university of groningen fluorescence molecular endoscopy

University of Groningen

Fluorescence molecular endoscopyHartmans, Elmire

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C H A P T e R 6

Data driven prioritization and review of

targets for molecular based

imaging and treatment strategies in

pancreatic cancer

Submitted, 2017

M Koller1*, E Hartmans2*, DJA de Groot3, XJ Zhoa3, GM van Dam4, WB Nagengast2†, RSN Fehrmann3†

*Contributed equally; † Shared last and corresponding authors

Department of 1 Surgery, 2 Gastroenterology and Hepatology, 3 Medical Oncology, 4 Surgery, Nuclear Medicine and Molecular

Imaging and Intensive Care, University of Groningen, University Medical Centre Groningen, Groningen, the Netherlands

C H A P T e R 6

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ABSTRACT The prognosis of pancreatic ductal adenocarcinoma (PDA) remains poor with current treat-ment strategies. Molecular targeted therapies may improve the prognosis by specifically targeting aberrant signaling-pathways in PDA tumor cells. In addition, molecular imaging may improve disease staging, leading to better selection of patients who are suitable for surgical resection. In order to adequately incorporate these strategies in PDA management, relevant molecular targets need to be identified. We collected expression profiles of normal pancre-atic tissue (n = 77) and patient derived PDA samples (n = 103) from the Gene Expression Omnibus database. We applied Functional Genomic mRNA (FGmRNA) profiling, to predict upregulation of targets on the protein level. An extensive literature search was performed to prioritize these targets based on 1) the known protein overexpression in PDA, 2) the known interaction with antineoplastic drugs, and 3) the current status of (pre)-clinical drug and imaging evaluation. We identified 213 significantly upregulated targets in PDA containing 41 therapeutic targets and 7 imaging targets. We prioritized MUC1, MSLN, GGT5 and CTSE, since studies demonstrated their potential for both therapeutic and imaging strategies. This study applied a new hybrid reviewing strategy that resulted in a molecular data and literature driven prioritization of therapeutic and imaging targets that will facilitate clinicians and drug developers in deciding which drug or imaging targets should be taken for further clinical evalu-ation in PDA. This might help to improve disease outcome of PDA patients in the short term.

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Data driven prioritization and review of targets for molecular based imaging and Treatment strategies in pancreatic cancer

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INTRODUCTION

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related mor-tality, causing more than 330 000 deaths per year worldwide.1 Despite extensive surgery and improved chemotherapeutic regimens, the prognosis of PDA remains poor. Since symptoms often occur late in the disease process, the majority of patients present with locally advanced or even metastatic disease, resulting in a 5 years overall survival rate of only ~7%.2 Solely patients with local disease are candidate for curative surgical treatment. Despite the curative intent, the 5 years survival in the surgical treated patients is still as low as 20%.3 This poor survival is partially caused by the rapid development of metastases shortly after surgery. Most likely, this is due to microscopic dissemination that was already present at the time of surgery. Once distant metastases are present, the best available palliative chemotherapy regimen with the best overall survival rate is fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX). However, the overall survival benefit is modest and the toxicity is significant.4

In contrast to the traditional working mechanism of chemotherapy, which has a cytotoxic effect on all rapidly dividing cells, molecular targeted therapies more selectively target aberrant cell signaling-pathways that drive tumor growth. Therefore, in general molecular targeted therapies are expected to be more tumor specific, which could enhance therapy efficacy and decrease side-effects.

In addition, identification of novel targets might improve PDA staging and thus patient stratification by the use of molecular imaging. Molecular imaging is a diagnostic approach in which highlighting of tumor specific proteins can be used to discriminate between benign and malignant tissue. In the context of PDA, molecular imaging could be used to better detect small metastases preoperatively and define surgical margins peroperatively, leading to improved selection of patients that are suitable for surgical resection with a curative intent. Therefore, in patients that will not benefit from surgery, such an extensive procedure with substantial morbidity and mortality will be prevented.

To achieve these goals for PDA patients, there is an unmet need for identification and prioritization of relevant targets. To this end, we used the recently developed method of functional genomic mRNA profiling (FGmRNA-profiling) to predict overexpression of target antigens on the protein level.5 FGmRNA-profiling is capable to correct a gene expression profile of an individual tumor for physiological and experimental factors, which are considered not to be relevant for the observed tumor phenotype and characteristics.

The aim of this study was to identify potential target antigens in PDC using FGmRNA-pro-filing. Subsequently, an extensive literature search was performed to prioritize these potential target antigens for both molecular targeted therapy and imaging techniques based on 1) the

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known protein overexpression in PDA, 2) the known interaction with antineoplastic drugs, and 3) the current status of drug or imaging development.

MATeRIAL AND MeTHODS

Data acquisition We searched publicly available raw microarray expression data in the Gene Expression Omni-bus (GEO) for the affymetrix HG-U133 plus 2.0 and the HG-U133A platforms.6 We used automatic filtering on relevant keywords with subsequent manual curation to include patient derived PDA samples and healthy pancreatic tissue. Cell line sample were deemed irrelevant and excluded for further analysis.

Sample processingNon-corrupted raw data CEL files were downloaded from GEO for the selected samples. After removal of duplicate CEL files, pre-processing and aggregation of CEL files was per-formed with Affymetrix Power Tools version 1.15.2, using apt-probe set-summarize and applying the robust multi-array average (RMA) algorithm. Sample quality control was per-formed using principal component analysis as previously described.7

Functional genomic mRNA profiling For a detailed description of FGmRNA-profiling we refer to Fehrmann et al.5 In short, we analyzed 77 840 expression profiles of publicly available samples with principal component analysis (PCA) and found that a limited number of ‘Transcriptional Components’ (TCs) cap-ture the major regulators of the mRNA transcriptome. Subsequently, we identified a subset of TCs that described non-genetic regulatory factors. We used these non-genetic TCs as covariates to correct microarray expression data and observed that the residual expression signal (i.e. FGmRNA-profile) captures the downstream consequences of genomic alterations on gene expression levels.

Class comparisonWe performed a genome-wide class comparison analysis (Welch’s T-test) between FGm-RNA-profiles of healthy pancreatic tissue and PDA to identify genes with upregulated FGmRNA-expression, which we considered a proxy for protein expression. To assess the degree of multiple testing, we performed this analysis within a multivariate permutation (MVP) test (1 000 permutations) with a false discovery rate of 1% and a confidence level of

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99%. This will result in a list of significant upregulated genes, which contains (with a confi-dence level of 99%) no more than 1% false positives.

Literature search – protein expressionPubMed was searched for articles published in English from conception until February 2017. The following search terms were used: HUGO gene symbol in combination with ’pancreatic cancer’, ‘expression’ and ‘immunohistochemistry’. The cellular location and function of the protein product of the gene was explored at http://www.genecards.org.

Drug-gene interaction database (DGIdb)Upregulated genes were explored in the Drug-Gene Interaction Database (DGIdb, accessable at dgibd.genome.wustl.edu) to get insight into which genes might be interesting for targeted PDA treatment strategies. The DGIdb integrates data from multiple resources that includes disease-relevant human genes, drugs, drug-gene interactions and potential druggability.8

Current knowledge about therapeutic efficacy - Clinicaltrials.gov and PubMedDrug-gene interaction reported by the DBIdb were reviewed in literature to determine the therapeutic efficacy of the drugs targeting these genes. PubMed was searched for articles published in English from conception until February 2017 and clinicaltrials.gov was explored for current (ongoing) clinical trials. PubMed was searched using the combination of 1) HUGO gene symbol; ‘pancreatic AND OR cancer’; and ‘therapy’ or 2) HUGO gene symbol; ‘pan-creatic AND OR cancer’.

An additional PubMed search was executed for articles published in English form concep-tion until February 2017 to determine if the downstream proteins of these genes are suitable as molecular based imaging targets. We used the following search combinations: ‘HUGO gene symbol’; ‘pancreatic AND OR cancer’; and ‘imaging’.

ReSULTS

FGmRNA profiling: identification of upregulated genes in PDA Table 1 shows the datasets that were obtained from GEO. In total, 180 pancreatic samples were identified, which are derived from 16 individual experiments; these samples consisted of 103 PDA and 77 normal pancreatic samples. Class comparison analysis, with multivariate permutation testing (FDR 1%, CI 99%, 1 000 permutations), resulted in a set of 213 unique genes with significant FGmRNA-overexpression in PDA.

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Table 1. GEO omnibus datasets included in the study

GSE Accession number profiling performed, year origin samples (n)

GSE1133 Walker et al, 2004 normal pancreatic tissue (2)

GSE12630 Buturovic et al, 2008 PDA (9)

GSE15471 Badea et al, 2009 normal pancreatic tissue (39)

PDA (39)

GSE17891 Sadanandam et al, 2009 PDA (1)

GSE18674 Miya et al, 2009 normal pancreatic tissue (1)

GSE19279 Chelala et al, 2009 normal pancreatic tissue (3)

PDA (9)

GSE19650 Hiraoka et al, 2009 normal pancreatic tissue (7)

GSE2109 Curley et al, 2004 PDA (16)

GSE22780 Chen et al, 2010 normal pancreatic tissue (8)

GSE2361 Ge et al, 2005 normal pancreatic tissue (1)

GSE32676 Tran et al, 2011 normal pancreatic tissue (7)

PDA (25)

GSE33846 Miya et al, 2011 normal pancreatic tissue (1)

GSE43288 Chelala et al, 2013 normal pancreatic tissue (3)

PDA (4)

GSE43346 Kaneda et al, 2013 normal pancreatic tissue (1)

GSE46385 Blais et al, 2013 normal pancreatic tissue (3)

GSE7307 Roth et al, 2007 normal pancreatic tissue (1)

Abbreviation: GSE, gene expression omnibus series; PDA, pancreatic ductal adenocarcinomaNote: GSE accession numbers can be used to query the data set in GEO (http://www.ncbi.nlm.nih.gov/geo/).

Literature based protein expression data for the top 50 upregulated PDA genesThe top 50 upregulated PDA genes were reviewed in literature to provide insight in the cur-rent knowledge on protein expression levels in human cancer patients (Table 2). We classified these 50 genes into three subcategories.

IHC subcategory 1 – Known protein overexpression in human PDA samples. Based on published immunohistochemistry results, 17/50 genes have a known downstream protein overexpression in human PDA samples. CLDN18 (Rank: 7/213) and its downstream protein is reported to be overexpressed in 50-80% of the PDA samples while no protein expression is observed in normal pancreatic tissue.9,10 Protein overexpression of CLDN18 has also been described for precursor lesions like pancreatic intraepithelial neoplasia (PanINs), intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs).11

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The downstream proteins of MUC1 and MSLN (Rank: 41/213 and 110/213 respectively) are localized on the cell membrane. MUC1 is reported to be overexpressed in 96% of the PDA cases, and MSLN overexpression has been described in up to 86-100%.12-14

IHC subcategory 2 – Protein overexpression in human cancer samples other than PDA. The downstream protein overexpression of 5/50 genes is described in other solid cancer types and could be of interest for PDA. Protein overexpression of KRT17 (rank 4/213) is found in 96% of cervical high-grade squamous cell carcinoma samples.15 Protein overexpression of PLA2G16 (rank 29/213) is described in 54,6% of human osteosarcoma samples and in 68% of human non-small cell lung cancer samples.16,17 Protein overexpression of TRAK1 (rank 38/213) is described in up to 81% in gastric cancer samples and 85% in colorectal cancer samples.18,19

IHC subcategory 3 – Unknown protein overexpression in human cancer samples. For 27/50 upregulated genes in PDA, no data is available on protein expression in human cancers. NPR3, HSD17B7, FXYD3, GJB3, GPRC5D, LRRC32, MRC and NTM are all cell membrane proteins and therefore of particular interest for molecular targeted strategies since they are easily accessible for imaging tracers.

Therapeutic potential of the FGmRNA-overexpressing genes We used the drug-gene interaction database to identify drugs that interact with the 213-upreg-ulated PDA genes. 94/213 upregulated genes in PDA have a known drug-gene interaction. Based on current knowledge at PubMed and clinicaltrials.gov, 41/94 genes are currently inves-tigated as a drug target for cancer treatment in clinical trials or in preclinical studies (Figure 1). We divided these 41 genes into four therapeutic subcategories (Figure 2).

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Tabl

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Figure 1. Study flowchart. A schematic overview of how the study results where obtained: firstly, FGmRNA profiling was used to select 209 upregulated genes in PDA. Secondly, to provide insight in the current knowledge on protein expression levels in human cancer patients, IHC results where assessed for the first fifty genes in liter-ature. Thirdly, the gene-drug interaction database was consulted to obtain a list of genes for which a therapeutic drug is yet available or genes that would be ‘potentially druggable’. And lastly, by performing an extensive literature search, these genes where divided into therapeutic and imaging subcategories to assess their priority and evaluate their relevance in future PDA management.

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I n t r a c e l l u l a r

C e l l m e m b r a n e

E x t r a c e l l u l a r

n u c l e u s

C a t e g o r y 1

C

ategory 2

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MUC1

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Figure 2. Potential therapeutic genes divided per cellular localization per subcategory. The 41 potential genes are divided into four therapeutic subcatego-ries per cellular localization. Therapeutic subcategory 1 shows antineoplastic drug targets investigated in clinical PDA trials Therapeutic subcategory 2 shows antineo-plastic drug targets in clinical trials involving other solid cancer types. Therapeutic sub-category 3 shows preclinical in vitro and in vivo experiments evaluating antineoplastic drugs in cancer-models. Therapeutic sub-category 4 shows potential targets based on preclinical (blocking) experiments.

Therapeutic subcategory 1 – antineoplastic drug targets in clinical pancreatic cancer trials. This subcategory includes 11/41 genes (Suppl. Table 1a). To date, the downstream proteins of MUC1 (rank: 41/213) and MSLN (Rank: 110/213) show the most interesting results in clinical trials and are therefore explained in more detail.

MUC1 (rank 41/213): The membrane bound protein MUC1 is being evaluated as a carrier for anti-cancer vaccines. In a clinical phase I study the cancer vaccine MUC1 100mer pep-tide with the SB-AS2 adjuvant was administered intramuscular in patients with resected or locally advanced pancreatic cancer. MUC1 specific immune responses were observed in 5/16 patients20,21. In a currently ongoing clinical phase I trial in patients with locally advanced or met-astatic pancreatic cancer, the cancer vaccine falimarev is being investigated (NCT00669734) which is a recombinant fowlpox viral vector encoding the carcinoembryonic antigen (CEA), MUC-1 and TRICOM, comprised of three co-stimulatory molecule transgenes (B7-1, ICAM-1 and LFA-3).

MSLN (Rank: 110/213): The GVAX-vaccine, which is given in combination with low dose cyclophosphamide (cy), and CRS-207-vaccine are cancer vaccines that can generate MSLN antigen-specific immune responses. In a phase II trial in metastatic PDA patients the combination of cy/GVAX and CRS-207 vaccines demonstrated an overall survival of 9.7 months versus 4.6 months compared to only cy/GVAX.22 An ongoing phase IIB study is investigating the cy/GVAX vaccine and CRS-207 compared to chemotherapy or CRS-207 alone in patients previously treated metastatic PDA patients (NCT0200426). Chimeric anti-

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gen receptor-engineered T cells (CART), potentially enhancing MLSN specific anti-cancer immune response, showed in a phase I clinical trial a partial response in the one patient with metastatic PDA,23 leading to a phase I/II clinical trial in PDA patients (NCT01583686, NCT02465983, NCT02159716).

Therapeutic subcategory 2 - Antineoplastic drug targets in clinical trials involving other solid cancer types. This subcategory includes 3/41 genes (Suppl. Table 1b): MST1R (rank 95/213), PTMA (rank 106/213) and PRLR (rank: 213/213). PTMA will be discussed in more detail, since clinical trial results are available for this gene.

PTMA (rank 106/213). Prothymosin alfa (PTMA) has been described to be involved in stimulating immune response. Thymosin alfa 1 is a synthetic formulated peptide fragment of PTMA which is evaluated as drug in a large randomized controlled trial in 488 stage IV metastatic melanoma patients. In this study, the addition of thymosin alfa 1 to conven-tional chemotherapy, increased the number of tumor response (11.2 vs 4.1%), increased the median overall survival with 2.8 months (9.4 vs 6.6 months) and increased the 6-month progression free survival from 9.1% to 21.1%.24 Currently, thymosin alfa 1 is being evaluated in other cancer types, such as esophageal cancer (NCT02545751), (non-) small cell lung cancer (NCT02542137; NCT02542930), colon cancer (NCT02535988) and hepatocellular car-cinoma (NCT02281266).

Therapeutic subcategory 3 – Antineoplastic drugs evaluated in preclinical in vitro and in vivo cancer-models. This third subcategory includes 12/41 genes (Suppl. Table 1c). CTSE (rank 8/213), GGT5 (rank 10/213) and ADAM8 (rank 141/213) are the three genes highest in rank that performed experiments in PDA and will be discussed in more detail below.

CTSE (rank 8/213). A preclinical study in a genetically engineered PDA mouse model demonstrated that Cathepsin E-expressing cancer cells could be effectively killed by pho-todynamic therapy using a CTSE-specific prodrug. 25 This prodrug gets activated by CTSE cleavage and releases the phototoxic drug 5-ALA. After light illumination 5-ALA generates highly reactive oxygen species, causing severe damage to all cells within the treated area. CTSE is highly upregulated in PDA compared to normal pancreatic tissue leading to tumor specific PDT with minimal damage to surrounding normal cells.

GGT5 (rank 10/213): The enzyme gamma-glutamyltransferase 5 (GGT5) can activate the prodrug GSAO (glutathione-S-conjugate of a trivalent arsenical) whereafter the drug can enter the tumor cell. In vitro and In vivo experiments showed that tumor growth was not affected by GSAO treatment in tumors expressing low levels of GGT, compared to a signifi-

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cant growth inhibition of tumors expressing high levels of GGT. 26 ADAM8 (rank 141/213): A Disintegrin And Metalloproteinase Domain 8 (ADAM8) is

involved in PDA tumor progression. In a PDA mouse model, the ADAM8 inhibitor BK-1361 demonstrated reduction of tumor load, infiltration and metastasis improving the survival to 24.2 weeks in the treatment group compared to 16 weeks in control group 27.

Therapeutic subcategory 4 – Suggested as potential targets. 15/41 selected genes belong within this fourth subcategory. This subcategory includes genes which are potentially druggable according to the DGIdb, but no antineoplastic drugs are cur-rently available that target the downstream proteins of these genes. Though, their involvement in tumor growth or cancer progression has been investigated in in vitro experiments, of which a summary can be found in Suppl. Table 1d.

Potential of the FGmRNA-overexpressing PDA genes in molecular imagingFurthermore, 7/94 genes that have a known drug-gene interaction are currently described in the context of molecular imaging. We divided the genes into three subcategories (Suppl. Table 2).

Imaging subcategory 1 – Molecular imaging targets in clinical pancreatic cancer trials. 2/7 genes, MUC1 and MSLN, are currently investigated as targets for molecular imaging in clinical trials that are including PDA patients.

MUC1 (rank 41/213): MUC1 can be targeted with the 111Indium labelled monoclonal anti-body PAM4, suitable for SPECT imaging. In a clinical phase I trial 111In-PAM4 showed specific uptake of pancreatic cancer lesions.28 More recently, the MUC1-specific optical imaging tracer Ab-FL-Cy5.5, which is a dual labeled MUC1-targeting antibody conjugated to both a far-red dye and a green dye, demonstrated specific uptake and in vivo visualization of ovarian cancer xenografts.29 The MUC1 aptamer-based tracer APT-PEG-MPA showed that tracer uptake in the tumor correlated well with MUC1 expression levels in MUC1-overexpressing hepatocel-lular carcinoma and lungcarcinoma cells in a xenograft mouse model.30

MSLN (rank: 110/213): In a clinical phase I trial, a 98zirconium labeled MSLN-antibody 89Zr-MMOT0530A was administered in 11 metastatic cancer patients, seven with PDA and four with ovarian cancer. In all patients at least one tumor lesion could be visualized.31 Beside this PET-tracer, a MSLN specific tracer have been developed for single-photon emission tomography (SPECT). 111Indium labelled amatuximab was investigated in six patients, four with malignant mesothelioma and two with PDA. In all patients at least one tumor lesion could be discriminated from its reference background. Tracer uptake was higher in mesothelioma

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than in pancreatic cancer lesions.32

Imaging subcategory 2 – Molecular imaging targets in clinical trials in other solid cancer types. 1/7 genes can be assigned to this category.

GGT5 (Rank: 10/213). Cell membrane bound enzyme GGT can be targeted by optical imaging probe γGlu-HMRG, which is only fluorescent after cleavage by GGT.33 γGlu-HMRG was topical applied on surgical breast cancer specimen to assess the surgical margin. Within 5 min after application of the tracer, tumors even smaller than 1 mm could be discriminated from normal mammary gland tissue.34 In preclinical mouse models for colon cancer and disseminated peritoneal ovarian cancer, tumors could be clearly visualized 1 min after topical administration.33,35

Imaging subcategory 3 – Molecular imaging targets in preclinical cancer-models. 4/7 genes fit into this third subcategory: THY1, CTSE, NPY1R and GPER. THY1 and CTSE are investigated as imaging targets in PDA models and are therefore described in more detail.

THY1 (Rank: 1/213). Cell membrane bound THY1 can be targeted by THY1 targeting microbubbles, which can be visualized by ultrasound molecular imaging. In pancreatic cancer mouse model it showed visualization of pancreatic cancer lesions with a minimum diameter of 1.6 mm.36

CTSE (Rank 8/213). Ritonavir tetramethyl-BODIPY (RIT-TMB) is an optical imaging agent based on a FDA-approved protease inhibitor. RIT-TMB showed CTSE specific imaging in a PDA cell line.37 A CTSE-activatable fluorescent imaging probe demonstrated specific detec-tion of CTSE activity in a PDA mouse model, in which the fluorescence signal in the tumour was 3-fold higher than in background tissue.38

DISCUSSION

In this study we were able to use FGmRNA-profiling on a substantial set of normal pancreatic tissue and PDCs to predict protein overexpression for a large set of targets and identified 213 upregulated targets in PDA, containing 41 therapeutic targets and 7 imaging targets.

Immunohistochemistry (IHC) is a widely used method for the determination of protein expression at a cellular level. Although IHC is considered the gold standard for protein evalu-ation, it is time consuming and it demands many resources including access to formalin-fixed and paraffin-embedded tissue samples of interest. Moreover, differences in execution of the staining protocol and scoring methods make it difficult to compare IHC results from differ-

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ent studies. Therefore, quantitative information concerning the expression level of proteins is currently lacking for many tumor types including PDA. Therefore, we believe that many potential target antigens are currently missed. To this end, we applied FGmRNA-profiling, which enabled us to predict upregulation on the protein level for a large set of targets utilizing publicly available data.5

FGmRNA-profiling has been applied previously for predicting protein overexpression of molecular targets in colorectal adenomas and the prioritization of solid cancers that overexpress the protein mesothelin.12,39 Hartmans et al. validated their FGmRNA results by demonstrating protein overexpression for the top-five identified target genes using IHC.39 Moreover, they demonstrated the applicability of a fluorescently labeled small peptide tar-geting the CD44 splice variant CD44v6 – which was identified by FGmRNA-profiling - for adenoma imaging purposes in a APCmin mouse model. Lamberts et al. compared mesothe-lin overexpression obtained with FGmRNA profiling with IHC data in literature. Agreement was found for MSLN overexpression in gastrointestinal and gynecological tumors, non-small cell lung cancer and synovial sarcomas, whereas overexpression of MSLN in thyroid cancers and renal cell cancers was not yet reported in literature with IHC.12 In the present study, the validity of the predicted target overexpression in PDA is strengthened by the finding that protein overexpression was described in literature for a large subset of the top 50 predicted overexpressing targets; 22/50 proteins encoded by the genes are known to be overexpressed in cancer, of which 17 are described to be PDA specific.

Adequate disease staging and selection of patients that would benefit from curative surgi-cal resection are currently challenging. Standard clinical preoperative imaging techniques (e.g. CT and MRI) are not able to sufficiently determine the extent of the primary tumor, nor detect micrometastases. Therefore, the extent of the disease is most likely underestimated, which might lead to overtreatment by performing extensive surgical resections with high mortality and morbidity in a group of patients that will not benefit from surgery. Molecular imaging might enhance disease staging by enabling visualization of small PDA lesions or it can be used to better assess the extent of the primary tumor lesion during PDA surgery and evaluate essential resection planes. To date, the most common used molecular imaging technique is nuclear imaging. However, the spatial resolution PET and SPECT imaging is most likely insufficient to adequately detect micrometastases. Furthermore it lacks the ability of real-time usage during surgery. Molecular fluorescence laparoscopy features a higher resolution; as such, micrometastases are more likely to be detected during staging laparoscopy leading to optimized patient selection. Clinical trials already demonstrated the feasibility of molecular fluorescence imaging in identifying micrometastases in peritoneal metastasized ovarian- and colon cancer patients by targeting the folate alpha receptor and Vascular Endothelial Growth

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Factor A receptor (VEGFA).40-42 In PDA patients, two clinical trials are currently registered that evaluate intraoperative molecular fluorescence imaging: in one trial the target VEGFA is evaluated (NCT02743975) and in the other trial the Epidermal Growth Factor Receptor (EGFR) is targeted (NCT02736578). Results of these trials are not yet published, but we predicted no protein overexpression of VEGFA and EGFR within this study in PDA.

Following prioritization, we consider MUC1, MSLN, GGT5 and CTSE as the most inter-esting target proteins. Published studies demonstrated the potential of these targets for both therapeutic and imaging strategies in PDA. By fluorescently or radioactively labeling of drugs, molecular imaging can provide insight in pharmacokinetics, tumor uptake and biodistribution that harbors the potential for drug development to support optimal dosing and determine uptake in critical organs to anticipate toxicity. Furthermore, these theranostic drugs might be used for clinical decision making by enabling visualization of molecular characteristics of the tumor. Clinicians might use this valuable information to stratify patients for the most optimal targeted therapy, besides, theranostics can aid in monitoring treatment effects helping clini-cians to adjust therapy dose or to switch to another targeted drug.

In conclusion, this study provides a data driven prioritization and overview of therapeutic and imaging targets. The presented data can facilitate clinicians and drug developers in decid-ing which drug or imaging targets should be taken for further clinical evaluation in PDA. This might help to improve disease outcome of PDA patients in the short term.

