Imaging the distribution of an antibody-drug conjugate constituent targeting mesothelin with Zr-89 and IRDye 800CW in mice bearing human pancreatic tumor xenografts

Mesothelin is a tumor differentiation antigen expressed by epithelial tumors, including pancreatic cancer. Currently, mesothelin is being targeted with an antibodydrug conjugate (ADC) consisting of a mesothelin-specific antibody coupled to a highly potent chemotherapeutic drug. Considering the toxicity of the ADC and reduced accessibility of pancreatic tumors, non-invasive imaging could provide necessary information. We therefore developed a zirconium-89 (Zr-89) labeled anti-mesothelin antibody (Zr-89-AMA) to study its biodistribution in human pancreatic tumor bearing mice. Biodistribution and dose-finding of Zr-89-AMA were studied 144 h after tracer injection in mice with subcutaneously xenografted HPAC. MicroPET imaging was performed 24, 72 and 144 h after tracer injection in mice bearing HPAC or Capan-2. Tumor uptake and organ distribution of Zr-89-AMA were compared with nonspecific 111In-IgG. Biodistribution analyses revealed a dose-dependent Zr-89-AMA tumor uptake. Tumor uptake of Zr-89-AMA was higher than 111In-IgG using the lowest tracer dose. MicroPET showed increased tumor uptake over 6 days, whereas activity in blood pool and other tissues decreased. Immunohistochemistry showed that mesothelin was expressed by the HPAC and CAPAN-2 tumors and fluorescence microscopy revealed that AMA-800CW was present in tumor cell cytoplasm. Zr-89-AMA tumor uptake is antigen-specific in mesothelin-expressing tumors. Zr-89-AMA PET provides non-invasive, real-time information about AMA distribution and tumor targeting

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), we transplanted the B. subtilis FBP-binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.status: publishe

Lapatinib and 17AAG Reduce Zr-89-Trastuzumab-F(ab')(2) Uptake in SKBR3 Tumor Xenografts

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with in vivo HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined in vitro using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied in vitro by Zr-89-trastuzumab-F(ab')(2) internalization. For in vivo evaluation, Zr-89-trastuzurnab-F(ab')(2) mu PET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second Zr-89-trastuzumab-F(ab')(2) injection. Imaging data were compared with ex vivo biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (P = 0.016) and HER2 by 76% (P = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of Zr-89-trastuzumab-F(ab')(2) with 25% (P = 0.0022). Zr-89-trastuzumab-F(ab')(2) tumor to blood ratio was lowered 32% by lapatinib (P = 0.00004), 34% by 17AAG (P = 0.0022) and even 53% by the combination (P = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce Zr-89-trastuzumab-F(ab')(2) tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation

Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single-cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), we transplanted the B. subtilis FBP-binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations

Bacterial persistence is an active σS stress response to metabolic flux limitation

While persisters are a health threat due to their transient antibiotic tolerance, little is known about their phenotype and what actually causes persistence. Using a new method for persister generation and high-throughput methods, we comprehensively mapped the molecular phenotype of Escherichia coli during the entry and in the state of persistence in nutrient-rich conditions. The persister proteome is characterized by σ(S)-mediated stress response and a shift to catabolism, a proteome that starved cells tried to but could not reach due to absence of a carbon and energy source. Metabolism of persisters is geared toward energy production, with depleted metabolite pools. We developed and experimentally verified a model, in which persistence is established through a system-level feedback: Strong perturbations of metabolic homeostasis cause metabolic fluxes to collapse, prohibiting adjustments toward restoring homeostasis. This vicious cycle is stabilized and modulated by high ppGpp levels, toxin/anti-toxin systems, and the σ(S)-mediated stress response. Our system-level model consistently integrates past findings with our new data, thereby providing an important basis for future research on persisters

Placental Growth Factor (PlGF)-Specific Uptake in Tumor Microenvironment of Zr-89-Labeled PlGF Antibody RO5323441

