Proteomic Analysis of Proteins Related to Prognosis of Lung Adenocarcinoma

We attempted to identify prognosis-related proteins expressed in early resection lung adenocarcinomas that had higher metastatic potential. Early resection of lung adenocarcinoma tissues were collected from patients who experienced recurrence within 5 years after surgery; these patients are defined here as the poor prognosis group. From these samples, we prepared frozen tissue sections and then isolated cancerous areas by laser capture microdissection to allow extraction of cancer tissue-derived soluble proteins. Shotgun LC–MS/MS analysis detected and identified a total of 875 proteins in these cancer tissues. Relative quantitative analysis revealed that 17 proteins were preferentially expressed in the poor prognosis group relative to the good prognosis group, which consisted of patients who did not exhibit recurrence. Among them, 14-3-3 beta/alpha and calnexin were reported to be potentially involved in tumor recurrence and the malignant properties of lung cancer. Here immunological analyses confirmed disease-associated expression of these proteins. In a cell-culture model using A549, targeted depletion of either 14-3-3 beta/alpha or calnexin reduced proliferation, invasion, and migration, suggesting that both proteins are involved in determining the malignant properties of lung cancer that contribute to poor prognosis

Summary of total cell area, <i>S</i>, total nucleus area, <i>S_n</i>, number of nuclei, <i>N_n</i>, and perimeter ratio, <i>R</i>, for each cluster size.

<p>Summary of total cell area, <i>S</i>, total nucleus area, <i>S_n</i>, number of nuclei, <i>N_n</i>, and perimeter ratio, <i>R</i>, for each cluster size.</p

Typical cell images for clustered cells composed of more than three cells in cancer cell-implanted and control blood.

<p>Each data count (<i>n</i>) indicates the image number having the same cluster size and <i>N_n</i>. Bars, 20 µm.</p

Typical cell images for single and double cells in cancer cell-implanted and control blood.

<p>Each data count (<i>n</i>) indicates the image number having the same cluster size and <i>N_n</i>. Bars, 20 µm.</p

Instrumental set-up.

<p>(a) Summary of the on-chip multi-imaging flow cytometry system. The system was composed of seven major modules: (i) microchip, (ii) bright-field (BF) imaging, (iii) fluorescent (FL) detection, (iv) multi-view, (v) CCD camera, (vi) sorting, and (vii) controller, as numbered in the figure. (b) Summary of the multi-view module. (c) A photograph of the system.</p

An example of cell sorting.

<p>Two photographs of the discarded reservoir (a) and the collection reservoir (b) indicated in the chip photograph are shown. Clustered cells are indicated by white arrows. Bars, 100 µm.</p

Summary of the number of nuclei, <i>N_n</i>.

<p>(a) A histogram of <i>N_n</i> obtained from cancer cell-implanted and control blood. (b) The relationship between <i>N_n</i> and cell cluster size.</p

Histograms of total cell area, S, for cancer cell-implanted (a and c) and control blood (b and d).

<p>Two threshold values (a) and (b) for cluster identifications are indicated as dotted and dashed lines.</p

Results of quantitative gene copy number assays.

<p>(a) Results of CGH assays performed for the MAT-LyLu cell line. Liver tissue of the rat was used as a reference. Gene amplifications for <i>csrp2</i> and <i>zdhhc17</i> located on chromosome 7q13 were found. (b) Results of TaqMan copy number assays performed with clusters larger than 300 µm² collected in the collection reservoir, and cells smaller than 300 µm² collected in the discarded reservoir. Results of the assays for the MAT-LyLu cell line (positive control) and liver tissue (negative control) are also shown.</p

Summary of image processing.

<p>Firstly, photographs of both a cell and the background were taken. Next, the background image was subtracted from the cell image and holes were filled. Finally, imaging biomarkers, <i>S</i>, <i>S_n</i>, <i>N_n</i>, and <i>R</i>, were calculated. Bars, 10 µm. The hole-filling procedure is explained as indicated by an asterisk. Bar, 10 µm.</p