Characterization of Anti-Angiogenic Chemo-Sensitization via Longitudinal Ultrasound Localization Microscopy in Colorectal Carcinoma Tumor Xenografts

Matthew Lowerison, Wei Zhang, Xi Chen, Timothy Fan, Pengfei Song, Matthew Lowerison, Wei Zhang, Xi Chen, Timothy Fan, Pengfei Song

Abstract

Objective: Super-resolution ultrasound localization microscopy (ULM) has unprecedented vascular resolution at clinically relevant imaging penetration depths. This technology can potentially screen for the transient microvascular changes that are thought to be critical to the synergistic effect(s) of combined chemotherapy-antiangiogenic agent regimens for cancer.

Methods: In this paper, we apply this technology to a high-throughput colorectal carcinoma xenograft model treated with either the antiangiogenic agent sorafenib, FOLFOX-6 chemotherapy, a combination of the two treatments, or vehicle control.

Results: Longitudinal ULM demonstrated morphological changes in the antiangiogenic treated cohorts, and evidence of vascular disruption caused by chemotherapy. Gold-standard histological measurements revealed reduced levels of hypoxia in the sorafenib treated cohort for both of the human cell lines tested (HCT-116 and HT-29). Therapy resistance was associated with an increase in tumor vascular fractal dimension as measured by a box-counting technique on ULM images.

Conclusion: These results imply that the morphological changes evident on ULM signify a functional change in the tumor microvasculature, which may be indicative of chemo-sensitivity.

Significance: ULM provides additional utility for tumor therapy response evaluation by offering a myriad of morphological and functional quantitative indices for gauging treatment effect(s).

Figures

Fig. 1.
Fig. 1.
Study design diagram. A) At EDD-09 a total of 52 CAMs were engrafted with the HCT-116 cell line and 47 CAMs were engrafted with the HT-29 cell line. B) The tumor engrafted CAMs were randomized, and a pretreatment ULM imaging session was performed on EDD-13. C) The tumors underwent their selected therapy as described in the methods section, and a post-treatment ULM imaging session was performed on EDD-17. Tumors were excised immediately after imaging and formalin fixed for histology.
Fig. 2.
Fig. 2.
ULM acquisition and processing pipeline. A) A reference transducer acquisition was used to estimate a noise equalization profile to correct for depth dependent attenuation. B) A total of 32000 contrast-enhanced IQ data frames were acquired and SVD filtered to generate isolated microbubble data and C) contrast-enhanced power Doppler images. The isolated microbubble data then went into the ULM processing pipeline, which included microbubble separation, microbubble localization, and microbubble pairing and tracking. D) The final super-resolved images were used to quantify the treatment effect in each CAM tumor therapy group.
Fig. 3.
Fig. 3.
Longitudinal changes in CAM tumors. A) Representative baseline contrast-enhanced power Doppler and super-resolution ULM images of CAM engrafted tumors. B) This same control tumor at endpoint imaging, which demonstrates both the growth of the tumor diameter and the continued development of intratumor vascularization over the course of the study. Tumor outlines and maximal diameter are demonstrated as cyan and orange lines on the B-mode image, respectively. C) Quantification of the tumor long-axis which reveals a general trend of tumor growth for all treatment groups in this study. D) Quantification of the contrast power of the tumor cross-sectional area.
Fig. 4.
Fig. 4.
Super-resolution images of treatment effect. A) Endpoint (EDD-17) control ULM images reveal a dense microvascular network throughout the tumor mass. B) In comparison to the control tumors, the sorafenib treated tumors exhibited slight vascular pruning of microvessels, with a more columnar appearance in vascular network structure (arrow). C) Chemotherapy treated tumors displayed avascular tumor regions, implying a more aggressive vascular pruning. D) Combination treated tumors also exhibit avascular tumor regions.
Fig. 5.
Fig. 5.
Histological comparison and quantifications. A) The super-resolution vascular map can be compared qualitatively to the B) histological staining in cases where the imaging plane was relatively aligned to the histology cross-section. Hypoxyprobe IHC, a marker of hypoxia, demonstrated darker staining in the tumor regions distant from the CAM membrane (left inset). In comparison, the regions closer to the membrane had reduced hypoxia staining (right inset). Ki-67 staining was prevalent across the depth of the tumor. C) Quantifications of the hypoxyprobe staining (top row) and Ki67 staining (bottom row) for both the HCT-116 and HT-29 cell lines for all treatment groups.
Fig. 6.
Fig. 6.
Quantification of tumor vascularization. A) HCT-116 tumors showed a trend toward reduced blood volume for the cohorts treated with sorafenib, with a significant difference detected in the combination therapy group. B) The intervessel distance for this cell line demonstrated significant decreases for the control group, increased for both chemotherapy treatments, and little change for the sorafenib monotherapy. C) Tumor mean blood velocity trended toward slower flow, in particular for the combination therapy group. D) HT-29 tumors also demonstrated a longitudinal trend of reduced blood volume, but no significance difference was detected. E) Intervessel distance was relatively static for the HT-29 cell line for all treatment groups. F) Mean blood velocity also demonstrated a trend of reduced velocity, but no significance was found.
Fig. 7.
Fig. 7.
Hausdorff box-counting of ULM images. The Hausdorff fractal dimension of ULM vascular maps was estimated using the slope generated using a multi-dimensional box-counting algorithm. Interestingly, all tumor groups treated with the AA agent sorafenib showed a significant increase in Hausdorff dimension. The HT-29 FOLFOX-treated tumors also demonstrated a significant increase in Hausdorff dimension but the opposite trend was noted in HCT-116 tumors treated with FOLFOX.
Fig. 8.
Fig. 8.
Measures of vascular tortuosity. Vascular tortuosity was measured using the distance metric (DM) and the sum of angle metric (SOAM). For the HCT-116 cell line, the A) DM was found to significantly increase in the combination therapy group and B) SOAM measures were found to significantly increase in both sorafenib containing therapies. The HT-29 cell line showed C) a significantly increased DM for FOLFOX treated tumors, and D) increased SOAM in the FOLFOX and combination therapy treated groups.

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