We have developed an in vivo immobilization strategy for systemic delivery of reporters to visualize the molecular targets in the extracellular microenvironment. Collagens were chosen as the base for immobilization because of their abundance in the extracellular matrix and the extremely small amount of reporters introduced (typically tens of pmol) should have minimal interference on their normal physiological function. The reporters immobilized on collagens are ubiquitously distributed in the body and retained for a long period of time. Importantly, the binding of the reporters to collagens will prevent them from being internalized into cells, and retain them extracellularly for long-term imaging. The protease-activatable reporters on collagens are well exposed and fully functional in the extracellular microenvironment of tumors and in the cutaneous inflammation site. The immobilization strategy offers an attractive solution to characterizing the extracellular microenvironment in vivo because it can provide local information on specific targets in the microenvironment, and also allows global mapping of the target activity in the whole body for comparison.
The MMP activity is usually analyzed in the tissue samples. However, the in vitro
analysis is hard to assess the exact MMP activity under in vivo
conditions due to tight regulation of the enzyme activity by the endogenous effectors such as tissue inhibitors of metalloproteinases (TIMPs).39,40
The variations on tissue sample preparation also limit accurately assessment of the enzyme activity in the different tissues.
A number of activatable probes have been previously reported for in vivo
imaging of enzyme activity in tumors.23–25
These probes preferentially accumulate at the tumor sites by taking advantage of the leaky tumor vasculature through the EPR effect. A potential problem of this type of probes is that they can also be activated by the enzyme present in other regions besides the tumor site, and the activated products may eventually translocate and accumulate in tumors, also through the EPR effect, as reported in a recent study.40
These issues make it difficult to reliably quantify the enzyme activity in tumors with the EPR based activatable probes. Furthermore, these probes will not be useful for studying other disease models where the EPR effect does not exist. Neither of these issues is a limiting factor for the collagen-based reporter immobilization strategy.
We observed slow activation of MMP reporters in the xenografted HT1080 tumor (). The zymogram of the tumor lysate, however, showed that over 50% of MMP-2 was in the active form (Supplementary Fig. 5
). Since the MMP activity is regulated by both the expression control and TIMP inhibition, it is thus likely that most of active MMP-2 in the tumor microenvironment was inhibited by TIMPs. For example, TIMP-1 inhibits MMP-mediated degradation of extracellular matrix,41
and can also stimulate cell growth 42
and inhibit apoptosis43
. In accompanying with the increased MMP expression in HT1080 cells, there may be a local increase in the TIMPs expressed from tumor or neighboring stroma cells to maintain the balance between the tumor growth and matrix degradation. This argument is consistent with the dual roles TIMPs play in the modulation of MMP activity: they are necessary to assist activation of pro-MMPs, but also inhibit the MMP activity.44
In the inflammation model, a burst of MMP-9 activation in the inflammation model was observed shortly after the injection of the reporters (). On the other hand, the zymogram of the tissue lysates showed that the proportion of active MMPs was much lower than that in the tumor (). It is thus likely that the MMP activity regulation in the inflammation site may be different from the tumor, and that there may not be an increased TIMPs expression. In light of the dual role of TIMPs in the modulation of MMP activity,44
like MMP-2, pro-MMP9 may similarly require TIMPs for its activation.
As an enzyme that catalyzes the hydrolysis of its substrates, both concentrations of the product and substrate have to be determined for quantitative imaging of the MMP activity. Previous activatable MMP probes only measure the signal from the product -- activated probe.23–25
Since the probe is often not evenly distributed after systematic introduction into the body, without prior knowledge of the amount of the probe localized at the tumor, it can result in over- or under-estimating the enzyme activity from only the activated product signal. Therefore, a second imaging modality should be introduced to provide an independent, quantitative measurement of the reporter (both activated and non-activated) concentration at all the tissues. The signal resulting from activated reporter is normalized against the total concentration determined from the PET imaging, and the normalized value thus truly reflects the MMP activity. For example, the MMP activity in the liver appeared as high as in the tumor before the normalization (), but after the normalization of the reporter concentration, the MMP activity in the liver is much lower than that in the tumor (). This result underscores the important consideration in designing and applying activatable probes for quantitative imaging enzyme activity in vivo
In summary, we have imaged the enzymatic activity of gelatinases in the extracellular microenvironment with immobilized activatable reporters, and demonstrated its general applicability for in vivo imaging extracellular microenvironment in two disease models. Reporters for sensing other molecular targets and events of interest may be similarly immobilized for the microenvironment imaging. The fact that many growth factors are present at very low concentrations in the microenvironment may also require activatable reporters with a mechanism of signal amplification for sensitive detection. We anticipate that the reporter immobilization will be a general strategy for long-term serial real-time imaging of biological targets in the microenvironment at the molecular level.