Three-dimensional microscale hanging drop arrays with geometric control for drug screening and live tissue imaging.

Existing three-dimensional (3D) culture techniques are limited by trade-offs between throughput, capacity for high-resolution imaging in living state, and geometric control. Here, we introduce a modular microscale hanging drop culture where simple design elements allow high replicates for drug screening, direct on-chip real-time or high-resolution confocal microscopy, and geometric control in 3D. Thousands of spheroids can be formed on our microchip in a single step and without any selective pressure from specific matrices. Microchip cultures from human LN229 glioblastoma and patient-derived mouse xenograft cells retained genomic alterations of originating tumors based on mate pair sequencing. We measured response to drugs over time with real-time microscopy on-chip. Last, by engineering droplets to form predetermined geometric shapes, we were able to manipulate the geometry of cultured cell masses. These outcomes can enable broad applications in advancing personalized medicine for cancer and drug discovery, tissue engineering, and stem cell research.

Chromoplectic TPM3-ALK rearrangement in a patient with inflammatory myofibroblastic tumor who responded to ceritinib after progression on crizotinib.

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) are rare sarcomas that can occur at any age. Surgical resection is the primary treatment for patients with localized disease; however, these tumors frequently recur. Less commonly, patients with IMTs develop or present with metastatic disease. There is no standard of care for these patients and traditional cytotoxic therapy is largely ineffective. Most IMTs are associated with oncogenic ALK, ROS1 or PDGFRß fusions and may benefit from targeted therapy. PATIENT AND METHODS: We sought to understand the genomic abnormalities of a patient who presented for management of metastatic IMT after progression of disease on crizotinib and a significant and durable partial response to the more potent ALK inhibitor ceritinib. RESULTS: The residual IMT was resected based on the recommendations of a multidisciplinary tumor sarcoma tumor board and analyzed by whole-genome mate pair sequencing. Analysis of the residual, resected tumor identified a chromoplectic TPM3-ALK rearrangement that involved many other known oncogenes and was confirmed by rtPCR. CONCLUSIONS: In our analysis of the treatment-resistant, residual IMT, we identified a complex pattern of genetic rearrangements consistent with chromoplexy. Although it is difficult to know for certain if these chromoplectic rearrangements preceded treatment, their presence suggests that chromoplexy has a role in the oncogenesis of IMTs. Furthermore, this patient's remarkable response suggests that ceritinib should be considered as an option after progression on crizotinib for patients with metastatic or unresectable IMT and ALK mutations.

Activation of TAK1 by MYD88 L265P drives malignant B-cell Growth in non-Hodgkin lymphoma.

Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHLs) including the rare lymphoplasmacytic lymphoma, Waldenström's macroglobulinemia (WM). Using whole-exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B-cell survival, therapeutic targeting of MYD88 signaling pathways may be clinically useful. However, we are lacking a thorough characterization of the role of intermediary signaling proteins on the biology of MYD88L265P-expressing B cells. We report here that MYD88L265P signaling is constitutively active in both WM and diffuse large B-cell lymphoma cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and NF-κB activation. Furthermore, we have identified the signaling protein, TAK1, to be an essential mediator of MYD88L265P-driven signaling, cellular proliferation and cytokine secretion in malignant B cells. Our studies highlight the biological significance of MYD88L265P in NHL and reveal TAK1 inhibition to be a potential therapeutic strategy for the treatment of WM and other diseases characterized by MYD88L265P.

ASCL1 and RET expression defines a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation.

ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine (NE) cell development, but its value as a biomarker of NE differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1 expression in lung cancer samples of varied histologic subtype, clinical outcome and smoking status and compared with expression of traditional NE markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers and other lung cancer subtypes. ASCL1 protein expression by immunohistochemical (IHC) analysis correlated best with synaptophysin compared with chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1-positive tumors (ASCL1(+)) compared with ASCL1(-) tumors (q-value <10(-9)). High levels of RET expression in ASCL1(+) but not in ASCL1(-) tumors was associated with significantly shorter overall survival (OS) in stage 1 (P=0.007) and in all AD (P=0.037). RET protein expression by IHC had an association with OS in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression defines a clinically relevant subgroup of ∼10% of AD characterized by NE differentiation.

DNA counterion current and saturation examined by a MEMS-based solid state nanopore sensor.

