Expansion Of Tumor Cells Research Articles

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens

POEMS Syndrome: A Multidisciplinary Diagnostic Challenge

POEMS syndrome is a paraneoplastic phenomenon associated with polyneuropathy and paraproteinemia that arises out of the clonal expansion of plasma tumor cells. Its other distinguishing features include sclerotic bone lesions, increased vascular endothelial growth factor levels, Castleman disease alterations in lymph node biopsy and association with organomegaly, endocrinopathy, cutaneous lesions, papilledema, extravascular volume overload and thrombocytosis. Although established diagnostic criteria exist, the fact that the disease is rare and shares similar clinical manifestations with non-neoplastic conditions means that in most cases patients are diagnosed at latestage, when multiple complications have appeared and treatments for other conditions have already been given. Therefore, a high degree of suspicion combined with a multidisciplinary approach are requisites for attaining precise diagnosis and providing timely treatment. Due to the wide differential diagnosis that the syndrome´s clinical features encompass as well as its subsequent favourable responses to bone marrow transplant and diverse chemotherapy treatments, clinicians from diverse fields should include the POEMS syndrome within polyneuropathy differential diagnoses that require specific diagnostic tests. Herein, we present our case study in which we emphasize diagnosis and treatment of patients treated at our institution.

Modeling Physiologic Microenvironments in Three-Dimensional Microtumors Maintains Brain Tumor Initiating Cells.

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity, including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy, because BTICs are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using the human-derived biomatrix HuBiogel™ and maintained with physiologically relevant microenvironments. BITC percentages were determined using cell surface marker expression, label retention, and neurosphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient-restricted microtumors significantly increased the percentage of both CD133+ and CFSEhigh cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintained neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Correlation Between Hypoxia Proteins and EPR-Detected Hypoxia in Tumors.

Rapid expansion of tumor cells that outpace existing vasculature essential for nutrient and oxygen support as well as waste removal, correlates with profound changes in the microenvironment including angiogenesis, vasodilation, glucose metabolism, and cell cycle perturbations. Since hypoxic cells are up to three times more radioresistant than normoxic cells, identification of hypoxic populations to predict radiotherapeutic outcome is important. The consequences of hypoxia and activated proteins contribute to radioresistant tumors and radiotherapeutic failure. Stereotactic MCa4 tumor tissue biopsies from mouse tumors that were guided by electron paramagnetic resonance (EPR) O2 imaging were examined for hypoxia-induced proteins. The oxygen broadening of narrow EPR spectral lines or, equivalently, the increase in relaxation rates of electron magnetization, report pO2 with 1-2torr resolution in image voxels less than 1 mm3. The pO2 reporter molecule OX063d64 (trityl) was used to acquire the data described here. Trityl appears to be selectively retained in tumors with a half-life of ~30 min. We used an inversion recovery electron spin echo (IRESE) to measure the T1 rate of the trityl inside the tumor bearing leg. We estimate our uncertainty in pO2 measurement to be 1-3torr per voxel. Three hypoxic cell biomarkers, hypoxic-induced factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), and carbonic anhydrase IX (CA9), were examined using the ELISA assay. Quantification of these proteins based on results from the ELISA immunoassay kits indicate a strong correlation between EPR pO2-identified hypoxic fractions (<10torr) and HIF-1α, VEGF, and CA9. We clearly demonstrate that hypoxic regions in tumors generate substantial amounts of HIF- 1α, VEGF, and CA9 protein.

TMOD-18. THREE-DIMENSIONAL MICROTUMORS IN PHYSIOLOGIC MICROENVIRONMENTS MAINTAIN BRAIN TUMOR INITIATING CELLS

Elucidation of mechanisms of brain tumor initiating cell (BTIC) maintenance and therapeutic resistance offers great promise for development of novel anti-tumor treatments. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined if three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were then determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. For example, fifteen percent of all brain tumor cells from a xenograft propagated as microtumors were CD133+, whereas only one percent of neurosphere cultured cells expressed the BTIC marker. Relative to CD133, CFSE was retained in a higher percentage of cells, but CFSE+ cells were still increased in the microtumor condition in comparison to neurospheres. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

Therapeutic resistance and cancer recurrence mechanisms: Unfolding the story of tumour coming back.

