The authors have declared that no competing interests exist.
Conceived and designed the experiments: KH JJ. Performed the experiments: KH MQ AS. Analyzed the data: KH JJ. Contributed reagents/materials/analysis tools: AS DH. Wrote the paper: KH JJ.
Pancreatic neuroendocrine tumors (PanNETs) are a relatively rare but clinically challenging tumor type. In particular, high grade, poorly-differentiated PanNETs have the worst patient prognosis, and the underlying mechanisms of disease are poorly understood. In this study we have identified and characterized a previously undescribed class of poorly differentiated PanNETs in the RIP1-Tag2 mouse model. We found that while the majority of tumors in the RIP1-Tag2 model are well-differentiated insulinomas, a subset of tumors had lost multiple markers of beta-cell differentiation and were highly invasive, leading us to term them poorly differentiated invasive carcinomas (PDICs). In addition, we found that these tumors exhibited a high mitotic index, resembling poorly differentiated (PD)-PanNETs in human patients. Interestingly, we identified expression of Id1, an inhibitor of DNA binding gene, and a regulator of differentiation, specifically in PDIC tumor cells by histological analysis. The identification of PDICs in this mouse model provides a unique opportunity to study the pathology and molecular characteristics of PD-PanNETs.
Pancreatic neuroendocrine tumors (PanNETs) are a rare but clinically challenging tumor type; a consequence of marked disease heterogeneity and limited understanding of the molecular basis for these cancers, among other factors. PanNETs arise from cells of the neuroendocrine system within the pancreas and include insulinomas, gastrinomas, glucagonomas, VIPomas and somatostatinomas
PanNETs are the second most common pancreatic neoplasms, representing approximately 1.3% of pancreatic cancers in incidence and 10% of cases in prevalence
It is currently unknown whether nonfunctioning tumors and PD-PanNETs arise from a different cell of origin to hormone-producing neoplasms, or reflect a more stem-like differentiation status
The RIP1-Tag2 (RT2) mouse model of islet-cell carcinoma has proven very instructive in studying neuroendocrine tumor progression, and in particular, in predicting clinical efficacy of new therapeutics
Several different classes of tumor grade have been reported in the RT2 model including encapsulated tumors, microinvasive and invasive carcinomas, which are thought to represent a multi-stage progression series
In this study, we have identified and characterized a previously undescribed class of poorly differentiated PanNETs in the RT2 model. We found that while the majority of tumors in the RT2 model are well-differentiated insulinomas, a subset of tumors were found to be poorly differentiated and highly invasive, leading us to term them poorly differentiated invasive carcinomas (PDICs). These tumors had lost expression of multiple markers of beta-cell differentiation. In addition, we found that these tumors exhibited a high mitotic index, resembling PD-PanNETs in human patients. We also identified the inhibitor of differentiation/inhibitor of DNA binding gene Id1 as being specifically expressed by these tumors by histological analysis, and also to be expressed in “met-like primary” tumors. The identification of PDICs provides a unique opportunity to study the pathology and molecular characteristics of PD-PanNETs in an experimental model.
During the course of analysis of serially sectioned pancreata from RT2 mice, we identified a previously undescribed class of tumors. These tumors were found histologically due to their high nuclear to cytoplasmic ratio, anaplastic morphology and extensive invasion into the surrounding normal exocrine pancreas (
(A) H&E staining of paraffin tissue sections demonstrates a tumor region with an anaplastic appearance and a high nuclear to cytoplasm ratio. 40× magnification of the boxed region is shown in the right panel. T = Tumor, E = Exocrine pancreas. Scale bars: 100 μm (left), 20 μm (right). (B) IHC for insulin was performed on paraffin sections from RT2 mice, and representative images are shown. While the majority of the tumors (labeled insulinoma) produce high levels of insulin (detected by DAB in brown), poorly differentiated invasive carcinomas (PDICs) are negative for insulin staining and are highly invasive. Scale bar: 100 μm. (C) Adjacent sections were stained for insulin and T-antigen. PDICs remained positive for T-antigen staining. Scale bar: 50 μm.
