Tumor Progenitor Cells Research Articles

Abstract 2853: Monoclonal antibodies derived in neuN mice following successful T cell therapy of spontaneous breast tumors are specific for tumor progenitor cells.

Abstract There is considerable interest in developing approaches to specifically target tumor progenitor cells. Our previous results demonstrated that adoptive transfer of CD4+ and CD8+ T cells primed with a breast cancer progenitor vaccine inhibited the progression of spontaneous primary tumors in neuN mice. Interestingly, post-treatment serum but not pre-treatment serum was reactive with surface molecules on the vaccine cells. This indicates that the transferred T cells resulted in uptake of tumor antigens and stimulation of host B cells. To characterize the antigenic targets, we generated hybridomas from spleen cells derived from treated mice. Three candidate clones which were strongly reactive with a neu-negative variant tumor line were subjected to two rounds of limiting dilution cloning. The mAb prepared from hybridoma #1 (IgG1) and hybridoma #2 (IgG2b) were each reactive against two independent tumor lines derived from spontaneous breast tumors in neuN mice (C2D1 and MMC5) when these cells were maintained in serum-free culture medium as tumorspheres. There was no reactivity against the same tumor cell lines when cultured as adherent monolayers in serum-containing medium nor was there reactivity against 3T3 adherent or tumorsphere cultures. Our previous analysis of tumorsphere cultures of C2D1 and MMC5 indicated that they express a panel of markers associated with tumor progenitor cells and have markedly enhanced per-cell tumorigenicity. Two of these antibodies have been purified by protein G or Protein A columns and retain binding to C2D1-tumorsphere cells, but not to C2D1 adherent cells. Importantly, these two antibodies can also bind to cultured 4T1-tumorsphere cells, an additional breast cancer cell line arising initially in BALB/c mice. These studies will allow us to identity corresponding antigens and determine expression on a broader array of spontaneous breast carcinomas in neuN mice. The epitopes recognized by these mAbs, which are expressed on progenitor cells from multiple tumor lines, might have importance as an immunotherapy target. Citation Format: Li-Xin Wang, Mona Patel, Michael Berk, Gregory E. Plautz. Monoclonal antibodies derived in neuN mice following successful T cell therapy of spontaneous breast tumors are specific for tumor progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2853. doi:10.1158/1538-7445.AM2013-2853

Abstract 365: Circulating bone marrow-derived VEGFR-2+ progenitor cells in benign and malignant soft tissue tumors.

Abstract Background : Circulating endothelial progenitor cells (cEPCs) as recruited to the angiogenic vascular system of malignant tumors have been evaluated as a biomarker of tumor remission or progression through therapy in lung and breast cancer. We were interested to analyze the presence of bone marrow derived (BMD) cEPCs in soft tissue malignancies and correlate the changes to treatment efficacy. Methods: 75 treatment-naïve patients have (41 sarcoma, 21 gastro-intestinal stromal tumors GIST, 12 desmoid) and 11 healthy controls have been enrolled to the study. 25 ml of blood samples were drawn prior to therapy and 4-6 weeks after treatment by 20G iv cannula, collected in tubes containing sodium citrate (0.105 M) and processed within 1 h. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation and cEPCs were characterized by quadruple staining using antibodies against CD133+, CD34, CD45dim and vascular endothelial growth factor-receptor (VEGF-R)-2+. The expression of cell-surface antigens was determined by four-color immunofluorescence. The frequency of cEPCs in peripheral blood was determined by a 2-dimensional side-scatter/fluorescence dot-plot analysis after exclusion of annexin V-positive cells and appropriate to exclude apoptotic cells. EPC counts are expressed as a percentage of total PBMCs. Serum-concentrations of VEGF and Angiopoetin-2 were determined by ELISA in triplicate samples from 5 ml serum. Results:The percentage of hematopoietic stem cells (CD34+, CD133+) was significantly increased in sarcoma and desmoid patients (0.44 ± 0.1 %) compared to GIST and healthy controls (0.13 ± 0.04 %; p=0.001). The percentage of VEGFR-2+ cells correspond to cEPC in sarcoma of 0.179 ± 0.190 % vs. healthy controls of 0.025 ± 0.018 %. Values for desmoid patients were similar to sarcoma whereas GIST patients showed values comparable to healthy patients. We found significantly increased mean VEGF levels in both sarcoma and desmoids (359 ± 157 pg/ml) in comparison to healthy controls (27 ± 13 pg/ml; p = 0.001). Ang-2 levels before therapy were significantly lower in healthy controls and desmoids (1665 ± 445 pg/ml) compared to sarcoma and GIST (2929 ± 960 pg/ml, p = 0.033) patients. Patients without signs of tumor recurrence showed normal cEPC values at post-treatment controls, whereas patients with progressive disease had increasing cEPC levels. Conclusion: This study demonstrates an increased mobilization of cEPCs in sarcoma and (non-metatasizing) desmoids tumors compared to healthy individuals and patients with GIST. cEPCs recruited for vasculogenesis and paracrine effects play a different role in the various subtypes of human soft tissue tumors. Citation Format: Kai Nowak, Elena Joas, Grietje Beck, Neysan Rafat, Peter Hohenberger. Circulating bone marrow-derived VEGFR-2+ progenitor cells in benign and malignant soft tissue tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 365. doi:10.1158/1538-7445.AM2013-365

