Super-resolution imaging methods promote tissue characterization beyond the spatial resolution limits of the devices and bridge the gap between histopathological analysis and non-invasive imaging. Here, we introduce motion model ultrasound localization microscopy (mULM) as an easily applicable and robust new tool to morphologically and functionally characterize fine vascular networks in tumors at super-resolution. In tumor-bearing mice and for the first time in patients, we demonstrate that within less than 1 min scan time mULM can be realized using conventional preclinical and clinical ultrasound devices. In this context, next to highly detailed images of tumor microvascularization and the reliable quantification of relative blood volume and perfusion, mULM provides multiple new functional and morphological parameters that discriminate tumors with different vascular phenotypes. Furthermore, our initial patient data indicate that mULM can be applied in a clinical ultrasound setting opening avenues for the multiparametric characterization of tumors and the assessment of therapy response.

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In clinical applications of super-resolution ultrasound imaging, it is often not possible to achieve a full reconstruction of the microvasculature within a limited measurement time. This makes the comparison of studies and quantitative parameters of vascular morphology and perfusion difficult. Therefore, saturation models were proposed to predict adequate measurement times and estimate the degree of vessel reconstruction. Here, we derive a statistical model for the microbubble counts in super-resolution voxels by a zero-inflated Poisson (ZIP) process. In this model, voxels either belong to vessels with probability P v and count events with Poisson rate , or they are empty and remain zero. In this model, P v represents the vessel voxel density in the super-resolution image after infinite measurement time. For the parameters P v and , we give Cramér-Rao lower bounds (CRLBs) for the estimation variance and derive maximum likelihood estimators (MLEs) in a novel closedform solution. These can be calculated with knowledge of only the counts at the end of the acquisition time. The estimators are applied to preclinical and clinical data and the MLE outperforms alternative estimators proposed before. The estimated degree of reconstruction lies between 38% and 74% after less than 90 s. Vessel probability P v ranged from 4% to 20%. The rate parameter was estimated in the range of 0.5-1.3 microbubbles/voxel. For these parameter ranges, the CRLB gives standard deviations of less than 2%, which supports that the parameters can be estimated with good precision already for limited acquisition times.

Purpose: Preclinical and clinical data indicate that contrast-enhanced ultrasound can enhance tumor perfusion and vessel permeability, thus, improving chemotherapy accumulation and therapeutic outcome. Therefore, we investigated the effects of high mechanical index (MI) contrast-enhanced Doppler ultrasound (CDUS) on tumor perfusion in breast cancer. Methods: In this prospective study, breast cancer patients were randomly assigned to receive either 18 minutes of high MI CDUS during chemotherapy infusion (n = 6) or chemotherapy alone (n = 5). Tumor perfusion was measured before and after at least six chemotherapy cycles using motion-model ultrasound localization microscopy. Additionally, acute effects of CDUS on vessel perfusion and chemotherapy distribution were evaluated in mice bearing triple-negative breast cancer (TNBC). Results: Morphological and functional vascular characteristics of breast cancer in patients were not significantly influenced by high MI CDUS. However, complete clinical tumor response after neoadjuvant chemotherapy was lower in high MI CDUS-treated (1/6) compared to untreated patients (4/5) and size reduction of high MI CDUS treated tumors tended to be delayed at early chemotherapy cycles. In mice with TNBC high MI CDUS decreased the perfused tumor vessel fraction (p < 0.01) without affecting carboplatin accumulation or distribution. Higher vascular immaturity and lower stromal stabilization may explain the stronger vascular response in murine than human tumors. Conclusion: High MI CDUS had no detectable effect on breast cancer vascularization in patients. In mice, the same high MI CDUS setting did not affect chemotherapy accumulation although strong effects on the tumor vasculature were detected histologically. Thus, sonopermeabilization in human breast cancers might not be effective using high MI CDUS protocols and future applications may rather focus on low MI approaches triggering microbubble oscillations instead of destruction. Furthermore, our results show that there are profound differences in the response of mouse and human tumor vasculature to high MI CDUS, which need to be further explored and considered in clinical translation.

Super-resolution imaging methods promote tissue characterization beyond the spatial resolution limits of the devices and bridge the gap between histopathological analysis and non-invasive imaging. Here, we introduce Ultrasound Bubble Tracking (UBT) as an easily applicable and robust new tool to morphologically and functionally characterize fine vascular networks in tumors at super-resolution. In tumor-bearing mice and for the first time in patients, we demonstrate that within less than one minute scan time UBT can be realized using conventional preclinical and clinical ultrasound devices. In this context, next to highly detailed images of tumor microvascularization and the reliable quantification of relative blood volume and perfusion, UBT provides access to multiple new functional and morphological parameters that showed superior performance in discriminating tumors with different vascular phenotypes.Furthermore, our initial clinical results indicate that UBT is a highly translational technology with strong potential for the multiparametric characterization of tumors and the assessment of therapy response.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.