Fig 2: Core-to-core comparison of clinical and CyCIF antibodies against ER, PR, and HER2.To qualify breast cancer-related antibodies, the BC03 TMA, representing 16 breast tumors in duplicate was used. a–c CyCIF was performed using the qualified CyCIF antibody against a single antibody commonly used in clinical practice as a reference for ER (a), PR (b), and HER2 (c). The left graph depicts a single-cell dot-plot between the clinical clone on the x axis and the validated CyCIF antibody on the y axis. Each dot represents single-cell fluorescent intensity values from the two antibodies. Dashed lines indicate the gating cutoffs. The middle graph shows the corresponding mean log intensity of the core-to-core analysis of the clinical and CyCIF antibodies. The single-cell data were collected for individual TMA cores, with a binary gate applied to obtain the positive signal of each core (range from 0–1). The X- & Y axis represent the positive score calculated from either clinical or CyCIF antibodies, respectively. The right graph shows positivity scores (number of positive cells over total cells) for the clinical and CyCIF antibodies by TMA case. d, e Cross-assay comparison of the clinical and CyCIF antibodies analyzed by CyCIF compared to the clinical antibody analyzed by IHC using Aperio software for ER (d) and HER2 (e). Left, dot-plot representation of two different scores obtained from CyCIF and from IHC-Aperio. CyCIF of clinical (green dots) and CyCIF antibodies (blue dots) were used on the same section, while IHC was performed on a different section from the same TMA block. Each dot represents a single core from BC03 TMA. CyCIF scores are plotted on y axis as positive ratio of immunofluorescence, IHC scores on x axis are plotted as the percent of positive cells. Right graph, quantitative assessment of ER and HER2 IHC versus CyCIF staining. IHC scores by Aperio were used to stratify (0–24, 25–49, 50–74, 75–100) different TMA cores/cases, and the mean intensities of CyCIF antibody staining from each TMA core are shown using boxplot analysis. CyCIF antibodies: ER (CST 74244 S) and HER2 (ab225510).

Fig 3: Overview of fluorescent CyCIF antibody qualification against antibodies used in the clinical laboratory.To qualify breast cancer-related antibodies HER2, ER, PR, AR, and p53, the BC03 tissue microarray (TMA), which represents 16 breast tumors in duplicate, was used. Multiple CyCIF antibodies were compared to a single antibody commonly used in clinical practice as a reference. a Schematic representation of the different levels of fluorescent antibody validation using the CyCIF method, starting from tissue staining (lowest level of validation) towards patient-level (highest level) inter-assay comparison (i.e., direct comparison of each patient tissue to itself between assays). b–f Representative CyCIF images of antibodies tested by CyCIF on the BC03 TMA. Asterisks indicate clinical antibodies (*) and qualified CyCIF antibodies (**) for each target. g Representative CyCIF image of HER2 (TF-MA5-14509; sp3) and ER (CS98710) staining, showing the majority of tumor cells are ER+, and some showing strong, membrane staining for HER2. Left image is a full TMA core (36× mag.); the right image corresponds to the left image (74× mag.).

Fig 4: FAXDC2 is downstream of Wnt/β-catenin in the regulation of RTK signaling.(A) Stabilized β-catenin prevents multiple RTKs from moving to the cell surface upon treatment with ETC-159. Cell surface abundance of EGFR, ERBB2, and EPHA2 was assessed as before in HPAF-II cells with or without stabilized β-catenin. Cells were treated with DMSO or 100 nM ETC-159 for 72 hours before staining with fluorescent tagged antibodies. Each histogram represents ~50,000 cells. Data are representative of 3 independent experiments, and 2 replicates are shown. (B) Knockdown of FAXDC2 prevents Wnt inhibition–mediated increase in EGFR cell surface abundance. HPAF-II cells were transfected with a pool of 4 siRNAs or 2 independent siRNAs (si6 and si7) against FAXDC2 for 24 hours, followed by treatment with ETC-159 for 72 hours. The cells were then stained with Alexa Fluor 488–conjugated anti-EGFR antibody and analyzed by flow cytometry. Data are representative of 3 independent experiments, and 2 replicates are shown. (C and D) Knockout of FAXDC2 in HPAF-II tumors prevents Wnt inhibition–mediated increase in tyrosine phosphorylation and p-EPHA2 levels. Protein lysates from HPAF-II or FAXDC2-KO tumor xenografts from vehicle- or ETC-159–treated mice were separated on 10% SDS gels and transferred to a PVDF membrane. Membranes were probed with anti-phosphotyrosine or –p-EphA2 antibodies. Each lane represents lysate from an individual tumor. (E) Overexpression of FAXDC2 in FAXDC2-KO tumors rescues the Wnt inhibition–mediated increase in tyrosine phosphorylation. Protein lysates from FAXDC2-KO or FAXDC2-OE tumor xenografts from vehicle- or ETC-159–treated mice were probed with phosphotyrosine antibodies. Each lane represents lysate from an individual tumor. (F–H) FAXDC2 knockout prevents the Wnt inhibition–mediated increase in EGFR and Eph family receptor tyrosine kinase abundance. Protein lysates from HPAF-II or FAXDC2-KO tumors from mice treated with vehicle or ETC-159 were analyzed as in C. Data are shown from 2 independent FAXDC2-KO clones, clone 3 (F and G) and clone 12 (H). Each lane represents lysate from an individual tumor.

Fig 5: Inter-assay analysis of HER2 enriched TMAs (TMAs 226 and 227).Following the selection of qualified ER, PR, and HER2 antibodies, two HER2-enriched TMAs, which included 567 tissue cores (representing 189 patients in triplicate), were used to further qualify CyCIF antibodies. a, b Percent of ER+ and HER2+ cells assessed through CyCIF (y axis) is compared to the score assigned by a clinical pathologist (x-axis) for each TMA. c Cross-assay comparison of the HER2 clinical and CyCIF antibodies analyzed by CyCIF compared to the clinical antibody analyzed by IHC using Aperio software. Left, dot-plot represents two different scores obtained from CyCIF and one obtained from IHC-Aperio. CyCIF of clinical (green dots) and CyCIF (blue dots) antibodies were used on the same section, while IHC was done on a different section from the same TMA block. Each dot represents a single core from BC03 TMA. CyCIF scores are plotted on y axis as positive ratio of immunofluorescence, IHC scores on x axis plotted as percent of positive cells. Right, quantitative assessment HER2 IHC versus CyCIF staining. IHC scores by Aperio were used to stratify (0–24, 25–49, 50–74, 75–100) different TMA cores/cases, and the mean intensities of CyCIF antibody staining from each TMA core are shown using boxplot analysis. d Clinically annotated HER2 FISH scores against IF/CyCIF staining using the SP3 antibody (Pearson r = 0.71) and HER2 FISH scores against IF/CyCIF staining using the CyCIF antibody, ab225510 (Pearson r = 0.65). Individual patients are shown in different colors, in triplicate. The triplicate cores tend to cluster together, indicating minimal variation.