Necrosis binding of Ac-Lys(0)(IRDye800CW)-Tyr(3)-octreotate:a consequence from cyanine-labeling of small molecules

BACKGROUND: There is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery. RESULTS: Binding of 800CW-TATE could be blocked with DOTA0-Tyr3-octreotate (DOTA-TATE) on cultured SSTR2-positive U2OS cells and was absent in SSTR2 negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE. CONCLUSION: This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents

Whole-body bioluminescence imaging of T-cell response in PDAC models

IntroductionThe location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo.MethodsHere, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically ‘hot’) or low T-cell infiltration (immunologically ‘cold’).ResultsDifferences in total T-cell bioluminescence could be seen between the ‘hot’ and ‘cold’ tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment.ConclusionsIn the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment

Erratum: Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging: Erratum

The authors regret to find an error in the published version of figure 1B, where the graph for 7D12-PS mistakenly was miscopied for 7D12-9G8-PS. During the review process a correct version of Figure 1B was included. The mistake was made while preparing the final text and figures in response to reviewers comments. The authors have revised Figure 1B, and confirm that the correction has no effect on the original data and conclusions. The authors apologize for any inconvenience that the errors may have caused

Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging: Erratum

Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging (Theranostics (2020) 10:5 (2436-2452) DOI: 10.7150/thno.37949

In Vivo Molecular Bioluminescence Imaging: New Tools and Applications

in vivo bioluminescence imaging (BLi) is an optical molecular imaging technique used to visualize molecular and cellular processes in health and diseases and to follow the fate of cells with high sensitivity using luciferase-based gene reporters. The high sensitivity of this technique arises from efficient photon production, followed by the reaction between luciferase enzymes and luciferin substrates. Novel discoveries and developments of luciferase reporters, substrates, and gene-editing techniques, and emerging fields of applications, promise a new era of deeper and more sensitive molecular imaging

Investigation of the Therapeutic Potential of Nanobody-Targeted Photodynamic Therapy in an Orthotopic Head and Neck Cancer Model

Photodynamic therapy (PDT) has a great therapeutic potential because it induces local cellular cytotoxicity upon application of a laser light that excites a photosensitizer, leading to toxic reactive oxygen species. Nevertheless, PDT still is underutilized in the clinic, mostly because of damage induced to normal surrounding tissues. Efforts have been made to improve the specificity. Nanobody-targeted PDT is one of such approaches, in which the variable domain of heavy-chain antibodies, i.e., nanobodies, are used to target photosensitizers selectively to cancer cells. In vitro studies are certainly very valuable to evaluate the therapeutic potential of PDT approaches, but many aspects such as bio-distribution of the photosensitizers, penetration through tissues, and clearance are not taken into account. In vivo studies are therefore essential to assess the influence of such factors, in order to gain more insights into the therapeutic potential of a treatment under development. This chapter describes the development of an orthotopic model of head and neck cancer, to which nanobody-targeted PDT is applied, and the therapeutic potential is assessed by immunohistochemistry one day after PDT

Investigation of the Therapeutic Potential of Nanobody-Targeted Photodynamic Therapy in an Orthotopic Head and Neck Cancer Model

Photodynamic therapy (PDT) has a great therapeutic potential because it induces local cellular cytotoxicity upon application of a laser light that excites a photosensitizer, leading to toxic reactive oxygen species. Nevertheless, PDT still is underutilized in the clinic, mostly because of damage induced to normal surrounding tissues. Efforts have been made to improve the specificity. Nanobody-targeted PDT is one of such approaches, in which the variable domain of heavy-chain antibodies, i.e., nanobodies, are used to target photosensitizers selectively to cancer cells. In vitro studies are certainly very valuable to evaluate the therapeutic potential of PDT approaches, but many aspects such as bio-distribution of the photosensitizers, penetration through tissues, and clearance are not taken into account. In vivo studies are therefore essential to assess the influence of such factors, in order to gain more insights into the therapeutic potential of a treatment under development. This chapter describes the development of an orthotopic model of head and neck cancer, to which nanobody-targeted PDT is applied, and the therapeutic potential is assessed by immunohistochemistry one day after PDT

Nanobody-targeted photodynamic therapy induces significant tumor regression of trastuzumab-resistant HER2-positive breast cancer, after a single treatment session