<p>Placental growth factor (PlGF) is a member of the proangiogenic vascular endothelial growth factor family, which is upregulated in many tumors. RO5323441, a humanized monoclonal antibody against PlGF, showed antitumor activity in human tumor xenografts. We therefore aimed to radiolabel RO5323441 and preclinically validate this tracer to study drug tumor uptake and organ distribution by PET imaging. Zr-89-RO5323441 was tested for stability and immunoreactivity in vitro. Methods: The tumor uptake and organ distribution for 10, 50, and 500 mu g of Zr-89-RO5323441 was assessed in mice bearing human PlGF-expressing hepatocellular cancer (Huh7) xenografts or human renal cell carcinoma (ACHN) xenografts without detectable human PlGF expression. The effect of pretreatment with RO5323441 (20 mg/kg) on Zr-89-RO5323441 tumor uptake was analyzed in Huh7 xenografts. In-111-IgG served as a control for nonspecific tumor uptake and organ distribution. Cy5-RO5323441 was injected to study the intratumor distribution of RO5323441 with fluorescence microscopy. Results: Zr-89-RO5323441 showed a time-and dose-dependent tumor accumulation. Uptake in Huh7 xenografts at 10 mu g of Zr-89-RO5323441 was 8.2% +/- 1.7% injected dose (ID)/cm(3) at 144 h after injection, and in ACHN xenografts it was 5.5 +/- 0.3 % ID/cm(3) (P = 0.03). RO5323441 pretreatment of Huh7 xenograft-bearing mice reduced Zr-89-RO5323441 tumor uptake to the level of nonspecific In-111-IgG uptake. Cy5-RO5323441 was present in the tumors mainly in the microenvironment. Conclusion: The findings show that RO5323441 tumor uptake is PlGF-specific and time-and dose-dependent.</p>

Lapatinib and 17AAG Reduce ⁸⁹Zr-Trastuzumab-F(ab′)₂ Uptake in SKBR3 Tumor Xenografts

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with <i>in vivo</i> HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined <i>in vitro</i> using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied <i>in vitro</i> by ⁸⁹Zr-trastuzumab-F­(ab′)₂ internalization. For <i>in vivo</i> evaluation, ⁸⁹Zr-trastuzumab-F­(ab′)₂ μPET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second ⁸⁹Zr-trastuzumab-F­(ab′)₂ injection. Imaging data were compared with <i>ex vivo</i> biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (<i>P</i> = 0.016) and HER2 by 76% (<i>P</i> = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of ⁸⁹Zr-trastuzumab-F­(ab′)₂ with 25% (<i>P</i> = 0.0022). ⁸⁹Zr-trastuzumab-F­(ab′)₂ tumor to blood ratio was lowered 32% by lapatinib (<i>P</i> = 0.00004), 34% by 17AAG (<i>P</i> = 0.0022) and even 53% by the combination (<i>P</i> = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce ⁸⁹Zr-trastuzumab-F­(ab′)₂ tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation

Lapatinib and 17AAG Reduce ⁸⁹Zr-Trastuzumab-F(ab′)₂ Uptake in SKBR3 Tumor Xenografts

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with <i>in vivo</i> HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined <i>in vitro</i> using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied <i>in vitro</i> by ⁸⁹Zr-trastuzumab-F­(ab′)₂ internalization. For <i>in vivo</i> evaluation, ⁸⁹Zr-trastuzumab-F­(ab′)₂ μPET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second ⁸⁹Zr-trastuzumab-F­(ab′)₂ injection. Imaging data were compared with <i>ex vivo</i> biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (<i>P</i> = 0.016) and HER2 by 76% (<i>P</i> = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of ⁸⁹Zr-trastuzumab-F­(ab′)₂ with 25% (<i>P</i> = 0.0022). ⁸⁹Zr-trastuzumab-F­(ab′)₂ tumor to blood ratio was lowered 32% by lapatinib (<i>P</i> = 0.00004), 34% by 17AAG (<i>P</i> = 0.0022) and even 53% by the combination (<i>P</i> = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce ⁸⁹Zr-trastuzumab-F­(ab′)₂ tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation

Lapatinib and 17AAG Reduce ⁸⁹Zr-Trastuzumab-F(ab′)₂ Uptake in SKBR3 Tumor Xenografts

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with <i>in vivo</i> HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined <i>in vitro</i> using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied <i>in vitro</i> by ⁸⁹Zr-trastuzumab-F­(ab′)₂ internalization. For <i>in vivo</i> evaluation, ⁸⁹Zr-trastuzumab-F­(ab′)₂ μPET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second ⁸⁹Zr-trastuzumab-F­(ab′)₂ injection. Imaging data were compared with <i>ex vivo</i> biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (<i>P</i> = 0.016) and HER2 by 76% (<i>P</i> = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of ⁸⁹Zr-trastuzumab-F­(ab′)₂ with 25% (<i>P</i> = 0.0022). ⁸⁹Zr-trastuzumab-F­(ab′)₂ tumor to blood ratio was lowered 32% by lapatinib (<i>P</i> = 0.00004), 34% by 17AAG (<i>P</i> = 0.0022) and even 53% by the combination (<i>P</i> = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce ⁸⁹Zr-trastuzumab-F­(ab′)₂ tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation