Article Figures & Data
Figures
- Fig. 1.
Expression of CA9 on outer cell membranes of SiHa cervical carcinoma cells grown as spheroids. Two-week-old SiHa spheroids, ∼600 μm in diameter, were incubated for 20 min with Hoechst 33342 to label the external cells of spheroids. After enzyme disaggregation, single cells were incubated with anti-CA9 antibodies and sorted on the basis of Hoechst 33342 or CA9 intensity. a and b show CA9 expression for external and internal cells of spheroids respectively. In c, SiHa cells were sorted on the basis of CA9 (•) or Hoechst 33342 (○) concentration and reanalyzed for the percentage of CA9-positive cells. The means for three independent experiments are shown; bars, SD. Dotted line, the response of unsorted cells.
- Fig. 2.
Comparison between the Hoechst 33342 sorting gradient (•) and the CA9 fluorescence-tagged antibody sorting gradient (▵) in SiHa xenografts. The means for 10 fractions from five tumors are shown; bars, SD. Dotted lines, the intensity of the unsorted populations.
- Fig. 3.
CA9 expression in tumor xenografts in relation to Hoechst 33342 fluorescence. a, the gated bivariate plot of the relation between Hoechst 33342 fluorescence intensity and CA9 antibody staining in the M006 glioma xenograft. b, the CA9 distribution in the brightest Hoechst staining fraction (i.e., the 10% of cells closest to the tumor blood vessels). c, CA9 in the dimmest Hoechst staining fraction (i.e., the 10% of cells most distant from the tumor blood vessels). d, the distribution of CA9 in unsorted cells.
- Fig. 4.
Pimonidazole binding in SiHa and MOO6 xenografts. a, the percentage of hypoxic cells, determined by pimonidazole staining, as a function of CA9 antibody staining intensity. Results are the means for 4 SiHa tumors (•) and 3 M006 tumors (○); bars, SD. b–d are representative distributions showing pimonidazole binding in a M006 tumor for sort fractions 1, 3, and 5, respectively. The hatched area shows the well-oxygenated population, and the filled area represents the hypoxic cells within the tumor.
- Fig. 5.
Viability and radiation response of CA9-positive cells in SiHa xenograft tumors. a, the plating efficiency of unirradiated SiHa xenograft cells sorted on the basis of the fluorescence concentration gradients for Hoechst 33342 (□) or CA9 (•). The means for four tumors are shown; bars, SD. b, the radiation response of SiHa xenograft cells sorted based on the concentration of Hoechst 33342 (▪, □) or CA9 (•, ○). □ and ○, the response of the 10% of cells that are least fluorescent within the tumor; ▪ and •, the response of the 10% of cells that are most intensely stained. The means for four tumors are shown; bars, SE.
- Fig. 6.
Comparison between the percentage of the tumor area that binds CA9 antibodies with percentage of tumor area, measured in a sequential section, that binds pimonidazole (data from Table 1<$REFLINK> ). The linear best-fit is shown.
- Fig. 7.
Immunohistochemical analysis of pimonidazole adduct binding and CA9 expression on sequential sections of a cervical carcinoma biopsy for tumor 1. The area highlighted by the squares in A and B is enlarged in C and D. A and C, CA9 antibody binding. Note the localization to the membrane. B and D, anti-pimonidazole antibody binding. The bar is D is 85 μm.
- Fig. 8.
A section of tumor 17 showing several regions staining for pimonidazole (B) with little or no CA9 staining (A), possibly indicating areas of transient hypoxia. The bar is ∼850 μm.
Tables
- Table 1
Comparison between CA9 expression and pimonidazole binding in cervical cancers
Patient Age Smoker FIGOa cervical cancer stage Histology % Pimopositive area % CA9- positive area 1 49 No IIb Squamous 13.6 27.7 2 71 No IVa Squamous 6.4 18.3 3 59 No IIb Adeno 3 14 4 34 Yes IIb Squamous 5.7 13 5 46 Yes IIIb Squamous 8.1 11.7 6 52 No 1b2 Squamous 5.7 11.4 7 59 No IIIb Squamous 5.1 10 8 33 Yes 1b2 Squamous 3.6 7.1 9 47 No 1b1 Adeno 4.3 7 10 42 No IIa Adeno 3.1 6.9 11 67 No 1b2 Adeno 4.6 6.2 12 33 Yes IIb Squamous 3.1 4.4 13 43 No IIb Squamous 2.3 4 14 38 No 1b2 Squamous 1.2 2 15 41 No 1b1 Adeno 1.1 2 16 41 No 1b1 Squamous 1.4 1.8 17 35 Yes 1b2 Squamous 5.1 1.6 18 41 Yes 1b2 Squamous 1.4 1.6 -
a FIGO, Fédération Internationale des Gynecologistes et Obstetristes; Pimo, pimonidazole; Adeno, adenocarcinoma.
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Volume 61, Issue 24