Abstract 4660: A randomized phase II study of the telomerase inhibitor imetelstat as maintenance therapy for advanced non-small cell lung cancer.

Abstract Tumor regrowth after chemotherapy may be driven by growth of tumor ‘stem cells’. Telomerase, required for indefinite replication, is upregulated in tumor progenitor cells. Imetelstat is a 13-mer oligonucleotide which is a potent and specific telomerase inhibitor. Progression-free survival (PFS) and the duration of responses after 1st-line chemotherapy for non-small-cell lung cancer (NSCLC) are short, which has led to an interest in developing active maintenance therapies. A randomized phase II study was conducted to assess whether imetelstat, given as maintenance therapy, prolongs PFS in NSCLC. Pts were eligible with advanced NSCLC not progressing after completing 4-6 cycles of platinum-based doublet induction chemotherapy, any NSCLC histology, performance status (PS) ECOG 0 or 1, and not scheduled to receive maintenance with pemetrexed or erlotinib. Pts were randomized 2:1 to imetelstat 9.4 mg/kg (d1 and 8 of a 21d cycle) or observation until progressive disease or unacceptable toxicity (concomitant use of bevacizumab was permitted). The 1o endpoint was PFS; safety/tolerability and objective response rate were 2o endpoints. 116 pts were enrolled between Jul 2010 to Apr 2012, with 114 completing a first visit (i.e. efficacy population). Baseline characteristics of age, gender, PS and number of induction cycles were well balanced. 18.4% had squamous histology; 31.6% received concomitant bevacizumab (bev). The median number of imetelstat maintenance cycles was 3. In the overall analysis of PFS, a non significant improvement in favor of the imetelstat arm was observed (HR = 0.77, P=0.295, 95% CI 0.48 - 1.25). Median PFS was 2.8m for imetelstat and 2.6m for control. In the subgroup of patients who did not receive bevacizumab, the HR was 0.59 (vs. 1.07 for the bevacizumab subgroup; interaction P=0.15). The objective response rate was 4.3% in the imetelstat arm and 2.8% in the control arm. Six month overall survival was 80% for imetelstat and 72% for control (HR = 0.86, P=0.642, 95% CI 0.44 - 1.66). Imetelstat was generally well tolerated although hematologic toxicity, predominantly neutropenia and thrombocytopenia, was increased in the imetelstat arm (grade 3/4 neutropenia 18% for imetelstat vs 0% for control, grade 3/4 thrombocytopenia 37% for imetelstat vs 0% for control). The most frequent non-hematologic toxicities were fatigue (imetelstat 42.1% vs control 18.4%), nausea (43.4% vs 7.9%), vomiting (25.0% vs 5.3%) and anaemia (19.7% vs 5.3%). The overall findings suggest that imetelstat has modest, but not clinically meaningful, activity as a maintenance therapy in pts with NSCLC. A pre-specified exploratory subgroup analysis of PFS by tumor telomere length is reported separately. Citation Format: Alberto Chiappori, Tatjana Kolevska, Bart Burington, David Spigel, Steven Hager, Mark Rarick, Shirish Gadgeel, Normand Blais, Joachim Von Pawel, Lowell Hart, Martin Reck, Joan Schiller. A randomized phase II study of the telomerase inhibitor imetelstat as maintenance therapy for advanced non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4660. doi:10.1158/1538-7445.AM2013-4660

Abstract 2512: Extreme value distributions describe cancer age-specific incidence data.

