Search

Additional file 1. Supplementary figures 1���6.

Additional file 2: Fig. S1. a Radio-TLC of purified antibody conjugates showing successful radiolabelling of DFO-Miltuximab® with 89Zirconium. b Radio-TLC of purified antibody conjugates showing successful radiolabelling of DOTA-Miltuximab® with 177Lutetium. Fig. S2. Immunoreactivity of a,c DFO-Miltuximab® and b,d DOTAMiltuximab® to cell surface GPC-1 on DU-145 cells measured via flow cytometry (a,b) and ELISA (c,d). For some tests, antibody was “mock” radiolabelled, i.e. treated in the same way as the radiolabelled antibody, but without the addition of radiolabel, to estimate the effect of radiolabelling conditions on immunoreactivity. Fig. S3. Quantitative flow cytometry analysis of DU-145 cells using MIL-38 binding for quantification. MIL-38 was used with the QIFIKIT antigen density analysis kit to determine GPC-1 density on the cell surface of a. prostate cancer cell line DU-145 and b. GPC1 negative lymphoma cell line Raji. Fig. S4. No effect of DOTA-Miltuximab® alone on in vivo tumour growth. DU-145-RFP-Luc cells (5x106 ) in matrigel were injected subcutaneously into the right flank of BALB/c/nude mice. When tumours reached ~100mm3 , mice were injected with saline (n=6) or DOTA-Miltuximab® (n=6) (80ug) intravenously. All mice were euthanised approximately 2 weeks thereafter. a Mean weekly mouse tumour volume. d Individual mouse tumour volume at endpoint. Data expressed as Mean ± SEM and statistical analysis performed using an unpaired t test. *p < 0.05. Fig. S5. Representative H&E staining of the mouse brain, heart, lung, liver, kidney, spleen, small intestine and testis tissue a 3 days, b 5 days, c 7 days and d 27 days post 6MBq [ 177Lu]Lu-DOTA-Miltuximab® treatment or e 27 days post DOTA-Miltuximab® treatment. Fig. S6. Individual weekly mouse tumour volumes of mice treated with a DOTAMiltuximab® (n=8) b 3MBq [ 177Lu]Lu-DOTA-Miltuximab® (n=9) or c 10MBq [ 177Lu]LuDOTA-Miltuximab® (n=9) treated DU-145 xenograft mice.

Additional file 1. Supplementary Methods.

External-stimuli controlled reversible formation of radical species is of great interest for synthetic and supramolecular chemistry, molecular machinery, as well as emerging technologies ranging from (photo)catalysis and photovoltaics to nanomedicine. Here we show a novel hybrid colloidal system for light-driven reversible reduction of chemical species that, on their own, do not respond to light. This is achieved by the unique combination of photo-sensitive plasmonic aggregates and temperature-responsive inorganic species generating radicals that can be finally accepted and stabilised by non-photo-responsive organic molecules. In this system Au nanoparticles (NPs) self-assembled via sub-nm precise molecular spacers (cucurbit[n]urils) interact strongly with visible light to locally accelerate the decomposition of dithionite species (S2O42-) close to the NP interfaces. This light-driven process leads to the generation of inorganic radicals whose electrons can then be reversibly picked up by small organic acceptors, such as the methyl viologen molecules (MV2+) used here. During light-triggered plasmon- and heat-assisted generation of radicals, the S2O42- species work as a chemical 'fuel' linking photo-induced processes at the NP interfaces with redox chemistry in the surrounding water environment. By incorporating MV2+ as a Raman-active reporter molecule, the resulting optically-controlled redox processes can be followed in real-time.

