Figure 1.
Cell membrane Trop-2 and internalization processes.
Breast MCF-7, ovarian OVCA-432 and colon HT29 cancer cells were analyzed. (A) Cancer cell membrane versus intracytoplasmic Trop-2 retention. OVCA-432 and HT29 cells were stained with the T16 mAb. Arrrowheads indicate intracytoplasmic Trop-2 deposits. (B) Immuno-gold electron microscopy analysis of MCF-7 breast cancer cells. Trop-2 internalization was analyzed after induction of signaling (two-step cross-linking with the T16 mAb, followed by rabbit anti-mouse pAbs [16]). Black dots are gold nanospheres conjugated to anti-Trop-2 antibodies. (left panel) internalization of Trop-2 in intracellular, membrane-delimited areas; (right panel) endosome-like localization of internalized Trop-2.
Table 1.
Association between Trop-2 functional states and tumor pathobiological parameters.
Figure 2.
Glycosylation-dependent Trop-2 transport and signaling.
(A) Binding of 2EF to fully-matured forms of Trop-2. Ample binding to the cell membrane (arrows) was revealed. Strong staining of the Golgi apparatus was also shown (arrowheads), consistent with recognition of glycosylated Trop-2. (B) Flow cytometry analysis of KM12SM cells stably transfected with glycosylation mutants. Living cells were analyzed for membrane-only staining. 2EF, T16 and 162–46.2: unconjugated anti-Trop-2 mAbs, followed by rabbit anti-mouse Alexa-488; control: irrelevant antibody stained cells. (C) Flow cytometry analysis of KM12SM stably transfected with wild-type Trop-2 or deglycosylated variant. Living cells were analyzed for membrane-only staining. 2EF: anti-Trop-2 Alexa-488 conjugated mAb; AF650: anti-Trop-2 goat pAb; control: irrelevant antibody-stained cells. Living cells were analyzed for membrane-only staining. (D) MTE 4–14 cells transfected with Trop-2 subjected to Ab-mediated capping. The T16 (left) and 2EF (right) mAbs were used for primary Ab incubation, followed by cross-linking with a secondary Ab conjugated with Alexa488.
Figure 3.
Immunohistochemistry analysis of Trop-2 expression in breast cancer.
Breast cancer samples were analyzed by immunohistochemistry using the 162–46.2 anti-Trop-2 mAb [32] for detection of the intracellular Trop-2 (A) and with the R&D AF650 goat pAb for detection of membrane-associated Trop-2 (B). Images are representative cases of ductal (top panels) and lobular (bottom panels) cancers. Arrows: normal breast ducts. Expression levels were classified as high and low/negative. Magnification is 40x.
Figure 4.
Impact of membrane versus intracellular Trop-2 on patient survival.
Cumulative incidence (CI) estimates of death from any cause were obtained as 1-Kaplan-Meier curves for distinct Trop-2 expression sub-groups (cell membrane; mAb-detected intracellular; pAb-detected intracellular). Trop-2 expression was categorized according to (top) intensity scores (0, 1+, 2+, 3+), (middle) intensity grouping, i.e. positive scores 1–12 (+) versus score 0 (−), (bottom) percentage of stained cells (low, ≤5%; intermediate, 6–85%; high, ≥86%), as indicated in the panels.
Figure 5.
Impact of membrane versus intracellular Trop-2 on disease relapse.
Crude cumulative incidence (CCI) estimates of disease relapse were obtained as 1-Kaplan-Meier curves for distinct Trop-2 expression sub-groups (cell membrane; mAb-detected intracellular; pAb-detected intracellular). Trop-2 expression was categorized according to (top) intensity scores (0, 1+, 2+, 3+), (middle) intensity grouping, i.e. positive scores 1–12 (+) versus score 0 (−), (bottom) percentage of stained cells (low, ≤5%; intermediate, 6–85%; high, ≥86%), as indicated in the panels. CCI were estimated accounting for death as a competing risk.
Table 2.
Proportional hazard Cox regression analysis.