High-throughput, label-free, single-cell photoacoustic microscopy of intratumoral metabolic heterogeneity

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Intratumoral heterogeneity, which is manifested in almost all of the hallmarks of cancer, including the significantly altered metabolic profiles of cancer cells, represents a challenge to effective cancer therapy. High-throughput measurements of the metabolism of individual cancer cells would allow direct visualization and quantification of intratumoral metabolic heterogeneity, yet the throughputs of current measurement techniques are limited to about 120 cells per hour. Here, we show that single-cell photoacoustic microscopy can reach throughputs of approximately 12,000 cells per hour by trapping single cells with blood in an oxygen-diffusion-limited high-density microwell array and by using photoacoustic imaging to measure the haemoglobin oxygen change (that is, the oxygen consumption rate) in the microwells. We demonstrate the capability of this label-free technique by performing high-throughput single-cell oxygen-consumption-rate measurements of cultured cells and by imaging intratumoral metabolic heterogeneity in specimens from patients with breast cancer. High-throughput single-cell photoacoustic microscopy of oxygen consumption rates should enable the faster characterization of intratumoral metabolic heterogeneity.

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Fig. 1: System schematic and working modes of SCM-PAM.
Fig. 2: SCM-PAM of single-cell trapping and oxygen sealing in a high-density microwell array.
Fig. 3: SCM-PAM of cellular metabolic heterogeneity in cultured cells.
Fig. 4: SCM-PAM of intratumoral metabolic heterogeneity in a breast cancer patient.
Fig. 5: Elevated and chaotic cellular metabolic heterogeneity in cancer measured by SCM-PAM.
Fig. 6: Oxygen consumption of cancer and normal cells in hypoxia measured by SCM-PAM.

Data availability

The authors declare that all data supporting the results in this study are available within the paper and its Supplementary information. The source data for the figures in this study are available in (identifier figshare)50.


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The authors appreciate the help received from J. Yao in the initial phase of the project and J. Liang in the data analysis while they were former members of our laboratory. We also thank J. Ballard for his close reading of the manuscript. This work was sponsored by National Science Foundation (grant nos 1255930 and 1255921) and National Institutes of Health (grant nos DP1 EB016986 (NIH Director’s Pioneer Award) and R01 CA186567 (NIH Director’s Transformative Research Award)).

Author information

P.H., J.Z. and L.V.W. conceived and designed the study. P.H. and T.I. developed the photoacoustic imaging system. S.X. fabricated the high-density microwell array. R.Z. cultured the cells. R.L.A. provided the excised breast cancer and normal tissues. P.H. and T.I. performed the experiments. P.H. and T.I. analysed the data. L.V.W. supervised the study. All authors wrote the manuscript.

Correspondence to Jun Zou or Lihong V. Wang.

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Competing interests

L.V.W. has a financial interest in Microphotoacoustics, Inc.; CalPACT, LLC and Union Photoacoustic Technologies, Ltd. These companies did not support this work.

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