Abstract The incidence of cancers varies with age, if normalized this is called the age-specific incidence. A mathematical model that describes this variation should provide a better understanding of how cancers develop. We suggest that the age-specific incidence should follow an extreme value distribution, based on three widely accepted assumptions: (1) a tumor develops from a single cell, (2) many potential tumor progenitor cells exist in a tissue, and (3) cancer is diagnosed when the first of these many potential tumor cells develops into a tumor. We tested this hypothesis by comparing the predicted distribution to the age-specific incidence data for colon and prostate carcinomas collected by the Surveillance, Epidemiology and End Results network of 17 cancer registries. We found that colon carcinoma age-specific incidence data is consistent with an extreme value distribution, while prostate carcinomas age-specific incidence data generally follows the distribution. This model indicates that both colon and prostate carcinomas only occur in a subset of the population (22% for prostate and 13.5% for colon.) Because of their very general nature, extreme value distributions might be applicable to understanding other chronic human diseases. Citation Format: Luis Soto-Ortiz, James P. Brody. Extreme value distributions describe cancer age-specific incidence data. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2512. doi:10.1158/1538-7445.AM2013-2512

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Use of pharmacogenomic modeling in pancreatic cancer for prediction of chemotherapy response and resistance.

142 Background: Pancreatic adenocarcinoma (PDAC) is uniformly lethal; despite this, modern cytotoxics (C) can induce tumor responses and extend life. Xenograft models have shown that pharmacogenomic (PGx) modeling of C can predict efficacy. Chemo-sensitivity and gene expression profiling of tumor progenitor (TP) cells isolated from peripheral blood may predict tumor response, progression and resistance. Methods: A prospective MSKCC study is ongoing with 46 of 60 planned patients accrued. 10 mL of peripheral blood is collected from patients with unresectable PDAC prior to C and at disease progression. Clinical data is prospectively collected. Circulating TP cells are isolated (Vita-Cap, Vitatex Inc.), total RNA is extracted and gene-expression analysis is performed. PGx models for twelve chemotherapy drug combinations used in PDAC were created from the GI50 data obtained from the NCI-60 cell lines (CellPath Therapeutics, Inc., Baltimore, MD). Expression data were normalized through GCRMA then probed to identify expression differences between the 35 patients with continued disease response and the 11 patients with disease progression. Analysis was performed at the pathway and individual gene level. Results: TP cells were isolated and gene expression and chemo-sensitivity patterns were obtained in all 46 evaluable patients prior to initiating C and in all 11 patients at 1st line disease progression. Preliminary analysis demonstrates clinical benefit for patients treated with C predicted to be effective versus C predicted to be ineffective (TTP 4.4 mo versus 2.7 mo, p=0.016). Changes in chemo-sensitivity patterns were evident at disease progression, reflecting treatment resistance. Hedgehog pathway over-expression was associated with resistance to gemcitabine but clinical response to 5-FU based treatment. Pathway analysis revealed that ErbB3, ARE/Nrf2 and Insulin pathways distinguished patients with disease progression. Conclusions: Isolation and gene expression profiling of TP cells can be performed reliably in unresectable PDAC. Preliminary analysis suggests that C profiling can predict response. Repeat PGx profiling identifies key pathways associated with treatment resistance. Clinical trial information: NCT01474564.

β4 Integrin signaling induces expansion of prostate tumor progenitors.

