Identification and Characterization of Poorly Differentiated Invasive Carcinomas in a Mouse Model of Pancreatic Neuroendocrine Tumorigenesis

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

Sensing cytosolic RpsL by macrophages induces lysosomal cell death and termination of bacterial infection.

The intracellular bacterial pathogen Legionella pneumophila provokes strong host responses and has proven to be a valuable model for the discovery of novel immunosurveillance pathways. Our previous work revealed that an environmental isolate of L. pneumophila induces a noncanonical form of cell death, leading to restriction of bacterial replication in primary mouse macrophages. Here we show that such restriction also occurs in infections with wild type clinical isolates. Importantly, we found that a lysine to arginine mutation at residue 88 (K88R) in the ribosome protein RpsL that not only confers bacterial resistance to streptomycin, but more importantly, severely attenuated the induction of host cell death and enabled L. pneumophila to replicate in primary mouse macrophages. Although conferring similar resistance to streptomycin, a K43N mutation in RpsL does not allow productive intracellular bacterial replication. Further analysis indicated that RpsL is capable of effectively inducing macrophage death via a pathway involved in lysosomal membrane permeabilization; the K88R mutant elicits similar responses but is less potent. Moreover, cathepsin B, a lysosomal protease that causes cell death after being released into the cytosol upon the loss of membrane integrity, is required for efficient RpsL-induced macrophage death. Furthermore, despite the critical role of cathepsin B in delaying RpsL-induced cell death, macrophages lacking cathepsin B do not support productive intracellular replication of L. pneumophila harboring wild type RpsL. This suggests the involvement of other yet unidentified components in the restriction of bacterial replication. Our results identified RpsL as a regulator in the interactions between bacteria such as L. pneumophila and primary mouse macrophages by triggering unique cellular pathways that restrict intracellular bacterial replication

PDICs and “met-like primary” tumors have high expression of Id1.

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

Identification of poorly differentiated invasive carcinomas (PDICs).

<p>(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.</p

Frequency of poorly differentiated invasive carcinomas (PDICs) in RT2 mice.

<p>RT2 pancreata were serially sectioned and every 10^th 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).</p

Id1 is expressed by tumor cells in PDICs.

<p>(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.</p

PDICs exhibit a high mitotic index.

<p>(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. <i>P</i> values were obtained using Student's unpaired t-test.</p