Discovery of a set of tumors that had lost insulin expression was surprising, as it had been previously thought that all RT2 tumors expressed insulin at high levels due to their origin from beta-cells. By H&E staining the PDICs appeared to be PanNETs, however there remained the possibility that these arose from a different cell of origin and were not PanNETs. To determine whether they were still driven by the SV40 T-antigen oncogene, which induces tumorigenesis in beta-cells under control of the rat insulin promoter (RIP)
High-grade PanNETs in patients are characterized by their high mitotic index
(A) Adjacent tumor sections were stained for insulin and Ki67. PDICs that do not express insulin (inset) were found to have a very large proportion of Ki67+ proliferating cells. Tumors outlined with dotted white lines on the right are insulinomas, showing a markedly lower degree of Ki67 staining. Scale bar represents 200 μm. (B) Tumor sections were stained by immunofluorescence for insulin and Ki67. Insulinomas (top right) exhibit significantly less Ki67 positive cells than adjacent PDICs (lower left). Scale bar: 20 μm. (C) Mitotic index was calculated by the number of Ki67+ cells over the total cells per tumor, and tumors were stratified into insulinomas or PDICs using insulin staining.
PDICs have not been previously described in the RT2 model and it was unknown whether these tumors were rare or present in all mice but had been previously missed. To thoroughly investigate the frequency of PDICs, we completely sectioned through the entire pancreas of RT2 mice and examined every 10th slide to ensure that each tumor throughout the tissue was represented and analyzed. When staining for insulin, we found that while all mice had seven to ten insulinomas each, 70% of WT RT2 mice examined also had at least one PDIC. Thus, our analysis has revealed that PDICs are actually not uncommon, constituting 10.3% of all tumors (
RT2 Mice | RT2 Tumors | |
10 | 86 | |
7 | 9 | |
RT2 pancreata were serially sectioned and every 10th slide was stained for insulin to identify PDICs, determined by loss of insulin expression. PDICs were confirmed by expression of Id1. RT2 mice have multiple tumors, thus the frequency of PDIC incidence per mouse and as a percentage of all tumors was calculated (70% and 10.3% respectively).
Given the loss of insulin expression in PDICs, we next investigated whether other markers of beta-cells and neuroendocrine cells were also absent in these tumors. Tissue sections containing both PDICs and insulinomas were stained for a panel of cell type-specific markers
Tissue sections were stained for the following markers of neuroendocrine differentiation: Synaptophysin, ChromograninA, MafA, Nkx6.1 and Pdx1, and representative images are shown. While insulinomas all stained positive for these markers (left panels, detected by DAB in brown), PDICs exhibited either complete or heterogeneous loss of these markers (right panels). T = tumor, E = exocrine pancreas. Scale bars: 50 μm.
As PDICs have lost expression of beta-cell specific genes, it raised the possibility that they are tumors that have arisen from one of the other neuroendocrine cell types that constitute pancreatic islets. While beta-cells represent the major cell type in islets, alpha-cells and delta-cells are also present, and tumors such as glucagonomas and somatostatomas can arise from these cells
(A, B) Tissues were stained for glucagon, to label alpha-cells, and somatostatin, to label delta-cells. Normal islets showed the expected expression pattern for these markers at the periphery of islets (detected by DAB in brown). PDICs were negative for these pancreatic endocrine cell markers, exhibiting staining in rare alpha-cells or delta-cells seen within the tumor and with non-specific background staining in exocrine cells. (C) Tissues were stained for elastase, which labels acinar cells in the exocrine pancreas. Positively stained cells are labeled in brown, and were only observed in the exocrine tissue (E), never in the PDIC tumors (T). Scale bar: 50 μm.