The contextual signals that regulate the expansion of prostate tumor progenitor cells are poorly defined. We found that a significant fraction of advanced human prostate cancers and castration-resistant metastases express high levels of the β4 integrin, which binds to laminin-5. Targeted deletion of the signaling domain of β4 inhibited prostate tumor growth and progression in response to loss of p53 and Rb function in a mouse model of prostate cancer (PB-TAg mice). Additionally, it suppressed Pten loss-driven prostate tumorigenesis in tissue recombination experiments. We traced this defect back to an inability of signaling-defective β4 to sustain self-renewal of putative cancer stem cells in vitro and proliferation of transit-amplifying cells in vivo. Mechanistic studies indicated that mutant β4 fails to promote transactivation of ErbB2 and c-Met in prostate tumor progenitor cells and human cancer cell lines. Pharmacological inhibition of ErbB2 and c-Met reduced the ability of prostate tumor progenitor cells to undergo self-renewal in vitro. Finally, we found that β4 is often coexpressed with c-Met and ErbB2 in human prostate cancers and that combined pharmacological inhibition of these receptor tyrosine kinases exerts antitumor activity in a mouse xenograft model. These findings indicate that the β4 integrin promotes prostate tumorigenesis by amplifying ErbB2 and c-Met signaling in tumor progenitor cells.

Tumor-Infiltrating Immune Cells: Triggers for Tumor Capsule Disruption and Tumor Progression?

Background: Our previous studies of human breast and prostate cancer have shown that aberrant immune cell infiltration is associated with focal tumor capsule disruption and tumor cell budding that facilitate invasion and metastasis. Our current study attempted to determine whether aberrant immune cell infiltration would have similar impact on colorectal cancer (CRC).Materials and Methods: Tissue sections from 100 patients with primary CRC were assessed for the frequencies of focal basement membrane (BM) disruption, muscularis mucosa (MM) fragmentation, and tumor cell dissemination in epithelial structures adjacent and distal to infiltrating lymphoid aggregates using a panel of biomarkers and quantitative digital imaging.Results: Our study revealed: (1) epithelial structures adjacent to lymphoid follicles or aggregates had a significantly higher (p<0.001) frequency of focally disrupted BM, dissociated epithelial cells in the stroma, disseminated epithelial cells within lymphatic ducts or blood vessels, and fragmented MM than their distal counterparts, (2) a majority of dissociated epithelial cells within the stroma or vascular structures were immediately subjacent to or physically associated with infiltrating immune cells, (3) the junctions of pre-invasive and invasive lesions were almost exclusively located at sites adjacent to lymphoid follicles or aggregates, (4) infiltrating immune cells were preferentially associated with epithelial capsules that show distinct degenerative alterations, and (5) infiltrating immune cells appeared to facilitate tumor stem cell proliferation, budding, and dissemination.Conclusions: Aberrant immune cell infiltration may have the same destructive impact on the capsule of all epithelium-derived tumors. This, in turn, may selectively favor the proliferation of tumor stem or progenitor cells overlying these focal disruptions. These proliferating epithelial tumor cells subsequently disseminate from the focal disruption leading to tumor invasion and metastasis.

Candidate Pathways for Promoting Differentiation or Quiescence of Oligodendrocyte Progenitor-like Cells in Glioma

Platelet-derived growth factor receptor alpha-positive oligodendrocyte progenitor cells (OPC) located within the mature central nervous system may remain quiescent, proliferate, or differentiate into oligodendrocytes. Human glioblastoma multiforme tumors often contain rapidly proliferating oligodendrocyte lineage transcription factor 2 (Olig2)-positive cells that resemble OPCs. In this study, we sought to identify candidate pathways that promote OPC differentiation or quiescence rather than proliferation. Gene expression profiling conducted in both normal murine OPCs and highly proliferative Olig2-positive glioma cells identified all the transcripts associated with the highly proliferative state of these cells and showed that among the various cell types found within the brain, Olig2-positive tumor cells are most similar to OPCs. We then subtracted OPC transcripts found in tumor samples from those found in normal brain samples and identified 28 OPC transcripts as candidates for promoting differentiation or quiescence. Systematic analysis of human glioma data revealed that these genes have similar expression profiles in human tumors and were significantly enriched in genomic deletions, suggesting an antiproliferative role. Treatment of primary murine glioblastoma cells with agonists of one candidate gene, Gpr17, resulted in a decreased number of neurospheres. Together, our findings show that comparison of the molecular phenotype of progenitor cells in tumors to the equivalent cells in the normal brain represents a novel approach for the identification of targeted therapies.

