Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging.

The tumor microenvironment plays a crucial role in determining response to treatment. This involves a series of interconnected changes in the cellular landscape, spatial organization, and extracellular matrix composition. However, assessing these alterations simultaneously is challenging from a spatial perspective, due to the limitations of current high-dimensional imaging techniques and the extent of intratumoral heterogeneity over large lesion areas. In this study, we introduce a spatial proteomic workflow termed Hyperplexed Immunofluorescence Imaging (HIFI) that overcomes these limitations. HIFI allows for the simultaneous analysis of > 45 markers in fragile tissue sections at high magnification, using a cost-effective high-throughput workflow. We integrate HIFI with machine learning feature detection, graph-based network analysis, and cluster-based neighborhood analysis to analyze the microenvironment response to radiation therapy in a preclinical model of glioblastoma, and compare this response to a mouse model of breast-to-brain metastasis. Here we show that glioblastomas undergo extensive spatial reorganization of immune cell populations and structural architecture in response to treatment, while brain metastases show no comparable reorganization. Our integrated spatial analyses reveal highly divergent responses to radiation therapy between brain tumor models, despite equivalent radiotherapy benefit.

Imaging and Molecular Annotation of Xenographs and Tumours (IMAXT): High throughput data and analysis infrastructure.

With the aim of producing a 3D representation of tumors, imaging and molecular annotation of xenografts and tumors (IMAXT) uses a large variety of modalities in order to acquire tumor samples and produce a map of every cell in the tumor and its host environment. With the large volume and variety of data produced in the project, we developed automatic data workflows and analysis pipelines. We introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located, instead of bringing data to their local computers. Here, we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumor models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT. The automatic pipelines are highly optimized and allow to obtain processed datasets several times quicker than current solutions narrowing the gap between data acquisition and scientific exploitation.

Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA.

BACKGROUND: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. METHODS: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. RESULTS: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. CONCLUSION: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Requirement for scleraxis in the recruitment of mesenchymal progenitors during embryonic tendon elongation.

The transcription factor scleraxis (Scx) is required for tendon development; however, the function of Scx is not fully understood. Although Scx is expressed by all tendon progenitors and cells, only long tendons are disrupted in the Scx-/- mutant; short tendons appear normal and the ability of muscle to attach to skeleton is not affected. We recently demonstrated that long tendons are formed in two stages: first, by muscle anchoring to skeleton via a short tendon anlage; and second, by rapid elongation of the tendon in parallel with skeletal growth. Through lineage tracing, we extend these observations to all long tendons and show that tendon elongation is fueled by recruitment of new mesenchymal progenitors. Conditional loss of Scx in mesenchymal progenitors did not affect the first stage of anchoring; however, new cells were not recruited during elongation and long tendon formation was impaired. Interestingly, for tenocyte recruitment, Scx expression was required only in the recruited cells and not in the recruiting tendon. The phenotype of Scx mutants can thus be understood as a failure of tendon cell recruitment during tendon elongation.

Cell fusion potentiates tumor heterogeneity and reveals circulating hybrid cells that correlate with stage and survival.

High lethality rates associated with metastatic cancer highlight an urgent medical need for improved understanding of biologic mechanisms driving metastatic spread and identification of biomarkers predicting late-stage progression. Numerous neoplastic cell intrinsic and extrinsic mechanisms fuel tumor progression; however, mechanisms driving heterogeneity of neoplastic cells in solid tumors remain obscure. Increased mutational rates of neoplastic cells in stressed environments are implicated but cannot explain all aspects of tumor heterogeneity. We present evidence that fusion of neoplastic cells with leukocytes (for example, macrophages) contributes to tumor heterogeneity, resulting in cells exhibiting increased metastatic behavior. Fusion hybrids (cells harboring hematopoietic and epithelial properties) are readily detectible in cell culture and tumor-bearing mice. Further, hybrids enumerated in peripheral blood of human cancer patients correlate with disease stage and predict overall survival. This unique population of neoplastic cells provides a novel biomarker for tumor staging, as well as a potential therapeutic target for intervention.

Microenvironment-Mediated Mechanisms of Resistance to HER2 Inhibitors Differ between HER2+ Breast Cancer Subtypes.

Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inhibitors (TKIs). To examine how microenvironmental signals influence resistance, we monitored TKI-treated breast cancer cell lines grown on microenvironment microarrays composed of printed extracellular matrix proteins supplemented with soluble proteins. We tested ∼2,500 combinations of 56 soluble and 46 matrix microenvironmental proteins on basal-like HER2+ (HER2E) or luminal-like HER2+ (L-HER2+) cells treated with the TKIs lapatinib or neratinib. In HER2E cells, hepatocyte growth factor, a ligand for MET, induced resistance that could be reversed with crizotinib, an inhibitor of MET. In L-HER2+ cells, neuregulin1-ß1 (NRG1ß), a ligand for HER3, induced resistance that could be reversed with pertuzumab, an inhibitor of HER2-HER3 heterodimerization. The subtype-specific responses were also observed in 3D cultures and murine xenografts. These results, along with bioinformatic pathway analysis and siRNA knockdown experiments, suggest different mechanisms of resistance specific to each HER2+ subtype: MET signaling for HER2E and HER2-HER3 heterodimerization for L-HER2+ cells.

Musculoskeletal integration at the wrist underlies the modular development of limb tendons.

The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. Here, we show that mouse limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.

The atypical homeodomain transcription factor Mohawk controls tendon morphogenesis.

The Mohawk homeobox (Mkx) gene encodes a new atypical homeodomain-containing protein with transcriptional repressor activity. Mkx mRNA exhibited dynamic expression patterns during development of the palate, somite, kidney, and testis, suggesting that it may be an important regulator of multiple developmental processes. To investigate the roles of Mkx in organogenesis, we generated mice carrying a null mutation in this gene. Mkx(-/-) mice survive postnatally and exhibit a unique wavy-tail phenotype. Close examination revealed that the mutant mice had smaller tendons than wild-type littermates and that the rapid postnatal growth of collagen fibrils in tendons was disrupted in Mkx(-/-) mice. Defects in tendon development were detected in the mutant mouse embryos as early as embryonic day 16.5 (E16.5). Although collagen fibril assembly initially appeared normal, the tendons of Mkx(-/-) embryos expressed significantly reduced amounts of collagen I, fibromodulin, and tenomodulin in comparison with control littermates. We found that Mkx mRNA was strongly expressed in differentiating tendon cells during embryogenesis and in the tendon sheath cells in postnatal stages. In addition to defects in tendon collagen fibrillogenesis, Mkx(-/-) mutant mice exhibited abnormal tendon sheaths. These results identify Mkx as an important regulator of tendon development.

Minimum altered regions in early prostate cancer progression identified by high resolution whole genome tiling path BAC array comparative hybridization.

BACKGROUND: Carcinoma of the prostate (CaP) is a serious health problem. The altered molecular mechanisms that lead to this disease are poorly understood. METHODS: Specimens from radical prostatectomies and blood were collected from 18 CaP surgery patients. For CGH studies, 20 CaP-related samples (16 Gleason grade 3, 3 higher grades, 1 BPH sample) and 18 samples of patient-matched normal epithelial cells were obtained by laser-assisted microdissection from frozen sections of the 18 prostatectomy specimens. High resolution SMRT aCGH was used to compare genomic profiles of prostatic samples to patient-matched blood and pooled female DNA. TMPRSS2-ERG fusion transcript analysis was performed by RT-PCR in relation to alterations detected at the TMPRSS2 locus. RESULTS: Our comprehensive aCGH approach allowed us to define 35 regions of recurrent alterations while excluding germline copy number polymorphisms. Novel regions identified include 2q14.2, containing INHBB, and 17q21.31. The TMPRSS2 locus at 21q22.3 may be a hotspot for rearrangements with 75% of the alterations resulting in the expression of a TMPRSS2-ERG fusion transcript. Differences in fusion expression in different areas in an individual tumor focus and expression in adjacent normal epithelium supported intrafocal heterogeneity and field cancerization, respectively. Both features challenge our efforts to develop more objective markers for diagnosis and prediction of the severity of CaP. CONCLUSION: The high-density array enabled precise mapping of genomic alterations and consequently definition of minimum altered regions smaller than previously reported thus facilitating identification of those genes that contribute to the cancer transformation process.

Recruitment and maintenance of tendon progenitors by TGFbeta signaling are essential for tendon formation.

