Limiting Dilution Experiments Research Articles

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-06.

Abstract 960: Improved cfDNA methylation profiling through correction of misrepaired jagged-ends

Abstract Background: Profiling cfDNA methylation enables early cancer detection and classification, but challenges arise from "jagged ends" (JEs) for ~90% of cfDNA molecules, as well as for FFPE samples and partially degraded genomic DNA. Repair of these jagged ends introduces erroneous methylation signals, especially in shorter cfDNA fragments enriched for tumor derived molecules. Separately, DNA conversions during 5mC profiling hamper accurate simultaneous ctDNA genotyping of somatic mutations. To meet these needs, we developed a method preserving double-stranded cfDNA molecules without error-prone end-repair at jagged ends. This enables superior methylation profiling including hemimethylation detection, and simultaneous ctDNA SNV genotyping. Methods: We present JEEPERS (Jagged-End Error Polishing of Enzymatically misRepaired Sequences), a novel method for correcting errors in double-stranded library preparation for methylation profiling. JEEPERS detects and quantifies enzymatic repair errors at JEs, correcting them in silico. Leveraging duplex UMIs, it utilizes complementary strands and sibling reads from cfDNA families without jagged ends. The method exploits the 5' to 3' polarity of ER-associated errors and JE length profiles for correction. JEEPERS introduces anchor CpGs for precise correction of rare signals and enables accurate SNV calling at non CpG sites, addressing the challenge of differentiating true SNVs from conversion artifacts leading to C>T/G>A changes in sequencing data. Results: Analyzing 248 cfDNA samples (139 NSCLC patients, 109 healthy controls) by methylation profiling (EM-Seq) and ctDNA mutation genotyping (CAPP-Seq), JEEPERS correction efficiently addressed distortions, even at low tumor concentrations. Limiting dilution experiments demonstrated JEEPERS achieving a LOD95 of ~0.2% with 95% specificity (n=300). Despite ~50% lower molecular depth, JEEPERS-corrected cfDNA EM-Seq enabled accurate simultaneous methylation profiling and ctDNA genotyping, correlating strongly with CAPP-Seq genotypes (R=0.998, n=30). As expected, mutant ctDNA molecules were shorter than wild-type in both CAPP-Seq and EM-Seq data. cfDNA methylation patterns varied by oncogene vs. tumor suppressor status; EGFR mutants showed increased methylation (p<0.01), while TP53 mutants had lower methylation levels (p<0.05). Conclusions: Our method effectively corrects cfDNA methylation data, even at low tumor concentration, ensuring enhanced methylation signal accuracy and proficient SNV calling capabilities. This integrated approach offers a powerful means for investigating the link between SNVs and DNA methylation, significantly enhancing the accuracy of cancer detection using cfDNA, especially for early-stage tumors. This paves the way for a deeper understanding of the molecular mechanisms driving cancer initiation and progression. Citation Format: Rui Wang, Emily G. Hamilton, Diego Almanza, Angela Hui, Maximilian Diehn, Ash A. Alizadeh. Improved cfDNA methylation profiling through correction of misrepaired jagged-ends [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 960.

Development of a 27-color panel for the detection of measurable residual disease in patients diagnosed with acute myeloid leukemia.

Acute myeloid leukemia (AML) measurable residual disease (MRD) evaluated by multiparametric flow cytometry (MFC) is a surrogate for progression-free and overall survival in clinical trials and patient management. Due to the limited number of detection channels available in conventional flow cytometers, panels used for assessing AML MRD are typically split into multiple tubes. This cripples the simultaneous and correlated assessment of all myeloblast measurements. In response, we prototyped a single-tube 27-color MFC assay for the evaluation of AML MRD, incorporating all recommended markers. Marrow aspirates from 22 patients were processed for analysis using full spectrum flow cytometry (FSFC). The signal resolution of each marker was compared between samples stained with single antibody vs. the fully stained panel. The analytical accuracy for quantifying hematopoietic cells between our established 8-color assay and the new 27-color method were compared. Variations within an operator and between separate operators were assessed to evaluate the assays reproducibility. The limited of blank (LOB), limit of detection (LOD), and lower limit of quantification (LLOQ) of the 27-color method were empirically determined using limiting dilution experiments. The stability of antibody cocktails over a period of 120 h was also studied using cryopreserved marrow cells. The stain indices for all antibodies were lower in the fully stained panel compared to cells stained with one antibody but clear separations between negative and positive signals were achieved for all antibodies. Our results demonstrated a high concordance between the established 8-color method and the new 27-color assay for enumerating myeloblasts and MRD interpretation within and between operators. The data further showed that the single-tube 27-color assay easily achieved the minimum required detection sensitivity of 0.1%. When antibodies were combined, however, expression intensity of some antigens deteriorated significantly when stored. Our single-tube 27-color panel is a suitable, high sensitivity flow cytometric approach that can be used for AML MRD testing, which improves the correlation of aberrant antigens and detection of asynchronous differentiation patterns. Based on the stability study, we recommend the full panel be made prior to staining.

