The bulk of cancer cell biology has been conducted using in vitro systems, with cells cultured in artificial environments. To better understand the origin and progression of cancer and chemotherapeutic drug responses we need in vivo imaging data, ideally at the single-cell level. Whole body imaging methods including optical, magnetic resonance imaging (MRI), positron emission tomography (PET) and computed tomography (CT) scans report on the state of tissues and diseases, but generally lack the resolution required for single cell analysis (1
). High-resolution images can be obtained from histology, but this requires invasive biopsies, or sacrificing animals at each time-point, without providing real-time data. Live-cell imaging in culture has revealed dynamic aspects of cancer cell biology and drug responses of single cells, but how these data apply to the situation in vivo is largely unknown. Thus, a clear need exists for sub-cellular resolution intravital microscopy (IVM) to correlate the acute responses of cells to drugs with the ultimate fates of cells, tumors and tissues in animal models of human disease. In rodents, IVM typically involves a glass window set into the animal, or an exteriorized organ (2
) to directly observe underlying tissues and tumors (6
). In optically favorable organisms such as zebra fish, drosophila and nematodes, IVM can visualize dynamic processes at the single cell level (10
). But, most applications of IVM in rodents follow cells or groups of cells at relatively low-resolution. The challenges facing sub-cellular IVM include physiological motion, low signal to noise ratios and slow image capture rates that limit directly studying rapid intracellular processes and transient events at a quality comparable to culture systems. Overcoming these limitations requires addressing issues including light penetration, phototoxicity, and especially motion caused by breathing, heartbeat and muscle movements. Here, we report optimized IVM that enables highly detailed, sub-cellular light microscopy to study formation of the mitotic spindle and chromosome dynamics before and after drug delivery in xenograft tumors (). Using this in vivo pharmacodynamic microscopy (IPDM), we analyzed the response of Paclitaxel (Ptx), an important anti-cancer mitotic drug, whose biology remains poorly understood at the whole-tumor level.
Figure 1 In vivo pharmacodynamic microscopy (IPDM). a) The imaging system. Xenograft tumor(s) in the DSC (i); anesthesia (ii); temperature-regulated holding bar (iii); temperature-regulated stage (iv); DSC holding plate and finger screws (v); laser scanning confocal (more ...)
Mitosis is central to tumor growth and aneuploidy due to mitotic errors contributes to both tumorigenesis and the progression of cancer toward more aggressive genotypes (13
). Anti-mitotic drugs that perturb microtubule dynamics are part of the chemotherapy regime for treating many cancers, and experimental drugs against other mitotic spindle proteins are in clinical trials (14
). Ptx binds to microtubules, interferes with polymerization dynamics, promotes mitotic arrest, and triggers apoptosis in cancer cells (15
). Time-lapse microscopy in culture has revealed important aspects of Ptx response dynamics and significant intra-cellular variability () (19
). At saturating Ptx (typically 100–300nM) cells rarely die without first entering mitotic arrest that can last 24h or longer depending on the cell line (19
). Post-arrest, cells either initiate apoptosis or exit without dividing into an abnormal G1-like state with multiple small nuclei; this abnormal mitotic exit is termed mitotic slippage () (20
). Once multinucleated most cells cannot recover normal nuclear morphology and remain arrested, die, or attempt another mitosis that is typically multipolar (e.g. tripolar, not bipolar). Which of these pathways a given cell follows is highly variable both within a cell line and between cell lines (19
Although the effects of Ptx on microtubules and mitosis are well understood in culture (22
), many questions remain as to how it promotes tumor regression in vivo, why patients with the same diagnosis respond differently, and how resistance arises (24
). One basic question is whether Ptx kills only cells that have entered mitosis in tumors, as in culture – i.e. is Ptx solely an anti-mitotic drug in the tumor context? Pharmacokinetic (PK; drug concentration vs. time) and PD (drug action vs. time) data are essential for understanding tumor responses. In contrast to the extensive information on the PK of Ptx (18
), its PD is complicated and requires better understanding via a development of biomarkers, ideally at multiple sequential steps in the drug action pathway. Two natural biomarkers for Ptx are mitotic arrest and cell death. These have been used as a PD biomarkers in rodent cancer models and man through scoring histology sections or stained biopsies (16
); multinucleation has been largely ignored. A dynamic readout of these biomarkers would be more powerful, especially if it could be calibrated to measure the relative Ptx concentration experienced by a tumor cell as a function of dose and time. Effects of Ptx on the morphology and duration of mitosis are concentration-dependent in cell culture (31
), suggesting these measurements might provide semi-quantitative PD biomarkers in tumors. At low concentrations in culture (≤5nM), mitosis is delayed and often exhibits errors in chromosome alignment and segregation; at moderate concentrations (5–20nM) mitotic delays are longer, and cells often exhibit spindle multipolarity and multipolar division; at high concentrations (>20nM) cells arrest for many hours and then die or slip. These concentration-dependent responses have not been studied in vivo.
The questions we sought to answer included: (i) can tumor cells be visualized at a high enough resolution to observe single-chromosome errors during mitosis, and to discriminate different morphological biomarkers of Ptx action? (ii) how do Ptx responses differ in culture and in tumors, and are all Ptx effects in tumors mediated via mitotic arrest? (iii) can we quantify PD for a small molecule drug using live, sub-cellular imaging? To address these questions, human tumor cell lines known to establish xenograft tumors in nude mice were engineered to stably express fluorescent protein fusions to histone-H2b (to visualize chromatin) and/or β1-tubulin (to visualize microtubules). These cell lines report on cell-cycle state (interphase or mitotic), spindle morphology, mitotic errors such as lagging chromosomes, mitotic arrest, passage through mitotic arrest and apoptosis. We used dorsal skin-fold chambers (DSCs), and developed multiple approaches for immobilization and stabilization. depicts the main features of the set-up. HT-1080 cells rapidly established vascularized tumors in DSCs (). Imaging with a 2X objective revealed overall tumor morphology and blood vessels, while our standard imaging conditions revealed detailed morphology ().