Tendons and ligaments mediate the attachment of muscle to bone and of bone to bone to provide connectivity and structural integrity in the musculoskeletal system. We show that TGFbeta signaling plays a major role in the formation of these tissues. TGFbeta signaling is a potent inducer of the tendon progenitor (TNP) marker scleraxis both in organ culture and in cultured cells, and disruption of TGFbeta signaling in Tgfb2(-/-);Tgfb3(-/-) double mutant embryos or through inactivation of the type II TGFbeta receptor (TGFBR2; also known as TbetaRII) results in the loss of most tendons and ligaments in the limbs, trunk, tail and head. The induction of scleraxis-expressing TNPs is not affected in mutant embryos and the tendon phenotype is first manifested at E12.5, a developmental stage in which TNPs are positioned between the differentiating muscles and cartilage, and in which Tgfb2 or Tgfb3 is expressed both in TNPs and in the differentiating muscles and cartilage. TGFbeta signaling is thus essential for maintenance of TNPs, and we propose that it also mediates the recruitment of new tendon cells by differentiating muscles and cartilage to establish the connections between tendon primordia and their respective musculoskeletal counterparts, leading to the formation of an interconnected and functionally integrated musculoskeletal system.

Tubulin polymerization-promoting protein family member 3, Tppp3, is a specific marker of the differentiating tendon sheath and synovial joints.

Tppp3, a member of the Tubulin polymerization-promoting protein family, is an intrinsically unstructured protein that induces tubulin polymerization. We show that Tppp3 is a distinct marker in the developing musculoskeletal system. In tendons, Tppp3 is expressed in cells at the circumference of the developing tendons, likely the progenitors of connective tissues that surround tendons: the tendon sheath, epitenon, and paratenon. These tissues form an elastic sleeve around tendons and provide lubrication to minimize friction between tendons and surrounding tissues. Tppp3 is the first molecular marker of the tendon sheath, opening the door for direct examination of these tissues. Tppp3 is also expressed in forming synovial joints. The onset of Tppp3 expression in joints coincides with cavitation, representing a molecular marker that can be used to indicate this stage in joint transition in joint differentiation. In late embryonic stages, Tppp3 expression highlights other demarcation lines that surround differentiating tissues in the forelimb.

Technical demonstration of whole genome array comparative genomic hybridization.

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in approximately 100 kilobase pair (kb) segments. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected--a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position. The SMRT array can detect alterations as small as 50 kb in size. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100 ng) this allows profiling of precious samples such as those produced by microdissection. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

Skewed X-chromosome inactivation is associated with primary but not secondary ovarian failure.

Premature ovarian failure (POF) is the occurrence of menopause before the age of 40, and may present with either primary or secondary amenorrhea. Numerous cases of POF in women with X-chromosome deletions or translocations have been reported; thus, it is possible that smaller rearrangements undetectable by conventional cytogenetics may contribute to POF in some patients. In females with an abnormal X chromosome, cells with inactivation of the normal X may be selected against, causing skewed X-chromosome inactivation (XCI). We therefore assessed XCI by methylation sensitive restriction digestion and PCR amplification at the androgen receptor (AR) locus, in 4 primary and 55 secondary POF patients and 109 control women. In samples heterozygous at AR and therefore informative for the skewing assay, the frequency of skewed XCI among the women with secondary amenorrhea was identical to that in control women, with 4 out of 48 (8.3%) secondary ovarian failure patients and 8 out of 97 (8.2%) control women having > or =90% skewing. Notably, all three primary amenorrhea patients that were informative at AR had skewed XCI > or =90% (P = 0.001 vs. control women; Fisher's exact test). To investigate whether X-chromosome copy number alterations were responsible, DNA from selected patients with skewed XCI was examined by high resolution DNA microarray, however no potential regions of DNA addition or deletion were confirmed by FISH or PCR. X-chromosome abnormalities undetectable by array, or reduced follicular pool due to an early trisomic rescue event, may explain the skewed XCI observed in POF patients presenting with primary amenorrhea.

Cytogenetically balanced translocations are associated with focal copy number alterations.

Current cytogenetic methods (e.g., G-banding and multicolor chromosomal painting) allow detection of translocation events but lack the resolution to (a) locate the breakpoints precisely at the chromosome band level or (b) discriminate balanced translocations from translocations with copy number alterations not previously reported, or imperfectly balanced translocations. In this study, we demonstrate that cytogenetically balanced translocations are in fact frequently associated with segmental gain or loss of DNA. The recent development of a whole genome tiling path BAC array has enabled tiling resolution analysis of genomic segmental copy number status. Combining tiling resolution BAC array comparative genomic hybridization (array CGH) with G-Banding analysis and multicolor chromosomal painting approaches such as spectral karyotyping (SKY) facilitates high-resolution mapping of genomic alterations associated with imperfectly balanced translocations. Using a refined version of our CGH array we have deduced the copy number status throughout the genomes of three cytogenetically well-characterized prostate cancer cell lines (PC3, DU145, LNCaP) to determine whether translocations are associated with focal gains and losses of DNA. At 78 kb tiling resolution we identified the boundaries of 170, 80, and 34 known and novel copy number alterations (CNA) in these cell line genomes, respectively. Thirty-three of the 36 known translocations (92%, P < 0.001) in DU145 were associated with segmental CNA. Likewise, 80% (P < 0.001) of the known translocations showed association in LNCaP. Although many translocation breakpoints exhibit segmental alteration in PC3, the pattern of chromosomal rearrangements is too complex for use in comprehensive association with CNA boundaries. Our results reveal that imperfectly balanced translocations in tumor genomes are a phenomenon that occurs at frequencies much higher than previously demonstrated.

Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold. RESULTS: We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar. CONCLUSION: Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

An integrated mBAND and submegabase resolution tiling set (SMRT) CGH array analysis of focal amplification, microdeletions, and ladder structures consistent with breakage-fusion-bridge cycle events in osteosarcoma.

Osteosarcoma (OS) is characterized by chromosomal instability and high-copy-number gene amplification. The breakage-fusion-bridge (BFB) cycle is a well-established mechanism of genomic instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. However, until now, there have been no high-resolution cytogenetic or genomic array studies of BFB events in OS. In the present study, multicolor banding (mBAND) FISH and submegabase resolution tiling set (SMRT) array comparative genomic hybridization (CGH) were used to identify and map genomic signatures of BFB events in four OS cell lines and one patient tumor. The expected intermediates associated with BFB-dicentric chromosomes, inverted duplications, and intra- and interchromosomal amplifications-were identified. mBAND analysis provided detailed mapping of rearrangements in 1p, 6p, and 8q and showed that translocation junctions were often in close proximity to fragile sites. More detailed mBAND studies of OS cell line MG-63 revealed ladderlike FISH signals of equally spaced interchromosomal coamplifications of 6p21, 8q24, and 9p21-p22 in a homogeneously staining region (hsr). Focal amplifications that concordantly mapped to the hsr were localized to discrete genomic intervals by SMRT array CGH. The complex amplicon structure in this hsr suggests focal amplifications immediately adjacent to microdeletions. Moreover, the genomic regions in which there was deletion/amplification had a preponderance of fragile sites. In summary, this study has provided further support for the role of the BFB mechanism and fragile sites in facilitating gene amplification and chromosomal rearrangement in OS.

A tiling resolution DNA microarray with complete coverage of the human genome.

We constructed a tiling resolution array consisting of 32,433 overlapping BAC clones covering the entire human genome. This increases our ability to identify genetic alterations and their boundaries throughout the genome in a single comparative genomic hybridization (CGH) experiment. At this tiling resolution, we identified minute DNA alterations not previously reported. These alterations include microamplifications and deletions containing oncogenes, tumor-suppressor genes and new genes that may be associated with multiple tumor types. Our findings show the need to move beyond conventional marker-based genome comparison approaches, that rely on inference of continuity between interval markers. Our submegabase resolution tiling set for array CGH (SMRT array) allows comprehensive assessment of genomic integrity and thereby the identification of new genes associated with disease.

Methods for high throughput validation of amplified fragment pools of BAC DNA for constructing high resolution CGH arrays.

BACKGROUND: The recent development of array based comparative genomic hybridization (CGH) technology provides improved resolution for detection of genomic DNA copy number alterations. In array CGH, generating spotting solution is a multi-step process where bacterial artificial chromosome (BAC) clones are converted to replenishable PCR amplified fragments pools (AFP) for use as spotting solution in a microarray format on glass substrate. With completion of the human and mouse genome sequencing, large BAC clone sets providing complete genome coverage are available for construction of whole genome BAC arrays. Currently, Southern hybridization, fluorescent in-situ hybridization (FISH), and BAC end sequencing methods are commonly used to identify the initial BAC clone but not the end product used for spotting arrays. The AFP sequencing technique described in this study is a novel method designed to verify the identity of array spotting solution in a high throughput manner. RESULTS: We show here that Southern hybridization, FISH, and AFP sequencing can be used to verify the identity of final spotting solutions using less than 10% of the AFP product. Single pass AFP sequencing identified over half of the 960 AFPs analyzed. Moreover, using two vector primers approximately 90% of the AFP spotting solutions can be identified. CONCLUSIONS: In this feasibility study we demonstrate that current methods for identifying initial BAC clones can be adapted to verify the identity of AFP spotting solutions used in printing arrays. Of these methods, AFP sequencing proves to be the most efficient for large scale identification of spotting solution in a high throughput manner.