Cancer recurrence is believed to be one of the major reasons for the failure of cancer treatment strategies. This biological phenomenon could arise from the incomplete eradication of tumour cells after chemo- and radiotherapy. Recent developments in the design of models reflecting cancer recurrence and in vivo imaging techniques have led researchers to gain a deeper and more detailed insight into the mechanisms underlying tumour relapse. Here, we provide an overview of three important drivers of recurrence including cancer stem cells (CSCs), neosis, and phoenix rising. The survival of cancer stem cells is well recognized as one of the primary causes of therapeutic resistance in malignant cells. CSCs have a relatively latent metabolism and show resistance to therapeutic agents through a variety of routes. Neosis has proven to be as an important mechanism behind tumour self-proliferation after treatment which gives rise to the expansion of tumour cells in the injured site via production of Raju cells. Phoenix rising is a prorecurrence pathway through which apoptotic cancer cells send strong signals to the neighbouring diseased cells leading to their multiplication. The mechanisms involved in therapeutic resistance and tumour recurrence have not yet been fully understood and mostly remain unexplained. Without doubt, an improved understanding of the cellular machinery contributing to recurrence will pave the way for the development of novel, sophisticated and effective antitumour therapeutic strategies which can eradicate tumour without the threat of relapse.

Melatonin downregulates nuclear receptor RZR/RORγ expression causing growth-inhibitory and anti-angiogenesis activity in human gastric cancer cells in vitro and in vivo.

An adequate supply of oxygen and nutrients, derived from the formation of novel blood vessels, is critical for the growth and expansion of tumor cells. It has been demonstrated that melatonin (MLT) exhibits marked in vitro and in vivo oncostatic activities. The primary purpose of the present study was to evaluate the in vitro and in vivo antitumor activity of MLT on the growth and angiogenesis of gastric cancer cells, and explore the underlying molecular mechanisms. The present results revealed that MLT inhibited the growth of gastric cancer SGC-7901 cells in a dose- and time-dependent manner. In addition, the present study demonstrated that low concentrations (0.01, 0.1 and 1 mM) of MLT had no clear effect on vascular endothelial growth factor (VEGF) secretion, whereas a high concentration (3 mM) of MLT suppressed VEGF secretion in SGC-7901 cells. Notably, administration of MLT caused suppression of gastric cancer growth and blockade of tumor angiogenesis in tumor-bearing nude mice. Furthermore, MLT treatment reduced the expression of the MLT nuclear receptor RZR/RORγ, SUMO-specific protease 1, hypoxia-inducible factor-1α and VEGF at transcriptional and translational levels within gastric cancer cells during tumorigenesis. In conclusion, MLT nuclear receptor RZR/RORγ may be of great importance in the MLT mediated anti-angiogenesis and growth-inhibitory effect in gastric cancer cells. Since RZR/RORγ is overexpressed in multiple human cancers, MLT may be a promising agent for the treatment of cancers.

Abstract 5139: Cell sorting strategies for the isolation of cancer cells and associated fibroblasts from tumor biopsies, surgical resections, and patient-derived xenografts