Reports of DNA translocation measurements have been increasing rapidly in recent years due to advancements in pore fabrication and these measurements continue to provide insight into the physics of DNA translocations through MEMS based solid state nanopores. Specifically, it has recently been demonstrated that in addition to typically observed current blockages, enhancements in current can also be measured under certain conditions. Here, we further demonstrate the power of these nanopores for examining single DNA molecules by measuring these ionic currents as a function of the applied electric field and show that the direction of the resulting current pulse can provide fundamental insight into the physics of condensed counterions and the dipole saturation in single DNA molecules. Expanding on earlier work by Manning and others, we propose a model of DNA counterion ionic current and saturation of this current based on our experimental results. The work can have broad impact in understanding DNA sensing, DNA delivery into cells, DNA conductivity, and molecular electronics.

Study design considerations in clinical outcome research of lung cancer using microarray analysis.

BACKGROUND: Prognosis following a diagnosis of primary lung cancer is very poor and varies significantly even after adjusting for known predictors. Inherent and acquired gene alterations could cause failure in lung cancer treatment and patient survival. To search for potential molecular markers with significant and independent predictive value in lung cancer survival, we applied oligo-nucleotide microarray analysis, along with patients' phenotypic profile, in a case-control study. The focus of this report is on the methodology used in the identification of potential genes as prognostic factors. METHODS: Selected from 304 patients at Mayo Clinic, 18 stage I squamous cell lung cancer patients who died within 2 years (high-aggressive) or lived beyond 5 years (low-aggressive) were included in this study. Both a one-to-one matched design (paired) and a two-group design (grouped) were utilized. Matching variables were age, gender, tumor size and grade, smoking status, and treatment. Two-GeneChip-array sets from Affymetrix (HG-U133) were used. We applied multiple analytic approaches including Dchip (Harvard University), SAM (Stanford University), ArrayTools (US National Cancer Institute), and MAS5 (Affymetrix); and integrated multiple results to generate the final candidate genes for further investigation. We evaluated the consistency across the methods and the effects of matched versus grouped design on the results. RESULTS: Using the same pre-processed data under the same criteria for type I error and fold-change in expression intensity, results are 94-100% concordant in the list of significant genes by Dchip and by ArrayTools, and 53% concordant between the paired and the grouped analysis. If using differently pre-processed data, the concordance rate is under 6% even by the same analytic tool. Combining results from all analyses, we found 23 potentially important genes that may distinguish the high- versus low-aggressive squamous cell tumors of the lung. CONCLUSION: Given the generally low consistency of results across analytic algorithms and study design, poor agreement is expected from different investigators reporting candidate genes for the same endpoint. A well-designed study with a carefully planned analytic strategy is critical. We are in the process of validating the 23 preliminary candidate genes found from this study among independent yet comparable cases.

Gal repressosome contains an antiparallel DNA loop.

Gal repressosome assembly and repression of the gal operon in Escherichia coli occurs when two dimeric GalR proteins and the histone-like HU protein bind to cognate sites causing DNA looping. Structure-based genetic analysis defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure. Stereochemical models of the four possible DNA loops compatible with the GalR tetramer configuration were constructed using the sequence-dependent structural parameters of the interoperator DNA and conformation changes caused by GalR and asymmetric HU binding. Evaluation of their DNA elastic energies gave unambiguous preference to a loop structure in which the two gal operators adopt an antiparallel orientation causing undertwisting of DNA.

ERGL, a novel gene related to ERGIC-53 that is highly expressed in normal and neoplastic prostate and several other tissues.

We have identified a new gene, that is highly expressed in normal and neoplastic prostate, and is also expressed in cardiac atrium, salivary gland, spleen and selective cells in the CNS. Database analyses of ESTs indicated prostate specificity but experimental results showed the expression in other tissues. The full length transcript is 1800 bp with an open reading frame of 526 aa. The amino-terminal 230 residues of the expressed protein has high homology to a family of lectins, especially to the sugar binding domain of ERGIC-53. We therefore designate the new gene ERGL (ERGIC-53-like). There is a transmembrane domain at amino acid positions 468-482 suggesting that the product of ERGL is a type-I membrane protein. In prostate there are two fully processed transcripts one of which is a splice variant with a deletion in the region of the transmembrane domain of the protein.

Identification of three new alternate human kallikrein 2 transcripts: evidence of long transcript and alternative splicing.

