Figure 2 from Anti-EGFR Antibody–Drug Conjugate Carrying an Inhibitor Targeting CDK Restricts Triple-Negative Breast Cancer Growth
Authors: Cheung Anthony , Chenoweth Alicia M. , Johansson Annelie , Laddach Roman , Guppy Naomi , Trendell Jennifer , Esapa Benjamina , Mavousian Antranik , Navarro-Llinas Blanca , Haider Syed , Romero-Clavijo Pablo , Hoffmann Ricarda M. , Andriollo Paolo , Rahman Khondaker M. , Jackson Paul , Tsoka Sophia , Irshad Sheeba , Roxanis Ioannis , Grigoriadis Anita , Thurston David E. , Lord Christopher J. , Tutt Andrew N. J. , Karagiannis Sophia N. 
Published: August 2024

<p>Single-cell RNA sequencing and spatial transcriptomic analyses reveal cellular and spatial co-expression of EGFR with CDK2/cyclin E in TNBCs. <b>A,</b> Dimensionality reduction <i>t</i>-distributed stochastic neighbor embedding (<i>t</i>-SNE) map of combined scRNA-seq transcriptomes of 150,290 cells from 27 untreated primary tumors (TNBC, <i>n</i> = 8 samples, 54,819 cells; HER2+, <i>n</i> = 6 samples, 31,917 cells; ER+, <i>n</i> = 13 samples, 63,554 cells) colored by cell cluster (<a href="#bib32" target="_blank">32</a>). <i>EpCAM</i> expression revealed two prominent <i>EpCAM</i>+ tumor epithelial clusters in each cancer subtype (dotted lines). <i>t</i>-SNE plots showing expression levels of <i>EGFR</i>, <i>CCNE1</i>, and <i>CDK2</i> genes for the same clusters. Red: high expression, gray: not detected. Overall expression of <i>CCNA1</i> was too low and was excluded from the analysis. <b>B,</b> Analyses of the transcriptomic data were conducted to evaluate expression of <i>EGFR</i>, <i>CCNE1</i>, and <i>CDK2</i> genes per patient and for each patient cohort (TNBC, HER2+, and ER+). Heatmap (top) with color boxes indicating normalized expression level; each column represents a patient tumor. Quantitative analysis (bottom) was calculated by the number of cells expressing <i>EGFR</i>, <i>CCNE1</i>, or <i>CDK2</i>, in proportion to <i>EpCAM</i>+ cells. <b>C,</b> Same analysis was performed on <i>EpCAM</i>+ <i>EGFR</i>+ cells for the expression of <i>CCNE1</i> and <i>CDK2</i>. <i>P</i> values determined by two-tailed unpaired <i>t</i> test against TNBC subtype. <b>D,</b> Spatial transcriptomic analysis of <i>EGFR</i>,<i> CCNE1</i>, and<i> CDK2 </i>expression in <i>EpCAM</i>+ cell clusters of 43 tumor sections from 22 patients with TNBC (<a href="#bib33" target="_blank">33</a>), where 26 sections were from 13 treatment-naïve patients and 17 sections were from nine residual TNBCs. Tissue architecture integrity in these sections was confirmed by hematoxylin and eosin staining (left). The color scale represents log-transformed normalized gene expression (red, highest; blue, lowest). Representative spatial mapping revealed a consistent pattern of <i>EGFR</i> expression in chemo-naïve and post-NAC-resistant TNBCs, and its spatial relationships with <i>CCNE1</i> and <i>CDK2</i> co-expression in tumor sections. Venn diagrams illustrate quantitative analyses for the relationships among <i>EGFR</i>, <i>CCNE1</i>, and <i>CDK2</i> co-expression (numbers in black represent the number of spatial clusters for all patients in the cohort, and % marked in red represent the proportion of these spatial clusters within all <i>EpCAM</i>+ clusters).</p>