The DNA methylation landscape of small cell lung cancer suggests a differentiation defect of neuroendocrine cells

Small cell lung cancer (SCLC) is a disease characterized by aggressive clinical behavior and lack of effective therapy. Owing to its tendency for early dissemination, only a third of patients have limited-stage disease at the time of diagnosis. SCLC is thought to derive from pulmonary neuroendocrine cells. Although several molecular abnormalities in SCLC have been described, there are relatively few studies on epigenetic alterations in this type of tumor. Here, we have used methylation profiling with the methylated-CpG island recovery assay in combination with microarrays and conducted the first genome-scale analysis of methylation changes that occur in primary SCLC and SCLC cell lines. Among the hundreds of tumor-specifically methylated genes discovered, we identified 73 gene targets that are methylated in >77% of primary SCLC tumors, most of which have never been linked to aberrant methylation in tumors. These methylated targets have potential for biomarker development for early detection and therapeutic management of SCLC. SCLC cell lines had a greater number of hypermethylated genes than primary tumors. Gene ontology analysis indicated a significant enrichment of methylated genes functioning as transcription factors and in processes of neuronal differentiation. Motif analysis of tumor-specific methylated regions identified enrichment of binding sites for several neural cell fate-specifying transcription factors including NEUROD1, HAND1, ZNF423 and REST. We hypothesize that two potential mechanisms, loss of cell fate-determining transcription factors by methylation of their promoters and functional inactivation of their corresponding genomic-binding sites by DNA methylation, can promote a differentiation defect of neuroendocrine cells thus enhancing the ability of tumor progenitor cells to transition toward SCLC.

Neuronatin in a Subset of Glioblastoma Multiforme Tumor Progenitor Cells Is Associated with Increased Cell Proliferation and Shorter Patient Survival

Glioblastoma multiforme is the most common and malignant primary brain tumor. Recent evidence indicates that a subset of glioblastoma tumor cells have a stem cell like phenotype that underlies chemotherapy resistance and tumor recurrence. We utilized a new “multidimensional” capillary isoelectric focusing nano-reversed-phase liquid chromatography platform with tandem mass spectrometry to compare the proteomes of isolated glioblastoma tumor stem cell and differentiated tumor cell populations. This proteomic analysis yielded new candidate proteins that were differentially expressed. Specifically, two isoforms of the membrane proteolipid neuronatin (NNAT) were expressed exclusively within the tumor stem cells. We surveyed the expression of NNAT across 10 WHO grade II and III gliomas and 23 glioblastoma (grade IV) human tumor samples and found NNAT was expressed in a subset of primary glioblastoma tumors. Through additional in vitro studies utilizing the U87 glioma cell line, we found that expression of NNAT is associated with significant increases in cellular proliferation. Paralleling the in vitro results, when NNAT levels were evaluated in tumor specimens from a consecutive cohort of 59 glioblastoma patients, the presence of increased levels of NNAT were found to be a an independent risk factor (P = 0.006) for decreased patient survival through Kaplan-Meier and multivariate analysis. These findings indicate that NNAT may have utility as a prognostic biomarker, as well as a cell-surface target for chemotherapeutic agents.

A theory of the cancer age-specific incidence data based on extreme value distributions

The incidence of cancers varies with age, if normalized this is called the age-specific incidence. A mathematical model that describes this variation should provide a better understanding of how cancers develop. We suggest that the age-specific incidence should follow an extreme value distribution, based on three widely accepted assumptions: (1) a tumor develops from a single cell, (2) many potential tumor progenitor cells exist in a tissue, and (3) cancer is diagnosed when the first of these many potential tumor cells develops into a tumor. We tested this by comparing the predicted distribution to the age-specific incidence data for colon and prostate carcinomas collected by the Surveillance, Epidemiology and End Results network of 17 cancer registries. We found that colon carcinoma age-specific incidence data is consistent with an extreme value distribution, while prostate carcinomas age-specific incidence data generally follows the distribution. This model indicates that both colon and prostate carcinomas only occur in a subset of the population (22% for prostate and 13.5% for colon.) Because of their very general nature, extreme value distributions might be applicable to understanding other chronic human diseases.

