Single Cell RNA Sequencing Reveals Increased Adhesion Signals in Treatment-Resistant Tumor Stem Cells in a Preclinical Mouse Model of Genetically Engineered Patient-Derived Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2630-2630
Author(s):  
Ebinger Sarah ◽  
Özdemir Erbey ◽  
Sebastian Tiedt ◽  
Christoph Ziegenhain ◽  
Catarina Castro Alves ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Risk ◽  
Tumor Cells ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Drug Resistant ◽  
Treatment Resistant ◽  
Dormant Cells ◽  
Resistant Cells

Abstract Treatment-resistant cells determine prognosis and outcome of cancer patients as they induce relapse with poor outcome. Novel therapeutic options are urgently needed to eradicate chemo-resistant tumor cells. Towards this aim, a deep understanding is required on mechanisms determining treatment-resistance in vivo and the ability to induce relapse. Here, we aimed to identify and characterize the challenging rare subpopulation of drug resistant cells which survive in vivo chemo-therapy and are able to induce relapse. As technical approach, we used the individualized mouse model of acute lymphoblastic leukemia (ALL) and amplified primary tumor cells in mice to generate patient-derived xenograft (PDX) cells. Upon genetic engineering by lentiviral transduction, PDX ALL cells expressed the three transgenes NGFR, a red fluorochrome and luciferase. While recombinant luciferase was used for in vivo imaging, a combined MACS/FACS procedure based on the expression of the transgenes enabled enriching PDX ALL cells from murine bone marrow by a factor above 10,000. Staining of PDX cells with CFSE was used to discriminate between highly proliferative and dormant tumor cells in vivo. We treated mice harboring triple-transgenic, CFSE labeled PDX ALL cells with conventional chemotherapy; while in vivo treatment decreased the number of highly proliferative cells by more than 1 order of magnitude, the amount of dormant cells remained completely unchanged. Isolated, drug resistant cells revealed leukemia propagating potential and induced leukemia upon transplantation into next generation mice. Thus and using dormancy as an anchor, we could identify, isolate and enrich a subpopulation of treatment-resistant PDX ALL cells which might mimic relapse-inducing cells at minimal residual disease in patients. We next aimed at characterizing the expression profile of these cells and were able to isolate single cells out of the low number of dormant cells to perform single cell RNA sequencing. Dormant, drug-resistant cells showed increased expression of several adhesion molecules suggesting an increased dependence on the bone marrow environment. Upon using gene set enrichment analyses, drug-resistant cells showed a highly similar expression profile to primary high risk leukemia subpopulations such as primary high risk ALL cells, the subpopulation of CD34 positive CML cells and leukemic or benign hematopoietic stem cells. Taken together, treatment-resistant PDX ALL stem cells isolated and enriched from mice showed increased expression of adhesion molecules and resemble primary tumor cells of high risk subpopulations. These cells represent valuable tools to increase our understanding of mechanisms in minimal residual disease and relapse in patients. Our model will help to develop novel therapies which eliminate treatment resistant cells, prevent disease relapse and increase the prognosis of patients with ALL. Disclosures No relevant conflicts of interest to declare.

Status of Minimal Residual Disease Determines Outcome of Autologous HSCT in Adults with High-Risk Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2321-2321
Author(s):  
Sebastian Giebel ◽  
Beata Stella-Holowiecka ◽  
Malgorzata Krawczyk-Kulis ◽  
Nicola Goekbuget ◽  
Dieter Hoelzer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Minimal Residual

