Figure 1.
Structures, molecular weight (MW) and maximum optical absorption and emission wavelengths (1×PBS) for HS680 and the control agent.
Figure 2.
In vitro characterization of HS680 in hypoxic and normoxic tumor cells.
A, Binding of HS680 to hypoxic and normoxic HeLa cells was shown with fluorescence microcopy. Cells labeled with the control agent cultured under hypoxic conditions provided a negative control. Blocking of HS680 binding by pre-incubating the hypoxic cells with AZ is shown in lower right panel. The images of cells cultured with control agent and HS680 with AZ blocking under normoxic conditions were similar to the images of HS680 cultured at nomoxic conditions, and therefore they were not shown. B, Representative histograms of HS680 binding to normoxic and hypoxic HeLa cells by flow cytometry with AZ blocking in hypoxic cells. C, Flow cytometry quantifications of binding of HS680 and control agent to normoxic and hypoxic HT-29, HeLa, HCT-116, and MDA-MB-231 cells with or without AZ blocking in hypoxic cells. D, The disassociation constant (Kd) of HS680 was determined in HeLa cells by flow cytometry to be 8.3 nM.
Table 1.
Inhibition data for CA II, IX, XII and XIV for the parent CA inhibitor AZ, HS680 and the control agent as determined by stopped-flow CO2 hydration method.
Figure 3.
In vivo imaging of HS680 clearance from HeLa tumors and terminal bio-distribution of HS680 in mouse tissues.
A, Representative images of tomographic imaging of tumor bearing mice at various times after IV injection of the control agent and HS680, showing progressive changes in tumor fluorescence signals. B, Tumor fluorescence concentrations were quantified by the FMT at various times after the agent injection, showing the highest contrast of HS680 over the control agent background signal at 24 hours post injection. C, Bio-distribution of the control agent and HS680 in mice bearing HeLa tumors was determined from the 2D FRI images of excised organ tissues, removed 24 hours post-agent injection. Representative images of tumors and organs are inset with arrows indicating tumors and kidneys. High mechanistic fluorescence signal was observed only within tumors of HS680 injected mice, and the high kidney fluorescence seen with both agents was attributed to renal clearance.
Figure 4.
In vivo imaging of HS680 and control agent in mice bearing CA IX positive (HT-29 and HeLa) and CA IX negative (HCT-116 and MDA-MB-231) tumors, with and without AZ competition.
A, Representative FMT images and B, Tomographic quantitative analysis of tumor bearing mice injected with control agent and HS680 showing significant accumulations of HS680 fluorescence signals within HT-29 and HeLa tumors, but not in HCT-116 and MDA-MB-231 tumors, and less in the mice that were pre-injected with 10 mg/kg AZ (HS680+AZ) 1 hour before HS680 injection. Mice injected with control agents did not show any appreciable tumor fluorescence signals. HT-29 tumors are at the flank regions of the mice while all other tumor types are implanted in the mammary fat pads, and all tumors are housed with a 3D region of interest (ROI) in each image. ND indicates “Not determined.” C, Representative FRI images of four types of tumors collected following the FMT imaging, showing ex-vivo validation of in vivo imaging results.
Figure 5.
The effect of breathing 8% O2 on HS680 tumor signal in HeLa xenografts.
Two groups of mice (n = 4 per group) were used to assess the effect of oxygen breathing levels on HS680 labeling of small tumors. A, Representative FMT 3D images of mice breathing normal air (top left) and breathing low oxygen (top right) showing the intensity differences of HS680 signals within the tumors. FRI 2D images of tumors excised from mice that were placed on normal air (bottom left) and 8% O2 (bottom right) showing again the intensity of fluorescence in tumors. B and C, Quantitative analysis (3D and 2D, respectively) of HS680 tumor signals in mice breathing normal or low oxygen, showing greater tumor HS680 signals in a mice breathing low oxygen than that of mice breathing normal air.
Figure 6.
Localization of HS680 and tumor hypoxia markers in HeLa xenografts.
A, The tissue staining patterns of the control agent, HS680, and HS680+AZ (red) from the same or adjacent tumor sections as staining with fluorescent CA IX antibody or pimonidazole (green) and the Hoechst perfusion stain (blue). H&E staining of tissue sections that were used for localization are shown on the left side of images. HS680 was specifically localized in regions with low Hoechst staining indicative of low oxygen (less perfused) but positive staining with both the CA IX antibody and Pimonidazole. Preinjection of the mice with unlabeled AZ blocked the binding of HS680 to control levels. B, Co-localization (overlay) of HS680 with CA IX antibody or pimonidazole (hypoxyprobe) is shown on the right side images indicating HS680 was clearly co-localized with both anti-CA IX antibody and hypoxyprobe (pimonidazole). Similar results were obtained using HT-29 tumors (Figure S4A and Figure S4B).