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Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive responses in subgroups of patients with cancer. PD-L1 expression in tumors seems to be a prerequisite for treatment response. However, PD-L1 is heterogeneously expressed within tumor lesions and may change upon disease progression and treatment. Imaging of PD-L1 could aid in patient selection. Previously, we showed the feasibility to image PD-L1+ tumors in immunodeficient mice. However, PD-L1 is also expressed on immune cell subsets. Therefore, the aim of this study was to assess the potential of PD-L1 micro single-photon emission tomography/computed tomography (microSPECT/CT) using radiolabeled PD-L1 antibodies to (i) measure PD-L1 expression in two immunocompetent tumor models (syngeneic mice and humanized mice harboring PD-L1 expressing immune cells) and (ii) monitor therapy-induced changes in tumor PD-L1 expression. We showed that radiolabeled PD-L1 antibodies accumulated preferentially in PD-L1+ tumors, despite considerable uptake in certain normal lymphoid tissues (spleen and lymph nodes) and nonlymphoid tissues (duodenum and brown fat). PD-L1 microSPECT/CT imaging could also distinguish between high and low PD-L1–expressing tumors. The presence of PD-L1+ immune cells did not compromise tumor uptake of the human PD-L1 antibodies in humanized mice, and we demonstrated that radiotherapy-induced upregulation of PD-L1 expression in murine tumors could be monitored with microSPECT/CT imaging. Together, these data demonstrate that PD-L1 microSPECT/CT is a sensitive technique to detect variations in tumor PD-L1 expression, and in the future, this technique may enable patient selection for PD-1/PD-L1–targeted therapy.

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Table S1. Overview of selected autophagy activating compounds, their structure, chemical and pharmacological classification and the supplier; Table S2. Significantly upregulated genes in all cell lines at all time points (fold change log2 {greater than or equal to} 1); Table S3. Significantly downregulated genes in all cell lines at all time points (fold change log2 {less than or equal to} -1); Table S4. Physicochemical properties of digitalis-like compounds predicted by ChemBioOf�ce software; Figure S1: Visualization of whole transcriptome data of BC-PAP treated with 50 µM proscillaridin A for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S2: Visualization of whole transcriptome data of FTC133 treated with 50 µM digoxin for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S3: Visualization of whole transcriptome data of FTC133 treated with 50 µM digoxigenin for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S4: Visualization of whole transcriptome data of FTC133 treated with 50 µM proscillaridin A for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S5: Visualization of whole transcriptome data of FTC133 treated with 50 µM strophantin K for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S6: Visualization of whole transcriptome data of TPC-1 treated with lanatoside C for 24, 48 or 72 hours, comprising principal component analysis and Venn diagrams of overlapping genes, either up- or downregulated, between 24, 48 and 72 hours time points; Figure S7: STRING protein interaction networks of significantly up- or downregulated genes by digitalis-like compounds in all three cell lines and at all three time points; Figure S8: Bonferroni corrected P-value enrichment scores of significantly upregulated pathways by digitalis-like compounds combined for all three cell lines and for all three time points. P-values are generated by using the Gene Ontology (GO) platform category Biological Processes; Figure S9: Expression of JUN after treatment for 48 and 72 hours of BC-PAP, FTC133 and TPC-1 with the indicated digitalis-like compounds. Data are obtained from three independent experiments. Data are means {plus minus} SD. Square boxes represent statistical output of Spearman's rho tests for degree of correlation between expression of JUN with hNIS expression at 48 and 72 hours time points; Figure S10: Gene expression of thyroid transcription factors TTF1, TTF2 and PAX8 in BC-PAP, FTC133 or TPC-1 after treatment with the indicated digitalis-like compounds for 48 or 72 hours. Data are obtained from three independent experiments. Data are means {plus minus} SD; Figure S11: Heatmaps depicting gene expression of ABC transporters and organic anion transporters in untreated and digitalis-like compound treated BC-PAP, FTC133 and TPC-1 (N=6); Figure S12: Full unedited Western blot pictures, cropped pictures are depicted in Figure 1B.

PDF file - 41K, Clinical protocol and immunization schedule.

Images from a kidney lamella after perfusion made with the clinical laparoscopic fluorescence camera showing normal kidney parenchyma (A): visible light image (B): corresponding fluorescence image.

Video made with the laparoscopic fluorescence camera during experiment #1.

