Ultrasound molecular imaging (UMI) has shown promise for assessing the expression levels of biomarkers for the early detection of various diseases. However, it remains difficult to simultaneously image multiple biomarkers in a single systemic administration, which is important for the accurate diagnosis of diseases and for understanding the dynamic intermolecular mechanisms that drive their malignant progression. The authors develop an ultrasound molecular imaging method by serial collapse of targeting microbubbles with distinct acoustic pressures for the simultaneous detection of two biomarkers. To test this, αvβ3‐targeting lipid microbubbles (L‐MBα) and VEGFR2‐targeting lipid‐PLGA microbubbles (LP‐MBv) are fabricated and simultaneously injected into tumor‐bearing mice at 7 and 14 days, followed by the low‐intensity acoustic collapse of L‐MBα and high‐intensity acoustic collapse of LP‐MBv. The UMI signals of L‐MBα and LP‐MBv are obtained by subtracting the first post‐burst signals from the first pre‐burst signals, and subtracting the second post‐burst signals from the first post‐burst signals, respectively. Interestingly, the signal intensities from UMI agree with the immunohistochemical staining results for αvβ3 and VEGFR2. Importantly, they find a better fit for the invasive behavior of MDA‐MB‐231 breast tumors by analyzing the ratio of αvβ3 integrin to VEGFR2, but not the single αvβ3 or VEGFR2 levels.

Epithelial mesenchymal transition (EMT) of tumor cells is recognized as the main driver to promote metastasis. Extensive researches suggest that gradually decreased E‐cadherin (E‐cad) and increased N‐cadherin (N‐cad) exist in the tumor cells during the EMT process. However, there still lacks suitable imaging methods to monitor the status of EMT for evaluating tumor metastatic potentials. Herein, the E‐cad‐targeted and N‐cad‐targeted gas vesicles (GVs) are developed as the acoustic probes to monitor the EMT status in tumor. The resulting probes have ≈200 nm particle size and good tumor cell targeting performance. Upon systemic administration, E‐cad‐GVs and N‐cad‐GVs can traverse through blood vessels and bind to the tumor cells, producing strong contrast imaging signals in comparison with the nontargeted GVs. The contrast imaging signals correlate well with the expression levels of E‐cad and N‐cad and tumor metastatic ability. This study provides a new strategy to noninvasively monitor the EMT status and help to evaluate tumor metastatic potential in vivo.

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