In a search for prostate-specific genes in the human expressed sequence tag (EST) database, we identified a seemingly unique EST cluster C81. Experimental data linked C81 to the human hKLK2 gene that encodes a prostate specific serine protease-human glandular kallikrein (hK2). We uncovered a full-length hKLK2 cDNA corresponding to a 3.0 kb hKLK2 mRNA by PCR and sequence analysis. The 3.0 kb transcript accounts for about 25% of the hKLK2 transcripts as compared to the previously known 1.5 kb transcript. We also identified a third spliced form of the hKLK2 gene produced by alternative splicing between intron III and exon 4. This spliced form was detected in normal prostate, prostate cancer and the prostate adenocarcinoma cell line LNCaP. The identification of long hKLK2 transcript and an alternative spliced form of the hKLK2 gene indicates that regulation of the gene is complex.

Reduction of the nonspecific animal toxicity of anti-Tac(Fv)-PE38 by mutations in the framework regions of the Fv which lower the isoelectric point.

Anti-Tac(Fv)-PE38, also called LMB-2, is a very active recombinant immunotoxin that has produced eight responses, including a durable clinical complete remission in a recently completed phase I trial of leukemias and lymphomas. Dose escalation was limited by liver toxicity. We have noted that the Fv of anti-Tac has an isoelectric point (pI) of 10.2. We hypothesize that the overall positive charge on the Fv portion of anti-Tac(Fv)-PE38 contributes to nonspecific binding to liver cells and results in dose-limiting liver toxicity. We have used a mouse model to investigate the basis of this toxicity and found that lowering the pI of the Fv of anti-Tac from 10.2 to 6. 82 by selective mutation of surface residues causes a 3-fold decrease in animal toxicity and hepatic necrosis. This change in pI did not significantly alter the CD25 binding affinity, the cytotoxic activity toward target cells, or antitumor activity, resulting in a 3-fold improvement in the therapeutic index. If this decreased toxicity occurs in humans, it should greatly increase the clinical utility of this immunotoxin.

High expression of a specific T-cell receptor gamma transcript in epithelial cells of the prostate.

We have identified expression of T-cell receptor gamma chain (TCRgamma) mRNA in human prostate and have shown that it originates from epithelial cells of the prostate and not from infiltrating T-lymphocytes. In contrast, the T-cell receptor delta chain (TCRdelta) gene is silent in human prostate. The major TCRgamma transcript in prostate has a different size than the transcript expressed in thymus, spleen, and blood leukocytes. It is expressed in normal prostate epithelium, adenocarcinoma of the prostate, and the prostatic adenocarcinoma cell line LNCaP. The RNA originates from an unrearranged TCRgamma locus, and it is initiated within the intronic sequence directly upstream of the Jgamma1.2 gene segment. The prostate-specific TCRgamma transcript consists of the Jgamma1.2 and Cgamma1 gene segments, and it has an untranslated sequence including a polyadenylation signal and poly(A) sequence at the 3'end. The finding that prostate epithelial cells express a high level of a transcript from a gene that was thought to by exclusively expressed by T-lymphocytes is highly unexpected.

GalR mutants defective in repressosome formation.

Transcription repression of the galactose operon of Escherichia coli requires (1) the binding of the GalR repressor to tandem operators flanking the promoters, (2) the binding of histone-like protein, HU, to a site between the GalR-binding sites, and (3) negatively supercoiled DNA. Under these conditions, protein-protein interactions mediate the formation of a nucleoprotein complex in the form of a DNA loop, which we have termed a repressosome. To analyze the structure of the repressosome, we have screened and isolated galR mutants in which single amino acid substitutions in GalR lead to defects in loop formation while the protein's operator-binding activity is retained. The mutant proteins were purified and their properties confirmed in vitro. We verified that in the case of the two stronger mutations, the proteins had secondary structures that were identical to that of wild-type GalR as reflected by circular dichroism spectroscopy. Homology-based modeling of GalR by use of the crystal structures of PurR and LacI has enabled us to place the three sites of mutation in a structural context. They occur in the carboxy-terminal subdomain of the GalR core, are surface exposed, and, therefore, may be involved in protein-protein interactions. On the basis of our model of GalR and its structural alignment with LacI and PurR, we have identified additional residues, the substitution of which leads to a specific defect in repression by looping. The effects of the mutations are the same in the presence of HMG-17, a eukaryotic protein unrelated to HU, which can also mediate GalR-dependent repression of the gal promoter. This observation suggests that the mutations define sites of GalR-GalR interaction rather than HU-GalR interaction in the repressosome.

Novel genes in the PAGE and GAGE family of tumor antigens found by homology walking in the dbEST database.

