Fig 2: ADAM8 is expressed in all breast cancer subtypes. Freshly cut consecutive TMA slides containing breast cancer patient primary tumor samples were subjected to IHC with ADP2 vs isotype-matched control IgG2b at 1:50 dilutions. H&E staining was used to confirm tissue quality and exclude depleted samples. A Representative images of ADP2-stained TNBC samples with increasing ADAM8 expression and associated H-scores are shown. Staining with IgG2b was negative as expected (not shown). Upper panels: whole core images, scale bar: 200 µm; lower panels: magnified images of same samples, scale bar: 50 µm. B Representative images of ADP2 IHC staining for ADAM8 in breast cancer patient samples of non-TNBC HR-/HER2+, HR+/HER2- and HR+/HER2+ subtypes. Specimens with high H-scores (e.g., 250–300) are shown. Upper panels: whole core images, scale bar: 200 µm; lower panels: magnified images of same samples, scale bar: 50 µm

Fig 3: The ADP2 IHC assay detects a range of ADAM8 levels with high specificity and reproducibility. A ADP2 staining is specific for ADAM8 in competition analyses. ADP2 was pre-incubated overnight at 4 °C at 1:1000 dilution in the absence or presence of 1×, 10× or 100× molar equivalents of purified recombinant human ADAM8 protein (rHuA8), and used in IHC of HEK-A8-2D and MB-231-3D cells. IgG2b was employed as isotype control. ADP2 staining was reduced in a dose-dependent manner in the presence of rHuA8, demonstrating the assay’s specificity for ADAM8. B ADP2 has excellent range and linearity of ADAM8 detection. IHC was performed using slides of the breast CCM and a range of ADP2 dilutions from 1:50 to 1:120,000 under the optimized ADP staining conditions vs isotype control IgG2b at 1:50. ADP2 detected both very low and very high levels of ADAM8 in a dose-dependent manner. C ADP2 detects ADAM8 in PDX tumor tissue samples at levels that are within the range of the CCM. IHC was performed using ADP2 at dilutions of 1:50, 1:100 and 1:500 vs isotype control IgG2b at 1:50 and ADAM8-expressing TNBC PDX samples 5998, 3561, and 4849 vs the breast CCM. All 3 PDX samples displayed strong ADAM8 staining that was in the range of that seen in the breast lines of the CCM, indicating the latter is suitable for scoring of more complex tissue samples. Representative images of staining with ADP2 1:50 dilution, which was identified as optimal for tissue sample analysis, are shown. D ADP2 reproducibly detects ADAM8 in PDX tumor tissue samples. Consecutively cut slide sets of each PDX were processed on different days (3–4 days apart), and demonstrated equal staining, indicating the reproducibility of the ADP2 IHC assay. Representative images of PDX3561 are shown. Images are at 40× magnification. MB-231, MDA-MB-231

Fig 4: Development of an anti-ADAM8 ADP2 IHC assay and breast CCM. A ADAM8 protein levels in breast lines. Protein extracts (30 µg) from breast lines MCF10A, SUM149, MB-231 and MB-231-LUC, and control lines HEK-A8 and HEK-EV, grown in 2D or 3D as indicated, were subjected to immunoblotting with an anti-ADAM8 antibody (LS-C20181), which detected precursor (Proform) and active ADAM8; ß-actin was used as loading control. A representative blot of two independent experiments with similar results is shown. To create a breast CCM with a gradient of active ADAM8, MCF10A-2D, MB-231-2D and MB-231-3D were selected; HEK-EV-2D and HEK-A8-2D were included as negative and positive controls, respectively. B ADPs did not detect ADAM8 in breast lines under IHC conditions optimized for the commercial anti-human ADAM8 LS-B4068 antibody. CCM slides were subjected to IHC using conditions for the rabbit LS-B4068 antibody and either LS-B4068 or our newly developed ADP antibodies. Representative images of staining in HEK-A8-2D and MB-231-3D cells are shown. Pink color is due to Matrigel used (only) in this early CCM. C–E ADP2 under ADP-optimized conditions provides superior ADAM8 detection compared to LS-B4068. CCM IHC staining was performed using the final optimized conditions for the ADP mAbs and either our top diagnostic mAb ADP2 (1:100) or IgG2b (1:100) isotype-matched control for non-specific staining (C). The CCM breast lines (red box) displayed ADAM8 levels consistent with those seen in Western blotting (Part A). Values given below were determined by densitometry of immunoblots with extracts from independent cultures loaded at various concentrations to ensure a linear range of quantitation, and are presented relative to MCF10A (set to 1.0). IHC of the CCM with LS-B4068 (1:50) and its optimal conditions shows weaker staining than that observed with ADP2 (D). IHC of CCM with LS-B4068 (1:100) using ADP conditions shows poorer staining than with its optimal conditions (E). Images are at 40× magnification. MB-231, MDA-MB-231