phenocycler instrument  (Akoya Biosciences)

Journal: bioRxiv

Article Title: Single-cell spatial multiomics identifies POSTN + CAFs mediating chemoradiotherapy resistance in rectal cancer

doi: 10.64898/2026.04.30.721803

Figure Lengend Snippet: a, Schematic illustration of the neoadjuvant chemoradiotherapy (CRT) schedule and timing of tumor sampling in the study cohort (N = 24). The CRT regimen consisted of capecitabine in combination with 50.4 Gy of radiation delivered in 28 fractions. “Pre” refers to the time point prior to CRT, “JustAfter” indicates within 2 weeks after the completion of CRT, and “Resection” represents three months post-CRT. Biopsy samples were collected from all 24 patients at the Pre time point and from 4 patients at the JustAfter time point, and surgical samples were obtained from all 24 patients at the Resection time point. Among the 24 patients, 8 achieved a pathological complete response (pCR), 8 achieved a major pathological response (MPR), and 8 were classified as no response (NR). b, Each sample obtained at the designated time points was serially sectioned into four parts (Sections 1–4). Section 1 was used for Xenium analysis, PhenoCycler analysis, and hematoxylin and eosin (H&E) staining. Section 2 was used for Xenium analysis and H&E staining. Section 3 was used for Visium HD spatial transcriptomics on adjacent serial sections. Section 4 was used for whole-exome sequencing. c, Summary matrix showing patient characteristics (age, sex, recurrence status) and the availability of each modality. The purple circles represent samples analyzed at both the Pre and Resection time points, whereas the dark circles indicate samples analyzed at all three time points: Pre, JustAfter, and Resection.

Article Snippet: Multiplexed immunofluorescence staining and imaging were performed using the PhenoCycler platform (Akoya Biosciences).

Techniques: Sampling, Staining, Spatial Transcriptomics, Sequencing

Journal: bioRxiv

Article Title: Single-cell spatial multiomics identifies POSTN + CAFs mediating chemoradiotherapy resistance in rectal cancer

doi: 10.64898/2026.04.30.721803

Figure Lengend Snippet: a, Line plots showing the number of cells adjacent to tumor cells across various subclusters. Each line color represents one of the eight NR patients. Subclusters on the x-axis are ordered by descending mean adjacency across the NR cohort. b, Schematic illustration of neighborhood enrichment analysis. Tumor microenvironment (TME) regions within 30 μm of tumor cells were extracted from Xenium data, and spatial neighborhood patterns were analyzed using the SpatialKNifeY (SKNY) algorithm . c, Upper triangular clustered heatmap representing the spatial proximity among subclusters within 30 μm of tumor cells. Hierarchical clustering was performed using Ward’s algorithm with the Euclidean distance. The heatmap color intensity (red scale) indicates the average proportion of adjacent cell pairs across the eight NR patients. The categorical annotation bar represents major cell types, whereas the grayscale bar indicates the average number of tumor-adjacent cells per subcluster in the NR group. The blue dendrogram shows the distance based on the similarity of adjacency counts among different cell types. d, Spatial localization of NR-enriched CAFs and myeloid cells identified by Xenium (left), corresponding protein expression patterns determined by PhenoCycler on the same tissue sections (middle), and magnified views (right). In the Xenium panel, purple indicates tumor cells, yellow indicates NR CAFs, and green indicates NR myeloid cells. PhenoCycler images show the expression of DAPI, EPCAM, αSMA, CD68, PDPN, and IDO1. e, Representative protein expression visualized with the PhenoCycler platform. The upper row shows CAF-related markers (αSMA, collagen I, galectin, vimentin, and PDPN), and the lower row shows macrophage-related markers (CD68, CD14, and IDO1).

Article Snippet: Multiplexed immunofluorescence staining and imaging were performed using the PhenoCycler platform (Akoya Biosciences).

Techniques: Expressing

Journal: bioRxiv

Article Title: Single-cell spatial multiomics identifies POSTN + CAFs mediating chemoradiotherapy resistance in rectal cancer

doi: 10.64898/2026.04.30.721803

Figure Lengend Snippet: a, Spatial visualization of nonresponder cancer-associated fibroblasts (NR CAFs) within both tumor and normal epithelial regions. The top-left panel shows a hematoxylin and eosin (H&E)-stained image merged with the spatial distribution of NR CAFs (red, orange, and yellow). The right panels show magnified views of the normal epithelial region (top) and the tumor region (bottom). The bottom-left violin plot displays the density of NR CAFs within 30 μm of tumor and normal epithelial cells (see Methods). Each line represents one of the eight NR patients. The P value was calculated using the paired t test. b, Spatial distribution of tumor cells (black), normal epithelial cells (gray), NR CAFs (red), and fibroblasts (blue) in patients Pt-2 and Pt-7. The data for the remaining six NR patients are shown in Supplementary Fig. 5. c, Summary schematic of the comparative analyses. To characterize the gene expression profiles and morphological features of NR CAFs, fibroblasts adjacent to the normal epithelia of NR group patients (n = 8) and pCR-dominant CAFs from pCR group patients (n = 8; see ) were used as controls. Comparative analyses included morphology (H&E staining), protein expression (PhenoCycler), gene expression and spatial distribution (Xenium), and survival outcomes (relapse-free survival and overall survival). d, Comparison among fibroblasts, NR CAFs, and pCR CAFs across multiple modalities: row 1, H&E staining; rows 2–3, Xenium-based spatial distribution; rows 4–8, protein expression by PhenoCycler. The green, red, and blue arrowheads indicate fibroblasts, NR CAFs, and pCR CAFs, respectively. e, f, Differential gene expression (DEG) analysis comparing fibroblasts (control) and NR CAFs (e) and pCR CAFs (control) and NR CAFs (f). Volcano plots show genes upregulated in NR CAFs (red) and controls (fibroblasts, green; pCR CAFs, blue). The DEG thresholds were set to P < 1×10⁻¹⁰ and |log₂ fold change| > 1. g, Pathway enrichment analysis using the Reactome pathway database. The color scale indicates -log₁₀(FDR). The size of the dots indicates the overlap between the upregulated genes and each pathway. h, Comparison of spatial localization across fibroblasts, NR CAFs, and pCR CAFs. Row 1: H&E staining; row 2: spatial distribution and merged images from Xenium; rows 3–5: Xenium-based spatial localization of SFRP1, POSTN, and MMP11 with H&E overlay. Fibroblast and pCR CAF regions were taken from adjacent areas within the same tissue section. i, Kaplan–Meier curve of relapse-free survival in patients grouped by total POSTN expression per tissue section (high versus low). j, Kaplan–Meier curve of overall survival in the TCGA CRC cohort stratified by POSTN expression. The red and blue lines indicate the high-and low-expression groups, respectively. P values were calculated using the log-rank test.

Article Snippet: Multiplexed immunofluorescence staining and imaging were performed using the PhenoCycler platform (Akoya Biosciences).

Techniques: Staining, Gene Expression, Expressing, Comparison, Control