Evaluation of CD98 light chain-LAT1 as a potential marker of cancer stem-like cells in glioblastoma

ObjectiveGlioma stem cells (GSCs) are a minority population of glioma cells that regarded as the cause of tumor formation and recurrence. Identifying new molecular strategies targeting GSCs must be urgently developed to treat glioblastoma. In this study, one of CD98 light chain-L type amino acid transporter 1 (LAT1) was found as a potential GSC marker. LAT1 served as EAA transporter has been shown to be closely related with tumor invasion, metastasis, angiogenesis, and radiosensitivity. MethodsLAT1+ and LAT1− glioma cells were sorted by flow cytometry. Cellular immunofluorescence, sphere-formation arrays, and in vitro limiting dilution experiments were used to identify cell stemness. Differentiated glioma stem cells were cultured, and the expressions of β-tubulinIII, GFAP, and LAT1 were detected by Western blot. Nude mouse models were constructed to observe tumor formation and metastasis in nude mice. ResultsLAT1+ glioma cells were testified a small percentage of all cells and selected as the subsequent sorting marker. LAT1+ cells were separated from U87 and U251 cells could express high level of stem cell markers, and possessed GSC properties including self-renewal ability and multi-directional differentiation potential. But LAT1− cells did not have these characteristics. In addition, LAT1+ cells were able to generate tumors in vivo, tumor size of LAT1+ cells formed were much bigger than that of LAT1− cells. ConclusionOur study, including molecular, cell, vitro and vivo experiments, has shown that LAT1+ cells possess GSC properties, and present for the first time that LAT1 can be used as a new marker for GSCs screening.

Cellular dormancy in minimal residual disease following targeted therapy

BackgroundBreast cancer mortality is principally due to tumor recurrence, which can occur following extended periods of clinical remission that may last decades. While clinical latency has been postulated to reflect the ability of residual tumor cells to persist in a dormant state, this hypothesis remains unproven since little is known about the biology of these cells. Consequently, defining the properties of residual tumor cells is an essential goal with important clinical implications for preventing recurrence and improving cancer outcomes.MethodsTo identify conserved features of residual tumor cells, we modeled minimal residual disease using inducible transgenic mouse models for HER2/neu and Wnt1-driven tumorigenesis that recapitulate cardinal features of human breast cancer progression, as well as human breast cancer cell xenografts subjected to targeted therapy. Fluorescence-activated cell sorting was used to isolate tumor cells from primary tumors, residual lesions following oncogene blockade, and recurrent tumors to analyze gene expression signatures and evaluate tumor-initiating cell properties.ResultsWe demonstrate that residual tumor cells surviving oncogenic pathway inhibition at both local and distant sites exist in a state of cellular dormancy, despite adequate vascularization and the absence of adaptive immunity, and retain the ability to re-enter the cell cycle and give rise to recurrent tumors after extended latency periods. Compared to primary or recurrent tumor cells, dormant residual tumor cells possess unique features that are conserved across mouse models for human breast cancer driven by different oncogenes, and express a gene signature that is strongly associated with recurrence-free survival in breast cancer patients and similar to that of tumor cells in which dormancy is induced by the microenvironment. Although residual tumor cells in both the HER2/neu and Wnt1 models are enriched for phenotypic features associated with tumor-initiating cells, limiting dilution experiments revealed that residual tumor cells are not enriched for cells capable of giving rise to primary tumors, but are enriched for cells capable of giving rise to recurrent tumors, suggesting that tumor-initiating populations underlying primary tumorigenesis may be distinct from those that give rise to recurrence following therapy.ConclusionsResidual cancer cells surviving targeted therapy reside in a well-vascularized, desmoplastic microenvironment at both local and distant sites. These cells exist in a state of cellular dormancy that bears little resemblance to primary or recurrent tumor cells, but shares similarities with cells in which dormancy is induced by microenvironmental cues. Our observations suggest that dormancy may be a conserved response to targeted therapy independent of the oncogenic pathway inhibited or properties of the primary tumor, that the mechanisms underlying dormancy at local and distant sites may be related, and that the dormant state represents a potential therapeutic target for preventing cancer recurrence.

ZIP4 Is a Novel Cancer Stem Cell Marker in High-Grade Serous Ovarian Cancer.

Simple SummaryOvarian cancer is the most deadly gynecologic cancer. The treatment options for ovarian cancer, and for the recurrent cancer in particular, are limited. One of the major obstacles is the presence of drug-resistant cancer stem cells. The aim of this study is to identify and characterize a new cancer stem marker, namely ZIP4. ZIP4 is a transporter for human essential element zinc. Our results have shown that ZIP4 is not only a novel and potent stem cell marker, but also a target for developing innovative treatment for ovarian cancer. In addition, ZIP4 is interacting with another oncogene, NOTCH3. Both ZIP4 and NOTCH3 play important roles in tumor development in ovarian cancer. This interaction may represent a useful target for ovarian cancer treatment.High-grade serous ovarian cancer (HGSOC) is one of the most deadly and heterogenic cancers. We have recently shown that ZIP4 (gene name SLC39A4), a zinc transporter, is functionally involved in cancer stem cell (CSC)-related cellular activities in HGSOC. Here, we identified ZIP4 as a novel CSC marker in HGSOC. Fluorescence-activated cell sorter (FACS)-sorted ZIP4+, but not ZIP4− cells, formed spheroids and displayed self-renewing and differentiation abilities. Over-expression of ZIP4 conferred drug resistance properties in vitro. ZIP4+, but not ZIP4− cells, formed tumors/ascites in vivo. We conducted limiting dilution experiments and showed that 100–200 ZIP4+ cells from both PE04 and PEA2 cells formed larger tumors than those from 100–200 ALDH+ cells in mice. Mechanistically, we found that ZIP4 was an upstream regulator of another CSC-marker, NOTCH3, in HGSOC cells. NOTCH3 was functionally involved in spheroid formation in vitro and tumorigenesis in vivo in HGSOC. Genetic compensation studies showed that NOTCH3, but not NOTCH1, was a critical downstream mediator of ZIP4. Furthermore, NOTCH3, but not NOTCH1, physically bound to ZIP4. Collectively, our data suggest that ZIP4 is a novel CSC marker and the new ZIP4-NOTCH3 axis represents important therapeutic targets in HGSOC.

CD319 (SLAMF7) an alternative marker for detecting plasma cells in the presence of daratumumab or elotuzumab.

Daratumumab is an anti-CD38 immunotherapeutic drug that has increasingly been used to treat patients with heavily pre-treated and relapsed/refractory multiple myeloma. In so doing, the detection of CD38 antigen on plasma cells by flow cytometry is impeded. We hypothesized that alternative markers can be used in place or in addition to CD38 when detecting plasma cells post-treated with daratumumab. A total of 16 alternative markers were tested using 22 bone marrow aspirates from patients with plasma cell neoplasm. The ability of selected markers to discern plasma cells from other hematopoietic cells were evaluated. The stability of tested markers when stored at 4 or 25°C after T=0, 24, 48, and 72 h was also established. Finally, selected markers were incorporated into a panel used for monitoring multiple myeloma measurable residual disease to test their utility to identify plasma cells in the presence of daratumumab and/or elotuzumab (anti-CD319) drugs. Out of the 16 tested markers, CD319, CD54, CD229, CD317, and p63 were expressed by >90% of the plasma cells. Only CD319, CD54, and CD229 achieved 100% detection sensitivity. Further analysis showed that CD319 was better than CD229 and CD54 at resolving plasma cells from background hematopoietic cells, with CD54 being the worst (resolution metric, mean ± SD: CD319 [2.04 ± 0.86]; CD229 [1.47 ± 0.45]; and CD54 [1.22 ± 0.60]). CD229 was expressed by >90% of T lymphocytes, whereas CD319 was expressed preferentially by the CD8+ T cells and less frequently in CD4+ T cells. Additionally, CD229 was found on >60% of B and NK cells, as well as minor subsets of monocytes and granulocytes. CD319 was expressed on most NK cells and a minor subset of B cells, granulocytes, and monocytes. Even though CD229 and CD319 were expressed by different leukocyte subsets, their expression levels were highest on plasma cells. The expression of CD138 on plasma cells was significantly lower after storage at 4°C, while the expression levels of CD38, CD229, and CD319 remained stable at 4 or 25°C. Using limiting dilution experiments, the treatment of cells with daratumumab severely impeded the detection of CD38 antigen on plasma cells, whereas elotuzumab treatment did not block detection of CD319 on plasma cells. CD319 is a suitable alternative to CD38 for identifying plasma cells. Our results showed that a panel used for monitoring multiple myeloma measurable residual disease could be modified by using CD319 alone or in combination with CD38 to detect PCs in daratumumab or elotuzumab treated patients.

Abstract 4794: Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells

Abstract Background: We previously reported that ONC201, a novel anti-tumor drug currently being tested in early phase clinical trials, impairs mitochondrial structure and function in breast cancer cells (Greer et al., 2018). Recent studies demonstrated that ONC201 and the related TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein to mitochondrial protein homeostasis (Graves et al. 2019). Cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we tested the impact of ClpP agonists-mediated mitochondrial dysregulation on breast CSCs. Methods: Mitochondrial DNA (mtDNA)-depleted (rho0) MDA-MB231 (MB231) cells were generated by ethidium bromide treatment. MtDNA copy numbers were measured by qPCR. Mitochondrial respiration was monitored by Seahorse XF analyzer. Cell viability was analyzed by CellTiter-Glo 2.0 and RealTime-Glo assay. Protein expression was examined by Western blotting. ClpP knockout (KO) cells were generated by CRISPR/Cas9 technology. Mammosphere formation assay was performed to evaluate CSC function. MB231 cells stably expressing SORE6 (OCT4/SOX2 response element)-mCherry reporter gene were used to monitor CSC numbers. In vivo limiting dilution analysis was performed to examine the effect of ClpP agonists on MB231 mammary fat pad tumor initiation capability in athymic nude female mice. Results: We observed that mammospheres formed by MB231were enriched in mtDNA compared to cells grown in 2D culture while rho0 cells lacking functional mitochondria failed to form mammospheres. This suggested that mitochondria are required for CSC function. The effects of ONC201 and TR compounds in breast cancer cells were tested. First, we confirmed that TR compounds (TR-65, TR-57) had ~60-250 fold higher potency compared to ONC201 in cell viability assays. Both ONC201 and TR-57 impaired mitochondrial structure, function, inhibited cell growth in 2D, and significantly inhibited mammosphere formation in multiple breast cancer cell lines. Importantly, ONC201 and TR-57 did not affect mitochondrial function, cell growth in 2D, nor mammosphere formation in CLPP KO cell lines, confirming ClpP is the target of these drugs. Flow cytometry analysis of MB231-SORE6-mCherry expressing cells demonstrated that ONC201 treatment significantly reduced the fraction of cells that were positive for the SORE6-mCherry activity, consistent with loss of CSCs. Finally, using in vivo limiting dilution experiments, CSC frequency was significantly lower (p<0.05) in the ONC201-treated group compared with the control group, indicating that ONC201 inhibits tumor initiation capability in breast cancers. Conclusion: Our studies suggest that targeting mitochondria by ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells. Citation Format: Yoshimi E. Greer, Lidia Hernandez, Voeller Donna, Raj Chari, Sam Gilbert, Binwu Tang, Christina Annunziata, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4794.

