CSCs and non-stem tumor cells prior to transplantation contain different fractions of stem-like and proliferating cells.

<p>Representative micrographs (<b>A</b>) and bar graph (<b>B</b>) of expanded cells prior to transplantation demonstrate Sox2 and PH3 expression (red) is higher in the CSC fraction of cells as compared with the non-stem tumor cells. Summary figure depicts marker expression from in vivo and in vitro analyses (<b>C</b>). Scale bar represents 50 µm. Data displayed as mean values +/− S.E.M. ***, p<0.001 and N.S. represents not significant (p>0.05) as assessed by one-way analysis of variance (ANOVA), nuclei counterstained with Hoechst 33342 (blue).</p

Multiphoton microscopy reveals cancer stem cell driven tumor propagation.

<p>Fractionated CSCs and non-stem tumor cells were labeled with different fluorescent proteins and transplanted into mice at a 10% cancer stem cell (YFP) to 90% non-stem tumor cell (CFP) ratio as shown in experimental design schematic (<b>A</b>). CSCs outgrew non-CSCs in vivo as shown in summary graph (<b>B</b>), which was calculated based on three-dimensional reconstructions of projection micrographs (<b>B, C</b>). Additionally, tumor populations did not intermingle in vivo (non-stem tumor population indicated by yellow oval). Fluorescent dextran (shown in purple) was injected into the circulation to illuminate blood vessels prior to imaging. Scale bar represents 100 µm.</p

Tumors contain fractions of stem-like and proliferating cells that originated from cancer stem cells.

<p>Histological examination was performed from resulting tumors in the cell mixing experiments (n = 3) to determine the fraction of stem-like cells as assessed by Sox2 expression and the presence of proliferating cells as confirmed by the M-phase marker phosphorylated histone 3 (PH3). Representative micrographs (<b>A</b>) and bar graph (<b>B</b>) demonstrate Sox2 expression (red) is associated with cancer stem cells and their descendants (green) but not with non-stem tumor cells and their descendants (blue). Representative micrographs (<b>C</b>) and bar graph (<b>D</b>) demonstrate PH3 expression (red) is associated with cancer stem cells and their descendants (green) but not with non-stem tumor cells and their descendants (blue). Scale bar represents 50 µm. Data displayed as mean values +/− S.E.M. ***, p<0.001 as assessed by one-way analysis of variance (ANOVA), nuclei counterstained with Draq5 (purple).</p

Histological evaluation reveals tumors contained cancer stem cells and their descendants that had association with blood vessels.

<p>Tumors from the cell mixing experiments (n = 3) were evaluated to determine their composition. Subsequent evaluation of resulting tumors demonstrates that the majority of the cells within the tumor mass was of human origin and derived from CSC as confirmed by Tra-1-85 staining and YFP expression, shown in representative micrographs (<b>A</b>) and bar graph (<b>B</b>). Peripheral transplanted tumor cells (YFP positive CSCs and their descendants) were observed to have an association with blood vessels. Micrograph from multiphoton imaging and three-dimensional reconstruction (<b>C</b>) depict close association of tumor cells (green) with adjacent blood vessel (purple, illuminated by fluorescent dextran injection into the circulation prior to imaging). Histological examination of resulting tumors confirms close association of peripheral tumor cells to the vasculature using CD31 immunostaining (<b>D</b>; CD31 in red, tumor cells in green, nuclei in purple). Scale bar represents 50 µm. Data displayed as mean values +/- S.E.M. ***, p<0.001 as assessed by one-way analysis of variance (ANOVA).</p

Multiple Administrations of Viral Nanoparticles Alter <i>in Vivo</i> BehaviorInsights from Intravital Microscopy

Multiple administrations of nanoparticle-based formulations are often a clinical requirement for drug delivery and diagnostic imaging applications. Steady pharmacokinetics of nanoparticles is desirable to achieve efficient therapeutic or diagnostic outcomes over such repeat administrations. While clearance through mononuclear phagocytic system is a key determinant of nanoparticle persistence <i>in vivo</i>, multiple administrations could potentially result in altered pharmacokinetics by evoking innate or adaptive immune responses. Plant viral nanoparticles (VNPs) represent an emerging class of programmable nanoparticle platform technologies that offer a highly organized proteinaceous architecture and multivalency for delivery of large payloads of drugs and molecular contrast agents. These very structural features also render them susceptible to immune recognition and subsequent accelerated systemic clearance that could potentially affect overall efficiency. While the biodistribution and pharmacokinetics of VNPs have been reported, the biological response following repeat administrations remains an understudied area of investigation. Here, we demonstrate that weekly administration of filamentous plant viruses results in the generation of increasing levels of circulating, carrier-specific IgM and IgG antibodies. Furthermore, PVX specific immunoglobulins from the serum of immunized animals quickly form aggregates when incubated with PVX <i>in vitro</i>. Such aggregates of VNP-immune complexes are also observed in the mouse vasculature <i>in vivo</i> following repeat injections when imaged in real time using intravital two-photon laser scanning microscopy (2P-LSM). The size of aggregates diminishes at later time points, coinciding with antibody class switching from IgM to IgG. Together, our results highlight the need for careful <i>in vivo</i> assessment of (viral) nanoparticle-based platform technologies, especially in studying their performance after repeat administration. We also demonstrate the utility of intravital microscopy to aid in this evaluation

Multiple Administrations of Viral Nanoparticles Alter <i>in Vivo</i> BehaviorInsights from Intravital Microscopy

Multiple administrations of nanoparticle-based formulations are often a clinical requirement for drug delivery and diagnostic imaging applications. Steady pharmacokinetics of nanoparticles is desirable to achieve efficient therapeutic or diagnostic outcomes over such repeat administrations. While clearance through mononuclear phagocytic system is a key determinant of nanoparticle persistence <i>in vivo</i>, multiple administrations could potentially result in altered pharmacokinetics by evoking innate or adaptive immune responses. Plant viral nanoparticles (VNPs) represent an emerging class of programmable nanoparticle platform technologies that offer a highly organized proteinaceous architecture and multivalency for delivery of large payloads of drugs and molecular contrast agents. These very structural features also render them susceptible to immune recognition and subsequent accelerated systemic clearance that could potentially affect overall efficiency. While the biodistribution and pharmacokinetics of VNPs have been reported, the biological response following repeat administrations remains an understudied area of investigation. Here, we demonstrate that weekly administration of filamentous plant viruses results in the generation of increasing levels of circulating, carrier-specific IgM and IgG antibodies. Furthermore, PVX specific immunoglobulins from the serum of immunized animals quickly form aggregates when incubated with PVX <i>in vitro</i>. Such aggregates of VNP-immune complexes are also observed in the mouse vasculature <i>in vivo</i> following repeat injections when imaged in real time using intravital two-photon laser scanning microscopy (2P-LSM). The size of aggregates diminishes at later time points, coinciding with antibody class switching from IgM to IgG. Together, our results highlight the need for careful <i>in vivo</i> assessment of (viral) nanoparticle-based platform technologies, especially in studying their performance after repeat administration. We also demonstrate the utility of intravital microscopy to aid in this evaluation