MyrAkt1 overexpression in endothelial cells in vivo leads to a transient increase in IgM^hiIgD^loCD24^intCD43^−/lo cells in the spleen.

<p>At 4 weeks off tetracycline, endothelial myrAkt1 mice displayed an increase in spleen weight (<b>A</b>) p<0.04. The total number of nucleated cells in the spleen increased transiently at 1–2 weeks but returned to normal at 3–4 weeks (<b>B</b>) p<0.03. The absolute numbers of CD45+ (<b>C</b>) and CD45+B220+ (<b>D</b>) cells displayed a transient increase at one week p<0.02. The relative percentage of IgM^loIgD^hi and IgM^hiIgD^lo cells was altered at one week (<b>E</b>) p<0.02. Absolute numbers of B220⁺IgM^hiIgD^loCD24^intCD43^−/lo cells were increased at one week (<b>F</b>) p<0.01. Representative plots of B220⁺ fractionation by IgM and IgD (<b>G</b>), and B220⁺IgM^hiIgD^lo fractionation by CD24 and CD43 (<b>H</b>).</p

Splenic erythropoiesis is not triggered by anemia.

<p>Hematocrit remained unchanged until 4 weeks off tetracycline (<b>A</b>) p<0.01, and no hemolysis was observed in peripheral blood smears of endothelial myrAkt1 animals at 2 weeks off tetracycline as compared with controls (<b>B</b>) (100x).</p

Initial splenic erythropoiesis is not mediated by erythropoietin or BMP4.

<p>Addition of BMP4 to methylcellulose cultures did not increase the number of colonies compared with erythropoietin alone (<b>A</b>). Levels of BMP4 mRNA (<b>B</b>) and protein (<b>C</b>) were unchanged in the spleen. Splenic BMP4+ macrophages recruited in response to chemotherapeutically-induced anemia (<b>D</b>) were not found in spleens of control (<b>E</b>) or myrAkt1 (<b>F</b>) animals (40x). Plasma erythropoietin protein was not significantly elevated until 4 weeks off tetracycline (<b>E</b>) p<0.0005, and levels of erythropoietin mRNA were not elevated until 3 weeks off tetracycline in the kidney (<b>F</b>) or liver (<b>G</b>) p<0.05. Endothelial cells isolated from myrAkt1 kidneys displayed downregulation of phosphorylated and total Akt1 in the presence of tetracycline (<b>J</b>). TER119⁺ erythroblasts showed no significant decrease in Annexin V positivity in spleen or bone marrow (<b>K</b>), but TUNEL staining showed a transient decrease in apoptotic nuclei in myrAkt1 spleens at one week p<0.006 (<b>L)</b>.</p

Increased spleen weight in myrAkt1 overexpressing animals is due to increased erythropoiesis.

<p>An increased percentage of reticulocytes was found in the blood at 3–4 weeks (<b>A</b>) p<0.02. Staining against TER119 revealed an increase in red cell area in the spleens of endothelial myrAkt1 compared with control mice (<b>B</b>) (5x). The total number of TER119^hi cells increased by 2 weeks and remained elevated in the spleen (<b>C</b>) p<0.04. Further characterization using CD71 and TER119 showed no differences in erythroid differentiation in the spleen (<b>D</b>). The total number of TER119^hi cells in the bone marrow remained constant (<b>E</b>), but myrAkt1 animals displayed an increase in stages I and II with a complementary decrease in III (<b>F</b>) p<0.007.</p

Splenic erythropoiesis in endothelial myrAkt1 mice is due to host and not stem cell genotype.

<p>Lethally irradiated control hosts rescued with endothelial myrAkt1 bone marrow did not display splenic erythropoiesis, in contrast with lethally irradiated endothelial myrAkt1 hosts rescued with control bone marrow, as measured by absolute number of TER119^hi cells in spleen (<b>A</b>) p<0.05 and number of BFU-Es from methylcellulose culture (<b>B</b>) p<0.005. Neither transplant resulted in an increase in TER119^hi cells in the bone marrow (<b>C</b>).</p

Tumor Cell Expression of Vascular Endothelial Growth Factor Receptor 2 Is an Adverse Prognostic Factor in Patients with Squamous Cell Carcinoma of the Lung