GRANT SUPPORT AND ACKNOWLeDGMeNTS

This work was financially supported by grants from the Dutch Cancer Society / Alpe d’HuZes (RUG 2013-5960 to R.S.N.F and RUG 2012-5416 to W.B.N.), the Dutch Organization for Scientific Research (NWO-VENI grant 916-16025), a Mandema Stipendium to R.S.N.F and W.B.N, MK and GMvD reports grants from the FP-7 Framework Programme BetaCure grant no. 602812, during the study.

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ReFeReNCeS

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103 Li L, Liang S, Wasylishen AR, Zhang Y, Yang X, Zhou B et al. PLA2G16 promotes osteosarcoma metas-tasis and drug resistance via the MAPK pathway. Oncotarget 2016; 7: 18021–18035.

104 Liu Y-F, Qu G-Q, Lu Y-M, Kong W-M, Liu Y, Chen W-X et al. Silencing of MAP4K4 by short hairpin RNA suppresses proliferation, induces G1 cell cycle arrest and induces apoptosis in gastric cancer cells. Mol Med Rep 2016; 13: 41–48.

105 Chakraborty PK, Xiong X, Mustafi SB, Saha S, Dhanasekaran D, Mandal NA et al. Role of cystathionine beta synthase in lipid metabolism in ovarian cancer. Oncotarget 2015; 6: 37367–37384.

106 Druzhyna N, Szczesny B, Olah G, Módis K, Asimakopoulou A, Pavlidou A et al. Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine β-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the exper-imental therapy of colon cancer. Pharmacol Res 2016; 113: 18–37.

107 Zougman A, Hutchins GG, Cairns DA, Verghese E, Perry SL, Jayne DG et al. Retinoic acid-induced protein 3: Identification and characterisation of a novel prognostic colon cancer biomarker. European Journal of Cancer 2013; 49: 531–539.

108 Liu S-L, Zhong S-S, Ye D-X, Chen W-T, Zhang Z-Y, Deng J. Repression of G protein-coupled receptor family C group 5 member A is associated with pathologic differentiation grade of oral squamous cell carcinoma. J Oral Pathol Med 2013; 42: 761–768.

109 Liu H, Zhang Y, Hao X, Kong F, Li X, Yu J et al. GPRC5A overexpression predicted advanced biological behaviors and poor prognosis in patients with gastric cancer. Tumor Biol 2015; : 1–8.

110 Jahny E, Yang H, Liu B, Jahnke B, Lademann F, Knösel T et al. The G Protein-Coupled Receptor RAI3 Is an Independent Prognostic Factor for Pancreatic Cancer Survival and Regulates Proliferation via STAT3 Phosphorylation. PLoS ONE 2017; 12: e0170390.

111 Nagahata T, Sato T, Tomura A, Onda M, Nishikawa K, Emi M. Identification of RAI3 as a therapeutic target for breast cancer. Endocr Relat Cancer 2005; 12: 65–73.

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Supplementary table 1a. Subcategory 1 - Targets in pancreatic cancer clinical trialsRank Gene name Protein

locationProtein function Antineoplastic drug Therapy type study population Phase conclusion / status study Reference /

clinicaltrial.gov identifier

41 MUC1 cell-surface Glycoprotein MUC1 100mer peptide with SB-AS2 adjuvant cancer vaccine unresectable PDA I feasible Ramanathan, 2005 17 (NCT00008099)

MUC1 100mer peptide cancer vaccine unresectable PDA I 1/6 SD Yamamoto, 200518

MUC1-DC and MUC1-CTL adoptive immunotherapy unresectable PDA I 1/20 CR 5/20 SD

Kondo, 200864

MUC1-DC adoptive immunotherapy Advanced PDA I 7/7 PD Rong, 201265

90Y-hPAM4 radio-immunotherapy Advanced PDA I/II 6/38 PR 16/38 SD

Ocean, 2012 66(NCT00603863)

Falimarev (fowlpox-CEA-MUC-1-TRICOM vaccine) Inalimarev (vaccinia-CEA-MUC1-TRICOM vaccine)

cancer vaccine unresectable PDA I recruiting NCT00669734

anti-MUC1 CAR T Cells immunotherapy advanced, refractory solid tumors

I/II recruiting NCT02587689

anti-MUC1 CAR-pNK cells immunotherapy Relapsed or Refractory Solid Tumor

I/II rectruiting NCT02839954

53 NQO1 intracellular Reductase APAZIQUONE, bioreductive prodrug activated by NQO1

Pancreatic cancer first line

II Antitumour activity was not observed.

Dirix, 199667

54 PSEN2 cell-surface Protease MK-0752 NOTCH inhibitor unresectable PDA I completed no results yet NCT01098344

57 TNFSF11 cell-surface Cytokine Lenalidomide immunotherapy metastatic PDA II PR: 8/72 SD: 26/72 PD: 22/72 MOS 4.7 months

infante, 201368

65 ITGB5 cell-surface Integrin Cilengitide anti-angiogenic therapy unresectable PDA II C+G MOS: 6.7 months gemcitabine MOS: 7.7 months

Friess, 200669

110 MSLN cell-surface Cell adhesion protein BAY94-9343 antibody drug conjugate advanced, refractory solid tumors

I recruiting NCT02485119

BMS-986148 antibody drug conjugate mesothelin positive pancreatic cancer


CART-meso immunotoxin metastatic mesothelin expressing cancers


Mesothelin expressing cancers


metastatic PDA I recruiting NCT02465983

I safe and feasible beatty, 2014 20

MetastaticPDA


PDA I recruiting NCT02706782

SS1P(dsFv)-PE38 immunotoxin unresectable or metastatic PDA



I SS1p is well tolerated Hassan, 200770

mesothelin experessing cancers

I SS1p is well tolerated kreitman, 200971

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Supplementary table 1a. Subcategory 1 - Targets in pancreatic cancer clinical trialsRank Gene name Protein

locationProtein function Antineoplastic drug Therapy type study population Phase conclusion / status study Reference /

clinicaltrial.gov identifier

41 MUC1 cell-surface Glycoprotein MUC1 100mer peptide with SB-AS2 adjuvant cancer vaccine unresectable PDA I feasible Ramanathan, 2005 17 (NCT00008099)

MUC1 100mer peptide cancer vaccine unresectable PDA I 1/6 SD Yamamoto, 200518

MUC1-DC and MUC1-CTL adoptive immunotherapy unresectable PDA I 1/20 CR 5/20 SD

Kondo, 200864

MUC1-DC adoptive immunotherapy Advanced PDA I 7/7 PD Rong, 201265

90Y-hPAM4 radio-immunotherapy Advanced PDA I/II 6/38 PR 16/38 SD

Ocean, 2012 66(NCT00603863)

Falimarev (fowlpox-CEA-MUC-1-TRICOM vaccine) Inalimarev (vaccinia-CEA-MUC1-TRICOM vaccine)

cancer vaccine unresectable PDA I recruiting NCT00669734

anti-MUC1 CAR T Cells immunotherapy advanced, refractory solid tumors


anti-MUC1 CAR-pNK cells immunotherapy Relapsed or Refractory Solid Tumor

I/II rectruiting NCT02839954

53 NQO1 intracellular Reductase APAZIQUONE, bioreductive prodrug activated by NQO1

Pancreatic cancer first line

II Antitumour activity was not observed.

Dirix, 199667

54 PSEN2 cell-surface Protease MK-0752 NOTCH inhibitor unresectable PDA I completed no results yet NCT01098344

57 TNFSF11 cell-surface Cytokine Lenalidomide immunotherapy metastatic PDA II PR: 8/72 SD: 26/72 PD: 22/72 MOS 4.7 months

infante, 201368

65 ITGB5 cell-surface Integrin Cilengitide anti-angiogenic therapy unresectable PDA II C+G MOS: 6.7 months gemcitabine MOS: 7.7 months

Friess, 200669

110 MSLN cell-surface Cell adhesion protein BAY94-9343 antibody drug conjugate advanced, refractory solid tumors


BMS-986148 antibody drug conjugate mesothelin positive pancreatic cancer


CART-meso immunotoxin metastatic mesothelin expressing cancers




metastatic PDA I recruiting NCT02465983

I safe and feasible beatty, 2014 20

MetastaticPDA


PDA I recruiting NCT02706782

SS1P(dsFv)-PE38 immunotoxin unresectable or metastatic PDA



I SS1p is well tolerated Hassan, 200770

mesothelin experessing cancers

I SS1p is well tolerated kreitman, 200971

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Morab-009 (amatuximab) antibody mesothelin expressing cancers

I safe and feasible Hassan, 201072

unresectable PDA II completed, no article published yet

NCT00570713

GVAX (GM-CSF) immunotherapy Advanced PDA I safe and feasible laheru, 200873

PDA, adjuvant; II PD: 17/60 MOS: 24.8 months

Lutz, 201174

ANZ-100 and CRS-207 cancer vaccine metastatic PDA I Safe and feasible OS: 3/7 > 15months

Le dung,201275

GVAX and CRS-207 cancer vaccine metastatic PDA II cy/GVAX and CRS-207: OS 9.7 months cy/GVAX: OS 4.6 months

Le, dung 201519

LMB-100 + Nab-Paclitaxel Immunotoxin combined with chemotherapy

Pancreatic Neoplasms I/II recruiting NCT02810418

Anetumab ravtansine Antibody drug conjugate Pretreated Advanced Pancreatic Cancer

II not yet recruiting NCT03023722

134 SLC2A1 cell-surface Transporter Glufosfamide vs F-5U chemotherapy metastatic PDA III recruiting NCT01954992

Glufosfamide Advanced PDA II PR: 2/34 SD: 11/35 MOS: 5.3 months

Briasoulis, 200376

Glufosfamide + gemcitabine metastatic PDA II PR: 5/28 SD: 11/28 MOS: 6 months

Chiorean, 201077

Glufosfamide vs best supportive care metastatic PDA III MOS glufosfamide: 105 daysMOS best supportive care: 84 days

Ciuleanu, 200978

148 PLK3 intracellular Kinase BI 2536 Polo-like kinase inhibitor, unresectable advanced PDA

II PR: 2/79 SD: 19/79 MOS: 149 days

Mross, 201279

184 TPSAB1 intracellular Protease nafamostat + gemcitabine protease inhibitor advanced or metastatic PDA

I PR: 3/12 SD: 7/12 PD: 2/7

Uwagawa, 200980

unresectable advanced or metastatic PDA

II PR: 6/35 SD: 25/34 PD: 4/35 MOS: 10 months

Uwagawa, 201381

186 MMP11 extracelluar Protease marimastat vs gemcitabine MMP inhibitor unresectable advanced or metastatic PDA

III MOS gemcitabine: 167 days MOS 25mg: 125 days MOS 10mg: 105 days MOS 5 mg: 110 days

bramhall, 200282

199 MMP28 extracellular Protease marimastat MMP inhibitor Advanced PDA II SD: 41/83 in 28 day study period PD: 42/83 in 28 day study period MOS: 113 days

Bramhall, 200183

SD: Stable disease, CR: complete response, PD: progressive disease, PR: partial response, MOS: median overall survival, MPFS: mean progression free survivall

Supplementary table 1a. Continued

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Morab-009 (amatuximab) antibody mesothelin expressing cancers

I safe and feasible Hassan, 201072

unresectable PDA II completed, no article published yet

NCT00570713

GVAX (GM-CSF) immunotherapy Advanced PDA I safe and feasible laheru, 200873

PDA, adjuvant; II PD: 17/60 MOS: 24.8 months

Lutz, 201174

ANZ-100 and CRS-207 cancer vaccine metastatic PDA I Safe and feasible OS: 3/7 > 15months

Le dung,201275

GVAX and CRS-207 cancer vaccine metastatic PDA II cy/GVAX and CRS-207: OS 9.7 months cy/GVAX: OS 4.6 months

Le, dung 201519

LMB-100 + Nab-Paclitaxel Immunotoxin combined with chemotherapy

Pancreatic Neoplasms I/II recruiting NCT02810418

Anetumab ravtansine Antibody drug conjugate Pretreated Advanced Pancreatic Cancer


134 SLC2A1 cell-surface Transporter Glufosfamide vs F-5U chemotherapy metastatic PDA III recruiting NCT01954992

Glufosfamide Advanced PDA II PR: 2/34 SD: 11/35 MOS: 5.3 months

Briasoulis, 200376

Glufosfamide + gemcitabine metastatic PDA II PR: 5/28 SD: 11/28 MOS: 6 months

Chiorean, 201077

Glufosfamide vs best supportive care metastatic PDA III MOS glufosfamide: 105 daysMOS best supportive care: 84 days

Ciuleanu, 200978

148 PLK3 intracellular Kinase BI 2536 Polo-like kinase inhibitor, unresectable advanced PDA

II PR: 2/79 SD: 19/79 MOS: 149 days

Mross, 201279

184 TPSAB1 intracellular Protease nafamostat + gemcitabine protease inhibitor advanced or metastatic PDA

I PR: 3/12 SD: 7/12 PD: 2/7

Uwagawa, 200980

unresectable advanced or metastatic PDA

II PR: 6/35 SD: 25/34 PD: 4/35 MOS: 10 months

Uwagawa, 201381

186 MMP11 extracelluar Protease marimastat vs gemcitabine MMP inhibitor unresectable advanced or metastatic PDA

III MOS gemcitabine: 167 days MOS 25mg: 125 days MOS 10mg: 105 days MOS 5 mg: 110 days

bramhall, 200282

199 MMP28 extracellular Protease marimastat MMP inhibitor Advanced PDA II SD: 41/83 in 28 day study period PD: 42/83 in 28 day study period MOS: 113 days

Bramhall, 200183


Supplementary table 1a. Continued

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Supplementary table 1b. Subcategory 2 - Targets in clinical trials in other cancer types

Rank Gene name Protein location

Protein function Antineoplastic drug Therapy type study population Phase conclusion / status study Reference / clinicaltrial.gov identifier

95 MST1R cell-surface Tyrosine kinase Foretinib small-molecule multikinase inhibitor

advanced or metastatic gastric adenocarcinoma

II PR: 0/69 SD: 15/65 lack of efficacy

shah, 201384

papillary renal cell carcinoma

II ORR: 13.5% MPFS: 9.3 month

Choueiri, 201385

MGCD265 Tyrosine kinase inhibitor Advanced metastatic or unresectable malignancy


advanced or metastatic non-small cell lung cancer

II recruiting NCT02544633

106 PTMA intracellular Prothymosin Thymalfasin / Thymosin 1 / ( T-alfa-1) Immunomodulatory polypeptide metastatic esophageal cancer


metastatic small cell lung cancer


metastatic non small cell lung cancer


metastatic colon cancer II not yet recruiting NCT02535988

hepatocellular carcinoma

IV not yet recruiting NCT02281266

metastatic melanoma patients

I MOS: 9.4 months vs. 6.6 months

maio 201021

213 PRLR cell-surface Receptor prolanta prolactine receptor antagonist Epithelial ovarian cancer I recruiting NCT02534922

LFA102 monoclonal antibody breast and prostate cancer

I completed, no results published NCT01338831


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Supplementary table 1b. Subcategory 2 - Targets in clinical trials in other cancer types

Rank Gene name Protein location

Protein function Antineoplastic drug Therapy type study population Phase conclusion / status study Reference / clinicaltrial.gov identifier

95 MST1R cell-surface Tyrosine kinase Foretinib small-molecule multikinase inhibitor

advanced or metastatic gastric adenocarcinoma

II PR: 0/69 SD: 15/65 lack of efficacy

shah, 201384

papillary renal cell carcinoma

II ORR: 13.5% MPFS: 9.3 month

Choueiri, 201385

MGCD265 Tyrosine kinase inhibitor Advanced metastatic or unresectable malignancy


advanced or metastatic non-small cell lung cancer

II recruiting NCT02544633

106 PTMA intracellular Prothymosin Thymalfasin / Thymosin 1 / ( T-alfa-1) Immunomodulatory polypeptide metastatic esophageal cancer


metastatic small cell lung cancer


metastatic non small cell lung cancer


metastatic colon cancer II not yet recruiting NCT02535988

hepatocellular carcinoma

IV not yet recruiting NCT02281266

metastatic melanoma patients

I MOS: 9.4 months vs. 6.6 months

maio 201021

213 PRLR cell-surface Receptor prolanta prolactine receptor antagonist Epithelial ovarian cancer I recruiting NCT02534922

LFA102 monoclonal antibody breast and prostate cancer

I completed, no results published NCT01338831


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Supplementary table 1c. Subcategory 3 - Drug available - Targets in preclinical in vitro and in vivo studiesRank Gene name Protein location Protein function Antineoplastic drug drug type Study type conclusion / status study Reference

8 CTSE intracellular Protease Cathepsin E-activatable 5-ALA prodrug photo dynamic therapy

in vivo - mouse PDA cells

Effectively targeting and killing cancer cells that express CTSE Abd-Elgaliel, 2013 22

10 GGT5 cell-surface Protease GSAO (glutathione-S-conjugate activated by γGT cleavage)

prodrug in vivo- PDA mouse model

Tumor γGT activity positively correlated with GSAO-mediated inhibition of pancreatic tumor angiogenesis and tumor growth in mice.

Ramsay, 201423

18 GJB3 cell-surface Gap junction protein Carbenoxolone gap junction blocker in vitro - Pancreatic stellate cells

Carbenoxolone inhibited platelet-derived growth factor-BB-induced proliferation and migration

Masamune 201386

73 TNK2 intracellular Tyrosine kinase AIM-100pyrazolopyrimidine derivative 2bALK inhibitor 5

TNK2 inhibitors in vitro - prostate cancer cells

AIM-100 treatment is leading to cell cycle arrest in the G1 phase causing significant decrease in the proliferation of pancreatic cancer cells and induction of apoptosis.