Abstract Abstract 2321 Poster Board II-298 The role of autologous hematopoietic stem cell transplantation (autoHSCT) in the treatment of adult acute lymphoblastic leukemia (ALL) is a subject of controversies as several prospective studies failed to prove its advantage over maintenance chemotherapy. Those studies, however, did not take into account the status of minimal residual disease (MRD), which is now recognized a potent predictor for relapse among patients treated with conventional-dose chemotherapy. The goal of this analysis was to determine the impact of MRD on outcome of autoHSCT. Data on 123 autoHSCT recipients collected from 6 study groups cooperating in the European Leukemia Net were analyzed. Median age of 77 B-lineage and 46 T-lineage high-risk ALL patients was 31 (16-59) years. Ph+ ALL was recognized in 20 cases. All patients were in first complete remission (CR) lasting 6 (1.5-22) months. Peripheral blood was used as a source of stem cells in 67 patients whereas bone marrow, in 56 cases. Conditioning was based on chemotherapy alone (n=76) or total body irradiation (n=47). MRD was evaluated in bone marrow with the use of either multiparametric flow cytometry (n=79) or molecular techniques (n=44). MRD level of 0.1% bone marrow cells was used as a cut-off point for the purpose of this study. At the time of autoHSCT MRD was &0.1% in 93 patients and ≧0.1% in 30 cases. With the median follow up of 5 years, the probability of leukemia-free survival (LFS) at 5 years for the whole group equaled 48% (+/-5). Three patients died of transplantation-related complications. The LFS rate was significantly higher for patients with the MRD level at transplantation &0.1% compared to those with MRD ≧0.1% (57% vs. 19%, p=0.0002). The difference was particularly pronounced for peripheral blood HSCT (66% vs. 20%, p=0.0006) and for T-lineage ALL (62% vs. 8%, p=0.001). In a multivariate analysis adjusted for other potential prognostic factors (age, CR duration, Ph+ ALL, immunophenotype, source of stem cells, type of conditioning), the MRD status &0.1% remained the only independent factor associated with increased LFS (HR=2.5, p=0.0009). CONCLUSIONS: MRD status is the most important predictor for LFS after autoHSCT in adults with ALL. More than half of patients with high risk disease and low MRD level at the time of transplantation may be cured. This observation may contribute to re-evaluation of the role of autoHSCT in the therapy of adult ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Discovery of Novel Doxorubicin Metabolites in MCF7 Doxorubicin-Resistant Cells

2019 ◽  
Vol 10 ◽  
Author(s):  
Xu Wang ◽  
Renjie Hui ◽  
Yun Chen ◽  
Wentao Wang ◽  
Yujiao Chen ◽  
...  
Keyword(s):  
Drug Metabolism ◽  
Stress Tolerance ◽  
Tumor Cells ◽  
Drug Resistant ◽  
Wild Type ◽  
High Drug ◽  
Metabolism Pathway ◽  
Show Difference ◽  
Resistant Cells

Doxorubicin (DOX) is metabolized to a variety of metabolites in vivo, which has been shown to be associated with cardiotoxicity. We speculate that metabolic processes are also present in tumor cells. A LC-MS/MS method was developed to detect intracellular metabolites. Drug resistant tumor cells with high drug stress tolerance and metabolically active are suitable as materials for this study. Our results show difference in drug metabolites between the wild-type and drug-resistant cells. Three novel doxorubicin metabolites were discovered after the LC-MS/MS analysis. All these metabolites and their profiles of metabolites are totally different from that in liver or kidney in vivo. Our results suggest that tumor cells and drug-resistant tumor cells have a unique drug metabolism pathway for doxorubicin.

TRAIL Is Active Against Leukemia Stem Cells and Leukemia-Initiating Cells of Children with Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2953-2953
Author(s):  
Catarina Castro Alves ◽  
Leticia Quintanilla-Martinez ◽  
Irmela Jeremias
Keyword(s):  
Stem Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Cancer Stem Cells ◽  
Tumor Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Stem Cells ◽  
Nsg Mice ◽  
Anti Cancer