CXCR4 Expression in Prostate Cancer Progenitor Cells

Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44+/CD133+ prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.

The origins of vacularization in tumors

Vascularization is crucial for tumor growth and metastasis. Angiogenesis and vasculogenesis are widely accepted processes of tumor vascularization, particularly for endothelium-dependent vessels. In both these processes, the tumor vascular endothelial cells are derived from the host cells, including cells in normal tissues around the tumor or endothelial progenitor cells. In addition, the mosaic vessels occur as a transitional pattern between endothelium-dependent vessels and vasculogenic mimicry (VM), wherein both host endothelium and tumor cells participate in tumor vascularization. VM provides a special passage not involving endothelial cells and is conspicuously different from angiogenesis and vasculogenesis. The biological features of the tumor cells that form VM remain unknown. Tumor stem cells may participate in VM. In this review, we discuss the patterns involved in the origin of vascularization in tumors.

T cells sensitized with breast tumor progenitor cell vaccine have therapeutic activity against spontaneous HER2/neu tumors

Cancer progenitor cells are critical for tumor initiation and recurrence so they are an important therapeutic target. We tested whether T cells could recognize tumor antigens expressed by breast cancer progenitor cells and acquire therapeutic activity against established metastases or delay onset of spontaneous tumors. Breast tumors were derived from HER2/neu transgenic mice and propagated in vitro under conditions that selected progenitor cells which were then used as an irradiated whole cell vaccine. A minor subset of recently sensitized T cells was isolated from vaccine-draining lymph nodes then activated in vitro to achieve numerical expansion. We show that the tumor progenitor cell vaccines reversed tolerance to a known HER2/neu epitope, otherwise inhibited by Treg cells. Additional shared tumor antigens were recognized because a Neuneg subclone also induced a Th1 type immune response against breast tumors. Adoptive transfer of in vitro activated lymph node T cells-mediated regression of established metastases from multiple independently derived breast tumor lines. Moreover, adoptive transfer of effector T cells into Neu-tolerant mice, months before the onset of spontaneous tumors, significantly postponed tumor development. Interestingly, T-cell-mediated lysis of metastases stimulated an IgG response to HER2/neu as well as other shared antigens. In summary, tumor progenitor cells contain shared antigens which can lead to a cross-protective T-cell response. Moreover, antigens acquired during immune-mediated tumor destruction are presented in a manner conducive to reversal of tolerance and Ig class switching. These complementary effector mechanisms might augment therapy by eliminating refractory breast cancer stem cells.

Profiling of 24 Standard of Care Drugs in a Panel of 20 Human Hematological Cell Lines Using Xenograft-Derived Three-Dimensional (3D) Cultures Ex Vivo and In Vivo

