Single‐Cell Intravital Microscopy of Trastuzumab Quantifies Heterogeneous in vivo Kinetics

Abstract

Cell-to-cell heterogeneity can substantially impact drug response, especially for monoclonal antibody (mAb) therapies that may exhibit variability in both delivery (pharmacokinetics) and action (pharmacodynamics) within solid tumors. However, it has traditionally been difficult to examine the kinetics of mAb delivery at a single-cell level and in a manner that enables controlled dissection of target-dependent and -independent behaviors. To address this issue, here we developed an in vivo confocal (intravital) microscopy approach to study single-cell mAb pharmacology in a mosaic xenograft comprising a mixture of cancer cells with variable expression of the receptor HER2. As a proof-of-principle, we applied this model to trastuzumab therapy, a HER2-targeted mAb widely used for treating breast and gastric cancer patients. Trastuzumab accumulated to a higher degree in HER2-over expressing tumor cells compared to HER2-low tumor cells (~5:1 ratio at 24 h after administration) but importantly, the majority actually accumulated in tumor-associated phagocytes. For example, 24 h after IV administration over 50% of tumoral trastuzumab was found in phagocytes whereas at 48 h it was >80%. Altogether, these results reveal the dynamics of how phagocytes influence mAb behavior in vivo, and demonstrate an application of intravital microscopy for quantitative single-cell measurement of mAb distribution and retention in tumors with heterogeneous target expression. © 2019 International Society for Advancement of Cytometry Key terms tumor associated macrophage; metastatic breast cancer; human epidermal growth factor receptor 2/HER2/ERBB2; Fc-receptor; antibody-dependent cellular phagocytosis

Macrophage imaging and subset analysis using single-cell RNA sequencing

“…Similar imaging studies with fluorescently-labeled anti-HER2 mAb trastuzumab have found initial accumulation on HER2+ cells, but by 2 days after injection, the localization was primarily in TAM rather than tumor cells ( Fig. 4) [81]. The mAb isotypes used in these studies were distinct, as were mouse strains, and FcR-dependencies therefore are likely to be different.…”

“…Scale bar, 50 μm. Adapted with permission from [81], copyright 2020. MΦ activation states have been transcriptionally defined, and IL12b and Arg1 have been used to define M1-like and M2-like MΦ, respectively, in mice [91,92].…”

In vivo microscopy reveals macrophage polarization locally promotes coherent microtubule dynamics in migrating cancer cells

“…In fact, MΦ polarization is likely playing a substantial role in shaping the extracellular matrix 61 . Our findings may be especially important for prodrug formulations of MTtargeted therapies based on NPs (e.g., Genexol PM, used clinically in Korea) and antibody-drug conjugation (e.g., trastuzumab emtansine/Kadcyla), as these can accumulate in TAMs 24,62 . Future studies examining MTs and MT-targeted drugs in vitro might evaluate their action in the context of relevant populations including TAMs to better gauge true in vivo effects.…”

Understanding the In Vivo Fate of Advanced Materials by Imaging

“…[71] SWC tumor models have also been utilized to study the PK/PD of small molecule drugs, [62] therapeutic NPs, [10] and antibodies. [14,15]…”