Subcutaneous Immunization of Leishmania HSP70-II Null Mutant Line Reduces the Severity of the Experimental Visceral Leishmaniasis in BALB/c Mice.

Leishmania infantum parasites cause a severe form of visceral leishmaniasis in human and viscerocutaneous leishmaniasis in dogs. Recently, we reported that immunization with an attenuated L. infantum cell line, lacking the hsp70-II gene, protects against the development of murine cutaneous leishmaniasis. In this work, we analyzed the vaccine potential of this cell line towards the long-term protection against murine visceral leishmaniasis. This model shows an organ-dependent evolution of the disease. The infection can resolve in the liver but chronically affect spleen and bone marrow. Twelve weeks after subcutaneous administration of attenuated L. infantum, Bagg Albino (BALB/c) mice were challenged with infective L. infantum parasites expressing the luciferase-encoding gene. Combining in vivo bioimaging techniques with limiting dilution experiments, we report that, in the initial phase of the disease, vaccinated animals presented lower parasite loads than unvaccinated animals. A reduction of the severity of liver damage was also detected. Protection was associated with the induction of rapid parasite-specific IFN-γ production by CD4+ and CD8+ T cells. However, the vaccine was unable to control the chronic phase of the disease, since we did not find differences in the parasite burdens nor in the immune response at that time point.

Breast Cancer Stem Cells with Tumor- versus Metastasis-Initiating Capacities Are Modulated by TGFBR1 Inhibition.

SummaryCancer stem cells (CSCs) are defined by their ability to regenerate a tumor upon transplantation. However, it is not yet clear whether tumors contain a single CSC population or different subsets of cells with mixed capacities for initiating primary and secondary tumors. Using two different identification strategies, we studied the overlap between metastatic stem cells and tumor-initiating cells (TICs) in the MMTV-PyMT model. Our results show that in the MMTV-PyMT model, Lin−CD90−ALDHhigh cells retained a high tumor-initiating potential (TIP) in orthotopic transplants, in contrast to Lin−CD24+CD90+, which retained higher metastatic capacity. Interestingly, suppression of TGFβ signaling increased TIC numbers. We here describe the existence of distinct populations of CSCs with differing capacities to initiate tumors in the primary or the secondary site. Inhibiting TGFβ signaling shifts the balance toward the former, which may have unanticipated implications for the therapeutic use of TGFβ/TGFBR1 inhibitors.

Catecholamine Exposure Accelerates In Vivo Leukemogenesis in Acute Myeloid Leukemia Patient Derived Xenografts