We have developed a computer-based screening strategy to search the dbEST database to find differentiation antigens that are expressed by cancers arising in nonessential normal tissues such as prostate, breast, and ovary (G. Vasmatzis et al., Proc. Natl. Acad. Sci. USA, 95: 300-304, 1998). Here, we report the identification of three new members of the GAGE/ PAGE family, termed XAGEs. XAGE-1 and XAGE-2 are expressed in Ewing's sarcoma, rhabdomyosarcoma, a breast cancer, and a germ cell tumor. We also describe the relationship of the XAGEs to the GAGE/ PAGE family. XAGE-1 and XAGE-2 should be evaluated as possible targets for vaccine-based therapies of cancer.

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus.

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Improved stability and yield of a Fv-toxin fusion protein by computer design and protein engineering of the Fv.

The conversion of the anti-mesothelin monoclonal antibody K1 to a single-chain Fv (scFv) that is fused to a truncated form of Pseudomonas exotoxin A (PE) results in a fusion protein (immunotoxin) that is unstable and refolds very inefficiently. We have devised a method that identifies candidate residues in the framework region of K1 Fv that, when mutated, improved the yield and stability of the protein. The method works by initially aligning the framework sequences of K1 VH and VL with those of other scFvs that are stable and give a good yield as immunotoxins. Then we assigned a character to each residue that indicates its state of exposure based on the known crystal structures of Fabs. This identifies residues that are not compatible with their environment in the folded state of the protein. Next we calculated the frequencies of different amino acids for each position of the Fvs based on the available sequence database. This identifies residues that are not commonly present in the conserved positions. If these residues are compatible with their exposure profile they are left unaltered. Otherwise, they are identified as candidate residues for mutation. We identified two such residues in the VH (T82 and A85) and two in the VL (H36 and V60) of K1 that did not seem appropriate for their respective positions. By mutating these residues in K1 into those that occur most commonly in the sequence database or in stable scFvs, we significantly improved the stability and yield of the K1 scFv immunotoxins. By making single and combined mutations we assessed the relative contribution of mutations at these four sites towards the stability and yield of K1 scFv immunotoxins. The method we devised is probably general and can be used to improve other scFvs.

Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: conformational influence of an engineered disulfide bond.

A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (BldsFv) crystallizes in space group P6(1)22 with the unit cell parameters a = b = 80.1 A, and c = 138.1 A. The crystal structure of the BldsFv has been determined at 2.1-A resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 A and in bond angle of 2.74 degrees. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv is a deep depression 10-A wide and 17-A long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab.

Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis.

A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.

Stabilization of protein structures.

The technique of protein stabilization has been improving steadily in recent years, but it is only in the past year or two that the stability of some protein molecules has been improved to the level of those from extreme thermophilic organisms. This was achieved by multiple mutations and often by utilizing the knowledge gained from the homologous protein structures from extreme thermophiles.

Determination of atomic desolvation energies from the structures of crystallized proteins.

We estimated effective atomic contact energies (ACE), the desolvation free energies required to transfer atoms from water to a protein's interior, using an adaptation of a method introduced by S. Miyazawa and R. L. Jernigan. The energies were obtained for 18 different atom types, which were resolved on the basis of the way their properties cluster in the 20 common amino acids. In addition to providing information on atoms at the highest resolution compatible with the amount and quality of data currently available, the method itself has several new features, including its reference state, the random crystal structure, which removes compositional bias, and a scaling factor that makes contact energies quantitatively comparable with experimentally measured energies. The high level of resolution, the explicit accounting of the local properties of protein interiors during determination of the energies, and the very high computational efficiency with which they can be assigned during any computation, should make the results presented here widely applicable. First we used ACE to calculate the free energies of transferring side-chains from protein interior into water. A comparison of the results thus obtained with the measured free energies of transferring side-chains from n-octanol to water, indicates that the magnitude of protein to water transfer free energies for hydrophobic side-chains is larger than that of n-octanol to water transfer free energies. The difference is consistent with observations made by D. Shortle and co-workers, who measured differential free energies of protein unfolding for site-specific mutants in which Ala or Gly was substituted for various hydrophobic side-chains. A direct comparison (calculated versus observed free energy differences) with those experiments finds slopes of 1.15 and 1.13 for Gly and Ala substitutions, respectively. Finally we compared calculated and observed binding free energies of nine protease-inhibitor complexes. This requires a full free energy function, which is created by adding direct electrostatic interactions and an appropriate entropic component to the solvation free energy term. The calculated free energies are typically within 10% of the observed values. Taken collectively, these results suggest that ACE should provide a reasonably accurate and rapidly evaluatable solvation component of free energy, and should thus make accessible a range of docking, design and protein folding calculations that would otherwise be difficult to perform.