Abstract A12: Heterogeneous accumulation of trastuzumab in Her2+ve tumor and metastases models
Authors: Baker Jennifer , Kyle Alastair H. , Moosvi Firas , Minchinton Andrew I. 
Published: May 2015

Despite significant success, the response of Her2+ patients to trastuzumab (Herceptin ®) is varied, with many still experiencing tumour progression. In previous studies we have shown that trastuzumab has limited distribution in MDA-435-LCC6-Her2 over-expressing breast cancer xenografts. We have used biomarkers derived from MRI and multiplexed immunohistochemistry to examine the limited distribution of trastuzumab in the context of the tumour microenvironment.

Methods: Endogenously over-expressing SKOV-3 ovarian carcinomas, MDA361, BT474 and vector-overexpressed Her2+ve MCF7 mammary (MCF7Her2) tumours were grown as xenografts in NOD/SCID mice in the sacral region, or were injected ip or iv to generate metastases in the peritoneal or lung compartments. Animals were administered 10 mg/kg trastuzumab with tissues harvested & frozen at 20-24h.

MRI: Tumours were imaged for vascular function using DCE-MRI (Gadovist, 60mM), then treated with trastuzumab. Implanted fiducial markers enabled cryosections that closely approximated MR imaged slices to be obtained for comparison to tumour maps. Area Under the Curve (AUC) was calculated for the first 60 seconds post contrast agent injection.

Tumour mapping: Multiplexed immunohistochemistry generated maps of whole tumour sections for quantitative and qualitative analysis. In addition to direct visualization of trastuzumab, features including Her2 expression (Her2), blood vessels (CD31), vascular perfusion (Dil18) and pericytes (SMA, desmin), basal lamina (CIV) and tight junctions (ZO-1) were stained and mapped relative to each other.

Findings: All models exhibit highly heterogeneous distribution of trastuzumab, with variation at the inter-vessel, inter-tumour and intra-tumoural levels. Accumulation of trastuzumab is not limited to the tumour margins, though sections obtained on the distal ends of tumours have greater average amounts of bound drug. Trastuzumab is bound to tumour cells up to 200 µm away from the nearest perfused blood vessels in some areas, while other areas containing perfused vessels have little to no binding despite Her2 expression.

The average amount of Her2+ve tissue positive for bound trastuzumab at 20-24h post administration is: MCF7-Her2 (68.6±7.2%), SKOV-3 (31.5±1.9%), BT474 (33.2%±1.6), MDA361 (21.5%+3.2). Small SKOV-3 metastatic lesions collected from the peritoneal cavity and lung tissues have an average of 68.5±3.5% of Her2+ve tissue bound for trastuzumab, although many have as little as 30%, and some very small lesions have no bound trastuzumab at all. For both the solid tumours and the metastatic lesions, no consistent, quantifiable difference or correlation was found between the amount of bound trastuzumab and the presence of greater ZO-1 labeled tight junctions, the density of nearest CD31 vessels, or the fraction of perfused, desmin +ve or SMA +ve vessels.

Maps derived from DCE-MRI data showed that in some cases a high AUC value matched with high trastuzumab saturation, however these patterns were not consistent; in every tumour significant areas of mismatched high and low regions were observed.

Summary: The highly heterogeneous microregional distribution of trastuzumab in these models appears to be independent of conventionally considered barriers to drug delivery, including ZO-1 tight junctions, microvessel density, blood flow and vascular maturity. The common occurrence of perfused vessels with no trastuzumab on the perivacular Her2+ve cells suggests that limited distribution is not a consequence of the binding site barrier hypothesis. The absence of a consistent pattern of greater accumulation of trastuzumab at the tumour margins suggests that interstitial fluid pressure (IFP) is not primarily responsible for the observed heterogeneity and limited distribution.

These data suggest an as yet unidentified barrier may be responsible for inadequate access of trastuzumab to all Her2+ve cells.

Citation Format: Jennifer Baker, Alastair H. Kyle, Firas Moosvi, Andrew I. Minchinton. Heterogeneous accumulation of trastuzumab in Her2+ve tumor and metastases models. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A12. doi:10.1158/1538-7445.CHTME14-A12