Supplementary Figure 1. Individual and expanded images of DAPI, Cytokeratin and CD45 from Figure 2. Supplementary Figure 2. Determining the thresholds for scoring PD-L1 expression in circulating cells. Supplementary Figure 3. Average PD-L1 and RAD50 changes in each individual patient before and after induction of radiotherapy, separated by cell type. Supplementary Figure 4. Confocal imaging of RAD50 within the nuclei of a cluster of EMTCTCs from Figure 1, imaged top to bottom. Supplementary Figure 5. Distribution of RAD50 foci in cells by stage and before treatment or after treatment. Supplementary Figure 6. Percent change in pixel intensity from the T0 time point to the T1 time point. Supplementary Table 1. Patient population overview Supplementary Table 2. Tracking the longevity of PD-L1 expression levels after completion of radiotherapy.
ARTICLE ABSTRACTPurpose: Evidence suggests that PD-L1 can be induced with radiotherapy and may be an immune escape mechanism in cancer. Monitoring this response is limited, as repetitive biopsies during therapy are impractical, dangerous, and miss tumor stromal cells. Monitoring PD-L1 expression in both circulating tumor cells (CTCs) and circulating stromal cells (CStCs) in blood-based biopsies might be a practical alternative for sequential, noninvasive assessment of changes in tumor and stromal cells.Experimental Design: Peripheral blood was collected before and after radiotherapy from 41 patients with lung cancer, as were primary biopsies. We evaluated the expression of PD-L1 and formation of RAD50 foci in CTCs and a CStC subtype, cancer-associated macrophage-like cells (CAMLs), in response to DNA damage caused by radiotherapy at the tumor site.Results: Only 24% of primary biopsies had sufficient tissue for PD-L1 testing, tested with IHC clones 22c3 and 28-8. A CTC or CAML was detectable in 93% and 100% of samples, prior to and after radiotherapy, respectively. RAD50 foci significantly increased in CTCs (>7×, P < 0.001) and CAMLs (>10×, P = 0.001) after radiotherapy, confirming their origin from the radiated site. PD-L1 expression increased overall, 1.6× in CTCs (P = 0.021) and 1.8× in CAMLs (P = 0.004): however, individual patient PD-L1 expression varied, consistently low/negative (51%), consistently high (17%), or induced (31%).Conclusions: These data suggest that RAD50 foci formation in CTCs and CAMLs may be used to track cells subjected to radiation occurring at primary tumors, and following PD-L1 expression in circulating cells may be used as a surrogate for tracking adaptive changes in immunotherapeutic targets. Clin Cancer Res; 23(19); 5948–58. ©2017 AACR.