Characterization of PDICs revealed that they have lost multiple markers of beta-cell differentiation, however, factors that were specifically expressed by these tumors remained unknown. We therefore undertook a candidate-based approach to determine a specific, positive marker of PDICs. Due to the apparent dedifferentiation of these tumors, we investigated the expression pattern of Id1, one of the inhibitor of DNA binding (Id) proteins. Id1 has been shown to inhibit differentiation, to stimulate proliferation and to be expressed by embryonic stem cells, adult stem cells and cancer stem cells
We stained tissue sections from RT2 mice and found that Id1 was expressed in all endothelial cells within tumors (
(A) Paraffin sections were stained for Id1 and Id3 and representative images are shown. PDIC tumor cells specifically expressed Id1 and Id3, while insulinoma tumor cells did not, with Id1 and Id3 staining only detectable in endothelial cells as expected. Scale bar: 50 μm. (B) Id1 and insulin expression are mutually exclusive by immunofluorescence staining. Scale bar: 20 μm. (C) Id1+ cells (brown) are evident at the invasive front of a tumor, and (D) Id1 staining is observed in exocrine cells adjacent to a PDIC (indicated by white arrows). Scale bar: 50 μm.
Additionally, we confirmed the expression of Id1 in protein lysates from a panel of RT2 tumors (
(A) A panel of protein lysates from individual RT2 tumors was blotted for Id1, insulin and MafA, with actin as a loading control. The tumor lysate #5 is a PDIC, as it has high Id1 levels with absence of insulin and MafA protein expression. (B) Expression of Id1 and Id2 were examined in RNA from invasive tumors (IT) and “met-like primary” tumors (MLP) from the RT2 model, and calculated compared to the housekeeping gene RPL13A. Both Id1 and Id2 are expressed at significantly higher levels in MLP compared to IT tumors.
A previous study has suggested that in the RT2 model, a subset of tumors may arise through a distinct pathway, termed “met-like primary” (MLP) tumors
To investigate whether MLPs exhibited similar markers to PDICs, we analyzed the expression of specific Id family members in RNA in tumors that had previously been identified as MLPs based on a previously reported gene signature (Olson et al., 2009). We found that expression of Id1 and Id2 were significantly upregulated in MLPs, as compared to invasive tumors (IT) (
We have described the discovery of a class of tumors, PDICs, which have lost markers of beta-cell differentiation, are highly proliferative and anaplastic. Intriguingly, this tumor type exhibits many of the characteristics of high grade PD-PanNETs, a tumor subset of which very little is known and for which patients have very poor prognosis. Studying these tumors may thus provide important insights into the molecular mechanisms of PD-PanNETs.
Having serendipitously discovered these tumors through their loss of insulin expression, we first further characterized these tumors histologically. We found that PDICs had not only lost insulin expression, but did not express many markers of beta-cell differentiation, including those that are expressed in progenitor cells during development.
We also excluded the possibility that these tumors were derived from, or trans-differentiated into, a different cell type within the pancreas by staining for markers of other pancreatic neuroendocrine cells and acinar cells. PDICs did not express markers of alpha-cells or delta-cells, and maintained expression of T-antigen. It is intriguing that tumors that have lost insulin expression still maintain T-antigen expression, as its expression is driven by the rat insulin promoter (RIP), which is controlled in a similar manner to that of the endogenous mouse insulin gene. The mechanism of this silencing of insulin expression remains an open question.
The inhibitor of DNA binding family member Id1 was the first protein that we identified as being specifically expressed in PDICs. Id1 has been shown to be expressed by adult neural stem cells and glioma stem cells
Having identified and characterized PDICs histologically, we were interested in understanding how they develop. There are several hypotheses as to how these tumors may arise. PDICs could result from a further progression from the invasive IC2 class of carcinomas, in which loss of differentiation markers has occurred during the progression to a high-grade tumor. Alternatively, PDICs could represent a separate tumorigenesis pathway, in which they progress without beta-cell markers in a distinct development pathway to insulinomas. Similarly, PDICs could represent a completely different tumor type, derived from a different cell of origin, whether it be a stem or progenitor cell or another pancreatic neuroendocrine cell.
