S5-3: Basement Membrane Localized Tumor Cells Are Protected from HER2−Targeted Therapy In Vivo.
Authors: Zoeller JJ , Bronson RT , Gilmer TM , Selfors LM , Lu Y , Mills GB , Brugge JS 
Published: June 2012

Abstract
Drug resistance compromises the efficacy of HER2−targeted therapy. Results from our laboratory, and previous reports from others, indicate that tumor cell attachment to basement membrane (BM) and other extracellular matrix (ECM) proteins may confer drug resistance. We have discovered a differential drug response between the outer, matrix-attached cells and inner matrix-deprived cells comprising 3-dimensional (3D) tumor spheroids grown in reconstituted basement membrane (T. Muranen and J. Brugge, unpublished data). The outer matrix-attached cells are resistant to multiple drug therapies. To address whether these observations are relevant in vivo, we utilized a previously described model of human-in-mouse HER2−positive ductal carcinoma in situ (DCIS), which involves the intraductal transplantation of human HER2−positive SUM225 tumor cells directly into the ductal network of the mouse mammary gland. The intraductal tumors generated are histologically indistinguishable from human DCIS lesions and recapitulate the architecture of the 3D tumor spheroids. The tumors are characterized by organized nests of noninvasive cells confined within a BM surrounded by ECM. These features permit a direct assessment of differential drug response within the tumor. We focused on the tumor cell response to short-term lapatinib monotherapy in vivo. A close examination of the tumor architecture revealed that cells closest to the basement membrane, and nearest to the vasculature, display a striking insensitivity to lapatinib whereas the remainder of the tumor undergoes extensive cell death in response to treatment. Further characterization also revealed that cells closest to the basement membrane largely maintain proliferative capacity despite an overall significant reduction in the total Ki67-positive cell population. These results provide in vivo evidence that basement membrane-attached tumor cells are protected from lapatinib. We confirmed that these cells maintain HER2 status and also observed an overall reduction in pHER2, pAKT and pERK throughout the tumor suggestive of adaptive response mechanisms, which support the proliferation and survival of these cell populations despite inhibition of the HER2 pathway. To further explore potential mechanisms of the adaptive response, we performed reverse phase protein array (RPPA) analysis on protein lysates prepared from tumor biopsy fragments following lapatinib monotherapy. RPPA profile analysis revealed an adaptive response composed of upregulation of multiple RTKs (HER2, IGFI-R) and altered apoptotic protein levels (Bcl-2, Bim, Bcl-xL) in addition to activation of AKT/S6K and ERK/p38 pathway components. These observations suggest that basement membrane-attached tumor cells may escape from lapatinib response via compensatory activation of these survival mechanisms. Each of these components will serve as targets for designing combined therapeutic approaches capable of targeting the protected basement membrane-attached tumor cells. Our results suggest that resistant populations may be a source of residual disease post-therapy, therefore identifying and characterizing these cells will be crucial to the prevention of disease recurrence in the clinic.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S5-3.