Mapping in vivo tumor oxygenation within viable tumor by 19F-MRI and multispectral analysis

Abstract

Quantifying oxygenation in viable tumor remains a major obstacle toward a better understanding of the tumor micro-environment and improving treatment strategies. Current techniques are often complicated by tumor heterogeneity. Herein, a novel in vivo approach that combines (19)F magnetic resonance imaging ((19)F-MRI) R 1 mapping with diffusion-based multispectral (MS) analysis is introduced. This approach restricts the partial pressure of oxygen (pO2) measurements to viable tumor, the tissue of therapeutic interest. The technique exhibited sufficient sensitivity to detect a breathing gas challenge in a xenograft tumor model, and the hypoxic region measured by MS (19)F-MRI was strongly correlated with histologic estimates of hypoxia. This approach was then applied to address the effects of antivascular agents on tumor oxygenation, which is a research question that is still under debate. The technique was used to monitor longitudinal pO2 changes in response to an antibody to vascular endothelial growth factor (B20.4.1.1) and a selective dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor (GDC-0980). GDC-0980 reduced viable tumor pO2 during a 3-day treatment period, and a significant reduction was also produced by B20.4.1.1. Overall, this method provides an unprecedented view of viable tumor pO2 and contributes to a greater understanding of the effects of antivascular therapies on the tumor's microenvironment.

Figure 1

Anatomic images of PFC uptake in an HM-7 xenograft tumor. (A) The ¹⁹F density images acquired in the same anatomic locations with ¹H density images reveal variable, albeit adequate PFC uptake. (B) A sample image of PFC uptake from the second slice in A is highlighted in red. The PFC uptake was heterogeneous. Some areas showed strong uptake of PFCs in the center of the tumor slice (green arrowheads). (C) The corresponding KM class map for the slice in B revealed that strong uptake of PFCs occurred in some areas of viable tumor, some areas of adipose tissue, and the low-T₂ necrosis class.

Figure 2

Repeated pO₂ measurements revealed no significant change over time in the HM-7 mouse xenograft tumor model. (A) Two consecutive pO₂ data sets were obtained under normoxia (black and blue). There was no significant change of mean pO₂ over time in all four tissue classes. The overall tumor response is a weighted average of all four tissue classes. Data are shown as means ± SEM. (B) An example of a pO₂ spatial map for the same tumor slice generated from the two consecutive measurements.

Figure 3

The change in mean pO₂ under hyperoxia challenge for the MS tissue classes. (A) pO₂ data were acquired under normoxia (black) and once again under carbogen (95% O₂, 5% CO₂) breathing gas challenge (red). The pO₂ in viable tumor class increased from 45.73 ± 4.30 to 61.06 ± 5.98 mm Hg, and the adipose tissue class and low-T₂ necrosis class also exhibited a significant increase of pO₂. The high-ADC necrosis class showed no change in pO₂. The overall response is a weighted average of the four tissue classes. Data are shown as means ± SEM. (B) An example of a pO₂ spatial map response to hyperoxia challenge. There was a heterogeneous response within the tumor, some areas increased dramatically, whereas other regions did not respond. *P < .05 and ***P < .001; NS, not significant.

Figure 4

MS ¹⁹F-MRI-identified hypoxic regions in viable tumor correlate with histology. (A) A one-to-one comparison between the hypoxic area in viable tissue for the center slice of each tumor measured by MS ¹⁹F-MRI and pimonidazole-positive area in viable tissue in the corresponding tumor slice measured by histology showed a significant correlation. (B) The MRI hypoxia region was shown in red, and the histologic hypoxia area was shown in brown. The images showed similar patterns of hypoxia (pointed by arrows).

Figure 5

At 24 hours posttreatment, B20.4.1.1 exhibited a significant decrease in viable tumor pO₂. (A) The B20.4.1.1-treated group showed no significant change in viable tumor volume (NS, P > .05). (B) The viable tumor pO₂ was significantly reduced in the B20.4.1.1 group relative to the pretreatment level (^###P < .001) as well as in comparison with the control group (*P < .05). (C) Images showing pO₂ change after 24 hours.

Figure 6

At 24 hours posttreatment, B20.4.1.1 significantly decreased vascular density and increased hypoxic fraction in the viable tumor. (A) Images show vessel density (brown, MECA-32 staining) at 24 hours posttreatment. Bar, 100 µm. (B) The B20.4.1.1 group showed a 40% reduction of vascular density in comparison with the control (**P < .01). (C) Images depict hypoxic cells (brown, hypoxyprobe staining) at 24 hours posttreatment. Bar, 100 µm. (D) The B20.4.1.1 group showed a 13% increase in the hypoxic fraction in comparison with the control (*P < .02).

Figure 7

(A) PFCs remained in the tumor throughout the course of the 4-day study following i.v. delivery. No significant loss of ¹⁹F signal was observed, which enabled a multiday longitudinal study of pO₂ change. Every effort was made to locate the same imaging slice in the tumor according to the distance and shape, but deviations may still exist due to the positioning of the animal on different days, as well as tumor growth. (B) The temporal evaluation of mean ΔpO₂ in viable tumor. Both the B20.4.1.1 and GDC-0980 groups exhibited a decrease in pO₂ in the viable tumor posttreatment relative to pretreatment levels. When compared with the control group, the GDC-0980 group exhibited a significant decrease in viable tumor pO₂, whereas the B20.4.1.1 group showed a trend toward a reduction of pO₂ (P = .14 on day 1). (C) The viable tumor volume change with time. Only the GDC-0980 group showed a significant reduction on day 3 in comparison with the control. *P < .05 in comparison with the control group. ^#P < .05 versus pretreatment; ^##P < .01 versus pretreatment. Data are shown as means ± SEM.

Figure 8

At 72 hours posttreatment, GDC-0980 significantly increased the hypoxic fraction in viable tumor as assessed by histology. (A) Images showed hypoxic cells at 72 hours posttreatment. Bar, 300 µm. (B) GDC-0980 showed a significant increase in hypoxic fraction (*P < .05), whereas the B20.4.1.1-treated group showed no significant change (P > .05).