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Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology

  • Authors:
    • Mai Matsumura
    • Hideaki Mitsui
    • Tetsukan Woo
    • Takehisa Suzuki
    • Hiromasa Arai
    • Chihiro Koike
    • Toshiaki Kataoka
    • Daisuke Motooka
    • Kiyoharu Fukushima
    • Koji Okudela
  • View Affiliations / Copyright

    Affiliations: Department of Pathology, Saitama Medical University, Moroyama‑cho, Saitama 350‑0495, Japan, Department of Pathology, Yokohama City University, School of Medicine, Yokohama, Kanagawa 236‑0004, Japan, Department of General Thoracic Surgery, Yokohama City University Medical Center Hospital, Yokohama, Kanagawa 232‑0024, Japan, Department of Surgery, Kanagawa Prefectural Cardiovascular and Respiratory Center Hospital, Yokohama, Kanagawa 236‑0051, Japan, Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Suita, Osaka 565‑0871, Japan, Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Osaka 565‑0871, Japan
    Copyright: © Matsumura et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 533
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    Published online on: September 18, 2025
       https://doi.org/10.3892/ol.2025.15278
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Abstract

The micropapillary histological subtype is a high‑grade element and a poor prognostic marker in lung adenocarcinoma (LUAD). This subtype develops through the lepidic‑filigree micropapillary (filigree)‑conventional/overt micropapillary (mPAP) pathway. The present study aimed to identify key molecules that promote this progression. To this end, gene expression profiles specific to lepidic, filigree and mPAP elements were investigated in histological sections obtained from 4 different LUAD cases. The 10x Genomics Visium Spatial Gene Expression Solution was used due to its superior resolution compared with conventional microdissection techniques. Cellular retinoic acid binding protein 2 (CRABP2), carcinoembryonic antigen‑related cell adhesion molecule 5 (CEACAM5) and mucin 21 (MUC21) were identified as common molecules with significantly elevated levels along the lepidic‑filigree‑mPAP pathway. Furthermore, the present findings indicated that CRABP2 may serve an important role in the early stage of this process, as its level significantly increases during the transition from the lepidic to the filigree substage. Immunohistochemical analysis of the expression of CRABP2, CEACAM5 and MUC21 proteins in 207 surgically resected LUAD samples (expanded sample size) was performed. The present study revealed an increase in the expression levels of CRABP2 between the lepidic and filigree elements, and between filigree and mPAP for CEACAM5 and MUC21. Thus, these three proteins were demonstrated to serve roles in the lepidic‑filigree‑mPAP pathway at different stages. Notably, these molecules were associated with poor prognosis, characterized by an elevated recurrence rate and poor survival rate. In conclusion, crucial molecules that promote the lepidic‑filigree‑mPAP pathway, and exhibit potential clinical utility as prognostic markers and molecular therapeutic targets, were identified.
View Figures

Figure 1

Representative H&E staining
images of patient 1 acquired using Visium profiling. (A) Loupe
slide of surgically resected lung adenocarcinoma. Tissue sections
in situ and invaded areas were analyzed using Visium
profiling. Scale bar, 5 mm. (B) Tumor cells consisted of lepidic
elements in situ. Scale bar, 2 mm. (C) High magnification
view of the green square in (B). The lepidic element was
characterized by neoplastic cell extension along the alveolar wall
surface. (C-1) Blue spots. (C-2) Serial section. Scale bar, 200 µm.
(D) Tumor cells in the invaded area comprised both filigree and
mPAP elements. Scale bar, 2 mm. (E) High magnification view of the
blue square in (D). Filigree element consisted of stacked tumor
cells extending from alveolar walls towards airspace without
fibrovascular cores. (E-1) Light green spots. (E-2) Serial section.
Scale bar, 100 µm. (F) High magnification view of the yellow square
in (D). mPAP elements were characterized by the formation of tufted
papillary structures with a central fibrovascular core and floated
in the alveolar space. (F-1) Orange spots. (F-2) Serial section.
Scale bar, 200 µm. mPAP, conventional/overt micropapillary.

Figure 2

Number of significantly
differentially expressed genes among histological elements in 4
cases. The numbers of changes in gene expression between lepidic
and mPAP elements were a few dozen to several thousand. Most of the
changes in gene expression during the transition from lepidic to
mPAP elements occurred between the lepidic and filigree elements
(approximately one hundred to several thousand). By contrast,
relatively few changes were observed between filigree and mPAP
elements (<50). mPAP, conventional/overt micropapillary.

Figure 3

Representative images of CRABP2,
CEACAM5 and MUC21 immunohistochemical staining. (A) Lepidic, (B)
filigree and (C) mPAP elements stained with H&E. (D) CRABP2,
(G) CEACAM5 and (J) MUC21 were rarely expressed in lepidic
elements. CRABP2 was expressed in (E) filigree and (F) mPAP
elements. (I) CEACAM5 and (L) MUC21 were expressed only in mPAP
elements. (H) CEACAM5 and (K) MUC21 were rarely expressed in
filigree elements. Scale bar, 250 µm (A, B, D, E, G, H, J and K) or
100 µm (C, F, I and L). CEACAM5, carcinoembryonic antigen-related
cell adhesion molecule 5; CRABP2, cellular retinoic acid binding
protein 2; mPAP, conventional/overt micropapillary; MUC21, mucin
21.