Abstract The ability of early passage patient-derived tumor xenografts (PDXs) to recapitulate human tumor biology makes them invaluable tools for pre-clinical drug development (1, 2). Similarly, clinically-annotated human tumor cell lines of low passage have potential as scalable biosimilar reagents for in vitro study. Here, we describe an integrated platform for the purification, expansion, and quality control of tumor cells and cancer-associated fibroblasts (CAFs) from patient biopsies, surgical resections, and mouse PDX models. Following dissociation of solid tumors, cells are cultured on Matrigel® in fibroblast conditioned media containing Y-27632 (a Rho-associated kinase inhibitor). Cells are then analyzed by FACS using a panel of species- or cell type-specific antibodies. This panel (anti-mCD9, hCD9, hCD24, hCD90, MHC, HLA, and hEPCAM) permits quantitation of the percentage of mouse/human cells, and the percentages of tumor cells and CAFs in each patient specimen. From these data, a cell sorting strategy can be selected (e.g. mCD9 and hCD90 with hEpCAM or CD24) that will permit purification of both tumor cells and CAFs, along with removal of any mouse fibroblast contamination (for PDX tumors). Samples are sorted multiple times until target populations reach 99.99% purity (typically 2-3 rounds). Purified cells are then returned to culture, expanded over several weeks, and frozen at low passage. The purity and identity of these cultures is quality-assured using 1) mouse-specific QPCR, 2) a cDNA array designed to differentiate fibroblasts from tumor cells, and 3) STR genotyping. Tumor lines and CAFs are then scaled and re-qualified at passages 10 and 20, an essential step given the propensity of low-frequency mouse and human fibroblasts to ‘overtake’ impure cultures with time. Lastly, cancer lines and CAF cultures undergo genomic characterization and are evaluated for in vivo tumorigenicity (or the lack thereof for CAFs). To date, the platform has been used to generate a growing number of purified cancer and CAF pairs from patient materials. In conclusion, our goal is to provide useful reagents to the extramural community that will accompany current efforts within the Division of Cancer Treatment and Diagnosis (DCTD, NCI) aimed at generating a large and extensively characterized repository of PDX models. Funded by NCI Contract No. HHSN261200800001E.

Can we be SMaRT-er in our approach to cancer therapy?

Can we be SMaRT-er in our approach to cancer therapy?

Chitinase-3-like-1/YKL-40 as marker of circulating tumor cells.

Ex vivo expansion of circulating tumor cells (CTCs) of small cell lung cancer (SCLC) patients enabled systematic screening of secreted cytokines. Permanent CTC cultures of different patients shared secretion of chitinase-3-like-1 (CHI3L1)/YKL-40, known to be upregulated in a range of tumor entities and to be associated with increased metastasis and decreased survival. This protein lacks enzymatic activity and its mechanism of promoting tumor dissemination has not been resolved. Results from SCLC CTC cultures suggest CHI3L1 as marker and important effector of tumor cell dissemination in the peripheral blood. Furthermore, this protein may link chronic inflammation of the lung, chronic obstructive pulmonary disease (COPD) and lung cancer.

Enhancing the Cytotoxicity of NK Cells Toward Multiple Myeloma Using an Antibody Specific to SLAMF7