Abstract 4995 Introduction:Leukemia and lymphoma account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of hematological malignancies induce unique therapeutic challenges. It is well known that pluripotent as compared to differentiated cells possess the potential for anchorage independent growth in semisolid medium. This can be monitored via clonogenic or colony formation assays, in which cells grow in vitro in a three-dimensional (3D) manner without adherence to plastic culture material support. These assays can be utilized to evaluate growth and drug sensitivity of tumor stem and progenitor cells (Fiebig HH et al. Eur J Cancer 40:802, 2004). In addition, these 3D cell culture assays often mimic the in vivo scenario better than 2D cell culture assays with adherent tumor cells. Material and methods:For our ex vivo anti-tumor efficacy profiling using clonogenic assays, we established a panel of 20 hematological cell lines comprising different entities like acute lymphoblastic leukemia (ALL, 4 cell lines), acute myeloid leukemia (AML, 6 cell lines), chronic myeloid leukemia (CML, 5 cell lines), Hodgkin- (1 cell line) and non-Hodgkin-lymphoma (NHL, 3 cell lines), as well as multiple myeloma (MM, 3 cell lines). Tumor cells were injected into the flanks of NOD/SCID mice in order to obtain subcutaneous tumor xenografts, which were kept at low passages (n <3). These xenografts served as starting material either to prepare single cell suspensions for ex vivo analysis, or to carry out in vivo efficacy tests using either subcutaneous or disseminated growing tumor xenografts. Results:Twenty-four standard of care agents were tested in terms of their ex vivo chemosensitivity (e.g. cytarabine, cyclophosphamide, dexamethasone, doxorubicin, etoposide, melphalan, prednisolone, vincristine), including selected targeted drugs also (e.g. bortezomib, imatinib, nilotinib, sorafenib). The drugs showed diverse patterns of selectivity and potency: vincristine, doxorubicin and cytarabine, but also the proteasome inhibitor bortezomib exhibited pronounced activity with IC50 values in the nanomolar range (mean IC50 = 1 – 100nM), not only in their respective clinical application, but also in various other tumor entities, such as in ALL and AML with use of bortezomib. Differential activity was determined e.g. for prednisolone and dexamethasone, which were active in a micromolar range (mean IC50 = 22 – 58μM) in the ALL cell lines CCRF-CEM and MOLT-4, AML cell lines NOMO-1, NHL DAUDI and U-937, as well as the MM cell line IM-9. All-trans-retinoic acid (mean IC50 = 1.3μM) as well as interferon-gamma-1b (mean IC50 = 0.43 μM) showed specific activity patterns with pronounced growth inhibition in AML (3/6 tested AML cell lines: KG-1, NOMO-1, OCI-AML2), but also in CML (1/5 tested CML cell lines: EM-2) and MM (1/3 tested MM cell lines: L-363). The strong correlation of both tyrosine kinase inhibitors imatinib and nilotinib (spearman coefficient: 0.73, p <0.001) and their differential activity restricted to bcr-abl-positive cells served as a positive control for the implemented test system. In vivo follow-up testing in defined tumor xenografts confirmed the results obtained ex vivo. For example, cyclophosphamide that showed strong antitumor activity with use of the NHL cell line DAUDI via clonogenic assay (IC50 = 0.3μM), also induced tumor remissions of 80% in xenografts with subcutaneously growing DAUDI cells as compared to untreated control animals. Moreover, an exceedingly promising antitumor activity of sorafenib in AML cells assessed via clonogenic assay (mean IC50 0.84μM in AML cells vs. mean IC50 4.0μM over all tested entities) could be confirmed in the disseminated in vivo model using HL-60 cells (reduction of 99% vs. untreated control; Schueler J. et al. Blood 116 (21):2141, 2010). Conclusions:The presented panel screen using clonogenic assays is of great value for time and cost effective profiling of traditional cytotoxic as well as new targeted anti-cancer agents which can be confirmed in tumor models of hematological malignancies and can thereby guide to more effectively designed in vivo experiments. Diverse activity and resistance patterns ex vivo and in vivo also contribute to create clinical development strategies of standard and novel compounds. Disclosures:No relevant conflicts of interest to declare.

OCT4 pseudogenes present in human leukemia cells

The transcription factor OCT4 is expressed in embryonic stem cells (ESCs) and has been considered as a main regulator in maintaining pluripotency of ESCs. Several studies have showed OCT4 expression in human solid tumor and adult stem or progenitor cells. However, whether OCT4 is expressed in normal hematopoietic system including the peripheral blood and bone marrow remains controversial. Furthermore, the functional expression of OCT4 in leukemia cells and its potential significance in leukemia studies have been poorly defined. The aim of this study was to examine and analyze the genuine expression of OCT4 in human leukemia cells by means of RT-PCR, flow cytometry, PCR product sequencing and alignment with NCBI BLAST and DNAMAN software. The full lengths of the putative OCT4 genes were amplified in 2/9 leukemia cell lines and 7/49 leukemia patients' samples. However, many base mutations in putative OCT4 positive samples were found. Sequence alignment analysis showed a higher similarity between the putative OCT4 PCR products and the pseudogenes in chromosomes 1 and 8. The positive rates of OCT4 protein detected with flow cytometry were low, and almost all of them were less than 10% of positivity. A very small fraction of leukemia stem cells with OCT4 protein expression was found. We conclude that OCT4 pseudogenes in chromosomes 1 and 8 present in the panel of leukemia cells tested and the OCT4 protein is rarely detected with flow cytometry in leukemia cells.

