Direct visualization of heterogeneous extravascular distribution of trastuzumab in human epidermal growth factor receptor type 2 overexpressing xenografts

Abstract

Purpose: The high molecular weight and binding affinity of trastuzumab, a monoclonal antibody in use for treatment of breast cancers overexpressing human epidermal growth factor receptor type 2 (HER2), in combination with microenvironmental factors, may limit its distribution and efficacy. We assessed and mapped the distribution of systemically given, unlabeled trastuzumab at micrometer resolution in tumor xenografts using immunohistochemistry.

Experimental design: Mice bearing MDA-435/LCC6(HER2) xenografts were given single doses of 4 or 20 mg/kg unlabeled trastuzumab with tumor harvest at various time points thereafter; bound trastuzumab was imaged directly in tumor cryosections using fluorescently tagged antihuman secondary antibodies. Combinations of additional markers, including HER2, 5-bromo-2-deoxyuridine, CD31, DioC(7)(3), desmin, and collagen IV were also mapped on the same tumor sections.

Results: Distribution of trastuzumab in MDA-435/LCC6(HER2) tumors is found to be heterogeneous, with tumor margins saturating more thoroughly in doses and times analyzed. Considerable intervessel heterogeneity is also seen. For example, in unsaturated tissues, there remain perfused vessels without any trastuzumab in addition to vessels with a few layers of positively stained perivascular cells, in addition to vessels with bound drug up to 150 microm away. This heterogeneity is independent of HER2 expression, microvessel density, and perfusion. A slightly greater proportion of vessels were associated with pericytes in sections with greater trastuzumab saturation, but this would not adequately account for observed heterogeneous trastuzumab distribution.

Conclusions: Complete penetration of trastuzumab in tumor tissue was not seen in our study, leaving the possibility that inadequate distribution may represent a mechanism for resistance to trastuzumab.