Background Triple-negative breast cancers (TNBC) have a relatively poor prognosis and responses to targeted therapies. Between 25 and 39% of TNBCs are claudin-low, a poorly differentiated subtype enriched for mesenchymal, stem cell and mitogen-activated signaling pathways. We investigated the role of the cell-surface co-receptor NRP1 in the biology of claudin-low TNBC. Methods The clinical prognostic value of NRP1 was determined by Kaplan–Meier analysis. GSVA analysis of METABRIC and Oslo2 transcriptomics datasets was used to correlate NRP1 expression with claudin-low gene signature scores. NRP1 siRNA knockdown was performed in MDA-MB-231, BT-549, SUM159 and Hs578T claudin-low cells and proliferation and viability measured by live cell imaging and DNA quantification. In SUM159 orthotopic xenograft models using NSG mice, NRP1 was suppressed by shRNA knockdown or systemic treatment with the NRP1-targeted monoclonal antibody Vesencumab. NRP1-mediated signaling pathways were interrogated by protein array and Western blotting. Results High NRP1 expression was associated with shorter relapse- and metastasis-free survival specifically in ER-negative BrCa cohorts. NRP1 was over-expressed specifically in claudin-low clinical samples and cell lines, and NRP1 knockdown reduced proliferation of claudin-low cells and prolonged survival in a claudin-low orthotopic xenograft model. NRP1 inhibition suppressed expression of the mesenchymal and stem cell markers ZEB1 and ITGA6, respectively, compromised spheroid-initiating capacity and exerted potent anti-tumor effects on claudin-low orthotopic xenografts (12.8-fold reduction in endpoint tumor volume). NRP1 was required to maintain maximal RAS/MAPK signaling via EGFR and PDGFR, a hallmark of claudin-low tumors. Conclusions These data implicate NRP1 in the aggressive phenotype of claudin-low breast cancer and offer a novel targeted therapeutic approach to this poor prognosis subtype.

External-stimuli controlled reversible formation of radical species is of great interest for synthetic and supramolecular chemistry, molecular machinery, as well as emerging technologies ranging from (photo)catalysis and photovoltaics to nanomedicine. Here we show a novel hybrid colloidal system for light-driven reversible reduction of chemical species that, on their own, do not respond to light. This is achieved by the unique combination of photo-sensitive plasmonic aggregates and temperature-responsive inorganic species generating radicals that can be finally accepted and stabilised by non-photo-responsive organic molecules. In this system Au nanoparticles (NPs) self-assembled via sub-nm precise molecular spacers (cucurbit[n]urils) interact strongly with visible light to locally accelerate the decomposition of dithionite species (S(2)O(4)(2–)) close to the NP interfaces. This light-driven process leads to the generation of inorganic radicals whose electrons can then be reversibly picked up by small organic acceptors, such as the methyl viologen molecules (MV(2+)) used here. During light-triggered plasmon- and heat-assisted generation of radicals, the S(2)O(4)(2–) species work as a chemical ‘fuel’ linking photo-induced processes at the NP interfaces with redox chemistry in the surrounding water environment. By incorporating MV(2+) as a Raman-active reporter molecule, the resulting optically-controlled redox processes can be followed in real-time.

Genetic variation at the 19q13.3 KLK locus is linked with prostate cancer susceptibility. The non-synonymous KLK3 SNP, rs17632542 (c.536T>C; Ile163Thr-substitution in PSA) is associated with reduced prostate cancer risk, however, the functional relevance is unknown. Here, we identify that the SNP variant-induced change in PSA biochemical activity as a previously undescribed function mediating prostate cancer pathogenesis. The 'Thr' PSA variant led to small subcutaneous tumours, supporting reduced prostate cancer risk. However, 'Thr' PSA also displayed higher metastatic potential with pronounced osteolytic activity in an experimental metastasis in-vivo model. Biochemical characterization of this PSA variant demonstrated markedly reduced proteolytic activity that correlated with differences in in-vivo tumour burden. The SNP is associated with increased risk for aggressive disease and prostate cancer-specific mortality in three independent cohorts, highlighting its critical function in mediating metastasis. Carriers of this SNP allele had reduced serum total PSA and a higher free/total PSA ratio that could contribute to late biopsy decisions and delay in diagnosis. Our results provide a molecular explanation for the prominent 19q13.3 KLK locus, rs17632542 SNP, association with a spectrum of prostate cancer clinical outcomes., Competing Interests: HL holds patents for free PSA, hK2, and intact PSA assays, and is named on a patent for a statistical method to detect prostate cancer. The marker assay patents and the patent for the statistical model has been licensed and commercialized as the 4Kscore by OPKO Diagnostics. HL receives royalties from sales of this test and owns stock in OPKO. All the other authors declare no conflict of interest.