RATIONALE: A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT. METHODS: Nanobodies targeting HER2 were obtained from phage display selections. Monovalent nanobodies were engineered into a biparatopic construct. The specificity of selected nanobodies was tested in immunofluorescence assays and their affinity was evaluated in binding studies, both performed in a panel of breast cancer cells varying in HER2 expression levels. The selected HER2-targeted nanobodies 1D5 and 1D5-18A12 were conjugated to the photosensitizer IRDye700DX and tested in in vitro PDT assays. Mice bearing orthotopic HCC1954 trastuzumab-resistant tumors with high HER2 expression or MCF-7 tumors with low HER2 expression were intravenously injected with nanobody-PS conjugates. Quantitative fluorescence spectroscopy was performed for the determination of the local pharmacokinetics of the fluorescence conjugates. After nanobody-PS administration, tumors were illuminated to a fluence of 100 J∙cm-2, with a fluence rate of 50 mW∙cm-2, and thereafter tumor growth was measured with a follow-up until 30 days. RESULTS: The selected nanobodies remained functional after conjugation to the PS, binding specifically and with high affinity to HER2-positive cells. Both nanobody-PS conjugates potently and selectively induced cell death of HER2 overexpressing cells, either sensitive or resistant to trastuzumab, with low nanomolar LD50 values. In vivo, quantitative fluorescence spectroscopy showed specific accumulation of nanobody-PS conjugates in HCC1954 tumors and indicated 2 h post injection as the most suitable time point to apply light. Nanobody-targeted PDT with 1D5-PS and 1D5-18A12-PS induced significant tumor regression of trastuzumab-resistant high HER2 expressing tumors, whereas in low HER2 expressing tumors only a slight growth delay was observed. CONCLUSION: Nanobody-PS conjugates accumulated selectively in vivo and their fluorescence could be detected through optical imaging. Upon illumination, they selectively induced significant tumor regression of HER2 overexpressing tumors with a single treatment session. Nanobody-targeted PDT is therefore suggested as a new additional treatment for HER2-positive breast cancer, particularly of interest for trastuzumab-resistant HER2-positive breast cancer. Further studies are now needed to assess the value of this approach in clinical practice

Nanobody-targeted photodynamic therapy induces significant tumor regression of trastuzumab-resistant HER2-positive breast cancer, after a single treatment session

Rationale: A substantial number of breast cancer patients with an overexpression of the human epidermal growth factor receptor 2 (HER2) have residual disease after neoadjuvant therapy or become resistant to trastuzumab. Photodynamic therapy (PDT) using nanobodies targeted to HER2 is a promising treatment option for these patients. Here we investigate the in vitro and in vivo antitumor efficacy of HER2-targeted nanobody-photosensitizer (PS) conjugate PDT. Methods: Nanobodies targeting HER2 were obtained from phage display selections. Monovalent nanobodies were engineered into a biparatopic construct. The specificity of selected nanobodies was tested in immunofluorescence assays and their affinity was evaluated in binding studies, both performed in a panel of breast cancer cells varying in HER2 expression levels. The selected HER2-targeted nanobodies 1D5 and 1D5-18A12 were conjugated to the photosensitizer IRDye700DX and tested in in vitro PDT assays. Mice bearing orthotopic HCC1954 trastuzumab-resistant tumors with high HER2 expression or MCF-7 tumors with low HER2 expression were intravenously injected with nanobody-PS conjugates. Quantitative fluorescence spectroscopy was performed for the determination of the local pharmacokinetics of the fluorescence conjugates. After nanobody-PS administration, tumors were illuminated to a fluence of 100 J∙cm-2, with a fluence rate of 50 mW∙cm-2, and thereafter tumor growth was measured with a follow-up until 30 days. Results: The selected nanobodies remained functional after conjugation to the PS, binding specifically and with high affinity to HER2-positive cells. Both nanobody-PS conjugates potently and selectively induced cell death of HER2 overexpressing cells, either sensitive or resistant to trastuzumab, with low nanomolar LD50 values. In vivo, quantitative fluorescence spectroscopy showed specific accumulation of nanobody-PS conjugates in HCC1954 tumors and indicated 2 h post injection as the most suitable time point to apply light. Nanobody-targeted PDT with 1D5-PS and 1D5-18A12-PS induced significant tumor regression of trastuzumab-resistant high HER2 expressing tumors, whereas in low HER2 expressing tumors only a slight growth delay was observed. Conclusion: Nanobody-PS conjugates accumulated selectively in vivo and their fluorescence could be detected through optical imaging. Upon illumination, they selectively induced significant tumor regression of HER2 overexpressing tumors with a single treatment session. Nanobody-targeted PDT is therefore suggested as a new additional treatment for HER2-positive breast cancer, particularly of interest for trastuzumab-resistant HER2-positive breast cancer. Further studies are now needed to assess the value of this approach in clinical practice