Evaluation of somatostatin receptor subtype expression in human neuroendocrine tumors using two sets of new monoclonal antibodies
Introduction
The expression and reliable detection of somatostatin receptor subtypes (SSTRs) is a prerequisite for the successful use of somatostatin analogs in neuroendocrine tumors (NETs). Two sets of monoclonal antibodies (mAbs) against human SSTR1, 2A, 3 and 5 have recently been independently developed by different laboratories using rabbit and mouse hybridomas [1], [2], [3], [4]. Currently available SSTR antibodies are polyclonal antibodies raised in different species and thus have the inherent problem of limited availability of a given batch. To date, five SSTRs have been described in various human tissues, SSTR1–5 [5], and their activation results in the inhibition of secretion and/or a reduction of proliferation in endocrine and non-endocrine cells, respectively [6], [7], [8], [9], [10]. Through different techniques, the over-expression of SSTRs and/or their mRNA has been demonstrated in most endocrine and non-endocrine tumors [11], [12]. In studies of expression in NET, the majority of tumors expressed SSTR1, 2A, 3 and 5, while SSTR4 was present in a minority of tumors. Additionally, SSTR4 is not targeted by any of the clinically used somatostatin analogs and little is known about its functional relevance [11], [13], [14].
Although expression levels of SSTRs vary widely between different tumors, it is a common result in these studies that a given tumor usually expresses several SSTRs and that the strong expression of a sole receptor subtype is an exception [11], [12]. In addition, the use of different technologies and cut-off levels in the measurement of SSTR expression has made the comparison of data between studies difficult. These findings, together with results showing that activation of two or more SSTRs has additive inhibitory effects on hormone secretion [15], triggered the development of a new somatostatin analog (Signifor®, pasireotide, SOM230) which has high affinity for four of the five receptor subtypes (SSTR1, 2A, 3 and 5). Although reverse transcription polymerase chain reaction (rtPCR) techniques allow for the reliable detection and quantification of mRNA for specific SSTRs, only immunohistochemical (IHC) detection from formaldehyde-fixed tumor tissues allows identification and cellular localization of SSTRs and thus determines the potential usefulness of somatostatin analogs as treatment options. The use of mAbs is preferred to the use of polyclonal Abs, as they are more specific and allow the unlimited production of mAbs of constant quality.
Our aim was to compare and evaluate the usefulness of two sets of mAbs against SSTR1, 2A, 3 and 5 in NET in order to determine SSTR expression in samples as they are routinely collected in clinical practice. Newly generated mouse and rabbit mAbs specific for human SSTR1, 2A, 3 and 5 were initially characterized in SSTR-transfected HEK293 cell lines, archival human pancreatic tissues and tissues from NET known to express SSTRs. Following this characterization, a comparative analysis of rabbit versus mouse mAbs was performed by immunostaining of a tissue microarray (TMA) composed of 75 cores from NET. The results were analyzed with an automated system to allow an unbiased quantification and, in addition, the staining intensity was evaluated and agreed upon by two independent investigators. Ultimately, this report will provide a common statement about the strengths and weaknesses, and thus the usefulness, of both sets of newly generated mAbs for detection of SSTR expression in formaldehyde-fixed human tissue samples.
Section snippets
Cell lines and tissues
HEK293 cells transfected with human SSTR1, 2A, 3 or 5 cDNA were cultured in Dulbecco's Modified Eagle's Medium (4.5 g/mL glucose, containing 1% l-glutamate; Amimed, Bioconcept, Switzerland) with 10% fetal calf serum (Amimed), 1% penicillin/streptomycin (Amimed) and 250 μg/mL G418 (Gibco, Invitrogen, Switzerland). Formalin-fixed paraffin-embedded (FFPE) cell blocks were prepared as previously described [4]. Human tissue specimens included normal pancreatic tissue and NET blocks provided by Asterand
Antibody specificities in HEK293-transfected cell lines
The specificity of the mouse mAbs has been shown in a recent publication by the authors [4]. Each mouse mAb was found to selectively recognize the human SSTR against which it was raised and did not cross-react with any of the other receptor subtypes in HEK293 cell lines overexpressing SSTR1, 2A, 3 and 5 (Fig. 2A). Similarly, each rabbit mAb strongly immunostained their cognate receptor in HEK293 cell lines, although weak cross-reactivity was observed with the other receptor subtypes for
Discussion
The goal of the present studies was to compare two sets of newly generated mAbs against SSTRs in FFPE human tissues and provide recommendations on the optimal use of these reagents for the characterization of SSTR expression in NET.
Declaration of interest
CL, PB, LD'A and HS are employees of Novartis Pharma AG. PN is a former employee of Novartis Pharma AG. SS has no conflict of interests to declare.
Funding
This study was funded in part by Novartis Pharma AG. Financial support for medical editorial assistance was provided by Novartis Pharmaceuticals Corporation.
Author contributions
HS and SS provided the mouse and rabbit mAbs, respectively. HS initiated the collaboration with SS. PN designed the comparative study. SS and PN evaluated the stainings performed in parallel with both sets of mAbs. PBR and LD'A operationally executed the comparative study by preparing cell samples and performing staining of cell pellets, tissues and TMA with both sets of antibodies.
Acknowledgments
The authors would like to thank Prof. Luigi Terraciano for providing the NET-TMA. We thank Keri Wellington, Mudskipper Business Ltd, for medical editorial assistance with this manuscript.
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