt5a−/− mice and littermate controls and seeded them with lin− HPCs. We observed that hematopoietic cells cultured on control fetal spleen stroma contained 12.5-fold more myeloid CFU (72.6 ± 21.6 CFU/104 cells) than cells cultured on Wnt5a−/− fetal spleen stroma (5.8 ± 5.8; n = 6, p < .001) after two weeks. These data indicate that the effect of Wnt5a on hematopoietic cells is independent of the developmental stage of the surrounding microenvironment. To determine if the effects of Wnt5a were due to regulation of proliferation or differentiation of hematopoietic cells, we cultured bone marrow cells in cytokine-supplemented methylcellulose in the presence of 300 ng/ml rmWnt5a. We observed a 5.5-fold decrease in the number of myeloid CFU formed in cultures with rmWnt5a (14.1 ± 3.8/104 cells) compared to control (77.6 ± 5.1 CFU; n = 3; p < .001), suggesting that Wnt5a could be regulating both processes. In the converse experiment, we cultured bone marrow cells for 4 days in cytokine-supplemented serum-free media with the same dose of rmWnt5a after which equal numbers of cells were plated in rmWnt5a-free methylcellulose. We did not observe any difference in CFU frequency between control (19.3 ± 4.2 CFU/104 cells) and rmWnt5a (24.0 ± 2.6 CFU; n = 3) cultures, indicating that treatment with rmWnt5a inhibited hematopoietic proliferation but not differentiation. To identify the mechanism by which Wnt5a regulates HSC and HPC proliferation, we analyzed potential Wnt5a-mediated signaling pathways. We observed that Wnt5a induced intracellular Ca2+ (iCa2+) flux in HSCs (defined as lin−, Sca-1HI, c-kitHI; LSK). Previous studies have shown that Wnt5a-mediated induction of iCa2+ can result in activation of the NFAT family of transcription factors. Since NFATc1 promotes quiescence of hair follicle stem cells and is expressed in HSCs, we hypothesized that the effects of Wnt5a required activation of NFAT family members. We cultured bone marrow cells with Wnt5a-CM in the presence of cyclosporine A (CsA), which inhibits activation of NFAT factors. In agreement with our earlier findings, we observed that culturing bone marrow cells in Wnt5a-CM increased the percentage of quiescent (defined as Ki-67-) LSKCD34− HSCs (79.7 ± 3.3%) compared to control-CM (55.0 ± 1.6%; n = 3; p < .001). This increase was inhibited by CsA (69.4 ± 2.6%; n = 3; p = .01 compared to Wnt5a-CM alone). In conclusion, our data point to a role for Wnt5a in regulating HSC and HPC proliferation and that this function may require the activation of NFAT transcription factors. Disclosures:No relevant conflicts of interest to declare.

Notch/Delta4 Interaction in Human Embryonic Liver CD34+ CD38− Cells: Positive Influence on BFU-E Production and LTC-IC Potential Maintenance

We investigated whether Notch signaling pathways have a role in human developmental hematopoiesis. In situ histochemistry analysis revealed that Notch1, 2, and 4 and Notch ligand (Delta1-4, and Jagged1) proteins were not expressed in the yolk sac blood islands, the para-aortic splanchnopleure, the hematopoietic aortic clusters, and at the early stages of embryonic liver hematopoiesis. Notch1-2, and Delta4 were eventually detected in the embryonic liver, from 34 until 38 days postconception. Fluorescence-activated cell sorter analysis showed that first-trimester embryonic liver CD34(+)CD38(low) cells expressed both Notch1 and Notch2. When these cells were cultured on S17 stroma stably expressing Delta4, a 2.6-fold increase in BFU-E number was observed at day 7, as compared with cultures with control stroma, and this effect was maintained for 2 weeks. Importantly, exposure of these cells to Delta4 under these conditions maintained the original frequency and quality of long-term culture-initiating cells (LTC-ICs), while control cultures quickly resulted in the extinction of this LTC-IC potential. Furthermore, short-term exposure of embryonic liver adherent cells to erythropoietin resulted in a dose-dependent increase in Delta4 expression, almost doubling the expression observed with untreated stroma. This suggests that Delta4 has a role in the regulation of hematopoiesis after a hypoxic stress in the fetus.

Role of the epithelial-stromal interaction during the development and expression of ovary-independent vaginal hyperplasia

The tissue interactions involved in the induction and perpetuation of ovary-independent vaginal hyperplasia were studied by growing recombinants prepared with vaginal epithelium and stroma from untreated and neonatally estrogenized mice. As expected, recombinants prepared with untreated tissues developed an atrophied epithelium, while those prepared with estrogenized epithelium and stroma exhibited epithelial hyperplasia in ovariectomized hosts. Recombinants prepared with estrogenized stroma and untreated epithelium and the reciprocal recombination of untreated stroma and estrogenized epithelium also exhibited ovary-independent hyperplasia in many cases. This suggests that the expression of ovary-independent hyperplasia is due to irreversible changes in vaginal epithelium and inductive activities in vaginal stroma. Development of ovary-independent hyperplasia in response to neonatal exposure to estradiol is facilitated when the epithelial-stromal association is maintained and is blocked if this association is disrupted. Finally, Takasugi's (1971, Proc. Japan Acad. 47, 193–198) hypothesis, that the age-dependent loss in sensitivity of the vagina to permanent, irreversible effects of estradiol at 5 days postpartum is due to maturational changes in the epithelium, was confirmed through analysis of the developmental response of heterochronal vaginal recombinants.

The effects of sex-steroids on the synthesis of uterine non-histone proteins

Newly synthesized non-histone proteins (NHP) labelled with [ 35S]methionine were isolated from uterine epithelium and stroma after different hormone treatments and examined by isoelectric focusing in polyacrylamide gels. Progesterone increased the proportion of basic NHP in the epithelium. This effect was detectable within 4 h and was maintained unchanged after oestrogen stimulation, which increased the synthesis of all classes of NHP. The proportion of basic NHP was higher in the untreated stroma than in the epithelium. Progesterone pretreatment did not increase the proportion of basic NHP in the stroma but prevented the increase in the more acidic NHP which followed treatment with oestrogen alone. The tendency to synthesize a higher proportion of basic NHP did not in any way correlate with the tissue's proliferative status.