Figure 4

Representative images of tumors with
various immunohistochemical scores. Red, green and blue dots
indicate tumor cells with strong, weak and no signals (intensity,
2, 1 and 0, respectively) (A) Lepidic elements (left) rarely
exhibited positive signals for each antibody (CRABP2, CEACAM5 and
MUC21 scores: 5.8, 2.9 and 1.8). Filigree elements (center) showed
some strong and weak CRABP2 signals in most neoplastic cells
(score, 118=1×34+2×42). Signals for CEACAM5 and MUC21 were rarely
positive (scores, 1.2 and 1.0, respectively). mPAP elements (right)
exhibited stronger signals in most neoplastic cells for CRABP2,
CEACAM5 and MUC21 than the other elements (lepidic and filigree)
(scores, 199.3=1×0.7+2×99.3, 184.5=1×8.5+2×88 and 55.1=1×1.1+2×27,
respectively). Scale bar, 250 µm. (B) All scores were low in
lepidic elements. Only the expression score for CRABP2 was high in
the filigree element, whereas the scores for all three proteins
were high in mPAP elements. CEACAM5, carcinoembryonic
antigen-related cell adhesion molecule 5; CRABP2, cellular retinoic
acid binding protein 2; mPAP, conventional/overt micropapillary;
MUC21, mucin 21.

Figure 5

Association between histological
elements and CRABP2, CEACAM5 and MUC21 immunohistochemical scores.
(A) The CRABP2 score was significantly higher in filigree elements
than in lepidic elements (P<0.0001) but no significant
difference was observed between mPAP elements and filigree elements
(P=0.8985). (B) The CEACAM5 score was significantly higher in
filigree elements than in lepidic elements (P=0.0174) and
significantly higher in mPAP elements than in filigree elements
(P<0.0001). This difference was more significant between
filigree and mPAP elements than between filigree and lepidic
elements. (C) The MUC21 score was significantly higher in filigree
elements than in lepidic elements (P=0.0094) and significantly
higher in mPAP elements than in filigree elements (P<0.0001).
This difference was more significant between filigree and mPAP
elements than between filigree and lepidic elements.
Box-and-whisker plots show immunohistochemical scores for 50
surgically resected lung adenocarcinoma cases (median, thick line;
25th-75th percentiles, box; 10th-90th percentiles, whiskers;
outliers, dots). P<0.05 (Kruskal-Wallis test with Dunn's
multiple comparison test). *Statistically significant. Aci, acinar;
CEACAM5, carcinoembryonic antigen-related cell adhesion molecule 5;
CRABP2, cellular retinoic acid binding protein 2; mPAP,
conventional/overt micropapillary; MUC21, mucin 21; Pap, papillary;
Sol, solid.

Figure 6

Kaplan-Meier RFS and OS curves of the
associations between CRABP2, CEACAM5 and MUC21 and disease
recurrence and survival. RFS rates were significantly worse in
samples with high (A) CRABP2, (C) CEACAM5 and (E) MUC21 expression
levels. OS rates were significantly worse in samples with high (B)
CRABP2 and (D) CEACAM5 expression levels. (F) No significant
difference in OS rates was observed between patients with high and
low MUC21 expression levels. P<0.05, log-rank test.
*Statistically significant. A total of 207 patients with lung
adenocarcinoma were followed up for a median of 84 (4–201) months. CEACAM5, carcinoembryonic
antigen-related cell adhesion molecule 5; CRABP2, cellular retinoic
acid binding protein 2; MUC21, mucin 21; n, number of tumors; OS,
overall survival; RFS, recurrence-free survival.
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Spandidos Publications style
Matsumura M, Mitsui H, Woo T, Suzuki T, Arai H, Koike C, Kataoka T, Motooka D, Fukushima K, Okudela K, Okudela K, et al: Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology. Oncol Lett 30: 533, 2025.
APA
Matsumura, M., Mitsui, H., Woo, T., Suzuki, T., Arai, H., Koike, C. ... Okudela, K. (2025). Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology. Oncology Letters, 30, 533. https://doi.org/10.3892/ol.2025.15278
MLA
Matsumura, M., Mitsui, H., Woo, T., Suzuki, T., Arai, H., Koike, C., Kataoka, T., Motooka, D., Fukushima, K., Okudela, K."Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology". Oncology Letters 30.5 (2025): 533.
Chicago
Matsumura, M., Mitsui, H., Woo, T., Suzuki, T., Arai, H., Koike, C., Kataoka, T., Motooka, D., Fukushima, K., Okudela, K."Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology". Oncology Letters 30, no. 5 (2025): 533. https://doi.org/10.3892/ol.2025.15278
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Spandidos Publications style
Matsumura M, Mitsui H, Woo T, Suzuki T, Arai H, Koike C, Kataoka T, Motooka D, Fukushima K, Okudela K, Okudela K, et al: Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology. Oncol Lett 30: 533, 2025.
APA
Matsumura, M., Mitsui, H., Woo, T., Suzuki, T., Arai, H., Koike, C. ... Okudela, K. (2025). Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology. Oncology Letters, 30, 533. https://doi.org/10.3892/ol.2025.15278
MLA
Matsumura, M., Mitsui, H., Woo, T., Suzuki, T., Arai, H., Koike, C., Kataoka, T., Motooka, D., Fukushima, K., Okudela, K."Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology". Oncology Letters 30.5 (2025): 533.
Chicago
Matsumura, M., Mitsui, H., Woo, T., Suzuki, T., Arai, H., Koike, C., Kataoka, T., Motooka, D., Fukushima, K., Okudela, K."Identification of key molecules in micropapillary progression of lung adenocarcinoma: A comprehensive gene expression analysis study using the spatial gene expression solution methodology". Oncology Letters 30, no. 5 (2025): 533. https://doi.org/10.3892/ol.2025.15278
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