We have made several interesting observations that could provide insight into these possibilities regarding the development of PDICs. The majority of PDICs were found to be entirely Id1+, suggesting that tumors arose from a single clone, consistent with the hypothesis that these tumors originated through a separate pathway or cell of origin to the other classes of RT2 tumor. However, we found that occasional tumors exhibited Id1+ cells only at the invasive edges of the tumor. This could be indicative of the progression of an invasive tumor cell population that has lost insulin expression, and gained Id1 expression. Alternatively, it could again represent a subset of tumor cells that have arisen from a PDIC clone, which is present at the tumor edge. The observation that PDICs exhibit a high mitotic index also brings into question the timing of their development. With high proliferation rates, it would be expected that PDICs would grow at a much faster rate than insulinomas. However, PDICs are not any larger on average than insulinomas, suggesting that they may arise later than insulinomas. Thus, the development of these tumors remains an interesting, open question and could provide important insights into the development of patient PD-PanNETs.
Due to the similarly low ∼10% frequency of PDICs found in our study and the previously described met-like primary (MLP) tumors
Surgical resection is the most effective treatment option for PanNETs; however, approximately 65% of patients present with unresectable or metastatic disease. PD-PanNETs respond very differently to therapeutic agents than well-differentiated PanNETs and thus are treated with a different regimen. PD-PanNETs are generally managed with surgery and platinum-based chemotherapy
The generation and characterization of RIP1-Tag2 (RT2)
Paraffin sections were stained using DAB detection with a Discovery XT automated staining processor (Ventana Medical Systems, Inc). For immunofluorescence staining, paraffin sections were processed with a Discovery XT automated staining processor, incubated with the primary antibody of interest overnight at 4°C, incubated with the corresponding fluorescently-tagged secondary antibody for 1 hour at room temperature, incubated with 46-diamidino-2-phenyl indole (DAPI) for 10 minutes and mounted with ProLong Gold (Invitrogen). Tissue sections were visualized under a Carl Zeiss Axioimager Z1 microscope and images were acquired with Axiovision using an Apotome (Zeiss) or with TissueFAXS (TissueGnostics, Vienna, Austria). For the proliferation analysis, the quantitation was performed using HistoQuest software (TissueGnostics) to determine the percentage of Ki67+ cells. The following antibodies were used for IF and IHC staining: rabbit anti-SV40 T antigen (Santa Cruz, 1∶500), guinea pig anti-insulin (DAKO, 1∶1000), rabbit anti-synaptophysin (DAKO, 1∶200), rabbit anti-Ki67 (Vector, 1∶200), rabbit anti-chromogranin A (Abcam 1∶250), rabbit anti-MafA (Abcam, 1∶500), anti-Nkx6.1, rabbit anti-Pdx1 (Abcam, 1∶500), rabbit anti-glucagon (Millipore 1∶1000), rabbit anti-somatostatin (DAKO, 1∶500), rabbit anti-elastase (Abcam, 1∶2000), rabbit anti Id1 (BioCheck, 1∶200) and rabbit anti-Id3 (BioCheck, 1∶200). Relevant species-specific IgG controls for each antibody were stained in parallel, and no non-specific staining was observed.
Protein lysates were made from dissected RT2 tumors using RIPA lysis buffer. 40 μg of protein was loaded onto SDS-PAGE gels and transferred to PVDF membranes for immunoblotting. Membranes were probed with antibodies against Id1 (BioCheck 1∶500), insulin (DAKO 1∶1000), MafA (Abcam 1∶1000) and actin (Sigma, 1∶5000) and detected using HRP-conjugated anti-guinea pig or anti-rabbit (Jackson Immunoresearch) antibodies using chemiluminescence detection (Amersham).
RNA was isolated from different tumors (invasive tumors (IT) and “met-like primary” tumors (MLPs)) from the RT2 mouse model and reverse transcription was performed as described
Throughout this study, means +/− SEM (standard error of the mean) are reported unless otherwise specified. For all two-way comparisons, unpaired t-tests were used and were considered statistically significant if
We thank Dr. Robert Benezra and members of the Joyce lab for insightful discussion on this topic. We gratefully acknowledge Xiaoping Chen and Kenishana Simpson for excellent technical assistance.