Multiple myeloma (MM) is a malignancy of plasma cells characterized by a monoclonal expansion of tumor cells in bone marrow and accumulation of a monoclonal protein in blood and urine, which ultimately leads to end-stage organ damage. Despite advances in therapy, MM remains an incurable disease. Recent investigations have revealed a new therapeutic target for MM, SLAMF7 (CS1), which is expressed at high levels on MM tumor cells. SLAMF7 is also found on NK, CD8+ T, CD4+ T and activated B cells. Elotuzumab (HuLuc63) is a humanized IgG1 anti-SLAMF7 antibody, which can facilitate killing of primary MM cells by NK cells via antibody-dependent cellular cytotoxicity (ADCC) and has shown promise in treating relapsed MM. A key question in understanding elotuzumab’s mechanism of action is whether it can directly activate NK cells via SLAMF7 binding, in addition to Fc-mediated activation via binding of CD16 (FcγRIIIA).We have examined the in vitro effect of elotuzumab on NK cells in fresh human peripheral blood from healthy donors and MM patients incubated with or without MM cell lines that have varied levels of SLAMF7 surface expression. Cytotoxic degranulation of NK cells was quantified by staining for CD107a (marker of degranulation) after 2 hours, and direct effects of elotuzumab on NK cells were assessed by measuring upregulation of the CD69 activation marker after 24 hours of antibody exposure.Degranulation of fresh primary NK cells toward SLAMF7-bearing MM target cell lines was stimulated by addition of elotuzumab. Intensity of the degranulation response was dependent upon the concentration of elotuzumab and the expression level of SLAMF7 on the MM target cells. While elotuzumab alone could stimulate low-level degranulation of NK cells in fresh PBL from healthy donors, the degranulation response was stronger if SLAMF7-expressing MM target cells were added in combination with elotuzumab. Addition of F(ab’)2 elotuzumab or an Fc mutant form of elotuzumab that cannot bind CD16 did not stimulate degranulation, implying that binding of the antibody to SLAMF7 on NK cells and MM cells, without concomitant engagement of NK cell Fc receptors, is insufficient to trigger degranulation.Soluble intact elotuzumab stimulated CD69 expression and degranulation by fresh primary NK cells in the absence of MM target cells, which peaked at 1 µg/ml and only modestly increased up to 100 µg/ml elotuzumab. In the same assays, we only observed modest stimulation of CD69 expression using soluble F(ab’)2 or Fc mutant elotuzumab. In contrast, plate-bound F(ab’)2 elotuzumab stimulated robust CD69 expression, indicating that the antibody can induce direct stimulation of NK cells through SLAMF7, but strong stimulation requires aggregation of the antibody. In soluble form, a non-fucosylated version of elotuzumab with greater affinity for CD16 stimulated significantly stronger degranulation and CD69 expression than intact elotuzumab, both in the presence or absence of MM target cells, and with both high and low SLAMF7 expression on target cells. Importantly, elotuzumab was found to induce similar degranulation by NK cells from freshly isolated peripheral blood of MM patients as compared to healthy donors.Taken together, our data support the hypothesis that binding of soluble elotuzumab to SLAMF7 can weakly activate NK cells, but strong activation and degranulation require antibody aggregation or engagement of the Fc domain with CD16. Elotuzumab variants with enhanced CD16 binding, such as a non-fucosylated version, can augment ADCC against myeloma cell lines, and warrant further study as a potential strategy to improve clinical efficacy in MM patients. DisclosuresCohen:Celgene: Member, Independent Response Adjudication Committee Other; Janssen: Advisory Board, Advisory Board Other; Bristol-Myers Squibb: Advisory Board, Advisory Board Other, Research Funding; Onyx: Advisory Board, Advisory Board Other. Campbell:Bristol-Myers Squibb: Advisory Board Other, Research Funding; Janssen: Advisory Board, Advisory Board Other; Conkwest: Consultancy, Patented NK-92 cell lines, Patented NK-92 cell lines Patents & Royalties, Research Funding.

In Vitro Efficient Expansion of Tumor Cells Deriving from Different Types of Human Tumor Samples

Obtaining human tumor cell lines from fresh tumors is essential to advance our understanding of antitumor immune surveillance mechanisms and to develop new ex vivo strategies to generate an efficient anti-tumor response. The present study delineates a simple and rapid method for efficiently establishing primary cultures starting from tumor samples of different types, while maintaining the immuno-histochemical characteristics of the original tumor. We compared two different strategies to disaggregate tumor specimens. After short or long term in vitro expansion, cells analyzed for the presence of malignant cells demonstrated their neoplastic origin. Considering that tumor cells may be isolated in a closed system with high efficiency, we propose this methodology for the ex vivo expansion of tumor cells to be used to evaluate suitable new drugs or to generate tumor-specific cytotoxic T lymphocytes or vaccines.

Metastasis in the Light

Cancer![Figure][1] CREDIT: © RISAMAY/ISTOCKPHOTO Cutaneous melanoma is less common than other forms of skin cancer but is far more deadly, especially if not detected at an early stage. Ultraviolet (UV) light from the Sun plays a key role in the initiation of melanoma by inducing mutations in the DNA of melanocytes, the pigment-producing cells in skin. A new study suggests that UV light does damage well beyond this early role: It appears to trigger a chain of pathologic events that facilitate metastasis. Studying a mouse model, Bald et al. found that repetitive exposure of primary melanomas to UV light did not increase tumor incidence or growth but rather increased the number of lung metastases. Closer examination revealed that UV light induced the recruitment of certain immune cells (neutrophils) to the primary tumor. This inflammatory response in turn activated endothelial cells (cells that line blood vessels) and triggered migration of the melanoma cells toward them, resulting in the expansion of tumor cells along blood vessels. This “angiotrophic” growth pattern is thought to increase the likelihood that tumor cells enter the bloodstream and metastasize. Consistent with this, human melanoma samples with high levels of neutrophils were more metastatic. Nature 10.1038/nature13111 (2014). [1]: pending:yes

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

Immune effects of high dose radiation treatment: implications of ionizing radiation on the development of bystander and abscopal effects

Tumors grow progressively when they escape from immune surveillance. Cancer progression is mainly driven by the expansion of tumor cells, but tumor microenvironment and anti-tumor immunity may also play a role. Ionizing radiation therapy (RT), either alone or in combination with additional immune stimulators, can render cancer cells visible to the immune system. In addition to the direct effects of radiation, the ensuing immune response promotes the expression of inflammatory and immunostimulatory mediators, which act on neighboring, non-irradiated, cells. Bystander effects induced by radiation are characterized by biological responses, which are observed in non-irradiated cells that are in the vicinity of irradiated cells. Bystander effects are mediated via cell-to-cell gap junctions or through secreted, diffusible signaling molecules into the local milieu. After treatment with localized radiation, systemic effects in non-irradiated area (out-offield) may also occur. These effects are named abscopal effects and appear to be immune mediated, particularly by adaptive immunity. It has been suggested that a high single dose of RT may induce an immune response that leads to the priming of antigen-specific dendritic cells (DCs). The targeted intraoperative radiotherapy (TARGIT) method, using INTRABEAM ® , could reduce tumor recurrence, modifying the wound microenvironment, and eradicating residual tumor cells when applied immediately after surgery procedure.

Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia

Parathyroid adenomas (PAs) causing primary hyperparathyroidism (PHPT) are histologically heterogeneous yet have been historically viewed as largely monotypic entities arising from clonal expansion of a single transformed progenitor. Using flow cytometric analysis of resected adenomatous parathyroid glands, we have isolated and characterized chief cells, oxyphil cells, and tumor-infiltrating lymphocytes. The parathyroid chief and oxyphil cells produce parathyroid hormone (PTH), express the calcium-sensing receptor (CASR), and mobilize intracellular calcium in response to CASR activation. Parathyroid tumor infiltrating lymphocytes are T cells by immunophenotyping. Under normocalcemic conditions, oxyphil cells produce ∼50% more PTH than do chief cells, yet display significantly greater PTH suppression and calcium flux response to elevated calcium. In contrast, CASR expression and localization are equivalent in the respective parathyroid cell populations. Analysis of tumor clonality using X-linked inactivation assays in a patient-matched series of intact tumors, preparatively isolated oxyphil and chief cells, and laser-captured microdissected PA specimens demonstrate polyclonality in 5 of 14 cases. These data demonstrate the presence of functionally distinct oxyphil and chief cells within parathyroid primary adenomas and provide evidence that primary PA can arise by both clonal and polyclonal mechanisms. The clonal differences, biochemical activity, and relative abundance of these parathyroid adenoma subpopulations likely reflect distinct mechanisms of disease in PHPT.

Targeting CD123 In Leukemic Stem Cells Using Dual Affinity Re-Targeting Molecules (DARTs®)

T-cell directed killing of tumor cells using bispecific reagents is a promising approach for the treatment of hematologic malignancies. In contrast to B-cell malignancies, this approach has been limited in AML by the lack of tumor-specific antigens/targets. CD123 (IL3RA) is highly and differentially expressed in AML blasts compared to normal hematopoietic stem and progenitor cells and is a potential target for immunotherapy. We investigated the ability of a DART constructed from an antibody to CD123 (7G3) and a MacroGenics' proprietary CD3 antibody to redirect T cells against CD123+ AML blasts. DARTs consist of 2 independent polypeptides, each comprising the VH of one antibody in tandem with the VL of the other antibody. The resultant heterodimer is stabilized by a disulfide bond at the carboxyl terminal domains of the 2 VH regions. This construct binds to both the N-terminal extracellular domain of human CD123 and to the extracellular domain of CD3 in the human T-cell receptor complex. Our in vitro studies demonstrate that the CD3xCD123 DART induces specific aggregation of TCR/CD3+ Jurkat or human T cells and human CD123-transduced K562 (K562CD123/GFP) cells compared to a control DART lacking specificity for one of the antigens (16±3.2% vs. 1.6±0.2%, p=0.0074) or when compared to CD3xCD123 DART incubated with control GFP-transduced K562 (K562GFP) cells. Incubation of human T cells (1:1 ratio) with K562CD123/GFP and CD3xCD123 DART vs. control DARTS (10 ng/ml) for 5 days in vitro resulted in profound T-cell activation (CD25 expression, 88.8±2.7% vs.1.2±0.2%; p=0.0009), T-cell proliferation (VPD-450 proliferation assay; 98.2 ± 0.4% vs. 2.27± 0.4%, p=0.0001), expansion of the central memory T cell compartment (TCM, 62.6±1.5% vs. 5±1.3%, p<0.0001), and killing of K562CD123/GFP targets as measured by 7-AAD FACS (97±0.9% apoptosis relative to the control; p<0.0001) and chromium release assays (28.5% vs.3.1%; p=0.0002). As expected, aggregation, T-cell activation and target cell killing was negligible when T cells and CD3xCD123 DART were incubated with control K562GFP cells. Similar results were seen when A20 targets (BALB-C/H-2d B lymphoma cell line) overexpressing CD123 were used (7-AAD+ after 18 hours, 91.1±2.01% vs. 28.1±0.76%, p=0.0012). In spite of very low E:T ratios (0.009:1-0.071:1), when primary frozen/thawed AML peripheral blood specimens (n=6; CD123+ blasts ranging from 34.7 to 87.2%) were used, there was a profound and universal CD3xCD123 DART-specific activation and expansion of the few human T cells present in these AML samples (ranging between 0.9 to 6.6% of cells). After 6 days of in vitro incubation (37°C w/o exogenous IL-2) of each AML sample (1x106/ml) with CD3xCD123 DART (0.1 ng/ml) or control DARTs, there was a CD3xCD123 DART-specific increase in both T cell numbers (median: 8 fold, range:1.7-15 fold vs. 0.6-1 fold for control DARTs), and in the percentage of T cells expressing CD25 (median: 69.4 fold, range: 21.4-152.8 fold vs. 0.2-6.5 fold for control DARTs). This was accompanied by a dose-dependent reduction in blasts by 35±25% at 0.1 ng/ml DART and 99.6±0.05% at 10 ng/ml DART (p=0.0063). In addition, AML colony-forming units (L-CFU) were also inhibited by 94 ±0.6% while CFU-GEMM, CFU-GM and BFU-E from cord blood and (G-CSF)-mobilized peripheral blood from normal donors were not affected by either CD3xCD123 DART or control DARTs (0.1-10 ng/ml), suggesting limited impact on normal human hematopoietic progenitors in vitro. Bioluminescence imaging of irradiated NSG mice (n= 5/group; 300cGy) performed on days 3, 12, 19, and 28 after the infusion of 1.5 x 106 Click Beetle Red (CBR) luciferase+- transduced K562CD123/GFP cells on day 0 and both CD3xCD123 DART (0.5 mg/kg IV) and human T cells (3 x 106) on day 3 revealed no expansion of tumor cells in sharp contrast to NSG mice receiving either control DARTs and/or no human T cells (1415- fold expansion; p<0.0001). Of interest is that at 6 weeks post-infusion of primary AML xenografts into NSG mice (E:T=0.009:1), there was near- complete elimination (>97%) of AML blasts from the peripheral blood even in the absence of exogenously added human T cells. Clearing of primary human AML blasts from the spleen and bone marrow was also significant (40-77.8%) but less than that seen in the blood. These results provide the basis for the CD3xCD123 DART as a novel reagent for the treatment of patients with CD123+ AML. Disclosures:Chichili:Macrogenics. Inc: Employment. Moore:Macrogenics. Inc.: Employment. Johnson:Macrogenics. Inc.: Employment. Bonvini:Macrogenics. Inc.: Employment.

