Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice

Abstract The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53, loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3 +/+ mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3 +/− and Phlda3 −/− littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced

Fractional blood volume.

<p>Comparison of FBVs calculated from CT scan 1 (based on gold) and CT scan 3 (based on iodine) for each of the organs. None of the differences were found to be statistically significant (p<0.05).</p

Dual-Energy Micro-CT Functional Imaging of Primary Lung Cancer in Mice Using Gold and Iodine Nanoparticle Contrast Agents: A Validation Study

<div><p>Purpose</p><p>To provide additional functional information for tumor characterization, we investigated the use of dual-energy computed tomography for imaging murine lung tumors. Tumor blood volume and vascular permeability were quantified using gold and iodine nanoparticles. This approach was compared with a single contrast agent/single-energy CT method. <i>Ex vivo</i> validation studies were performed to demonstrate the accuracy of <i>in vivo</i> contrast agent quantification by CT.</p><p>Methods</p><p>Primary lung tumors were generated in <i>LSL-Kras^G12D; p53^FL/FL</i> mice. Gold nanoparticles were injected, followed by iodine nanoparticles two days later. The gold accumulated in tumors, while the iodine provided intravascular contrast. Three dual-energy CT scans were performed–two for the single contrast agent method and one for the dual contrast agent method. Gold and iodine concentrations in each scan were calculated using a dual-energy decomposition. For each method, the tumor fractional blood volume was calculated based on iodine concentration, and tumor vascular permeability was estimated based on accumulated gold concentration. For validation, the CT-derived measurements were compared with histology and inductively-coupled plasma optical emission spectroscopy measurements of gold concentrations in tissues.</p><p>Results</p><p>Dual-energy CT enabled <i>in vivo</i> separation of gold and iodine contrast agents and showed uptake of gold nanoparticles in the spleen, liver, and tumors. The tumor fractional blood volume measurements determined from the two imaging methods were in agreement, and a high correlation (R² = 0.81) was found between measured fractional blood volume and histology-derived microvascular density. Vascular permeability measurements obtained from the two imaging methods agreed well with <i>ex vivo</i> measurements.</p><p>Conclusions</p><p>Dual-energy CT using two types of nanoparticles is equivalent to the single nanoparticle method, but allows for measurement of fractional blood volume and permeability with a single scan. As confirmed by <i>ex vivo</i> methods, CT-derived nanoparticle concentrations are accurate. This method could play an important role in lung tumor characterization by CT.</p></div

Thick slab maximum intensity projections of the 80 kVp CT datasets following bilateral filtration.

<p>CT scan 1 is shown in (A), CT scan 2 in (B) and CT scan 3 in (C). Tumor is marked “T,” liver is marked “L,” spleen is marked “S.” and kidneys are marked “K.” The images are all windowed between −300 and 1200 HU. The scale bar represents 1 cm in all panels.</p

Average tissue enhancement.

<p>Tissue enhancement (average of 5 mice) is shown at 40 and 80 kVp for tumor, blood, spleen, liver, and kidneys in each of three CT scans. Pre-contrast values are the average tissue enhancement of the mice from pre-experiment monitoring scans.</p

Comparison of CT-measured concentrations with validation methods.

<p>(A) Concentrations of accumulated gold in the tumors and other organs for the single-material method (based on CT scan 1 and 2), the two-material method (based only on CT scan 3) and ICP-OES. (B) Measured CT concentrations of gold in the blood on day 1 and day 3, iodine concentration in the blood on day 3, and corresponding measurements by ICP-OES and UV-Vis. None of the differences were found to be statistically significant (p<0.05).</p

Thick slab maximum intensity projections of the lungs.

<p>Coronal CT images showing the filtered 80 kVp dataset from CT scan 1 (A), CT scan 2 (B), CT scan 3 (C), and the final overlay of the DE decomposition of CT scan 3 (D) with bones segmented out (bones in white). (E) is a zoomed-in image of the right side of CT scan 3, and (F) is a zoomed in image of the right side of the DE overlay of CT scan 3. The boxes in (C) and (D) show the regions that are enlarged in (E) and (F), respectively. 80 kVp images are windowed from –300 to 1200 HU. The overlays are windowed from 0.25 to 6 mg/mL gold, and 0.25 to 15 mg/mL iodine. The scale bar represents 0.5 cm in all images.</p

Dual-energy decomposition process diagram.

<p>Raw 40 kVp and 80 kVP datasets were acquired, and then they underwent affine registration and joint bilateral filtration to produce filtered data sets. Dual-energy decomposition was performed on these filtered images, which resulted in two independent images (maps) representing the iodine and gold concentration in each voxel. After obtaining the two maps, the images were overlaid and the bones were segmented out (colored white) to form the final concentration map overlay. These images were taken from CT scan 3, in which both iodine and gold were present in the blood stream. The scale bar represents 1 cm in all images. The 40 and 80 kVp images are windowed from −300 to 1200 HU, the iodine maps are windowed from 0.25 to 15 mg/mL, and gold maps are windowed from 0.25 to 6 mg/mL.</p