Supplementary Materials- figure legends

Purpose: Antibodies labeled with both a near-infrared fluorescent dye and a radionuclide can be used for tumor-targeted intraoperative dual-modality imaging. Girentuximab is a chimeric monoclonal antibody against carbonic anhydrase IX (CAIX), an antigen expressed in 95% of clear cell renal cell carcinoma (ccRCC). This study aimed to assess the feasibility of targeted dual-modality imaging with 111In-girentuximab-IRDye800CW using ex vivo perfusion of human tumorous kidneys.Experimental Design: Seven radical nephrectomy specimens from patients with ccRCC were perfused during 11 to 15 hours with dual-labeled girentuximab and subsequently rinsed during 2.5 to 4 hours with Ringer's Lactate solution. Then, dual-modality imaging was performed on a 5- to 10-mm-thick lamella of the kidney. Fluorescence imaging was performed with a clinical fluorescence camera set-up as applied during image-guided surgery. The distribution of Indium-111 in the slice of tumor tissue was visualized by autoradiography. In two perfusions, an additional dual-labeled control antibody was added to demonstrate specific accumulation of dual-labeled girentuximab in CAIX-expressing tumor tissue.Results: Both radionuclide and fluorescence imaging clearly visualized uptake in tumor tissue and tumor-to-normal tissue borders, as confirmed (immuno)histochemically and by gamma counting. Maximum uptake of girentuximab in tumor tissue was 0.33% of the injected dose per gram (mean, 0.12 %ID/g; range, 0.01–0.33 %ID/g), whereas maximum uptake in the normal kidney tissue was 0.04 %ID/g (mean, 0.02 %ID/g; range, 0.00–0.04 %ID/g).Conclusions: Dual-labeled girentuximab accumulated specifically in ccRCC tissue, indicating the feasibility of dual-modality imaging to detect ccRCC. A clinical study to evaluate intraoperative dual-modality imaging in patients with ccRCC has been initiated. Clin Cancer Res; 22(18); 4634–42. ©2016 AACR.

Purpose: To investigate whether indium-111–labeled R1507 (111In-R1507) immuno-SPECT (single—photon emission computed tomography), a novel noninvasive, in vivo screening method to visualize membranous insulin-like growth factor 1 receptor (IGF-1R) expression and accessibility, can be used to predict IGF-1R treatment (R1507) response in bone sarcomas.Experimental Design: BALB/c nude mice were subcutaneously implanted with IGF-1R–expressing human bone sarcoma xenografts (OS-1, EW-5, and EW-8) which showed high, modest, or no response, respectively, to R1507, a monoclonal antibody targeting the extracellular domain of IGF-1R. An IGF-1R–negative tumor (OS-33), unresponsive to IGF-1R inhibitors, was examined as well. Mice were injected with 111In-R1507. Biodistribution and immuno-SPECT/computed tomography imaging studies were carried out 1, 3, and 7 days p.i. in mice with OS-1 and EW-5 xenografts and 3 days p.i. in mice with EW-8 and OS-33 xenografts.Results: Biodistribution studies showed specific accumulation of 111In-R1507 in OS-1 and EW-5 xenografts (27.5 ± 6.5%ID/g and 14.0 ± 2.8%ID/g, 3 days p.i., respectively). Most importantly, 111In-R1507 uptake in IGF-1R positive, but unresponsive, EW-8 xenografts (6.5 ± 1.5%ID/g, 3 days p.i.) was similar to that of the IGF-1R–negative OS-33 tumor (5.5 ± 0.6%ID/g, 3 days p.i.). Uptake in normal tissues was low and nonspecific. Corresponding immuno-SPECT images clearly discriminated between high, modest, and nonresponding tumors by showing a homogeneous (OS-1), heterogeneous (EW-5), or nonspecific (EW-8 and OS-33) tumor uptake of 111In-R1507.Conclusions:111In-R1507 immuno-SPECT is an excellent method to visualize membranous IGF-1R expression and target accessibility in vivo in human bone sarcoma xenografts and may serve as an independent marker to predict IGF-1R therapy (R1507) response in bone sarcoma patients. Clin Cancer Res; 17(24); 7693–703. ©2011 AACR.

PDF file - 55K, Consort flow chart.

Purpose: It is unknown whether the route of administration influences dendritic cell (DC)-based immunotherapy. We compared the effect of intradermal versus intranodal administration of a DC vaccine on induction of immunologic responses in melanoma patients and examined whether concomitant administration of interleukin (IL)-2 increases the efficacy of the DC vaccine.Experimental Design: HLA-A2.1+ melanoma patients scheduled for regional lymph node dissection were vaccinated four times biweekly via intradermal or intranodal injection with 12 × 106 to 17 × 106 mature DCs loaded with tyrosinase and gp100 peptides together with keyhole limpet hemocyanin (KLH). Half of the patients also received low-dose IL-2 (9 MIU daily for 7 days starting 3 days after each vaccination). KLH-specific B- and T-cell responses were monitored in blood. gp100- and tyrosinase-specific T-cell responses were monitored in blood by tetramer analysis and in biopsies from delayed-type hypersensitivity (DTH) skin tests by tetramer and functional analyses with 51Cr release assays or IFNγ release, following coculture with peptide-pulsed T2 cells or gp100- or tyrosinase-expressing tumor cells.Results: In 19 of 43 vaccinated patients, functional tumor antigen–specific T cells could be detected. Although significantly more DCs migrated to adjacent lymph nodes upon intranodal vaccination, this was also highly variable with a complete absence of migration in 7 of 24 intranodally vaccinated patients. Intradermal vaccinations proved superior in inducing functional tumor antigen–specific T cells. Coadministration of IL-2 did not further augment the antigen-specific T-cell response but did result in higher regulatory T-cell frequencies.Conclusion: Intradermal vaccination resulted in superior antitumor T-cell induction when compared with intranodal vaccination. No advantage of additional IL-2 treatment could be shown. Clin Cancer Res; 17(17); 5725–35. ©2011 AACR.

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