Multimodality labeling strategies for the investigation of nanocrystalline cellulose biodistribution in a mouse model of breast cancer

Methods We have developed a nuclear and fluorescence labeling strategy for nanocrystalline cellulose (CNC), an emerging biomaterial with versatile chemistry and facile preparation from renewable sources. We modified CNC through 1,1′-carbonyldiimidazole (CDI) activation with radiometal chelators desferrioxamine B and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), allowing for the labeling with zirconium-89 (t½ = 78.41 h) and copper-64 (t½ = 12.70 h), respectively, for non-invasive positron emission tomography (PET) imaging. The far-red fluorescent dye Cy5 was added for ex vivo optical imaging, microscopy and flow cytometry. The multimodal CNC were evaluated in the syngeneic orthotopic 4T1 tumor model of human stage IV breast cancer. Results Modified CNC exhibited low cytotoxicity in RAW 264.7 macrophages over 96 h, and high radiolabel stability in vitro. After systemic administration, radiolabeled CNC were rapidly sequestered to the organs of the reticulo-endothelial system (RES), indicating immune recognition and no passive tumor targeting by the enhanced permeability and retention (EPR) effect. Modification with NOTA was a more favorable strategy in terms of radiolabeling yield, specific radioactivity, and both the radiolabel and dispersion stability in physiological conditions. Flow cytometry analysis of Cy5-positive immune cells from the spleen and tumor corroborated the uptake of CNC to phagocytic cells. Conclusions Future studies on the in vivo behavior of CNC should be concentrated on improving the nanomaterial stability and circulation half-life under physiological conditions and optimizing further the labeling yields for the multimodality imaging strategy presented. Advances in knowledge Our studies constitute one of the first accounts of a multimodality nuclear and fluorescent probe for the evaluation of CNC biodistribution in vivo and outline the pitfalls in radiometal labeling strategies for future evaluation of targeted CNC-based drug delivery systems. Implications for patient care Quantitative and sensitive molecular imaging methods provide information on the structure–activity relationships of the nanomaterial and guide the translation from in vitro models to clinically relevant animal models.Peer reviewe

Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models

Background: Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. Results: Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t½ = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 ± 0.1%ID/g for cetuximab and 1.5 ± 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. Conclusions: We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end

Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models

Pretargeting-based approaches are being investigated for radioimmunoimaging and therapy applications to reduce the effective radiation burden to the patient. To date, only a few studies have used short-lived radioisotopes for pretargeting of antibodies, and such examples with internalizing antibodies are even rarer. Herein, we have investigated pretargeting methodology using inverse electron-demand Diels-Alder (IEDDA) for tracing two clinically relevant, internalizing monoclonal antibodies, cetuximab and trastuzumab. Bioorthogonal reaction between tetrazine and trans-cyclooctene (TCO) was used for tracing cetuximab and trastuzumab in vivo with a fluorine-18 (t (A 1/2) = 109.8 min) labelled tracer. TCO-cetuximab or TCO-trastuzumab was administered 24, 48, or 72 h prior to the injection of tracer to A431 or BT-474 tumour-bearing mice, respectively. With cetuximab, the highest tumour-to-blood ratios were achieved when the lag time between antibody and tracer injections was 72 h. With trastuzumab, no difference was observed between different lag times. For both antibodies, the tumour could be clearly visualized in the PET images with the highest tumour uptake of 3.7 +/- 0.1%ID/g for cetuximab and 1.5 +/- 0.1%ID/g for trastuzumab as quantified by ex vivo biodistribution. In vivo IEDDA reaction was observed in the blood for both antibodies, but with trastuzumab, this was to a much lower degree than with cetuximab. We could successfully visualize the tumours by using cetuximab and trastuzumab in pretargeted PET imaging despite the challenging circumstances where the antibody is internalized and there is still some unbound antibody circulating in the blood flow. This clearly demonstrates the potential of a pretargeted approach for targeting internalizing antigens and warrants development of pharmacokinetic optimization of the biorthogonal reactants to this end.Peer reviewe

PET Imaging of Extracellular pH in Tumors with \u3csup\u3e64\u3c/sup\u3eCu- and \u3csup\u3e18\u3c/sup\u3eF-Labeled pHLIP Peptides: A Structure–Activity Optimization Study

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides (64Cu and 18F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with 64Cu or [18F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either 18F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or 64Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on 18F- or 64Cu-labeled NO2A-cysVar3

Additional file 1: of Pretargeting of internalizing trastuzumab and cetuximab with a 18F-tetrazine tracer in xenograft models

Supporting information contains the PET images of 89Zr-radiolabelled cetuximab and trastuzumab; description of the purification of mAbs, immunoreactivity testing of modified mAbs, cell uptake assay, in vitro pretargeting assay, and radiosynthesis of [18F]TAF; and ex vivo biodistribution results for pretargeted experiments and 89Zr-radiolabelled cetuximab and trastuzumab. (PDF 1832 kb

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology

PET Imaging of Extracellular pH in Tumors with ⁶⁴Cu- and ¹⁸F‑Labeled pHLIP Peptides: A Structure–Activity Optimization Study

pH (low) insertion peptides (pHLIP peptides) target acidic extracellular environments in vivo due to pH-dependent cellular membrane insertion. Two variants (Var3 and Var7) and wild-type (WT) pHLIP peptides have shown promise for in vivo imaging of breast cancer. Two positron emitting radionuclides (⁶⁴Cu and ¹⁸F) were used to label the NOTA- and NO2A-derivatized Var3, Var7, and WT peptides for in vivo biodistribution studies in 4T1 orthotopic tumor-bearing BALB/c mice. All of the constructs were radiolabeled with ⁶⁴Cu or [¹⁸F]-AlF in good yield. The in vivo biodistribution of the 12 constructs in 4T1 orthotopic allografted female BALB/c mice indicated that NO2A-cysVar3, radiolabeled with either ¹⁸F (4T1 uptake; 8.9 ± 1.7%ID/g at 4 h p.i.) or ⁶⁴Cu (4T1 uptake; 8.2 ± 0.9%ID/g at 4 h p.i. and 19.2 ± 1.8% ID/g at 24 h p.i.), shows the most promise for clinical translation. Additional studies to investigate other tumor models (melanoma, prostate, and brain tumor models) indicated the universality of tumor targeting of these tracers. From this study, future clinical translation will focus on ¹⁸F- or ⁶⁴Cu-labeled NO2A-cysVar3