Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
[Preprint]. 2024 Aug 10:2024.08.09.607341.
doi: 10.1101/2024.08.09.607341.

dia-PASEF Proteomics of Tumor and Stroma LMD Enriched from Archived HNSCC Samples

Aswini Panigrahi  1 , Allison L Hunt  2 , Diego Assis  3 , Matthew Willetts  3 , Bhaskar V Kallakury  4 , Bruce Davidson  5 , Thomas P Conrads  2 , Radoslav Goldman  1   6
Affiliations

dia-PASEF Proteomics of Tumor and Stroma LMD Enriched from Archived HNSCC Samples

Aswini Panigrahi et al. bioRxiv. .

Update in

Abstract

We employed laser microdissection to selectively harvest tumor cells and stroma from the microenvironment of formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. The captured HNSCC tissue fractions were analyzed by quantitative mass spectrometry-based proteomics using a data independent analysis approach. In paired samples, we achieved excellent proteome coverage having quantified 6,668 proteins with a median quantitative coefficient of variation under 10%. We observed significant differences in relevant functional pathways between the spatially resolved tumor and stroma regions. Our results identified extracellular matrix (ECM) as a major component enriched in the stroma, including many cancer associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from very low starting input in a scalable format that is useful to decipher the alterations in tumor and the stromal microenvironment. Correlating such results with clinical features or disease progression will likely enable identification of novel targets for disease classification and interventions.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest AP, ALH, BVK, BD, TPC, RG declare that they have no known competing financial interests. DA, MW are employees of Bruker Scientific.

Figures

Figure 1.
Figure 1.
Representative (A) H&E stained FFPE tissue section, and (B) image after LMD capture of the tumor regions. Base peak chromatograms showing the overlay of triplicate runs of an enriched (C) tumor and (D) paired stroma sample. (E) Graphical representation of number of proteins identified with 2 or more peptide matches in the enriched tumor and stroma samples. The median is indicated by a line. (F) Schematic representation of CV value spread of quantitation for each sample, median (bold line) and upper and lower quartile (thin line) are indicated.
Figure 2.
Figure 2.
Functional association of upregulated proteins. (A) Unsupervised hierarchical cluster analysis of stroma and tumor region proteins based on abundance, and (B) Volcano plot of protein abundance. In volcano plot the box dot plots of significantly changed proteins between the regions are indicated, and selected candidates are in red. Selected enriched functional pathways (String analysis) in stroma (B) and tumor (C) are shown with enrichment score and false discovery rate (FDR).
Figure 3.
Figure 3.
High confidence STRING network (A) functional and physical association of upregulated CAF markers in stroma, physical association of upregulated stromal (B) heparin binding and (C) GAG binding proteins.
See this image and copyright information in PMC

References

    1. Barsouk Adam, Aluru JS, Rawla P, Saginala K, Barsouk Alexander. 2023. Epidemiology, Risk Factors, and Prevention of Head and Neck Squamous Cell Carcinoma. Med Sci (Basel) 11:42. doi:10.3390/medsci11020042 - DOI - PMC - PubMed
    1. Demichev V, Szyrwiel L, Yu F, Teo GC, Rosenberger G, Niewienda A, Ludwig D, Decker J, Kaspar-Schoenefeld S, Lilley KS, Mülleder M, Nesvizhskii AI, Ralser M. 2022. dia-PASEF data analysis using FragPipe and DIA-NN for deep proteomics of low sample amounts. Nat Commun 13:3944. doi:10.1038/s41467-022-31492-0 - DOI - PMC - PubMed
    1. Huang C, Chen L, Savage SR, Eguez RV, Dou Y, Li Y, da Veiga Leprevost F, Jaehnig EJ, Lei JT, Wen B, Schnaubelt M, Krug K, Song X, Cieślik M, Chang H-Y, Wyczalkowski MA, Li K, Colaprico A, Li QK, Clark DJ, Hu Y, Cao L, Pan J, Wang Y, Cho K-C, Shi Z, Liao Y, Jiang W, Anurag M, Ji J, Yoo S, Zhou DC, Liang W-W, Wendl M, Vats P, Carr SA, Mani DR, Zhang Z, Qian J, Chen XS, Pico AR, Wang P, Chinnaiyan AM, Ketchum KA, Kinsinger CR, Robles AI, An E, Hiltke T, Mesri M, Thiagarajan M, Weaver AM, Sikora AG, Lubiński J, Wierzbicka M, Wiznerowicz M, Satpathy S, Gillette MA, Miles G, Ellis MJ, Omenn GS, Rodriguez H, Boja ES, Dhanasekaran SM, Ding L, Nesvizhskii AI, El-Naggar AK, Chan DW, Zhang H, Zhang B, Clinical Proteomic Tumor Analysis Consortium. 2021. Proteogenomic insights into the biology and treatment of HPV-negative head and neck squamous cell carcinoma. Cancer Cell 39:361–379.e16. doi:10.1016/j.ccell.2020.12.007 - DOI - PMC - PubMed
    1. Hunt AL, Bateman NW, Barakat W, Makohon-Moore S, Hood BL, Conrads KA, Zhou M, Calvert V, Pierobon M, Loffredo J, Litzi TJ, Oliver J, Mitchell D, Gist G, Rojas C, Blanton B, Robinson EL, Odunsi K, Sood AK, Casablanca Y, Darcy KM, Shriver CD, Petricoin EF, Rao UNM, Maxwell GL, Conrads TP. 2021. Extensive three-dimensional intratumor proteomic heterogeneity revealed by multiregion sampling in high-grade serous ovarian tumor specimens. iScience 24:102757. doi:10.1016/j.isci.2021.102757 - DOI - PMC - PubMed
    1. Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. 2020. Head and neck squamous cell carcinoma. Nat Rev Dis Primers 6:1–22. doi:10.1038/s41572-020-00224-3 - DOI - PMC - PubMed

Publication types

LinkOut - more resources