Mahajan, 201287

(R)-9bMS small-molecule inhibitor

triple negative breast cancer (TNBC)

In vitro inhibition significantly compromised TNBC proliferation

Xinyan Wu, 201788

92 NPY1R cell-surface GPCR BIBP3226 peptide-drug conjugate

in vitro - neuroblastoma cells

The active compund BIBP3226 is able to release the drug intracellular

Langer, 200189

107 TRIO intracellular Kinase TRIP-E32G peptide aptamer In vivo - NIH 3T3 cells TRIPE32G reduces the formation of TRIO-induced tumors. bouquier 200990

118 GPER cell-surface GPCR Gefitinib Tyrosine Kinase inhibitor

In vitro – Triple-negative breast cancers cells

Reduction of GPER expression is a promising therapeutic approach for TNBC

Girgert, 201791

agonist G-1 GPER-receptor-agonist

In vitro – nonsmall cell lung cancer cells

G-1 treatment rapidly decreased the phosphorylation, nuclear translocation, and promoter activities of NF-κB, which will help to better understand the roles and mechanisms of GPER as a potential therapy target

Zhu, 201692

141 ADAM8 cell-surface Protease BK-1361, ADAM8 inhibitor in vitro - PDA cells BK-1361 decreased tumour burden and metastasis of implanted pancreatic tumour cells in mice

Schlomann, 201524

142 CDC42BPA intracellular Kinase DJ4 small molecule inhibitor

in vitro - (PDA) cells DJ4 treatment significantly blocked stress fiber formation and inhibited migration and invasion of multiple cancer cell lines

pralhad, 201493

161 PRKCi intracellular Kinase aPKC-PSP pseudosubstrate peptide

In vivo -glioblastomaStem-like cells(GSC)

Targeting PKCι in the context of Notch signaling could be an effective way of attacking the GSC population in GBM

Phillips, 201694

180 SULF1 extracellular/cell surface

Sulfatase IQ2-S radioactive prodrug

in vitro - PDA cells Quinazolinone-based radiopharmaceuticals can lead to the development of a novel noninvasive approach for imaging and treating pancreatic cancer.

Pospisil, 201295

188 S100P intracellular calcium-binding protein cromolyn cromolyn analog, C5OH

in vivo - PDA mouse C5OH blocked the S100P-mediated growth and antiapoptotic effect in PDA and improved the animal survival.

Arumugam, 201396

2H8 S100P antibody in vivo - mouse - PxPC3 cells

2H8 antibody decreased tumor growth and liver metastasis formation in a subcutaneous and orthotopic BxPC3 tumor model.

Dakhel, 201497

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Supplementary table 1c. Subcategory 3 - Drug available - Targets in preclinical in vitro and in vivo studiesRank Gene name Protein location Protein function Antineoplastic drug drug type Study type conclusion / status study Reference

8 CTSE intracellular Protease Cathepsin E-activatable 5-ALA prodrug photo dynamic therapy

in vivo - mouse PDA cells

Effectively targeting and killing cancer cells that express CTSE Abd-Elgaliel, 2013 22

10 GGT5 cell-surface Protease GSAO (glutathione-S-conjugate activated by γGT cleavage)

prodrug in vivo- PDA mouse model

Tumor γGT activity positively correlated with GSAO-mediated inhibition of pancreatic tumor angiogenesis and tumor growth in mice.

Ramsay, 201423

18 GJB3 cell-surface Gap junction protein Carbenoxolone gap junction blocker in vitro - Pancreatic stellate cells

Carbenoxolone inhibited platelet-derived growth factor-BB-induced proliferation and migration

Masamune 201386

73 TNK2 intracellular Tyrosine kinase AIM-100pyrazolopyrimidine derivative 2bALK inhibitor 5

TNK2 inhibitors in vitro - prostate cancer cells

AIM-100 treatment is leading to cell cycle arrest in the G1 phase causing significant decrease in the proliferation of pancreatic cancer cells and induction of apoptosis.

Mahajan, 201287

(R)-9bMS small-molecule inhibitor

triple negative breast cancer (TNBC)

In vitro inhibition significantly compromised TNBC proliferation

Xinyan Wu, 201788

92 NPY1R cell-surface GPCR BIBP3226 peptide-drug conjugate

in vitro - neuroblastoma cells

The active compund BIBP3226 is able to release the drug intracellular

Langer, 200189

107 TRIO intracellular Kinase TRIP-E32G peptide aptamer In vivo - NIH 3T3 cells TRIPE32G reduces the formation of TRIO-induced tumors. bouquier 200990

118 GPER cell-surface GPCR Gefitinib Tyrosine Kinase inhibitor

In vitro – Triple-negative breast cancers cells

Reduction of GPER expression is a promising therapeutic approach for TNBC

Girgert, 201791

agonist G-1 GPER-receptor-agonist

In vitro – nonsmall cell lung cancer cells

G-1 treatment rapidly decreased the phosphorylation, nuclear translocation, and promoter activities of NF-κB, which will help to better understand the roles and mechanisms of GPER as a potential therapy target

Zhu, 201692

141 ADAM8 cell-surface Protease BK-1361, ADAM8 inhibitor in vitro - PDA cells BK-1361 decreased tumour burden and metastasis of implanted pancreatic tumour cells in mice

Schlomann, 201524

142 CDC42BPA intracellular Kinase DJ4 small molecule inhibitor

in vitro - (PDA) cells DJ4 treatment significantly blocked stress fiber formation and inhibited migration and invasion of multiple cancer cell lines

pralhad, 201493

161 PRKCi intracellular Kinase aPKC-PSP pseudosubstrate peptide

In vivo -glioblastomaStem-like cells(GSC)

Targeting PKCι in the context of Notch signaling could be an effective way of attacking the GSC population in GBM

Phillips, 201694

180 SULF1 extracellular/cell surface

Sulfatase IQ2-S radioactive prodrug

in vitro - PDA cells Quinazolinone-based radiopharmaceuticals can lead to the development of a novel noninvasive approach for imaging and treating pancreatic cancer.

Pospisil, 201295

188 S100P intracellular calcium-binding protein cromolyn cromolyn analog, C5OH

in vivo - PDA mouse C5OH blocked the S100P-mediated growth and antiapoptotic effect in PDA and improved the animal survival.

Arumugam, 201396

2H8 S100P antibody in vivo - mouse - PxPC3 cells

2H8 antibody decreased tumor growth and liver metastasis formation in a subcutaneous and orthotopic BxPC3 tumor model.

Dakhel, 201497

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Supplementary table 1d. Subcategory 4 - suggested as potential targetsRank Gene name Protein location Protein function Cancer type Conclusion Reference

9 TMPRSS4 cell-surface Protease breast cancer tissue IHC Prognostic marker Liang, 2013 98

Non-small cell lung cancer (NSCLC) In vitro treatment with demethylating agent significantly increased TMPRSS4 levels

Potential therapeutic target Villalba, 201699

Gastric cancer Upregulation of TMPRSS4 enhances the invasiveness of gastric cancer cells

Potential therapeutic target Jin, 2016100

16 FXYD3 Cell membrane Protein Breast cancer Suppression of FXYD3 by transfection with siRNA

Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.

Liu, 2016101

26 CPB1 intracellular Protease Metastasis in Low Grade Breast Cancer samples IHC Biomarker bouchal 2015102

29 PLA2G16 Intracellular Protein Osteosarcoma In vitro and in vivo functional analyses Potential therapeutic target Li, 2016103

30 MAP4K4 Intracellular Protein kinase Gastric cancer In vitro siilencing of MAP4K4 by shRNA Potential therapeutic strategy Liu, 2016104

42 CBS intracellular Lysase in vitro - mouse CBS silencing CBS silencing resulted in reduced tumor cells proliferation, blood vessels formation and lipid content.

chakraborty, 2015105

Colon cancer In vitro, in vivoxenograft

Benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo

Druzhyna, 2016106

70 GPRC5A cell-surface GPCR colon cancer samples IHC Prognostic biomarker zougman 2013107

oral squamus cell carcinoma IHC Prognostic biomarker Liu, 2013108

gastric cancer samples mRNA expression levels Prognostic biomarker Liu, 2015109

PDAC cells siRNA Suppression of GPRC5a results in decreased cell growth, proliferation and migration

Jahny, 2017110

breast cancer cell line siRNA Transfection of siRNA suppressed RAI3 mRNA and growth of the cancer cells.

