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Additional file 1: Supplemental Figure 1. Example Distribution of Biomarker Expression. A) Each symbol represents the expression level of each individual cell identified in the final well after CTC capture and staining. Lines on graphs indicate thresholds used to define positive expression. Red symbols indicate cells identified as CTCs, and black or grey symbols represent background non-CTCs that carried over into the final well. Data represents all cells identified from one sample from patient 22. B) Autofluorescent signal detected on all cells after processing with hoechst only and C) side-by-side comparison of the same two patients with all stains included. Supplemental Figure 2. Assessment of Spectral Overlap. Absence of spectral overlap was confirmed by acquiring images of either compensation beads (UltraComp eBeads) or cell line cells (H358) that were stained with each of the fluorophores associated with the patient sample antibody set. Briefly, beads were stained with one of each fluorescent antibody in addition to a BV421 tag to enable automated algorithm-based image analysis. Cell line H358 cells were stained with hoechst to evaluate any spectral overlap due to the hoechst stain. Images of beads and cells were acquired with all wavelengths used in the patient sample antibody set, acquiring the wavelengths in the same order as patient sample imaging. Each symbol represents the average intensity of all beads within one image, with n = 3 replicate images acquired for each condition. Controls of beads with BV421 alone, beads without any fluorescent tag, and cells without hoechst were used to quantify background intensity. Supplemental Figure 3. Accuracy of Quantifying Positive Biomarker Expression. LNCaP and H358 cell line cells were mixed together at different ratios prior to staining and image analysis (100:0, 60:40, 40:60, 0:100), then quantified for the number of either PD-L1+ or HLA I+ cells within each condition. The number of expected positive cells was calculated by multiplying the number of each cell line in the final mixture by the frequency of positivity quantified from the pure populations. The observed number showed a linear relationship with the expected number (R2 = 0.9981 for PD-L1 and 0.9993 for HLA I). Supplemental Table 1. Serial CTC Evaluation From Multiple Patients. Serial evaluation of CTC number and protein expression compared to radiographic assessment in three additional patients. DP: Double-positive, DN: Double-negative, Nivo: Nivolumab, Atezo: Atezolizumab, Pembro: Pembrolizumab, R: Responding, S: Stable, P: Progressive Disease. Supplemental Table 2. Clinical Details. Patient clinical characteristics are summarized here, including age, histology, treatments rendered (including systemic therapy, surgical resection, and/or radiotherapy), pertinent genomic alterations, tissue tumor mutation burden (if known), and tissue PD-L1 expression. All patients in our cohort had a diagnosis of stage IV metastatic non-small cell lung cancer. Np indicates that the test was not performed. An asterisk in the other genomic alterations column indicates that alterations were evaluated using a limited lung-cancer specific panel – alterations in all other patients were identified using broad next-generation sequencing panels. Adeno: adenocarcinoma, NOS: not otherwise specified, N: Nivolumab, A: Atezolizumab, P: Pembrolizumab, Ch: Chemotherapy, Ch/IO: Chemoimmunotherapy, TKI: Tyrosine kinase inhibitor, XRT: radiation therapy, LN: lymph node. Supplemental Figure 4. Correlation Between Liquid and Solid Biopsy. CTC characterization at baseline compared to the tissue biopsy results in our patient cohort. Percentage of PD-L1 positive tumor cells on tissue biopsy were compared to percentage of PD-L1 positive CTCs (A) or the log mean fluorescence intensity of PD-L1 on CTCs (B). There is no correlation between tissue and CTC PD-L1 expression on either parameter. The number of CTCs detected in each patient sample was labeled next to each symbol. Supplemental Figure 5. Clinical Correlation of Tissue or Baseline CTC Evaluation on Prolonged Response. Solid tumor biopsy PD-L1 levels and baseline liquid biopsy PD-L1 and HLA I parameters were compared for the ability to predict progression-free survival (PFS) of less than or greater than 6 months. T-tests (all assuming normal distribution except total #CTC which did not) were used to assess the ability of each of the different characteristics to differentiate between patients with shorter or longer PFS (> 6 months). Kaplan-Meier curves were generated for tissue PD-L1, with a cutoff of 1% positivity, and for average CTC PD-L1, with a cutoff of 1.5.

Supplemental material, supplemental_material_for_SASCA_by_Tokar_and_Stahlfeld_et_al for Pairing Microwell Arrays with an Affordable, Semiautomated Single-Cell Aspirator for the Interrogation of Circulating Tumor Cell Heterogeneity by Jacob J. Tokar, Charlotte N. Stahlfeld, Jamie M. Sperger, David J. Niles, David J. Beebe, Joshua M. Lang and Jay W. Warrick in SLAS Technology