We have shown that the myeloid cells activated in the brain and infiltrating the tumor upon OV treatment induce MPO, thus allowing MR-imaging of their inflammatory activity through a gadolinium-based molecular imaging agent (27
). Our data indicate that the kinetics of intracerebral/intratumoral inflammation, verified by histopathology of CD68+ and CD163+ cells in a rat orthotopic glioma model treated with an oncolytic herpes simplex virus, corresponds to the kinetics of MPO activity measured on excised brains through an enzymatic assay. These changes can be tracked in vivo through MPO-Gd-MRI. The specificity of the contrast induced by MPO-Gd was proved by comparing MPO-Gd-MRI with standard DTPA-Gd-MRI. Moreover, previously published works have shown that there is no detectable MPO-Gd-MRI contrast in mice knock-out for MPO, thus emphasizing the specificity of this MRI agent (26
). The levels of viral LacZ and host MPO mRNAs display similar kinetics. However, whereas MPO mRNA is only detectable during the acute phase of this inflammatory response, a baseline of MPO activity is always present. This could be due to a general low abundance of MPO mRNA molecules or to a dual MPO regulation system: genetic and enzymatic (42
). Because MPO-Gd-MRI is reproducible in mouse and rat gliomas established in the respective syngeneic animal model, and because we have shown that activation of CD68+ and CD163+ cells is not virus-, tumor-, or species-specific and that these cells infiltrate the tumor of patients treated with OV (19
) and secrete MPO, there is high translational potential for this technology. This is strengthened by the lower toxicity of MPO-Gd compared to many clinically-approved MRI agents (30
An important advantage of MPO-Gd-MRI is its ability to distinguish between inflammation and tumor. While inflammation decreases between days 1 and 7 post viral dosing, the tumor size increases. Moreover, comparison of animals treated with two different doses of virus show stronger inflammation and smaller tumors for animals receiving the highest viral dose. Indeed, even though OV induce other oxidases in cancer cells, MPO-Gd is specific to MPO which is expressed only in myeloid cells. Current MRI strategies are not able to distinguish between inflammatory areas from tumor re-growth. This is a recurrent diagnostic dilemma that prevents the ability of the oncologists to provide the timeliest and most suitable treatment plan to the patient. Thus, this MRI technology can solve a critical diagnostic problem for the treatment of brain tumors. To establish the broad diagnostic applicability of this technology, it is important to test its capacity to detect inflammation during other therapeutic strategies such as radiation, immunotherapies, and treatment with anti-inflammatory and anti-angiogenic drugs.
Attempts to image intracerebral inflammation through MRI were previously done using MIONs (41
), which detect all phagocytic cells. However, even though this strategy can detect OV-induced intratumoral macrophages (19
), it does not enhance the peritumoral inflammation, nor provides information on tumor size. Conversely, MPO-Gd-MRI can identify spatiotemporal changes of active inflammatory cells infiltrating into the cancer as well as in the brain parenchyma surrounding the tumor. Fusion of MION and MPO-Gd MRI scans in the same tumor model suggests that these two strategies detect three different cellular subsets: 1) intratumoral phagocytic cells not making MPO, 2) peritumoral MPO-secreting cells that are not phagocytic and 3) intratumoral phagocytic cells that also produce MPO. It is not clear if the peritumoral cells are not enhanced by MION-MRI because MIONs do not reach them or because they are not phagocytic. We have previously published that CD68+ microglia surrounding the tumor infiltrate into the tumor region and engulf the infectious OV (20
), suggesting that MIONs do not diffuse to the peritumoral parenchyma. However, the phagocytic activity of these cells may be acquired only after their infiltration into the tumor, indicating that the different cellular subsets enhanced by MION and MPO-Gd-MRI are the same cells at different stages of inflammatory activation. Further comparison and combination of these imaging technologies will allow a deeper understanding of the distinct role of individual innate immune cells in the inflammatory responses (31
Imaging inflammation is also useful in ways other than simply recognizing it in a re-occurring tumor after a specific treatment. As our understanding of cancer biology advances, increased relevance is being given to this host defense response. Even though the role of inflammation in the ultimate therapeutic outcome is unclear (being described both as controlling and promoting tumor recurrence and progression), it is accepted that it influences tumor treatment one way or another. It is therefore crucial to establish in vivo techniques that allow understanding of the relationship between inflammation and the outcome of tumor treatment. Demand for these techniques is further emphasized by the current trend of examining drugs that modulate immune processes (such as COX-2 inhibitors and NSAIDs) as anti-cancer agents. Finally, inflammation poses a crucial dilemma in OV-treatment. Even though it is established that host innate immunity is detrimental for OV lytic activity, inflammatory cells can also kill cancer cells and synergize with OV in tumor treatment (47
). Virotherapies aimed at increasing and suppressing OV-induced immunity were studied and both strategies presented positive results (47
). In this respect, our data indicate that increasing the viral dose, temporally augments also the infiltration/activation of MPO+ cells. Even though the higher viral dose transiently decreases the tumor size, it is impossible to establish from these data whether the temporal lysis of the tumor is mediated by OV or immune cells alone, or by the combination of these two factors. Further studies comparing MPO-Gd-MRI and MION-MRI during OV-treatment in presence of anti-inflammatory or pro-inflammatory agents will bring more insights into the role that different innate immune cells may have in the therapeutic outcome.
In conclusion, MPO-Gd MRI will strongly improve our diagnostic ability of the effects of cancer treatment on the tumor versus tumor micro-environment. Imaging inflammation during pro-inflammatory and immunosuppressive therapies will allow understanding of its role in cancer treatment and on side-effects of current therapies.
Statement of translational relevance
Glioma management induces intracerebral inflammation undistinguishable from tumor re-growth with current MRI technologies. This poses a recurrent diagnostic dilemma that prevents the ability of the oncologists to provide the patients with a suitable treatment plan in a timely fashion. Moreover, it is accepted that inflammation influences the outcome of the treatment, but the role of this host response remains controversial, being described to control as well as promote tumor recurrence and progression. Because of the lack of means to monitor inflammation in vivo, this host response is not taken in consideration by current therapies, thus preventing further understanding of its clinical significance. Herein we have addressed this diagnostic problem and have established a molecular MRI technology that tracks the spatiotemporal evolution of peri- and intra-tumoral inflammation while monitoring glioma response to treatment. Because this technology utilizes a stable gadolinium-based compound, it has a strong translational potential.