Insights into therapeutic resistance from whole-genome analyses of circulating tumor DNA

The selection and expansion of tumor cells with de novo genetic alterations in specific genes has been described as a mechanism of resistance to targeted therapy. Unfortunately, in the metastatic setting it is often not possible to examine the multiple lesions present in an individual to determine the presence and mechanism of acquired resistance during therapy. Circulating tumor DNA (ctDNA) can be used to analyze the genomic characteristics of tumors in a non-invasive manner [1]. We have previously reported that tumor-specific chromosomal changes can be detected in plasma from patients through integrated whole-genome analyses of cell-free DNA [1]. We have now applied whole-genome sequencing to analyze plasma ctDNA from a patient with chemotherapy-refractory colorectal cancer that had become resistant to EGFR-blockade with cetuximab. A total of 630 Gb of sequence data were obtained, corresponding to 145x sequence coverage of cell-free genomic DNA, and analyzed to identify sequence and structural alterations. These and other analyses revealed emergence of a Q61H mutation in KRAS as well as focal high-level (>9 fold) amplification and rearrangement of the MET locus that were not detected in pre-treatment tumor samples (Figure ​(Figure1).1). Alterations in these genes have been reported in tumors with secondary resistance to EGFR-directed therapy and are consistent with clinical progression [2-5]. In addition to its role in resistance to targeted therapy, MET amplification has been shown to confer sensitivity to the tyrosine kinase inhibitor crizotinib and other inhibitors [6, 7], and may provide a new therapeutic target in patients with resistance to EGFR blockade. These results describe the first whole-genome analysis of ctDNA to identify genetic alterations associated with mechanisms of resistance to a targeted therapy. Our analyses suggest that genome-wide analyses of ctDNA can be used for discovery of tumor-specific alterations in the in the context of disease monitoring, detection of molecular resistance, and identification of new therapeutic targets. Figure 1 Identification of MET amplification through analyses of plasma DNA

Using a systems biology approach to understand and study the mechanisms of metastasis

Metastasis remains the main cause for cancer-related deaths due to the lack of effective therapy. The clonal selection model has long been thought to be the primary mechanism of metastatic progression but many different mechanisms have been hypothesized for the progression from tumorigenesis to the successful dissemination and expansion of tumor cells at the secondary site. MicroRNAs, germline polymorphisms in combination with the tumor microenvironment are few of the different pathways to explain the metastatic cascade. Technological advances for high-throughput screening of cells such as expression profiling, next generation sequencing, as well as global network analyses have advanced the studies of these mechanisms. Combined with new insights into the various mechanisms of metastasis a systems biology approach has also been shown to be useful in identifying metastasis-specific gene signatures as well as predicting disease outcome. Furthermore, the results of these studies have been relevant for identifying biomarkers for metastatic disease.

Aptamers as Targeting Delivery Devices or Anti-cancer Drugs for Fighting Tumors

Aptamer researches applied to the treatment of human cancers have increased since their discovery in 1990. This is due to different factors including: 1) the technical possibility to select, by SELEX-based procedures, specific aptamers targeting virtually any given molecule, 2) the aptamer favorable bio-activity in vivo, 3) the low production costs and 4) the ease synthesis and storage for the marketing. In the field of cancer treatments, aptamers have been studied as tumor-specific agents driving drugs into cancer cells; additionally they have been used as anti-neoplastic agents, able to inhibit tumor cell growth and dissemination when administered alone or in combination with conventional anti-neoplastic drugs. Aptamers are gaining an increased interest for pharmaceutical companies and some of them are under clinical evaluation trials. In this review we update the findings about the use of aptamers as "escort" molecules able to drive drugs into the cells and as antineoplastic drugs. Current anti-neoplastic treatments suffer from the intrinsic toxicity related to the un-specific targeting of both normal and tumorigenic proliferating cells. The aptamers could be useful to improve: 1) the selective targeting of molecules essential for the viability and expansion of tumor cells and/or the selective driving of chemotherapies into tumor cells, thus resulting in higher effectiveness and lower systemic side-effects compared to conventional anti-neoplastic drugs alone and 2) to improve the therapeutic index of currently used chemotherapies. Even if some problems related to the in vivo stability and pharmacokinetic/dynamics of aptamers remain to be improved, their potential use in the treatment of different human cancers is getting closer and closer to a practical therapeutic use.