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Article Open Access

Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients

  • Authors:
    • Alexander T. Sougiannis
    • Harrison B. Taylor
    • Stephen C. Zambrzycki
    • Lindsey Conroy
    • Rachel Strubler
    • Christin Edge
    • Richard R. Drake
    • Kristin Wallace
    • Derek Allison
    • Eun Lee
    • Ramon C. Sun
    • Peggi M. Angel
  • View Affiliations / Copyright

    Affiliations: College of Medicine, Medical University of South Carolina, Charleston, SC 29403, USA, Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29403, USA, Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40506, USA, Department of Public Health Services, College of Medicine, Medical University of South Carolina, Charleston, SC 29403, USA, Department of Pathology and Laboratory Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY 40506, USA
    Copyright: © Sougiannis et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 413
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    Published online on: June 26, 2025
       https://doi.org/10.3892/ol.2025.15159
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Abstract

Emerging evidence reports that regulation of the extracellular matrix influences the progression of colorectal cancer (CRC). The present study investigated regulation of the extracellular matrix proteome in colorectal malignancy within a high‑risk Appalachian population compared with non‑Appalachian populations. A targeted mass spectrometry imaging proteomic method directed at collagen regulation was used. Tissue microarrays (TMAs) comprising of matched CRC with adjacent normal to tumor (NAT) from 45 patients were constructed into 86 samples to evaluate the extracellular matrix proteome (ECM). A total of five specific peaks were discovered to differ between NAT and tumor with high sensitivity and specificity by receiver operating characteristic (AUROC) ≥0.7, Wilson/Brown P<0.0002. Evaluation of patient TMA cores showed increased levels of combined ECM peptides in advanced stage Appalachian CRC (III + IV) compared with early staged CRC (I + II) (AUROC 0.8595; 95% confidence interval, 0.8190‑0.8999; Wilson/Brown P<1.0x10‑15), contrasting with the non‑Appalachian tumors, which showed a decreased ability to discriminate between early and late stage (AUROC 0.6618; 95% confidence interval, 0.6126‑0.7110; Wilson/Brown P<1.0x10‑9). Comparison of advanced stage CRCs between Appalachian and non‑Appalachian populations showed high sensitivity and specificity in distinguishing the populations (AUROC 0.7612; 95% confidence interval, 0.7109‑0.8114; Wilson/Brown P<3.0x10‑15). History of smoking, sex and tumor origin location did not show significant ability to distinguish by AUROC. A combination of high mass resolution, high mass accuracy spatial proteomics and sequencing proteomics by liquid chromatography coupled to tandem mass spectrometry revealed that fibrillar collagens were spatially regulated within the CRC tumor microenvironment. Fibrillar collagen post‑translational modifications of hydroxylated proline revealed distinct spatial separation based on the presence of a number of hydroxylated proline sites. The present study highlighted that the targeted mass spectrometry imaging of the ECM proteome may provide new insight and novel predictive tools for understanding CRC, particularly among Appalachian patients.
View Figures

Figure 1

Study design and workflow. (A)
Regional Appalachian and non-Appalachian counties inform which
samples were collected in the study. Map was constructed from the
publicly available U.S. Geological Survey, National Geospatial
Program (https://www.usgs.gov/programs/national-geospatial-program).
(B) Previously banked patient matched tumors and NAT from men and
women were used in the study (panel 1). Cores were selected by a
pathologist from the tumor or NAT and formatted into a TMA (panel
2). TMAs were sectioned for proteomic imaging analysis. Tumors were
further annotated by stage. Pathologist-designated cores were used
to create a formalin-fixed, paraffin-embedded tissue microarray
(panel 3). Tissue sections of the TMA prepared for collagen
targeted proteomic analysis were scanned by mass spectrometry; Each
core was sampled ~225 times (panel 4). Data analysis used peak
intensities as a total score per core (panel 5). Images created
using Biorender (Biorender.com). NAT, normal adjacent to tumors;
TMA, tissue microarray.

Figure 2

Evaluation of NAT versus tumor
independent of Appalachian status and independent of tumor stage.
(A) Peptide peaks showed distinct clusters based on peptide
intensity. (B-F) Five peptide peaks were altered in NAT vs. tumor
per P≤0.001. (B) Peptide 827.431, collagen α-1(I) chain amino acid
domain 562–570, showed significant increases in tumor with AUROC of
0.7420 (P=0.00001); (C) Peptide 843.394, collagen α-1(I) chain
amino acid domain 506–514, showed significant decreases in tumor
with an AUROC of 0.7580 (P=0.0001); (D) Peptide 870.404, collagen
α-1(I) chain amino acid domain 557–565, decreased in tumor with an
AUROC of 0.7373 (P=0.0002); (E) Peptide 1041.540, collagen α-1(I)
chain amino acid domain 347–358, decreased in tumor with an AUROC
of 0.7334, (P=0.0002); (F) Peptide 1172.520, collagen α-1(III)
chain amino acid domain 795–806 decreased in tumor with an AUROC of
0.7518 (P<0.0001). AA designates amino acid domain per each
collagen. Peptides showed sensitive and specific discrimination
between NAT and tumor by area under the receiver operating curve
(AUROC) ≥0.7 and Wilson/Brown P≤0.0001. ***P<0.001 and
****P<0.0001. NAT, normal adjacent to tumors; Col, collagen; LN,
natural log.

