Optical molecular imaging detects changes in extracellular pH with the development of head and neck cancer

Abstract

Non-invasive localized measurement of extracellular pH in cancer tissues can have a significant impact on the management of cancer. Despite its significance, there are limited approaches for rapid and non-invasive measurement of local pH in a clinical environment. In this study, we demonstrate the potential of noninvasive topical delivery of Alexa-647 labeled pHLIP (pH responsive peptide conjugated with Alexa Fluor® 647) to image changes in extracellular pH associated with head and neck squamous cell carcinoma using widefield and high resolution imaging. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of Alexa-647 labeled pHLIP in intact fresh human tissue specimens using widefield and high-resolution fluorescence imaging. Using topical delivery, Alexa-647 labeled pHLIP can be rapidly delivered throughout the epithelium of intact tissues with a depth exceeding 700 microns. Following labeling with Alexa-647 labeled pHLIP, the mean fluorescent contrast of increased 4 to 8 fold higher in clinically abnormal tissues as compared to paired clinically normal biopsies. Furthermore, the imaging approach showed significant differences in fluorescence contrast between the cancer and the normal biopsies across diverse patients and different anatomical sites (unpaired comparison). The fluorescence contrast differences between clinically abnormal and normal tissues were in agreement with the pathologic evaluation. Topical application of fluorescently labeled pHLIP can detect and differentiate normal from cancerous tissues using both widefield and high resolution imaging. This technology will provide an effective tool to assess tumor margins during surgery and improve detection and prognosis of head and neck cancer.

Targeting acidity in diseased tissues: Mechanism and applications of the membrane-inserting peptide, pHLIP

“…Given the close correspondence of the pH of the tumor microenvironment to pHLIPs’ pK ins , pHLIPs preferentially accumulate in tumors relative to healthy tissues, which generally exhibit local environments of pH 7.4. Fluorescently labeled pHLIPs have so far been shown in eleven published studies to accumulate in tumors with a high degree of contrast to healthy tissues (6–12, 15–18). …”

“…pH-Low Insertion Peptides (pHLIPs) are being developed for applications in several biomedical fields to exploit their specific biodistribution into tumors and other acidic tissue microenvironments, including drug delivery (1–5), in vivo imaging (6–17) and diagnostic histology (18), as well as in basic research (19–24). pHLIP peptides target acidic tissues such as tumors due to a serendipitous biophysical coincidence.…”

Comparative Study of Tumor Targeting and Biodistribution of pH (Low) Insertion Peptides (pHLIP® Peptides) Conjugated with Different Fluorescent Dyes

“…We have introduced family of pHLIP ® peptides with pK of insertion varying from 4.5 to 6.5 [10]. The targeting of primary tumors by pHLIP ® peptides and delivery imaging and therapeutic agents has been demonstrated on more than 10 varieties of human and murine tumors [15–17], including lymphoma [18], pancreatic [19], breast [20–21] and prostate [22] transgenic mouse models and human head/neck biopsy samples [23–24]. It was confirmed that tumor targeting by pHLIP ® peptides is indeed pH-dependent and could be modulated by bicarbonate and glucose, which affects tumor acidity [15, 20–21, 25–26].…”

The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions

“…Clinical applications have focused on the topical application of pHLIP onto biopsies from human head and neck cancer patients, where pHLIP detected cancerous tissue within the samples (46,47).…”