Nagahata 2005111

79 KLK10 extracellular Peptidase Breast cancer RNA-Sequencing analysis Predictive biomarker for trastuzumab resistance and potential therapeutic target for reversing trastuzumab resistance

Wang, 2016112

93 COPS5 intracellular Peptidase Breast cancer Integrated genomic and functional studies COPS5 overexpression causes tamoxifen-resistance in preclinical breast cancer models in vitro and in vivo >potential therapeutic approach for endocrine-resistant breast cancer

Lu, 2016113

97 GTSE1 intracellular unknown Gastric cancer cells shRNA GTSE1 knockout Biomarker. Potential therapeutical target. subhash, 2014114

hepatocellular carcinoma cells shRNA GTSE1 silencing GTSE1 is aberrantly overexpressed in HCC cell lines and cancerous tissues >Potential therapeutic target

Guo, 2016115

104 KMT2B nucleus Lysine Breast cancer cells siRNA knockdown Inhibition of IL-20 and KMT2B may have therapeutic benefits in ERα-positive breast cancer

Su, 2016116

160 SPN cell-surface sialoglycoprotein HPB-ALL lymphoblastoid T cells in mice UN1 monoclonal antibody UN1 mAb is leading to natural killer–mediated cytotoxicity causing growth inhibition

Tuccillo,2014117

mouse model - breast cancer siRNA SPN knockdown Reduction in primary tumour growth in vivo Fu, 2014118

166 RAMP1 cell-surface receptor activity modifying protein

prostate cancer Potential molecular target logan, 2013119

167 HNF1A nucleus Transcription factor PDA tissue and cells siRNA HNF1A knockdown siRNA HNF1A knockdown reduced apoptosis in pancreatic cancer cell lines. HNF1A is a possible tumor suppressor

Luo, 2015 120

181 MYBL2 nucleus Transcription factor In vivo - mouse Breast cancer xenografts Si-RNA B-myb plays a role in cell cycle progression and tumorigenesis.Potential diagnostic / therapeutical target

deyou tao121

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Supplementary table 1d. Subcategory 4 - suggested as potential targetsRank Gene name Protein location Protein function Cancer type Conclusion Reference

9 TMPRSS4 cell-surface Protease breast cancer tissue IHC Prognostic marker Liang, 2013 98

Non-small cell lung cancer (NSCLC) In vitro treatment with demethylating agent significantly increased TMPRSS4 levels

Potential therapeutic target Villalba, 201699

Gastric cancer Upregulation of TMPRSS4 enhances the invasiveness of gastric cancer cells

Potential therapeutic target Jin, 2016100

16 FXYD3 Cell membrane Protein Breast cancer Suppression of FXYD3 by transfection with siRNA

Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.

Liu, 2016101

26 CPB1 intracellular Protease Metastasis in Low Grade Breast Cancer samples IHC Biomarker bouchal 2015102

29 PLA2G16 Intracellular Protein Osteosarcoma In vitro and in vivo functional analyses Potential therapeutic target Li, 2016103

30 MAP4K4 Intracellular Protein kinase Gastric cancer In vitro siilencing of MAP4K4 by shRNA Potential therapeutic strategy Liu, 2016104

42 CBS intracellular Lysase in vitro - mouse CBS silencing CBS silencing resulted in reduced tumor cells proliferation, blood vessels formation and lipid content.

chakraborty, 2015105

Colon cancer In vitro, in vivoxenograft

Benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo

Druzhyna, 2016106

70 GPRC5A cell-surface GPCR colon cancer samples IHC Prognostic biomarker zougman 2013107

oral squamus cell carcinoma IHC Prognostic biomarker Liu, 2013108

gastric cancer samples mRNA expression levels Prognostic biomarker Liu, 2015109

PDAC cells siRNA Suppression of GPRC5a results in decreased cell growth, proliferation and migration

Jahny, 2017110

breast cancer cell line siRNA Transfection of siRNA suppressed RAI3 mRNA and growth of the cancer cells.

Nagahata 2005111

79 KLK10 extracellular Peptidase Breast cancer RNA-Sequencing analysis Predictive biomarker for trastuzumab resistance and potential therapeutic target for reversing trastuzumab resistance

Wang, 2016112

93 COPS5 intracellular Peptidase Breast cancer Integrated genomic and functional studies COPS5 overexpression causes tamoxifen-resistance in preclinical breast cancer models in vitro and in vivo >potential therapeutic approach for endocrine-resistant breast cancer

Lu, 2016113

97 GTSE1 intracellular unknown Gastric cancer cells shRNA GTSE1 knockout Biomarker. Potential therapeutical target. subhash, 2014114

hepatocellular carcinoma cells shRNA GTSE1 silencing GTSE1 is aberrantly overexpressed in HCC cell lines and cancerous tissues >Potential therapeutic target

Guo, 2016115

104 KMT2B nucleus Lysine Breast cancer cells siRNA knockdown Inhibition of IL-20 and KMT2B may have therapeutic benefits in ERα-positive breast cancer

Su, 2016116

160 SPN cell-surface sialoglycoprotein HPB-ALL lymphoblastoid T cells in mice UN1 monoclonal antibody UN1 mAb is leading to natural killer–mediated cytotoxicity causing growth inhibition

Tuccillo,2014117

mouse model - breast cancer siRNA SPN knockdown Reduction in primary tumour growth in vivo Fu, 2014118

166 RAMP1 cell-surface receptor activity modifying protein

prostate cancer Potential molecular target logan, 2013119

167 HNF1A nucleus Transcription factor PDA tissue and cells siRNA HNF1A knockdown siRNA HNF1A knockdown reduced apoptosis in pancreatic cancer cell lines. HNF1A is a possible tumor suppressor

Luo, 2015 120

181 MYBL2 nucleus Transcription factor In vivo - mouse Breast cancer xenografts Si-RNA B-myb plays a role in cell cycle progression and tumorigenesis.Potential diagnostic / therapeutical target

deyou tao121

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Supplementary table 2. Targets for PDA imagingRank Gene

nameProtein location

Protein function

Tracer name Study type Cancer type Conclusion Reference

1 THY1 cell-surface Glycolipid Thy1-Targeted Microbubbles (MBThy1) in vivo - mouseultrasound molecular imaging

pancreatic cancer xenofgrafts Thy1 targeted ultrasound molecular imaging is feasible Foygel, 201333

8 CTSE intracellular Protease CTSE-activatable optical molecular probe in vivo - mouseoptical imaging

pancreatic cancer xenofgrafts CTSE-activatable probe can be detected by confocal laser endomicroscopy (CLE)

hui Li, 2014122

ritonavir tetramethyl-BODIPY (RIT-TMB ) in vivo - mouseoptical imaging

pancreatic cancer orthotopic tumors RIT-TMB imaging is feasible in vitro and demonstrated good co- localization with CTSE in both humand and mouse PDA samples

keliher, 201334

CTSE-activatable optical molecular probe in vivo - mouseoptical imaging

pancreatic cancer xenofgrafts The Cath E-activatable probe was able to highlight the Cath E-positive tumors; control imaging probe confirmed the superior selectivity and sensitivity

abd-elgaliel, 201135

10 GGT5 Cell-surface Protease gGlu-HMRG ex vivooptical imaging EUS-FNA

Human pancreatic samples gGlu-HMRG did not clearly differentiate pancreatic tumor tissues from normal pancreatic ones because GGT activity was not different between tumor cells and normal cells.

gGlu-HMRG ex vivo breast cancer samples Breast cancer fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors from normal mammary gland tissues, with 92% sensitivity and 94% specicity

ueo, 201531

BODIPY-GSH In vitro Ovarian cancer cells FIST probes enable monitoring the GGT activity in living cells,which showed differentiation between ovarian cancer cells and normal cells.

Feiyi Wang, 2015 123

gGlu-HMRG Ex vivo colon carcinoma samples Topically spraying gGlu-HMRG enabled rapid and selective fluorescent imaging of colorectal tumors owing to the upregulated GGT activity in cancer cells.

sato, 2015124

gGlu-HMRG In vivo - mouse Colon cancer mouse model fluorescence endoscopic detection of colon cancer was feasible. All fluorescent lesions contained cancer or high-grade dysplasia, all non-fluorescent lesions contained low-grade dysplasia or benign tissue.

Mitsunaga, 2013 32

gGlu-HMRG In vivo - mouse disseminated peritoneal ovarian cancer model

Activation of gGlu-HMRG occurred within 1 min of topically spraying the tumor, creating high signal contrast between the tumor and the background.

urano, 201130

41 MUC1 cell-surface Glycoprotein aptamer-PEG-near- infrared fluorescence probe (APT-PEG-MPA)

in vivo - mouseoptical imaging

breast cancer, non-small cell lung carcinoma, hepatocellular carcinoma xenografts

MUC1 aptamer-based NIR fluorescence probe has a high tumor-targetinga ability and low accumulation in normal tissue

chen, 201527

MN-EPPT (iron oxide nanoparticles (MN), labeled with Cy5.5 dye conjugated to peptides (EPPT)

in vivo - mouseoptical imaging/MRI

breast cancer transgenic mouse model changes in uMUC-1 expression during tumor development and therapeutic intervention could be monitored non-invasively using molecular imaging approach with the uMUC-1-specific contrast agent (MN-EPPT) detectable by magnetic resonance and fluorescence optical imaging

Ghosh, 2013125

(111)In-labeled PAM4 phase I clinical trialPET-scan

pancreatic cancer radiolabeled PAM4 selectively targets pancreatic cancer in both the experimental animal model and clinical studies.

gold, 200125

[64Cu]-DOTA-PR81 in vivo - mousePET-scan

breast cancer xenografts The biodistribution and scintigraphy studies showed the accumulation of 64Cu-DOTA-PR81 at the site of tumors with high sensitivity and specificity for MUC1 compared to control probes.

