Oncologic surgery is practiced today as it has been for millennia; the surgeon looks for and sees the tumor, and executes a strategy for the most complete and efficient removal. Intraoperatively surgeons still largely rely on inspection and palpation to determine surgical margins in real-time. Given this inherently imprecise technique, positive tumor margins are encountered often. For example, a second operation, due to inadequate tumor removal during breast cancer surgery, is required for 20 – 25 percent of patients, delaying recovery, causing patient trauma, and adding to healthcare costs as well as worsening patient prognosis. Techniques that can improve the intraoperative visualization of tumor margins would improve the completeness of surgical resection while minimizing the removal of normal tissue.

Surgeons rely heavily on preoperative imaging with modalities such as computed tomography (CT) and magnetic resonance imaging (MRI), but these do not provide real-time information to the surgeon during the operation and do not easily mesh with the optical and tactile methods that surgeons have used since the earliest days of surgery. To integrate imaging into the surgical workflow, optical imaging strategies are being developed using fluorescent probes, targeting tumor markers 1,2. One limitation of the marker-targeting strategy is the lack of broad tumor applicability in cancer patients. For example, although the folate receptor is widely expressed in ovarian cancer, expression in other cancers, such as head-and-neck cancer, is limited. Even within a given type of cancer, expression can be variable; < 25 percent breast cancer patients have Her2 expression.

To achieve universal tumor targeting, our group has been working with ubiquitous tumor markers, or “hallmarks of cancer”. We have previously shown that targeting two such hallmarks, angiogenesis and the acidic tumor extracellular microenvironment, allows for imaging of a wide variety of cancers using fluorescence. We have subsequently discovered that the acidic cancer microenvironment, first described by Otto Warburg, can be imaged in a unique way that enhances the information provided to surgeons.

One important reason for the slow adoption of image-guided surgery is that variable target expression results in variable fluorescence. This ambiguity does not provide sufficient additional data to the surgeon to help with the decision of whether to remove tissue. To overcome this ambiguity, we turned to a library of pH sensitive nanoprobes with binary on/off fluorescent responses that are finely tuneable in a broad range of physiological pH (4.0–7.4). The ultra pH-sensitive property is a unique nanoscale phenomenon arising from a catastrophic phase transition during pH-triggered self-assembly of amphiphilic copolymers. At the molecular level, an all-or-nothing protonation distribution in cationic unimers or neutral micelles, respectively, was observed, in contrast to what occurs with conventional polymeric bases.

The resulting pH cooperativity results in a dramatically sharpened pH response (∆pHoff/on < 0.2 pH), compared to 2 pH units for small molecular pH sensors. We adopted a nanoprobe with a pKa of 6.9 as a pH threshold sensor to image the acidic tumor microenvironment. This nanoprobe stays completely dark at physiologic pH, and is activated to full fluorescence in a variety of tumors, because most tumors have an average extracellular pH around 6. A nearly binary tumor fluorescence was achieved over the muscle background for margin delineation that correlates with the histologic boundary of the tumor. This sharp tumor fluorescence allowed real-time image-guided surgery with significantly improved long-term survival in mice bearing breast and head-and-neck cancers.

Surgeons use complex pattern recognition (acquired by surgical experience) to distinguish tumor from normal tissue. The final decision, however, is binary; to remove or not to remove tissue. To help with that decision, an imaging agent must minimize ambiguous signals. The binary response of the nanoprobes achieves this when compared to probes that respond linearly to target concentrations. The demonstration of the value of that binary response is an exciting and tremendously important step for image-guided surgery.