Mouse xenografts are routinely used for in vivo studies on human acute myeloid leukemia (AML) cells. However, a significant proportion of primary AML samples (~50-60% of cases) are not able to repopulate mice, or respectively require a particularly long time (up to 9 months) to show detectable engraftment in such models. Here we report that changing the transplantation time-point from day (5:00 pm) to night (4:00 am) strongly improved the engraftment of human AML cells in NOD/SCID/IL2Rgnull (NSG) mice. Sublethally irradiated gender-matched NSG mice of 6-8 weeks of age (n=40) were transplanted with primary AML cells (n=5 patients). For each sample, equal numbers of freshly thawed AML cells were injected via the tail vein at both time-points; transplanted mice were afterwards monitored for human leukemic engraftment in peripheral blood (PB) and bone marrow (BM) using multicolor flow cytometry and immunohistochemistry. In all n=5 analyzed AML patient samples, transplantation at night strongly promoted leukemogenesis, with 2/5 samples showing engraftment only in the night transplant condition and 3/5 samples showing engraftment in both but significantly higher leukemic burden in night vs. day transplanted mice (Fig. 1a), although similar leukemia-specific mutations were retrieved in mice of the two conditions by next generation sequencing analysis (Fig. 1b). Limiting dilution experiments (n=2 AML samples) further confirmed these findings and showed that in fact lower numbers of AML cells were required to initiate leukemia when cells were transplanted at night versus day times.Given that the immediate process that happens after intravenous injection of AML cells into mice involves homing to BM niches, we hypothesized that the time-point of transplantation influences engraftment by modulating homing. Therefore, we performed homing assays as described for healthy hematopoietic stem/progenitor cells (HSPCs); we injected equally treated human CFSE labeled AML cells via tail vein injection at day versus night time points and analyzed murine BMs 8 hours later for the presence of human leukemic cells. Indeed, significantly higher BM colonization by leukemic cells was observed after night vs. day time injections (0.31±0.09 vs. 0.13±0.18, p<0.001, n= 7 AML patient samples).Since mice transplanted at night showed stress-related behavioral abnormalities, we hypothesized that this transplantation method might induce catecholamine release. ELISAs indeed revealed enhanced epinephrine levels in PB (318.00±12.96 vs. 122.20±47.11 ng/mL, p<0.001) as well as BM (21.08±1.04 vs. 13.50±3.57 ng/mL, p<0.05) of night vs. day transplanted animals. The role of catecholamines influencing leukemia initiation is underinvestigated; b-adrenergic receptors are expressed on several cell types in the BM including HSCs, osteoblasts and mesenchymal stem cells and catecholamine release was shown to regulate HSC egress from BM niches. We hypothesized that enhanced adrenergic signaling may promote leukemogenesis by enhancing leukemic cell homing to BM niches, which in this setting are more readily available due to enhanced healthy HSC egress. To explore this hypothesis, we treated mice with the b1/2-blocker propranolol prior to night transplantation (10mg/kg, intraperitoneal (i.p) injection, 1h pre-transplant, to reverse the “night effect”) and respectively with epinephrine prior to day transplantation (2mg/kg, i.p injection, 1h pre-transplant, to mimic the “night effect”). Indeed, propranolol treatment reduced homing and delayed leukemogenesis in night transplanted mice. Vice-versa, epinephrine enhanced AML cell homing and accelerated long-term leukemia induction from patient derived AML cells and cells of the EOL-1 cell line, and these effects could be counteracted by co-treatment with propranolol. Interestingly, in vitro co-cultures with epinephrine versus empty carrier control enhanced AML cell but not healthy HSPC adhesion to MS5 stroma cells (shown for n=3 cell lines and n=8 primary AML), suggesting that enhanced catecholamine levels may specifically induce AML cell adhesion.We conclude that enhanced catecholamine levels can accelerate leukemia development from AML cells transplanted in xenograft models by facilitating BM niche colonization by AML cells. Catecholamines might in general promote leukemia progression by favoring the retention of leukemic versus healthy HSCs in BM niches. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 5001: Identification of a Six2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization

Abstract Although significant progress has been made in understanding the molecular mechanisms that lead to metastatic breast cancer, it remains the overwhelming cause of death for patients. Current studies primarily focus on the prevention of early stages of metastasis, such as migration and invasion. But at the time of diagnosis tumor cells have likely left the primary tumor, suggesting that inhibition of the early stages of metastasis may not be the most effective means of inhibiting metastatic burden. Instead, the identification and targeting of molecules required for establishment and survival of cells at secondary sites is imperative for advancing therapies. We are examining the role of the developmental transcription factor Six2 in promoting metastatic burden. Six2 is a member of the Six family of transcription factors and is responsible for the maintenance of stem/progenitor cells during nephrogenesis. Using knockdown (KD) and overexpression (OE) models in murine mammary cells, we demonstrated that Six2 promotes specifically late-stage metastasis (growth at the secondary site). RNA-Seq analysis on control and Six2 OE cells showed a dramatic enrichment in stem cell transcriptional programs downstream of Six2 in mammary/breast cancer cells. Flow cytometry analyses and tumorsphere assays demonstrate that Six2 OE leads to an increase, whereas Six2 KD leads to a decrease, in the mammary stem cell population and tumorsphere formation, respectively. Using an immune competent mouse model, in vivo orthotopic limiting-dilution experiments reveal that Six2 regulates tumor initiation. In addition, we show that Six2 regulates late-stage metastasis of human breast cancer cells in vivo, as measured by metastatic burden after tail vein injection. Thus, our data demonstrate that Six2 similarly promotes stem phenotypes in breast cancer cells as it does during kidney development, and suggest that this attribute may be critical for its ability to promote metastatic outgrowth. To determine the molecular mechanism by which Six2 mediates stem phenotypes and metastasis in breast cancer, we interrogated our RNA-seq data, which suggested that Six2 may control master regulators of stemness. We demonstrate that both Sox2 and Nanog are regulated by Six2. In human breast cancer gene expression datasets, Six2 significantly positively correlates with both Sox2 and Nanog, and combined OE of Six2 with either Sox2 or Nanog results in poor prognosis. Preliminary data suggest that loss of Sox2 downstream of Six2 inhibits metastatic outgrowth, and that Sox2 is upstream of Nanog in the pathway. Together, our data demonstrate that Six2 regulates cancer stem phenotypes to promote metastatic outgrowth, uncovering a novel Six2/Sox2/Nanog axis that is critical for metastatic colonization. Citation Format: Michael U. Oliphant, Ahwan Pandey, Katherine Johnson, Rani Powers, Matthew Galbraith, James Costello, Heide L. Ford. Identification of a Six2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5001.