Abstract Abstract 2953 Cancer stem cells represent the most important target cells for anti-tumor therapy as they are uniquely capable to maintain tumor growth and to induce relapse. For sustained anti-tumor activity, anti-cancer therapy has to address and eliminate cancer stem cells. Unfortunately in acute lymphoblastic leukemia (ALL), cancer stem cells / leukemia stem cells (LSCs) can not be reliably defined by cell surface markers complicating studies on cancer stem cells. To overcome this obstacle, we used the functional definition of CSCs as surrogate and studied leukemia-initiating cells (LICs) by their name-defining capability to initiate leukemia in immune-compromised mice. TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family which induces apoptosis in a wide variety of tumor cells while sparing normal cells. TRAIL represents an important candidate anti-cancer agent which is currently tested in phase I and II clinical trials, yet its ability to target cancer stem cells is currently unknown. Here we tested the effect of TRAIL on LSCs and LICs using tumor cells from children with precursor B-cell acute lymphoblastic leukemia (pre-B ALL). Primary ALL cells were engrafted and passaged in NSG mice. Cells were freshly isolated from NSG mice and stimulated with TRAIL in vitro for 2 days and afterwards re-engrafted into mice in dilution curves upon limiting dilution transplantation assay comparing non-treated with TRAIL-treated cells and using about 25 mice per stimulation. After 12–16 weeks, mice were evaluated for leukemic engraftment by staining for leukemia cells in organs like bone marrow, spleen, liver and blood using FACscan analysis and immune histochemistry. In all 3 pre-B ALL samples tested, in vitro treatment with TRAIL prior to transplantation of cells into mice significantly reduced their engraftment capability. TRAIL disabled leukemic engraftment by > 95 % in all 3 samples. Accordingly, in vitro TRAIL treatment significantly increased the time to engraftment and completely disabled engraftment below a defined threshold. In a second, additive approach, TRAIL was used in a preclinical mouse model. Pre-B ALL samples were engrafted in NSG mice and leukemia-bearing mice were treated with TRAIL systemically at 7.5 mg / kg daily i.p. for 10 days. In this preclinical in vivo model, TRAIL treatment completely cured a proportion of animals harbouring patient-derived pre-B ALL xenografts. Taken together, TRAIL significantly disabled the leukemia-initiating function of LICs from patient-derived pre-B ALL xenografts in vitro and that TRAIL eliminated leukemia together with its LSCs in vivo. Although the methods used do not allow the study of signalling mechanisms, TRAIL might most probably have induced apoptosis in LICs and LSCs. Our data show that it is feasible, although technically demanding, to test the apoptosis sensitivity of LICs and LSCs. We conclude from these data that TRAIL constitutes an attractive future drug for treatment of ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Co-culture model of B-cell acute lymphoblastic leukemia recapitulates a transcription signature of chemotherapy-refractory minimal residual disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Stephanie L. Rellick ◽  
Gangqing Hu ◽  
Debra Piktel ◽  
Karen H. Martin ◽  
Werner J. Geldenhuys ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Tumor Cells ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Adherent Cells ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Minimal Residual

AbstractB-cell acute lymphoblastic leukemia (ALL) is characterized by accumulation of immature hematopoietic cells in the bone marrow, a well-established sanctuary site for leukemic cell survival during treatment. While standard of care treatment results in remission in most patients, a small population of patients will relapse, due to the presence of minimal residual disease (MRD) consisting of dormant, chemotherapy-resistant tumor cells. To interrogate this clinically relevant population of treatment refractory cells, we developed an in vitro cell model in which human ALL cells are grown in co-culture with human derived bone marrow stromal cells or osteoblasts. Within this co-culture, tumor cells are found in suspension, lightly attached to the top of the adherent cells, or buried under the adherent cells in a population that is phase dim (PD) by light microscopy. PD cells are dormant and chemotherapy-resistant, consistent with the population of cells that underlies MRD. In the current study, we characterized the transcriptional signature of PD cells by RNA-Seq, and these data were compared to a published expression data set derived from human MRD B-cell ALL patients. Our comparative analyses revealed that the PD cell population is markedly similar to the MRD expression patterns from the primary cells isolated from patients. We further identified genes and key signaling pathways that are common between the PD tumor cells from co-culture and patient derived MRD cells as potential therapeutic targets for future studies.

scholarly journals Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia

Blood ◽  
2020 ◽  
Vol 136 (2) ◽  
pp. 210-223 ◽  
Author(s):  
Eun Ji Gang ◽  
Hye Na Kim ◽  
Yao-Te Hsieh ◽  
Yongsheng Ruan ◽  
Heather A. Ogana ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Underlying Mechanism ◽  
Conditional Knockout

Abstract Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

scholarly journals The Ribosomal Protein L28 Gene Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma

2021 ◽  
Author(s):  
Yi Shi ◽  
Xiaojiang Wang ◽  
Qiong Zhu ◽  
Gang Chen
Keyword(s):  
Hepatocellular Carcinoma ◽  
Drug Resistance ◽  
Hepg2 Cells ◽  
Cell Model ◽  
Resistant Cell ◽  
Drug Resistant ◽  
Resistant Gene ◽  
Key Genes ◽  
Resistant Cells

Abstract Background: Sorafenib is the first molecular-targeted drug for the treatment of advanced hepatocellular carcinoma (HCC). However, its treatment efficiency decreases after a short period of time because of the development of drug resistance. This study investigates the role of key genes in regulating sorafenib-resistance in hepatocellular carcinoma and elucidates the mechanism of drug resistance. Methods: The HCC HepG2 cells were used to generate a sorafenib-resistant cell model by culturing the cells in gradually increasing concentration of sorafenib. RNA microarray was applied to profile gene expression and screen key genes associated with sorafenib resistance. Specific targets were knockdown in sorafenib-resistant HepG2 cells for functional studies. The HCC model was established in ACI rats using Morris hepatoma3924A cells to validate selected genes associated with sorafenib resistance in vivo. Results: The HepG2 sorafenib-resistant cell model was successfully established. The IC50 of sorafenib was 9.988mM in HepG2 sorafenib-resistant cells. A total of 35 up-regulated genes were detected by expression profile chip. High-content screening technology was used and a potential drug-resistant gene RPL28 was filtered out. After knocking down of RPL28 in HepG2 sorafenib-resistant cells, the results of cell proliferation and apoptosis illustrated that RPL28 is the key drug-resistant gene in the cells. Furthermore, it was found that both RNA and protein expression of RPL28 increased in HepG2 sorafenib-resistant specimens of Morris Hepatoma rats. In addition, the expression of functional proteins Ki-67 increased in sorafenib-resistant cells. Conclusion: Our study suggested that RPL28 was a key gene for sorafenib resistance in HCC both in vitro and in vivo.

scholarly journals LncRNA-42060 Regulates Tamoxifen Sensitivity and Tumor Development via Regulating the miR-204-5p/SOX4 Axis in Canine Mammary Gland Tumor Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Enshuang Xu ◽  
Mengxin Hu ◽  
Reidong Ge ◽  
Danning Tong ◽  
Yuying Fan ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mammary Gland ◽  
Tumor Cells ◽  
Luciferase Reporter ◽  
Drug Resistant ◽  
Mammary Gland Tumor ◽  
Luciferase Reporter Gene ◽  
Gland Tumor ◽  
Canine Mammary Gland ◽  
Resistant Cells

Tamoxifen is the drug of choice for endocrine therapy of breast cancer. Its clinical use is limited by the development of drug resistance. There is increasing evidence that long non-coding RNAs (lncRNAs) are associated with tumor drug resistance. Therefore, we established two TAM-resistant cell lines, CHMpTAM and CHMmTAM. The different expression levels of lncRNA and miRNA in CHMmTAM and CHMm were screened by RNA sequencing, and the lncRNA-miRNA interactions were analyzed. LncRNA ENSCAFG42060 (lnc-42060) was found to be significantly upregulated in drug-resistant cells and tumor tissues. Further functional validation revealed that the knockdown of lnc-42060 inhibited proliferation, migration, clone formation, restoration of TAM sensitivity, and reduction of stem cell formation in drug-resistant cells, whereas overexpression of lnc-4206 showed opposite results. Bioinformatics and dual-luciferase reporter gene assays confirmed that lnc-42060 could act as a sponge for miR-204-5p, further regulating SOX4 expression activity and thus influencing tumor cell progression. In conclusion, we screened lncRNAs and miRNAs associated with TAM resistance in canine mammary gland tumor cells for the first time. lnc-42060 served as a novel marker that may be used as an important biomarker for future diagnosis and treatment.

scholarly journals IKZF1 Deletion Is An Independent Predictor of Outcome In Pediatric Acute Lymphoblastic Leukemia Treated According to the ALL-BFM 2000 Protocol

