Fig 2: Multi-scale imaging of t-CyCIF specimens.(A) Bright-field H&E image of a metastasectomy specimen that includes a large metastatic melanoma lesion and adjacent benign tissue. The H&E staining was performed after the same specimen had undergone t-CyCIF. (B) Representative t-CyCIF staining of the specimen shown in (A) stitched together using the Ashlar software from 165 successive CyteFinder fields using a 20X/0.8NA objective. (C) One field from (B) at the tumor-normal junction demonstrating staining for S100-postive malignant cells, α-SMA positive stroma, T lymphocytes (positive for CD3, CD4 and CD8), and the proliferation marker phospho-RB (pRB). (D) A melanoma tumor imaged on a GE INCell Analyzer 6000 confocal microscope to demonstrate sub-cellular and sub-organelle structures. This specimen was stained with phospho-Tyrosine (pTyr), Lamin A/C and p-Aurora A/B/C and imaged with a 60X/0.95NA objective. pTyr is localized in membrane in patches associated with receptor-tyrosine kinase, visible here as red punctate structures. Lamin A/C is a nuclear membrane protein that outlines the vicinity of the cell nucleus in this image. Aurora kinases A/B/C coordinate centromere and centrosome function and are visible in this image bound to chromosomes within a nucleus of a mitotic cell in prophase (yellow arrow). (E) Staining of a melanoma sample using the GE OMX Blaze structured illumination microscope with a 60X/1.42NA objective shows heterogeneity of structural proteins of the nucleus, including as Lamin B and Lamin A/C (indicated by yellow arrows) and part of the nuclear pore complex (NUP98) that measures ~120 nm in total size and indirectly allows the visualization of nuclear pores (indicated by non-continuous staining of NUP98). (F) Staining of a patient-derived mouse xenograft breast tumor using the OMX Blaze with a 60x/1.42NA objective shows a spindle in a mitotic cell (beta-tubulin in red) as well as vesicles staining positive for VEGFR2 (in cyan) and punctuate expression of the EGFR in the plasma membrane (in green).

Fig 3: Spatial distribution of immune infiltrates and checkpoint proteins.(A) Low-magnification image of a clear cell renal cancer subjected to 12-cycle t-CyCIF (see Supplementary file 4 for a list of antibodies). Regions high in α-smooth muscle actin (α-SMA) correspond to stromal components of the tumor, those low in α-SMA represent regions enriched for malignant cells. (B) Representative images from selected t-CyCIF channels are shown. (C) Quantitative assessment of total lymphocytic cell infiltrates (CD3+ cells), CD8+ T lymphocytes, cells expressing PD-1 or its ligand PD-L1 or the VEGFR2 for the entire tumor or for α-SMAhigh and α-SMAlow regions. VEGFR2 is a protein primarily expressed in endothelial cells and is targeted in the treatment of renal cell cancer. The error bars represent the S.E.M. derived from 100 rounds of bootstrapping. (D) Density plot for CD3 and CD8 expression on single cells in the tumor (left) or stromal domains (right). (E) Centroids of CD3+ or CD3+CD8+ cells in blue or dark blue as well as cells staining as SMAhigh or SMAlow (gray and light-gray, respectively) used to define the stromal and tumor regions. (F) Centroids of PD-1+ and PD-L1+ cells are shown in red and green, respectively. (G) Results of a K-nearest neighbor algorithm used to compute areas in which PD-1+ and PD-L1+ cells lie within ~10 µm of each other and with high spatial density (in yellow) and thus, are potentially positioned to interact at a molecular level.10.7554/eLife.31657.029Figure 9—source data 1.Immune cell counts from bootstrapping in tumor and stroma regions (Figure 9C).10.7554/eLife.31657.030Figure 9—source data 2.Single-cell intensity data used in Figure 9.

Fig 4: The effects of aprepitant, ERS-Apr, and PLGA-Apr concentrations in M2c co-culture on HUVEC VEGFR-2 protein levels (objective: 10X) (A). The slope graph drawn is based on immunofluorescence intensity measurement results obtained from morphological imaging (B). The graph represents the mean ± standard deviation of immunofluorescence intensity data (n = 3), normalized to cell numbers. Statistical analysis was conducted using a one-way ANOVA followed by a post hoc Tukey test for multiple comparisons (significant difference compared to control: ****p < 0.0001).