Abstract 1115: A CRISPR/Cas9 kinome screen identifies GRK5 as a novel regulator of rhabdomyosarcoma (RMS) self-renewal

Abstract Rhabdomyosarcoma (RMS) is a devastating pediatric soft tissue sarcoma. Clinical outcome for patients with relapsed or metastatic disease remains poor. However, current treatment options remain relatively unchanged over the last two decades, so there remains an urgent need for more effective treatment options for disease relapse. Cancer stem cells (CSCs), also known as tumor-propagating cells (TPCs), have the capacity for self-renewal and have been proposed to be responsible for tumor relapse and metastasis. RMS has been shown to follow the CSC model of tumor progression. We have previously performed an siRNA library screen against the human kinome (714) and a secondary CRISPR/Cas9 screen of top candidates to narrow down to 11 novel kinase candidates that are potentially essential for self-renewal of human RMS cells. GRK5, a G-protein coupled receptor kinase, was among the top candidate genes. Preliminary data from in vitro cell-based assays and in vivo limiting-dilution experiments in xenografts showed that CRISPR/Cas9-mediated targeted disruption of GRK5 resulted in significant reduction in the self-renewal capacity of RMS cells. GRK5-deficient RMS cells also displayed significant reduction in growth secondary to cell cycle arrest at the G2/M checkpoint. The functional effects on tumor growth and self-renewal appeared unique to GRK5 among the GRK kinase subfamily, as targeted disruption of closely-related GRK4 and GRK6 did not significantly affect RMS tumor cell growth and self-renewal. We propose that targeted disruption of GRK5 by CRISPR/Cas9 results in elevated p53 protein stability and activity, leading to inhibition of proteins required for cell cycle progression. We are also characterizing the loss-of-function effects of GRK5 in a RMS TPC reporter cell line created using a CRISPR/Cas9 knockin approach that will allow for direct assessment of GRK5 and other candidate gene function within the TPC population. By characterizing the biologic role of TPC as well as the function of GRK5 in RMS, we hope to provide new biologic insights into cancer stem cell biology and GRK5 as a novel therapeutic target for the treatment of advanced RMS. Citation Format: Thao Q. Pham, Terra Vleeshouwer-Neumann, Eleanor Chen. A CRISPR/Cas9 kinome screen identifies GRK5 as a novel regulator of rhabdomyosarcoma (RMS) self-renewal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1115.

Metastasis: Slipping Control

Metastasis: Slipping Control

Abstract B24: Patient-derived tumor xenograft to study cancer stem cells is pediatric sarcomas

Abstract Tumor heterogeneity is recognized as an important hallmark in cancer. This characteristic allows cancer cells to evolve in time, to survive radiation and cyotoxic therapies, and to thrive in different organ microenvironments. Therefore, the presence of distinct tumor subpopulations, as mainstay feature of tumor heterogeneity, may play a role in tumor progression and treatment resistance. Patient-derived xenografts (PDXs) have been used to discover the molecular signaling pathways involved in tumorigenesis, metastasis formation and radiation/chemotherapy resistance in different epithelial-adult cancers. Our research interest in pediatric sarcomas is to understand the processes that drive tumor heterogeneity, to isolate and characterize cancer stem cells (CSCs), and to elucidate the mechanisms of chemoresistance using a PDX model. Pediatric patients with suspected or established diagnosis of sarcoma evaluated at Texas Children's Hospital signed an informed consent to provide tumor samples for subcutaneous implantation of tumor explants in the flank of nonobese diabetic severe combined immunodeficient gamma (NSG) mice. Tumor explants were implanted on the same day of collection when was possible. First and subsequent PDX generations were harvested for tumor propagation, tumor histology, tumor cell karyotyping , RNA/DNA extraction for genetic studies and fluorescent activated cell sorting (FACS) analysis for ALDH, CD133, EphA2 and CD47. Forty-six tumor samples have been collected since June 2013. Half of tumor samples (n=21) corresponded to osteosarcoma patients and 59% of all tumor specimens were obtained at the time of the initial biopsy. Approximately 66% of biopsies have been performed by Interventional Radiology in our institution. All PDXs maintained the same histological characteristics that original tumors, and also presented specific genetic translocations associated with the different sarcoma types. Chemotherapy resistant and very aggressive tumors (metastatic) had short tumor latency and generate tumors in all transplanted mice. We did not observe the formation of metastases (lungs or liver) in any of the transplanted PDX mice. PDXs significantly increased the amount of tumor material necessary to perform cell culture under stem-like cell conditions and to analyze the expression of CSC markers by FACS. These studies would be very difficult to perform directly from patient tumor biopsies. All tumors harvested (n=15) contained 2-9% of ALDH Hi or stem-like cells. None of the PDXs analyzed expressed CD133 but synovial sarcoma. We found that &amp;gt;90% of sarcoma cells expressed CD47 and EphA2 at different intensity levels. Whole genome sequencing of different tumor cell subpopulations and limiting dilution experiments to validate the presence of CSCs are ongoing. Core needle biopsies would represent the most common source to obtain tumor specimens before treatment in pediatric sarcomas. The development of PDXs allows the propagation of tumor cell subpopulations, including CSCs, without the biological changes impose by cell culture. PDXs resemble the original tumor and constitute a powerful tool to study tumor heterogeneity. We plan to perform orthotopic transplantation and tail vein injection of PDX cells in an attempt to develop a PDX model of metastatic disease in pediatric sarcomas. Citation Format: Nino Rainusso, Jose Hernandez, Rex Marco, Sanjeev Vasudevan, Norma Quintanilla, John Hicks, Wendy Allen-Rhoades, Matteo Trucco, Jason Yustein. Patient-derived tumor xenograft to study cancer stem cells is pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B24.