<div><p>A robust immunohistochemical (IHC) assay for VEGFR2 was developed to investigate its utility for patient tailoring in clinical trials. The sensitivity, specificity, and selectivity of the IHC assay were established by siRNA knockdown, immunoblotting, mass spectrometry, and pre-absorption experiments. Characterization of the assay included screening a panel of multiple human cancer tissues and an independent cohort of non-small cell lung carcinoma (NSCLC, n = 118) characterized by TTF-1, p63, CK5/6, and CK7 IHC. VEGFR2 immunoreactivity was interpreted qualitatively (VEGFR2 positive/negative) in blood vessels and by semi-quantitative evaluation using H-scores in tumor cells (0–300). Associations were determined among combinations of VEGFR2 expression in blood vessels and tumor cells, and clinico-pathologic characteristics (age, sex, race, histologic subtype, disease stage) and overall survival using Kaplan-Meier analyses and appropriate statistical models. VEGFR2 expression both in blood vessels and in tumor cells in carcinomas of the lung, cervix, larynx, breast, and others was demonstrated. In the validation cohort, 99/118 (83.9%) NSCLC tissues expressed VEGFR2 in the blood vessels and 46/118 (39.0%) showed high tumor cell positivity (H-score ≥10). Vascular and tumor cell expression were inversely correlated (<i>p</i> = 0.0175). High tumor cell expression of VEGFR2 was associated with a 3.7-fold reduction in median overall survival in lung squamous-cell carcinoma (SCC, n = 25, <i>p</i> = 0.0134). The inverse correlation between vascular and tumor cell expression of VEGFR2 and the adverse prognosis associated with high VEGFR2 expression in immunohistochemically characterized pulmonary SCC are new findings with potential therapeutic implications. The robustness of this novel IHC assay will support further evaluation of its utility for patient tailoring in clinical trials of antiangiogenic agents.</p></div

VEGFR2 immunoreactivity in areas of squamous differentiation.

<p>(A) Tumor cell-derived cytoplasmic and nuclear immunoreactivity (black arrowhead) in a focus of squamous differentiation on a background of pulmonary ADC. (B) tumor cell-derived cytoplasmic and nuclear immunopositivity in SCC of the cervix. Keratin pearls (open arrowhead) are not immunoreactive. Slides were counterstained with hematoxylin. Original magnification ×200. Scale bar: 50 µm, applicable to both panels.</p

Vascular endothelial cell and tumor cell-derived VEGFR2 immunoreactivity on representative cases on a multi-tumor survey.

<p>Left panels, H? right panels, VEGFR2 IHC. (A) VEGFR2 IHC on renal cell carcinoma of the kidney showed endothelial cell immunoreactivity (×400). (B) VEGFR2 IHC on ADC of the colon showed endothelial cell immunoreactivity in the stromal mucosa. Tumor cells were negative for VEGFR2 (×400). (C) VEGFR2 IHC on SCC of the lung showed endothelial cell and a range of tumor cell-derived nuclear, cytoplasmic, and membranous immunoreactivity (×200). (D) VEGFR2 IHC showed vascular endothelial cell immunoreactivity and a range of tumor cell cytoplasmic and nuclear immunoreactivity on SCC of the cervix (×200). Immunoreactivity in endothelial cells lining vessels (white and black arrows). Slides were counterstained with hematoxylin. Scale bars: 50 µm.</p

Refinement of IHC assay parameters was integral to detect subcellular immunoreactivity in tumor cells.

<p>Representative immunoreactivity is shown on serial sections of a pulmonary SCC. Slides were subjected to heat induced epitope retrieval (HIER) conditions using buffers of differing pH, followed by an optimized IHC protocol using 55B11: primary antibody reagent negative control (A), Tris buffer (B), citrate buffer (C), and EDTA buffer (D). VEGFR2 immunoreactivity in endothelial cells lining blood vessels (black arrows) and membranous (black arrowheads), cytoplasmic, and nuclear compartments of malignant tumor cells was found to be most optimal with EDTA buffer (panel D). Slides were counterstained with hematoxylin. Original magnification ×200. Scale bar: 50 µm, applicable to all panels.</p

VEGFR2 IHC is specific and selective against other VEGFR family members.

<p>The anti-VEGFR2 55B11 antibody specifically recognizes a unique epitope in the sequence HSDDTDTTVYSSEEA that is harbored within the c-terminal region. (A) HIS-tagged recombinant proteins corresponding to the intracellular domains of VEGFR1, VEGFR2, and VEGFR3 were incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of ductal carcinoma of the breast. VEGFR2 and not VEGFR1 or VEGFR3 pre-absorbed the antibody leading to negative immunoreactivity. (B) Recombinant 21 or 22-mer peptides corresponding to contiguous stretches of the immunogenic sequence of 55B11 were generated as indicated in the schematic (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080292#pone.0080292.s002" target="_blank">Table S2</a>). Each peptide was incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of pulmonary SCC. Pre-incubation with peptides 6 and 12 abolished immunoreactivity, while pre-incubation with all other peptides had no effect. The results shown for peptides 5 and 13 are representative of peptides 1–5, 7–11, and 13. (C) Recombinant 10 or 11-mer peptides corresponding to contiguous stretches of the consensus sequence of peptides 6 and 12 were generated as indicated in the schematic. Each peptide was incubated at 200-fold molar excess with 55B11 prior to IHC on serial sections of pulmonary SCC, a unique patient sample than in panel B. Pre-incubation with peptides 6 (data not shown), 12 and 15 abolished all immunoreactivity, while pre-incubation with peptides 14, 17 and 18 had no effect. Pre-incubation with peptide 16 showed an attenuated effect. All slides were counterstained with hematoxylin. Original magnification, ×200. Scale bars: 50 µm.</p