Figure 3

CRC tumors show alterations based on
stage and regionalized Appalachian or non-Appalachian county
origins. Stages are combined as Stage I + II (early) and Stage II +
IV (late). (A) A total of sixteen peptides reported potential
differences compared between Appalachian/non-Appalachian early/late
stage within the same population group. Patient data is an average
from a minimum of two cores per patient. AA designates amino acid
domain per each collagen. (B) Combined results for area under the
receiver operating curve showing significant discriminatory
difference in Appalachian stage I + II compared with stage III + IV
CRC. (C) Lower discriminatory difference in non-Appalachian stage I
+ II compared with stage III + IV CRC. (D) Combined results for
area under the receiver operating curve showing no discriminatory
difference in Appalachian compared with non-Appalachian in stage
I/II CRC. (E) Significant discriminatory differences when comparing
between Appalachian and non-Appalachian stage III + IV CRC.
*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. CRC,
colorectal cancer; App., Appalachian; non-App., Appalachian; ns,
not significant; Col, collagen; LN, natural log.

Figure 4

Complex peptide gradient patterns in
CRC resections by collagen targeted mass spectrometry imaging. (A)
Workflow took tissue sections from colorectal resections for
experiments by high mass accuracy, high mass resolution imaging
mass spectrometry. Image created using Biorender (biorender.com).
(B) Photomicrograph of sections from four colorectal resections
demonstrating different CRC features. (C) Total ion current over
all four sections showed 4,696 peptides. (D) A high level of
peptide complexity was found within a narrow mass window
representing unique image patterns within the four sections (scale
bar, 3 cm). (E) Heuristic peptide clustering of the 4,696 peak set
by image segmentation shows high definition of pathologically
defined regions found by hematoxylin and eosin pathology staining
(scale bar, 1 cm). (F) Principal components analysis of all spectra
(4,696 peak set) from pathological regions of tumor, mucosa and
muscularis demonstrates separation based on pathological region.
Component 1 represents 25.6% of variance derived from pathological
location. (G) Spatial mapping of components 1 through 3 further
confirmed distinct pathological regions defined by collagen peptide
regulation (scale bar, 1 cm). CRC, colorectal cancer; LC-MS/MS,
liquid chromatography-mass spectrometry.

Figure 5

Variation in CRC pathology dependent
on collagen peptide status of hydroxylated proline modification.
(A) Case studies of example peptides identified by sequencing
proteomics on the same tissue sections (G=grade). Each peptide
reports unique spatial patterns following tissue pathology. Images
are from high mass accuracy, high mass resolution imaging
experiments by Fourier Transform Ion Cyclotron Resonance mass
spectrometer. Parenthesis refers to site probability found by high
mass accuracy, high mass resolution sequencing proteomics. (B)
Peptide peak intensity varying by potential proline statin in
pathological regions or across each section. Peaks are defined by
difference in hydroxylated proline status within each peptide.
Window for peak selection from image data was ± 5 ppm. (C) Example
mapping of peaks defined by potential differences in proline status
defined by high mass accuracy. (D) Combined ion image showing
complementary peak distribution of a Col peptide with unmodified
status or hydroxylated proline status. CRC, colorectal cancer; Col,
collagen; HYP, hydroxylated proline.
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Copy and paste a formatted citation
Spandidos Publications style
Sougiannis AT, Taylor HB, Zambrzycki SC, Conroy L, Strubler R, Edge C, Drake RR, Wallace K, Allison D, Lee E, Lee E, et al: Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients. Oncol Lett 30: 413, 2025.
APA
Sougiannis, A.T., Taylor, H.B., Zambrzycki, S.C., Conroy, L., Strubler, R., Edge, C. ... Angel, P.M. (2025). Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients. Oncology Letters, 30, 413. https://doi.org/10.3892/ol.2025.15159
MLA
Sougiannis, A. T., Taylor, H. B., Zambrzycki, S. C., Conroy, L., Strubler, R., Edge, C., Drake, R. R., Wallace, K., Allison, D., Lee, E., Sun, R. C., Angel, P. M."Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients". Oncology Letters 30.3 (2025): 413.
Chicago
Sougiannis, A. T., Taylor, H. B., Zambrzycki, S. C., Conroy, L., Strubler, R., Edge, C., Drake, R. R., Wallace, K., Allison, D., Lee, E., Sun, R. C., Angel, P. M."Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients". Oncology Letters 30, no. 3 (2025): 413. https://doi.org/10.3892/ol.2025.15159
Copy and paste a formatted citation
x
Spandidos Publications style
Sougiannis AT, Taylor HB, Zambrzycki SC, Conroy L, Strubler R, Edge C, Drake RR, Wallace K, Allison D, Lee E, Lee E, et al: Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients. Oncol Lett 30: 413, 2025.
APA
Sougiannis, A.T., Taylor, H.B., Zambrzycki, S.C., Conroy, L., Strubler, R., Edge, C. ... Angel, P.M. (2025). Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients. Oncology Letters, 30, 413. https://doi.org/10.3892/ol.2025.15159
MLA
Sougiannis, A. T., Taylor, H. B., Zambrzycki, S. C., Conroy, L., Strubler, R., Edge, C., Drake, R. R., Wallace, K., Allison, D., Lee, E., Sun, R. C., Angel, P. M."Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients". Oncology Letters 30.3 (2025): 413.
Chicago
Sougiannis, A. T., Taylor, H. B., Zambrzycki, S. C., Conroy, L., Strubler, R., Edge, C., Drake, R. R., Wallace, K., Allison, D., Lee, E., Sun, R. C., Angel, P. M."Differential extracellular matrix proteomic signatures in colorectal tumors from Appalachian and non‑Appalachian patients". Oncology Letters 30, no. 3 (2025): 413. https://doi.org/10.3892/ol.2025.15159
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