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1.
Figure 3

Figure 3. PDICs exhibit loss of multiple neuroendocrine markers.. From: Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis.

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.

Karen E. Hunter, et al. PLoS One. 2013;8(5):e64472.
2.
Figure 5

Figure 5. Id1 is expressed by tumor cells in PDICs.. From: Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis.

(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.

Karen E. Hunter, et al. PLoS One. 2013;8(5):e64472.
3.
Figure 6

Figure 6. PDICs and “met-like primary” tumors have high expression of Id1.. From: Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis.

(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. n = 4 tumors for each group. P values were obtained using Student's unpaired t-test.

Karen E. Hunter, et al. PLoS One. 2013;8(5):e64472.
4.
Figure 4

Figure 4. PDICs do not express alpha-cell, delta-cell or exocrine cell markers.. From: Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis.

(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.

Karen E. Hunter, et al. PLoS One. 2013;8(5):e64472.
5.
Figure 2

Figure 2. PDICs exhibit a high mitotic index.. From: Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis.

(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. P values were obtained using Student's unpaired t-test.

Karen E. Hunter, et al. PLoS One. 2013;8(5):e64472.
6.
Figure 1

Figure 1. Identification of poorly differentiated invasive carcinomas (PDICs).. From: Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis.

(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.

Karen E. Hunter, et al. PLoS One. 2013;8(5):e64472.

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