Intra-Bone Marrow Transplantation Confers Superior Multilineage Engraftment of Murine Aorta-Gonad Mesonephros Cells Over Intravenous Transplantation.

Hematopoietic stem cell (HSC) engraftment has been achieved using single-cell transplantation of prospectively highly purified adult HSC populations. However, bulk transplants are still performed when assessing the HSC potential of early embryonic hematopoietic tissues such as the aorta-gonad mesonephros (AGM) due to very low HSC activity content early in development. Intra-bone marrow transplantation (IBMT) has emerged as a superior administration route over intravenous (IV) transplantation for assessing the reconstituting ability of human HSCs in the xenotransplant setting since it bypasses the requirement for homing to the BM. In this study, we compared the ability of IBMT and IV administration of embryonic day 11.5 AGM-derived cells to reconstitute the hematopoietic system of myeloablated recipients. IBMT resulted in higher levels of AGM HSC long-term multilineage engraftment in the peripheral blood, BM, spleen, and thymus of primary and secondary recipients, and in limiting dilution experiments. The administration route did not skew the multilineage contribution pattern, but IBMT conferred higher Lineage(-)Sca-1(+)c-kit(+) long-term engraftment, in line with the superior IBMT reconstitution. Therefore, IBMT represents a superior administration route to detect HSC activity from developmentally early sources with limited HSC activity content, such as the AGM.

Abstract 1910: Evolution of preleukemia stem cells to lymphoma initiating cells requires thymus in msh2-/- mice

Abstract Thymic lymphomas have been thought to develop from disregulated differentiation and proliferation of developing thymocytes. T cell progenitors are generated from hematopoietic stem cells (HSCs) in the bone marrow (BM). Recent evidence strongly suggests that whereas transformation events may occur at different developmental stages, the initial mutation originates in the HSCs, and creates a preleukemic stem cell (PLSC). Subsequent mutations at either stem cell or progenitor cell levels would transform the PLSC into lymphoma/leukemia initiating cells (LIC). Maturation and proliferation of T cells depend on regulatory mechanisms in the thymus where the T-progenitors must interact with the microenvironment. Here we investigate the requirement of thymus environment for lymphoma development. Defects in the mismatch repair system (MMR) underlie hereditary non-polyposis colorectal cancer (HNPCC)/Lynch syndrome. Individuals with no MMR function present with childhood onset of hematological and brain malignancies. Mice carrying a null allele for the MMR gene, Msh2, are preferentially prone to develop thymic lymphomas and to a lesser extent, gastrointestinal tumors. Consistent with these findings in mice, MMR defects have also been observed in sporadic and hereditary hematological malignancies. In this study, we use MSH2-/- mice as a model to investigate the existence of PLSC and the evolution of PLSC to LIC. Using bone marrow transplantation, we found that limiting dilutions of MSH2-/- HSCs from young mice are able to reconstitute lethally irradiated wild-type recipients, and contribute to development of multiple hematopoietic lineages. However, all the recipients develop thymic lymphomas after a latency of 3-4 months post transplantation. Transplantation of different fractions of bone marrow cells or thymocytes from young MSH2-/- mice showed that only the HSC enriched fraction leads to lymphoma development. The lymphomas are transplantable, limiting dilution experiments showed that even 40 lymphoma cells could initiate T cell leukemia in sublethally irradiated secondary recipients within a month. In contrast, transplantation of the HSC enriched LSK fraction from the BM of lymphoma bearing mice into secondary recipients resulted in thymic lymphomas after a latency of 5 months. However, lymphoma development following transplant of HSC required an intact thymus and was not observed for more than 9 months if recipients were thymectomized, whereas this stromal requirement was not observed when transplanting T cell leukemicthymic lymphoma cells. These results suggested that MSH2-/- HSCs are PLSCs. While they retain full hematopoietic potential, their T-cell progeny gain lymphomagenic potential in the thymic microenvironment and become LICs. In this model, the evolution from PLSC to LIC is stromal-dependent. Citation Format: Yulan Qing, Stanton L. Gerson. Evolution of preleukemia stem cells to lymphoma initiating cells requires thymus in msh2-/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2014-1910

A Pyrosequencing-Based Assay for the Rapid Detection of the 22q11.2 Deletion in DNA from Buccal and Dried Blood Spot Samples