Matrix metalloproteinase 12 overexpression in myeloid lineage cells plays a key role in modulating myelopoiesis, immune suppression, and lung tumorigenesis

AbstractMatrix metalloproteinase 12 (MMP12) is a macrophage-secreting proteinase. To fully understand the function of MMP12 in myeloid lineage cells, a myeloid-specific c-fms-rtTA/(TetO)7-CMV-MMP12 bitransgenic mouse model was created. In this bitransgenic system, induction of MMP12 abnormally elevated frequencies and numbers of common myeloid progenitor (CMP) and granulocyte/macrophage progenitor (GMP) populations, and decreased the frequency and number of the megakaryocyte/erythrocyte progenitor (MEP) population in the bone marrow (BM). The CD11b+/Gr-1+ immature cell population was systemically increased in multiple organs. Both in vitro and in vivo studies showed an immunosuppressive function on T-cell proliferation and function by CD11b+/Gr-1+ immature cells from MMP12-overexpressing bitransgenic mice. MMP12 directly stimulated lineage-negative (Lin−) progenitor cells to differentiate into CD11b+/Gr-1+ immature cells that showed immunosuppression on T-cell proliferation and function in vitro. Regulatory T cells (Tregs) were increased. In the lung, the concentration of IL-6 was increased, which aberrantly activated oncogenic Stat3 and increased expression of Stat3 downstream genes in epithelial tumor progenitor cells. Spontaneous emphysema and lung adenocarcinoma were sequentially developed after MMP12 overexpression. BM chimeras confirmed that the MMP12-induced myeloid cell autonomous defect led to abnormal myelopoiesis, immune suppression, and lung adenocarcinoma.

Corticosteroids in brain cancer patients: benefits and pitfalls

Glucocorticoids have been used for decades in the treatment of brain tumor patients and belong to the most powerful class of agents in reducing tumor-associated edema and minimizing side effects and the risk of encephalopathy in patients undergoing radiation therapy. Unfortunately, corticosteroids are associated with numerous and well-characterized adverse effects, constituting a major challenge in patients requiring long-term application of corticosteroids. Novel anti-angiogenic agents, such as bevacizumab (Avastin®), which have been increasingly used in cancer patients, are associated with significant steroid-sparing effects, allowing neuro-oncologists to reduce the overall use of corticosteroids in patients with progressive malignant brain tumors. Recent experimental studies have revealed novel insights into the mechanisms and effects of corticosteroids in cancer patients, including modulation of tumor biology, angiogenesis and steroid-associated neurotoxicity. This article summarizes current concepts of using corticosteroids in brain cancer patients and highlights potential pitfalls in their effects on both tumor and neural progenitor cells.

Omental milky spots in screening gastric cancer stem cells

The existence of cancer stem and progenitor cells in solid tumors has been widely postulated. However, neither the cancer stem cells nor the cancer progenitor cells have been definitively identified and functionally characterized. Here we propose a new strategy to identify and isolate gastric cancer stem cells -using omental milky spots to screen gastric cancer stem cells in peritoneal metastasis mouse models of gastric cancer. In this study, we used the property that the macrophages in omental milky spots are cytotoxic against tumor cells and so able to screen and collect cancer stem cells. Our findings suggest that macrophages in omental milky spots have not only cytotoxic properties against tumor cells but also provide a microenvironment within milky spots in which cancer stem cells are capable to survive and grow into micrometastasis. Omental milky spot become a cancer stem cell niche in this situation. Further we studied the omental milky spots for screening gastric cancer cells (OMSS-GCCs) and found that omental milky spot enriched the volume of gastric cancer stem cells. Tumors were consistently generated after an injection of 1×103 OMSS-GCCs. OMSS-GCCs high express CD133 and low express CD324. Omental milky spots are a highly efficient "natural filter" for screening gastric cancer stem cells.