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 409-409
Author(s):  
Petra Breithaupt ◽  
Barbara Meissner ◽  
Martin Zimmermann ◽  
Anja Möricke ◽  
André Schrauder ◽  
...  
Keyword(s):  
Treatment Outcome ◽  
High Risk ◽  
Cox Regression ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Risk Group ◽  
Independent Predictor ◽  
Lymphoblastic Leukemia ◽  
Time Points ◽  
Pediatric All

Abstract Abstract 409 Alteration of the IKZF1 gene – encoding the transcription factor IKAROS, a key player in lymphoid development and tumor suppression – has been reported to be associated with a poor outcome in pediatric precursor B-cell ALL, especially in cases positive for the BCR-ABL1 fusion gene. In order to assess the prognostic value of IKZF1 deletions in a representative cohort of pediatric ALL patients treated on the German ALL-BFM 2000 study protocol, we screened 409 patients by applying a multiplex ligation-dependent probe amplification (MLPA) assay covering all eight IKZF1 exons (P335-A3 ALL-IKZF1 probemix; MRC-Holland, Amsterdam, The Netherlands). In ALL-BFM 2000, risk group stratification (standard, SR; intermediate, MR; high, HR) was based on minimal residual disease (MRD) analysis at two different time points (TP) and required two MRD targets with sensitivities of ≤10−4 (Flohr et al. Leukemia 2008). SR patients were MRD-negative on treatment days 33 (TP1) and 78 (TP2). HR patients had residual disease (≥10−3) at TP2. MRD MR patients had positive MRD detection at either one and or both time points but at a level of <10−3 at TP2. Although MRD-based stratification criteria were introduced in ALL-BFM 2000, established high-risk parameters were also retained: patients with prednisone poor-response or ≥5% leukemic blasts in the bone marrow on day 33 or positivity for a t(9;22) or t(4;11) or their molecular equivalents (BCR/ABL1 or MLL/AF4 fusion RNA) were stratified into the high-risk group independent of their MRD results. First results on MRD and outcome were published earlier (Conter et al. Blood 2010). Out of the 409 patients analyzed in our study, 46 (11%) displayed a deletion in at least one of the eight IKZF1 exons. Forty-three out of the 46 cases showed heterozygous deletions, while 3 patients displayed homozygous loss of IKZF1 exons. MLPA results of 11 patients were validated with results derived from copy number/LOH analyses using Affymetrix SNP 6.0 arrays. IKZF1 deletion was significantly more common in precursor B compared to T cell ALL (13% vs. 4%, P = 0.03) and less frequent in TEL/AML1-positive ALL (3% vs. 13%, P = 0.004). Out of 11 BCR/ABL1-positive samples, only two were characterized by an IKZF1 deletion. Forty-four patients with IKZF1-deleted ALL had results of MRD analyses available for both informative time points (day 33 after induction and day 78 after consolidation). Despite a trend towards increasing incidence of IKZF1 deletion in patients with slow response, the distribution of IKZF1-deleted ALL patients over the risk groups was not significantly different from non-deleted ALL (SR: 40.9 vs. 41.9; MR: 45.5 vs. 52.3; HR: 13.6 vs. 5.7%; P = 0.153). Regarding treatment outcome, patients with an IKZF1 deletion had a significantly lower 5-year event-free survival (EFS) compared to non-deleted patients (0.78±0.06 vs. 0.86±0.02; P = 0.015). This result was due to a higher cumulative incidence of relapses in IKZF1-deleted patients (0.16±0.05 vs. 0.10±0.02; P = 0.031). In multivariate Cox regression analyses including known prognostic variables (gender, immunophenotype, WBC count at diagnosis, TEL/AML1 status, risk group criteria of ALL-BFM 2000), IKZF1 deletion conferred a risk of 2.16 (95% confidence interval 1.14 – 4.10; P = 0.018) for an event when compared to non-deleted patients. We conclude that IKZF1 deletion is an independent predictor of treatment outcome for patients enrolled on the ALL-BFM 2000 protocol and represents a candidate marker to be integrated in future algorithms for early risk stratification in pediatric ALL. Disclosures: No relevant conflicts of interest to declare.

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