The 22q11.2 deletion syndrome is one of the most common deletion syndromes in newborns. Some affected newborns may be diagnosed shortly after birth because of the presence of heart defects, palatal defects, or severe immune deficiencies. However, diagnosis is often delayed in patients presenting with other associated conditions that would benefit from early recognition and treatment, such as speech delays, learning difficulties, and schizophrenia. Fluorescence in situ hybridization (FISH) is the gold standard for deletion detection, but it is costly and time consuming and requires a whole blood specimen. Our goal was to develop a suitable assay for population-based screening of easily collectible specimens, such as buccal swabs and dried blood spots (DBS). We designed a pyrosequencing assay and validated it using DNA from FISH-confirmed 22q11 deletion syndrome patients and normal controls. We tested DBS from nine patients and paired buccal cell and venous blood specimens from 20 patients. Results were 100% concordant with FISH assay results. DNA samples from normal controls (n = 180 cell lines, n = 15 DBS, and n = 88 buccal specimens) were negative for the deletion. Limiting dilution experiments demonstrated that accurate results could be obtained from as little as 1 ng of DNA. This method represents a reliable and low-cost alternative for detection of the common 22q11.2 microdeletions and can be adapted to high-throughput population screening.

Switching and emergence of CTL epitopes in HIV-1 infection.

BackgroundHuman Leukocyte Antigen (HLA) class I restricted Cytotoxic T Lymphocytes (CTLs) exert substantial evolutionary pressure on HIV-1, as evidenced by the reproducible selection of HLA-restricted immune escape mutations in the viral genome. An escape mutation from tyrosine to phenylalanine at the 135th amino acid (Y135F) of the HIV-1 nef gene is frequently observed in patients with HLA-A*24:02, an HLA Class I allele expressed in ~70% of Japanese persons. The selection of CTL escape mutations could theoretically result in the de novo creation of novel epitopes, however, the extent to which such dynamic “CTL epitope switching” occurs in HIV-1 remains incompletely known.ResultsTwo overlapping epitopes in HIV-1 nef, Nef126-10 and Nef134-10, elicit the most frequent CTL responses restricted by HLA-A*24:02. Thirty-five of 46 (76%) HLA-A*24:02-positive patients harbored the Y135F mutation in their plasma HIV-1 RNA. Nef codon 135 plays a crucial role in both epitopes, as it represents the C-terminal anchor for Nef126-10 and the N-terminal anchor for Nef134-10. While the majority of patients with 135F exhibited CTL responses to Nef126-10, none harboring the “wild-type” (global HIV-1 subtype B consensus) Y135 did so, suggesting that Nef126-10 is not efficiently presented in persons harboring Y135. Consistent with this, peptide binding and limiting dilution experiments confirmed F, but not Y, as a suitable C-terminal anchor for HLA-A*24:02. Moreover, experiments utilizing antigen specific CTL clones to recognize endogenously-expressed peptides with or without Y135F indicated that this mutation disrupted the antigen expression of Nef134-10. Critically, the selection of Y135F also launched the expression of Nef126-10, indicating that the latter epitope is created as a result of escape within the former.ConclusionsOur data represent the first example of the de novo creation of a novel overlapping CTL epitope as a direct result of HLA-driven immune escape in a neighboring epitope. The robust targeting of Nef126-10 following transmission (or in vivo selection) of HIV-1 containing Y135F may explain in part the previously reported stable plasma viral loads over time in the Japanese population, despite the high prevalence of both HLA-A*24:02 and Nef-Y135F in circulating HIV-1 sequences.

CD3 Antibody Treatment Prevents Gvhd and Allows For Rapid, Long Term, Multilineage Engraftment Of Unfractionated UCB Into Immunodeficient Mice

NSG mice are currently the best hosts for human hematopoietic stem cell xenografts, due in large part to their ability to initiate robust grafts comprised of diverse human blood cell lineages. Unfortunately, NSG mice are susceptible to GVHD induced by contaminating T cells. Therefore, stem and progenitor cells must be selected with a high degree of purity. This not only complicates the procedure, but also prevents modeling of whole umbilical cord blood (WUCB) and bone marrow transplantation. Here, we have explored both in vivo and in vitro methods to prevent xenogenic GVHD utilizing the anti-T cell antibodies ATG and OKT3. IP injection of either antibody within the first 72 hours of transplant proved effective at preventing T cell expansion. Alternatively, a simple short-term incubation of the cell suspension with antibody was equally effective. ATG, but not OKT3, had significant non-specific activity resulting in reduction and even prevention of the non-T cell portion of the graft, including SCID repopulating cells, using either the in vivo or in vitro approach. However, using the OKT3 in vitro incubation protocol, we were able to initiate stable, long term, multi-lineage engraftment from whole, unselected UCB without GVHD. Additionally, WUCB transplanted mice engrafted more readily, with a wider array of human cell types in the peripheral blood as early as 2 weeks, relative to CD34+ engrafted mice. Stem cell engraftment was confirmed by successful transplant of the human graft to secondary recipients. Limiting dilution experiments showed that significant human grafts could be generated with as few as 1 million total WBCs per mouse, indicating that very large cohorts of mice can be generated with a single UCB sample. This protocol not only has the potential to significantly streamline hematopoietic xenograft studies while greatly reducing the cost and time commitments involved, but also to allow for whole cord blood transplantation modeling and generation of unique subsets of human cells in vivo. Disclosures:No relevant conflicts of interest to declare.