Alirezapour, 2015 126

Ab-FL-Cy5.5 in vivo - mousedual labelled optical imaging

ovarian cancer xenografts Ab-FL-Cy5.5 probe can be used for in vivo imaging of MUC1 expressing tumors

Zhang, 2015 26

92 NPY1R cell-surface GPCR [Lys(M/DOTA)4]BVD15 in vitro Breast cancer cells [Lys(DOTA)4]BVD15 is a potent and specific ligand for NPY1R Guerin . 2010 127

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Supplementary table 2. Targets for PDA imagingRank Gene

nameProtein location

Protein function

Tracer name Study type Cancer type Conclusion Reference

1 THY1 cell-surface Glycolipid Thy1-Targeted Microbubbles (MBThy1) in vivo - mouseultrasound molecular imaging

pancreatic cancer xenofgrafts Thy1 targeted ultrasound molecular imaging is feasible Foygel, 201333

8 CTSE intracellular Protease CTSE-activatable optical molecular probe in vivo - mouseoptical imaging

pancreatic cancer xenofgrafts CTSE-activatable probe can be detected by confocal laser endomicroscopy (CLE)

hui Li, 2014122

ritonavir tetramethyl-BODIPY (RIT-TMB ) in vivo - mouseoptical imaging

pancreatic cancer orthotopic tumors RIT-TMB imaging is feasible in vitro and demonstrated good co- localization with CTSE in both humand and mouse PDA samples

keliher, 201334

CTSE-activatable optical molecular probe in vivo - mouseoptical imaging

pancreatic cancer xenofgrafts The Cath E-activatable probe was able to highlight the Cath E-positive tumors; control imaging probe confirmed the superior selectivity and sensitivity

abd-elgaliel, 201135

10 GGT5 Cell-surface Protease gGlu-HMRG ex vivooptical imaging EUS-FNA

Human pancreatic samples gGlu-HMRG did not clearly differentiate pancreatic tumor tissues from normal pancreatic ones because GGT activity was not different between tumor cells and normal cells.

gGlu-HMRG ex vivo breast cancer samples Breast cancer fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors from normal mammary gland tissues, with 92% sensitivity and 94% specicity

ueo, 201531

BODIPY-GSH In vitro Ovarian cancer cells FIST probes enable monitoring the GGT activity in living cells,which showed differentiation between ovarian cancer cells and normal cells.

Feiyi Wang, 2015 123

gGlu-HMRG Ex vivo colon carcinoma samples Topically spraying gGlu-HMRG enabled rapid and selective fluorescent imaging of colorectal tumors owing to the upregulated GGT activity in cancer cells.

sato, 2015124

gGlu-HMRG In vivo - mouse Colon cancer mouse model fluorescence endoscopic detection of colon cancer was feasible. All fluorescent lesions contained cancer or high-grade dysplasia, all non-fluorescent lesions contained low-grade dysplasia or benign tissue.

Mitsunaga, 2013 32

gGlu-HMRG In vivo - mouse disseminated peritoneal ovarian cancer model

Activation of gGlu-HMRG occurred within 1 min of topically spraying the tumor, creating high signal contrast between the tumor and the background.

urano, 201130

41 MUC1 cell-surface Glycoprotein aptamer-PEG-near- infrared fluorescence probe (APT-PEG-MPA)

in vivo - mouseoptical imaging

breast cancer, non-small cell lung carcinoma, hepatocellular carcinoma xenografts

MUC1 aptamer-based NIR fluorescence probe has a high tumor-targetinga ability and low accumulation in normal tissue

chen, 201527

MN-EPPT (iron oxide nanoparticles (MN), labeled with Cy5.5 dye conjugated to peptides (EPPT)

in vivo - mouseoptical imaging/MRI

breast cancer transgenic mouse model changes in uMUC-1 expression during tumor development and therapeutic intervention could be monitored non-invasively using molecular imaging approach with the uMUC-1-specific contrast agent (MN-EPPT) detectable by magnetic resonance and fluorescence optical imaging

Ghosh, 2013125

(111)In-labeled PAM4 phase I clinical trialPET-scan

pancreatic cancer radiolabeled PAM4 selectively targets pancreatic cancer in both the experimental animal model and clinical studies.

gold, 200125

[64Cu]-DOTA-PR81 in vivo - mousePET-scan

breast cancer xenografts The biodistribution and scintigraphy studies showed the accumulation of 64Cu-DOTA-PR81 at the site of tumors with high sensitivity and specificity for MUC1 compared to control probes.

Alirezapour, 2015 126

Ab-FL-Cy5.5 in vivo - mousedual labelled optical imaging

ovarian cancer xenografts Ab-FL-Cy5.5 probe can be used for in vivo imaging of MUC1 expressing tumors

Zhang, 2015 26

92 NPY1R cell-surface GPCR [Lys(M/DOTA)4]BVD15 in vitro Breast cancer cells [Lys(DOTA)4]BVD15 is a potent and specific ligand for NPY1R Guerin . 2010 127

R1

R2

R3

R4

R5

R6

R7

R8

R9

R10

R11

R12

R13

R14

R15

R16

R17

R18

R19

R20

R21

R22

R23

R24

R25

R26

R27

R28

R29

R30

R31

R32

R33

R34

R35

R36

R37

R38

R39

Part 2 Chapter 6

146

110 MSLN cell-surface Cell adhesion protein

89Zr-MMOT0530A phase I clinical trialPET-scan

pancreatic cancer and ovarian cancer 89Zr-MMOT0530A-PET pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized.

lamberts, 201528

64Cu-NOTA-amatuximab in vivo - mousePET-scan

epithelial carcinoma cells 64Cu-NOTA-amatuximab enables quantification of tumor and major organ uptake values using PET scanning

lee, 2015 128

Indium-CHX-A amatuximab phase I clinical trialSPECT-scan

mesothelin overexpressing tumors 111In-amatuximab localizes to mesothelin expressing cancers with a higher uptake in mesothelioma than pancreatic cancer.

NCT01521325

Me-F127COOH-QD nanomicelles in vivo - mouse pancreatic cancer xenofgrafts anti-mesothein antibody conjugated carboxylated F127 nanomicelles accumulated specifically at the pancreatic tumor site 15 min after intravenous injection with low toxicity

ding, 2011129

anti-mesothelin antibody-conjugated PEGlyated liposomal ultrasmall superparamagnetic iron oxides

in vivo - mouseMRI

pancreatic cancer xenofgrafts M-PLDUs specically targets MSLN and could well improve the therapeutic efficacy of DOX chemotherapy in vivo and could be visualized by MRI in vivo.

deng, 2012130

118 GPER cell-surface GPCR 99mTc(I)-labeled nonsteroidal GPER-specific ligands

in vivo - mouseSPECT-scan

human endometrial and breast cancer cell xenografts

99mTc-labeled-GPER-specific radioligands are tumor specific nd could be cleary visualized using SPECT-scan

nayak, 2014131

Supplementary table 2. Continued

R1

R2

R3

R4

R5

R6

R7

R8

R9

R10

R11

R12

R13

R14

R15

R16

R17

R18

R19

R20

R21

R22

R23

R24

R25

R26

R27

R28

R29

R30

R31

R32

R33

R34

R35

R36

R37

R38

R39


147

6

110 MSLN cell-surface Cell adhesion protein

89Zr-MMOT0530A phase I clinical trialPET-scan

pancreatic cancer and ovarian cancer 89Zr-MMOT0530A-PET pancreatic and ovarian cancer lesions as well as antibody biodistribution could be visualized.

lamberts, 201528

64Cu-NOTA-amatuximab in vivo - mousePET-scan

epithelial carcinoma cells 64Cu-NOTA-amatuximab enables quantification of tumor and major organ uptake values using PET scanning

lee, 2015 128

Indium-CHX-A amatuximab phase I clinical trialSPECT-scan

mesothelin overexpressing tumors 111In-amatuximab localizes to mesothelin expressing cancers with a higher uptake in mesothelioma than pancreatic cancer.

NCT01521325

Me-F127COOH-QD nanomicelles in vivo - mouse pancreatic cancer xenofgrafts anti-mesothein antibody conjugated carboxylated F127 nanomicelles accumulated specifically at the pancreatic tumor site 15 min after intravenous injection with low toxicity

ding, 2011129

anti-mesothelin antibody-conjugated PEGlyated liposomal ultrasmall superparamagnetic iron oxides

in vivo - mouseMRI

pancreatic cancer xenofgrafts M-PLDUs specically targets MSLN and could well improve the therapeutic efficacy of DOX chemotherapy in vivo and could be visualized by MRI in vivo.

deng, 2012130

118 GPER cell-surface GPCR 99mTc(I)-labeled nonsteroidal GPER-specific ligands

in vivo - mouseSPECT-scan

human endometrial and breast cancer cell xenografts

99mTc-labeled-GPER-specific radioligands are tumor specific nd could be cleary visualized using SPECT-scan

nayak, 2014131

Supplementary table 2. Continued

Pa r t 3

Pa r t 3FUTURe THeRANOSTIC

APPROACH

university of groningen fluorescence molecular endoscopy

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