Oxygen—odorless, colorless, and tasteless—makes up about 65% of the human body.1 Oxygen regulates all of the human body's functions. For example, it energizes our cells, fights aging, and burns up toxins. In fact, almost all diseases are linked to abnormal oxygen metabolism. Cancers are especially starved for oxygen, so measuring tissue oxygenation is a key tool in cancer diagnosis and prognosis.2,3 At a very early stage of carcinogenesis, epithelial (i.e., inner lining) cells become hyperproliferative and consume much more oxygen to maintain their fast growth.4 This hypermetabolism of oxygen is a hallmark of early stage cancer.5
The ability to accurately map oxygen metabolism would be useful for early cancer detection, which greatly increases the success rate of treatment and hence also the chances of patients' survival.6 The metabolic rate of oxygen (MRO2) directly reflects actual oxygen consumption instead of static oxygen concentration, so it is a superior oxygenation index of biological tissue compared to other existing indices (oxygen saturation of hemoglobin sO2, and partial oxygen pressure pO2).7 MRO2 can quantify cancer hypermetabolism and brain functions in absolute units.
Figure 1. (a) Schematic of metabolic photoacoustic microscopy (mPAM) system. A transverse resolution of 5μm and an axial resolution of 15μm are achieved. AL: Acoustic lens; UT: Ultrasonic transducer; WT: Water tank; MS: Motorized scanner. (b) Total concentration of hemoglobin (CHb) in a mouse ear by mPAM. Scale bar: 500μm. (c) Magnified mPAM of area in dash box in (b) of the oxygen saturation, sO2, and (d) blood flow. Scale bar: 125μm.
Figure 2. Label-free mPAM of hypermetabolism and hyperoxia of early stage cancer. (a) Photograph (scale bar: 2mm) of a mouse ear bearing a xenographed glioblastoma on Day 7. (b) Color-coded (blue: superficial to red: deep) mPAM image (scale bar: 200μm) of the vasculature of the mouse ear. (c) An mPAM image (scale bar: 200μm) of the tumor region in (b) of the sO2 shows clearly that the tumor region is hyperoxic. (d) Quantification of the MRO2change induced by tumor growth shows a 100% increase in MRO2that indicates the tumor hypermetabolism. (e) Quantification of the averaged sO2inside and outside the tumor further confirms the hyperoxia of the tumor region.
Current techniques for MRO2 quantification, such as positron emission tomography and functional magnetic resonance imaging, either lack the needed spatial resolution for studying the microenvironment of tumors, rely on radioactively labeled exogenous tracers, or both.8 We have developed label-free metabolic photoacoustic microscopy (mPAM) with capillary resolution to quantify MRO2 noninvasively in vivo in absolute units.9 The mPAM technique provides a means to measure optical contrast ultrasonically through the photoacoustic (PA) effect by pinpointing the microvasculature, exciting the hemoglobin molecules with a laser, and then listening to the sound generated by the excited molecules (see Figure 1a). The method is unique for simultaneously imaging all five anatomical, chemical, and fluid-dynamic parameters required for such quantification: tissue volume, vessel cross-section, concentration of hemoglobin, oxygen saturation of hemoglobin, and blood flow speed.
Using endogenous contrast, mPAM has shown a robust capability to non-invasively image microvasculature with high spatial resolution (lateral: ∼5μm; axial: ∼15μm). For MRO2 quantification, the anatomic parameters (tissue volume and vessel cross-section) are quantified from the structural mPAM image. Functional parameters (total concentration and oxygen saturation of hemoglobin) are measured by laser excitation at two wavelengths: see Figure 1(b and c). We estimate blood flow speed on the basis of photoacoustic bandwidth broadening of the PA signal which is induced by circulating red blood cells: see Figure 1(d).
To validate mPAM, we studied the MRO2 responses to hyperthermia and cryotherapy, two common therapeutic techniques for cancers.2 We further used mPAM to image the MRO2 change of melanoma (skin cancer) and glioblastoma (brain cancer) longitudinally, demonstrating its capability for early cancer detection.2
We found that under hyperthermia (42°C), the MRO2 of the mouse ear increased by 28%, which indicated elevated oxygen metabolism in response to an increased rate of enzymatic reactions. We also found that after cryotherapy, the MRO2 of the treated area decreased by 56% due to induced cell death. After one month, the MRO2 of the treated area returned to the baseline, reflecting improved tissue viability. Therefore, we believe that mPAM could be used to evaluate hyperthermia and cryotherapy cancer treatments.
We then considered whether mPAM is capable of early cancer detection on the basis of MRO2 measurement. The hypermetabolism of melanoma and glioblastoma was reflected by 36% and 100% increases in MRO2 in the first week, respectively, which proves the early cancer detection capability of mPAM: see Figure 2(a) and (b). However, for early-stage cancer, such hypermetabolism did not lead to tumor hypoxia: see Figure 2(c) and 2(d). In fact, the sO2 in the intratumoral vasculature was even higher than that of the surrounding normal tissue, directly indicating early-stage tumor hyperoxia: see Figure 2(e). This finding suggests that hypoxia-based diagnosis may not apply to early-stage cancer.
In summary, we demonstrated the power of mPAM as the only noninvasive label-free imaging modality that can measure all of the parameters required for the quantification of MRO2 in absolute units at the resolution of single capillaries, making it well suited for MRO2 quantification in tumor microenvironments. Our future work will pursue two directions. The first is to improve mPAM to image tissue beyond the optical diffusion limit. The second is to apply mPAM to prospective applications related to MRO2, such as diabetes, mini-strokes, neuro-vascular coupling, and epileptic seizures.
The authors thank Professor James Ballard for manuscript editing. This research was supported by the National Institutes of Health Grants R01 EB000712, R01 EB008085, R01 CA134539, U54 CA136398, R01 EB010049, and 5P60 DK02057933.
Junjie Yao, Lihong Wang
Department of Biomedical Engineering
Washington University in St. Louis
Saint Louis, MO
Junjie Yao is a graduate student whose research interests are the development of novel biomedical imaging techniques including photoacoustic imaging, diffused optical imaging, and ultrasonic imaging.
Lihong Wang currently holds the Gene K. Beare Distinguished Professorship. He is an SPIE Fellow and also a fellow of the American Institute for Medical and Biological Engineering, the Optical Society of America, and the Institute of Electrical and Electronics Engineers.
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